ISSN isflD-3iss Pages 1-245 Year 2019, Vol. 66, No. 1 Acta ChimicaSlc Acta Chimica Slc Slovenica ActaC 66/2019 http://acta.chem-soc.si EDITOR-IN-CHIEF ASSOCIATE EDITORS KSENIJA KoGEJ Slovenian Chemical Society, Hajdrihova 19, SI-1000 Ljubljana, Slovenija, E-mail: ACSi@fkkt.uni-lj.si, Telephone: (+386)-1-479-8538 GUEST-EDITOR Janez Cerkovnik, University of Ljubljana, Slovenia Krištof Kranjc, University of Ljubljana, Slovenia Ksenija Kogej, University of Ljubljana, Slovenia Franc Perdih, University of Ljubljana, Slovenia Aleš Podgornik, University of Ljubljana, Slovenia Helena Prosen, University of Ljubljana, Slovenia Damjana Rozman, University of Ljubljana, Slovenia Melita Tramšek, Jožef Stefan Institute, Slovenia Irena Vovk, National Institute of Chemistry, Slovenia Vito Turk, Jožef Stefan Institute, Slovenia Borut Štrukelj, Faculty of Pharmacy, Slovenia Aleš Berlec, Jožef Stefan Institute, Slovenia ADMINISTRATIVE ASSISTANT Marjana Gantar Albreht, National Institute of Chemistry, Slovenia EDITORIAL BOARD Wolfgang Buchberger, Johannes Kepler University, Austria Alojz Demšar, University of Ljubljana, Slovenia Stanislav Gobec, University of Ljubljana, Slovenia Marko Goličnik, University of Ljubljana, Slovenia Günter Grampp, Graz University of Technology, Austria Wojciech Grochala, University of Warsaw, Poland Danijel Kikelj, Faculty of Pharmacy, Slovenia Janez Košmrlj, University of Ljubljana, Slovenia Blaž Likozar, National Institute of Chemistry, Slovenia Mahesh K. Lakshman, The City College and The City University of New York, USA Janez Mavri, National Institute of Chemistry, Slovenia Friedrich Srienc, University of Minnesota, USA Walter Steiner, Graz University of Technology, Austria Jurij Svete, University of Ljubljana, Slovenia Ivan Švancara, University of Pardubice, Czech Republic Jiri Pinkas, Masaryk University Brno, Czech Republic Gašper Tavčar, Jožef Stefan Institute, Slovenia Christine Wandrey, EPFL Lausanne, Switzerland Ennio Zangrando, University of Trieste, Italy Chairman Branko Stanovnik, Slovenia Members Josef Barthel, Germany Udo A. Th. Brinkman, The Netherlands Attilio Cesaro, Italy Dusan Hadzi, Slovenia Vida Hudnik, Slovenia Venceslav Kaucic, Slovenia ADVISORY EDITORIAL BOARD Željko Knez, Slovenia Radovan Komel, Slovenia Janez Levec, Slovenia Stane Pejovnik, Slovenia Anton Perdih, Slovenia Slavko Pečar, Slovenia Andrej Petrič, Slovenia Boris Pihlar, Slovenia Milan Randic, Des Moines, USA Jože Škerjanc, Slovenia Miha Tišler, Slovenia Durda Vasic-Rački, Croatia Marjan Veber, Slovenia Gorazd Vesnaver, Slovenia Jure Zupan, Slovenia Boris Žemva, Slovenia Majda Žigon, Slovenia Acta Chimica Slovenica is indexed in: Chemical Abstracts Plus, Current Contents (Physical, Chemical and Earth Sciences), PubMed, Science Citation Index Expanded and Scopus. 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V primeru posvečenih številk izhaja revija tudi v tiskani obliki v omejenem številu izvodov. Acta Chimica Slovenica appears quarterly in electronic form on the web site http://acta.chem-soc.si. In case of dedicated issues, a limited number of printed copies are issued as well. Transakcijski račun: 02053-0013322846 Bank Account No.: SI56020530013322846-Nova Ljubljanska banka d. d., Trg republike 2, SI-1520 Ljubljana, Slovenia, SWIFT Code: LJBA SI 2X Oblikovanje ovitka - Design cover: KULT, oblikovalski studio, Simon KAJTNA, s. p. Grafična priprava za tisk: Majanafin, d. o. o. © Copyright by Slovenian Chemical Society ActaChimicaSlovenica Memorial Issue Dedicated to the memory of the late Prof. Dr. Igor Kregar (1937-2017) Igor Kregar, Slovenian and an internationally renowned biochemist in the field of proteolysis and plant molecular biology, unexpectedly passed away on April 11, 2017 in Ljubljana at the age of 79. After almost 40 years of conducting excellent research, teaching and mentoring of undergraduate and graduate students at the Jožef Stefan Institute and Biotechnical faculty at Ljubljana University, he had retired in year 1997 to enjoy his family, friends, travel and some hobbies. Igor was born on September 11, 1937 in Ljubljana, the son of architect Rado and mother Antonija. In his family reigned an intelectual atmosphere, which resulted in his broad interests for reading books, enjoying traveling, classical music, art and culture in general. Interestingly, as a boy he was highly motivated to read adventure novels set in the American Wild West written by German writer Karl May. Many of May's novels were translated into the Slovenian language, but unfortunately those editions were not accessible in our libraries after World War II for unknown reasons, most probably political. However, Igor did have access to the German editions, which stimulated him to learn the German language. At the same time Igor enjoyed competitive swimming at a local swimming pool. After completing high school with an excellent degree, he decided to study chemistry at the University of Ljubljana. At that time we first met. Soon we established a friendship that included my classmate Franček Gubenšek. Igor was one of the best students in his class. He chose organic chemistry for his diploma at the Faculty of Chemistry at Ljubljana University. Then Igor joined the Department of Radiobiology at the Nuclear Institute Jožef Stefan (now Jožef Stefan Institute) by enrolling in a master's degree program and completed his PhD dissertation in December 1965 under the mentorship of Prof. Drago Lebez, working on the characterization of intracellular proteinases from the small intestinal mucosa of the rat. At that time intracellular proteases were poorly understood. The main enzyme studied was an acidic hemoglobin-splitting enzyme, called Anson's enzyme in laboratory jargon after the pioneering protein biochemist M. L. Anson. After the lysosome organelle was discovered by C. de Duve, it became clear that this acid protease is a lysosomal enzyme, and in 1960 it was characterized and named cathepsin D. Igor and the author started to collaborate primarily on cathepsin D and also on cathepsin E soon after its discovery. This was at that time an almost unexplored field of research and we were one of the pioneering groups to work in this field. In 1968 Igor received a Fulbright scholarship to study in Professor John A. Rupley's laboratory in the Department of Chemistry at the University of Arizona, Tucson, USA. Coming from Yugoslavia at that time was for Igor (and later for the author) a completely new world. The Rupley laboratory was modern and very well equipped with adequate funding to buy chemicals. Igor studied the pH dependence of lysozyme-catalyzed hydrolysis of the N-acetylglucosamine hexasaccharide. These kinetic experiments were time consuming and required precise measurements, but later resulted in a high quality publication (1973). Rupley was an excellent biochemist with a great and warm but serious personality. Not to forget, Rupley and his lovely wife, Ila, organized nice parties in their home, thus keeping and promoting friendly relations in his international laboratory. I can say as one of Rupley's postdocs that those working with him were very fortunate and learned to enjoy hard work and at the same time to be happy. Igor established many friendships in the USA, especially with Rupley's graduate student, Karl Kramer and his wife Virginia, which lasted until Igor's death. In 1968 Igor joined the Kramers on a unique train trip to Mexico where they visited Guadalajara, Mexico City and Taxco. On that trip the train passed through many small Mexican communities, some with bullet-riddled buildings, which were perhaps a reflection of past political conflicts. Igor learned to love Mexican cuisine in part through his many visits to the Kramer's apartment, where Virginia made mouth-watering dishes such as enchiladas, refried beans, flautas, green chiles and spicy meats. Upon his return to Yugoslavia, these experiences motivated Igor to learn how to replicate Mexican meals and to share them with his family and good friends. There was a problem, however, in that many of the ingredients required for Mexican cooking were not readily available in Yugoslavia in the 1970s. Nevertheless, the Kramers were able to supply Igor with Mexican food items such as masa harina, a special red chile powder and various types of chiles in care packages until he was later able to obtain them locally. Returning to our Department at the Institute after his Fulbright-sponsored study in the USA, Igor continued his research on cathepsins. Development of the first purification of cathepsin D by affinity chromatography allowed him to obtain large quantities of the pure enzyme for numerous biochemical studies including structural and de-naturation studies. Almost at the same time an acid sul-phydryl protease was discovered (1973) and later named cathepsin S. This discovery was not immediatelly recognized and many believed that cathepsin S and L were identical enzymes. Ultimately, this problem was solved by isolating both proteins from the same species and determining their amino acid sequences. Cathepsin S is involved in antigen processing and presentation, degradation of the extracellular matrix, tumor progression, cardiovascular disease and obesity among other roles. A new method for cathepsin B purification from bovine lymph nodes yielded sufficient enzyme for biochemical characterization. All advances in the field resulted in the Second International Symposium on Intracellular Protein Catabolism organized in Ljubljana in 1975. A brief document was prepared by Alan Barrett, Fred Woessner, Igor Kregar and Vito Turk, which highlighted all known 23 intracellular proteases. This modest beginning resulted finally with the establishment of a new classification system and the MEROPS database, which now includes well over 1000 peptidases (Handbook of Proteolytic Enzymes, N.D. Rawlings and G. Salvesen, Preface, Acad. Press, 2013). Igor then extended his research on proteases to those present in the ergot fungus Claviceps purpurea and bacterium Streptomyces rimo-sus as well as proteases and their inhibitors from plants such as the potato. Igor and his coworkers discovered the potato cysteine proteinase inhibitor gene family and characterized some of the expressed recombinant proteins. Igor was a highly respected scientist and personality. His research resulted in approximately 80 original research papers and reviews published primarily in international journals. He attended many domestic and international symposia and congresses as an invited lecturer. In parallel to his contributions to science, he was an excellent teacher and popular among many students. Most of his students who obtained their PhD degree under his guidance went on to become successful researchers and professors at different universities and institutes. Igor served on various committees at the Institute. He was also the first Secretary General of the Slovenian Biochemical Society (1972-1987) and then Secretary General of the Union of Biochemical Societies of Yugoslavia (1987-1992) until the collapse of Yugoslavia. In 1987 the 18th meeting of the Federation of European Biochemical Societies (FEBS) was held for the first time in Ljubljana, which was a great success from the organizational and scientific points of view. Igor contributed greatly to this success, serving as secretary of this prestigious meeting with more than 1800 scientists attending from all over the world. For his achievements in science, Igor received several distinguished recognitions including the Yugoslav Order of Labour with Silver Wreath Award (1978), Slovenian Boris Kidrič Fund Award (1979) and Lapanje Plaque from the Slovenian Biochemical Society (2009). Igor's remarkable wife, Anka, died several months after he did. He is survived by two sons, Zlato and Robert, three grandchildren and their families. For us and for those who knew him, Igor always will be remembered. His scientific legacy will live on. Vito Turk Jožef Stefan Institute Graphical Contents ActaChimicaSlo ActaChimicaSlo SlovenicaActaC Year 2019, Vol. 66, No. 1 REviEw 5—17 Biochemistry and molecular biology Characteristics, Structure, and Biological Role of Stefins (Type-1 Cystatins) of Human, Other Mammals, and Parasite Origin Vito Turk, Dušan Turk, Iztok Dolenc and Veronika Stoka SCIENTIFIC PAPER 18—27 Biochemistry and molecular biology Surface Anchoring on Lactococcus lactis by Covalent Isopeptide Bond Tina Vida Plavec and Aleš Berlec 28—36 Biochemistry and molecular biology ^-Trefoil Protease Inhibitors Unique to Higher Fungi Jerica Sabotič, Miha Renko and Janko Kos Graphical Contents 37—44 Biochemistry and molecular biology Epitope Mapping of Major Ragweed Allergen Amb a 1 Abida Zahirovic, Borut Štrukelj, Peter Korošec and Mojca Lunder 45—49 Biochemistry and molecular biology The Titre of the Virus in the Inoculum Affects the Titre of the Viral RNA in the Host Plant and the occurrence of the Disease Symptoms Maruša Pompe-Novak, Maja Križnik and Kristina Gruden 50—57 Biochemistry and molecular biology Characterization of Legumain Degradome Confirms Narrow Cleavage Specificity Robert Vidmar, Matej Vizovišek, Dušan Turk, Boris Turk, and Marko Fonovic 62—69 Analytical chemistry Spectrophotometry Determination of Ru(IV) Using 5-Hydroxyimino-4-imino-1,3-thiazolidin-2-one as a Novel Analytical Reagent Oleksandr Tymoshuk, Lesia Oleksiv, Liudmyla Khvalbota, Taras Chaban and Ihor Patsay Fig. 4. Absnrbancespectra [>f reagent and complex RmT'A 70—77 Analytical chemistry Atmospheric Solids Analysis Probe with Mass Spectrometry for Chlorpyrifos and Chlorpyrifos-Gxon Determination in Apples Mirjana R. Cvijovic, Valerio Di Marco, Srboljub J. Stankovic, Zoran P. Nedic, Ljubinka G. Joksovic and Nevena R. Mihailovic 78—84 Analytical chemistry Solid-Phase Extraction Method by Magnetic Nanoparticles Functionalized with Murexide for Trace U(VI) from Sea Water Prior to Spectrophotometry Determination Tulay Oymak The magnetic sorbent was T-H g-,-1 85-102 Materials science Dawson-Type Polyoxometalate Incorporated into nanoporous MIL-101(Cr): Preparation, Characterization and Application for Ultrafast Removal of organic Dyes Afsoon Jarrah and Saeed Farhadi P2Wl8/MIL101(Cr) 103-111 Orga nic chemistry Synthesis, Anti-Inflammatory and Antioxidant Activities of novel 3ff-Thiazolo[4,5-fr]Pyridines Taras I. Chaban, Volodymyr V. Ogurtsov, Vasyl S. Matiychuk, Ihor G. Chaban, Inna L. Demchuk and Ihor A. Nektegayev 112-122 Phy sical chemistry A Novel Environmentally Friendly Synergistic Mixture for Steel Corrosion Inhibition in 0.51 M naCl Stojan Božovic, Sanja Martinez and Veselinka Grudic 123-136 Chem ical, biochemical and environmental engineering Adsorptive Removal of Fluoride from Aqueous Solution by Biogenic Iron Permeated Activated Carbon Derived from Sweet Lime Waste Mohd. Ibrahim, Adil Siddique, Lata Verma, Jiwan Singh and Janardhan Reddy Koduru i«,) 136-144 Chemical, biochemical and environmental engineering Fabrication of Silver-Modified Halloysite Nanotubes and their Catalytic Performance in Rhodamine 6G and Methyl Orange Reduction Maryam Akhondi and Effat Jamalizadeh 145-154 Orga nic chemistry Directed Search of Biologically Active Compounds among Hydrogenated Isoindolylalkyl(alkylaryl-,aryl-) carboxylic Acids with Quinazoline Fragment that ... Yulya Victorivna Martynenko, Oleksii Mykolayovich Antypenko, Oleksandr Anatolievich Brazhko, Irina Borisivna Labenska and Sergii Ivanovich Kovalenko 155-162 Phy sical chemistry Investigation of the Electrochemical Reduction Process of the Molybdate Ions in the Tartaric Electrolytes Vusala Asim Majidzade, Akif Shikhan Aliyev, Dunya Mahammad Babanly, Mahmood Elrouby and Dilgam Babir Tagiyev 163-167 Analyt ical chemistry Kinetic-Spectrophotometric Determination of Thiocyanate in Human Saliva Based on Landolt Effect in Presence of Astrafloxine FF Alexander Chebotarev, Vitaliy Dubovyi, Dmytro Barbalat, Elena Guzenko, Kateryna Bevziuk and Denys Snigur 168-172 i norganic chemistry Synthesis and Crystal Structure of a Polymeric Copper(II) Complex Derived from 2-Hydroxy-5-methylbenzaldehyde Oxime with Antibacterial Activities Ya-Li Sang and Xue-Song Lin 173-181 Materials science Density Functional Approach: To Study Copper Sulfide Nanoalloy Clusters Prabhat Ranjan and Tanmoy Chakraborty m 182-189 Phy sical chemistry Voltammetric Investigation of Inclusion Complexes of the Selected Succinimides with 0-Cyclodextrin and (2-Hydroxypropyl)-P-Cyclodextrin Jelena Lovič, Milka Avramov Ivič, Bojan Božič, Jelena Ladarevič and Dušan Mijin 190-195 norganic chemistry Synthesis, Crystal Structures, and Antimicrobial Activity of Zinc(II) and Manganese(II) Complexes Derived from N'-(1-(Pyridin-2-yl)Ethylidene)Isonicotinohydrazide Ling-Wei Xue, Hui-Jie Zhang and Pan-Pan Wang 196-207 i norganic chemistry Evaluation of DNA/BSA Binding and Chemical Nuclease Activity of L-Tyrosine-Based Mn(III) and Fe(III) Metallo-Intercalators Biju Bennie Rajaretnam, Joel Chellappa, Daniel Abraham Solomon, Iyyam Pillai Subramanian and Theodore David Manickam Selvanayagam 208-216 Chem ical, biochemical and environmental engineering Adsorption of Cr (III) and Cd (II) Ions using Mesoporous Cobalt-Ferrite Nanocomposite from Synthetic Wastewater Iman Khoshkerdar and Hossein Esmaeili i 217-228 chemical, biochemical and environmental engineering Release of Halophilic Extremozymes by Mechanical Cell Disruption Mateja Primožič, Maja Čolnik, Željko Knez, and Maja Leitgeb 229-238 phy sical chemistry Characterization and Atmospheric Implication of Hydrotrioxy Radical-Water-Methylamine-Formic Acid-Sulphuric Acid Complexes Matjaž Dlouhy and Antonija Lesar 239—246 chemical, biochemical and environmental engineering Determination of Dissolved Iron Redox Species in Freshwater Sediment using DGT Technique Coupled to BDS Hanna Budasheva, Aleksander Kravos, Dorota Korte, Arne Bratkic, Yue Gao and Mladen Franko 247-254 Analytical chemistry Development of a Dispersive Liquid-Liquid Microextraction Followed by LC-MS/MS for Determination of Benzotriazoles in Environmental Waters Ida Krasevec and Helena Prosen SHORT communication 1—4 chemical, biochemical and environmental engineering My Times with Professor Igor Kregar and some Hydrolytic Enzymes Karl J. Kramer Graphical Contents 58—61 Biochemistry and molecular biology The Carboxypeptidase Activity of Cathepsin X is not Controlled by Endogenous Inhibitors Urša Pečar Fonovic, Milica Perišic Nanut, Nace Zidar, Brigita Lenarčič and Janko Kos DOI: 10.17344/acsi.2019.4981 Acta Chim. Slov. 2019, 66, 1-4 ©commons , Short communication My Times with Professor Igor Kregar and some Hydrolytic Enzymes Karl J. Kramer* Department of Biochemistry and Molecular Biophysics, Chalmers Hall, Kansas State University, Manhattan, Kansas 66506 USA * Corresponding author: E-mail: kkramer1@cox.net; Tel: +1 785 5395975 Received: 01-21-2019 Dedicated to the memory of Prof. Dr. Igor Kregar Abstract The late Professor Igor Kregar and this author had several overlapping biochemical research interests. One focus was the glycoside hydrolase, lysozyme, and the other was proteolytic enzymes and their inhibitors, in particular those present in tissues from insects and plants. Regarding lysozyme our results helped to understand its catalytic mechanism and the carboxylic acid functional groups involved. Another area was insect-plant interactions involving defensive responses of plants to insect feeding via proteolytic enzyme inhibitors. Those results can be utilized in transgenic plant and seed biotechnological applications, which would help to reduce damage to plants and seeds caused by coleopteran and other insect pests. Also described are some cultural and travel interactions that the author benefited from his friendship with Professor Kregar. Keywords: Lysozyme; catalysis; mechanism; proteases; inhibitors; beetles; potato; resistance 1. Introduction My interactions with the late Slovenian scientist Professor Igor Kregar began more than 50 years ago. We met in Tucson, Arizona, USA, where he had arrived in the late 1960s from Ljubljana, Slovenia (then Yugoslavia) to begin working in the Department of Chemistry at the University of Arizona with Professor John A. Rupley. Igor was the second of three postdoctoral researchers from the Jozef Stefan Institute in Ljubljana to work in Rupley's laboratory during my tenure there as a graduate student. Franc Gubensek was the first postdoctoral scholar to work there and Vito Turk the third. After each initial meeting, valued friendships were established with all three Slovenes, all of which were maintained long thereafter, and in Igor's case, our friendship lasted a very long time until his untimely death in 2017. 2. Research Time Igor had a long list of scientific publications.1 In Ru-pley's laboratory Igor and I both worked on protein chemistry projects focused on the structure and function of a glycosidic enzyme, hen's egg white lysozyme, he on the dependence of pH on the hydrolysis of the chitohexasac-charide substrate2 and myself on the relative chemical reactivity in aqueous solution and acidic methanol of its ten carboxylic acids, several of which are involved in substrate binding and the catalytic mechanism.3,4 Results from Igor's research supported the hypothesis that non-productive enzyme-substrate complexes are of kinetic importance and that the structure of the productive complex and the catalytic mechanism were those suggested by results obtained from the x-ray crystallographic enzyme and enzyme-substrate complex structures. Results from my experiments revealed that the two carboxyl groups present in the active site of lysozyme, which were indicated by the crystallographic structures to participate in the catalytic mechanism, were more reactive to nucleophilic chemical modification than any of the other eight carboxyl groups. During our subsequent scientific careers, both Igor and I became interested in another type of hydrolytic enzyme, the proteolytic enzymes and also their inhibitors. He studied several kinds of proteases and inhibitors obtained from a wide array of sources such as microbes, cows, rats, pigs, sheep, marine animals, plants and insects. My labo- Kramer: My Times with Professor Igor Kregar and Acta Chim. Slov. 2019, 66, 1-4 2 ratory was more focused on proteases and their inhibitors that were present in insect and plant tissues. Our interests overlapped in the area of insect-plant interactions involving defensive responses of plants to insect feeding via proteolytic enzyme inhibitors. Igor published several papers dealing with proteolytic enzymes and inhibitors in the potato, Solanum tuberosum as well as the cysteine proteases in the gut of Colorado potato beetle, Leptinotarsa decem-lineata.5-10 He and his collaborators found that the beetle's cysteine proteolytic activity was insensitive to potato protease inhibitors, enabling the insects to overcome this defense mechanism of potato plants. They then looked for inhibitors from other sources that would be more effective against the beetle's proteases.5 After testing several pro-teinaceous inhibitors of different structural types for their ability to inhibit the beetle's proteolytic activities in vitro, most members of the cystatin superfamily of inhibitors11 were found to be poor inhibitors of the beetle's induced endoproteolytic activities except for the third domain of human kininogen, which was a fairly strong inhibitor. The best inhibitor of the beetle's induced endoproteolytic activity was one of the structurally different thyroglobulin type-1 domain-like inhibitors, equistatin, a protease inhibitor from the sea anemone, Actinia equine.12-14 The stefin family of inhibitors in the cystatin superfamily was inhibitory of the beetle's induced aminopeptidase-like activity. In in vivo feeding experiments, larvae of the Colorado potato beetle fed on equistatin-coated potato leaves were strongly retarded in their growth and development with about half of the larvae dying after only a few days. Igor's results demonstrated the potential of using thyroglobulin type-1 domain-like and stefin family-like inhibitors as potential field crop protectants against insect attack.5 A related insect control project involved a collaboration between not only Slovenian and American scientists but also several Japanese scientists on a study of the effects of proteinase inhibitors on the growth and digestive proteolysis of another coleopteran, the red flour beetle, Tri-bolium castaneum.15 Inhibitors of T. castaneum digestive proteinases were examined to identify potential biopesticides for incorporation into transgenic plants and seeds thereof. Cysteine proteinase inhibitors from potato, Job's tears and sea anemone (equistatin) were effective inhibitors of in vitro casein hydrolysis by T. castaneum proteinases (Fig. 1). Similar to Igor's findings with the Colorado potato beetle, the thyroglobulin type-1 domain was the inhibitor domain most active toward red flour beetle gut proteases. Casein hydrolysis was inhibited weakly by chymosta-tin and soybean trypsin inhibitor. The soybean trypsin inhibitor had no significant effect on growth when it was bioassayed alone, but it was effective when used in combination with a potato cysteine proteinase inhibitor. In oral feeding bioassays with single inhibitors, larval growth was suppressed by the cysteine proteinase inhibitors from potato, Job's tears and sea anemone. Levels of inhibition were similar to that observed with a low molecular weight model cysteine protease inhibitor, E-64, although the moles of proteinaceous inhibitors tested were many fold less. These proteinaceous inhibitors were identified to be candidates Figure 1. Relative inhibition (as a percent of the control activity) of casein hydrolysis by Tribolium castaneum larval gut proteinases with selected proteinase inhibitors15 including E-64: L-trans-epoxysuccinylleucylamido w4-guanidinox butane; JCPI, Job's tears cysteine proteinase inhibitor; TLCK, tosyl-L-lysine chloromethyl ketone; TPCK, N-tosyl-L-phenylalanine chloromethyl ketone; PCPI, potato cysteine proteinase inhibitor and STI, soybean trypsin inhibitor (Kunitz). Leupeptin, E-64, equistatin and TLCK were the most potent caseinolytic enzyme inhibitors. In feeding bioassays, equistatin and PCPI were the most potent peptides in inhibiting larval growth of the red flour beetle.15 Kramer: My Times with Professor Igor Kregar and 3 Acta Chim. Slov. 2019, 66, 1-4 for transgenic plant and seed technology to reduce damage by coleopteran pests such as the red flour beetle and Colorado potato beetle. Overall, from a practical viewpoint, Igor's and other findings further illustrated the striking diversity of possible protease-inhibitor interactions in insect-plant systems and underline the relevance of protease inhibitor activity-based protease profiling to characterize the inhibitory range of recombinant inhibitors considered for insect pest control. For example, a straightforward way to use cys-tatin-like inhibitors against coleopteran insects might be to combine them with serine protease inhibitors in such a way as to significantly broaden the overall spectrum of target protease types and minimize the effectiveness of any physiological compensatory processes in the herbi-vores.16,17 3. Cultural and Travel Times In addition to our scientific interactions, I gained much information from not only Igor but also Vito and Franc about the history, politics and geography of Slovenia/Yugoslavia and the Balkan region. Until that time, I knew very little about their country, but because of their generosity, I came to appreciate its greatness and wonderful citizens. All three are/were very good ambassadors for Slovenia. One of the more memorable times spent with Igor was when he joined my wife Virginia and myself on a Mexican railroad trip south to Guadalajara and Mexico City where we enjoyed many cultural attractions and savored many kinds of delicious Mexican dishes. Afterwards, Igor became quite the connoisseur of Mexican cooking and loved to prepare Mexican food for his family and friends in Slovenia. We would even mail him care packages containing Mexican food items that at that time were hard to find in Slovenia. A very special travel time with Igor for me was a visit to Ljubljana when he helped to host me and to enjoy the natural beauty and culture of that area of Slovenia. Another happy travel memory of Igor was one regarding the car he had bought and drove in Tucson. He was very delighted to be the owner of a 1961 white Buick LeSabre convertible and enjoyed cruising around Arizona very much with the top down while taking in the warm southwestern sunshine. 4. Quality of Life Time One of the greatest gifts of life is friendship, and I received that and more from Professor Kregar. He added much to my quality of life overall. As we grew older and in spite of living far apart, we spent many hours visiting via Skype on the internet, updating our activities, sharing stories and trying to find solutions to the world's problems. Not only did I benefit scientifically from our friendship, I also became better educated in international history, geography, politics and culture because of Igor. I will always remember him as a wonderful colleague and friend with great love and respect. 5. References 1. PubMed, 2018, https://www.ncbi.nlm.nih.gov/pubmed?cm-d=Search&term=Kregar%20I%5BAuth%5D 2. S. K. Banerjee, I. Kregar, V. Turk and J. A. Rupley, J. Biol. Chem. 1973, 248, 4786-92. 3. K. J. Kramer and J. A. Rupley, Arch. Biochem. Biophys. 1973, 156, 414-25. DOI: 10.1016/0003-9861(73)90290-7 4. K. J. Kramer and J. A. Rupley, Arch. Biochem. Biophys. 1973, 158, 566-76. DOI:10.1016/0003-9861(73)90549-3 5. K. Gruden, B. Štrukelj, T. Popovic, B. Lenarcic, T. Bevec, J. Brzin, I. Kregar, J. Herzog-Velikonja, W. J. Stiekema, D. Bosch and M. A. Jongsma, Insect Biochem. Mol. Biol. 1998, 28, 54960. DOI:10.1016/S0965-1748(98)00051-4 6. K. Gruden, B. Štrukelj, M. Ravnikar, M. Poljsak-Prijatelj, I. Mavric, J. Brzin, J. Pungercar and I. Kregar, Plant Mol. Biol. 1997, 34, 317-23. DOI:10.1023/A:1005853026333 7. S. Kreft, M. Ravnikar, P. Mesko, J. Pungercar, A. Umek, I. Kregar and B. Strukelj, Phytochemistry 1997, 44, 1001-6. DOI:10.1016/S0031-9422(96)00668-1 8. D. B. Maganja, B. Strukelj, J. Pungercar, F. Gubensek, V. Turk and I. Kregar, Plant Mol. Biol. 1992, 20, 311-3. DOI:10.1007/BF00014499 9. U. Schluter, M. Benchabane, A. Munger, A. Kiggundu, J. Vorster, M. C. Goulet, C. Cloutier and D. Michaud, J. Exp. Bot. 2010, 61, 4169-83. DOI:10.1093/jxb/erq166 10. B. Strukelj, J. Pungercar, P. Mesko, D. Barlic-Maganja, F. Gubensek, I. Kregar and V. Turk, Biol. Chem. Hoppe Seyler 1992, 373, 477-82. DOI:10.1515/bchm3.1992.373.2.477 11. V. Turk, V Stoka and D. Turk, Front. Biosci. 2008, 13, 540620. DOI: 10.2741/3089 12. B. Lenarcic, A. Ritonja, B. Strukelj, B. Turk and V Turk, J. Biol. Chem. 1997, 272, 13899-903. DOI:10.1074/jbc.272.21.13899 13. T. Bevec, V. Stoka, G. Pungercic, I. Dolenc and V. Turk, J. Exp. Med. 1996, 183, 1331-8. DOI:10.1084/jem.183.4.1331 14. T. Ogrinc, I. Dolenc, A. Ritonja and V. Turk, FEBS Lett. 1993, 336, 555-9. DOI:10.1016/0014-5793(93)80875-U 15. B. Oppert, T. D. Morgan, K. Hartzer, B. Lenarcic, K. Galesa, J. Brzin, V. Turk, K. Yoza, K. Ohtsubo and K. J. Kramer, Comp. Biochem. Physiol. C Toxicol. Pharmacol. 2003, 134, 481-90. 16. B. Oppert, T. D. Morgan, K. Hartzer and K. J. Kramer, Comp. Biochem. Physiol. C Toxicol. Pharmacol. 2005, 140, 53-8. 17. J. Vorster, A. Rasoolizadeh, M. C. Goulet, C. Cloutier, F. Sainsbury and D. Michaud, Insect Biochem. Mol. Biol. 2015, 65, 10-9. DOI:10.1016/j.ibmb.2015.07.017 Kramer: My Times with Professor Igor Kregar and 4 Acta Chim. Slov. 2019, 66, 1-4 Povzetek Nekateri interesi pokojnega profesorja Igorja Kregarja ter avtorja tega prispevka so se na področju biokemijskih raziskav prekrivali. Na eni strani glikozidne hidrolaze in lizocim na drugi strani pa proteolizni encimi in njihovi inhibitorji, prisotni v tkivih insektov in rastlin. V primeru lizocima so naši rezultati doprinesli k razumevanju njegovega katalitskega mehanizma, pri katerem funkcionalne skupine karboksilne kisline sodelujejo. Drugo področje pa zajema interakcije med insektom in rastlino, ki vključuje obrambne odgovore rastlin na prehrano insektov s pomočjo inhibitorjev proteoliznih encimov. Dobljeni rezultati so lahko uporabni pri biotehnološki aplikaciji transgenih rastlin in semen, kar naj bi pripomoglo k zmanjšanju škode, ki jo povzročajo Coleoptere in drugi insekti. Iz prikazanih skupnih interesov na področju kulture in potovanj avtor tega prispevka ocenjuje, da je veliko pridobil s prijateljstvom s profesor Kregarjem. Kramer: My Times with Professor Igor Kregar and dol i0.i7344/acsi.20i8.4639 Acta (Mm. Slov. 2019, 66, 5-17 ©ccwnmons Review Characteristics, Structure, and Biological Role of Stefins (Type-1 Cystatins) of Human, Other Mammals, and Parasite Origin Vito Turk,1,2* Dušan Turk,1,3* Iztok Dolenc1 and Veronika Stoka1,2* 1 Department of Biochemistry and Molecular and Structural Biology, J. Stefan Institute, Jamova 39, SI-1000 Ljubljana, Slovenia 2 Jožef Stefan International Postgraduate School, Jamova 39, SI-1000 Ljubljana, Slovenia 3 Centre of Excellence CIPKEBIP, Jamova 39, SI-1000 Ljubljana, Slovenia * Corresponding author: E-mail: vito.turk@ijs.si; dusan.turk@ijs.si; veronika.stoka@ijs.si Tel: +386 1 4773 365 Fax: + 386 1 4773 984 Received: 08-02-2018 Dedicated to the memory of Prof. Dr. Igor Kregar 5 Abstract The majority of lysosomal cysteine cathepsins are ubiquitously expressed enzymes. However, some of them differ in their specific cell or tissue distribution and substrate specificity, suggesting their involvement in determining normal cellular processes, as well as pathologies. Their proteolytic activities are potentially harmful if uncontrolled. Therefore, living organisms have developed several regulatory mechanisms such as endogenous protein inhibitors of the cystatin family, including the group of small cytosolic proteins, the stefins. The main focus of this review is stefins of various origins and their properties, structure, and mechanism of interaction with their target enzymes. Furthermore, oligomerization and fibrillogenesis in stefins and/or cystatins provide insights into conformational diseases. The present status of the knowledge in this field and current trends might contribute to identifying novel therapeutic targets and approaches to treat various diseases. Keywords: Mammalian stefins; parasite stefins; cystatins; cysteine cathepsins; mechanism of interaction; oligomerization 1. Introduction The discovery of the lysosome was crucial for understanding intracellular protein degradation processes.1 Clearly, this finding subsequently contributed to the rapid progress in studies on lysosomal proteases and led to the discovery of a great variety of their endogenous protein inhibitors and small-molecule inhibitors. Among lyso-somal proteases are cysteine cathepsins, the most thoroughly studied enzymes that require a slightly acidic and reducing lysosomal environment. There are 11 human cysteine cathepsins, cathepsins B, C, F, H, K, L, O, S, V, X, and W, and they were identified at the sequence level and later confirmed by bioinformatic analysis of the genome sequence and mRNA expression levels.2 Some of the cathep-sins, such as cathepsins B, H, L, C, and O, are ubiquitously expressed in a wide variety of cells and tissues, whereas cathepsins F, K, S, V, X, and W show a more restricted cell-or tissue-specific distribution and expression.2-4 Most of the cathepsins exhibit predominantly endopeptidase activity, while cathepsins B, C, H, and X are exopeptidases. Lysosomal cysteine cathepsins resemble the papain family of cysteine peptidases (C1A). The crystal structure of papain includes two adjacent structural domains separated by a V-shaped active site cleft, with Cys25, His159, and Asn175 residues essential for catalysis. Compared to the crystal structures of true endopeptidases such as cathep-sins L5, S6, and K7, the additional features found in the crystal structures of exopeptidases enable their exopepti-dase activity by modifying the active-site cleft of these enzymes, such as an occluding loop in cathepsins B and X or an additional exclusion domain in cathepsin C and an oc-tapeptide in cathepsin H.8-11 The determined crystal structures of cathepsins and their substrate specifici- Turk et al.: Characteristics, Structure, and Biological Role 6 Acta Chim. Slov. 2019, 66, 5-17 ties12-14 can provide clues about the biological function of these enzymes. Human cysteine cathepsins, in addition to intracellular protein degradation, participate and control many important physiological processes such as antigen presentation,3 aging,15 bone remodeling,16 apoptosis,17,18 prohormone activation,19 and cell signaling.13,20 However, more recent studies demonstrated the presence of the lysosomal cathepsins in the extracellular environment, nucleus, nuclear and plasma membrane, and cytosol, where they play a crucial role in the pathogenesis of cardiovascular diseases,21 cancer,22,23 neurodegeneration,15,24 and other diseases. Cathepsins are synthesized as inactive precursors; however, once activated, these enzymes are potentially hazardous to their environment.25 Therefore, their proteolytic activities in vivo must be strictly regulated at multiple levels by various control mechanisms, including pH, zy-mogen activation, and endogenous protein inhibitors, to prevent improper cleavage of signaling molecules.26,27 The nature of zymogen activation was elucidated from proca-thepsin structures,28,29 which revealed that the propeptide folds on the surface of the enzyme and runs through the active site cleft, thus blocking the access of the substrate. In the final step, the propeptide unfolds at acidic pH and opens the catalytic site of the mature enzyme.30 Propeptides differ in their length. Cathepsin X propeptide contains only 38 residues,31 while cathepsin C and F propep-tides contain 206 and 251 residues, respectively.32,33 In most cathepsins, the N-terminal propeptide is proteolyti-cally removed by various proteases34 or autocatalytically under acidic conditions.30,35,36 Very recently was demonstrated that procathepsin H is not autoactivated but requires other proteases, such as endopeptidase cathepsin L for its activation.37 It was found that glycosaminoglycans (GAGs) can accelerate the autocatalytic removal of the propeptide and subsequent activation of cathepsin B38 and some other cathepsins.28 The released propeptides from endopeptidases exhibit a limited selectivity of inhibition against their cognate cathepsins in vitro,39 whereas the true exopeptidases cathepsins C and X require endopeptidases, such as cathepsins L and S, for their activation but not au-tocatalytic processing.40 The main regulators of cysteine cathepsins and other papain-like enzymes are their endogenous protein inhibitors, cystatins. The main function of cystatins is to protect the organism against their endogenous enzymes when released from the lysosomes to the extracellular environment, as well as to serve as a defense mechanism against proteases of invading pathogens. In the immune system, parasite stefins and cystatins modulate host's cysteine cathepsin activities by inhibiting processing of exogenous antigens and the MHC class II - Ii, carried out by lysosomal cysteine cathepsins and legumain.41-43 Stefins and cystatins upregulate nitric oxide (NO) production by interferon y-activated murine macrophages. NO inhibits cysteine proteases, particularly those from parasitic protozoa.44-46 Parasite inhibitors contribute to the innate and adaptive immunity by targeting host's cysteine peptidases. It is evident that cystatin thus exert several immunomodulary functions.41 The cystatins are generally non-selective, competitive, reversible, and tight-binding inhibitors.28 They are widely found in all living organisms, from humans, animals, plants, parasites, bacteria, and archaea.47 Based on their protein sequences and tertiary structure, the cystatin family (clan IH) is divided into three inhibitory subfamilies: the stefins (type-1 cystatins or I25A), cystatins (type-2 cystatins or I25B), and kininogens (type-3 cystatins or I25C), as seen in the MEROPS database (http://merops. sanger.ac.uk). However, the classification of protein pepti-dase inhibitors, including the cystatin family, is continually being revised.48 Stefins are primarily intracellular single-chain proteins of about 100 amino acid residues that lack carbohydrate and disulfide bonds. Cystatins are extracellular single-chain proteins of about 115 amino acid residues and contain a signal peptide for secretion and two intracellular disulfide bridges, with the exception of human cystatin F, which contains an additional disulfide bridge. The most well-studied member is human cystatin C.49 The third subfamily of inhibitors is the kininogens, also known as kinin precursor proteins.50 They are large multifunctional and multi-domain proteins and are predominantly found in the blood plasma, with different biological functions attributable to each different domain. In humans, there are two types of kininogens: high-molecular-weight kininogen (HK) and low-molecular-weight inhibitor (LK). Both HK and LK are composed of three tan-demly repeated type-2 cystatin domains (designated 1, 2, and 3) containing eight disulfide bridges. Only domains 2 and 3 of HK and LK bind and inhibit various cysteine proteases, including cathepsins and cruzipain.51-53 Additional information about cystatins can be found in a recent re-view28 and in several older review papers.54-57 In addition to the cystatins, there are other known protein inhibitors of papain-like enzymes. Structurally unrelated to cystatins are thyropins, which are assigned according to the MEROPS database to the family I31 of clan IX48 and show significant homology to thyroglobulin type-1 domains.58 The main representatives are the p41 fragment of the invariant chain of MHC class II molecules59,60 and the equistatin from the sea anemone Actinia equina.61,62 The equistatin is composed of the three structurally related domains; the N-terminal domain inhibits cyste-ine cathepsins,63 whereas the second domain inhibits lysosomal cathepsin D.61,63 The p41 fragment strongly inhibits various cysteine cathepsins64 and cruzipain.65 The crystal structure of the cathepsin L-p41 inhibitory fragment complex possesses a novel fold of p41, which enables specificity to their target enzymes, in contrast to rather non-selective cystatins.66 It was demonstrated that mammalian serpins are involved in cross-class inhibition with cysteine proteases. Thus, the serpin endopin 2C demon- Turk et al.: Characteristics, Structure, and Biological Role 7 Acta Chim. Slov. 2019, 66, 5-17 strates selective inhibition of cathepsin L and elastase-like serine protease,67 while the serpin squamous cell carcinoma antigen (SCCA) inhibits cathepsins K, L, and S,68 suggesting a novel inhibitory pathway. Many small-molecule protease inhibitors of clinical significance and applicability were synthesized using a number of reactive groups, which interact with enzymes. For example, the pioneering group of Elliott Shaw exploited, among others, the diazomethyl ketone functional group to inhibit irreversibly cysteine proteases, including cathepsins.69,70 The discovery of the epoxysuccinyl-based inhibitor E-6471 as a non-selective irreversible inhibitor of cysteine cathepsins led to its wide use in a variety of biological studies and as a diagnostic tool to assess the proteo-lytic activity of cysteine cathepsins and some other related enzymes. E-64 does not inhibit aspartic, serine, and metal-lo-proteases. Many E-64 derivatives were systematically synthesized by Katunuma's group that targeted various cysteine cathepsins such as CA-030, CA-074, and several CLIK inhibitors (reviewed in72). It was reported that CA-074 as a specific inhibitor of cathepsin B suppressed the degradation of collagen in rheumatoid arthritis fluid.73 The crystal structure of cathepsin B in complex with CA030 revealed for the first time a substrate-like interaction in the S1' and S2' sites of the active site cleft of the enzyme.74 The binding geometry of the double-headed inhibitors was confirmed by the crystal structure of the pa-pain-CLIK complex75 and the cathepsin B-NS-134 com-plex.76 More details about small-molecule inhibitors can be found in previous reviews28,77,78 and in numerous original publications. Recent advances in the field of cysteine cathepsins as suitable drug targets are providing valuable research avenues for the treatment of various diseases that result from uncontrolled elevated cathepsin activity.79 In this review, after the introduction to the lysosomal cysteine cathepsins and the regulation of their activities by various protein and chemically synthesized inhibitors, we discuss the current knowledge of the properties, structural characteristics, and oligomerization of protein inhibitors belonging to the stefin subfamily (type-1 cystatins) of the cystatin family. 2. Stefins 2. 1. Human and Other Mammalian Stefins The first protein inhibitor of papain-like cysteine protease was isolated and characterized from chicken egg white, and later, the name "cystatin'' was proposed to designate its function.80 The first intracellular protein inhibitors were isolated and partially characterized from pig leu-cocytes,81 human epidermis,82 and human spleen.83 The stefin inhibitor (later named stefin A) was isolated from human polymorphonuclear granulocytes, and the ami-no-acid sequence was determined84,85 as well as that of chicken cystatin from egg white.86 Both sequences con- firmed structural differences between these two homologous protein inhibitors. In addition, a protein inhibitor of cysteine cathepsins was isolated from the sera of patients with Balkan endemic nephropathy, and the first 47 residues of the N-terminal sequence86 was identical to that of human y-trace,87 and the name human cystatin was proposed.86,88 Soon after, it was renamed human cystatin C.89 These and other accumulated data were of great importance for the nomenclature and classification of the cysta-tin superfamily, comprising three families.90 The inhibitor cystatin B/stefin B was isolated from human liver91 and human spleen,92 and the resulting sequences of the first 65 residues were identical, thus strongly suggesting that both inhibitors, isolated from different tissues, are structurally identical proteins.92 The stefin B dimer was confirmed for the first time from human spleen.92 Structurally homologous inhibitors to human stefins A and B have been identified and characterized in mammals, such as rats93,94 and mice.95 Stefin A,96 stefin B,97 and stefin C98 are found in bovines. Interestingly, bovine stefin C was identified as the first tryptophan-containing stefin with a prolonged N-ter-minus. Four different porcine stefin-type inhibitors, namely A, B, D1, and D2, have been isolated and characterized.99 Porcine stefins A, B, and D1 were sequenced, revealing that porcine D1 and the previously characterized pig leukocyte cysteine proteinase inhibitor-PLCPI100 were identical proteins. Most of the stefins occur in multiple isoelectric forms in acidic or close to neutral pH and are mostly stable in the pH range 3-10 and temperatures up to 80 °C, thus avoiding protein denaturation.54 Among mammals, human stefins A and B are clearly the main representatives and most studied protein inhibitors of the stefin subfamily. However, homologues of both human stefins have been found in various mammals, as mentioned above. Human stefins are intracellular proteins that are present in the cytosol of many cell types and tissues, but they also appear extracellularly in body fluids.101 They are synthesized without signal peptides. It seems that stefin B is generally more widely spread in various cell types and tissues than stefin A. Stefins are the smallest among the members of the cystatin family of inhibitors. 2. 2. Stefins from Parasite Origin Little is known about stefins, cystatins, and other protease inhibitors in parasites and their role to protect themselves from degradation by host proteases and to manipulate the host response to the parasite.102 Stefins have been identified and characterized in a wide range of organ-isms.47,103,104 Currently, about 700 members of the stefin subfamily can be found in the MEROPS database. They are involved in the regulation of their own proteolytic activities and processing of their host proteins. Two inhibitors were isolated from the liver fluke Clonorchis sinensis, CsStefin-1 and CsStefin-2, which have sequence similarities to human stefins.105,106 It was suggested that both in- Turk et al.: Characteristics, Structure, and Biological Role 8 Acta Chim. Slov. 2019, 66, 5-17 hibitors share functionally redundant regulatory functions to modulate activity and processing of CsCathepsin F. In addition, two inhibitors were isolated from the tropical liver fluke Fasciola gigantica, FgStefin-1107 and FgStefin-2, which contain a signal peptide.107,108 The cystatin B homologue SmCytB from turbot Scophthalmus maximus enhances macrophage bactericidal activity.109 Three different stefins, designated rFhStf-1, rFhStf-2, and rFhStf-3, expressed by the trematode Fasciola hepatica exhibited differences in their inhibition profile against various tested enzymes.110 Immunomodulatory properties of FhStefins could be used in order to evaluate their therapeutic potential against inflammatory diseases. The inhibitors FhStf-2 and FhStf-3 fall into an atypical subgroup of stefins due to the presence of a signal peptide, similar to the previously mentioned FgStefin-2.108 The cysteine protease inhibitor AcStefin was identified and characterized from Acan-thamoeba, the causative agent of granulomatous amoebic encephalitis and amoebic keratitis.111 The human stefin homolog as SmCys expressed by Schistosoma mansoni is involved in hemoglobin degradation and its regulation112. Very recently, the novel stefin-type inhibitor EnStef was found in the sanguinivorous fish parasite Eudiplozoon nip-ponicum, and it was found to inhibit endogenous cathep-sins and, surprisingly, legumain, asparaginyl endopepti-dase (family C13), from the Ixodes ricinus tick.113 Notably, only limited knowledge about the characteristics and roles of fish stefins and other endogenous inhibitors are avail-able.114-116 It is well known that fish and shellfish quality depend on the meat texture, which is mainly controlled by proteolysis and autolysis and storage conditions. Endogenous proteases and their inhibitors play crucial roles in these processes, as do fish parasite proteases and their inhibitors. Therefore, more biochemical and molecular biology studies in this direction are of great economic importance in order to improve and ensure the quality of fish and their products.117 3. Stefin Inhibitory Profile 3. 1. Mammalian Stefins Members of the stefin subfamily are rather non-specific inhibitors of mammalian cysteine cathepsins. They are competitive, reversible inhibitors that form tight, equi-molar complexes with their target enzymes.54,55 However, they are able to differentiate between endopeptidases and exopeptidases because of the differences in the structures of the interacting regions of the enzymes. Human and other mammalian stefins mostly act as fast and tight-binding inhibitors of typical endopeptidases, cathepsins L and S, papain, and cruzipain, inhibiting with Ki values in the pM to nM range.28,51 However, human stefin B is generally a weaker inhibitor than stefin A. In contrast, the exopeptidases cathepsins B, X, C, and H possess structural features that restrain the binding of the inhibitors to the parts of the active site cleft.118 In mice, there are at least three variants of stefin A (Stfal, Stfa2, and Stfa3); the first two are a result of polymorphisms.95 Two variants, Stfal and Stfa2, act as fast and tight-binding inhibitors of endopeptidases such as cathepsins L and S (Ki values ranging 0.07-0.16 nM); however, their interaction with the exopeptidases cathepsins B, C, and H is several orders of magnitude weaker compared to that of human, porcine, and bovine stefins, suggesting that in mice, stefin A variants are involved predominantly in the regulation of endopeptidases. Bovine stefin A binds tightly and rapidly to cathepsin L (Ki = 0.03 nM), binds weaker to cathepsin H (Ki = 0.4 nM), and binds to cathepsin B slower but still tight (Ki = 1.9 nM), indicating different mechanisms of inhibition of various cathepsins by stefin A.96 Bovine stefin B strongly inhibits cathepsin S (Ki = 8.0 pM) as a tight-binding inhibi-tor.97 Similar to bovine stefins A and B, bovine stefin C strongly inhibits cathepsin L and papain (Ki of about 0.18 nM) and weakly inhibits exopeptidase cathepsin B.98 Interestingly, porcine stefins A and B bind tightly and rapidly to exopeptidase cathepsin H (Ki = 0.02 and 0.07 nM, respectively), stefins D1 and D2 are poorer inhibitors of the same enzyme (Ki = 102-125 nM) and weak inhibitors of cathepsin B (Ki = 335 and 195 nM, respectively), and all four stefins (A, B, D1, and D2) are fast-acting and tight-binding inhibitors to the endopeptidases cathepsins L and S and papain (Ki values ranging 0.01-0.19 nM), as expected.99 These results suggest that in addition to the differences in the enzyme active sites, which are used to classify proteases as endo- and exopeptidases, minor specific structural features of the porcine stefins, in particular, play an important role in binding. 3. 2. Stefins of Parasite Origin In non-mammalian species, there are some important differences in the potency and selectivity of their target enzymes compared to human and other mammalian ste-fins. There are several examples listed in this context. Two stefins (CsStefin-1 and CsStefin-2) from the parasite Clo-norchis sinensis almost equally inhibit the endopeptidase plant papain, human cathepsin L, two endogenous cathep-sin F variants (CsCF-4 and CsCF-4-6), and surprisingly human cathepsin B. All enzymes are inhibited in the range of Ki 0.03-0.06 nM.105,106 Nanomolar inhibitions of bovine cathepsins B and L, human cathepsin S, and the released cysteine protease of the parasite were observed with the fluke Fasciola gigantica inhibitors FgStefin-1 and Fg-Stefin-2.107,108 The Fasciola hepatica recombinant stefin inhibitors rFhStf-1, rFhStf-2, and rFhStf-3 strongly inhibit two variants of endogenous cathepsin L (FhCL-1,-3) and human cathepsin L (Ki 1.52-52 nM); variants rFhStf-1 and rFhStf-2 inhibit human cathepsin C (Ki 35-57 nM); and human cathepsin B is inhibited only by rFhStf-2 (Ki = 15 nM).108 The S.mansoni inhibitor SmCys strongly inhibits papain (Ki = 0.065 nM).112 However, the N-terminally Turk et al.: Characteristics, Structure, and Biological Role 9 Acta Chim. Slov. 2019, 66, 5-17 truncated forms of SmCys with deletions of10 and 20 amino acid residues resulted in much weaker papain inhibition (Ki = 0.739 nM and 4.915 nM, respectively). A similar effect was observed in truncated forms of human cystatin C of the first ten residues119 and chicken cystatin upon deletion of the first eight residues preceding Gly9.120 In summary, it is evident that some stefin-type parasite inhibitors are strong and tight-binding inhibitors of their endogenous cysteine proteases-cathepsins as well as human and other mammalian cathepsins, suggesting their involvement in the immune regulation and inflammatory diseases.121-125 Furthermore, they demonstrate different inhibitory potencies against their endogenous cathepsins with endo- and exopeptidases activities compared to those of human and other mammalian cathepsins. This might be of importance for the successful accommodation and reproduction of parasites in their host organisms. 4. Structure of Stefins and the Mechanism of Interaction with Their Target Enzymes 4. 1. Interaction Between Stefins and Cathepsin Endopeptidases Based on the known 3D structures of the chicken egg white cystatin126 and human stefin B-papain complex,127 the Figure 1. Multiple sequence alignment of stefins and cystatins. The alignment was performed by Clustal Omega using sequences obtained from Uni-Prot.128 Conserved residues in comparison to human stefin A (HU_ST_A, P01040) are marked using BoxShade as black background, and similar residues have gray background. Asterisks represents conserved residues forming tripartite wedge-shaped edge interacting with the active site of an enzyme. Signal sequences are underlined. Secondary structure elements alpha-helices (H) and beta-sheets (B), and loops (L) are indicated for stefin A (above) and for chicken cystatin (below). Five-stranded antiparallel beta-sheets are numbered. Other aligned inhibitors are: human stefin B (HU_ST_B, P04080), bovine stefins A (BOV_ST_A, P80416), B (BOV_ST_B, P25417), and C (BOV_ST_C, P35478), stefins 1 (CS_ST_1, A6YID9) and 2 (CS_ ST_2, A6YE0) from Clonorchis sinensis, Type-1 cystatin cysteine protease inhibitor from Fasciola gigantica (FG_T1_CYS, K4P3W9), cystatin B (stefin type) from Schistosoma mansoni (SM_SMCYS, Q7YW72), human cystatin C (HU_CYS_C, P01034), and chicken cystatin (CHI_CYS, P01038). Turk et al.: Characteristics, Structure, and Biological Role 10 Acta Chim. Slov. 2019, 66, 5-17 amino acid sequences of several stefins of mammalian and parasite origin have been aligned. The conserved residues in equivalent positions confirmed the relationships between stefin and cystatin subfamilies, although some differences are evident (Figure 1). Moreover, the correct alignment of the stefins and the cystatins revealed that the previous sequence alignments were partly incorrect because of the deletion of the shorter a-helical segment in the stefins. The first and the most important step in the elucidation of the mechanism of inhibition of cysteine proteases was the determination of the crystal structure of chicken cystatin.126 The chicken cystatin molecule consists mainly of a five-stranded antiparallel ^-pleated sheet that is twisted and wrapped around a long central a-helix and an appending shorter a-helical segment. The partially flexible N-terminal highly conserved GG residues, an exposed first hairpin loop with the sequence QLVSG (the prototype of the highly conserved QVVAG sequence in almost all stefins), and a second hairpin loop with PW residues form a wedge-shaped hydrophobic tripartite edge that has high complementarity to the V-shaped active site cleft of papain, as shown in a docking experiment.126 Based on this docking model, the mechanism of interaction between cysteine proteases and their cystatin-like inhibitors was proposed126 and later essentially confirmed by the crystal structure of the recombinant human stefin B-papain complex.127 The main-chain interactions are provided by the N-terminal segment occupying the non-primed subsites S3 to S1 of the enzyme in a substrate-like manner, but the peptide segment afterwards turns away at P1 from the ac- tive site preventing cleavage. The two hairpin loops bind to the primed-sites (S1' to S4') of the enzyme (Figure 2). In stefin B, there are only minor contributions from the second hairpin loop, but the carboxyl terminus provides an additional interaction region compared to chicken cysta-tin. These results provide firm evidence that the inhibition by the protein inhibitors of cysteine proteases is fundamentally different from that obtained with serine protease inhibitors.129 4. 2. Interaction Between Stefins and Cathepsin Exopeptidases Binding of the cystatin-type inhibitors to cathepsin exopeptidases cannot be explained by the stefin B-papain complex.127 Cathepsin H acts as an aminopeptidase and endopeptidase; however, it exhibits strong aminopeptidase activity and is inhibited by various cystatins, including the tight-binding inhibitor stefin A, with Ki = 0.31 nM.28 The crystal structure of native porcine cathepsin H shows a typical papain fold.11 In addition, it contains the octapep-tide EPQNCSAT derived from the propeptide, called a mini-chain, which is covalently attached to the main body of the enzyme by the disulfide bond to the narrowed active site cleft in the substrate-binding direction in non-primed binding sites from S2 backwards (Figure 3). The major reason for the narrowing feature is a unique insertion loop of four residues. The carbohydrate moiety attached to the main body of the enzyme participates in the positioning of the mini-chain in the active-site cleft. The displacement of the residues in the active site cleft results in the exopeptidase activity of cathepsin H. From the crystal structure of the stefin A-cathepsin H complex,127 it is evident that stefin A binds to the active site cleft of the en- Figure 2. Papain - stefin B complex127 (1STF). Stefin B fold is shown in red bound to papain shown as white surface with catalytic Cys area in yellow. All images (Figure 2-6) were made with MAIN soft- Figure 3. Cathepsin H11 (8PCH). Cathepsin H is shown as white surface with catalytic Cys area in yellow. Mini chain and carbohydrate rings responsible for its stabilization are shown as sticks in dark and bright blue, respectively. Turk et al.: Characteristics, Structure, and Biological Role 11 Acta Chim. Slov. 2019, 66, 5-17 Figure 4. Cathepsin H - stefin A complex131 (1NB5). Cathepsin H is shown as in the figure 3, whereas stefin A fold is shown as red ribbon. zyme. However, the N-terminal residues of stefin A adopt the form of a hook, which pushes away the cathepsin H mini-chain residues and distorts the structure of an insertion loop that is unique to cathepsin H (Figure 4). The crucial role of the human cathepsin H mini-chain was further confirmed by the expression of the recombinant cathepsin H in Escherichia coli as a nonglyco-sylated protein lacking the mini-chain after autocatalytic processing.132 Removal of the mini-chain resulted in en-dopeptidase activity only. The recombinant cathepsin H was inhibited by human stefins A and B with Ki values in the range of 0.05-0.1 nM, which is stronger than the inhibition of native cathepsin H. Another example that possesses both exopeptidase and endopeptidase activities is human cathepsin B8 (Figure 5). Although its overall structure and the arrangement of the active site residues are similar to those of endopeptidase papain, there are several insertion loops on the surface of the molecule that modify its properties. Some of the primed subsites are occluded by a novel 20 residue peptide segment, termed the occluding loop with two histidine residues (H110 and H111), which provide positively charged anchors for the C-terminal carboxylate group of the polypep-tide substrates. The occluding loop restricts access to the active site cleft of cathepsin B by occupying part of the active site cleft on the primed side and blocking access to the active site cleft beyond the S2' substrate binding site.8,12 These structural features explain the unique peptidyl-di-peptidase activity of exopeptidase cathepsin B. Deletion of the occluding loop by site-directed mutagenesis resulted in an enzyme with endopeptidase activity but completely lacking exopeptidase activity.134 The crystal structure of the human stefin A-human cathepsin B complex revealed that occluding loop residues are displaced, thus allowing the in- Figure 5. Cathepsin B - stefin A complex133 (3K9M). Cathepsin B is shown as white surface with the catalytic Cys area yellow and occluding loop displaced from the active site cleft in blue. Stefin A is shown as red ribbon. teraction with inhibitors in the binding region133 and indicating that the occluding loop flexibility must be responsible for the cathepsin B endopeptidase activity. Most of the protein structures were determined by X-ray crystallography with comparisons to NMR spectroscopy. Structures determined by both techniques, in the solid state and in solution, are usually very similar. However, two NMR structures of chicken cystatin, the native phosphorylated and recombinant non-phosphorylated variants,135,136 showed the same overall fold and the flexible N-terminal part, but there were also some significant differences in the structurally variable segments of the polypeptide chain compared to the crystal structure.126 The NMR analysis revealed that the second a-helix determined in the crystal is not present in the solution. Similarly, the solution structure of human stefin A137 showed similarity to the homologous protein stefin B in complex with papain,127 but some important differences in the binding regions such as in the mobile N-terminal region and the second binding loop were observed. The crystal structure of the stefin B type inhibitor CsStefin-1 from the liver fluke C. sinensis was just reported, indicating some minor structural differences to human stefin B such as a four-stranded antiparallel ^-pleated sheet and an additional short a-helix not present in human stefin B.138 4. 3. Oligomerization and Fibrillogenesis of Stefins and Cystatins Small-sized proteins, also termed mini-proteins, represent a useful and relatively simple model for studies on Turk et al.: Characteristics, Structure, and Biological Role 12 Acta Chim. Slov. 2019, 66, 5-17 oligomeric proteins. They are composed of two or more subunits, and most of them are symmetrical homo-oligo-mers, which are on average tetramers.139 Oligomerization results from a variety of mechanisms and can provide insights into the evolution of proteins. Suitable examples are stefins (I25A) and cystatins (I25B), members of the cystatin family of inhibitors. The phyletic distribution of the cystatin family indicates the presence of only two ancestral lineages, stefins and cystatins, in eukaryotes and prokaryotes.47 Stefins are present as single copy genes or small multigene families throughout the eukaryotes and underwent small changes in function during evolution. In contrast to stefins, the cystatins went through a more complex evolution involving numerous gene and domain duplications. Stefins and cystatins share a rather high sequence similarity and nearly the same fold, as already discussed. The early finding that human stefin B92 and rat TPI-2 (cystatin p/stefin B)140 form dimers indicated for the first time the possible appearance of the oligomerization of these proteins. Later, it was found that the trematode parasitic C. sinensis native stefin-type inhibitor CsStefin-2 exists in monomer, dimer, and tetramer forms, which are not the result of interchain disulfide bond interactions.105 Similarly, the oligomerization from monomers (10 kDa) to oligomers of various sizes (over 100 kDa) in F. hepatica ste-fin-type inhibitors (rFhStf-1, rFhStf-2, and rFhStf-3) was reported very recently.110 An important step in elucidating the oligomeriza-tion of cystatins was determining the crystal structure of dimerized domain-swapped human cystatin C141 and of chicken cystatin and human stefin A in solution.142 Then, it was demonstrated that the domain-swapped dimer of chicken cystatin oligomerizes to a tetramer as a transient intermediate prior to oligomerization.143 Furthermore, it was shown that human cystatin C oligomers are intermediates in fibrillogenesis, indicating that the propagation of three-dimensional domain swapping is crucial to oligom-erization processes.144 A variant of human cystatin C (L68Q mutant) found in patients with hereditary cystatin C amyloid angiopathy (HCCAA) causes massive amyloi-dosis as a result of amyloid fibrils in the cerebral arteries, with fatal consequences for young adults145,146). It has just been reported that the conformational destabilization of human cystatin E (I25B) results in a domain-swapped di-mer that can convert to amyloid fibrils.147 This dimer inhibits legumain by forming a trimeric complex but does not inhibit papain and human cathepsin S. Furthermore, it was shown that recombinant human stefin B, in contrast to stefin A, dimerizes, oligomerizes, and forms amyloid fibrils under in vitro conditions.148,149 Soon afterwards, the crystal structures of the tetrameric human stefin B and of stefin B in solution were determined.150 The structures revealed that the formation of the stefin B tetramer is not a further domain swapping process, as it was proposed earlier for cystatins,151,152 but a new mechanism, termed hand shaking, through which 3D domain-swapped dimers be- Figure 6. Stefin A tetramer150 (2OCT). Each chain in the tetramer is shown as ribbon with own color. The dimers of the two domain swapped dimers are shown at the top and bottom of the figure. The two dimers interlink with loop handshake shown in the middle. come entwined as a consequence of concurrent trans to cis isomerization of proline 74,150 as can be seen in Figure 6. This proline residue is widely conserved throughout the stefins and cystatins. It was found that the tetrameric structure of stefin B in solution correlates with that of the crystal. These and other experimental data suggest that the isomerization of proline residues is a crucial component in tetramerization and very likely involved in other steps of amyloid formation. Taken together, the similarities in structure, sequence, and oligomerization processes between stefins and cystatins suggest that in addition to domain swapping there is an additional mechanism called, hand shaking, in which the trans to cis isomerization of proline 74 is leading from may be on the path of formation of the mature fibrils. Additional information about oligo-merization and amyloid formation can be found in previous reports.151-153 The recent progress in sample preparation due to their polymorphic purity, as well as solid state NMR and cryo-EM methods, recently provided insight in high-resolution 3D structures of amyloids.154,155 5. Conclusions and Future Trends Lysosomal cysteine cathepsins and the precise regulation of their harmful proteolytic activities are of crucial importance to prevent improper cleavage(s) of signaling molecules.55,156 There are several means for this regulation, one of which is the use of endogenous protein inhib- Turk et al.: Characteristics, Structure, and Biological Role 13 Acta Chim. Slov. 2019, 66, 5-17 itors, such as stefins and cystatins. We understand a great deal about the mechanisms of interaction with their target enzymes. However, low specificity of inhibitors for their target proteases indicates that we still do not understand their exact individual physiological roles. On the other hand, most of the cathepsins are ubiquitously expressed, exhibit relatively wide specificity, and have multiple functions. Therefore, it is crucial to understand the diseases in which cathepsins play critical roles and the roles of individual cathepsins in these diseases. Interestingly, mutations in two endogenous protein inhibitors of cysteine cathepsins, stefin B, and cystatin C, are critical for the development of two neurological disorders, such as Unver-richt-Lundborg disease-EPM1157,158 and Hereditary cystatin C amyloid angiopathy (HCCAA).159,143,160 Insight into the interplay of stefins and cathepsins may encourage the development of selective cathepsin inhibitors as candidates for clinical studies and eventually new drugs.79 Furthermore, studies on parasites induced immune regulation and inflammatory diseases should also be encouraged in order to develop new therapeutic drugs.161-163 Another important area is the identification of physiological substrates using proteomic strategies and chemical tools.164,165 Although the understanding of the complexity of the numerous vital biological processes, both physiological and pathological, is best illustrated by the current trends in a number of ongoing research projects, it is likely that studies on the regulation of proteolysis in the light of the structure-function relationship will reveal valuable information in the near future. 6. 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Lehtinen, E. Mervaala, M. Simonato and A. Vaarmann, Epileptic Disord. 2016, 18, 28-37. DOI: 10.1684/epd.2016.0841 158. T. Joensuu, M. Kuronen, K. Alakurtti, S. Tegelberg, P. Haka-la, A. Aalto, L. Huopaniemi, N. Aula, R. Michellucci, K. Eriksson and A. E. Lehesjoki, Eur. J. Hum. Genet. 2007, 15, 185-93. DOI: 10.1038/sj.ejhg.5201723 159. W. Mi, M. Pawlik, M. Sastre, S. S. Jung, D. S. Radvinsky, A. M. Klein, J. Sommer, S. D. Schmidt, R. A. Nixon, P. M. Mathews and E. Levy, Nat. Genet. 2007, 39, 1440-2. DOI: 10.1038/ng.2007.29 160. I. Olafsson and A. Grubb, Amyloid 2000, 7, 70-9. 161. C. Klotz, T. Ziegler, E. Danilowicz-Luebert and S. Hartmann, Adv. Exp. Med. Biol. 2011, 712, 208-21. DOI: 10.1007/978-1-4419-8414-2_13 162. S. L. Ranasinghe and D. P. McManus, Trends Parasitol. 2017, 33, 400-413. DOI: 10.1016/j.pt.2016.12.013 163. S. Coronado, L. Barrios, J. Zakzuk, R. Regino, V. Ahumada, L. Franco, Y. Ocampo and L. Caraballo, 2017, 39. DOI: 10.1111/pim.12425 164. L. E. Sanman and M. Bogyo, Annu. Rev. Biochem. 2014, 83, 249-73. DOI: 10.1146/annurev-biochem-060713-035352 165. M. Vizovisek, R. Vidmar, M. Drag, M. Fonovic, G. S. Salves-en and B. Turk, Trends Biochem. Sci. 2018, 43, 829-44. DOI: 10.1016/j.tibs.2018.07.003 Turk et al.: Characteristics, Structure, and Biological Role Acta Chim. Slov. 2019, 66, 5-17 17 Povzetek Večina lizosomskih cisteinskih katepsinov je splošno izražena. Razlikujejo pa se v specifični celični in/ali tkivni porazdelitvi ter substratni specifičnosti. Vse to vpliva na njihovo delovanje v normalnih celičnih procesih, kot tudi v patologiji. Če njihova proteolizna aktivnost ni kontrolirana postane potencialno nevarna. V ta namen so živi organizmi razvili regulatorne mehanizme, med katere spadajo tudi endogeni proteinski inhibitorji družine cistatinov, ki vključujejo skupino majhnih citozolnih proteinov, imenovanih stefini. Pregledni članek obravnava predvsem stefine različnih izvorov, njihove lastnosti, strukturo ter mehanizme interakcij z njihovimi tarčnimi encimi. Nadalje obravnava oligomerizacijo in tvorbo fibrilov pri stefinih in cistatinih, kar omogoča boljši vpogled v konformacijske bolezni. Obstoječe znanje na tem področju ter sedanje usmeritve naj bi prispevale k identifikaciji novih terapevtskih tarč in pristopov pri zdravljenju različnih obolenj. Turk et al.: Characteristics, Structure, and Biological Role DOI: 10.17344/acsi.2018.4422 Acta Chim. Slov. 2019, 66, 18-27 /^creative ^commons Scientific paper Surface Anchoring on Lactococcus lactis by Covalent Isopeptide Bond Tina Vida Plavec1,2 and Aleš Berlec2^ 1 Faculty of Pharmacy, University of Ljubljana, Aškerčeva 7, Ljubljana, Slovenia 2 Department of Biotechnology, Jožef Stefan Institute, Jamova 39, Ljubljana, Slovenia * Corresponding author: E-mail: ales.berlec@ijs.si + 386 1477 3754 Received: 04-24-2018 Dedicated to the memory of Prof. Dr. Igor Kregar Abstract Display of recombinant proteins on the bacterial surface is an emerging research area with wide range of potential bio-technological applications. Because of its GRAS (generally recognized as safe) status, lactic acid bacterium Lactococcus lactis represents an attractive host for surface display and promising vector for in situ delivery of bioactive proteins. The present study focused on finding a new alternative approach for surface display on Lactoccocus lactis. We developed a system that enables the formation of irreversible isopeptide bonds on the surface of Lactococus lactis. This was achieved through the following two protein/peptide pairs, SpyCatcher/SpyTag and SnoopCatcher/SnoopTag.1-3 Attachment of tagged model protein B domain to the cell surface of Lactococccus lactis displaying the corresponding catcher protein was demonstrated using flow cytometry. We demonstrated effective use of aforementioned protein anchors which thus represent a promising alternative to established approaches for surface display on Lactoccocus lactis. Keywords: Surface display; Lactococcus lactis; isopeptide bond 1. Introduction Display of recombinant proteins on bacterial surface offers a variety of possible biotechnological applications. Proteins-displaying bacteria can act as bioadsorbents, biosensors, biocatalysts or oral vaccines. They can be used in antibody production and in peptide screening.4-6 Several lactic acid bacteria (LAB) are probiotics and are therefore considered valuable hosts in biotechnology due to their beneficial influence on health.7,8 Because of the "generally recognized as safe" (GRAS) status which confirms their safety, LAB are attractive not only for industrial application but also therapeutically.9 Display of heterologous proteins on the surface of LAB has already been exploited in therapy for the preparation of mucosal vaccines.10-12 Moreover, beneficial effects in inflammatory bowel disease could be achieved when displaying binding molecules directed against pro-inflammatory molecules such as TNFa.13-17 Different approaches can be exploited for displaying a protein on the bacterial surface. The protein to be dis- played is usually fused to an anchoring motif.14,18 Five different types of surface anchoring domains have been described for LAB: transmembrane domains, LPXTG-type domains, lysin motif (LysM) domains, surface layer proteins and lipoprotein anchors.19-21 The most frequently applied surface anchoring domains in prototype LAB, Lac-tococcus lactis, are the C-terminal part of endogenous AcmA, enabling non-covalent anchoring through pepti-doglycan binding LysM repeats,22-25 and the LPXTG sequence of M6 protein of Streptococcus pyogenes enabling covalent anchoring.26,27 Despite these available options, alternative surface display approaches are being sought. Recently, two peptide/protein pairs known as Spy-Tag/SpyCatcher, from Streptococcus pyogenes, and Snoop-Tag/SnoopCatcher, from Streptococcus pneumoniae have been developed.1-3 Interaction between the peptide and the protein leads to the formation of an irreversible iso-peptide bond. The reaction is high-yielding and fast while the bond is highly stable. It can survive extreme pH, high ionic strength and exposure to detergents.1,28,29 Stable bond formation enables combinatorial assembly of multi- Plavec and Berlec: Surface Anchoring on Lactococcus lactis 19 Acta Chim. Slov. 2019, 66, 18-27 a) L. lactis AcmA SpyC + SpyT| AcmA SpyT AcmA SnC Spy Bdom AcmA SpyC SpyC AcmA SpyT Bdom AcmA SnC Sn b) pSDBA3b pSD_SpyC_AcmA pSD_SnC_AcmA pSD_SpyT_AcmA pSD_SpyT_Bd pSD_SnT_Bd pSD_SpyC_Bd pSD_Bd pET_SpyT_Bd USP B dom AcmA USP SpyC AcmA USP SnC AcmA USP SpyT USP B dom SpyT USP B dom SnT USP B dom SpyC B dom SpyT Fig. 1. The principle of surface display of B domain on L. lactis by the formation of isopeptide bond (A), and the gene constructs prepared for its implementation (B). A: Surface displayed SpyCatcher (SpyC), SpyTag (SpyT), or SnoopCatcher (SnC), all anchored via AcmA on L. lactis, bind fusion proteins consisting of SpyT, SpyC, or SnoopTag (SnT), respectively, and B domain. The fusion proteins were produced by L. lactis co-culture, were extracted from L. lactis conditioned medium, or were isolated from E. coli. B: Gene constructs for lactococcal surface display and isopeptide bond formation. USP: gene for Usp45 signal peptide for secretion to the growth medium (84 bp). B dom: gene for reporter protein B domain of staphylococcal protein A (174 bp). SpyC: gene for protein SpyCatcher which binds SpyT (348 bp). SnC: gene for protein SnoopCatcher which binds SnT (336 bp). SpyT: gene for peptide SpyTag (39 bp). SnT: gene for peptide SnoopTag (36 bp). AcmA: gene for C-terminal part of AcmA protein-containing 3 LysM repeats for surface anchoring to L. lactis (642 bp). protein constructs, and opens an opportunity to use this approach in vaccine production, enzyme substrate channeling, antibody polymerization, cell signaling activation, and biomaterials.1,30 The key properties of isopeptide binding are simple and fast procedure, irreversible and stable bond, specificity, and cysteine independence; the latter offering the possibility to use the approach in reducing en-vironment.3 The goal of the present study was to develop a system for surface display on recombinant LAB L. lactis by applying the isopeptide bond formation (Fig. 1a). This was achieved by preparing genetic constructs consisting of surface anchor, elements of SpyTag/SpyCatcher or SnoopTag/ SnoopCatcher pairs and model passenger protein B domain (Fig. 1b).13 B domain is one out of five antibody-binding domains of staphylococcal protein A that can bind antibodies via their Fc region. 13 The fusion proteins were expressed either in L. lactis or in E. coli, and assembled on the surface of L. lactis, as confirmed by surface localization of B domain. 2. Experimental 2. 1. Bacterial Strains, Media and Culture Conditions Bacterial strains used in this study are listed in Table 1. E. coli strains DH5a and BL21 (DE3) were grown at 37 °C, with aeration in lysogeny broth (LB) medium supplemented with either ampicillin (100 ^g/mL) or kanamycin (50 ^g/ mL). L. lactis NZ9000 was grown in M-17 medium (Mer- ck) supplemented with 0.5% glucose (GM-17) and chloramphenicol (10 ^g/mL) at 30 °C without aeration. 2. 2. Molecular Cloning Plasmid DNA was isolated with NucleoSpin Plasmid (Macherey and Nagel, Düren, Germany), with an additional lysozyme treatment step for L. lactis. Lactococci were transformed with electroporation using a Gene Pulser II apparatus (Biorad, Hercules, USA) according to the MoBiTec GmbH (Goettingen, Germany) instructions. Nucleotide sequencing was performed by GATC (Constance, Germany). Gene for SpyTag in fusion with B domain for expression in E. coli was amplified from pSDBA3b by PCR using primers B-F-NcoI-Spy and B-R-XhoI, cloned to pGEM-T Easy and then to pET28a via restriction enzymes NcoI/ XhoI, yielding pET_SpyT_Bd. Gene for SpyTag in fusion with B domain for secretion from L. lactis was amplified from pSDBA3b by PCR using primers B-F-BamHI and B-R-Kpn-Sy-Xba. Gene for SnoopTag in fusion with B domain was prepared likewise using primers B-F-BamHI and B-R-Kpn-So-Xba. Both were first cloned to plasmid pGEM-T Easy and then to plasmid pSDBA3b via restriction enzymes BamHI/XbaI, yielding pSD_SpyT_Bd and pSD_SnT_Bd. Gene for B domain for secretion from L. lactis was amplified from pSDBA3b by PCR using primers B-F-BamHI and B-R-Xba, first cloned to plasmid pGEM-T Easy and then to plasmid pSDBA3b via restriction enzymes BamHI/XbaI, yielding pSD_Bd (Table 2-4). Genes for SpyCatcher and SnoopCatcher were designed and synthesized de novo as gBlocks (Table 4) by Plavec and Berlec: Surface Anchoring on Lactococcus lactis 20 Acta Chim. Slov. 2019, 66, 18-27 Table 1. Strains used in this study Strain Relevant features or sequence Reference E. coli DH5a endAl glnV44 thi-1 recAl relAl gyrA96 deoR F- ®80dlacZAM15 A(lacZYA-argF)U169, Invitrogen hsdR17(rK- mK+), L. lactis NZ9000 MG1363 nisRK ApepN NIZO Table 2. Plasmids used in this study Plasmid Relevant features or sequence Reference pET28a Kanr, E. coli expression vector Novagen pGEM-T Easy Apr, cloning vector for PCR products Promega pSDBA3b pNZ8148 containing gene fusion of spUsp45, b-dom and acmA3b 31 pNZ8148 pSH71 derivative, PnisA, Cmr, nisin-controlled expression 32-34 pET_SpyT_Bd pET28a containing gene fusion of spytag and b-dom This work pSD_SpyC_AcmA pNZ8148 containing gene fusion of spUsp45, spycatcher and acmA3b This work pSD_SnC_AcmA pNZ8148 containing gene fusion of spUsp45, snoopcatcher and acmA3b This work pSD_Bd pNZ8148 containing gene fusion of spUsp45 and b-dom This work pSD_SpyT_Bd pNZ8148 containing gene fusion of spUsp45, spytag and b-dom This work pSD_SnT_Bd pNZ8148 containing gene fusion of spUsp45, snooptag and b-dom This work pSD_SpyC_Bd pNZ8148 containing gene fusion of spUsp45, spycatcher and b-dom This work pSD_SpyT_AcmA pNZ8148 containing gene fusion of spUsp45, spytag and acmA3b This work Table 3. Primers used in this study Primer Relevant features or sequence Reference B-F-NcoI-Spy 5'-CCATGGCTCATATTGTAATGGTCGATGCATATAAACCAACCAAAGCTGATAA CAAATTCAACAAAGAAC-3' This work B-R-XhoI 5'-CTCGAGTTTTGGTGCTTGTGCATC-3' This work B-F-BamHI 5'-AGGATCCGCTGATAACAAATTCAAC-3' This work B-R-Kpn-Sy-Xba 5'-TTCTAGATTATTTGGTTGGTTTATATGCATCGACCATTACA This work ATATGAGCGGTACCTTTTGGTGCTTGTGCATC-3' B-R-Kpn-So-Xba 5'-TTCTAGATTATTTGTTAACTTTAATAAATTCGATGTCACCCA This work ACTTGGTACCTTTTGGTGCTTGTGCATC-3' B-R-Xba 5'-TTCTAGATTATTTTGGTGCTTGTGCATC-3' This work SpyC-F-Kpn 5'-AGGTACCGGAGCTATGGTTGATACATTG -3' This work SpyC-R-Xba 5'-TTCTAGATTAAATATGAGCATCACCTTTTGTTG-3' This work AcmA-F-Bam-SpyT 5'-AGGATCCGCTCATATTGTAATGGTCGATGCATATAAACCAACCAAA This work TCTGGTGGCTCGACAACC-3' AcmA-R-Xba 5'-TTCTAGATTATTTTATTCGTAGATACTGACC-3' This work Spy-F-Bam 5'-AGGATCCGGAGCTATGGTTGATACATTG-3' This work Spy-R-Eco 5'-AGAATTCAATATGAGCATCACCTTTTGTTG-3' This work Sno-F-Bam 5'-AGGATCCAAACCTTTGCGTGGTGCAG-3' This work Sno-R-Eco 5'-AGAATTCCTTTGGTGGGATTGGTTCGTTC-3' This work IDT (Leuven, Belgium). Gene for secretion of SpyCatch-er-B domain fusion from L. lactis was amplified from gBlock by PCR using primers SpyC-F-Kpn and SpyC-R-Xba, cloned to pGEM-T Easy and then to pSD_SpyT_Bd via restriction enzymes KpnI/XbaI, yielding pSD_SpyC_ Bd (Table 1-4). Genes for the surface display of SpyCatcher and SnoopCatcher were amplified from gBlocks using primer pairs Spy-F-Bam/Spy-R-Eco and Sno-F-Bam/Sno-R-Eco, respectively, and were cloned first to pGEM-T Easy and then to plasmid pSDBA3b via restriction enzymes EcoRI/ BamHI, yielding pSD_SpyC_AcmA and pSD_SnC_ AcmA, respectively. Gene for the display of SpyTag on the L. lactis surface was amplified from pSDBA3b by PCR using primers AcmA-F-Bam-SpyT and AcmA-R-Xba, cloned to pGEM-T Easy and then to pSD_SpyC_AcmA via re- Plavec and Berlec: Surface Anchoring on Lactococcus lactis Acta Chim. Slov. 2019, 66, 18-27 21 Table 4. Genes used in this study Gene Relevant features or sequence Reference spycatcher snoopcatcher spytag snooptag GGATCCGGAGCTATGGTTGATACATTGTCAGGTTTATCATCAGAACAA This work GGACAAAGTGGAGATATGACTATTGAAGAAGATTCTGCTACACATATTAAA TTTTCAAAACGTGATGAAGATGGAAAAGAATTAGCAGGTGCTACTATGGA ATTGCGTGATTCATCAGGTAAAACAATTTCAACTTGGATTTCAGATGGACAA GTTAAAGACTTTTATCTGTACCCTGGAAAATATACTTTCGTTGAAACAGCAGCA CCTGACGGATACGAAGTTGCTACTGCTATCACTTTTACAGTTAACGAACAAGG TCAAGTTACAGTTAATGGTAAAGCAACAAAAGGTGATGCTCATATTGAATTC GGATCCAAACCTTTGCGTGGTGCAGTCTTCTCATTACAAAAACAACATCC This work AGACTACCCTGATATTTATGGTGCCATTGATCAAAATGGTACTTATCAGAA TGTTCGAACTGGTGAAGACGGAAAATTGACTTTTAAGAATTTGAGTGACGG TAAATATCGTTTATTCGAAAACAGTGAACCAGCTGGATATAAGCCAGTA CAAAATAAACCTATTGTCGCATTTCAAATTGTAAACGGTGAAGTTAGAGACG TTACTTCTATTGTACCTCAGGATATTCCTGCTACTTATGAATTTACTAA TGGAAAACATTATATTACGAA CGAACCAATCCCACCAAAGGAATTC GCTCATATTGTAATGGTCGATGCATATAAACCAACCAAA This work AAGTTGGGTGACATCGAATTTATTAAAGTTAACAAA This work striction enzymes BamHI/XbaI, yielding pSD_SpyT_ AcmA (Table 2-4). 2. 3. Expression of SpyTag-B Domain Fusion in E.coli 100 ^L of overnight culture of E. coli BL21 (DE3) harboring plasmid pET_SpyT_Bd was diluted (1:100) in 10 mL of fresh LB medium and, to determine optimal expression conditions, various parameters were tested: incubation temperature 37 °C or 25 °C, induction at optical densities (A6Qo) 0.5 or 1.0, induction with IPTG in concentration of 0.5 and 1.0 mM. Large-scale expression of SpyTag-B domain fusion was performed by diluting 10 mL of overnight culture of E. coli BL21 (DE3) harboring plasmid pET_SpyT_Bd in 1 L of fresh LB medium. The culture was grown to optical density A600 = 0.5 at 37 °C. At that point, the expression of SpyTag-B domain fusion, additionally tagged with hexa-histidine (H6), was induced by the addition of 0.5 mM IPTG for 3 h at 37 °C; the conditions that were found to be the most effective in preliminary screen. 2. 4. Purification of SpyTag-B Domain With Hexa-histidine (H6) Tag The E. coli culture expressing SpyTag-B domain with hexa-histidine (H6) tag was centrifuged at 5000 x g for 20 min and the pellet resuspended in 20 mL of equilibration/ wash (Eq/W) buffer (50 mM NaH2PO4, 300 mM NaCl, pH 7.0). The cells were lysed with a cycle of freezing and thawing, and with 3 cycles of 5 min sonication with a UPS200S sonifier (Hielscher, Teltow, Germany). After cell lysis, the suspension was centrifuged at 15000 x g for 20 min and the supernatant stored. SpyTag-B domain with H6 tag was iso- lated with BD Talon metal affinity resin (BD Biosciences), using batch/gravity-flow column purification and imidazole elution (elution buffer: 50 mM NaH2PO4, 300 mM NaCl, 150 mM imidazole, pH 7.0) according to the manufacturer's instructions. Eluted fractions were analyzed by SDS-PAGE, pooled and concentrated by ultrafiltration using Amicon Ultra 1 KDa cut off (Merck Millipore; Darmstadt, Germany). Purified fusion protein was dialyzed against PBS. 2. 5. Expression of Fusion Proteins in L. lactis Overnight cultures of L. lactis NZ9000 harboring pSD_SpyC_AcmA, pSD_SnC_AcmA, pSD_SpyT_AcmA, pSD_SpyT_Bd, pSD_SnT_Bd, pSD_SpyC_Bd, or pSD_Bd were diluted (1:100) in 10 mL of fresh GM-17 medium and grown to optical density A600 = 0.8-1.0. Fusion protein expression was induced with 25 ng/mL nisin (Fluka AG, Buchs, Switzerland) for 3 h at 30 °C. After incubation, 1 mL of culture was stored at 4 °C for flow cytometric analysis. The remaining cell culture was centrifuged at 5000 x g for 10 min. 2. 6. Formation of Isopeptide Bond Between SpyTag and SpyCatcher / SnoopTag and SnoopCatcher In order to enable binding of E. coli-expressed Spy-Tag-B domain to L. lactis with surface displayed SpyCatcher, we centrifuged 20 ^L of the cell culture of L. lactis with surface displayed SpyCatcher for 5 min at 5000 x g at 4 °C, resuspended the pellet in 500 ^L of purified E. coli-ex-pressed SpyTag-B domain with concentration of 0.4 mg/ mL and incubated for 2 h at RT with constant shaking. To enable binding of SpyTag-B domain from L. lactis conditioned medium to L. lactis with surface displayed Plavec and Berlec: Surface Anchoring on Lactococcus lactis 22 Acta Chim. Slov. 2019, 66, 18-27 SpyCatcher, we separately cultured the SpyTag-B domain-secreting L. lactis and SpyCatcher-displaying L. ¡actis. The producer cells of SpyTag-B domain were removed and the conditioned medium containing SpyTag-B domain fusion protein was stored. 20 ^L of L. lactis cell culture with surface displayed SpyCatcher was centrifuged for 5 min at 5000 x g at 4 °C, resuspended in 500 ^L of conditioned medium containing SpyTag-B domain and incubated overnight at RT with constant shaking. Binding of SpyTag-B domain secreted from L. lactis to SpyCatcher displayed on L. lactis was also achieved during co-culturing of the two strains. 100 ^L of overnight cultures of L. lactis NZ9000 harboring pSD_SpyT_Bd and pSD_SpyC_AcmA were concomitantly added to 10 mL of fresh GM-17 medium. Simultaneous expression of the two fusion proteins was induced with nisin. Similarly, binding of SpyCatcher-B domain secreted from L. lactis to SpyTag displayed on L. lactis was achieved by co-culturing L. lactis NZ9000 harboring pSD_SpyC_Bd and pSD_SpyT_AcmA, as well as binding of SnoopTag-B domain secreted from L. lactis to SnoopCatcher displayed on L. lactis by co-cultur-ing L. lactis NZ9000 harboring pSD_SnC_AcmA and pSD_SnT_Bd. 2. 7. SDS-PAGE and Western Blot SDS PAGE was performed with a Mini-Protean II apparatus (Bio-Rad, Hercules, USA). Samples were thawed in an ice bath, briefly sonicated with UPS200S sonicator (Hielscher, Teltow, Germany), mixed with 2x Laemmli Sample buffer and DTT, and denatured by heating at 100 °C before loading. Page Ruler Plus (Fermentas, St. Leon-Rot, Germany) pre-stained standard was used for molecular weight comparison. Proteins were stained with Coomassie Brilliant Blue or transferred to polyvinylidene fluoride (PVDF) membranes (Immobilon-P, Millipore) using wet transfer at 100 V for 90 minutes. Membranes were blocked in 5% non-fat dried milk in TBS with 0.05% Tween-20 (TBST; 50 mM Tris-HCl, 150 mM NaCl, 0.05% Tween 20, pH 7.5) and incubated overnight at 4 °C with goat anti-protein A antibody (1:2000, Abcam) in 5% non-fat dried milk in TBST. Following three washes with TBST, membranes were incubated for 2 h with HRP conjugated secondary donkey anti-goat IgG (1:5000, Jackson ImmunoResearch) in 5% non-fat dried milk in TBST. After three further washes with TBST, membranes were incubated with Lumi-Light chemiluminescent reagent (Roche). Images were acquired using ChemiDoc MP Imaging System (BioRad). 2. 8. Flow Cytometry For flow cytometry 20 ^L of cell culture in stationary phase was added to 500 ^L of Tris-buffered saline (TBS; 50 mM Tris-HCl, 150 mM NaCl, pH 7.5) and centrifuged for 5 min at 5000 x g at 4 °C. The pellet was resuspended in 500 ^L of TBS with 1 ^L of fluorescein-5-isothiocyanate (FITC)-conjugated human IgG antibody (Jackson ImmunoResearch, West Grove, USA) that binds the B domain via Fc region. After 2 h of incubation at RT with constant shaking at 100 rpm, cells were washed three times with 200 ^L 0.1% TBST and finally resuspended in 500 ^L TBS. Samples were analyzed with a flow cytometer (FACS Calibur; Becton Dickinson, Franklin Lakes, USA) using excitation at 488 nm and emission at 530 nm in the FL1 channel. The geometric mean fluorescence intensity (MFI) of at least 20 000 bacterial cells in the appropriate gate was measured. The average of at least three independent experiments was considered. All the samples went through the same procedures of preparation for flow cytometry analysis. 2. 9. Statistical Analyses Statistical analyses were performed with GraphPad Prism 5.0 software. Student's t test was used to compare the significance of differences between B domain-displaying bacteria and control. a) 37°C 25°C OD6o 1000 490 ± 18 > 1000 125 ± 10 ND ND Cathepsin H C1 n.i. 100 ± 10 24 ± 5 32 ± 6 ND ND Legumain C13 21.5 ± 2.81 3.38 ± 1.44 9.17 ± 1.09 > 1000 n.i. n.i. Caspase 3, 6, 9 C14 n.i. ND ND ND ND ND Trypsin S1 n.i. n.i. n.i. 160 ± 14 3.10 ± 0.66 0.022 ± 0.002 Chymotrypsin S1 ND ND ND ND 120 ± 20 116 ± 8 Kallikrein S1 ND ND ND ND > 1000 > 1000 Thrombin S1 ND ND ND ND n.i. n.i. Subtilisin S8 ND ND ND ND > 1000 > 1000 Pepsin A1 n.i. n.i. n.i. n.i. n.i. n.i. Table 4. Inhibition of various proteases by natural mycocypins. The kinetic data are as reported.17 Standard deviations are given where appropriate. nMcp' natural macrocypin; nClt' natural clitocypin; n.i., no inhibition. Enzyme Protease family Ki (nM) nClt nMcp Papain C1 2.5 ± 0.94 5.04 ± 0.98 Cathepsin L C1 0.03 ± 0.002 3.81 ± 1.66 Cathepsin V C1 0.14 ± 0.01 12.6 ± 3.77 Cathepsin S C1 3.2 ± 0.3 47.1 ± 3.1 Cathepsin K C1 0.02 ± 0.005 4.5 ± 0.5 Cathepsin B C1 > 1000 515 ± 36 Cathepsin H C1 n.i. 370 ± 11 Legumain C13 7.1 ± 1.12 110 ± 23 Trypsin S1 n.i. n.i. Pepsin A1 n.i. n.i. 2. 3. Structural Plasticity Mycocypins and mycospins both possess a (-trefoil fold,23,31 thus classifying them to clan IC in the MEROPS classification, together with Kunitz serine protease inhibitors from plants (family I3).13 The (-trefoil fold consists of a (-barrel composed of three pairs of antiparallel (-strands. An additional three pairs of (-strands cover the (-barrel. The strands are connected by loops of different shapes and compositions (Figure 3). The large surface area of the loops, that accounts for approximately 70% of the proteins total solvent accessible area, enables these proteins to interact with many different binding partners, including proteins, carbohydrates and DNA.23,31,32 For the inhibition of papain-like proteases, mycocypins utilize two loops ((1-(2 and (3-(4) that bind to either side of the active site cleft with several hydrogen bonds, occluding the catalytic cysteine. The binding is associated with a glycine-glycine peptide-bond flip (Gly-24-Gly25 in clitocypin) that occurs before or concurrently with inhibitor docking.31 The mode of cysteine protease inhibition, as revealed by the cathepsin V - clitocypin complex, is unique in utilizing two loops to achieve inhibition, while other known modes of inhibition by cysteine protease inhibitors, like those of cystatins and thyropins, utilize three loops to achieve active site occlusion.31,32 Inhibition of asparaginyl peptidase/legumain by mycocypins is achieved via a second inhibitory active site in the (5-(6 loop. Site-directed mutagenesis has confirmed that residues Asn72 in macrocypins and Asn69 in clitocypin mediate inhibition of asparaginyl peptidase.31 Furthermore, the same inhibitory active site mediates the inhibition of trypsin when Asn replaces Lys or Arg in different macrocypins but not in clitocypin.31 The inhibitory mechanism of the cysteine protease asparaginyl peptidase (family C13) and of the serine protease trypsin (family S1) appears to involve a similar substrate-like binding inhibiti- 32 on.32 Cospin and cnispin are classic canonical inhibitors that bind to the active site in a substrate-like manner and form a tight and stable complex with trypsin. The trypsin -cospin complex is stable for weeks at 37 °C while the trypsin-cnispin complex is degraded within 24 h. This is also reflected in the stronger inhibition of trypsin by cos-pin as opposed to that by cnispin (Table 3). Inhibition of trypsin by mycospins is achieved through the different inhibitory reactive sites in cospin and cnispin. The reactive site residue P1 of cospin is Arg27 in the (2-(3 loop, while Lys127 in the (311-^12 loop of cnispin fulfils the same role (Figure 2 & 3).23,30 Inhibitory activity can be mediated by Sabotič et al.: fi-Trefoil Protease Inhibitors Unique to Higher Fungi Acta Chim. Slov. 2019, 66, 28-36 33 Figure 3. Structures of fungal cysteine protease inhibitors. Ribbon diagrams of the cysteine protease inhibitors macrocypin 1 (PDB code 3H6Q) in orange and clitocypin (PDB code 3H6S) in magenta are shown next to that of the serine protease inhibitor cospin (PDB code 3N0K9) in cyan. Loops with the following inhibitory reactive sites are marked with arrows: light grey arrows indicate asparaginyl protease/legumain inhibition, dark grey arrows indicate papain-like protease inhibition while white and black arrows indicate trypsin inhibition. A schematic representation of the (3-trefoil fold loops involved in protease inhibition is shown bottom right. The protease family inhibited by individual loops of the (3-trefoil fold in these fungal inhibitors is indicated by coloured arrows as follows: the asparaginyl protease/legumain with orange, papain-like proteases with magenta and trypsin with cyan. both (2-^3 and (311—^12 loops in the two inhibitors; engineering of various P1 residues in the aforementioned loops yielded a strong or weak trypsin inhibitor, a chymot-rypsin-specific inhibitor, a double headed trypsin inhibitor or a double-headed trypsin and chymotrypsin inhibitor. Other serine proteases are only weakly or not at all inhibited. Cysteine and aspartic proteases were not inhibited and asparaginyl peptidase/legumain inhibition was not achieved by introducing Asn as the P1 residue.30 2. 4. Functional Variability The proposed function of mycocypins and mycos-pins is to regulate both the endogenous proteolytic system and the defence against predators and pathogens. Mycocy-pins and mycospins are cytoplasmic proteins that are not secreted. In addition to the absence, in all the identified genes, of a signal sequence for the classical secretion and to the low number of cysteine residues and lack of glycosyla-tion in natural samples, no secretion of cnispin and clitocypin has been detected from cultivated mycelium.22,23,25 An obvious biological role for protease inhibitors is the regulation of the endogenous proteolytic system. Activity of several cysteine proteases from fruiting bodies of different basidiomycetes is inhibited by clitocypin.11 Similarly, cnispin and cospin inhibit the activity of various serine proteases from their origin species as well as from other basidiomycetes.11,22,23 Regulation of the complex developmental and temporal expression of both mycocypins and mycospins indicates potentially different biological roles for different inhibitors.22,6 Another possible role for protease inhibitors, that is indirectly but strongly supported, is in defence against various antagonists. The strong toxicity of cnispin and cospin against Drosophila melanogaster larvae, their cytoplasmic localization and the higher expression in fruiting bodies Sabotič et al.: fi-Trefoil Protease Inhibitors Unique to Higher Fungi 34 Acta Chim. Slov. 2019, 66, 28-36 compared to that in mycelium indicates their role in the defence of the spore-bearing fruiting body against dipte-ran larvae that hatch and feed on fruiting bodies.22,23 Protection of reproductive tissues from pests and diseases has also been suggested as a defensive role of mycocypins. Thus, cysteine proteases are predominant digestive proteases in many insects and slugs and could be targeted, like trypsin-like digestive proteases in dipteran insects are by mycospins. Furthermore, cysteine proteases are important virulence factors of different pathogenic bacteria, parasites and mycoviruses. These could be targeted by different mycocypins, as indicated by their different expression profiles. Clitocypin, which is constitutively expressed in large amounts in fruiting bodies, represents one line of defence, while the specific pattern of expression localized to the outer layer of the developing fruiting body of some macrocy-pins constitutes another defence strategy for protection against external attacks by predators or pathogens. The inducible expression of clitocypin analogs in L. bicolor when challenged by an antagonistic soil bacterium supports their role in defence. A defensive role is further supported by the high thermal and pH stability and the resistance to proteolytic degradation of these proteins, as well as by their high sequence diversity and versatile inhibitory profile.17,22,23,25,26,28 Another layer of regulation of the ^-trefoil fungal protease inhibitors mycocypins and mycospins, is indicated by their interaction in vitro with ^-trefoil lectins MpL and CNL from the same species, which are also expressed intra-cellularly and involved in fruiting body defence.21,27, 33-36 2. 5. Diverse Applications Based on their unique characteristics indicating their function in defence, as described in the previous section, mycocypins have been evaluated for their potential in protecting plants against herbivores. A major pest, Colorado potato beetle (Leptinotarsa decemlineata), that utilizes cysteine proteases for protein digestion, has been used as a model. Clitocypin and macrocypins were shown to exhibit adverse effects on Colorado potato beetle larvae, both when expressed as proteins in potato leaves and when recombinant proteins produced in a bacterial expression system were added to the diet. Clitocypin and macrocypins reduced the weight gain of larvae and delayed their development. The effect was linked to inhibition of the special adaptive cysteine proteases, intestains, in larval guts. Moreover, dietary mycocypins did not induce the expression of known adaptation-related genes of digestive enzymes in guts of Colorado potato beetle larvae, as was the case with all other dietary inhibitors from other sources.37,38 The potential of mycocypins and mycospins in bio-technological applications has been confirmed by their use as ligands in affinity chromatography for isolating proteases from various sources. Trypsin was isolated from a complex, partially purified, trypsin sample using cnispin-affi- nity chromatography. Active cysteine proteases of families C1 (papain-like) and C13 (legumain/asparaginyl protease) have been isolated from plant and animal sources using macrocypin affinity chromatography. Their superior characteristics in terms of stability to pH and temperature make them ideal candidates for use as affinity chromato-graphy ligands. They withstand the harsh conditions during immobilization procedures. For example, following 24h incubation at neutral pH and 45 °C for covalent binding to a monolithic disk, the unoccupied groups are inactivated by incubation for 1 h in 0.5 M H2SO4 at 50 °C and macrocypins then retain their inhibitory activity through several elution cycles of extreme pH changes.39 The distinct inhibitory profiles of mycocypins and the highly specific inhibitory profile of mycospins makes them valuable tools in medical research, since many cell processes depend on appropriately regulated proteolytic activity. Protease inhibitors are being studied as promising therapeutic drugs for many types of disease by targeting a variety of deregulated proteases, including those involved in cancer and autoimmune, neurodegenerative, inflammatory, and cardiovascular diseases; secreted bacterial, fungal, and parasite proteases; and viral polyprotein processing proteases.7,40 3. Conclusions and Future Perspectives Protein protease inhibitors from a number of higher fungi exhibit a variety of unique characteristics when compared to similar protease inhibitors from plant, animal or microbial sources. Distinct inhibitory specificity profiles, coupled with structural plasticity of the ^-trefoil fold, afford these inhibitors their diverse functions. Further, their noteworthy stability enables wide-ranging application of mycocypins and mycospins. The uniqueness of these protease inhibitors lies in the combination of the mentioned exceptional features in one protein family promoted by lack of homologues outside of the fungal kingdom. The protease inhibitors from higher fungi described here just scratch the surface of the immense potential of the fungal proteolytic defence system hidden in forests in Slovenia and in forests worldwide. In addition to the cysteine protease inhibitors represented by mycocypins and the serine protease inhibitors represented by mycospins, there are most probably protease inhibitors waiting to be identified from higher fungi, possibly with the ^-trefoil fold, that inhibit other catalytic classes of proteases, including aspartic and metallo-prote-ases. Another direction for research, involving the ^-trefo-il fold proteins with other functionalities such as carbohydrate binding, lies via the ^-trefoil fold lectins that have been shown to be versatile and to support unique characteristics. It will be interesting to find more novel multifunctional proteins with both carbohydrate binding and Sabotič et al.: fi-Trefoil Protease Inhibitors Unique to Higher Fungi Acta Chim. Slov. 2019, 66, 28-36 35 inhibitory activities. Knowledge of the different functionalities evolved naturally in the ^-trefoil scaffold will facilitate the engineering of customized multifunctional proteins. 4. Acknowledgements We dedicate this paper to prof. Igor Kregar, who was the leader of our group for many years and initiated studies on plant and fungal biochemistry and molecular biology. We are grateful to dr. Jože Brzin who initiated the research on protease inhibitors from mushrooms and guided our investigations until recently. We thank dr. Tatjana Popovič who meticulously determined their kinetics and passed on her knowledge brilliantly. We are indebted to dr. Marjetka Kidrič and prof. Roger H. Pain for numerous discussions and invaluable input. We would also like to acknowledge the many other researchers and students from the Jožef Stefan Institute, the National Institute of Biology, the ETH Zürich and University of Bristol who, over the years, have contributed pieces of the puzzle to the knowledge we now have on fungal protease inhibitors. 4. 1. Funding This work was supported by the Research Agency of the Republic of Slovenia Grant P4-0127 (to J.K.). 5. References 1. C. Lopez-Otin and J. S. Bond, J Biol Chem 2008, 283, 3043330437. DOI:10.1074/jbc.R800035200 2. M. B. Rao, A. M. Tanksale, M. S. Ghatge and V. V. Deshpande, Microbiol Mol Biol Rev 1998, 62, 597-635. 3. H. Maeda, Microbiol Immunol 1996, 40, 685-99. DOI:10.1111/j.1348-0421.1996.tb01129.x 4. A. Schaller, Planta 2004, 220, 183-97. DOI:10.1007/s00425-004-1407-2 5. I. Yike, Mycopathologia 2011, 171, 299-323. DOI:10.1007/s11046-010-9386-2 6. N. D. Rawlings, D. P. Tolle and A. J. Barrett, Biochem J 2004, 378, 705-16. DOI:10.1042/bj20031825 7. J. Sabotic and J. 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DOI:10.1099/mic.0.032805-0 23. J. Sabotič, S. Bleuler-Martinez, M. Renko, P. Avanzo Caglič, S. Kallert, B. Štrukelj, D. Turk, M. Aebi, J. Kos and M. Kunzler, J Biol Chem 2012, 287, 3898-907. DOI: 10.1074/jbc.M111.285304 24. S. Odani, K. Tominaga, S. Kondou, H. Hori, T. Koide, S. Hara, M. Isemura and S. Tsunasawa, Eur J Biochem 1999, 262, 91523. DOI:10.1046/j.1432-1327.1999.00463.x 25. J. Sabotič, D. Gaser, B. Rogelj, K. Gruden, B. Štrukelj and J. Brzin, Biol Chem 2006, 387, 1559-66. DOI:10.1515/BC.2006.194 26. J. Sabotič, S. Kilaru, M. Budič, M. B. Gašparič, K. Gruden, A. M. Bailey, G. D. Foster and J. Kos, Biochimie 2011, 93, 16851693. DOI:10.1016/j.biochi.2011.05.034 27. J. Sabotič, R. A. Ohm and M. Kunzler, Appl Microbiol Biot 2015, submitted. 28. A. Deveau, M. Barret, A. G. Diedhiou, J. Leveau, W. de Boer, F. Martin, A. Sarniguet and P. Frey-Klett, Microbial ecology 2015, 69, 146-59. DOI:10.1007/s00248-014-0445-y 29. T. Lukanc, J. Brzin, J. Kos and J. Sabotič, Acta Biochim Pol 2017, 64, 21-24. DOI:10.18388/abp.2015_1187 30. P. Avanzo Caglič, M. Renko, D. Turk, J. Kos and J. Sabotič, Biochimica et Biophysica Acta 2014, 1844, 1749-56. 31. M. Renko, J. Sabotič, M. Mihelič, J. Brzin, J. Kos and D. Turk, J Biol Chem 2010, 285, 308-16. DOI:10.1074/jbc.M109.043331 32. M. Renko, J. Sabotič and D. Turk, Biological chemistry 2012, 393, 1043-54. DOI:10.1515/hsz-2012-0159 Sabotič et al.: fi-Trefoil Protease Inhibitors Unique to Higher Fungi 36 Acta Chim. Slov. 2019, 66, 28-36 33. J. Pohleven, J. Brzin, L. Vrabec, A. Leonardi, A. Čokl, B. Štrukelj, J. Kos and J. Sabotič, Appl Microbiol Biotechnol 2011, 91, 1141-8. DOI:10.1007/s00253-011-3236-0 34. J. Pohleven, N. Obermajer, J. Sabotič, S. Anžlovar, K. Sepčic, J. Kos, B. Kralj, B. Štrukelj and J. Brzin, Biochim Biophys Acta 2009, 1790, 173-81. 35. J. Pohleven, M. Renko, S. Magister, D. F. Smith, M. Kunzler, B. Štrukelj, D. Turk, J. Kos and J. Sabotič, J Biol Chem 2012, 287, 10602-12. DOI:10.1074/jbc.M111.317263 36. S. Žurga, J. Pohleven, M. Renko, S. Bleuler-Martinez, P. Sos-nowski, D. Turk, M. Kunzler, J. Kos and J. Sabotič, FEBS J 2014, 281, 3489-506. D0I:10.1111/febs.12875 37. I. Šmid, K. Gruden, M. Buh Gašparič, K. Koruza, M. Petek, J. Pohleven, J. Brzin, J. Kos, J. Žel and J. Sabotič, J Agr Food Chem 2013, 61, 12499-509. D01:10.1021/jf403615f 38. I. Šmid, A. Rotter, K. Gruden, J. Brzin, M. Buh Gašparič, J. Kos, J. Žel and J. Sabotič, Pestic Biochem Physiol 2015, 122, 59-66. D0I:10.1016/j.pestbp.2014.12.022 39. J. Sabotič, K. Koruza, B. Gabor, M. Peterka, M. Barut, J. Kos and J. Brzin, in: S. Magdeldin (Ed.): Affinity Chromatography, InTech, Rijeka, 2012, pp. 307-332. 40. J. Sabotič and J. Kos, in: J.-M. Merillon and K. G. Ramawat (Eds.): Fungal Metabolites, Springer International Publishing, Switzerland, 2016. Povzetek Inhibitorji cisteinskih proteaz, klitocipin in makrocipini iz višjih gliv (mikocipini), skupaj s serinskima proteaznima inhibitorjema, ki sta zelo specifična za tripsin, kospin in knispin iz višjih gliv (mikospini), kažejo številne značilnosti, ki jih razlikujejo od inhibitorjev proteaz iz drugih virov. Visoka genetska raznolikost ima vpliv na funkcionalnost in / ali stabilnost proteinov in privede do številnih proteinskih variant z nekoliko različnimi inhibitornimi profili, ki vplivajo na vrsto tarčne proteaze in / ali moč inhibicije. Imajo p-triperesno zvitje, ki kaže visoko plastičnost pri uporabi 11 različnih zank za inhibicijo različnih družin proteaz z različnimi mehanizmi inhibicije. Njihova vsestranskost se kaže tudi v regu-latorni in obrambni funkciji ter široki potencialni uporabi v biotehnologiji, kmetijstvu in medicini. Sabotič et al.: fi-Trefoil Protease Inhibitors Unique to Higher Fungi DOI: 10.17344/acsi.2018.4516 Acta Chim. Slov. 2019, 66, 37-44 /^creative ^commons Scientific paper Epitope Mapping of Major Ragweed Allergen Amb a 1 Abida Zahirovic,1 Borut Štrukelj,1 Peter Korošec2 and Mojca Lunder1'* 1 Faculty of Pharmacy, University of Ljubljana, Ljubljana, Slovenia 2 University Clinic of Respiratory and Allergic Diseases Golnik, Golnik, Slovenia * Corresponding author: E-mail: mojca.lunder@ffa.uni-lj.si Tel.: +386 1 47 69 570; Fax.: +386 1 42 58 031 Received: 06-01-2018 Dedicated to the memory of Prof. Dr. Igor Kregar Abstract Ragweed is a prominent cause of seasonal allergies. Thus far, information on IgE-binding sites of major allergen in ragweed pollen, Amb a 1, is very limited. A powerful experimental method to gain insights on the allergen epitopes is the selection of peptides from biological libraries that bind to anti-allergen antibodies. In this work, we aimed to map IgE epitopes of Amb a 1 using epitope-mimicking short peptides - mimotopes that were affinity-selected from phage-dis-played random peptide libraries. The peptides weakly aligned with the Amb a 1 primary sequence, thus suggesting that the epitopes are conformational. When the peptides were mapped onto the surface of Amb a 1 homology model, the EpiSearch analysis predicted the location of four potential epitopic sites on surface patches centred at residues K104, S110, H214, and W312. The peptides matching to the predicted epitopes bound selectively to the IgE from pool of ragweed-allergic patients' sera and therefore represent mimetics of Amb a 1 IgE epitopes. The knowledge of IgE epitopes is a prerequisite for the rational design of molecular-based approaches to diagnosis and immunotherapy of allergic diseases. Keywords: Ragweed allergy; Amb a 1; epitope mapping; phage display; mimotopes 1. Introduction Short ragweed (Ambrosia artemisiifolia) is one of the most important allergen source in North America.1 Because of its fast spreading, sensitization rates are also increasing in Central and Southeastern Europe, ranging from 15% to ~80%.2 Current therapeutic options for ragweed allergy involve symptomatic treatment and allergen-specific immunotherapy. Conventional immunotherapy with crude pollen extracts is the only available curative treatment. However, it may induce undesired IgE-mediat-ed side effects and long-term therapy is required, which often hampers patient compliance.3,4 Therefore, new approaches to immunotherapy that include well-defined therapeutic molecules with reduced or abolished IgE binding capacity are being investigated. Ragweed pollen allergy is especially suited for molecule-based immunotherapeutic strategies due to the dominance of one allergen. Molecule-based vaccines are based on individual allergen proteins, allergen-derived peptides containing relevant epitopes or epitope-mim-icking peptides (mimotopes).5 Among 14 allergens described in ragweed pollen, Amb a 1 has been identified as the major disease-causing agent, which reacts with IgE of more than 90% of the ragweed-sensitized patients.6,7 It is a non-glycosylated 38 kDa protein that belongs to the family of pectate lyases and accounts for up to 15% of total proteins in the ragweed pollen.8,9 It has been demonstrated that Amb a 1 in a form of a conjugate with toll-like receptor agonist can replace the whole pollen extract in immunotherapy.10 Five different isoforms of Amb a 1 with about 80% sequence identity have been found.11 They display distinct patterns of IgE binding and immu-nogenicity with limited B- and T-cell cross-reactivity patterns.12 Amb a 1 is cross-reactive with its homologous allergen in mugwort pollen Art v 6, a pectate lyase with 65% of sequence identity with Amb a 1.13-15 The identification of relevant epitopes can reveal the molecular basis of allergenic cross-reactivity. Therefore, detailed studies of T- and B- cell epitopes are necessary for rational de- Zahirovic et al.: Epitope Mapping of Major Ragweed 38 Acta Chim. Slov. 2019, 66, 37-44 sign of molecule-based reagents for diagnosis and immunotherapy. Investigation of T-cell response to Amb a 1 revealed multiple dominant T-cell epitopes (Amb a 1 176-191, 200-215, 280-295, 304-319, 320-335, and 344-359).16,17 However, the data on the conformational B-cell epitopes is still scarce. Screening of random peptide phage libraries against anti-allergen antibodies is a fast and relatively inexpensive alternative compared to other methods for epitope mapping and in combination with computer-based algorithms can lead to the identification of conformational allergen epitopes.18,19 Successful application of this technology provides peptide mimotopes that are able to bind IgE antibodies and are not necessarily identical to original epitope, but rather mimic its essential features.20,21 Mimo-topes are considered to have similar physicochemical characteristics and spatial organization as their corresponding epitopes. As such, they may be employed for the development of safer immunotherapies either based on carrier-bound peptide vaccines or based on hypoallergen-ic recombinant allergens.22,23 In this study, we screened phage display libraries of random peptides using Amb a 1-specific IgG to define peptides mimicking Amb a 1 epitopes. Best mimotope candidates were tested for their IgE reactivity with sera of ragweed-allergic patients. Using computational epitope mapping tools affinity-selected peptide sequences were analyzed to predict the location of epitopes on Amb a 1. 2. Experimental 2. 1. Purification and Evaluation of Target Antibodies Anti-Amb a 1 IgG were affinity-purified from rabbit antiserum (Indoor Biotechnologies, Cat# PA-AM1, RRID:AB_2728628) using natural allergen Amb a 1 (INDOOR Biotechnologies) immobilized on Dynabeads M-280 Tosylactivated (Thermo Fisher Scientific, Waltham, Massachusetts, USA) following the manufacturer's instructions. 2. 2. Biopanning of Phage Display Libraries Affinity-purified rabbit IgG specific for Amb a 1 were immobilized alternatingly onto 0.45 mg of protein G or protein A coupled magnetic beads (Dynabeads, Thermo Fisher Scientific) by incubation in PBS/0.05% Tween 20 for 30 min at room temperature and used as a target in biopanning. Three phage display libraries (New England Biolabs, Ipswich, Massachusetts, USA) of linear heptam-er, linear dodecamer, and cyclic heptamer random peptides were panned as described in the manufacturer's manual. The libraries contain approximately 109 unique peptide sequences fused to the pIII minor coat protein of the M13 filamentous phage. Bound phages were eluted from the target antibodies either with 0.1 M glycine-HCl (pH 2.2) for 10 min followed by immediate neutralization with 1 M Tris (pH 8.0) or competitively with Amb a 1 at the final concentration of 12 ^g/ml. After three rounds of affinity selection, 24 individual clones from each elution method (total 144 clones) were amplified and screened for binding to target antibodies by monoclonal phage ELISA. 2. 3. Monoclonal Phage ELISA MaxiSorp microtiter plates (Thermo Fisher Scientific, Waltham, Massachusetts, USA) were coated with 2 ^g/ ml of anti-Amb a 1 IgG (RRID:AB_2728628) in PBS overnight at 4 °C. Plates were blocked with 5% skimmed milk in PBS for 1.5 h at room temperature and washed three times with PBS/0.1% Tween 20. A separate set of wells was covered with blocking buffer only, to determine background binding. The amplified phage clones or control (wild-type phage clone with no peptide displayed on its surface) in LB were then loaded into the wells. After 60 min of incubation, the wells were washed five times with PBS/0.1% Tween 20. For detection, HRP-conjugated an-ti-M13 monoclonal antibodies (GE Healthcare Cat# 27942101 RRID: AB_2616587) diluted 1:5000 were added to the wells and incubated for 1 h. The colour was developed with TMB Super Tracker substrate (ImmunoO4, Westminster, UK) supplemented with 0.006% H202. After terminating the reaction with 2 M H2SO4, absorbance was measured at 450 nm with the microtiter plate reader (Te-can Safire, Tecan Group AG, Mannedorf, Switzerland). Phage clones with the highest target to background ab-sorbance ratio were subjected to DNA sequencing (GATC Biotech, Konstanz, Germany). 2. 4. Characterization of Phage-displayed Peptides Binding to Target Antibodies Seventeen phage clones displaying unique peptides were purified by PEG-precipitation, resuspended in PBS and quantified by spectrophotometry. Binding of the phage-displayed peptides to the target antibodies was assessed by semiquantitative ELISA. The suspensions containing 2 x 109 pfu of individual phage clones or control phage in PBS were loaded into the wells. To detect possible binders to antibody constant regions, the wells were coated with control rabbit antiserum raised against procathepsin X (Biogenes, Berlin, Germany) diluted 1:1000 in PBS overnight at 4 °C. For competition ELISA selected clones were added to the wells together with the allergen in three different concentrations (1 ^g/ml, 5 ^g/ml, 10 ^g/ml) and incubated with target antibodies for 1 h at room temperature. The following steps in the assays were as described above for monoclonal phage ELISA. All experiments were carried out in triplicates. Zahirovic et al.: Epitope Mapping of Major Ragweed 39 Acta Chim. Slov. 2019, 66, 37-44 2. 5. Linear Alignment of Peptides with Amb a 1 Sequence and Mapping to the 3D Homology Model of Amb a 1 Obtained amino acid sequences were checked for the presence of target-unrelated peptides using the MimoDB 2.0 database. Peptides were compared among themselves and aligned with Amb a 1 sequence using multiple sequence alignment program, Clustal Omega, to find a consensus pattern of amino acids.24 The 3D model structure of Amb a 1 was generated using a protein fold recognition server Phyre2.25 The EpiSearch method was used to define the potential epitope sites on the surface of Amb a 1.26 2. 6. Isolation of pIII-Fused Peptides from E. coli Periplasm Six peptides showing the highest specific binding to target antibodies were extracted from the periplasm of E. coli ER2738 as fusions with pill phage coat protein. Host bacteria were infected with individual peptide-displaying phage clone and grown for 2 h at 37 °C with agitation. Bacterial pellets were spun down at 5000 xg for 10 min and resuspended in 1 ml of an ice-cold solution consisting of 20% sucrose, 200 mM Tris-HCl (pH 8.0) and 1 mM EDTA supplemented with protease inhibitor cocktail (EZBlock™, BioVision, San Francisco, USA) at a dilution of 1:200. Following 1 h incubation on ice with occasional stirring, su-pernatants were harvested by centrifugation at 12000 xg for 20 min at 4 °C. The resulting periplasmic extracts were concentrated (4-5 fold) and the extraction buffer was exchanged for PBS by ultrafiltration using 10 kDa cut-off membranes (Microsep Advance Centrifugal Device, Pall Corporation, New York, USA). Extracts of non-infected bacteria and bacteria infected with phage clone carrying unrelated pill-fused control peptide (linear GTFDHPQ targeting streptavidin) were prepared in the same way and used as negative controls. 27 2. 7. Sera of Ragweed-Allergic Patients Serum samples from ragweed-allergic patients with positive sigE to Amb a 1 were collected at the University Clinic of Respiratory and Allergic Diseases, Golnik, Slovenia prior to starting immunotherapy. Patients' characteristics are shown in Table 1. The study was approved by National Medical Ethics Committee of Republic of Slovenia (No. 35/06/14). All patients willingly donated their blood for research. 2. 8. Immunodot Assay: Binding of pIII-Fused Peptides to Patients' IgE Two microliters of each sample of pill-fused peptides were spotted onto 0.45 ^m nitrocellulose membrane (GE Healthcare). The membrane was blocked with 5% Table 1. Characteristics of ragweed-allergic patients. sIgE* Amb a 1 (kU/L) Patient 1 0.77 Patient 2 41.4 Patient 3 1.51 Patient 4 0.61 Patient 5 0.62 Patient 6 0.5 Patient 7 1.6 Patient 8 1.71 sIgE* - specific immunoglobulin E were measured by using CLIA Immulite (Siemens, Erlangen, Germany). skimmed milk in Tris-buffered saline/0.05% Tween 20 (0.05% TBST) for 3 h at room temperature and then incubated overnight at 4 °C with a pool of sera from ragweed allergic-patients (patients 1 to 8, Table 1) diluted 1:10 in 0.05% TBST. After triple washing with 0.1% TBST, membranes were incubated with HRP-conjugated goat anti-human IgE antibodies (Thermo Fisher Scientific Cat# A18793 RRID: AB_2535570) diluted 1:2000 in 1% BSA/0.1% TBST for 2 h at room temperature. The reactive dots were visualized with CCD image analysis system (G-Box, Syngene, United Kingdom) using SuperSignal West Dura Extended Duration Substrate (Thermo Fisher Scientific). 3. Results and Discussion In this study, we sought to map epitopes of major ragweed allergen Amb a 1 by panning phage-displayed random peptide libraries. A thoughtful choice of biopan-ning conditions is crucial to overcome the limitations of commercially available peptide libraries and to improve the selection success rate.28,29 To avoid recovery of target-unrelated binders we used protein A or protein G coupled beads alternatingly for immobilization of target antibodies. Biopanning of three phage displayed-libraries was carried out with affinity-purified rabbit IgG specific for Amb a 1 using specific and non-specific elution. Given that the antibodies of IgG isotype were used as target, the reactivity of selected peptides with serum IgE was tested in order to evaluate whether a conserved epitope specificity between the IgG and IgE exist and thus to determine whether the identified peptides are also mimetics of IgE epitopes in ragweed-allergic patients. Forty-two phage clones reactive with Amb a 1-specific target antibodies but not with the components of background buffer in preliminary phage ELISA were selected for sequencing. Peptides RVVELMDWTVLH, CLFSQGNRC, MRTDMVI, and CIMSLVGTC were the most strongly enriched (number of isolated identical sequences is shown in brackets alongside these peptides in Figs. 1 and 2). Overall, sequencing yielded 17 different peptides. Zahirovic et al.: Epitope Mapping of Major Ragweed 40 Acta Chim. Slov. 2019, 66, 37-44 Binding of individual peptides displayed on phage to target antibodies was ranked in semiquantitative ELISA assay (Fig 1, A). In contrast to control phage (with no peptide displayed), six peptide-displaying phage clones demonstrated at least two-fold higher binding to the target antibodies compared to the background. Other 11 peptide-dis-playing phage clones showed lower binding to target antibodies. Potential non-specific interaction with anti- body constant region was checked by ELISA with control antibodies that were produced in the same species as target antibody and thus contain identical Fc fragment. Binding to control rabbit antisera was low and comparable between peptide-displaying phage clones and control phage (Fig 1, B). Therefore, the interaction of peptides with antibody constant region was excluded. Six phage-displayed peptides that exhibited the best binding to target antibodies (RV- Figure 1: Characterization of the phage-displayed peptides affinity-selected with anti-Amb a 1 rabbit IgG. (A) Binding of phage-displayed peptides to the immobilized target antibodies and background (5% milk). Values in brackets represent the frequency of the selected peptides. (B) Binding of phage-displayed peptides to the immobilized control rabbit antiserum (anti-procathepsin X). Wild-type phage with no displayed peptide served as negative control. The data are presented as the mean ± standard deviation of three individual experiments. (C) Displacement of six selected phage-displayed peptides from target antibodies with 1 ^g/ml, 5 ^g/ml and 10 ^g/ml of natural Amb a 1. Zahirovic et al.: Epitope Mapping of Major Ragweed Acta Chim. Slov. 2019, 66, 37-44 41 Sequences --sqdlasglepyf--- - -syniiatgihpv---- ---tmvatglmpvli— --kpdscrgclpvl--- ----EMAVTRWDVLAI- ---rvvelmdwtvlh—x5 ------------gsamtwgmlaae ---CSQGNRNWC------------ -CLFSOGNRC----------- -CALTQGNRC----------- -CLVTQGNKC----------- ----------qdfddil---- x9 mipisvgpttkr------------ - cvlglvgpc--------------- cimslvgtc--------------- MRTDMVI-----------------x8~ MRLDMEI----------------- - Group 1 Group 2 Group 3 Group 4 Group 5 Group 6 Figure 2: Sequence alignment of isolated peptides obtained using multiple sequence alignment program Clustal Omega. Consensus residues within each group are marked in different colours. Bolded amino acids are exact match; not bolded amino acids have similar physicochemical properties. Underlined peptides from groups 1-4 showed the highest binding to target antibodies. VELMDWTVLH, GSAMTWGMLAAE, SYNIIATGIHPV, TMVATGLMPVLI, QDFDDIL, and CLFSQGNRC) were further examined for competitive binding to target antibody paratopes in the presence of allergen (Fig 1, C). After addition of 5 or 10 ^g/ml of Amb a 1, the signal for phage-displayed peptide GSAMTWGMLAAE substantially decreased. The signal drop, albeit less pronounced, was also observed for Cys constrained phage-displayed peptide CLFSQGNRC. Peptide displacement from the target antibody by allergen indicates that they competed with allergen for the same paratopes on target antibodies i.e. the peptides and allergen share the same binding sites. Interestingly, other tested peptides showed no change or even slight increase in signal after addition of allergen (Fig 1, C). This may imply that they have a higher affinity for target antibodies than the allergen. Enhanced binding of phages to target antibodies occur possibly due to the conformational stabilization of target antibodies upon allergen binding that makes the paratopes more accessible for phages. Isoforms of Amb a 1 display distinct patterns of IgE binding. In previous study, Amb a 1.01 showed higher IgE-binding activity compared to Amb a 1.02 or 03 iso-forms.12 Therefore, we used Amb a 1.01 isoform for in silica linear alignment and conformational epitope mapping. The sequences of 17 different peptides were arranged into six groups according to their degree of similarity (Fig. 2). Despite common amino acid motifs within individual groups, not all peptides exhibited high binding to the target antibodies. For example, motif SQGNR appeared in 12 out of14 peptides from the cyclic library but only one pep- tide sequence (CLFSQGNRC) bound significantly to the target antibodies (Fig. 1A). This indicated that other residues outside the motif were important for target antibody binding as well. Fig. 3 depicts linear alignment of the peptides with Amb a 1.01 sequence. The peptides were either aligned with shorter segments consisting of only a few matching residues within Amb a 1 sequence or not aligned at all (Fig. 3). Matching amino acids were also not the same as those included in common motifs within individual groups. This suggests that the epitopes of Amb a 1 are con-formational. Indeed, it has been previously shown that in-halational allergens contain mainly conformational epitopes consisting of amino acids that are distributed over the protein sequence and come into close contact upon protein folding.30-32 For in silica mapping of conformational epitopes on Amb a 1, we created the 3D structural model of Amb a 1 since its crystal structure is not yet available. Using a protein fold recognition server Phyre2, we generated a high confidence close homology model of Amb a 1 with sequence coverage of 92%.25 The model is based on structural template d1pxza of Jun a 1, the major allergen from cedar pollen, as the closest homolog with known structure that contains a single-stranded right-handed beta-helix fold. Jun a 1 is a member of the pectate lyase family of allergens and provides a reliable homology model since it has a sequence identity of 47% with a FFAS score -93.1 with Amb a 1.33 EpiSearch method was used to reveal the location of epitopes on surface of Amb a 1 model.26 This approach uses patch analysis and solvent accessible surface area of amino acids to map peptides obtained from phage display experiments onto the 3D structure of a protein. The best match between the amino acid composition of the peptides and surface-exposed areas on the 3D model of allergen is predictive of epitope. We used six representative peptides from groups 1-4 that showed the highest binding to target antibodies (Fig. 1C, Fig. 2; underlined peptides) as input sequences for Episearch. The natural allergen Amb a 1 is composed of two non-covalently associated subchains, the N-terminal P chain (amino acids 26-180) and the C-terminal a chain (amino acids 181-396). Two representatives from group 1 were mapped to the loop on P chain at the N terminus containing residues V72, A73, N74, L102, Km V107, Gm, VU8, N149, P150, G151, G152, L153, S156, A161, A162, P163, A165, G166, and S167 with center residue at K104 (score: 1.000) (Fig. 4A). Two representatives from group 2 were mapped to the beta strand on a chain at the C terminus containing residues R253, H254, A276, S277, T279, L281, L299, G300, R301, H302, G303, E304, A305, A306, E308, S309, M310, W312, R315, V328, A329, S330, and, G331 with center residue at W312 (score: 0.950) (Fig. 4B). A representative from group 3 was mapped to the loop on P chain at the N terminus containing residues Q86, N87, R88, L90, N109, S110, N131, G132, N135, N157, G159, G176, S178, and Q179 with center residue at S110 (score: 1.000) Zahirovic et al.: Epitope Mapping of Major Ragweed 42 Acta Chim. Slov. 2019, 66, 37-44 10 20 30 40 50 Amb a 1.01 aedlqeilpwetrklttsgayniidgcwrgkadwaenrkaladcaqgfg Peptide QDFDDIL CSQGNRNWC 60 70 80 90 100 110 kgtvggkdgdiytvtseldddvanpkegtlrfgaaqnrplwiiferdmvirldkemvvns mrtdmvi MRLDMEI 120 130 140 150 160 170 dktidgrgakveiinagftlngvknviihninmhdvkvnpggliksndgpaapragsdgd MIPISVGPTTKR 180 190 200 210 220 230 aisisgssqiwidhcslsksvdglvdaklgttrltvsnslftqhqfvllfgagdeniedr CIMSLVGTC 240 250 260 270 280 290 gtllatvafntftdnvdqrmprcrhgffqvvnnnydkwgsyaiggsasptilsqgnrfcap RVVELMDWTVLH EMAVTRWDVLAI GSAMTWGMLAAE CLFSQGNRC CALTQGNRC CLVTQGNKC 300 310 320 330 340 350 derskknvlgrhgeaaaesmkwnwrtnkdvlengaifvasgvdpvltpeqsagmipaepg CVLGLVGPC SYNIIATGIHPV TMVATGLMPVLI SQDLASGLEPYF KPDSCRGCLPVL 360 371 esalsltssagvlscqpgapc Figure 3: Alignment of isolated peptide sequences to the sequence of Amb a 1.01 isoform (the accession number in the gene bank of NCBI: P27759). Amino acids in individual peptides are coloured corresponding to groups 1-6 (Fig. 2). Bolded amino acids are exact match to residues in Amb a 1; not bolded amino acids have similar physicochemical properties as residues in Amb a 1.01. (Fig. 4C). A representative from group 4 was mapped to the beta strand on a chain at the C terminus containing residues D^, D^o, D^, L210, Q213. H214, Q215. F216> D243. D246, Q247, and D265 with center residue at H214 (score: 1.000) (Fig. 4D). p chain was suggested to contain IgE binding sites based on its higher IgE reactivity compared to a subchain in the previous study.34 In this study, Epi-search analysis predicted two epitopes to be located on p chain at the N terminus (mapped by peptides from groups 1 and 3) and also predicted two epitopes on a chain at the C terminus (mapped by peptides from groups 2 and 4). Predicted epitopes are located on the solvent-exposed loops and beta strands of Amb a 1 model structure. These results agree with the known fact that conformational B-cell epitopes are usually associated with turns or loops and exposed regions protruding from protein surfaces and suggest that residues in these areas are involved in antibody binding.35,36 Given that the biopanning was carried out with antibodies of IgG isotype as target, in order to determine the relevance of deduced epitopes in ragweed-allergic patients we tested the conserved epitope specificity between IgG and IgE by evaluating the reactivity of identified peptides with patient sera. Six representative peptides from groups 1-4 that showed the highest binding to target (Fig. 1C, Fig. 2; underlined peptides) were isolated as fusions with pIII phage coat protein from E. coli and tested for binding to IgE from sera pool of ragweed-allergic patients (patients 1-8, Table 1). Extract from noninfected bacteria and an unrelated pIII-fused peptide (linear GTFDHPQ targeting streptavidin) were used as controls.27 Sera pool showed IgE binding to the six pIII-fused peptides (Fig. 5). Protein pIII used as a carrier of peptides ensured the correct conformation of the peptides during the assay and allowed efficient immobilisation on the membrane. Signals were not detected with the control samples (Fig. 5). Therefore, binding was attributed only to the IgE epitope-mimicking peptides (mimotopes). Thus, the mimotopes showed binding with target IgG as well as with patients' IgE. This indicates that the identified epitopes are relevant for both anti- Zahirovic et al.: Epitope Mapping of Major Ragweed Acta Chim. Slov. 2019, 66, 37-44 43 Figure 4: Mapping epitopes on a 3D structure of Amb a 1 homology model. The Amb a 1 model was generated using a protein fold recognition server Phyre2. (A) Two representatives from group 1 were mapped to the loop on | chain at the N terminus containing residues V72, A73, N74, L102, K104, V107, G127, V148, N149, P150, Gm, 6152, S^, A^, P163, AW5, G^, and S167 (violet). (B) Two representatives from group 2 were mapped to the beta strand on a chain at the C terminus containing residues R253, H254, A276, S277, T279, L281, L299, G300, R301, H302, G303, E304, A305, A306, E308, S309, M310, W312, R315, V328, A329, S330, and, G331 (green). (C) A representative from group 3 was mapped to the loop on | chain at the N terminus containing residues Q86, N87, R88, L90, N109, S110, N131, G132, N135, N157, G159, G176, S178, and Q179 (orange). (D) A representative from group 4 was mapped to the beta strand on a chain at the C terminus containing residues D145, D170, D192, L210, Q213, Q215, H214, F216, D243, D246, Q247, and D265 (blue). body isotypes. Since the mimotopes were selected with IgG antibodies, additional IgE epitopes might still be present on Amb a 1. Figure 5. Binding of pill-fused representative peptides from groups 1-4 to IgE from sera pool of eight ragweed-allergic patients. (1) pIII-RVVELMDWTVLH, (2) pill - GS AMTWGML A AE, (3) pIII-SYNIIATGIHPV, (4) pIII-TMVATGLMPVLI, (5) pIII-QDFDDIL, and (6) pIII-CLFSQGNRC. Controls: (C1) non-infected E. coli and (C2) unrelated pIII-fused peptide pIII-GTFDHPQ. 4. Conclusions In biopanning experiments against the polyclonal Amb a 1-specific rabbit IgG, we enriched peptides and arranged them into six groups according to their degree of similarity. The peptides weakly matched with shorter segments of Amb a 1 sequence, thus suggesting that the epitopes of Amb a 1 are conformational. Conformational mapping of the six representative peptides to the surface of the structural model of Amb a 1 predicted the location of four epitopic sites on surface patches centred at residues K1o4, S110, H214, and W312. The representative peptides bound to IgE from ragweed-allergic patients and are therefore mimetics of Amb a 1 IgE epitopes. These results pave the way towards the identification of conformational epitopes of Amb a 1. The structure and location of each new epitope increase our knowledge and, hence, the probability of identifying common features of cross-reactive allergen-antibody recognition sites, which would ultimately help to reveal the characteristics of the cross-reac- tive allergens and their underlying mechanism of action. In the context of immunotherapy, the identification of allergen epitopes and their mimotopes provides the basis for the rational design of immunotherapeutic constructs either based on recombinant allergen derivatives with reduced allergenic activity or mimotope-based carrier-bound vaccine for more defined and safer allergen-specific immunotherapy. Conflict of interest Authors declare no conflict of interest. 5. References 1. F. Ihler, M. Canis, J. Asthma Allergy 2015, 8, 15-24. DOI: 10.2147/JAA.S47789 2. M. L. Oswalt, G. D. Marshall, Allergy Asthma Clin. Immunol. 2008, 4, 130-135. DOI:10.1186/1710-1492-4-3-130 3. H. L0wenstein, Chem. Immunol. Allergy 2014, 100, 323-332. DOI: 10.1159/000359989 4. M. Turkalj, I. Banic, S. A. Anzic, Patient Prefer. Adherence 2017, Volume 11, 247-257. DOI:10.2147/ppa.s70411 5. B. Linhart, R. Valenta, Curr. Opin. Immunol. 2005, 17, 646655. DOI:10.1016/j.coi.2005.09.010 6. M. Wallner, U. Pichler, F. Ferreira, Immunotherapy 2013, 5, 1323-1338. 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D0I:10.1016/j.jaci.2010.05.038 18. P. Molek, B. Strukelj, T. Bratkovic, Molecules 2011, 16, 857887. D0I:10.3390/molecules16010857 19. T. Bratkovic, Cell. Mol. Life Sci. 2010, 67, 749-767. D0I:10.1007/s00018-009-0192-2 20. E. Ganglberger, FASEB J. 2000, 14, 2177-2184. D0I:10.1096/fj.99-1000com 21. J. M. Davies, E. O'Hehir R, C. Suphioglu, J. Allergy Clin. Immunol. 2000, 105, 1085-1092. 22. E. Jensen-jarolim, A. Leitner, H. Kalchhauser, A. Zurcher, E. Ganglberger, B. Bohle, O. Scheiner, G. Boltz-nitulescu, H. Breiteneder, FASEB J. 1998, 12, 1635-1642. 23. X. Chen, S. C. Dreskin, Mol. Nutr. Food Res. 2017, 61, 1600568. D0I:10.1002/mnfr.201600568 24. F. Sievers, A. Wilm, D. Dineen, T. J. Gibson, K. Karplus, W. Li, R. Lopez, H. McWilliam, M. Remmert, J. Soding et al., Mol. Syst. Biol. 2011, 7, 539. D0I:10.1038/msb.2011.75 25. L. A. Kelley, S. Mezulis, C. M. Yates, M. N. Wass, M. J. Sternberg, Nat. Protoc. 2015, 10, 845-858. D0I:10.1038/nprot.2015.053 26. R. Tiwari, S. S. Negi, B. Braun, W. Braun, A. Pomes, M. D. Chapman, R. M. Goldblum, T. Midoro-Horiuti, Int. Arch. Allergy Immunol. 2012, 157, 323-330. D0I:10.1159/000330108 27. P. Molek, T. Bratkovic, Acta Chim. Slov. 2016, 63, 914-919. D0I:Molek-2016-4 [pii] 28. S. S. Sidhu, C. R. Geyer: Phage Display In Biotechnology and Drug Discovery, Second Edition, CRC Press. Boca Raton, 2015. 584 p. 29. M. Vodnik, U. Zager, B. Strukelj, M. Lunder, Molecules 2011, 16, 790-817. D0I:10.3390/molecules16010790 30. A. Pomes, Int. Arch. Allergy Immunol. 2010, 152, 1-11. D01:10.1159/000260078 31. S. Padavattan, S. Flicker, T. Schirmer, C. Madritsch, S. Randow, G. Reese, S. Vieths, C. Lupinek, C. Ebner, R. Valenta et al., J. Immunol. 2009, 182, 2141-2151. D0I:10.4049/jimmunol.0803018 32. M. Lombardero, P. W. Heymann, T. A. Platts-Mills, J. W. Fox, M. D. Chapman, J. Immunol. 1990, 144, 1353-1360. 33. L. Jaroszewski, L. Rychlewski, Z. Li, W. Li, A. Godzik, Nucleic Acids Res. 2005, 33, W284-288. D0I:10.1093/nar/gki418 34. N. Wopfner, B. Jahn-Schmid, G. Schmidt, T. Christ, G. Hubinger, P. Briza, C. Radauer, B. Bohle, L. Vogel, C. Ebner et al., Mol. Immunol. 2009, 46, 2090-2097. D0I:10.1016/j.molimm.2009.02.005 35. A. Pomes, M. Li, J. Glesner, S. Wunschmann, E. M. King, M. D. Chapman, A. Wlodawer, A. Gustchina, J. Allergy Clin. Immun. 2009, 123, S229-S229. D0I:10.1016/j.jaci.2008.12.881 36. S. Padavattan, T. Schirmer, M. Schmidt, C. Akdis, R. Valenta, I. Mittermann, L. Soldatova, J. Slater, U. Mueller, Z. Mark-ovic-Housley, J. Mol. Biol. 2007, 368, 742-752. D0I:10.1016/j.jmb.2007.02.036 Povzetek Ambrozija je pomemben vzrok sezonskih alergij. Do sedaj imamo zelo malo informacij o IgE vezavnih mestih na glavnem alergenu iz cvetnega prahu ambrozije, Amb a 1. Afinitetna selekcija peptidov iz bioloških knjižnic z uporabo specifičnih protiteles proti alergenu je uporabna laboratorijska metoda za določevanje epitopov. V tej raziskavi smo s pomočjo mimotopov, kratkih peptidov, ki posnemajo epitope, izoliranih iz bakteriofagnih knjižnic naključnih peptidov, določali IgE epitope Amb a 1. Izbrani peptidi so se le šibko ujemali s primarnim zaporedjem Amb a 1, kar je nakazovalo, da so epitopi konformacijski. Da bi jih določili, smo izdelali homologni model tridimenzionalne strukture Amb a 1, na katerega smo prilegali izbrane peptide. S pomočjo programa EpiSearch smo identificirali štiri potencialne epitope na površini alergena okoli aminokislinskih ostankov K104, S110, H214 in W312. Peptidni mimetiki predvidenih epitopov so se specifično vezali na IgE iz zmesi serumov za ambrozijo alergičnih pacientov in tako predstavljajo mimetike IgE epitopov Amb a 1. Poznavanje IgE epitopov je predpogoj za racionalno načrtovanje molekularnih pristopov v diagnostiki in imu-noterapiji alergijskih bolezni. Zahirovic et al.: Epitope Mapping of Major Ragweed DOI: 10.17344/acsi.2018.4585 Acta Chim. Slov. 2019, 66, 45-49 ^creative ^commons Scientific paper The Titre of the Virus in the Inoculum Affects the Titre of the Viral RNA in the Host Plant and the Occurrence of the Disease Symptoms Maruša Pompe-Novak,* Maja Križnik and Kristina Gruden National Institute of Biology, Ljubljana, Slovenia * Corresponding author: E-mail: marusa.pompe.novak@nib.si Received: 07-04-2018 Dedicated to the memory of Prof. Dr. Igor Kregar Abstract Potato virus Y (PVY) is the most economically important potato virus, therefore extensive research is focusing on elucidation of its interaction with the host. To obtain repeatable results, strict standardization of research methods is crucial. Mechanical inoculation by rubbing sap from a PVY infected plant onto the leaf surface together with a fine abrasive powder is the most convenient way of experimental transmission of PVY to host plants. However, factors determining reproducibility of this process need to be determined. In the present study, it was shown that higher titre of the virus in the inoculum resulted in faster increase of PVYntn RNA titre in the inoculated leaves, as well as in faster translocation of PVYntn from inoculated leaves into upper non-inoculated leaves. The final titre of PVYNTN RNA in upper non-inoculated leaves was independent of the virus titre in the inoculum. In addition, the occurrence of the disease symptoms was followed and the dependence to the titre of the virus in the inoculum was observed. Keywords: Potato virus Y; PVY; potato; mechanical inoculation; inoculum; virus titre; symptoms 1. Introduction Potato virus Y (PVY) is the most economically important potato virus affecting potato production worldwide1 and it was classified as one of ten most important plant viruses overall.2 In sensitive potato cultivars, it can cause potato tuber necrosis ring spot disease.3 On the green part of a plant, local lesions, chlorosis, mosaic, crinkling, systemic necrosis, leaf drop and plant death can appear.4 The outcome of the interaction depends on genotypes of both, potato host plant and PVY pathogen. In addition, the response of a potato towards PVY infection can vary with various environmental conditions and various developmental and physiological growth stages of a plant.5 PVY infected potato plants can exhibit either a compatible or an incompatible response.6 Plants exhibiting a compatible interaction are susceptible and the virus can replicate and invade the plants.7 Susceptible potato plants can be either sensitive or tolerant to PVY infection. Sensitive potato plants develop disease symptoms, while tolerant plants develop no or very mild symptoms, although they can accumulate high titre of the virus.8 In an incompatible interaction, plants resist the virus by restricting cell invasion, virus replication and/or virus spread. Plants can respond to virus infection with an extreme resistance (ER) or a hypersensitive response (HR). In the case of ER, potato plants show no symptoms or very limited necrosis in the form of pinpoint lesions.9 Virus titres remain extremely low, below the limit of detection also in the initially infected leaf.9,10,11,12 In the case of HR, virus translocation from the initially infected leaf to other parts of the plant is prevented, although the virus replication and initial cell-to-cell movement are not blocked. Later, most of the infected cells die, which result in a localised necrotic lesion at the site of infection.12,13 In the natural environment, PVY is transmitted by sap-feeding aphid vector, or vegetatively through propagated potato tubers. Experimentally, PVY can be transmitted by mechanical means such as grafting or rubbing of a sap from a PVY infected plant onto the leaf surface together with a fine abrasive powder, e.g. carborundum.14 The latter method is a convenient mean for experimental transmission of the PVY to host plants, although strict standardization of the method is crucial for repeatable re- Novak et al.: The Titre of the Virus in the Inoculum Affects 46 Acta Chim. Slov. 2019, 66, 45-49 suits. The aim of the present study was to investigate the relation between the titre of the virus in the inoculum and the titre of the viral RNA in the host plant, and additionally to determine whether there is the correlation between PVY titre in the inoculum and induction of disease symptom development in inoculated plants. 2. Experimental 96 virus free potato plants (Solanum tuberosum L.) of cultivar Desiree and 105 virus free plants of cv. Igor from stem node tissue culture (cultivated for 2 weeks) were planted in soil and kept at 21 ± 2 °C in growth chambers, with illumination at 70 ^Mm-2s-1 (Osram L36W/77 lamp), a photoperiod of 16 h and relative humidity 70%. After 4 weeks, three bottom leaves of each plant were mechanically inoculated by rubbing the inoculum onto the leaf surface together with a fine abrasive powder of carborundum (0.037 mm). Inoculum was prepared by grinding PVYNTN (NIB-NTN isolate, AJ585342, referred also as NTN-Slo Isolate15) infected plants of cv. Pentland Squire from node tissue culture in the grinding buffer (0.02 M phosphate buffer, 0.01 M DETC, pH 7.6) in the ratios (w/v) 1:5, 1:10, 1:50 and 1:100. For control mock inoculation, healthy plants of cv. Pentland Squire from node tissue culture were ground in the grinding buffer in the ratio (w/v) 1:5. After 10 min incubation, the inoculum was washed from the leaf surface by tap water. On potato plants of cv. Igor disease symptom development was monitored at 0, 3, 4, 5, 6, 7, 10 and 14 days post inoculation (dpi). At the same time points, samples from three plants per treatment were collected for the quantification of PVY RNA. For that purpose, the right distal quarter of the second inoculated leaf and the right distal quarter of the second upper non-inoculated leaf were collected and further analysed separately from each plant of cv. Desiree.. Collected leaf tissues were frozen immediately in liquid nitrogen and stored at -80 °C for further analysis. Plant tissue was homogenised by Tissue Lyser (Qia-gen, Hilden, Germany). RNA was isolated by MagMAX™ Plant RNA Isolation Kit (Applied Biosystems, Foster City, CA, USA) including DNAse treatment. RNA concentration, quality, and purity were assessed using agarose gel electrophoresis and NanoDrop ND-1000 spectrophotom-eter (Thermo Fisher Scientific, Waltham, Massachusetts, USA) . RT-qPCR reactions were performed on 2 ^l of extracted RNA in a total volume of 10 ^l using the Ag-Path-IDTM One-Step RT-PCR Kit (Applied Biosystems, Foster City, CA, USA) according to the manufacturer's instructions. Primer and probe concentrations, together with PCR amplification conditions, were as in Kogovsek et al.16 Two dilutions of RNA were analysed, each in duplicate. Reactions were carried out using the ABI 7900HT Sequence Detection System (Applied Biosystems, Foster City, CA, USA). RT-qPCR conditions included a reverse transcription step (10 min at 48 °C) followed by 40 amplification cycles (1 min at 60 °C, 15 s at 95 °C). Data were analysed using the SDS v 2.3 software (ABI, Foster City, CA, USA). PVY RNA amount was quantified using a relative standard curve method by normalization to the endogenous control cox mRNA by quantGenius.17 The significance of differences (p < 0.05) between mean values was determined by the Student's t-test. 3. Results and Discussion The titre of PVYNTN RNA was studied in potato host plants of tolerant cv. Désireé after inoculation with the inocula obtained by grinding PVYNTN infected plant tissue in grinding buffer in different ratios (w/v), 1:5, 1:10 and 1:100, and therefore containing different titres of PVYNTN. The amount of PVYNTN RNA detected immediately after inoculation (0 dpi) correlated with the titre of PVYNTN in the inoculum, indicating that the remnants of PVYNTN RNA on the leaf surface after washing the inoculum was detected. From 0 dpi to 4 dpi the detected amount of PVYNTN RNA decreased (Figure 1) due to degradation of PVYNTN RNA on the leaf surface. It was already demonstrated in previous studies18 that it is possible to detect the traces of the inoculum on the leaf surface of extremely resistant cv. Santé (in which PVY cannot multiply) even at 14 dpi; and to detect viral RNA in dry inoculum 12 days after its preparation. Double-stranded RNA products shown to arise during viral replication of Potyviridae19,20 can explain the stability of viral RNA. It is hypothesised that PVY enters the initial potato cells for further multiplication through broken leaf hairs 25 - „ ■ 1:S • 1:10 —t—1:100 —O control 20 - •101234567B time (days post inoculation) Figure 1. The relative amount of PVYNTN RNA measured by RT-qPCR in the inoculated leaves of cv. Desiree from 0 to 7 dpi. Error bars represent the standard error (n = 3). Statistical comparison was made between plants inoculated with the inoculum in which PVYNTN infected potato sap was diluted in buffer in the ratio 1:100 and plants inoculated with the inocula in which PVYNTN infected potato sap was diluted in buffer in the ratios 1:5 or 1:10. Statistically significant differences (p < 0.05) are indicated with.* Novak et al.: The Titre of the Virus in the Inoculum Affects ... 47 Acta Chim. Slov. 2019, 66, 45-49 (broken by rubbing at mechanical inoculation). At 5 dpi the amount of newly multiplied PVYNTN RNA exceeded the amount of PVYNTN RNA in the remnants of the inoculum on the leaf surface of the plants inoculated with the inocula in which PVYNTN infected potato sap was diluted in buffer in the ratios 1:5 or 1:10 (Figure 1). From 5 to 7 dpi, higher PVYNTN RNA titres were observed in plants inoculated with more concentrated inoculum (Figure 1). In leaves, inoculated with the inoculum in which PVYNTN infected potato sap was diluted in buffer in the ratio 1:100, the titre of PVYNTN probably increased later than 7 dpi, as also the translocation of PVYNTN from inoculated into upper noninoculated leaves occurred later (Figure 2). In inoculated leaves of potato the concentrical distribution of PVY around the point of infection was shown during HR.12 In inoculated leaves of tobacco (Nicotiana tabacum L.) cv. Xanthi movement of PVY from initially inoculated cells to neighbour cells in concentrical circles, loading of PVY into the veins and translocation of PVY into upper non-inoculated leaves was shown in tolerant interaction.21 Our results in tolerant potato-PVY interaction showed that the translocation of PVYNTN from inoculated into upper non-inoculated leaves of potato cv. Desiree occurred regardless of the virus titre in the inoculum, while the speed of the process was dependent on the virus titre in the inoculum (Figure 2). Although the multiplication of PVYntn in inoculated leaves and the translocation of PVYntn from inoculated into upper non-inoculated leaves was slower in the case of lower viral titre in the inoculum, the final titre of PVYNTN RNA in upper non-inoculated leaves 14 dpi was independent of the virus titre in the inoculum (Figure 2). Even much longer times (a few months) are required for the spread of PVYNTN from nutrient solution containing extremely low titre of PVYNTN, through the roots, to the green parts of the plants as shown in experiments using a hydroponic system.22 Additionally, the disease symptom development after inoculation with different PVYNTN titre was monitored on potato plants of cv. Igor, which is one of the most susceptible and sensitive potato varieties to PVYNTN. A few days after infection, local lesions appeared on inoculated leaves, followed by leaf chlorosis (yellowing of the leaves) and leaf drop of inoculated leaves. In general, the severity of the disease symptoms correlated with the titre of PVYN-TN in the inoculum. The appearance and the development of disease symptoms was slower in the plants inoculated with lower titre of PVYNTN in the inoculum (Figure 3), what is shown also by the correlation between the titre of PVYntn in the inoculum and the leaf drop (Figure 4). By 14 dpi, all 3 inoculated leaves dropped in plants inoculated with the inocula diluted in the ratios 1:5 or 1:10. At the same time, in plants inoculated with the inocula diluted in the ratios 1:50 or 1:100 only the 1st and the 2nd inoculated leaf dropped, while the 3rd inoculated leaf showed more severe symptoms in plants inoculated with the inoculum diluted in the ratio 1:50 as compared to the plants inoculated with the inoculum diluted in the ratio 1:100 (Figure 3). No symptoms appeared on control potato plants, where also no leaf drop was observed at 14 dpi (Figure 3, right panel). Besides the high titre of PVYNTN in the inoculum, also too much carborudum or too excessive rubbing at mechanical inoculation promotes the leaf drop on inoculated plants (data not shown). Early leaf drop of inoculated leaves can prevent the spread of the virus to upper non-inoculated leaves and the development of symptoms in upper non-inoculated leaves. In PVYNTN - cv. Igor interaction, the absence of symptoms in upper non-inoculated leaves indicates with a high probability the absence of the virus in upper non-inoculated leaves. Although the plants are kept in the same growth chamber controlled environment with the same tempera- time idays post inoculation) Figure 2. The relative amount of PVYNTN RNA measured by RT-qPCR in upper non-inoculated leaves of cv. Désireé from 6 to 14 dpi. Error bars represent the standard error (n = 3). Statistical comparison was made between plants inoculated with the inoculum in which PVYNTN infected potato sap was diluted in buffer in the ratio 1:100 and plants inoculated with the inocula in which PVYNTN infected potato sap was diluted in buffer in the ratios 1:5 or 1:10. None of the differences was statistically significant (p < 0.05). Novak et al.: The Titre of the Virus in the Inoculum Affects ... 48 Acta Chim. Slov. 2019, 66, 45-49 Ir m w Figure 3. Disease symptoms on potato plants of cv. Igor at 14 days after inoculation with the inocula in which PVYNTN infected potato sap was diluted in buffer in the ratios 1:5 (left), 1:10 (second from the left), 1:50 (middle) or 1:100 (second from the right); and control potato plants (right). Figure 4. The average number of dropped inoculated leaves per plant in cv. Igor after infection with PVYNTN from 0 dpi to 14 dpi. Error bars represent the standard error (n = 3). Statistical comparison was made between plants inoculated with the inoculum in which PVYNTN infected potato sap was diluted in buffer in the ratio 1:100 and plants inoculated with the inocula in which PVYNTN infected potato sap was diluted in buffer in the ratios 1:50, 1:10 or 1:5. Statistically significant differences (p < 0.05) are indicated with.* ture, relative humidity, illumination and photoperiod, the plant's response to viral infection (including the development of symptoms) can vary between experiments (biological repetitions) due to yet unknown parameters (e.g. physiological state of the plant, season...). 4. Conclusions We can conclude that higher titre of PVYNTN in the inoculum resulted in faster increase of PVYNTN RNA titre in the inoculated leaves and in faster translocation of PVYntn from inoculated leaves into upper non-inoculated leaves. The final amount of PVYNTN RNA in upper noninoculated leaves was independent of PVYNTN titre in the inoculum. In addition, the occurrence of the disease symptoms was slower in the plants inoculated with lower titre of PVYntn in the inoculum as compared to the plants inoculated with higher titre of PVYNTN in the inoculum. Thus to obtain reproducible results of any biochemical, physiological or molecular study of potato - PVY interaction one should use standardised titre of the virus in the inoculum. 5. Acknowledgment The research was financially supported by the Slovenian Research Agency (research core funding No. P4-0165). The authors thank Tjaša Cof, Mihaela Leupušček and Živa Marinko, who joined to the research as students; and Neža Turnšek for technical help. 6. References 1. A. V. Karasev, S. M. Gray, Annu. Rev. Phytopathol. 2013, 51, 571586. DOI: 10.1146/annurev-phyto-082712-102332 2. K. B. Scholthof, S. Adkins, H. Czosnek, P. Palukaitis, E. Jac-quot, T. Hohn, B. Hohn, K. Saunders, T. Candresse, P. Ahl-quist, C. Hemenway, G.D. Foster. Mol Plant Pathol. 2011, 12, 938-954. DOI:10.1111/j.1364-3703.2011.00752.x 3. L. Beczner, J. Horvath, I. Romhanyi, H. Forster, Potato Res. 1984, 27, 339-352. DOI:10.1007/BF02357646 4. M. Kus, in: M. Recnik, M. Kus (Eds.) Proceedings of the 9th EAPR Virology Section Meeting, Ribno, Bled, Slovenia, 1995, pp. 135-138. 5. M. Pompe-Novak, C. Lacomme, in: C. Lacomme, L. Glais, D. Novak et al.: The Titre of the Virus in the Inoculum Affects ... Acta Chim. Slov. 2019, 66, 45-49 49 Bellstedt, B. Dupuis, A.V. Karasev, E. Jacquot (Eds.), Potato virus Y-bio diversity, pathogenicity, epidemiology and management, Cham, Springer, 2017, pp. 22-42. 6. J. Hinrichs-Berger, M. Harford, S. Berger, H. Buchenauer, Physiol. Mol. Plant Pathol. 1999, 55, 143-150. DOI:10.1006/pmpp.1999.0216 7. J. I. Cooper, A. T. Jones, Phytopathology 1983, 73, 127-128. DOI: 10.1094/Phyto-73-127 8. M. Ravnikar, in: J. Freitag (Ed.) ETNA Plant genomics and bioinformatics: expression microarrays and beyond: a course book, Postdam, 2005, pp. 66-71. 9. J. P. T. Valkonen, R. A. C. Jones, S. A. Slack, K. N. Watanabe, Plant Breed. 1996, 115, 433-438. DOI:10.1111/j.1439-0523.1996.tb00952.x 10. J.P.T. Valkonen, Plant Breed. 1994, 112, 1-16. DOI:10.1111/j.1439-0523.1994.tb01270.x 11. R.M. Solomon-Blackburn, H. Barker, Heredity 2001, 86, 1735. DOI:10.1046/j.1365-2540.2001.00799.x 12. J. P. T. Valkonen, Breed. Sci. 2015, 65, 69-76. DOI:10.1270/jsbbs.65.69 13. T. Lukan, S. Baebler, M. Pompe-Novak, K. Gucek, M. Zagorscak, A. Coll, K. Gruden, Front. Plant Sci. 2018, 9, 168. DOI:10.3389/fpls.2018.00168 14. C. Lacomme, J. Pickup, A. Fox, L. Glais, B. Dupuis, T. Steinger, J.-L. Rolot, J. P. T. Valkonen, K. Kruger, X. Nie, S. Modic, N. Mehle, M. Ravnikar, M. Hullé, in: C. Lacomme, L. Glais, D. Bellstedt, B. Dupuis, A.V. Karasev, E. Jacquot (Eds.), Potato virus Y-biodiversity, pathogenicity, epidemiology and man- agement, Cham, Springer, 2017, pp. 141-176. DOI:10.1007/978-3-319-58860-5_6 15. H. Barker, K. McGeachy, N. Toplak, K. Gruden, J. Žel, I. Browning, Am. J. Potato Res. 2009, 86, 227-238. DOI:10.1007/s12230-009-9076-0 16. P. Kogovšek, L. Gow, M. Pompe-Novak, K. Gruden, G.D. Foster, N. Boonham, M. Ravnikar, J. Virol. Methods 2008, 149, 1-11. DOI:10.1016/j.jviromet.2008.01.025 17. Š. Baebler, M. Svalina, M. Petek, K. Stare, A. Rotter, M. Pom-pe-Novak, K. Gruden, BMC Bioinformatics 2017, 18, 276. DOI:10.1186/s12859-017-1688-7 18. N. Mehle, M. Kovač, N. Petrovič, M. Novak Pompe, Š. Baebler, H. Krečič-Stres, K. Gruden, M. Ravnikar, Physiol. Mol. Plant Pathol. 2004, 64, 293-300. DOI:10.1016/j.pmpp.2004.10.005 19. I.P. Gadh, V. Hari, Virology 1986, 150, 304-307. DOI: 10.1016/0042-6822(86)90292-8 20. V. Hari, in: R.P. Singh, U.S. Singh, K. Kohmoto (Eds.), Pathogenesis and host specificity in plant diseases. Histopatholog-ical, biochemical, genetic and molecular bases. Viruses and viroids, vol. III. Great Britain, Elsevier, 1995, pp. 1-18. 21. M. Rupar, F. Faurez, M. Tribodet, I. Gutierrez-Aguirre, A. Delaunay, L. Glais, M. Križnik, D. Dobnik, K. Gruden, E. Jacquot, M. Ravnikar, Mol. Plant Microbe. Interact. 2015, 28, 739-750. DOI:10.1094/MPMI-07-14-0218-TA 22. N. Mehle, I. Gutierrez-Aguirre, N. Prezelj, D. Delic, U. Vidic, M. Ravnikar, Applied and environmental microbiology 2014, 80, 1455-1462. DOI:10.1128/AEM.03349-13 Povzetek Virus krompirja Y (Potato virus Y, PVY) je ekonomsko najpomembnejši virus krompirja, zato se številne raziskave osre-dotočajo na študij interakcije PVY z gostiteljsko rastlino. Za zagotavljanje ponovljivosti rezultatov je pomembna standardizacija raziskovalnih metod. Najpogostejši način prenosa PVY na gostiteljsko rastlino v eksperimentalnih pogojih je mehanska inokulacija, pri kateri na površino lista nanesemo abrazivno sredstvo in sok okužene rastline, pri čemer pa je potrebno določiti dejavnike, ki so ključni za ponovljivost tega postopka. V raziskavi smo pokazali, da se je višji titer virusa v inokulumu odražal v hitrejšem dvigu titra RNA PVYNTN v inokuliranih listih ter v hitrejšem širjenju PVYNTN iz inukuliranih listov v zgornje neinokulirane liste. Končni titer RNA PVYNTN v zgornjih neinokuliranih listih pa ni bil odvisen od titra virusa v inokulumu. Poleg tega smo opazili tudi, da je bila hitrost pojava bolezenskih znamenj odvisna od titra virusa v inokulumu. Novak et al.: The Titre of the Virus in the Inoculum Affects DOI: 10.17344/acsi.2018.4632 Acta Chim. Slov. 2019, 66, 50-57 /^creative ^commons Scientific paper Characterization of Legumain Degradóme Confirms Narrow Cleavage Specificity Robert Vidmar,1 Matej Vizovišek,1 Dušan Turk,1,2 Boris Turk,1,3 and Marko Fonovic1^ 1 Jožef Stefan Institute, Department of Biochemistry and Molecular and Structural Biology, Jamova 39, SI-1000 Ljubljana, Slovenia 2 Centre of Excellence for Integrated Approaches in Chemistry and Biology of Proteins, Ljubljana, Slovenia, Jamova 39, SI-1000 Ljubljana, Slovenia 3 Faculty of Chemistry and Chemical Technology, University of Ljubljana, Večna pot 113, SI-1000 Ljubljana, Slovenia * Corresponding author: E-mail: marko.fonovic@ijs.si Phone: +386 1 477 3474 Received: 08-02-2018 Dedicated to the memory of Prof. Dr. Igor Kregar Abstract Legumain or asparagine endopeptidase is a unique cysteine endopeptidase with a distinctive specificity for the hydrolysis of peptide bonds after asparagine and to a lesser extent after aspartate. It is ubiquitously expressed in various tissues and besides its involvement in immune response and other physiological processes, it was also shown to play a role in pathological states such as inflammation and cancer. In order to improve our understanding of legumain substrate recognition we have performed proteomic profiling of legumain specificity on native proteins derived from MDA-MB-231 cells using two different N-terminal labelling methodologies (FPPS and ISIL). Our data revealed narrow cleavage specificity for P1 position combined with clear cleavage preference for unstructured secondary regions in the substrate proteins. No extended cleavage specificity on native proteins was observed. Moreover, a limited number of identified cleavages on individual substrates suggest its primary role in precision proteolysis and regulatory proteolytic events. Keywords: Proteolysis; proteases; degradome; cleavage specificity; legumain 1. Introduction Legumain or asparagine endopeptidase (AEP) is a lysosomal cysteine protease (C13 family, EC number 3.4.22.34) that catalyses the hydrolysis of peptide bonds after asparagines and to a lower extent also after aspartates.1-2 The catalytic site of legumain is composed of a catalytic dyad (His148 and Cys189). Consistent with its lysosomal localization, legumain has a pH optimum for substrate hydrolysis at acidic pH (pH = 5.8). In mammals, legumain is expressed in various organs and tissues, most abundantly in kidneys, testis and antigen-presenting cells. In contrast to other lysosomal proteases such as cathepsins with a high degree of functional redundancy, legumain exhibits unique substrate specificity and evolutionary highly conserved primary structure, which suggests that legu- main may be involved in specific physiological processes. However, the exact physiological role of legumain is still not well understood. Although it was reported to regulate immune response through the activation of TLR receptors and antigen presentation it has not been shown to be crucial in these processes.3-5 In pathological conditions legu-main is strongly associated with tumorigenesis, where its overexpression was shown in a number of human solid tumours such as carcinomas of the breast, colon and prostate.6-7 Recent reports have also shown its possible involvement in atherosclerosis8 and ischemic brain injury.9 Legumain has been also linked to the development of neu-ropathology in Alzheimer's disease, where it cleaves amyloid precursor protein and tau protein.10-11 Nevertheless, the pathophysiological functions of le-gumain, its substrates and its association with disease de- Vidmar et al.: Characterization of Legumain Degradome Acta Chim. Slov. 2019, 66, 50-57 51 velopment and progression remain poorly understood. Conservation of legumain between evolutionary divergent plants and animals suggest that there is a biological need for legumain that has yet to be identified. A better understanding of features that govern the legumain substrate recognition is therefore expected to provide a novel insight in its biological role. Processing of legumain substrates in vitro was usually performed on peptides or denatured proteins and although these studies revealed its primary ami-no acid preference, it did not account for the possible influence of secondary and tertiary structure of the substrate.12-13 In this work, we studied legumain processing on a complex pool of structurally native proteins. We treated native human proteome with recombinant legu-main and used in-gel stable isotope labelling (ISIL) and fast profiling of protease specificity (FPPS) for the identification of substrate cleavages. This enabled identification of primary cleavage specificity of legumain on native proteins as well as determination of secondary structure cleavage preference. 2. Experimental 2. 1. Materials Antibodies against human prelamin-A/C (ab8984) were purchased from Abcam, United Kingdom. Recombinant human legumain was expressed in the baculovirus expression system as previously described.13 Ac(D3)-NHS was synthesized at the Faculty of Pharmacy (University of Ljubljana, Slovenia) according to the established proto- col.14 2. 2. Cells and Cell Lysate Preparation MDA-MB-231 breast cancer cells were routinely maintained in DMEM medium (Lonza) supplemented with 10% FBS (Sigma-Aldrich), 2 mM glutamine (Gibco) and 1% antibiotic stock solution (10.000 U/ml penicilin and 10 mg/ml streptomycin) (Sigma-Aldrich) at 37 °C and 5% CO2. Before the harvesting, the cells were grown to confluency and washed with DPBS (Lonza). The cells were detached with enzyme-free dissociation solution (Milli-pore). After the centrifugation, the cell pellet was lysed on ice for 15 min in 20 mM sodium phosphate buffer pH 6.0, containing 150 mM NaCl, 0.05% NP-40, 1 mM EDTA, 25 ^M E-64, 1 mM PMSF. The insoluble material was removed by centrifugation at 14 000 g for 10 min and the cleared cell lysate was used to determine protein concentration with the Bradford assay and portioned into aliquots containing 0.5 mg of total protein. 2. 3. In vitro Processing of the Cell Lysate Recombinant prolegumain was first activated in 50 mM citric buffer pH 4.0, supplemented with 5 mM DTT for 30 min at 37 °C. The active concentration of legumain was determined to be 16.7 ^M.12 In our degradomic workflow, recombinant human legumain was added to each aliquot of the cell lysate at 0.2 ^M and 1.0 ^M final concentration and the samples were incubated at 37 °C for 1h before further processing. Immunological detection of prelamin-A/C was performed under identical conditions except that additional time-points were used for the in vitro processing (0, 10, 30 and 60 min). Western blot analysis was performed on nitrocellulose membrane using mouse monoclonal antibodies against prelamin-A/C according to manufacturer's recommendations. 2. 4. N-terminal Labelling with Trideutero-Acetylation 2. 4. 1. In-Gel Stable Isotope Labelling Protocol After the in vitro processing, the samples were labelled with Ac(D3)-NHS according to the ISIL protocol described earlier with some modifications.15 Briefly, the samples were incubated in 6x SDS-PAGE loading buffer at 95 °C for 5 min and separated on a 12.5% SDS-PAGE gel (Lonza). The gel was stained with Comassie brilliant blue and each of the protein lanes was cut into six bands and destained with 25 mM NH4HCO3 in 50% acetonitrile/ dH20. The proteins were reduced with 10 mM DTT (56 °C, 45 min) and alkylated with 55 mM iodoacetamide in the dark at room temperature for 30 min. In-gel stable isotope labelling was performed with an addition of 1 mg Ac(D3)-NHS per sample (40 mM final concentration). Prior to the labelling step Ac(D3)-NHS was dissolved in 100 mM phosphate buffer, pH 8.5. The reaction was performed at 30 °C for 1h and the labelling step was repeated one more time. To reverse any potential threonine and serine O-acetylation 1 mg hydroxylamine was added to each sample and the samples were incubated at room temperature for 20 min. The excess Ac(D3)-NHS reagent was quenched by adding 1 mg of glycine per sample followed by an incubation at 30 °C for 1h. The gel pieces were washed with acetonitrile and vacuum dried before rehy-drating in 80 ^l 25 mM NH4HCO3 solution containing 1 ^g of sequencing-grade modified porcine trypsin per sample, and the trypsinization was then performed over night at 37 °C. The next day the peptides were extracted from the gel using the extraction solution (50% acetonitrile, 5% formic acid). The samples were desalted before LC-MS/MS analysis on C18 tips as described elsewhere.12 2. 4. 2. Fast-Profiling of Protease Specificity Protocol The samples after in vitro processing were prepared according to the FPPS protocol described elsewhere.16 Briefly, the samples were transferred to a 500 ^l micro-fil- Vidmar et al.: Characterization of Legumain Degradome ... 52 Acta Chim. Slov. 2019, 66, 50-57 52 ter device with a cut-off of 3000 Da (Millipore) and the buffer was exchanged with 100 mM phosphate buffer pH 8.5. Subsequently, 2 mg of Ac(D3)-NHS reagent was dissolved in the sample followed by 1 hour incubation at 30 °C. Afterwards, the labelling step was repeated. To reverse partial labelling of serines, threonines and tyrosines, hydroxyl-amine was added to the sample at 1 mM concentration and left at room temperature for 20 min. Afterwards, 8 M urea was added and proteins were reduced with 10 mM DTT for 1 hour at room temperature before addition of iodoacetamide at 50 mM final concentration for 1 hour in the dark at room temperature. After the free cysteines were alkylated, the unreacted iodoacetamide was quenched with 50 mM DTT for 30 min at room temperature before the buffer was exchanged with 25 mM ammonium bicarbonate pH 7.8. The sample volume was set to 250 ^l before the overnight trypsinization at a 1:100 (w/w, enzyme/substrate) ratio at 30 °C. The peptide-rich flow-through was collected the next day by spinning the micro-filters in the centrifuge and concentrated to 50 ^l. The peptide samples were fractionated using a SAX-C18 stage tip protocol as previously described.17-18 Accordingly, the samples were mixed with Britton & Robinson buffer (20 mM acetic acid, 20 mM phosphoric acid and 20 mM boric acid, pH 11) and the pH was set to 11 with 1 M NaOH. The SAX tips were prepared by stacking 6 discs of Empo-re/Disk Anion Exchange (Varian) in a 200 ^l pipet tip (Eppendorf) and the C18 tips were prepared by stacking 4 discs of Empore/C18 (Varian). The samples were applied to the SAX-C18 tip and the peptide fractions were eluted using buffers at pH 11, 10, 9, 8, 7, 6, 5, 4 and 3. The eluting peptides were captured on C18 tips and subjected to LC-MS/MS analysis. 2. 5. Mass Spectrometric Analysis The LC-MS/MS analysis of the samples was performed using an Orbitrap LTQ Velos mass spectrometer (Thermo Fischer Scientific) coupled to an EASY-nanoLC II HPLC (Thermo Fischer Scientific) operated automatically via XCalibur software (Thermo Fischer Scientific). The samples containing 0.1% FA were loaded onto a C18 trapping column (Proxeon Easy-column, Thermo Fischer Scientific) and separated on a C18 PicoFrit Aquasil analytical column (New Objective). The peptides were eluted using a 5-40% (v/v) 50 min linear gradient of acetonitrile with 0.1% FA at a constant flow rate of 300 nl/min. The full MS mass spectra were acquired with the Orbitrap mass analyzer in the mass range of 300 to 2,000 m/z at resolution of 30,000 in the profile mode. The MS/MS spectra were obtained by HCD fragmentation of the nine most intense MS precursor ions and recorded at a resolution of 7,500 in the centroid mode. Only the precursor ions with assigned charge states (> 1) were chosen for MS/MS fragmentation. The dynamic exclusion was set to repeat count of 1, repeat duration of 30 s, and exclusion duration of 20 s. 2. 6. Database Search and Data Processing For the identification of peptides we used the MaxQuant proteomic software19 and performed the database searches against the human proteome deposited in the Un-iProt/Swiss-Prot database (UniProtKB, Homo sapiens, canonical database, 20 336 entries). The settings for the database searches applied trideutero-acetylation of peptide N-termini (+45.029 Da) and methionine oxidation (+15.995 Da) as variable modifications, trideutero-acetyl-ation of lysines (+45.029 Da) and carbamidomethylation of cysteines (+57.021 Da) as fixed modifications, Semi-Ar-gC/P as the enzyme specificity setting while allowing for one missed cleavage, precursor ion and fragment ion mass tolerances were set to 20 ppm and 0.5 Da, respectively. A reversed database search was performed and the false discovery rate (FDR) was set at 1% for peptide and protein identifications. Raw data and database search files are available via ProteomeXchange with identifier PXD010466. After the database searches, the identified modification specific peptides were filtered to obtain the true positive hits for the legumain cleavage sites. Peptides with trideute-ro-acetylated N-termini identified only in the legumain-treat-ed samples were considered to be the result of legumain cleavages. Reverse and contaminant peptides were removed from the peptide list. Additionally, the peptides were filtered for posterior error probability (PEP values above 0.05 were discarded) and score (values below 40 were discarded) as described previously.20 The P1'-P4' positions were determined from the peptide N-terminus, whilst the P4-P1 positions were determined bioinformatically. The iceLogo representations were generated using the frequencies of positional ami-no acid occurrences normalised to the natural amino acid abundances in the human Swiss-Prot database.21 2. 7. Analysis of Structural Determinants of Legumain Substrates To analyse the structural determinants of legumain substrate recognition we first prepared a list of the most reliable legumain substrates. Accordingly, we analysed the identified cleavages with ISIL and FPPS protocol and constructed Venn diagrams to identify the overlapping substrates and cleavages (identified in both experiments). These substrates were selected for further analysis to assign the identified cleavages to the secondary structure of each substrate using PSIPRED.22-24 3. Results and Discussion 3. 1. Determination of Legumain Cleavage Specificity For specificity profiling of the native proteome, we prepared protein lysates under mild lysis conditions to Vidmar et al.: Characterization of Legumain Degradome ... Acta Chim. Slov. 2019, 66, 50-57 53 Figure 1. Legumain cleavage specificity profiling. (a) ISIL and (c) FPPS specificity profile of legumain for P4 to P4' positions presented as iceLo-go21with the representative number of determined cleavages used for the iceLogo construction (n-value). Venn diagrams of unique and shared legumain (b) substrates and (d) cleavages sites between ISIL and FPPS approaches. preserve the protein fold. During the lysis, we inhibited proteolytic background with general protease inhibitors in order to minimise potential false positive hits as a result of endogenous proteases. After the treatment of the native proteome with recombinant human legumain we applied two different approaches for chemical labelling of neo-N-terminal peptides in order to identify the corresponding cleavage events (Fig 1). Using ISIL we identified 57 cleavage sites in 50 proteins, FPPS enabled us to identify 356 cleavages in 238 legumain substrates (Fig. 1b, c). This difference can be attributed to a better performance of the in-solution approach and the extensive peptide fractionation using the anion exchange resin. Our results confirmed that P1 asparagine is the main determinant of legumain substrate specificity and that even cleavages of native protein substrates showed no extended cleavage specificity. Both tested approaches resulted in similar cleavage specificity profiles with a strong preference for asparagine in the P1 position (ISIL 68.4% and FPPS 52.2% of cleavages, respectively) and less prominently for the P1 aspartate (ISIL 8.8% and FPPS 9.0% of cleavages, respectively). These results are consistent with the canonical specificity of legumain reported in the CutDB25 and PMAP26 databases, which is based on 15 proteolytic events deposited in both databases. The cleavage specificity determined is also in a good agreement with previously reported profiling experiments performed on the denaturat-ed protein samples, where primary structure was shown to be the leading factor of protease-substrate recognition.12 In both experimental setups the specificity for P1 asparagine was highly similar under native (52.2-68.4%) as well as under denaturing conditions (85%). This led us to the conclusion that regardless of the native/denatured state of the substrates, legumain has a relatively stringent requirement for an Asn in the P1 position. Such observations were reported also by studies using combinatorial peptide substrate libraries which concluded that the P1 position is crucial for recognition of legumain substrates, while other positions did not have a significant role.13 3. 2. Validation of Substrate Processing in the Case of Prelamin-A/C Our specificity profiling of legumain showed a considerable overlap of identified cleavages between the ISIL and FPPS approach (Fig. 1d). Among the 19 overlapping cleavage events (33% of the total ISIL-determined cleavage sites) we selected the cleavage after Asn283 in human prelamin-A/C (Uniprot code P02545, gene LMNA) for further validation. Prelamin-A/C is an important constituent of the nuclear lamina that provides framework for nuclear envelope. Maturation of prelamin-A/C involves several steps, including farnesylation of C-terminal -CAAX motif and subsequent proteolytic maturation by zinc metalloprotease ZMPSTE24.27 Failure of prelamin-A/C proteolytic maturation due to protease absence or mutations was shown to result in its accumulation and consequently to development of several diseases.28 We identified prelamin-A/C cleavage after Asn283 using both labelling approaches. The incubation with in- Vidmar et al.: Characterization of Legumain Degradome ... Acta Chim. Slov. 2019, 66, 50-57 54 Table 1. The ion intensity of prelamin-A/C peptide AcD3-LVGAAHEELQQSR determined by ISIL and FPPS approach. Approach PEP Score Ion intensity (*106 a.u.) Control 0.2 ^M legumain 1.0 |iM legumain ISIL 1.28E-16 265.61 0.0 19.3 31.1 FPPS 6.21E-12 164.33 0.0 14.5 43.5 creased concentration of legumain led to an increased MS peak intensity of trideutero-acetylated peptide LVGAA-HEELQQSR, as shown in Table 1. This additionally confirmed that generation of the labeled peptide was directly related to the legumain activity. To verify these results, we next performed immunological detection of prelamin-A/C processing (Figure 2). We confirmed that legumain processed prelamin-A/C at a single site, since the intensity of the single band corresponding to the cleaved prelamin-A/C fragment increased with incubation time. The theoretical molecular weight of prelamin-A/C fragments is 32.5 kDa (N-terminal fragment, amino acid sequence 1-283) and 39.7 kDa (C-ter-minal fragment, amino acid sequence 284-664), respectively. Since the antibodies used in this assay recognized the epitope between residues 319 and 566, we concluded that the band observed at a molecular weight around 40 kDa corresponded to the C-terminal cleavage product (284-664) of prelamin A/C. Identification of prelamin-A/C as legumain substrate is based on in vitro experiments Figure 2. Legumaim cleavage of prelamin-A/C. (a) Schematic representation of prelamin-A/C cleavage with legumain. (b) Immunological detection of prelamin-A/C processing with legumain. (c) MS/MS spectrum of prelamin-A/C peptide AcD3-LVGAAHEELQQSR. y and b ions are shown in red and blue, respectively. Vidmar et al.: Characterization of Legumain Degradome ... Acta Chim. Slov. 2019, 66, 50-57 55 that investigate sequence and structure specific features of legumain substrates. Further in vivo studies are therefore required to determine possible physiological relevance of identified cleavages and since legumain has been shown to be localized in the nucleus29 there is a potential spatial link present in living cells. 3. 3. Structural Preferences for Legumain Cleavages In addition to the sequence-based substrate specificity interpretation of the legumain substrate repertoire we also investigated the structural features of the cleaved sub- Table 2. Structural determinants of 19 identified legumain cleavage sites determined using disordered regions and secondary structure prediction algorithms. Gene Uniprot neo-N-terminal Cleavage 2D structure at 3D structure at name code peptide sequence site cleavage site cleavage site ACTN4 043707 GTLEDQLSHLKQYER N680 unstructured n.a. FLNB 075369 ETSSILVESVTR N2483 unstructured 2EEC CSDE1 075534 IMLLKKKQAR N175 a-helical 2YTX PNP P00491 STVPGHAGR R58 unstructured 1RSZ LMNA P02545 LVGAAHEELQQSR N283 a-helical n.a. NPM1 P06748 DENEHQLSLR N35 unstructured n.a. HSP90AB1 P08238 ASDALDKIR N46 unstructured 3NMQ HNRNPL P14866 YDDPHKTPASPVVHIR N91 unstructured n.a. EEF1D P29692 EEEDKEAAQLR N164 unstructured 2MVM RPL12 P30050 EIKVVYLR N8 unstructured n.a. RPL22 P35268 LGGGVVTIER N55 unstructured n.a. RPS16 P62249 GRPLEMIEPR N35 unstructured n.a. RPL7A P62424 FGIGQDIQPKR N38 unstructured n.a. EIF5A P63241 GFVVLKGR N28 unstructured 3CPF ACTG1 P63261 SYVGDEAQSKR D51 unstructured n.a. TUBA1B P68363 AAIATIKTKR N329 a-helical n.a. PCBP1 Q15365 STAASRPPVTLR N89 unstructured n.a. CNN3 Q15417 KIASKYDHQAEEDLR N18 unstructured n.a. RPL36 Q9Y3U8 KGHKVTKNVSKPR N12 unstructured n.a. n.a. - not available Figure 3. Example of a legumain cleavage site in a 3D protein structure. (a) The structure of eukaryotic translation initiation factor 5A-1 with annotated scissile site at asparagine 28 (For preparing this image the structure with PDB 3CPF was used). (b) An example of secondary structure prediction algorithm output for the region around Asn28 in eukaryotic translation initiation factor 5A-1 (marked in blue). Vidmar et al.: Characterization of Legumain Degradome ... 56 Acta Chim. Slov. 2019, 66, 50-57 56 strates. We performed a detailed structural analysis on 19 substrates identified by both ISIL and FPPS approaches (Table 2, Appendix 1) and combined the exact cleavage site information to the secondary structural characteristics of each substrate using a 2D structure and disordered region prediction tool (i.e. PSIPRED protein sequence analysis workbench).22 The close inspection of individual secondary structure features has shown that the large majority of legumain cleavage sites are not located in the structured regions like alpha-helices or beta-sheets (84%). Since the unstructured elements are devoid of regular secondary structures they often serve as flexible linkers or loops connecting the organized secondary structure elements. Our observation that unstructured elements are more easily accessible to legumain proteolysis is in good agreement with the central proteolytic paradigm stating that most cleavages occur in easily accessible protein regions. Moreover, similar observations were also reported by other studies showing that largest number of proteolytic cleavages occurs in the loops, followed by alpha-helices and be-ta-sheets.30-33 Among the identified substrates, 6 have known 3D structures of the cleavage site regions and we were able to confirm our observation, that cleavage sites are mainly located on unstructured regions characteristic for loops between protein domains or exposed tails near protein termini (Supplementary Figures 1-5). For example, in the crystal structure of eukaryotic translation initiation factor 5A-1 legumain cleaved the protein after an asparagine located in the unstructured region connecting two neighbouring beta-sheets (Fig 3), consistent with our predictions. For a more precise analysis a larger pool of cleavage sites and 3D structures would be required. 4. Conclusions Determination of protease specificity provides the most basic information about protease-substrate interactions and helps us to understand why certain proteins are cleaved or processed by proteases. Whether a substrate cleavage will occur depends on several factors: (i) subsite cleavage specificity of a protease that recognizes an amino acid motif in a protein substrate, (ii) structural arrangement of a potential cleavage site(s) in a substrate (iii), presence of potential protease's exosites located outside the active site cleft and (iv), spatiotemporal co-localization of a protease and a corresponding substrate. Our study was focused on the first two factors and it showed that P1 asparagine (or less preferably aspartate) is the most important determinant of legumain specificity even in the processing of native proteins. The identified protein cleavages also revealed that legumain has a high preference for cleavages outside secondary structure elements, since only a minor portion of cleavages was found in the alpha-helical regions and none in the beta-sheets. The low number of identified cleavage events in individual substrates showed that legumain is a highly selective protease, which promotes limited proteolysis rather than general protein degradation. Within the lysosomes, such limited proteolytic processing could serve as the first step in protein degradation, where legumain cleavage would increase exposure of proteins to other proteases. However, since legumain is also known to be secreted to extracellular space and translocated to the cell nucleus, its limited proteolysis could play an important role in cell signalling and other highly specific cellular processes. 5. Acknowledgments The work was funded by the Slovenian Research Agency (P1-0140, J1-3602, and N1-0022 to B.T.; J1-0185 and J1-5449 to M.F.; and I0-0048 to D.T.) and the COST Action PROTEOSTASIS BM1307. The mass spectrometry proteomics data have been deposited to the Proteome-Xchange Consortium via the PRIDE34 partner repository with the dataset identifier PXD010466. 6. References 1. Dall, E.; Brandstetter, H., Proc Natl Acad Sci 2013, 110 (27), 10940-5. DOI:10.1073/pnas.1300686110 2. Dall, E.; Brandstetter, H., Biochimie 2016, 122, 126-50. DOI:10.1016/j.biochi.2015.09.022 3. Sepulveda, F. E.; Maschalidi, S.; Colisson, R.; Heslop, L.; Ghirelli, C.; Sakka, E.; Lennon-Dumenil, A. M.; Amigorena, S.; Cabanie, L.; Manoury, B., Immunity 2009, 31 (5), 737-48. DOI:10.1016/j.immuni.2009.09.013 4. Maschalidi, S.; Hassler, S.; Blanc, F.; Sepulveda, F. E.; Tohme, M.; Chignard, M.; van Endert, P.; Si-Tahar, M.; Descamps, D.; Manoury, B., PLoS Pathog2012, 8 (8), e1002841. 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Staes, A.; Impens, F.; Van Damme, P.; Ruttens, B.; Goethals, M.; Demol, H.; Timmerman, E.; Vandekerckhove, J.; Gevaert, K., Nat Protoc 2011, 6 (8), 1130-41. DOI:10.1038/nprot.2011.355 15. Asara, J. M.; Zhang, X.; Zheng, B.; Christofk, H. H.; Wu, N.; Cantley, L. C., JProteome Res 2006, 5 (1), 155-63. DOI:10.1021/pr050334t 16. Vizovisek, M.; Vidmar, R.; Van Quickelberghe, E.; Impens, F.; Andjelkovic, U.; Sobotic, B.; Stoka, V.; Gevaert, K.; Turk, B.; Fonovic, M., Proteomics 2015, 15 (14), 2479-90. DOI: 10.1002/pmic.201400460 17. Wisniewski, J. R.; Zougman, A.; Mann, M., J Proteome Res 2009, 8 (12), 5674-8. DOI:10.1021/pr900748n 18. Wisniewski, J. R.; Zougman, A.; Nagaraj, N.; Mann, M., Nat Methods 2009, 6 (5), 359-62. DOI:10.1038/nmeth.1322 19. Cox, J.; Mann, M., Nat Biotechnol 2008, 26 (12), 1367-72. DOI:10.1038/nbt.1511 20. Vizovisek, M.; Vidmar, R.; Fonovic, M., Methods Mol Biol 2017, 1574, 183-195. DOI:10.1007/978-1-4939-6850-3_13 21. Colaert, N.; Helsens, K.; Martens, L.; Vandekerckhove, J.; Ge-vaert, K., Nat Methods 2009, 6 (11), 786-7. DOI: 10.1038/nmeth1109-786 22. Buchan, D. W.; Minneci, F.; Nugent, T. C.; Bryson, K.; Jones, D. T., Nucleic Acids Res 2013, 41, W349-57. DOI:10.1093/nar/gkt381 23. McGuffin, L. J.; Bryson, K.; Jones, D. T., Bioinformatics 2000, 16 (4), 404-5. DOI: 10.1093/bioinformatics/16.4.404 24. Jones, D. T.; Cozzetto, D., Bioinformatics 2015, 31 (6), 857-63. DOI:10.1093/bioinformatics/btu744 25. Igarashi, Y.; Eroshkin, A.; Gramatikova, S.; Gramatikoff, K.; Zhang, Y.; Smith, J. W.; Osterman, A. L.; Godzik, A., Nucleic Acids Res 2007, 35 (Database issue), D546-9. DOI:10.1093/nar/gkl813 26. Igarashi, Y.; Heureux, E.; Doctor, K. S.; Talwar, P.; Gramatikova, S.; Gramatikoff, K.; Zhang, Y.; Blinov, M.; Ibragimova, S. S.; Boyd, S.; Ratnikov, B.; Cieplak, P.; Godzik, A.; Smith, J. W.; Osterman, A. L.; Eroshkin, A. M., Nucleic Acids Res 2009, 37 (Database issue), D611-8. DOI:10.1093/nar/gkn683 27. Casasola, A.; Scalzo, D.; Nandakumar, V.; Halow, J.; Recil-las-Targa, F.; Groudine, M.; Rincon-Arano, H., Nucleus 2016, 7 (1), 84-102. DOI: 10.1080/19491034.2016.1150397 28. Rankin, J.; Ellard, S., Clin Genet 2006, 70 (4), 261-74. DOI: 10.1111/j.1399-0004.2006.00677.x 29. Haugen, M. H.; Johansen, H. T.; Pettersen, S. J.; Solberg, R.; Brix, K.; Flatmark, K.; Maelandsmo, G. M., PLoS One 2013, 8 (1), e52980. DOI:10.1371/journal.pone.0052980 30. Mahrus, S.; Trinidad, J. C.; Barkan, D. T.; Sali, A.; Burlingame, A. L.; Wells, J. A., Cell 2008, 134 (5), 866-76. DOI:10.1016/j.cell.2008.08.012 31. Timmer, J. C.; Zhu, W.; Pop, C.; Regan, T.; Snipas, S. J.; Eroshkin, A. M.; Riedl, S. J.; Salvesen, G. S., Nat Struct Mol Biol 2009, 16 (10), 1101-8. DOI:10.1038/nsmb.1668 32. Kazanov, M. D.; Igarashi, Y.; Eroshkin, A. M.; Cieplak, P.; Ratnikov, B.; Zhang, Y.; Li, Z.; Godzik, A.; Osterman, A. L.; Smith, J. W., J Proteome Res 2011, 10 (8), 3642-51. DOI:10.1021/pr200271w 33. Ratnikov, B. I.; Cieplak, P.; Gramatikoff, K.; Pierce, J.; Eroshkin, A.; Igarashi, Y.; Kazanov, M.; Sun, Q.; Godzik, A.; Osterman, A.; Stec, B.; Strongin, A.; Smith, J. W., Proc Natl Acad Sci 2014, 111 (40), E4148-55. DOI:10.1073/pnas.1406134111 34. Vizcaino, J. A.; Csordas, A.; Del-Toro, N.; Dianes, J. A.; Griss, J.; Lavidas, I.; Mayer, G.; Perez-Riverol, Y.; Reisinger, F.; Ternent, T.; Xu, Q. W.; Wang, R.; Hermjakob, H., Nucleic Acids Res 2016, 44 (22), 11033. DOI:10.1093/nar/gkw880 Povzetek Legumain je po svoji specifičnosti edinstvena lizosomalna proteaza, ki cepi C-terminalno od asparagina in v redkih primerih tudi za aspartatom. V eksperimentalnem delu smo izvedli profiliranje specifičnosti legumaina s proteomskim pristopom na osnovi kemijskega označevanja N-koncev z uporabo trideuteroacetilacije. Raziskava predstavlja prvo globalno analizo nativnih legumainskih substratov pri čemer smo potrdili visoko specifičnost legumaina za cepitev peptidne vezi za asparaginom. Na osnovi proteomskih rezultatov smo z bioinformatsko analizo podatkov raziskovali tudi povezavo med identificiranimi proteolitičnimi cepitvami ter sekundarno in tridimenzionalno strukturo substratov legumaina. Vidmar et al.: Characterization of Legumain Degradome ... DOI: 10.17344/acsi.2018.4427 Acta Chim. Slov. 2019, 66, 58-61 /^creative ^commons Short communication The Carboxypeptidase Activity of Cathepsin X is not Controlled by Endogenous Inhibitors Urša Pečar Fonovic,1'* Milica Perišic Nanut,2 Nace Zidar,1 Brigita Lenarčič3 and Janko Kos1,2 1 Faculty of Pharmacy, University of Ljubljana, Aškerčeva 7, 1000 Ljubljana, Slovenia 2 Department of Biotechnology, Jožef Stefan Institute, Jamova 39, 1000 Ljubljana, Slovenia 3 Faculty of Chemistry and Chemical Technology, University of Ljubljana, Večna pot 113, 1000 Ljubljana, Slovenia * Corresponding author: E-mail: ursa.pecarfonovic@ffa.uni-lj.si Tel.: +386 1 4769 500; Fax: +386 1 4258 031 Received: 04-26-2018 Dedicated to the memory of Prof. Dr. Igor Kregar Abstract Cysteine cathepsins are peptidases with housekeeping functions that play different specific roles in different tissues. Endogenous peptidase inhibitors, such as cystatins and thyropins are the ultimate way of controlling their activity. It appears, however, that cathepsin X, a monocarboxypeptidase, whose overexpression is associated with several pathological processes, is not under the control of endogenous inhibitors. Inhibitors belonging to various groups inhibit other cathepsins tested, but none decrease the carboxypeptidase activity of cathepsin X. This absence of inhibitor control is another feature that distinguishes cathepsin X from other members of the cysteine peptidases. Keywords: Cathepsin X; inhibitor; cystatin; stefin; thyropin 1. Introduction Lysosomal cysteine cathepsins are expressed in the form of inactive precursors. When activated, their activity is controlled by endogenous inhibitors.1 These inhibitors are divided into superfamilies of cystatins (which includes stefins, cystatins, kininogens and non-inhibitory fetuins) and thyropins.2 Physiologically, endogenous inhibitors can act as emergency inhibitors that usually act on peptidases released from cells or lysosomes, from infecting microorganisms, or as regulatory inhibitors modulating peptidase activity. Regulatory inhibitors are further divided into 1) the threshold inhibitors that act when there is accidental activation of peptidases, 2) buffer type inhibitors that keep peptidases inactive in the absence of substrate or 3) delay type inhibitors that bind slowly to the active enzyme, allowing its action for a certain period of time.2 Cathepsin X is a carboxypeptidase expressed predominantly in immune and neuronal cells.3 It regulates the migration, adhesion, proliferation, and maturation of cells, together with phagocytosis and signal transduction.4 Its overexpression, increased activation or mis-localization may trigger pathological processes such as cancer,5-9 neurodegenerative disorders10 or inflammatory diseases.11 Studies describing the inhibitory action of endogenous peptidase inhibitors towards cathepsin X are rare and contradictory. While some did not report inhibition of cathepsin X by cystatins C and F12-15, others reported its potent inhibition by cystatin C16 as well as by stefins A and B and chicken cystatin, but not by L-kininogen. The aim of this study was to evaluate the inhibitory effect of a broader range of endogenous protease inhibitors from the cystatin and thyropin families on cathepsin X in comparison to the related cathepsins B and L. In the assay a recombinant cathepsin X, void of contamination with other cathepsins, and its specific substrate Abz-FEK(Dnp) OH were used. 2. Experimental Activity assays were performed with recombinant cathepsins X (prepared in P.pastoris),17 B and L ( both prepared in E.coli).18,19 Assay buffers were 100 mM sodium acetate buffer, pH 5.5 (for cathepsins X and L), 60 mM Pečar Fonovič et al.: The Carboxypeptidase Activity of Cathepsin X is Acta Chim. Slov. 2019, 66, 58-59 61 acetate buffer pH 5.0 (for cathepsin B exopeptidase activity) and 100 mM phosphate buffer pH 6.0 (for cathepsin B endopeptidase activity). All assay buffers contained 5 mM cysteine, 1.5 mM EDTA and 0.1% PEG 8000. Enzymes were activated in the assay buffer at 37 °C for 5 min before assay.16 Cystatin F was activated for 15 min at 37 °C in the appropriate assay buffer containing 100 mM cysteine.15 The fluorogenic substrates Abz-FEK(Dnp)-OH,20 Z-FR-AMC, Abz-GIVRAK(Dnp)-OH21 and Z-RR-AMC were used for determining cathepsins X, L and B exo- and endopeptidase activities, respectively. Cystatin F (wild type and truncated form A15N) was expressed in FreeStyle 293F cells,22 chicken cystatin was isolated from chicken egg white,23 recombinant cystatin C and stefins A and B were expressed in E. coli,24,25 thyropins were from the laboratory of B. Lenarčič. Enzymes, substrates and inhibitor concentrations are summarized in Supplementary Table S1. 90 ^L of activated enzyme (20 nM cathepsin X or 0.5 nM cathepsin L or 0.6 nM/5.5 nM cathepsin B for its exo/ endopeptidase activity) was incubated for 30 min with 5 ^L of the inhibitor (100-fold molar excess over the enzyme) at 37 °C, then added to a black 96-well plate together with 5 ^L of substrate. The reaction was monitored continuously, at 37 °, at 420 x 10 nm with excitation at 320 x 20 nm for Abz substrates and at 460 nm x 10 nm with excitation at 380 nm x 20 nm for AMC substrates. 3. Results and Discussion Various peptidase inhibitors were tested in a standard kinetic assay to determine inhibition of cathepsin X. Previous reports of their inhibitory activities towards cathepsin X are ambiguous, probably due to the use of native cathepsin X, isolated from human liver, later shown to be contaminated with cathepsin B12 or of recombinant cathepsin X contaminated with cathepsin L which is needed for the activation of procathepsin X.26 The substrates used, such as FR-AMC, were not specific, being also degraded by other cathepsins. Since most of the cystatins used in these studies (cystatin C, chicken cystatin, stefins A and B)16 potently inhibit cathepsins L and B, it is very likely that the measured inhibition was in fact that of contaminating cathepsin B or L. In our study we used recombinant cathepsin X, containing cathepsin L below the detection limit of western blot and ELISA assays,17 and cathepsin X specific substrate Abz-FEK(Dn-p)-OH.26 The tested inhibitors (in 100-fold molar excess) did not significantly inhibit cathepsin X (Figure 1). The irreversible inhibitor of cysteine peptidases, E-64, was used as a control. Inhibitors from the cystatin and thyropin families were included in the study, namely cystatins C, F (wild type and N-terminally truncated form15), chicken cystatin, stefins A and B, p41, equistatin, testicans 1 and 2 and thy-roglobulin domains of nidogens 1 and 2 (domain 1 from nidogen 1 and domains 1 and 2 from nidogen 2). The results demonstrate the absence of inhibition, even at high inhibitor to enzyme ratios and are in line with studies on cystatins C and F.12-15 Inhibitory activity of thyropins has not yet been probed for cathepsin X. Like cystatins, they did not inhibit cathepsin X. The same set of inhibitors was tested on endo- and exopeptidase (carboxypeptidase) activities of cathepsin B Figure 1: Cathepsin X is not inhibited by endogenous peptidase inhibitors. Relative inhibition of cathepsin X by various inhibitors was tested in a kinetic assay. Concentrations of endogenous inhibitors were 2 |iM (100-fold higher than that of the enzyme), while E-64 was tested at 10 |iM concentration. Assay buffer was used in a control experiment. Values are the means of two or three independent experiments, each performed in duplicate ± SEM. Pečar Fonovič et al.: The Carboxypeptidase Activity of Cathepsin X is ... 6G Acta Chim. Slov. 2019, 66, 58-72 61 Figure 2: Comparison of the activities of endogenous inhibitors towards cathepsins X, B (exo- and endopeptidase activity) and L. The relative inhibitiory activities of cysteine cathepsins with various inhibitors was tested in a kinetic assay (p41 was not tested on cathepsin B endopeptidase activity). Concentrations of inhibitors were 100-fold higher than those of the enzymes, E-64, used as a positive control, was tested at 10 |M concentration. Assay buffers were used in control experiments. Values are the means of two or three independent experiments, each performed in duplicate. (Figure 2). Cystatin C, stefin A and equistatin potently inhibit both, endopeptidase and exopeptidase activities of cathepsin B, whereas stefin B, cystatin F and testican 1 are better inhibitors of endopeptidase activity. We also determined their inhibitory activities towards cathepsin L (Figure 2). As reported, cathepsin L was inhibited by the majority of inhibitors except testican 2 and nido-gen 2.27,28 Nidogen 1 was not very effective in inhibiting cathepsin L. 4. Conclusions Our results show that endogenous inhibitors of cysteine peptidases do not inhibit cathepsin X. Thus cathepsin X carboxypeptidase activity escapes this ultimate control. The possible physiological and pathological consequences of this observation have, however, not yet been addressed. 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Jerala, E. Zerovnik, K. Lohner, V. Turk, Protein Eng 1988, 7, 977-984. DOI:10.1093/protein/7.8.977 26 L. Puzer, S. S. Cotrin, M. H. Cezari, I. Y. Hirata, M. A. Juliano, I. Stefe, D. Turk, B. Turk, L. Juliano, A. K. Carmona, Biol Chem 2005, 386, 1191-1195. 27 A. Leonardi, B. Turk, V. Turk, Biol Chem Hoppe Seyler. 1996, 377, 319-21. 28 P. Meh, M. Pavsic, V. Turk, A. Baici, B. Lenarcic, Biol Chem 2005 386, 75-83. DOI:10.1515/BC.2005.010 Povzetek Cisteinski katepsini so peptidaze z vzdrževalno vlogo in z različnimi specifičnimi vlogami v različnih tkivih. Endogeni inhibitorji peptidaz, kot so cistatini in tiropini, so bistveni za nadzor njihove aktivnosti, vendar pa rezultati kažejo, da katepsin X, monokarboksipeptidaza, katere prekomerno izražanje je povezano z več patološkimi procesi, ni pod nadzorom endogenih inhibitorjev. Inhibitorji, ki pripadajo različnim skupinam, so zavirali druge preizkušene katepsine, nobeden pa ni zmanjšal karboksipeptidazne aktivnosti katepsina X. 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Slov. 2019, 66, 62-69 /^creative ^commons Scientific paper Spectrophotometric Determination of Ru(IV) Using 5-Hydroxyimino-4-imino-1,3-thiazolidin-2-one as a Novel Analytical Reagent Oleksandr Tymoshuk,1 Lesia Oleksiv,1,* Liudmyla Khvalbota,2 Taras Chaban3 and Ihor Patsay1 1 Department of Chemistry, Ivan Franko National University of Lviv, Kyryla and Mefodiya Str., 6, 79005 Lviv, Ukraine; 2 Faculty of Chemical and Food Technology, Slovak University of Technology in Bratislava, Radlinskeho 9, 812 37 Bratislava, Slovakia 3 Faculty of Pharmacy, Danylo Halytsky Lviv National Medical University, Pekarska Str., 69, 79010 Lviv, Ukraine * Corresponding author: E-mail: l_lozynska@ukr.net Received: 07-17-2018 Abstract The interaction of Ru(IV) ions with a novel analytical reagent - 5-hydroxyimino-4-imino-1,3-thiazolidin-2-one, by spectrophotometric method was investigated. The complex is formed at pH 5.0 in acetate buffer medium after heating in the boiling water bath (~371 K) for 25 min. The complex has maximum absorption at 350 nm and is stable for 24 h. Beer's law is valid over the concentration range of 0.5-6.1 |ig mL-1 for Ru(IV). The molar absorptivity at \ = 350 nm is 6.21 x 103 L mol-1 cm-1. The limit of detection of this method is 0.2 |g mL-1. The interfering effect of various cations and anions on the spectrophotometric determination of the Ru(IV) were investigated. The proposed method was successfully applied to the determination of Ru(IV) in alloys. Keywords: Spectrophotometry; ruthenium(IV); 5-hydroxyimino-4-imino-1,3-thiazolidin-2-one. 1. Introduction Ruthenium and its compounds are widely used in various fields: as a catalyst in chemical, petroleum, and pharmaceutical industries, in electrical contacts, resistors, as electrode materials for supercapacitors, and it improves the corrosion properties of alloys, etc.1-3 There is a need to develop sensitive, simple and inexpensive methods for the quantitative determination of ruthenium in different samples. For the determination of ruthenium, different analytical techniques such as: voltammetry, atomic absorption spectrometry, high performance liquid-chromatography, thin-layer chromatography, inductively coupled plasma atomic emission, inductively coupled plasma mass spec-troscopy are used.3 But spectrophotometric methods have been widely used thanks to their simplicity, versatility and cheapness.3-12 For spectrophotometric determination of ruthenium, S and N containing organic compounds are widely used.3-6,8-12 Azolidones belong to such compounds. This class of organic compounds was discovered in the middle of the 20th century.13 These compounds exhibit different biological activity, in particular hypoglycemic, antimicrobial, fungicidal, antiviral, antiinflammatory, and antitumor.14-16 In our previous studies, the physicochemical properties of the new reagents: 5-hydroxyimino-4-imino-1,3-thiazolidin-2-one (HITO), 4-[N'-(4-imino-2-oxo-thiazo-lidin-5-ylidene)-hydrazino]-benzenesulfonic acid and 1-(5-benzylthiazol-2-yl)azonaphthalen-2-ol, which belong to the class of azolidones, and the interaction of Pd(II), Rh(III), Pt(IV), Ir(IV), Cu(II), Zn(II), Cd(II), Ni(II), and Hg(II) ions with these reagents were investiga-ted17-28 and the methods of their determination with good metrological characteristics were proposed.18-25,27,28 Therefore in this paper, we investigated the interaction of ruthenium(IV) ions with 5-hydroxyimino-4-imino-1,3-thiazolidin-2-one. Tymoshuk et al.: Spectrophotometric Determination of Ru(IV) Acta Chim. Slov. 2019, 66, 62-63 69 2. Experimental 2. 1. Apparatus A ULAB 108-UV computerized spectrophotometer equipped with 1.0 cm quartz cells was used for absorbance measurements. Absorption spectra were obtained in the wavelength range of 240-600 nm with the step of 2 nm using distilled water as a blank. The pH measurements and adjustments were performed using pH-meter model pH-150 M equipped with a combination glass electrode. Voltammetric measurements were carried out using oscillopolarograph CLA-03 and computerized polarographic setup equipped with linear potential scan in three-electrode cell (indicator electrode - dropping mercury electrode, reference electrode - saturated calomel electrode and auxiliary electrode - platinum). Spectrometer Bruker Avance (400 MHz) was used for registration 1H NMR spectra of compounds dissolved in DMSO-d6, internal reference of TMS. 2. 2. Reagents All chemicals used in the research were analytically pure and all aqueous solutions were prepared using distilled water. The stock solution of Ru(IV) ([RuCl6]2-) was prepared by sintering the exact mass of pure metallic ruthenium (99.99%) with the oxidizing mixture of NaOH and NaNO3 (3:1) in a corundum crucible at 900 K (45-60 min). The obtained fusion was dissolved in 3.0 mol L-1 HCl. The form of Ru(IV) in the obtained stock solution was identified by the comparison of its absorption spectra with the ones described in the literature.29 Because of the possible losses during sintering, the obtained solution of Ru(IV) was additionally standardized using iodometric titration.3,5 Working solutions of Ru(IV) were prepared by suitable dilution of the stock solution with 1.0 mol L-1 hydrochloric acid. The stock solution of 5-hydroxyimino-4-imino-1,3-thiazolidin-2-one was prepared by the dissolution of an exact mass of the reagent in ethanol (96% v/v). Working solution of HITO was prepared by dilution the stock solution with water. Synthesis of HITO was carried out according to the methodology given in the literature,30 the only difference was the use of 10% HCl instead of 5%, that increased the practical yield of the reagent: 5.8 g (50 mmol) 4-iminothiazo-lidine-2-one in 10% HCl was placed in a 100 mL round-bottomed flask with a mechanical stirrer and was cooled to 273 K. Then the solution of 10.5 g sodium nitrite in 20 mL of water was added dropwise by stirring for 3 h and the resulting mixture was left for 12 h at room temperature. The precipitate was filtered, washed with water and acetone, and dried at 60 °C. The yield was 70%. The melting point is 477-480 K. After that the HITO was recrystallized from the ethanol. 1H NMR (400 MHz, DMSO-d6; 5, ppm): 8.98 s (H, NH), 9.34 s (H, NH), 13.10 s (H, OH). The purity of HITO was determined chromatographically with a mass spectro-metric detection and it was 100%. The solutions of CH3COONa and NaOH were prepared by dissolving of appropriate amount of CH3COONa and NaOH in water. The solutions of HCl and CH3COOH were obtained by dilution of concentrated HCl and CH3COOH with water. The universal buffer solutions (UBS) were prepared by mixing H3PO4, H3BO3 and CH3COOH.31 2. 3. Procedure Spectral and protolytic characteristics of the HITO In a series of 25.0 mL calibrated flask the volumes of HITO (8.0 x 10-3 mol L-1), NaCl (2.0 mol L-1), UBS (1.5 mol L-1) and distilled water (~15 mL) were added. Then pH was adjusted in the acceptable range of 2.0-12.0 by NaOH (4.0 mol L-1) and distilled water was added up to the mark. For pH 1.0 the solution was prepared in a similar way, but without adding the UBS and pH was adjusted by HCl (6.0 mol L-1). Then the absorption spectra were measured against the distilled water. Recommended procedure for determination of Ru(IV) by using HITO An aliquot of the solution that contained 12.5-152.5 ^g of Ru(IV) was transferred into a 25.0 mL volumetric flask. To this solution 0.125 mL of 8.0 x 10-3 mol L-1 HITO, 1.25 mL of 2.0 mol L-1 NaCl, 2.0 mL of 4.0 mol L-1 CH3COONa, and distilled water (~15 mL) were added. Then the pH (pH 5.0) was adjusted by means of CH3COOH or CH3COONa solutions and distilled water was added to complete the volume. After that the solutions were heated in the boiling water bath (~371 K) for 25 min and cooled to room temperature (~293-295 K). The absorbance was measured at 350 nm against a reagent blank, prepared in the same way but without the addition of Ru(IV). Determination of ruthenium in the alloys The Ce45Ru25Ga2o and TbRuGa5 alloys were prepared by art melting of pure components (not less than 99.9%) under an argon atmosphere. Then the alloys were annealed in evacuated quartz ampoules at 870 K for 720 h.32 0.05-0.1 g of the sample of alloy were dissolved in 10 mL of HCl + HNO3 (1:1) mix. The obtained solution was evaporated to wet salts and dissolved in 50.0 mL of 3.0 mol L-1 HCl solution. If the alloy dissolved incompletely sintering of the new sample with oxidative mixture of NaOH and NaNO3 (1:3) was carried out. The obtained solution was dissolved in HCl of 3.0 mol L-1 and the content of the beaker was quantitatively transferred into a 250.0 mL volumetric flask and diluted to the mark with distilled water. If necessary, the solution with lower concentration of ruthenium was prepared by dilution of stock solution with 1.0 mol L-1 hydrochloric acid. For ruthe- Tymoshuk et al.: Spectrophotometric Determination of Ru(IV) 64 Acta Chim. Slov. 2019, 66, 62-64 69 nium determination, the aliquots of alloys (0.5-1.0 mL) were analyzed as described previously in "Recommended procedure for determination of Ru(IV) by using HITO". The content of ruthenium was calculated using the method of a normal calibration curve. Voltammetric determination of ruthenium(IV) ions using pyrocatechol violet at pH 4.0 (polarizing range of -0.5 to -1.5 V and the potential sweep rate of 0.5 V s-1) was used as the reference method.33 3. Results and Discussion 3. 1. Spectral and Protolytic Characteristics of HITO in Water Medium The new analytical reagent, 5-hydroxyimino-4-imi-no-1,3-thiazolidin-2-one (Fig. 1), is a sand color powder, which is poorly soluble in water (0.05 g L-1), but soluble in ethanol, methanol, «-propanol, isoamyl alcohol, ethylene glycol, glycerol, dimethylformamide and dimethylsulfoxide. Previous research of the spectral characteristics of HITO18-22 has shown that its absorbance spectra depend on pH (Fig. 2). At pH 1.0-6.0 HITO has a maximum at the wavelength of 264 nm, pH 7.0-267 nm and at pH 8.0-12.0 two absorption bands: X = 278 nm, X = 330 nm. Beer's law is applicable at 256 nm (pH 5.0) over a wide concentration range (Fig. 3). The molar absorptivity at X = 256 nm is 1.60 x 104 L mol-1 cm-1 and at X = 278 nm - 1.24 x 104 L mol-1 cm-1, X = 330 nm - 1.02 x 104 L mol-1 cm-1. The acid dissociation constant of HITO was found (pK"a = 7.1). This indicates that HITO is a weak acid. NH Fig 1. Structural formula of the HITO. Fig 3. Absorbance spectra of HITO of different molar concentration. The experimental results show that the absorbance spectra of HITO change over time in the range of pH 6.0-12.0. The influence of oxygen on the shape of absorption spectra of HITO over time at pH 8.0 and pH 9.0 was checked and it was found that oxygen does not affect the shape of absorption spectra. Therefore, we consider that changes in absorbance spectra of a reagent in the range of pH 6.0-12.0 are related to the hydrolysis of the imino group, which lasts for 60 min (Scheme 1). This process is irreversible, since after acidification of solutions to pH 2.0 spectrum of the reagent did not coincide with the spectrum of HITO in acidic media. Wavelength /nm Fig 2. Absorbance spectra of HITO at different pH. Scheme 1. Hydrolysis of 5-hydroxyimino-4-imino-1,3-thiazoli-din-2-one. 3. 2. Interaction of Ru(IV) Ions with HITO HITO forms with Ru(IV) ions a complex compound of sandy color in weakly acidic medium, just like with Pd(II), Pt(IV), Rh(III), and Ir(IV)18-22. The absorbance spectra of the reagent itself and Ru(IV)-HITO complex are shown in Fig. 4. According to Fig. 4, the maximum difference in absorbance of the HITO and complex compound is at 350 nm. Thus all futher absorbance measurements were carried out at 350 nm wavelength against reagent blank (where the absorbance of the reagent is negligible). Tymoshuk et al.: Spectrophotometric Determination of Ru(IV) Acta Chim. Slov. 2019, 66, 62-65 69 Fig 4. Absorbance spectra of reagent and complex Ru(IV) with HITO. The effect of pH on the absorbance of Ru(IV)-HITO complex compound was investigated over the range 1.07.0 in order to find the optimum conditions (Fig. 5). The optimum pH range for complex formation lies between pH 4.5-5.5. So, further investigations were carried out at pH 5.0. Fig 5. Effect of acidity on the maximum yield of Ru(IV) with HITO colored complex. The investigation of the influence of ionic strength on the maximum yield of the complex is necessary for the study of optimal conditions for complexation. Therefore, the influence of the concentration and nature of anions of sodium salts (NaCl, NaNO3, NaClO4, Na2SO4, CH3COONa) was investigated. The yield of the complex practically does not depend on the nature of anion. The SO42- and NO3-ions practically do not affect the yield of complex, ClO4- -in large surpluses slightly decreases the yield of the complex. The yield of the complex insignificantly increases in the presence of Cl- and CH3COO- ions. Therefore, NaCl and CH3COONa were chosen as a reaction medium. The interaction of Ru(IV) ions with HITO at room temperature (~291-296 K) occurs very slowly. So the possibility of accelerating this process by heating the system in the boiling water bath (~371 K) was investigated (Fig. 6). The results show the maximum yield of the complex is observed after heating for 25 min in the boiling water bath (~371 K). The rapid formation of the Ru(IV)-HITO complex during heating can be related to reduction of the kinetic inertness of the [RuCl6]2- complex. The Ru(IV)-HITO complex is stable for 24 h. Fig 6. Effect of heating time on the maximum yield of Ru(IV) with HITO colored complex. The composition of the complex was determined by the mole-ratio method and the method of continuous variations (Fig. 7). The results of both methods agree and show that the molar ratio of Ru(IV) to HITO is 1:1. It was found that a 1.5-2-fold molar excess of the reagent is suffi- Fig 7. The method of continuous variations. Tymoshuk et al.: Spectrophotometric Determination of Ru(IV) 66 Acta Chim. Slov. 2019, 66, 62-66 69 Scheme 2. The proposed scheme of Ru-HITO complex formation. cient to obtain maximum absorbances of the Ru(IV)-HI-TO complex. The formal stability constant is 5.1 x 106. Based on the experimental results obtained from the study of the interaction of Ru(IV) ions with HITO and the data given in the literature,34,35 it can be assumed that the complex formation most probably takes place as shown in Scheme 2. Under the optimum conditions a calibration graph for Ru(IV) was obtained. The Beer's law is obeyed in the range from 0.5 to 6.1 ^g mL-1 for ruthenium(IV). The molar absorptivity is 6.21 x 103 L mol-1 cm-1. The metrologi-cal characteristics of spectrophotometric determination of ruthenium(IV) with HITO are given in Table 1. Table 1. The metrological characteristics of spectrophotometric determination of the Ru(IV) with HITO (C(HITO) = 8.0 x 10-4 mol L-1; C(CH3COOH + CH3COONa) = 0.32 mol L-1; C(NaCl) = 0.1 mol L-1; pH = 5.0; X = 350 nm; l = 1.0 cm; n = 5; P = 0.95) Characteristic Value Limits of Beer's law, CRu(IV), |g mL-1 0.5-6.1 Calibration equation, CRu(IV), |g mL-1 AA = 0.02 + 0.06 x C Limit of detection, CRu(IV), |g mL-1 0.2 Limit of quntification, CRu(IV), |g mL-1 0.6 Correlation coefficient, R 0.9993 3. 3. Effect of Foreign Ions on the Determination of Ru(IV) The effect of some potentially interfering ions on the determination of ruthenium(IV) was investigated (Table 2). According to the obtained results most metals, which often accompany ruthenium, such as Ni(II), Co(II), Zn(II), Cd(II), Pb(II), Mn(II), REE, and other metals, do not interfere on the determination of Ru(IV). Instead, the technique is characterized by low selectivity concerning the noble metals or Cu(II) and Fe(III). Ag(I) intereferes significantly, even with tolerance limits up to 0.1, which is due to the influence of chloride ions that are in the solution. The presence of many anions, which were investigated, does not interfere (except EDTA) with the determination of Ru(IV). The selectivity to some cations can be increased using masking agents. For example, Cu(II) and Fe(III) were masked with tartrate ions (the tolerance limits reach 2 for Cu(II) and 4 for Fe(III)), citrate ions (the tolerance limits reach 10 for Cu(II) and 15 for Fe(III)) and phosphate ions (the tolerance limits reach 5 for Cu(II) and 10 for Fe(III)). 3. 4. Application of the Method In order to verify the possibility of the proposed method usage, initially the determination of ruthenium in synthetic samples was carried out. These solutions were prepared by adding known amount of the Ru(IV). The results of analysis Ru(IV) in synthetic mixture are given in Table 3. The method was also applied to the determination of Ru(IV) in two samples of three-component alloys (Table 4). When ruthenium is added into alloys, mechanical strength increases significantly, while anticorrosion pro- Table 2. Tolerance limits of foreign ions in Ru(IV) spectrophotometric determination using HITO (C(HITO) = 8.0 x 10-4 mol L-1; C(Ru(IV)) = 2.0 x 10-5 mol L-1; C(NaCl) = 0.1 mol L-1; C(CH3COOH + CH3COONa) = 0.32 mol L-1; pH = 5.0; X = 350 nm; l = 1.0 cm) Ion Cion;CRu(IV) Ion Cion;CRu(IV) Ion Cion;CRu(IV) Pd(II) 0.2 Cu(II) 0.4 Al(III) 30 Ir(IV) 0.15 Pb(II) 100 Ba(II) >200 Rh(III) 0.15 Cd(II) 100 Mg(II), Ca(II) >200 Pt(IV) 0.14 Zn(II) 100 C2O42- 100 Au(III) 5 Yb(III) >200 F- >200 Ag(I) <0.1 Tb(III) >200 SiO32- >200 Hg(II) 5 Ho(III) >200 EDTA 5 Co(II) 50 Gd(III) >200 Sal- 45 Ni(II) 150 Ga(III) 10 Citr3- >200 Fe(III) 0.6 Ce(IV) 10 Tart2- 50 Mn(II) 100 Sn(II) 3 PO43- 100 Tymoshuk et al.: Spectrophotometric Determination of Ru(IV) Acta Chim. Slov. 2019, 66, 62-67 69 Table 3. Determination of Ru(IV) with HITO in synthetic solutions (n = 3; P = 0.95) Modeling solution CRu* Cjon Added Ru(IV), ^g Found Ru(IV), RSD, % Ru-Pd-Ni 1:0.1:100 38.0 39.9 ± 3.8 4.0 Ru-Pt-Co 1:0.1:30 38.0 39.2 ± 2.9 3.1 Ru-Rh-Zn 1:0.1:50 38.0 39.6 ± 1.9 2.0 Table 4. Determination of ruthenium(IV) in alloys (n = 3; P = 0.95) Intermetallides CORu , % 0)R„ Spectrophotometry Sxt -{- a O/ v« ' " RSD, % Voltammetry — pr Sxt„ ^ '% RSD, %% Ce45Ru25Ga20 23.1 23.0 ± 0.9 1.6 23.2 ± 1.2 2.1 TbRuGa5 16.6 16.4 ± 0.8 2.0 16.7 ± 0.6 1.4 perties, conductivity, etc. are improved. For these reasons, it is necessary to control the content of ruthenium in mul-ticomponent systems. The results are in perfect agreement with the ones obtained by voltammetric method. According to the data presented in Table 4 and Table 5, the presented method can be applied to real samples with good reproducibility and accuracy. Table 5. Comparison of the proposed method with other spectrophotometric methods for the determination of ruthenium(IV) Reagent Conditions ^max, nm ^max> L mol-1 cm-1 Linear range ^g mL1 , Interfering ions N,N'-diphenylthiourea4 HCl + C2H5OH, 6 mol L-1 HCl, 5 min (358 K); CHCl3, 30 min (373 K) 630; 650 -; 6-18; 1.5-7.5 Pt(IV), Fe(II) Quercetin7 0.01-0.1 mol L-1 HCl, CH3OH+ H2O (1:1) 291 5.0 x 103 0.11-30 - 1,10-Phen anthroline 4,8 pH 6.0, 2 h (373 K) 448 1.9 x 104 0.1-1.5 Platinoids 2-[(5-Bromo-2-pyridylazo)]-5-diethylaminophenol and N-hydroxy-N,W-diphenyl-benzamidine9 CH2Cl2, sodium citrate dihydrate, sodium acetate 560 2.99 x 104 0.2-2.8 Pd(II), Mn(II), Pb(II) 1,4-(2,4-Diphenylthio-semicarbazide)4 5.5-6.5 mol L-1 HCl, CHCl3, 10-15 min (373 K) 565 1.0 x 104 0.5-15.0 Oxidants p-Nitrosodimethylaniline4 pH 4.0, 50 min (323 K) 610 5.7 x 104 0.3-3.0 Platinoids Sulpho chlorophen olazo-rhodanine5'10'11 2 mol L-1 HCl + 10 mol L-1 CH3COOH, 30 min (353 K) 500 1.6 x 104 0.5-30 Pt(IV), Rh(III) Sulphobenzolazophenol-azorodanin10 pH 1.0-3.0, surfactants 540 4.0 x 104 - - Xylenol orange12 pH 6.0 575 3.23 x 103 0.2-2.5 Al(III), V(IV), Cd(II), Cu(II), Ni(II), Zr(IV), Fe(III), Hf(IV), Zn(II), Co(II), Pb(II), Pd, Pt, Rh 5-hydroxyimino-4-imino-1,3-thiazolidin-2-one pH 5.0 350 6.21 x 103 0.5-6.1 Platinoids, Cu(II), Fe(III), Ag(I) Tymoshuk et al.: Spectrophotometric Determination of Ru(IV) 68 Acta Chim. Slov. 2019, 66, 62-68 69 3. 5. Comparison with other Spectrophotometric Methods The proposed method has a better selectivity relative to Ni(II), Co(II), Mn(II), Cd(II), Zn(II), Pb(II), REE, etc. in comparison with other spectrophotometric methods for Ru determination mentioned in Table 5. There is no need to use highly acidic media and organic solvents. 4. Conclusions This work presents the spectral and protolytic characteristics of the new analytical reagent - HITO and a new spectrophotometric method for the ruthenium(IV) ions determination with this reagent. The developed method is simple, reproducibile, accurate, sufficiently sensitive and selective. It does not require the separation of the matrix and the use of toxic solvents. This method was successfully used for the determination of Ru(IV) in alloys and synthetic mixtures. 5. References 1. Q. Li, S. Zheng, Y. Xu, H. Xue, H. Pang, Chem. Eng. J. 2018, 333, 505-518. D01:10.1016/j.cej.2017.09.170 2. L. Wang, T. Liu, Chin. J. Catal. 2018, 39, 327-333. D0I:10.1016/ S1872-2067(17)62994-2 3. M. Balcerzak, Crit. Rev. Anal. Chem. 2002, 32, 181-226. D0I:10.1080/10408340290765524 4. S. I. Ginzburg, N. A. Yezerskaya, I. V. Prokof'eva, N. V. Fe-dorenko, V. I. Shlenskaya, N. K. Belsky, Analiticheskaya Khi-miya Platinovykh Metallov, Nauka, Moscow, Russia, 1972, p. 613. 5. Ya. A. Zolotov, G. M. Varshal, V. M. Ivanov, Analiticheskaya Khimiya Metallov Platinovoi Gruppy, Editorial URSS, Moscow, Russia, 2003, p. 592. 6. T. D. Avtokratova, Analiticheskaya Khimiya Ruteniya, IAN SSSR, Moscow, Russia, 1972, p. 264. 7. M. Balcerzak, M. Kopacz, A. Kosiorek, E. Swiecicka, S. Kus, Anal. Sci. 2004, 20, 1333-1337. D0I:10.2116/analsci.20.1333 8. A. V. Bashilov, N. M. Kuzmin, V. K. Runov, Zh. Anal. Khim. 1998, 53, 738 -743. 9. P. Ratre, D. Kumar, Int. J. ChemTech Res. 2014, 6, 236-247. 10. R. F. Gur'eva, S. B. Savvin, Usp. Khim. 1998, 67, 236-251. D0I:10.1070/RC1998v067n03ABEH000375 11. R. F. Gur'eva, S. B. Savvin, Zh. Anal. Khim. 1995, 50, 11501157. 12. T. Vrublevs'ka, O. Solovey, O. Volyanyk, Visnyk of the Lviv University. Series Chemistry 2003, 43, 126-129. 13. R. S. Lebedev, Russ. Phys. J. 2002, 45, 822-830. D0I:10.1023/A:1021928817028 14. D. Kaminskyy, B. Zimenkovsky, R. Lesyk, Eur. J. Med. Chem. 2009, 44, 3627-3636. D0I:10.1016/j.ejmech.2009.02.023 15. R. R. Panchuk, V. V. Chumak, M. R. Fil', D. Ya. Havrylyuk, B. S. Zimenkovsky, R. B. Lesyk, R. S. Stoika, Biopolym. Cell 2012, 28, 121-128. DOI: 10.7124/bc.00003D 16. T. I. Chaban, O. V. Klenina, B.S. Zimenkovsky, I.G. Chaban, V. V. Ogurtsov, L. S. Shelepeten, Pharma Chem. 2016, 8, 534542. 17. A. Tupys, J. Kalembkiewicz, Y. Ostapiuk, V. Matiichuk, O. Ty-moshuk, E. Woznicka, L. Byczynski, J. Therm. Anal. Calorim. 2017, 127, 2233-2242. D0I:10.1007/s10973-016-5784-0 18. L. Lozynska, O. Tymoshuk, Chem. Chem. Technol. 2013, 7, 391-395. D0I:10.23939/chcht07.04.391 19. L. Lozynska, O. Tymoshuk, In: O.L. Berezko, The Interaction of 5-hydroxyimino-4-imino-1,3-thiazolidin-2-one with Plati-num(IV) Ions,3rd International Conference of Young Scientists CCT-13, Lviv, Ukraine, 2013, 166. 20. L. V. Lozynska, O. S. Tymoshuk, T. I. Chaban, Metody Ob'ekty Khim. Anal. 2014, 9, 50-54. D0I:10.17721/moca.2014.50-54 21. L. V. Lozyns'ka, O. S. Tymoshuk, T. Ya. Vrublevs'ka, Materials Science 2015, 6, 870-876. D0I:10.1007/s11003-015-9795-y 22. L. V Lozynska, O. S. Tymoshuk, Voprosy Khimii i Khimich-eskoi Tekhnologii, 2014, 1, 80-85. 23. L. Lozynska, O. Tymoshuk, T. Chaban, Acta Chim Slov. 2015, 62, 159-167. D0I:10.17344/acsi.2014.866 24. A. Tupys, O. Tymoshuk, P. Rydchuk, Chem. Chem. Technol. 2016, 10, 19-25. D0I:10.23939/chcht10.01.019 25. A. Tupys, J. Kalembkiewicz, Y. Bazel, L. Zapala, M. Dranka, Y. Ostapiuk, O. Tymoshuk, E. Woznicka, J. Mol. Struct. 2017, 1127, 722-733. D0I:10.1016/j.molstruc.2016.07.119 26. M. Fizer, V. Sidey, A. Tupys, Y. Ostapiuk, O. Tymoshuk, Y. Bazel, J. Mol. Struct. 2017, 1149, 669-682. D0I:10.1016/j.molstruc.2017.08.037 27. Y. Bazel, A. Tupys, Y. Ostapiuk, O. Tymoshuk, V. Matiychuk, J. Mol. Liq., 2017, 242, 471-477. D0I:10.1016/j.molliq.2017.07.047 28. Y. Bazel, A. Tupys, Y. Ostapiuk, O. Tymoshuk, J. Imricha, J. Sandrejova, RSC Adv., 2018, 8, 15940-15950. D01:10.1039/C8RA02039F 29. P. Wehner, J. Hindman, J. Phys. Chem. 1952, 56, 10-15. D0I:10.1021/j150493a003 30. I. D. Komaritsa, Chem. Heterocycl. Compd. 1968, 4, 324-325. D0I:10.1007/BF00755270 31.Yu.Yu. Lur'e, Spravochnik po analiticheskoi khimii, Khimiya, Moscow, Russia, 1971, p. 456. 32. O. R. Myakush, A. A. Fedorchuk, A. V. Zelinskii, Neorganiche-skiye materialy, 1998, 34, 688-691. 33. T. Vrublevska, O. Tymoshuk Voprosy Khimii i Khimicheskoi Tekhnologii, 2001, 2, 9-11. 34. R. S. Correa, M. M. da Silva, A. E. Graminha, C. S. Meira, J. A. F. dos Santos, D.R.M. Moreira, M. B. P. Soares, G. Von Poelh-sitz, E. E. Castellano, C. B. Jr, M. R. Cominetti, A. A. Batista, J. Inorg. Biochem. 2016, 156, 153-163. D0I:10.1016/j.jinorgbio.2015.12.024 35. H. Meyer, M. Brenner, S. P. Höfert, T. O. Knedel, P.C. Kunz, A. M. Schmidt, A. Hamacher, M. U. Kassack, C. Janiak, Dalton Trans., 2016, 45, 7605-7615. D0I:10.1039/C5DT04888E Tymoshuk et al.: Spectrophotometric Determination of Ru(IV) Acta Chim. Slov. 2019, 66, 62-69 69 Povzetek Raziskali smo interakcijo Ru(IV) ionov z novim analiznim reagentom 5-hydroxyimino-4-imino-1,3-thiazolidin-2-one za uporabo v spektrofotometrični metodi. Kompleks nastane pri pH 5,0 v acetatnem pufru po segrevanju v vreli vodni kopeli (~371 K) 25 min. Kompleks ima maksimum absorpcije pri 350 nm in je stabilen 24 h. Beerov zakon velja v koncentracijskem območju 0,5-6,1 |ig mL-1 Ru(IV). Molarna absorptivnost pri \ = 350 nm je 6,21 x 103 L mol-1 cm-1. Meja zaznave te metode je 0,2 |g mL-1. Raziskali smo tudi moteči učinek različnih kationov in anionov na spektrofoto-metrično določitev Ru(IV). Predlagano metodo smo uspešno uporabili za določitev Ru(IV) v zlitinah. Tymoshuk et al.: Spectrophotometric Determination of Ru(IV) DOI: 10.17344/acsi.2018.4468 Acta Chim. Slov. 2019, 66, 70-77 /^creative ^commons Scientific paper Atmospheric Solids Analysis Probe with Mass Spectrometry for Chlorpyrifos and Chlorpyrifos-Oxon Determination in Apples Mirjana R. Cvijovic,1'* Valerio Di Marco,2 Srboljub J. Stankovic,3 Zoran P. Nedic,4 Ljubinka G. Joksovic5 and Nevena R. Mihailovic5 1 Faculty of Medicine, Institute for Microbiology and Immunology, University of Belgrade, Dr Subotica 1, 11000 Belgrade, Serbia 2 Dipartimento di Scienze Chimiche, University of Padova, Via Marzolo 1, 35131 Padova, Italy 3 Department of Radiation and Environmental Protection, Vinca Institute of Nuclear Sciences, University of Belgrade, Mike Petrovica 12-14, 11000 Belgrade, Serbia 4 Faculty of Physical Chemistry, University of Belgrade, Studentski trg 12-16, 11000 Belgrade, Serbia 5 Faculty of Science, Department of Chemistry, University of Kragujevac, Radoja Domanovica 12, 34000 Kragujevac, Serbia * Corresponding author: E-mail: mirjanacvijovic6@gmail.com; Phone/fax: +381 11 3643366 Received: 05-15-2018 Abstract Chlorpyrifos (CPS) is a toxic pesticide present in several pesticide formulations, with low degradability by natural processes. The degradation leads to the toxic metabolite chlorpyrifos-oxon (CPO). The analytical techniques used for the CPS and CPO analysis, like UPLC-PDA and GC-MS, are accurate but also expensive and time consuming, and they need sample pretreatment. In the search of a more rapid and simple analytical procedure, atmospheric solids analysis probe with mass spectrometry (ASAP-MS) was optimized for the determination of CPS and CPO in apples (Malus domestica „Idared"). The identification of the analytes was based on protonated ion and isotopic pattern, while the quantification was based on peak intensities. The obtained results were confirmed by re-validated UPLC-PDA and GC-MS techniques. CPS and CPO concentrations determined by ASAP-MS and UPLC-PDA showed moderate discrepancies (on average by 10-20%), thus demonstrating that ASAP-MS can be a semiquantitative tool for the quantification of these compounds. As additional goal of this work, the efficiency of a gamma irradiation treatment to remove CPS and CPO from apples was tested by analyzing their content before and after the irradiation: 89-99% of CPS and CPO were degraded with doses of 3.5-3.8 kGy and 66-72 h of irradiation per sample. Identical degradation results were obtained by UPLC-PDA and ASAP-MS, indicating that the latter technique is well suitable to rapidly check pesticide degradation in apples. Keywords: GC-MS; UPLC-PDA; ASAP-MS; gamma irradiation 1. Introduction Chlorpyrifos (0,0-diethyl-0-3,5,6-trichloro-2-pyri-dylphosphorothioate, CPS, Figure 1) is an organophosphorous insecticide used in agriculture formulations and in the military field. It is a very persistent (resistant to degradation) and non-volatile compound at room temperature. CPS may be detected in fruit, vegetables, water, soil, and body fluids, even months to years after its application. This occurs especially after unprofessional application of agricultural formulations, like EC (emulsifiable concentrate) and EW (emulsion in water). EC contains 30-50% of active substance, organic solvents (such as high-boiling mineral oils, 40-60%), and emulsifiers, whereas EW contains active substances (about 30%), emulsifier (15%), antifreeze (10%), antifoam, thickener, biocide, stabilizer, buffer, and water. There are also other formulations based on this pesticide.1-3 Cvijovič et al.: Atmospheric Solids Analysis Probe with Mass Acta Chim. Slov. 2019, 66, 70-71 77 The degradation process of CPS leads to the formation of chlorpyrifos-oxon (0,0-diethyl-0-3,5,6-trichloro-2-pyridylphosphate, CPO) and 3,5,6-trichloro-2-pyridinol (TCP). The formation of CPO (Figure 1) occurs in animal liver, where a phosphorus-sulfur bond of CPS is replaced by a phosphorus-oxygen bond.4,5 TCP is non-toxic, but CPS and CPO are very toxic to humans and cause tens of thousands of deaths per year worldwide because of many reasons: type of pesticide application, short waiting period between treatments, higher doses than allowed, and high toxicity. Both CPS and CPO can cause chemical injury leading to serious damages of human hepatocytes (cells of main liver parenchyma), immune, cognitive, and reproductive systems, in the gastrointestinal tract, and to the hormonal status. The most affected patients are children, elderly, and occupationally exposed people.6 K. Choi et al. and J. Choi et al. demonstrated that CPO inhibits the esterase in human liver.7,8 In case of highly intoxicated liver, metabolic paths change (due to enzyme inhibition) and carcinoma can appear in humans (colorectal carcinoma, liver, lung).9 It is known that hepatocytes in humans with polymorphisms do not have the ability to metabolize, detoxify, and inactivate exogenous compounds such as insecticides and drugs.10 Thus, both European Commission (EU) and U.S. Environmental Protection Agency (EPA) restricted the use of CPS and are planning to forbid it. The CPS and CPO concentrations in different matrices (food, water, agricultural formulation, blood, urine) should be monitored continuous-ly.11 Figure 1. 0,0-diethyl-0-3,5,6-trichloro-2-pyridylphosphorothio-ate (CPS, left) and 0,0-diethyl-0-3,5,6-trichloro-2-pyridylphos-phate (CPO, right). CPS and CPO concentrations in food and other matrices are most often monitored by chromatographic techniques like high pressure liquid chromatography with photodiode detection (UPLC(HPLC)-PDA), liquid chromatography - mass spectrometry (LC-MS), liquid chromatography - tandem mass spectrometry (LC-MS/MS)12, gas chromatography with mass spectrometric detector (GC-MS), gas chromatography with nitrogen phosphorus thermoionic detector (GC-NPD), gas chromatography with flame ionization detector (GC-FID),13,14 2D-chroma-tography with simultaneous analysis by two GC columns (GCxGC-FPD).15 Other, less used techniques are spectro- scopic ones like infrared spectroscopy (IR), Fourier transform with infrared spectroscopy (FTIR), and attenuated total reflection - Fourier transform with infrared spectroscopy (ATR-FTIR).16 Almost all of these techniques are highly expensive, and they require toxic solvents and long time for validation and sample preparation. These techniques are also not suitable to rapidly check the presence or absence of CPS and CPO in food or other matrices. Atmospheric solids analysis probe with mass spectrometry (ASAP-MS) represents a possible alternative method for the analysis of such analytes.17 ASAP-MS utilizes heated nitrogen to vaporize the sample and corona discharge to ionize it. This technique is capable of ionizing low polarity compounds, such as CPS and CPO, not amenable to electrospray (ESI), to atmospheric pressure photoionization (APPI), or to atmospheric pressure chemical ionization (APCI), and it is possible to analyze samples via direct introduction at atmospheric pressure, thus shortening the analysis time and simplifying the procedure. In this paper, therefore, an ASAP-MS method was optimized for the rapid and simple determination of CPS and CPO in plant origin food, such as apples. The obtained results were confirmed by values obtained through standard sample preparation methods (extractions) and chromatographic techniques, i.e. gas chromatography with mass spectrometric detection (GC-MS) and reverse-phase ultra-high performance liquid chromatography with photodiode array detector (RP UPLC-PDA), re-validated for these purposes. Furthermore, as natural processes (sun, air, microbiological) are not effective enough in real time for the removal of CPS from food, forced processes like chemical (acid and base hydrolysis, oxidation by H2O2) or physical (gamma irradiation) are necessary for the removal of this pesticide or its conversion into nontoxic compound such as TCP. Therefore, another goal of this work was to evaluate if a gamma irradiation treatment is able to efficiently remove CPS and CPO from apples treated by EC and EW formulations. Analytical measurements were performed with both RP UPLC-PDA and ASAP-MS. 2. Experimental 2. 1. Materials The reference materials for the analysis of CPS (98.0±1.0%) and CPO (98.0±1.0%) were purchased from Dr Ehrenstorfer GmbH (Augsburg, Germany). Multiresi-due standard mix with CPS in acetonitrile, Pestanal mix 101 with 20 pesticides and CPS (50 ng/^L, 99.0±0.5%) was purchased from Dr Ehrenstorfer GmbH (Augsburg, Germany). The 30% H2O2 solution was purchased from MerckCo (Germany). Radar EC and Radar EW, plant protection formulations (active substance CPS 300 g/L), were purchased from Galenika, Phitopharmacia (Belgrade, Serbia). Apples "Idared" were received from domestic pro- Cvijovic et al.: Atmospheric Solids Analysis Probe with Mass ... 72 Acta Chim. Slov. 2019, 66, 70-72 77 duction and bought in supermarkets. Methanol, acetoni-trile, hexane, petroleum ether, acetone (HPLC grade) were purchased from J. T. Baker (Netherlands). Dichlorometh-ane (HPLC grade) was purchased from Across Organic (New York, USA). Formic acid (98%) was obtained from J. T. Baker (Netherlands). Glacial acetic acid (99.8%, HPLC grade) was a Fisher Chemicals (United Kingdom) product. Deionized water was prepared by Purite Select Fusion System. Multi-walled carbon nanotubes (MWCNT) were a Sigma Aldrich product. QuEChERS kit was composed of two extraction mixtures: a mixture of 1.5 g CH3COONa and 6 g MgSO4, and a PSA sorbent. Cartriges used for an-alytes separation were Oasis® HLB (hydrophilic-lipophilic balance) from Waters (Milford, MA, USA). 2. 2. Solutions Standard stock solutions of CPS and CPO were prepared by dissolving 10 mg of CPS and 5 mg of CPO in 1000 [L of methanol (or dichloromethane, depending on the instrumental technique used), and then put in a volumetric Eppendorf tube. The stock solutions were diluted in hexane or in deionized water (pH 6.8) to prepare the desired concentrations for the calibration curves for GC-MS or UPLC-PDA, respectively. EW and EC formulations stock solutions were prepared by dissolving 100 [L of formulation in a 10 mL volumetric flask with dichloromethane (or in methanol/water mixture 1:3, v/v), at a concentration of 3000 mg/L calculated on active component, and stored at -18 °C when not in use. 2. 3. Instrumentations and Methods 2. 3. 1. ASAP-MS ASAP-MS with triple quadrupole analyzer (TQD, Acquility, Waters, USA) was used for the mass spectrome-try analysis of CPS and CPO. The ASAP compartment consists of set probes and removable sample insertions, corona pin, and glass sample capillaries. The capillary glass tubes were sealed at the end and baked at 500 °C to remove possible contaminations. The whole assembly (100 mm in length) was inserted in the mass spectrometer. Positive ions were recorded between 50 and 700 Da. The ASAP probe with sample was inserted into the sealed source enclosure and desolvation gas was rapidly heated to 300 °C. The heated nitrogen volatilizes the sample from a glass capillary tip, which is then ionized with a corona discharge pin, and MS spectrum is generated. The mass accuracy of the data was based on the instrument internal calibration. Instrumental parameters were varied to avoid CPS and CPO fragmentation. Values accepted after optimization were: corona voltage 3.8 kV, extraction cone 2.0 V, source temperature 150 °C, desolvation gas flow 650 L h-1, sampling cone voltage 35 V, desolvation temperature 300 °C. Data were processed by the MassLynx software, version 4.1. 2. 3. 2. GC-MS The GC-MS QP2010 Ultra instrument (Shimadzu, Kyoto, Japan) was used. A Rtx®-1 (RESTEK, Crossbond® 100% dimethylpolysiloxane, 30 m x 0.25 mm I.D., 0.25 ^m film thickness) column was used. The injector temperature was 290 °C. The GC temperature program was held at 50 °C for 1 min, then elevated to 140 °C at rate of 20 °C/min, and to 300 °C at rate 10 °C/min, with a hold time of 6 min. Helium was used as the carrier gas at approximately 1.0 mL/min pulsed in splitless mode. The mass spectrometric detector was operated in an electron impact ionization mode with an ionizing energy of 70 eV and scanning range from 50 to 500 m/z. All measurements have been done in triplicate. Quantification of CPS and CPO has been done by external calibration curves. The calibration ranges of CPS and CPO explored in this work were 0.2-5 mg/L and 0.3-3.0 mg/L, respectively; linearity in the signal/concentration ratio was obtained also up to 30 mg/L for CPS and 25 mg/L for CPO. The correlation coefficient R2 was higher than 0.99. The limit of detection (LOD) for CPS was 0.08 mg/L and it was 0.1 mg/L for CPO, calculated for samples injected three times. The limit of quantification (LOQ) was 0.2 mg/L for CPS and 0.3 mg/L for CPO. Relative standard deviations (RSD) were determined from ten measurements of a CPS standard solution (C = 2.5 mg/L) and a CPO standard solution (C = 1.5 mg/L). The RSD was 4.1% for CPS and 4.8% for CPO. 2. 3. 3. RP-UPLC-PDA Reversed-phase ultra high performance liquid chromatography (RP UPLC) was performed with an Acquility system with PDA detector (Waters, USA). Samples were injected automatically; the volume of injected sample was 10 [L. Acquity BEH Reverse Phase C18 (1.7 [m x 100 mm x 2.1 mm) column was used as a stationary phase for chro-matographic separations. Mobile phases contained 0.1% formic acid in water (v/v) (A) and 0.1% formic acid in ace-tonitrile (v/v) (B). The following gradient elution conditions were used: 0-1.5 min 45% A; 1.5-2 min 40% B; 2-2.5 min 20% B; 2.5-4.5 min 2% B; 4.5-4.8 min 55% B; 4.8-6.0 min 50% B. Flow rate was set at 1 mL/min, and column temperature was 30 °C. The chosen absorption wavelengths were 230 and 280 nm. Quantification of CPS and CPO has been done by external calibration curves. All measurements have been done in triplicate. The calibration ranges of CPS and CPO were 0.1-5 mg/L and 0.03-2.5 mg/L, respectively; linearity in the signal/concentration ratio was obtained also up to 30 mg/L for CPS and 20 mg/L for CPO. The correlation coefficient R2 was higher than 0.99. The LOD for CPS was 0.03 mg/L and it was 0.01 mg/L for CPO, calculated for samples injected three times. The LOQ was 0.10 mg/L for CPS and 0.03 mg/L for CPO. The accuracy and precision were evaluated by means of the recovery: values obtained by Cvijovic et al.: Atmospheric Solids Analysis Probe with Mass ... Acta Chim. Slov. 2019, 66, 70-73 77 post-extraction addition of standard to treated apples were 98-108% and 96-104% for the two compounds, respectively. RSD values were determined from ten measurements of CPS standard solution (C = 0.3 mg/L) and CPO standard solution (C = 0.8 mg/L). The RSD was 2.2% for CPS and 3.3% for CPO. 2. 4. Gamma Irradiation Experiments Gamma irradiation experiments were carried out in the metrology dosimetry laboratory at the Institute of Nuclear Sciences (Belgrade). The radiation unit IRPIK-B was used as a functional generator with a source of gamma radiation Co-60. The measured value of the absorbed dose in the air at the reference point for the irradiation was 48.27 Gy/h. At the point at which samples were positioned, the absorbed dose in water was 53.51 Gy/h. After each individual campaign irradiation, the total value of the absorbed dose in each sample was 3.5-3.8 kGy, depending on time of irradiation. In general, the relation between the absorbed dose in air and absorbed dose in water as the material that is irradiated with gamma rays is: (1) where DW and Dair are the absorbed dose in the water and absorbed dose in the air, respectively, and [^j and^'' are the energy absorption coefficients for air and water, respectively. It was assumed that absorption of irradiation in apple samples is equivalent to that in water. 2. 5. Sample Preparation The plant protection formulations based on CPS were applied to 20 samples of apples "Idared" bought in supermarkets, and to 20 samples of the same variety obtained from orchards. The supermarket samples were sprayed by 2 mL of EC formulation (dissolved in a water methanol mixture, 3:1, v/v) under 100 kPa air pressure from a Potter precision laboratory spray tower (Burkard Scientific, UK). Spraying was performed under controlled conditions of temperature (17 °C) and relative humidity (45%). The treated samples were collected in plastic bags after treatment and stored at -18 °C until further analysis. Some apples were further treated with 30% H2O2 solution to cause initial degradation of EC formulation to form CPO. For ASAP-MS analysis, the samples were brought to room temperature. Apples were peeled, and their skin was cut into small pieces (about 1x1 cm), and the ASAP-MS probe was dipped into the apple pieces. The skin was weighted before and after dipping the ASAP-MS probe. The sample was then inserted into the ionization source chamber. No particular sample pretreatment was needed for the ASAP-MS analysis, and MS data for each sample were collected in few seconds. For the chromatographic analysis, apples were homogenized (15 g in 30 mL acetone), vortexed and centri-fuged for 10 min at 3500 rpm. The procedure was repeated three times, and acetone extract was collected. The remaining apple homogenate was further mixed with 20 mL of petrol ether and 10 mL of dichloromethane. After cen-trifugation, the upper organic layer was collected and added to acetone extract by this modified Luke procedure.18 Combined extracts were evaporated on a water bath at 42-62 °C and reconstituted with methanol for UPLC-PDA analysis or dichloromethane for GC-MS analysis. For some apples, the skin was separated from the mesoderm, and each sample was treated separately. Three extraction techniques, other than the described Luke procedure, have been tested: liquid phase extraction followed by analytes separation in Oasis® HLB cartridge, extraction with multiwall carbon nano-tubes (MWCNT) and extraction with QuEChERS kit. For the liquid phase extraction, 5 g of apples have been homogenized and extracted with 20 mL of acetonitrile; 5 mL of extract were diluted to 100 mL with a 50:50 water/ methanol mixture, and eluted through the Oasis® HLB cartridge (previously conditioned with methanol). Eluted solutions were evaporated to dryness at 42-64 °C, and reconstituted with methanol for UPLC-PDA analysis or dichloromethane for GC-MS analysis. For the extraction with MWCNT, 6 mL PTFE tubes (Strata) were filled with 0.035 g MWCNT, sample was added, and pH was then set at 6-7.5 to enhance the analyte extraction. Analytes were eluted with a methanol solution, and then samples were evaporated to dryness and reconstituted as described above for the liquid phase extraction. As regards the QuEChERS kit extraction, 15 g of homogenized sample were loaded into an empty 50 mL tube, and 15 mL of a 1% CH3COOH in acetonitrile solution were added. The first QuEChERS mixture was then added. After centrifugation at 1500 rpm for 5 min, 3 mL of supernatant solution were transferred into another tube containing the second QuEChERS mixture. After cen-trifugation at 3000 rpm for 5 min, 100 ^L of the final extract were transferred into a vial and diluted with methanol. The extraction efficiency q (%) was calculated for each method. The combined Luke (Dutch) procedure gave an efficiency of 94% ± 8%, which was the highest among all obtained efficiencies. For the liquid phase extraction it was 84% ± 13%, for the MWCNT it was 88% ± 9%, and for the QuEChERS kit it was 90% ± 8%. Therefore, the combined Luke (Dutch) procedure was chosen for apple preparation for the UPLC-PDA analyses. Cvijovic et al.: Atmospheric Solids Analysis Probe with Mass ... 74 Acta Chim. Slov. 2019, 66, 70-74 77 3. Results and Discussion 3. 1. ASAP-MS The interpretation of ASAP-MS spectra of CPS and CPO was performed in accordance to m/z values and iso-topic pattern. The protonated CPS is at m/z 351.5±0.5 Da, and the protonated CPO is at m/z 335.5±0.5 Da. The nontoxic metabolite TCP can also be observed at m/z 198 with very low abundance. CPS and CPO are chlorinated compounds with three chlorine atoms which are excellently suitable for interpretation by isotope distribution. For example, the isotopic pattern for protonated CPS is 350:351:352:353:354:355:356, and calculated relative ion intensities are 97:11:100:11:36:4:5, respectively. In this pattern the even m/z values (350, 352...) stem mainly from the chlorine isotopes 35Cl (75.8% natural abundance) and 37Cl (24.2% natural abundance), and the uneven values (351, 353...) from the carbon isotopes 12C (98.9% natural abundance) and 13C (1.1% natural abundance). Theoretical ratio is in good agreement with ion ratio detected experimentally from standard and from apple samples (an example of experimental pattern is reported in Figure 1S in the Appendix A - Supporting In- formation).7,19 The ASAP-MS spectra of a blank apple and of an apple treated by 50 mg/L EC formulation and with H2O2 are shown in Figure 2. If the two spectra are compared, the appearance of two new intense signals (10-fold more intense than other m/z signals) due to CPS and CPO can be observed: CPS was added with the EC formulation, whereas CPO was produced by the H2O2 addition which simulates CPS degradation. a) ASAP-MS might be used also for the detection of these additional compounds. 3. 2. Results of Chromatographic Analyses Chromatographic techniques (GC-MS and UP-LC-PDA) were validated and applied for accurate measurements of CPO and CPS in apples, in order to acquire the "true" concentrations of these compounds. The identity of CPS and CPO in GC-MS has been confirmed from the electron impact (EI) spectra which gave confirmatory product ions, as shown in the Supporting Information (Figures 2S and 3S). The same ions are reported in Wiley database libraries. As well, the retention times of CPS and CPO were identical to those of the standard materials. The identification by UPLC-PDA techniques has been done by the retention time, which was identical to that of the standard material. The retention times were 4.23 min for CPS, 3.08 min for CPO, and 2.01 min for TCP. From the validation data reported in the Experimental section, UPLC-PDA demonstrated to have slightly better analytical performances (LOD, LOQ, recovery) than GC-MS. For this reason, the subsequent quantitative analyses have been performed using UPLC-PDA. Some quantitative results obtained on apples (blank or treated with pesticide formulations) are shown in Table 1S in the Supporting Information. These results show that CPS residues remained on apple skin, while apple mesoderm did not contain the pesticide. Untreated samples (domestic apple) were also CPS free. 309.4167 323.8167 405.5465 397.5076 IrJlJ^I i r ^ | J ^ ^r ^iL ^ r I ^ J ^ r Jf J ^ »r f i ^ r »f ^ r»1^ J ^ r ^ r^ J^r ^ * J|V r^ r ^ ^ f J ^J ^ fr J ^»'f rt J ^ ^ J ^»'f^r # # >J f Ji'J f ri-* f J f iVri-f > J f »V .....irfn'iWrfrtnytol^Vrfri'fi / 1 |L.......ji 453.5898 ' .454.6107 Scan AP+ 8.25e6 485.5586 488.5908 l|l|t'i'ftyiwiViW^ m/z 150 160 170 180 190 200 210 220 230 240 250 260 270 280 290 300 310 320 330 340 350 360 370 380 390 400 410 420 430 440 450 460 470 480 490 500 b) apple treated by EC formulation 335.3972 337 4055 192 1197 205.2307 ____ 253.2889 9ß, 281.3232 ^gna I 169.1897 \ i I 225.2265 \ 263.3122 . ¿¿l.JbüöO 339.3232 / 351.4334 Scan AP+ 6.02e7 A.........m 365.4381 366.4316 407.5929 397.5037 L408.5775 425.4743 Jfr............................................. ............................................... 400 410 420 430 440 450 460 470 150 160 170 180 190 200 210 220 230 240 250 260 270 280 290 300 310 320 330 340 350 360 370 Figure 2. ASAP-MS spectra of a control apple (a) and of an apple treated with 50 mg/L EC formulation and with H2O2 (b). According to literature data,20-22 some additional compounds can be likely identified in Figure 2: naringenin pentose (m/z 405), p-coumaric pentose (m/z 326), phlo-ridzin (m/z 435), hyperoside (quercetin 3-O-galactoside) or isoquercetin (m/z 463), ferulic acid (m/z 368), cyanidin pentosine (m/z 419), cyanidin hexoside (m/z 449), and quercetin pentoside (m/z 435). In principle, therefore, 3. 3. Comparison Between ASAP-MS and Chromatographic Measurement of CPS and CPO in Apples The results obtained in the analysis of two samples for both CPS and CPO by using two different techniques, UPLC-PDA and ASAP-MS, are reported in Table 1. The first column reports the concentrations of EC or EW formulations used for apples spraying. Although results obtained by ASAP-MS method were different than those obtained by UPLC-PDA meth- Cvijovic et al.: Atmospheric Solids Analysis Probe with Mass ... Acta Chim. Slov. 2019, 66, 70-75 77 od, the order of magnitude of the values given by the two techniques was the same, and relative differences were not very large (around 10-20%). Indeed, ASAP-MS results were obtained in few minutes, whereas UPLC-PDA values required a 10-100-fold longer time. This indicated that ASAP-MS technique can represent a rapid and semiquantitative method for the analysis of CPS and CPO. The quantitative results for the measure of the degradation efficiency were obtained with UPLC-PDA and are shown in Table 2. The last column reports the removal efficiency of CPS (if applicable also of CPO), as a function of the gamma irradiation doses reported in the previous column and/or of the H2O2 pretreatment. The pretreatment with H2O2 caused only a limited Table 1. Comparison between results from UPLC-PDA and from ASAP-MS in the analysis of two apple samples. sample technique treated with 50 - EC treated with 50 - EC treated with 50 - EW treated with 50 - EW UPLC-PDA ASAP-MS UPLC-PDA ASAP-MS CPS [mg/kg] CPO [mg/kg] 0.65±0.02 0.58±0.03 0.60±0.03 0.54±0.02 0.06±0.01 0.05±0.01 0.26±0.02 0.21±0.02 3. 4. Effect of Gamma Ray Treatment on the Removal of CPS and CPO from Treated Apples The GC-MS chromatogram (recorded as total ion chromatogram, TIC) obtained in EC formulations (5 mg/L) before gamma irradiation is presented in Figure 4Sa in the Supporting Information. The peak of CPS was observed at tR = 18.27 min. Other minor formulation ingredients can be seen at different tR values. When the analysis was repeated after gamma irradiation, the CPS peak almost disappeared (Figure 4Sb): CPS peak area is around 10-15% of the same peak in Figure 4Sa. Gamma irradiation caused the appearance of the CPO peak at tR = 18.03 min and of TCP at tR = 12.77 min. Both compounds were produced from the degradation of CPS. Figure 3 shows the ASAP-MS spectra of a blank apple gamma irradiated (the same sample of Figure 2a), and of an apple treated with 50 mg/L EC formulation, with H2O2 and then gamma irradiated (the same sample of Figure 2b). Both apples show an almost identical spectrum, indicating that the degradation was effective, and that the treated apple was almost identical to a blank (i.e. not polluted) apple. degradation of CPS (around 35-40%), and it caused the production of large amounts of CPO due to chemical oxidation of CPS. The exposure to gamma rays caused the removal of CPS and of CPO depending on the irradiation dose and on the irradiation time. 2.5 kGy for 66 h was not sufficient for pesticide removal, as the removal percentage was below 50%. A good efficiency was achieved for a 3.5 kGy dose and for a 66 h treatment, as the removal percentage of CPS reached 90%. The upper dose of 3.8 kGy for 72 h gave even better results, but the increase with respect to 3.5 kGy for 66 h was not very large. Little amounts of CPO appear as a degradation product of CPS after gamma irradiation. However, if CPO is present on apples in large amounts, it is degraded as well by gamma irradiation, as demonstrated in samples pretreated with H2O2. The parameters of gamma irradiation and their effects are in agreement with literature data for interaction of gamma irradiation with water medium.23-25 The removal efficiency of gamma irradiation can be easily checked also by ASAP-MS, by measuring the absolute intensities of the relevant pesticide peaks before and after irradiation. For example, with reference to Figures 2 and 3, in apples treated with 50 - EC the average intensi- a) control apple after gamma irradiation ,211.2493 ii|ii ii|i iii|iiii|iii i| iiii|iiii|ini|iiii|iiin 283-3300 y ■ I... I.I.I-1 .I- 365.4565 iii|iiii|iiii|iiii|iiii Scan AP+ 1.48e6 407.5035 423.5336 , ---— 4854582 "|iiii|iiii|mi'iiiii|iiii|iiii|iiii|iiii|ii'ii|iiii|iiiilMii|'ilii|iiii|iiii| m/z 150 160 170 180 190 200 210 220 230 240 250 260 270 280 290 300 310 320 330 340 350 370 380 390 400 410 420 430 440 450 460 470 480 490 500 b) apple treated by EC formulation, after gamma irradiation ,211.2474 239.3224 Ji|jlii|llllfllJ|lHli|il)J|lljlfiL)|^lrjJ)l|) 95%) for the uranium ions studied was obtained at a pH of 6-7. Therefore, a pH of 6.5 was chosen as an optimum pH for subsequent experiments. Fig. 4. Effect of pH on recovery % U(VI) with Fe3O4/SiO2/APTES/ Mu. 3. 3. Effect of Eluent Concentration and Volume In this study the elution of uranium was studied to find the optimum amount of HClO4 in the range of 2-5 M and volume of 0.5 to 2 mL. 1 mL of 5 M HClO4 was found to be satisfactory for elution of uranium (recovery > 95%). Therefore, 1 mL of 5 M HClO4 as eluent was chosen for the following experiments. 3. 4. Effect of Matrix Components The effects of matrix ions, which are found at high concentrations in real samples, on the recovery of metal ions were studied. Various concentrations of Fe3+, Cd2+, Pb2+, Co2+, Ni2+, Cu2+, Cr3+, Al3+, and Zn2+, as their chloride, nitrate and sulfate salts, were added individually to a model solution of 50 mL containing 0.05 mg L-1 U(VI). The described method was applied under optimum conditions. The results are given in Table 1. The most significant interferences were found with 1 mg L-1 of Cr3+ and Ni2+ when determining the presence of uranium. These interferences were prevented by using 0.02 M EDTA. Besides, EDTA can be used as a masking agent for many elements such as Th, Zr, because EDTA forms stable complex with these elements, and unstable complex with U(VI).34 Table 1. Tolerance limits for interference ions on the determination of U(VI) (n = 3 0,05 mg L-1 U, 1 mg L-1 of metal ions) Ion Interference ion to metal ion/ratio Recovery %, U(VI) Zn2+ 20 101.4 ± 4.2 Cd2+ 20 97.S ± 1.8 Pb2+ 20 99.2 ± 0.2 Fe3+ 20 100.2 ± 4.3 Al3+ 20 96.0 ± 2.3 Cr3+ 20 81.1 ± 0.7 Cr3+ + 0.02 M EDTA 20 96.2 ± 0,4 Ni2+ 20 86.8 ± 2.4 Ni+2 + 0.02 M EDTA 20 97.8 ± 0,S Cu+2 20 9S.4 ± 1.2 3. 5. Effect of Adsorption and Elution Time The rate of U(VI) adsorption by Fe3O4/SiO2/APTES/ Mu was studied (50 mL, 0.05 mg L-1) with 25 mg of the sorbent over a series of varying shaking times (5-30 min). The results showed that the extraction percentage of U(VI) at 15 min was higher than 98%. The rate of elution of U(VI) by Fe3O4/SiO2/APTES /Mu was studied (50 mL, 0.05 mgL-1) with 25 mg of the sorbent and an adsorption of15 min over a series of varying shaking times (1-5 min). Therefore, 15 min and 2 min, respectively, were used in all subsequent experiments for quantitative sorption and elution of U(VI). 3. 6. Sorption Capacity The maximum sorption capacity of Fe3O4/SiO2/ APTES/Mu was obtained from the batch methods. A total of 25 mg of Fe3O4/SiO2/APTES/Mu was added to a 40-mL Oymak: Solid-Phase Extraction Method by Magnetic Nanoparticles ... 82 Acta Chim. Slov. 2019, 66, 78-84 solution containing different amounts of U(VI) ions (0.88 mg) at pH 6.5. After shaking for 1 h, the mixture was separated with the use of a magnet. The supernatant solutions were then measured by UV-Vis spectrophotometry after dilution. Many isotherm models have been proposed to explain the adsorption equilibrium, such as the Lang-muir and Freundlich isotherms, which are the most commonly used for the clarification of adsorption of molecules from the liquid phase. The Langmuir equation is given as follows: where Qmax (mg g-1) is the maximum adsorption capacity; Qe is the amount of solute adsorbed per unit weight of adsorbent (mg g-1) at equilibrium; Ce is the equilibrium solute concentration (mg L-1) in solution and K is the Langmuir constant (L mg-1). The Freundlich isotherm equation is given be-low:35,36 where Qe is the amount of adsorbed U(VI) per mass of adsorbent, Kf is the Freundlich constant, Ce is the equilibrium U(VI) concentration and 1/n is a constant related to the adsorption intensity.37 As shown in Table 2, the adsorption mechanism was well-suited to the Langmuir model, with a correlation coefficient of 0.9997. The Qmax value was found to be 77.51 mg g-1. The n value was 3.87, calculated from the Freundlich isotherm, which is higher than 1. The n value indicated the favourable adsorption of U(VI) on Fe3O4/SiO2/ APTES/Mu. The Langmuir and Freundlich isotherm parameters are shown in Table 2. 3. 7. Analytical Performance and Applications to Real Sea Water Sample The limit of detection (LOD) study was performed by applying the described method to ten blank solutions of 40 mL. The limit of detection calculated as the ratio of the three standard deviations of the blank to the slope of plot was 0.001 mg L-1 with a preconcentration factor of 40. The relative standard deviation was calculated as 3.0% at 0.05 mg L-1 of U(VI) (n = 7) and the linear range in final eluate was 0.02-4.0 mg L-1 of uranium(VI). The method was successfully applied to sea water. The accuracy of the developed method for sea water was tested by adding the known amounts of U(VI). After applying the separation/ preconcentration procedure, quantitative recovery (>95%) was found for U(VI). The results of the analysis of sea water samples are shown in Table 3. Table 3. The results for determination of U(VI) in sea water Sample Added Found Recovery, (^g L-1) (^g L-1) % Sea water from 0 2.7±0.1 the Aegean Sea 20 22.1±0.4 97.0±2.0 40 44.2±0.5 104.0±1.2 Sea water from 0
1396 -I000 Î000 2000 1000 500 Wavenumber (cm1 ) Figure 1. FT-IR spectra of (a) MIL-101 (Cr), (b) P2W18 and (c) P2W18@MIL101(Cr) samples. cated around 1635, 1398, and 760 cm-1 were all observed in the FT-IR spectrum of P2W18@MIL-101(Cr) nanocom-posite, which demonstrates the presence of P2W18 and MIL-101 in the P2W18@MIL-101 composite. However, there are slightly shifted peaks compared with the pure P2W18. These shifts imply that a strong interaction exists between the Dawson-type anion and MIL-101(Cr) MOF.37 XRD patterns of the MIL-101(Cr) and P2W18@MIL-10 (Cr) samples are presented in Figure 2. The XRD pattern of the pristine MIL-101(Cr) is presented in Figure 2(a). The main diffraction peaks corresponding to reflection planes are indexed, which are consistent with the peak positions reported for standard MIL-101(Cr) structure, indicating that the synthesized material has the MIL-101(Cr) phase with good crystallinity.41 The peak positions of P2W18@MIL-10(Cr) in Figure 2(b) are matched well with the parent MIL-101(Cr). As can be seen, some diffraction peaks related to P2W18 are observed albeit with very low intensities, confirming the homogeneous distribution of the P2W18 molecules within the porous structure of MIL-101(Cr). Moreover, the XRD and FT-IR data demonstrate that the structure of the MOF does not de-struct during the incorporation of P2W18 molecules. The existence of the Dawson-type polyoxotungstate (P2W18) in the MIL-101(Cr) framework was further recognized by FT-Raman spectrum of the nanohybrid material in comparison with those of the isolated compounds as shown in Figure 3(a)-(c). It can be seen that the FT-Ra-man spectrum of the nanohybrid material reveals the typical bands of the porous material MIL-101 and some vibra- • MIL-101 1 ♦ * PiWis 2 10 20 30 40 45 20 (degrees) Figure 2. XRD patterns of (a) MIL-101(Cr) and (b) P2W18@MIL-101(Cr) samples. Jarrah and Farhadi: Dawson-Type Polyoxometalate Incorporated into Nanoporous ... Acta Chim. Slov. 2019, 66, 85-102 89 ♦ ♦ Zj A ii. A. t 1700 1500 1300 1100 900 700 500 Rvunan sluft (cm') Figure 3. FT-Raman spectra of (a) MIL-101 (Cr), (b) P2W18, and (c) P2W18@MIL101 (Cr) samples. tional modes of the P2W18. In particular, the strong band located at about 965 cm-1 assigned to W=O symmetrical stretching mode is clearly evident in the spectrum of the composite material, P2W18@MIL-101(Cr).42 The characteristic bands verify the existence of the both P2W18 and MIL-101 in the P2W18@MIL-101(Cr) nanocomposite. The morphology and microstructure of samples were clarified by FE-SEM observations. The SEM images of the samples are indicated in Figure 4. Figure 4(a)-(c) exhibits FE-SEM images of the pristine MIL-101(Cr) particles. It shows that the MIL-101(Cr) particles are irregular polyhedral in shape with high porosity. The sizes of these polyhedral crystals are in the range of 0.5-2 ^m. The SEM images of the P2W18@MIL-101(Cr) composite in Figure 4(d)-(f) clearly show that its shape and morphology are similar with those of the MIL-101(Cr). Confirming that the crystalline structure of MIL-101(Cr) remains unchanged after incorporating P2W18 anions. However, the porosity of MIL-101(Cr) decreased with the incorporation of P2W18 anions. Figure 5 show the EDX spectrum and a representative SEM image of the hybrid nanomaterial with corresponding EDX elemental mappings. The presence of C, O, P, K, Cr, and W elements in the composite can be proved by the EDX spectrum in Figure 5(a). As presented in Figure 5(b)-(h), the corresponding elemental mapping distribution shows the existence of C, O, P, K, Cr, and W. From the maps, it can be seen that the elements are uniformly distributed over the hybrid nanomaterial, confirming the Figure 4. SEM images of (a-c) MIL-101 (Cr) and (d-f) P2W18@MIL-10 (Cr) at different magnifications. Jarrah and Farhadi: Dawson-Type Polyoxometalate Incorporated into Nanoporous 90 Acta Chim. Slov. 2019, 66, 85-102 Kev (d) "V"*^ " - s • " -.«» ^ o-lui •• 1 • * - y*. '.^t* • • v- •.;■:' ■ ' . • - A L ' - _ _ O clément •" ' Mf/ • (g) i .■'■'. s» ••'.■■;•. " Cr element Figure 5. (a) EDX spectrum, and (b)-(h) a representative SEM image of the nano-hybrid with corresponding elemental mappings. homogeneity of the sample. The K, P and W elements were from P2W18, thus confirming the uniform incorporation of P2W18 anions into the porous MIL-101(Cr) framework. The BET surface area and pore volume of the samples were determined by N2 adsorption-desorption isotherms at 77 K and the results are indicated in Figure 6. On the basis of the IUPAC classification, the N2 adsorption-desorption isotherms of the samples in Figure 6(a) reveal mixed type I/IV isotherms with H4-type hysteresis loop, which is characteristic of solids with mesoporous and mi-croporous cages.36 The textural properties of these materials were summarized in Table 1. Compared with the pris- tine MIL-101, the encapsulated sample demonstrated a significant decrease in surface area, pore volume, and pore diameter owing to the insertion of P2W18 polyanion into the cages of the MOF.43 The pores of P2W18@MIL-101(Cr) were further occupied by the P2W18 polyanion which resulted in a further decrease in pore volume and surface area. The Barrett-Joyner-Halenda (BJH) pore size distributions in Fig. 6(b) also confirmed the mesoporous cages of MIL-101 and reflected the volume changes during the encapsulation process. These findings confirm that the P2W18 polyanion had been encapsulated within the channels of MIL-101(Cr) rather than outside the surfaces. Table 1. The textural properties of MIL-101(Cr) and P2W18@MIL-101(Cr) samples. Sample BET surface Langmuir surface Total pore V • * micro V * meso Average pore area (m2/g) area (m2/g) volume (cm3 /g) diameter (nm) MIL-101 Cr 2692.403 3455 2.481 0.045 2.436 0.768 P2W18@MIL-101(Cr) 1167.413 1671 1.108 0.029 1.079 0.504 Vmicro: Micropore volume calculated using t-plot method. Vmeso: Mesopore volume calculated using BJH method. Jarrah and Farhadi: Dawson-Type Polyoxometalate Incorporated into Nanoporous ... Acta Chim. Slov. 2019, 66, 85-102 91 Figure 6. (a) Nitrogen adsorption-desorption isotherms curve at 77 K and (b) pore size distributions obtained by the BJH method for nanohybrid. Zeta potential of materials is another key factor to influence their adsorption capacity, and thus was tested to understand further why P2W18/MIL-101(Cr) sample can remove cationic dyes MB and RhB more effectively than for anionic MO dye. As shown in Figure 7, zeta potential of MIL-101 (Cr) and P2W18/MIL-101(Cr) samples are about -2.7 and -12.8 mV, respectively. This illustrates that the higher efficiency removal of cationic dye is attributed to the electrostatic attraction interactions between the adsorbents and cationic dye. After modification with P2W18, the obtained P2W18/MIL-101 (Cr) showed higher negative zeta potential than that of pristine MIL-101 (Cr), thus capable of adsorbing more cationic dye MB, whereas it is unfavorable for P2W18/MIL-101 (Cr) to adsorb anionic dye MO. The obtained results on the selective adsorption of cationic dyes as well as studies on the Zeta potential of adsorbents clearly suggest that the adsorption of MB dye on the P2W18/MIL-101 (Cr) nanohybrid has been associated with electrostatic interaction between cationic dye molecules and the highly negative charged P2W18/MIL-101 (Cr) nanohybrid. 3. 2. Adsorption Capability of Nanohybrid Toward Organic Dyes In this work, the removal of dyes from contaminated aqueous solutions by using P2W18@MIL-101(Cr) nanohy- Figure 7. Zeta potential distribution curves of (a) pristine MIL-101 and (b) P5W30/MIL-101 (Cr) hybrid in aqueous solutions. brid as a novel adsorbent was investigated. Three organic dyes were used herein: MB and RhB as cationic dyes and MO as an anionic dye. From contaminated water, three organic pollutants (MB, RhB, and MO) with different sizes and charges were selected for experiments. The adsorption was monitored using the characteristic absorption peak, which is 664, 553 and 463 nm for MB, RhB, and MO, respectively. The process of adsorption was specified with the fading of the blue, red and orange colors of MB, RhB, and MO, respectively. The time dependent UV-Vis absorption spectra of dyes in the presence of P2W18@MIL-101(Cr) are shown in Figure 8(a)-(c). As shown in Figure 8(a) and (b), the digital images and UV-Vis spectroscopic results show that the characteristic absorption peaks of cationic MB and RhB dyes at 664 and 553 nm almost completely disappeared within 0.5 and 2 min, respectively. The result in Figure 8(c) confirms that the nanohybrid is a poor absorbent for anionic MO dye from aqueous solution even after 30 min. To further demonstrate the role of anionic P2W18 POM in the composite material, a set of control experiments was designed using pristine MIL-101(Cr) MOF and pure P2W18 POM samples as absorbents in removing the different types of organic dyes involving cationic dye MB, Jarrah and Farhadi: Dawson-Type Polyoxometalate Incorporated into Nanoporous 92 Acta Chim. Slov. 2019, 66, 85-102 X! 1.0 0.5 0.0-t (C) MO Original / \ 30 Seconds / \ 1 min / \ 6 min / \ 16 min / \ 31 min / \ 350 400 450 500 Wavelength (nm) 550 600 Figure 8. Time dependent UV-Vis spectra during adsorption of dyes over P2W18@MIL-101: (a) MB, (b) RhB, (c) MO. [dye]0 = 25 mgL-1 ; 30 mL, adsorbent amount = 30 mg, temp.= 25 °C. RhB and anionic dye MO. Figure 9 shows the adsorption abilities of Pristine MIL-101(Cr) MOF and pure P2W18 POM samples toward MB, RhB, and MO dyes under our reaction conditions. By using Pristine MIL-101(Cr) adsorbent, it is clear from Figure 9(a) and (b) the decrease in intensities of characteristic UV-Vis absorption bands of cationic MB and RhB dyes is almost negligible within 60 min, indicating that it has no considerable ability to adsorb cationic dyes even after long contact times whereas it adsorbs completely anionic MO dye within a very short time of 7 min. Figure 9(d)-(f) shows that the pure P2W18 sample has different adsorption ability towards the dyes. It can be seen that the intensity of the absorption bands of MB and RhB decreases with increasing contact time. The adsorption efficiencies of the pure P2W18 sample toward these two dyes are moderate, albeit after long adsorption times of 40-60, (Figure 9(d) and (e)). On the other hand, as can be seen in Figure 9(f), the noticeable decrease in intensity of characteristic absorption band of MO dye was not observed after a long time of 60 min. In Figure 9(g) the adsorption abilities of pure P2W18 and pristine MIL-101(Cr) and P2W18@MIL-101(Cr) samples toward MB, RhB, and MO dyes were compared. The removal percentages of three dyes in the presence of pure P2W18 sample as an adsorbent were within 42-66% in 60 min. The removal rates of pristine MIL-101(Cr) sample for MB and RhB cat-ionic dyes are 28 and 22% after long adsorption time of 60 min and the removal of MO anionic dye is almost complete (100%) in 7 min. It is clear that with respect to recov-erability and removal percentages and times, the P2W18@ MIL-101(Cr) nanocomposite is more suitable and superior for cationic dyes removal. Thus, pristine MIL-101(Cr) represented superior adsorption capacity for anionic dye MO, whereas the P2W18@MIL-101(Cr) nanocomposite exhibited high adsorption capacity for cationic dye MB and RhB. The reason for this phenomenon is that the framework of the pristine MIL-101 is cationic. Amazingly, after the introduction of P2W18 polyoxoanions into the cavity of MIL-101, the adsorption capacity of P2W18@ MIL-101 has changed a lot comparing with that of the isolated MIL-101 (Figures 8 and 9). This is because that P2W18 polyanions encapsulated in MIL-101 MOF can quickly and effectively adsorb MB and RhB cationic molecules, whereas strongly reject MO anions. 3. 2. 1. Effect of pH One of the important parameters affecting the adsorption of dyes is the pH of solution. The effect of pH on dye elimination by P2W18@MIL-101 (Cr) is shown in Figure 10(a). The results showed that the pH dependency specifications represent adsorption independent of the entire pH range because nanohybrids have a high negative charge, which allows them to interact with the positive charges of the dyes in all ranges of pHs. 3. 2. 2. Effect of Initial Dye Concentration As shown in Figure 10(b), with an increment in the initial dye concentration, the dye removal decreases. With the increment of the initial MB concentration in the solution more dye was adsorbed onto P2W18@MIL-101 (Cr), in the event that the dosage of adsorbent remained unchanged. Jarrah and Farhadi: Dawson-Type Polyoxometalate Incorporated into Nanoporous ... Acta Chim. Slov. 2019, 66, 85-102 93 Figure 9. The adsorption capabilities of (a)-(c) pristine MIL-101(Cr) and (d)-(f) pure P2W18 toward MB, RhB, and MO dyes. (g) Adsorption efficiency (%) of the dyes in the presence of different adsorbent samples. Jarrah and Farhadi: Dawson-Type Polyoxometalate Incorporated into Nanoporous 94 Acta Chim. Slov. 2019, 66, 85-102 The reason for this is that with the increased initial dye concentration, an increment occurs in the driving force of the concentration gradient. The adsorption of dye by P2W18@ MIL-101 (Cr) in a low initial concentration is very intense and a balance is reached quickly. In fact, for the initial concentration of MB in 25 mg L-1, the nanohybrid has a removal efficiency of 100%, in 30 seconds. This demonstrates that P2W18 was probably distributed homogeneously and uniformly within the porosities of the MIL-101 (Cr). At a constant adsorbent dosage, with an increasing concentration of the solution, the amount of adsorbed dye increased while the adsorption efficiency of the nanohybrid decreased. 3. 2. 3. Effect of Temperature The effect of temperature is another significant phys-icochemical process parameter because the temperature can change the adsorption capacity of the adsorbent.44 The adsorption investigations were carried out at different temperatures (25, 35, 45, 55, 65, 75, 85 and 95 °C) (Figure 10(c)). Decreasing the adsorption time with the increase of temperature shows that the adsorption is a spontaneous and endothermic process. With an increase in the temperature, the mobility of the dyes molecules increase and more molecules will be able to penetrate the inner structure of the nanohybrid.45 3. 2. 4. Effect of Adsorbent Dosage For the purpose of wastewater purification, determining a suitable adsorbent dosage is very important.46 The influence of POM@MOF dosage on dye elimination was investigated with fixed conditions such as dye solution volume of 30 mL, dye concentration of 100 mgL-1, pH = 6, and temperature of 25 °C for MB. The adsorbent dosage was applied in different amounts within a range of 10 to 40 mg. Following this, the dye solution was centrifuged and samples were investigated by a spectrophotometer, as Figure 10(d) shows. With increasing adsorbent dosage, the removal of MB dye increases which could be related to the more adsorption sites. In the result, the suitable adsorbent dosage was found to be 30 mg. To show the advantages of the present adsorbent, we have compared the obtained results in the removal of MB Figure 10. Effects of (a) pH, (b) dye concentration, (c) different temperatures and (d) adsorbent dosage on dyes removel by the P2W18@MIL-101(Cr) sample. Conditions: Co (MB) = 100 mg/L in 30 mL dye solution at 25 °C. Jarrah and Farhadi: Dawson-Type Polyoxometalate Incorporated into Nanoporous ... Acta Chim. Slov. 2019, 66, 85-102 95 Table 2. Comparison of the results of the newly synthesized nanohybrid with some reported adsorbents for the removal of dye pollutants. Adsorbent Dye pollutant Concentration Removal efficiency Time Ref of dye (mg/L) (%) (min) H6P2W18O62@Cu3(BTC)2 MB 20 80 60 [12] PV2Mo10/M (membrane) MB 20 100 48h [47] H3PW12O40/ ZIF-8 MB 60 100 30 [48] LaMnO3@SiO2/PW12 MB 25 98 30 [49] PW11V@ MIL-101(Cr) MB 10 98 60 [26] PW11V@ MIL-101(Cr) RhB 10 60 60 [26] MoS2@ MIL-101 (Cr) RhB 20 90 10 [52] Fe3O4/reduced graphene oxide RhB 5 91 120 [50] PW12@MIL-101(Fe) MB 100 100 30 [37] Fe3O4/MIL-101(Cr) MO 25 25 150 [51] P2W18@MIL-101 (Cr) MB 25 100 0.5 This work P2W18@MIL-101 (Cr) RhB 25 100 2 This work P2W18@MIL-101 (Cr) MO 25 31 73 This work Figure 11. The selective adsorption ability of P2W18@MIL-101 (Cr) toward the mixed dyes of (a) MB/MO, (b) MB/RhB, (c) RhB/MO and (d) MB/ MO/RhB. Conditions: C0 (MB) = C0 (RhB) = C0 (MO) = 25 mg/L; adsorbent dose = 25 mg in 30 mL mixed dye solution at 25 °C. Jarrah and Farhadi: Dawson-Type Polyoxometalate Incorporated into Nanoporous 96 Acta Chim. Slov. 2019, 66, 85-102 from aqueous solution by the P2W1g@MIL-101(Cr) nano-hybrid with some reported similar adsorbents in the literature.12,20,37,47-52 From Table 2, it is clear that with respect to the adsorption conditions (adsorption time, initial dye concentration and adsorption efficiency), the present method is more suitable and/or superior. We can see that the adsorption process in the presence of most reported adsorbents required longer reaction time compared to the P2W1g@MIL-101(Cr) nanohybrid. The P2W1g polyanions with a large number of negative charges incorporated in the hybrid have a stronger attraction force with the positive charges of cationic dyes (e.g. MB). In fact, higher adsorption efficiency of the P2W1g@MIL-101(Cr) is due to synergistic effect between MIL-101(Cr) and P2W1g poly- 3. 2. 5. Selective Adsorption Ability of the Nanohybrid for the Mixed Organic Dyes The selective adsorption of dyes from their mixture is more attractive and challenging. To verify the selective adsorption property of the P2W1g@MIL-101(Cr) nanohybrid adsorbent, the dye molecules with different sizes and charges were selected for the experiments, considering that the charge and size of organic dyes are the two major factors controlling the adsorption process. In this work, cationic MB (molecule size 1.3g nm x 0.64 nm x 0.21 nm), cationic RhB (molecule size 1.56 nm x 1.35 nm x 0.42 nm) and anionic MO (molecule size 1.54 nm x 0.4g nm x 0.2g nm) were selected, considering that they possess different sizes and different charges. As shown in Figure 10(a)-(c), the selective uptake of dyes was tested using the MB/MO mixture (30 mL, C0MB = C0MO = 25 mg/L), MB/RhB mixture (30 mL, C0MB = C0RhB = 25 mg/L) and MO/RhB mixture (30 mL, C0MO = C0RhB = 25 mg/L) with 25 mg of the P2W1g@ MIL-101(Cr) absorbent, and then the process was monitored by UV-vis spectroscopy. As MB and MO are similar in molecule size, the preferable uptake of MB from MB/ MO mixture may be assigned to the anionic nature of P2W1g@MIL-101(Cr), as shown in Figure 11(a). For comparison, cationic RhB was selected to mix with cationic MB, and the results revealed that MB was also preferably adsorbed on P2W1g@MIL-101(Cr) from the MB/RhB mixture as illustrated in Figure 11(b), which may imply that the uptake of dyes is heavily influenced by molecule size along with charges. Also, for the binary MO/RhB mixture, cati-onic RhB dye was preferably adsorbed (Figure 11(c)). It can be anticipated that the composite material may also have an outstanding adsorption and separation behavior in treatment of a ternary mixture of MB, RhB and MO. As exhibited in Figure 11(d), the representative peaks of MB and RhB all disappeared quickly in mixed dye, only the characteristic absorption peaks of MO left, suggesting that the P2W1g@ MIL-101(Cr) could selectively capture cationic dyes when is utilized in the corresponding ternary mixture. 3. 2. 6. Adsorption Kinetics According to the adsorption kinetics, several models for a mechanism of solution adsorption onto an adsorbent can be expressed. To investigate the controlling mechanisms and the rate controlling steps in the overall adsorption process, such as diffusion control, chemical reaction, and mass transfer, different kinetic models (intraparticle diffusion, pseudo-first-order, and pseudo-second-order) are utilized. To study the empirical data and investigate the probability of intraparticle diffusion that can influence the adsorption process, the equation below is used:53 (3) The values I and kp demonstrate the intraparticle dissemination rate constant and intercept, respectively. The parameters I, kp and R2 were computed for several dye concentrations and listed in Table 4. The parameter I indicates the thickness of the frontier layer. The results show that the surface adsorption can be performed by the dissemination of the particles. Generally, a pseudo-first-order equation is illustrated as follows:54 (4) The parameters qe and k indicate the value of dye adsorbed at equilibrium (mg/g) and the equilibrium rate constant of the pseudo-first-order kinetic (1/min), respectively. The pseudo-second-order kinetic equation is expressed as follows:55,56 (5) q, k2qe qe Table 3. Kinetic models parameters for MB dye adsorption at 150, 175 and 200 mg/L dye concentrations. Co (mg/L) qeexp (mg/g) Pseudo-first-order kinetic Pseudo-second-order kinetic Intraparticle diffusion model k, (min-1 qecal ') (mg/g) R,2 k2 (g/mg min) qecal (mg/g) R22 kp I R32 150 146.1 0.07 7.4 0.9892 0.003 151. 5 0.9992 12.1 66.7 0.6937 175 169.47 0.07 12.2 0.9891 0.003 175.4 0.9993 13.8 59.8 0.6819 200 192 0.04 18.1 0.9849 0.002 200 0.9989 15.8 50.9 0.6937 Jarrah and Farhadi: Dawson-Type Polyoxometalate Incorporated into Nanoporous ... Acta Chim. Slov. 2019, 66, 85-102 97 Figure 12. Kinetic adsorption data plots for MB: (a) plot of removal rate qt vs. t and (b) pseudo-second- order adsorption kinetics on nanomaterial. To adsorb dyes onto P2W18@MIL-101(Cr) at the different dye concentration amounts (150, 175, and 200 mg/L) linear plots of t/qt versus t are indicated in Figure 12. The parameters of K1, K2, R12 (correlation coefficient for pseudo-first-order adsorption kinetics), R22 (correlation coefficient for pseudo-second-order adsorption kinetics) KP I, and R32 (correlation coefficient for intra-par-ticle diffusion model) were calculated and have been presented in Table 3. As it can be seen in Table 3, the values of the qe(exp) (mg/g) are lower than the calculated qe(cal) (mg/g) according to pseudo-second-order equation, which further supports that the pseudo-second-order equation can be utilized to describe the adsorption of dye onto the P2W18@MIL-101 (Cr). 3. 2. 7 Adsorption Isotherm The design of an adsorption system for an adsorbent provides the most suitable correlation for the equilibrium curves. Different adsorption isotherms were investigated such as the Freundlich, Langmuir, and Temkin isotherms. The basic supposition in the Langmuir theory is that the adsorption in the adsorbent sample occurs at particular homogeneous sites. This equation can be expressed linearly as bellow:57 le 1 c. (6) KLQm Qm The parameters Ce, KL> and qm are the equilibrium concentration of the dye solution (mg/L), the Langmuir constant (L/mg), and the maximum adsorption capacity (mg/g), respectively. By assuming a non-homogenous surface with a non-uniform diffusion of heat of adsorption over the surface the Freundlich equation is derivative. The Freundlich equation can be represented linearly as follows:58 -llogC. \n) log qe =log Kf + (7) Where KF and 1/n indicate the adsorption capacity at unit concentration and the adsorption severity as Freundlich constants, respectively. The isotherm type is determined according to the values of 1/n, that is, unfavorable (1/n > 1), favorable (0 < 1/n < 1), and irreversible (1/n = 0).59 Some indirect adsorbate/adsorbate interactions studied by Temkin in relation to adsorption isotherms were examined, and it was suggested that for all the molecules, the heat of adsorption increases linearly with coverage. The Temkin equation can be expressed linearly as follows:60 — (8) The Temkin isotherm expresses that pending some maximum binding energy, the adsorption is characterized by a uniform distribution of binding energies. The isotherm constants B1 and KT were determined using the values qe and Ln Ce, which represent the slope and the intercept, respectively. The constant B1 represents the heat of adsorption and the constant KT indicates the maximum binding energy. The correlation coefficients for the Lang-muir, Freundlich and Temkin isotherms were obtained from Figures 13(a)-(b) are listed in Table 4. Based on the results obtained, the Langmuir isotherm is most suitable for studying MB dye. 3. 2. 8. Adsorption Thermodynamics In the adsorption process, energy and entropy are very significant. The value of MB adsorbed at equilibrium Jarrah and Farhadi: Dawson-Type Polyoxometalate Incorporated into Nanoporous 98 Acta Chim. Slov. 2019, 66, 85-102 Table 4. Isotherm constants for MB dye adsorption at 150, 175 and 200 mg/L dye concentrations Dye Langmuir isotherm model Freundlich isotherm model Temkin isotherm model MB R2 Kl (L mg-1) Qm (mg g-1) 1 0.3 270 R2 Kf (mg g-1) n (mgL-1) 0.9944 7 2.6 R2 Kt BJ 0.9987 2.6 63.83 Figure 13. (a) Isotherm plot of dye elimination, (b) Langmuir isotherm, (c) Freundlich isotherm, (d) Temkin isotherm of dye removal by P2W18@ MIL-101 (Cr). for determining the thermodynamic parameters at the different temperatures was examined. The enthalpy (AH°: kJ/ mol), entropy (AS°: kJ/mol K), and change in Gibbs energy (AG°: kJ/mol) were obtained using the following equations: 61 ln Kc = V R V r aH0^ yRT; AG0 = AH - TAS (9) (10) The parameters Kc (L/g), T, and R (8.314 J/mol K) are the standard thermodynamic equilibrium constant, the equilibrium concentration of the dye in the solution (Ce), the value of dye adsorbed on the adsorbent of the solution at equilibrium (qe), the absolute temperature, and the gas constant, respectively. The values AH° and AS° can be determined by drawing curves of ln Kc versus 1/T from the slopes and intercepts, respectively (Figure 14). Table 5 indicates the negative values of AG° and positive AH°. The data demonstrate that the MB adsorption process is an en-dothermic and spontaneous process. The increase randomly at the solid/solution interface based on the positive amount of AS° occurs in the internal structure of the adsorption of MB dye onto P2W18@MIL-101 (Cr). Also, the positive amount of AH° indicates the presence of an ener- Jarrah and Farhadi: Dawson-Type Polyoxometalate Incorporated into Nanoporous ... Acta Chim. Slov. 2019, 66, 85-102 99 gy impediment in the adsorption process and the endo-thermic process.62,63 According to the negative amounts of AG°, the adsorption process is a spontaneous process. Moreover, the increase in temperature which leads to a decrease in the amounts of AG° indicates that the adsorption process is endothermic. Given the values obtained from AG°, of between 20 and 0 kJ/mol, the dominating mechanism in this study is the physisorption mechanism (Table 5).64 Table 5. Thermodynamic parameters of dye adsorption on P2W18@ MIL-101 (Cr). Temperature °C Thermodynamic parameters MB AG° AH° AS° (kJ/mol) (kJ/mol) (J/mol K) 25 35 45 55 65 75 -9.63 -10.44 -11.78 -13.1 -14.43 -15.76 30.52 133.02 ».0028 0.0029 0.003 0.0051 0.0032 0.0033 0.0034 I T (1/K) Figure 14. Plots ln Kq versus 1/T for adsorption of dyes. 3. 2. 9. Stability and Recyclability of Hybrid Nanomaterial The stability and reusability of P2W18@MIL-101 (Cr) are important aspects of the practical removal of dye pol- 4000 3500 3000 2500 2000 150D 1000 500 YVaveiuimber (cm ) « t Vi ■—< — ( 300 °C with dec.; 1H NMR: 5 2.33 (s, 3H, CH3), 6.31 (s, 1H, Py), 10.84 (s, 1H, OH), 12.18 (s, 1H, NH); 13C NMR: 5 19.85, 104.73, 107.84, 144.59, 147.91, 162.91, 169.55; ESI-MS: m/z 183 [M+H]+; anal. calcd. for C7H7N2O2S: C 46.15, H 3.32, N 15.37; found: C 46.24, H 3.38, N 15.44. General Procedure for the Synthesis of 6-Alkyl-5-hydroxy-7-methyl-3H-thiazolo[4,5-b]pyridin-2-ones 2b-c Metallic sodium (200 mmol) was dissolved in anhydrous methanol (100 mL), and to the solution obtained 4-iminothiazolidin-2-one (50 mmol) and alkyl derivatives acetoacetic ester (50 mmol) were added at 20 °C. The mixture was left standing for 7 days with the intermittent stirring, then it was acidified with acetic acid to pH ~5, five-fold diluted with water. The precipitate was filtered off, washed with water, and dried at 100-110 °C. The obtained compounds were re-crystallized from acetic acid. 5-Hydroxy-7-methyl-6-propyl-3H-thiazolo[4,5-b] pyridin-2-one (2b) White solid; yield: 60%; mp 249-250 °C; 1H NMR: 5 0.90 (t, J = 5.1 Hz, J = 7.2 Hz, 3H, CH2-CH2-CH3), 1.43 (d, J = 7.3 Hz, 2H, CH2-CH2-CH3), 2.19 (s, 3H, CH3), 2.48 (d, J = 7.4 Hz, 2H, CH2-CH2-CH3), 11.48 (s, 1H, OH); 13C NMR: 5 14.42, 17.63, 22.39, 28.02, 108.56, 116.20, 141.73, 144.83, 160.88, 169.48; ESI-MS: m/z 225 [M+H]+; anal. calcd. for C10H12N2O2S: C 53.55, H 5.39, N 12.49; found: C 53.30, H 5.44, N 12.56. 6-Benzyl-5-hydroxy-7-methyl-3H-thiazolo[4,5-b] pyridin-2-one (2c): White solid; yield: 75%; mp 277 °C; 1H NMR: 5 2.18 (s, 3H, CH3), 3.93 (s, 2H, CH2-C6H5), 7.15-7.19 (m, 3H, C6H5), 7.25 (t, J = 7.4 Hz, 2H, C6H5), 10.96 (s, 1H, OH), 12.18 (s, 1H, NH); 13C NMR: 5 18.02, 31.46, 108.87, 114.86, 126.27, 128.44, 128.77, 140.73, 142.60, 145.62, 161.16, 169.48; ESI-MS: m/z 273 [M+H]+; anal. calcd. for C14H12N2O2S: C 61.75, H 4.44, N 10.29; found: C 62.02, H 4.38, N 10.36. General Procedure for the Synthesis Sodium Salts 5-Hydroxy- 7-methyl-3H-thiazolo[4,5-b]pyridin-2-one and 6-Benzyl-5-hydroxy-7-methyl-3H-thiazolo[4,5-b]pyridin-2-one 3a-b To 20 mL of water and (10 mmol) of potassium hydroxide was added (10 mmol) of compounds 2a or 2b, and the mixture was heated to complete dissolution. The solution obtained was evaporated to dryness. The residue was dried at 100 °C. 5-Hydroxy-7-methyl-3H-thiazolo[4,5-b]pyridin-2-one sodium salt (3a) White solid; yield: 60%; mp 280 °C; 1H NMR: 5 1.99 (s, 3H, CH3), 5.41 (s, 1H, Py), 11.07 (s, 1H, NH); 13C NMR: Chaban et al.: Synthesis, Anti-Inflammatory and Antioxidant. Acta Chim. Slov. 2019, 66, 103-111 105 8 21.47, 104.13, 104.18, 144.71, 154.52, 162.27, 162.31; ESI-MS: m/z 205 [M+H]+; anal. calcd. for C7H5N2NaO2S: C 41.18, H 2.47, N 13.72; found: C 41.25, H 2.52, N 13.66. 6-Benzyl-5-hydroxy-7-methyl-3H-thiazolo[4,5-b]pyri-din-2-one sodium salt (3b) White solid; yield: 86%; mp 298 °C; 1H NMR: 8 2.00 (s, 3H, CH3), 3.79 (s, 2H, CH2), 7.11 (t, J = 7.0 Hz, 1H, C6H5), 7.17-7.23 (m, 4H, C6H5), 11.07 (s, 1H, NH); 13C NMR: 8 18.06, 31.45, 108.87, 114.86, 126.27, 128.43, 128.78, 140.72, 142.61, 145.61, 161.15, 169.49; ESI-MS: m/z 295 [M+H]+; anal. calcd. for C14H11N2NaO2S: C 57.14, H 3.77, N 9.52; found: C 57.30, H 3.74, N 9.48. General Procedure for the Synthesis 5-yl Esters Aliphatic Derivatives 7-Methyl-3H-thiazolo[4,5-b]pyridin-2-ones 4a-d Compounds 2a or 2b or 2c (5 mmol), an appropriate aliphatic chloroanhydride (5 mmol), and triethylamine (5 mmol) were added to dioxane (20 mL). The reaction mixture was refluxed 15 min. On cooling the crystalline precipitate was filtered off, washed with methanol and dried. The obtained compounds were re-crystallized from methanol. Acetic acid 7-methyl-2-oxo-2,3-dihydro-thiazolo[4,5-b] pyridin-5-yl ester (4a) White solid; yield: 92%; mp 240-241 °C; 1H NMR: 8 2.30 (s, 3H, CH3), 2.36 (s, 3H, CH3-CO), 6.88 (s, 1H, Py), 12.69 (s, 1H, NH); 13C NMR: 8 12.13, 18.45, 117.33, 118.36, 143.89, 146.07, 154.01, 168.76, 169.30; ESI-MS: m/z 225 [M+H]+; anal. calcd. for C9H8N2O3S: C 48.21, H 3.60, N 12.49; found: C 48.66, H 3.55, N 12.51. Butyric acid 7-methyl-2-oxo-2,3-dihydro-thiazolo[4,5-b] pyridin-5-yl ester (4b) White solid; yield: 56%; mp 164 °C; 1H NMR: 8 0.99 (t, J = 7.3 Hz, 3H, CH3-CH2-CH2-CO), 1.64-1.69 (m, 2H, CH3-CH2-CH2-CO), 2.36 (s, 3H, CH3), 2.59 (t, J = 7.1 Hz, 3H, CH3-CH2-CH2-CO), 6.87 (s, 1H, Py), 12.69 (s, 1H, NH); 13C NMR: 8 14.81, 19.81, 22.08, 28.15, 116.84, 118.37, 144.52, 148.15, 154.07, 166.14, 169.23; ESI-MS: m/z 253 [M+H]+; anal. calcd. for C11H12N2O3S: C 52.37, H 4.79, N 11.10; found: C 52.50, H 4.74, N 11.25. Acetic acid 7-methyl-2-oxo-6-propyl-2,3-dihydro-thi-azolo[4,5-b]pyridin-5-yl ester (4c) White solid; yield: 91%; mp 212 °C; 1H NMR: 8 1.21 (t, J = 7.3 Hz, 3H, CH3-CH2-CH2), 1.41-1.45 (m, 2H, CH3-CH2-CH2), 2.32-2.36 (m, 2H, CH3-CH2-CH2), 2.34 (s, 3H, CH3), 2.47-2.49 (m, 3H, CH3-CO), 12.56 (s, 1H, NH); 13C NMR: 8 14.39, 17.99, 21.06, 22.74, 28.37, 117.61, 122.45, 143.69, 146.20, 154.23, 168.79, 169.64; ESI-MS: m/z 267 [M+H]+; anal. calcd. for C12H14N2O3S: C 54.12, H 5.30 N 10.52; found: C 54.18, H 5.10, N 10.46. Acetic acid 6-benzyl-7-methyl-2-oxo-2,3-dihydro-thi-azolo[4,5-b]pyridin-5-yl ester (4d): White solid; yield: 90%; mp 216 °C; NMR: 8 2.26 (s, 3H, CH3), 2.28 (s, 3H, CH3-CO), 3.95 (s, 2H, CH2-C6H5), 7.12 (d, J = 7.1 Hz, 2H, C6H5), 7.18 (t, J = 6.2 Hz, J = 7.0 Hz, 1H, C6H5), 7.27 (t, J =7.3 Hz, J = 7.1 Hz, 2H, C6H5), 12.65 (s, 1H, NH); 13C NMR: 8 18.45, 21.06, 31.84, 117.80, 121.08, 126.62, 128.49, 128.91, 139.38, 144.22, 146.77, 154.54, 168.72, 169.31; ESI-MS: m/z 315 [M+H]+; anal. calcd. for C16H14N2O3S: C 61.13, H 4.49, N 8.91; found: C 61.10, H 4.45, N 8.88. General Procedure for the Synthesis 5-yl Esters Aromatic Derivatives 7-Methyl-3H-thiazolo[4,5-b]pyridin-2-ones 4e-f To a solution of pyridine (20 mL) and an appropriate aromatic chloroanhydride (5 mmol) was added compound Ila or lib (5 mmol). The reaction mixture was refluxed 30 min. On cooling, the crystalline precipitate was filtered off, washed with acetic acid and dried. The obtained compounds were re-crystallized from acetic acid. 4-Nitrobenzoic acid 7-methyl-2-oxo-2,3-dihydro-thi-azolo[4,5-b]pyridin-5-yl ester (4e) White solid; yield: 72%; mp 207 °C; 1H NMR: 8 2.41 (s, 3H, CH3), 7.12 (s, 1H, Py), 8.38 (d, J = 8.7 Hz, 2H, C6H4), 8.44 (d, J = 8.7 Hz, 2H, C6H4), 12.78 (s, 1H, NH); 13C NMR: 8 19.98, 111.78, 116.85, 127.67, 129.85, 132.20, 139.94, 145.60, 148.69, 155.86, 164.04, 168.88; ESI-MS: m/z 332 [M+H]+; anal. calcd. for C14H9N3O5S: C 50.76, H 2.74, N 12.68; found: C 51.00, H 2.69, N 12.85. Benzoic acid 7-methyl-2-oxo-6-propyl-2,3-dihydro-thi-azolo[4,5-b]pyridin-5-yl ester (4f) White solid; yield: 74%; mp 203 °C; 1H NMR: 8 0.86 (t, J = 5.1 Hz, J = 7.2 Hz, 3H, CH2-CH2-CH3), 1.46-1.51 (m, 2H, CH2-CH2-CH3), 2.38 (s, 3H, CH3), 2.54 (d, J = 7.0 Hz, 2H, CH2-CH2-CH3), 7.66 (t, J =6.4 Hz, J = 7.3 Hz, 2H, C6H5), 7.81 (t, J =7.0 Hz, J = 6.5 Hz, 1H, C6H5), 8.16 (d, J = 7.5 Hz, 2H, C6H5), 12.65 (s, 1H, NH); 13C NMR: 8 14.44, 18.08, 21.21, 22.74, 117.33, 118.31, 126.25, 128.34 131.15, 138.75, 144.05, 146.18, 154.25, 167.71, 168.11; ESI-MS: m/z 329 [M+H]+; anal. calcd. for C17H16N2O3S: C 62.18, H 4.91, N 8.53; found: C 62.22, H 4.84, N 8.50. General Procedure for the Synthesis 6-Arylazo-5-hydroxy-7-methyl-3H-thiazolo[4,5-b]pyridin-2-ones 5a-i Metallic sodium (0.2 mol) was dissolved in anhydrous methanol (100 mL), and to the solution obtained 4-iminothiazolidin-2-one (50 mmol) and a-arylazo derivatives acetoacetic ester (50 mmol) were added at 20 °C. The mixture was left standing for 7 days with the intermittent stirring, then it was acidified with acetic acid to pH ~5, five-fold diluted with water. The precipitate was filtered off, washed with water, and dried at 100-110 °C. The obtained compounds were re-crystallized from acetic acid. Chaban et al.: Synthesis, Anti-Inflammatory and Antioxidant. 106 Acta Chim. Slov. 2019, 66, 103-111 5-Hydroxy-7-methyl-6-phenylazo-3H-thiazolo[4,5-b] pyridin-2-one (5a) Red solid; yield: 97%; mp 265 °C; 1H NMR: 5 2.40 (s, 3H, CH3), 7.27 (t, J = 7.4 Hz, 1H, Ph), 7.48 (t, J = 7.9 Hz, J = 7.4 Hz, 2H, Ph), 7.67 (d, J = 7.5 Hz, 2H, Ph), 13.30 (s, 1H, OH), 14.56 (s, 1H, NH); 13C NMR: 5 19.96, 112.44, 118.33, 128.87, 129.96, 133.44, 138.95, 143.98, 147.07, 163.11, 168.25; ESI-MS: m/z 287 [M+H]+; anal. calcd. for C13H10N4O2S: C 54.54, H 3.52, N 19.57; found: C 54.61, H 3.49, N 19.66. 5-Hydroxy-7-methyl-6-p-tolylazo-3H-thiazolo[4,5-b] pyridin-2-one (5b) Red solid; yield: 73%; mp 255 °C with dec.; 1H NMR: 5 2.33 (s, 3H, C6H4-CH3), 2.40 (s, 3H, CH3), 7.28 (d, J = 7.0 Hz, 2H, C6H4), 7.54 (d, J = 7.0 Hz, 2H, C6H4), 13.26 (s, 1H, OH), 14.52 (s, 1H, NH); 13C NMR: 5 14.44, 19.98, 112.23, 117.84, 128.15, 129.42, 132.15, 138.77, 144.32, 147.11, 163.07, 168.66; ESI-MS: m/z 301 [M+H]+; anal. calcd. for C14H12N4O2S: C 55.99, H 4.03, N 18.65; found: C 55.84, H 4.00, N 18.59. 5-Hydroxy-7-methyl-6-m-tolylazo-3H-thiazolo[4,5-b] pyridin-2-one (5c) Red solid; yield: 97%; mp 233 °C; 1H NMR: 5 5 2.35 (s, 3H, C6H4-CH3), 2.38 (s, 3H, CH3), 7.07 (d, J = 7.6 Hz, 1H, C6H4), 7.35 (t, J = 7.8 Hz, J = 7.6 Hz, 1H, C6H4), 7.44 (d, J = 10.0 Hz, 2H, C6H4), 13.28 (s, 1H, OH), 14.58 (s, 1H, NH); 13C NMR: 5 19.89, 21.54, 104.71, 107.70, 112.56, 114.58, 117.66, 119.61, 122.67, 130.06, 144.61, 148.11, 162.89, 169.8; ESI-MS: m/z 301 [M+H]+; anal. calcd. for C14H12N4O2S: C 55.99, H 4.03, N 18.65; found: C 55.87, H 4.08, N 18.68. 6-(2,4-Dimethyl-phenylazo)-5-hydroxy-7-methyl-3H-thi-azolo[4,5-b]pyridin-2-one (5d) Yellow solid; yield: 97%; mp 282 °C; 1H NMR: 5 2.32 (s, 3H, C6H3-CH3), 2.38 (s, 3H, CH3), 2.42 (s, 3H, C6H3-CH3), 7.18-7.20 (m, 2H, C6H3), 7.69-7.71 (m, 1H, C6H3), 13.37 (s, 1H, OH), 15.10 (s, 1H, NH); 13C NMR: 5 13.15, 16.23, 18.21, 102.08, 106.75, 112.12, 115.07, 118.71, 120.96, 125.12, 130.42, 144.77, 148.05, 162.44, 169.55; ESI-MS: m/z 315 [M+H]+; anal. calcd. for C15H14N4O2S: C 57.31, H 4.49, N 17.82; found: C 57.45, H 4.44, N 17.77. 5-Hydroxy-6-(2-hydroxy-phenylazo)-7-methyl-3H-thi-azolo[4,5-b]pyridin-2-one (5e) Orange solid; yield: 99%; mp > 280 °C with dec.; 1H NMR: 5 2.40 (s, 3H, CH3), 6.95-6.98 (m, 2H, C6H4), 7.12 (t, J = 7.3 Hz, 1H, C6H4), 7.71 (d, J = 7.7 Hz, 1H, C6H4), 10.75 (s, 1H, C6H4-oH), 13.28 (s, 1H, OH), 14.89 (s, 1H, NH); 13C NMR: 5 19.44, 103.12, 107.07, 113.44, 115.35, 119.33, 121.25, 124.71, 130.33, 145.12, 148.11, 163.08, 169.79; ESI-MS: m/z 303 [M+H]+; anal. calcd. for C13H10N4O3S: C 51.65, H 3.33, N 18.53; found: C 51.59, H 3.40, N 18.58. 3-(5-Hydroxy-7-methyl-2-oxo-2,3-dihydro-thiazolo [4,5-b]pyridin-6-ylazo)-benzenesulfonic acid (5f) Yellow solid; yield: 99%; mp > 280 °C with dec.; 1H NMR: 5 2.41 (s, 3H, CH3), 7.44-7.51 (m, 2H, C6H4), 7.60 (d, J = 7.0 Hz, 1H, C6H4), 7.86 (s, 1H, C6H4), 13.31 (s, 1H, OH), 14.61 (s, 1H, NH); 13C NMR: 5 17.35, 114.27, 117.95, 124.07, 125.74, 129.79, 137.54, 141.28, 141.58, 150.24, 160.62; ESI-MS: m/z 367 [M+H]+; anal. calcd. for C13H10N4O5S2: C 42.62, H 2.75, N 15.29; found: C 42.33, H 2.65, N 15.38. 5-Hydroxy-6-(4-methoxy-phenylazo)-7-methyl-3H-thi-azolo[4,5-b]pyridin-2-one (5g) Red solid; yield: 99%; mp > 258 °C with dec.; 1H NMR: 5 2.41 (s, 3H, CH3), 3.80 (s, 3H, O-CH3), 7.05 (d, J = 9.0 Hz, 2H, C6H4), 7.67 (d, J = 8.0 Hz, 2H, C6H4), 13.14 (s, 1H, OH), 14.78 (s, 1H, NH); 13C NMR: 5 16.44, 19.15, 111.97, 117.75, 128.17, 129.23, 132.42, 138.92, 143.98, 146.88, 162.65, 168.07; ESI-MS: m/z 317 [M+H]+; anal. calcd. for C14H12N4O3S: C 53.16, H 3.82, N 17.71; found: C 53.25, H 3.88, N 17.64. 4-(5-Hydroxy-7-methyl-2-oxo-2,3-dihydro-thiazolo [4,5-b]pyridin-6-ylazo)-benzenesulfonamide (5h) Orange solid; yield: 100%; mp > 280 °C with dec.; 1H NMR: 5 2.36 (s, 3H, CH3), 7.38 (s, 2H, NH2), 7.76 (d, J = 8.5 Hz, 2H, C6H4), 7.86 (d, J = 8.5 Hz, 2H, C6H4), 13.32 (s, 1H, OH), 14.32 (s, 1H, NH); 13C NMR: 5 17.44, 116.88, 123.19, 125.82, 127.62, 141.33, 142.11, 146.10, 160.55, 166.84, 177.99; ESI-MS: m/z 366 [M+H]+; anal. calcd. for C13H11N5O4S2: C 42.73, H 3.03 N 19.17; found: C 42.82, H 3.14, N 19.25. N-(5-Ethyl-[1,3,4]thiadiazol-2-yl)-4-(5-hydroxy-7-me-thyl-2-oxo-2,3-dihydro-thiazolo[4,5-b]pyridin-6-yla-zo)-benzenesulfonamide (5i) Orange solid; yield: 100%; mp 276-278 °C; 1H NMR: 5 1.21 (t, J = 7.5 Hz, 3H, CH3-CH2-thiadiazole), 2.36 (s, 3H, CH3), 2.80-2.85 (m, 2H, CH3-CH2-thiadiazole), 7.76 (d, J = 8.7 Hz, 2H, C6H4), 7.83 (d, J = 8.7 Hz, 2H, C6H4), 13.67 (s, 1H, OH), 14.32 (s, 1H, NH); 13C NMR: 5 12.78, 17.43, 24.17, 117.44, 126.79, 128.03, 138.78, 140.71, 144.96, 145.01, 160.55, 168.03, 168.06; ESI-MS: m/z 478 [M+H]+; anal. calcd. for C17H15N7O4S3: C 42.76, H 3.17, N 20.53; found: C 42.79, H 3.33, N 20.46. 2. 3. Anti-Inflammatory Activity Evaluation Assays Anti-inflammatory activity24 was evaluated using carrageenan induced rat paw edema method in rats. Out-bred (male/female) white rats weighing 180-220 g were used for the edema test. Animals were divided into 13 groups comprising five rats per group. One group was kept as the control and remaining 12 groups (test groups) were used to determine the anti-inflammatory activity Chaban et al.: Synthesis, Anti-Inflammatory and Antioxidant. Acta Chim. Slov. 2019, 66, 103-111 107 elicited by the 10 drug candidates, respectively. Rats were kept in the animal house under standard conditions of light and temperature on the general diet prior to the experiment. The standard drug, Ibuprofen (50 mg/kg body weight) and the test drugs (50 mg/kg body weight) were dissolved in DMSO and administered through intraperitoneal route. DMSO was injected to the control group; 30 minutes later, 0.1 mL of 2% carrageenan solution in saline was injected in the sub-plantar region of the right hind paw of each rat. After 4 h of the carrageenan injection, the volume of paw edema (in mL) was measured using water plethysmometer and paw edema decreasing was compared between control group and drug-tested groups. Danylo Halytsky Lviv National Medical University ethics committee, constituted by the Ministry of Health of Ukraine, approved the experimental protocol. The inflammatory reaction inhibition was expressed as percent of paw volume reduction and it was calculated using the following formula: V -V % Inhibition - COI,"°1 -100% (1) where Vcontrol is the increase in paw volume in control group animals; F is the increase in paw volume in animals injected with the test substances. 2. 4. Free Radical Scavenging Assays The antioxidant activity was determined on the basis of free radical scavenging activity of stable 2,2-diphe-nyl-1-picrylhydrazyl radical (DPPH). The effect of the studied compounds on DPPH radicals were estimated according to the method of Blois25-26 with minor modifications. The solution of DPPH in ethanol with the concentration of 150 ^mol/L (4 mL) was mixed with the compound or control solution in ethanol, its concentration being 250 ^mol/L (0.2 mL). The reaction mixture was vortex mixed thoroughly and incubated at room temperature in the dark for 60 min. Simultaneously, a control was prepared as ascorbic acid solution in ethanol (0.2 mL) mixed with DPPH solution in ethanol (4 mL) without sample fraction. Reduction in the absorbance of the mixture was measured at 517 nm using ethanol as blank. Ascorbic acid was used as the standard. Also the absorbance of DPPH solution was measured. Percentage of free-radical-scavenging activity was expressed as percent inhibition and it was calculated using the following formula: (2) where ADPPH is the absorbance of DPPH free radicals solution, As is the absorbance of the sample. Each experiment was performed in triplicate and average values were recorded. Results are expressed as the means ± S.D. 3. Results and Discussion 3. 1. Chemistry One of the efficient synthetic approaches for thi-azolo[4,5-fo]pyridine system construction used in modern medical chemistry is the protocol based on [3+3] cyclocondensation of 4-iminothiazolidin-2-one27 (1) on account of its N,C-binucleophylic properties when reacting with dielectrophylic reagents.28 We used 4-iminothiazolidin-2-one (1) as the initial compound that was reacted with acetoacetic ester. We optimized the conditions of this reaction that made it possible to obtain 5-hydroxy-7-methyl-3H-thiazolo[4,5-fo]pyri-din-2-one (2a) in good yield (Scheme 1). The best results were observed in the case keeping when methanol was used and the reagents mixture was kept in the presence of sodium methylate over 7 days. The next stage of our strategy includes the core het-erocycle structural modification at its C6 position. The direct functionalization procedure has been shown to be of a small synthetic utility owing to the low nucleophilic activity of the compound 1 at the C6 position. We studied the behavior in reaction of [3+3] cyclo condensation of 4-iminothiazolidin-2-one with alkylated acetoacetic ester derivatives. Under the chosen reaction conditions the corresponding 6-alkyl-5-hydroxy-7-methyl-3H-thi-azolo[4,5-fo]pyridin-2-ones 2b-c were isolated in good yields (Scheme 1). Some properties of the obtained compounds were investigated. The proton at the position 5 retained the acidic properties and the reaction with sodium hydroxide furnished the corresponding salts 3a-b (Scheme 1). Chloroacetamides are highly reactive chemicals which are being involved in alkylation reactions forming the basis for creating building blocks of wide utility for combinatorial chemistry, including biologically active substances, to design combinatorial libraries on their basis. The hydroxy group present at C5 position in compounds 2a-c provides an entry to 5-yl ester derivatives 4a-f. Anhydride was found to be the most suitable medium for the reaction of compounds 2a-c with aliphatic chloroanhydrides 4a-d. Reaction of compounds 2a-b with chloroanhydrides of aromatic acids readily took place in pyridine medium and enabled the preparation of compounds 4e,f (Scheme 1). Literature survey revealed that some antimicrobial drugs like sulfasalazine, salazopyridazine etc. contain arylazo group.29 In its turn, arylazo moiety introduction into thiazolidine ring system led to antimicrobial activity enhancement possessed by the respective thiazolidine ar-ylazo derivatives. In view of the results mentioned above, 5-hydroxy-7-methyl-6-arylazo-3H-thiazolo[4,5-fo]pyridin-2-ones 5a-i were synthesized following the same protocol as for a-ar-ylazo derivatives of acetoacetic esters, by treatment with 4-iminothiazolidin-2-one (Scheme 1). Powders of these Chaban et al.: Synthesis, Anti-Inflammatory and Antioxidant. 108 Acta Chim. Slov. 2019, 66, 103-111 5a-i 4a-f 2a-c: R = H (a), C6H5-CH2 (b), C3H7 (c); 3a-b: R= H (a), C6H5-CH2 (b); 4a-f: R= H, R1 = CH3 (a), R=H, R1 = C3H7 (b), R = C3H7, R1 = CH3 (c), R = C6H5-CH2, R1 = CH3 (d), R = H, R1 = 4-N02-C6H4 (e), R = C3H7, R, = C6H5 (f), 5a-i R2 = C6H5 (a), 4-CH3-C6H4 (b),3-CH3-C6H4 (c), 2,4-(CH3)2C6H3 (d), 2-OH-C6H4 (e), 3-S03H-C6H4 (f), 4-OCH3-C6H4 (g), 4-S02NH2C6H4 (h), 4-(N-(5-ethyl-[l,3,4]thiadiazol-2-yl)-benzenesulfonamide) (i). Scheme 1. Synthesis of novel 3H-thiazolo[4,5-fo]pyridines substances are well soluble in DMF and DMSO, sparingly soluble in water and in other organic solvents. The structures of the obtained compounds were confirmed by 1H and 13C NMR spectroscopy and elemental analysis. All these new compounds gave spectroscopic data in accordance with the proposed structures. The 1H NMR spectra of all compounds show the protons signals of methyl group in pyridine ring as singlets in the 1.992.41 ppm. Endocyclic NH groups of basic scaffold are apparent in the region of weak magnetic field at 11.07-15.10 ppm. Proton signals of pyridine cycle for compounds are evident at 5.41-7.12 ppm and are represented by correspondent singlets. Absence of given signals confirms the origin of reaction [3+3]cyclocondensation with corresponding alkyl as well as with arylazo derivatives of acetoacetic ester. OH group of basic scaffold is represented by relatively broad singlets at 10.84-13.67 ppm. The corresponding salts obtained are characterized by the absence of OH group signals. Conducting acylation reaction is characterized by the absence of OH group signal at position C5 as well as by the presence of a set of singlets, doublets and multiplets at 0.99-8.44 ppm that confirm the formation of the corresponding 5-acyl derivatives. 3. 2. Anti-inflammatory Activity in vivo Evaluation Carrageenan-induced paw edema is the most widely used animal model of acute inflammation. In vivo studies Chaban et al.: Synthesis, Anti-Inflammatory and Antioxidant. Acta Chim. Slov. 2019, 66, 103-111 109 of novel thiazolo[4,5-fr]pyridine-2-one derivatives were carried out for anti-inflammatory activity employing the carrageenan-induced rat paw edema method. Marked paw edema was produced in rats with sub-planter injection of 0.1 mL of 2% carrageenan. The test compounds were dissolved in DMSO and injected intraperitoneally in the dose of 50 mg/kg body weight 0.5 h prior to carrageenan injection. The NSAID drug Ibuprofen in its effective therapeutic dose was tested in parallel as an activity reference. Anti-inflammatory activity was defined by measuring the paw edema volume 4 h after the carrageenan injection. Results of paw edema decreasing are expressed as the mean ± standard deviation and compared statistically with the control group using Student's t-test. A level of p < 0.05 was adopted as the test of significance (Table 1). The percentage protection against inflammation was calculated as % inhibition by comparison between DMSO injected control group and drugs-tested groups. dicating the nature and position of the substituted groups did not influence notably their anti-inflammatory activity. 3. 3. In vitro Antioxidant Assay The antioxidant activity was determined on the basis of free radical scavenging activity of 2,2-diphenyl-1-pic-rylhydrazyl (DPPH) free radical. DPPH radical has found many applications due to its high stability in the metha-nolic solutions and its intense purple color. In its oxidized form, the DPPH radical has an absorbance maximum centered at a wavelength about 540 nm. The absorbance decreases when the radical is reduced by antioxidants. Its reduction affords 2,2-diphenyl-1-picrylhydrazine (DPPH-H), or the corresponding anion (DPPH-) in basic medium. The DPPH radical acts as a scavenger for other odd-electron species which afford para-substitution products at phenyl rings. Table 1. Anti-inflammatory effect of thiazolo[4,5-fo]pyridine-2-ones on carrageenan-induced rat paw edema (mL) in vivo evaluation, % protection from inflammation Compound Paw edema % Inhibition Compound Paw edema % Inhibition volume (mL) ± SEM* volume (mL) ± SEM* Control 2.20 ± 0.050 - 4f 1.29 ± 0.025 41.2 2a 1.41 ± 0.040 36.2 5a 1.54 ± 0.040 30.1 2c 1.64 ± 0.035 25.3 5g 1.60 ± 0.035 27.2 3a 1.44 ± 0.040 34.5 5h 1.25 ± 0.020 43.1 4a 1.57 ± 0.035 28.5 5i 1.09 ± 0.025 50.5 4c 1.22 ± 0.020 44.5 ibuprofen 1.32 ± 0.035 40.2 Evaluation of anti-inflammatory activity indicated that six compounds (i.e. 2a, 2c, 3a, 4a, 5a and 5g) showed no significant decrease in edema, the inhibition rate for them was observed at the level of 25.3-36.2% as compared to control group. The compounds 4c, 4f and 5h possessed the anti-inflammatory activity in the range of 41.2-44.5% which is comparable to the effect of Ibuprofen. The antiinflammatory evaluation test for compound 5i gave the result as 50.5% inhibition indicating the compound was more potent than Ibuprofen. The results of the pharmacological tests were analyzed with respect to the compounds structure. For ^-substituted 5-hydroxy-7-methyl-3H-thiazolo[4,5-fr]pyridin-2-one derivatives prepared by [3+3] cyclo condensation of 4-iminothiazolidin-2-one with arylazo derivatives of acetoacetic ester comparison of the substituents nature on the C6 position indicated that N-(5-ethyl-[1,3,4]thiad-iazol-2-yl)-methanesulfonamide presence in the phenyl ring contributed to the inflammation inhibition efficiency. The presence of the acetyl or phenyl groups at the C5 position of the basic scaffold and a propyl group at the C6 position caused the anti-inflammatory activity which is comparable to the effect of Ibuprofen. Among the remaining test compounds no active compounds were evaluated in- The DPPH method is described as a simple, rapid and convenient method for screening of many samples for radical scavenging activity. These advantages make the DPPH method interesting for testing newly synthesized compounds to scavenge radicals and to find out promising antioxidant drug candidates. In the present paper we demonstrate a modified spectrophotometric method that makes use of the DPPH radical and its specific absorbance properties. The free radical scavenging activities of each compound were assayed using a stable DPPH and were quantified by decolorization of the solution being mixed with DHHP as observed at the wavelength of 540 nm. The absorbance of DPPH solution in ethanol (150 ^mol/L) was measured as 0.770. The ab-sorbances and free-radical-scavenging activities % inhibitions of standard (ascorbic acid) and each compound are listed in Table 2. The antioxidant activity evaluation results showed that, in general, most of the tested compounds showed that their free radical scavenging effect was insignificant being in the range of 5.50-21.10%. The pharmacological screening allowed identification of only one lead compound, namely 5i, whose free radical scavenging activity (65.80%) exceeded that of ascorbic acid. Thus, the presence of N-(5- Chaban et al.: Synthesis, Anti-Inflammatory and Antioxidant. 110 Acta Chim. Slov. 2019, 66, 103-111 Table 2: Values of Absorbance and % Inhibition of 3H-thiazolo[4,5-fo]pyridine-2-ones Compound or Standard Absorbance of a Sample, As % Inhibition Compound or Standard Absorbance of a Sample, As % Inhibition ascorbic acid 0.406 ± 0.015 47.30 4f 0.654 ± 0.025 15.10 2a 0.682 ± 0.025 11.50 5a 0.670 ± 0.025 13.00 2b 0.654 ± 0.020 15.10 5b 0.725 ± 0.030 5.80 2c 0.608 ± 0.020 21.10 5c 0.715 ± 0.030 7.10 3a 0.704 ± 0.030 8.50 5d 0.728 ± 0.030 5.50 3b 0.672 ± 0.025 12.70 5e 0.662 ± 0.020 14.00 4a 0.697 ± 0.025 9.50 5f 0.700 ± 0.025 9.10 4b 0.697 ± 0.025 9.50 5g 0.711 ± 0.020 7.70 4c 0.706 ± 0.030 8.30 5h 0.691 ± 0.025 10.30 4d 0.704 ± 0.030 8.50 5i 0.263 ± 0.010 65.80 4e 0.684 ± 0.025 11.20 ethyl-[1,3,4]thiadiazol-2-yl)-methanesulfonamide in the phenyl ring of the core scaffold at C6 position was essential to the antioxidant activity of these compounds. 4. Conclusions A series of 5-hydroxy-7-methyl-3H-thiazolo[4,5-fo] pyridin-2-one derivatives possessing anti-inflammatory and antioxidant activities were prepared by the structural modification of the core heterocycle at C5 and C6 positions. Therefore, we have shown that the proposed approaches and developed synthetic protocols provided the possibility to synthesize diverse 5-hydroxy-7-methyl-3H-thi-azolo[4,5-fo]pyridin-2-ones with a considerable chemical novelty involving alkylation, Japp-Klingemann condensation, [3+3] cyclocondensation and acylation reactions. Anti-inflammatory activity evaluated in vivo and free radicals scavenging effect determined in vitro allowed to identify some lead compounds causing significant decrease in edema formation or considerable antioxidant effect. The present results suggest that the core fused heterocycle can be developed as a promising scaffold for anti-inflammatory and antioxidant drug candidates. 5. References 1. I. S. Shin, J. W. Park, N. R. 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Lelyukh, Pharmacia 2018, 65(2), 54-70. 16. T. I. Chaban, V. V. Ogurtsov, I. G. Chaban, O. V. Klenina, J. D. Komarytsia, Phosphorus, Sulfur Silicon Relat. Elem. 2013, 188, 1611-1620. DOI: 10.1080/10426507.2013.777723. 17. O. Klenina, I. Drapak, T. Chaban, V. Ogurtsov, I. Chaban, I. Golos, Chem. & Chem. Techn. 2013, 7, 397-404. 18. O. Klenina, T. Chaban, B. Zimenkovsky, S. Harkov, V. Ogurtsov, I. Chaban, I. Myrko, Pharmacia 2017, 64(4), 4971. 19. T. Chaban, O. Klenina, I. Drapak, V Ogurtsov, I. Chaban, V. Novikov, Chem. & Chem. Techn. 2014, 89, 287-292. 20. T. I. Chaban, R. R. Panchuk, O. V. Klenina, N. R. Skorokhyd, V. V. Ogurtsov, I. G. Chaban, Biopolym. Cell 2012, 28, 389396. DOI:10.7124/bc.000075 21. T. Chaban, O. Klenina, B. Zimenkovsky, I. Chaban, V. Chaban et al.: Synthesis, Anti-Inflammatory and Antioxidant. Acta Chim. Slov. 2019, 66, 103- 111 111 Ogurtsov, L. Shelepeten, Der Pharma Chemica. 2016, 8, 534-542. 22. T. Chaban, O. Klenina, S. Harkov, V. Ogurtsov, I. Chaban, I. Nektegaev, Pharmacia 2017, 64(4), 16-30. 23. S. Marzoog, Al-Thebeiti, Il Farmaco 2000, 55, 109-118. DOI: 10.1016/S0014-827X(99)00130-5 24. A. D. Pillai, P. D. Rathod, P. X. Franklin, H. Padh, K. K. Vasu, V. Sudarsanam, Biochem. Biophys. Res. Commun. 2004, 317, 1067-1074. DOI: 10.1016/j.bbrc.2004.03.148 25. M. S. Blois, Nature 1958, 181, 1199-1200. D0I:10.1038/1811199a0 26. P. Molyneux, J. Sci. Technol. 2004, 26, 211-219. 27. I. D. Komaritsa, Chem. Heterocycl. Compd. 1970, 4, 324-325. DOI: 10.1007/BF00755270 28. T. I. Chaban, B. S. Zimenkovskii, J. D. Komaritsa, I. G. Chaban, Rus. J. Org. Chem. 2012, 48, 268-272. D0I:10.1134/S1070428012020170 29. B. Combe, C. Codreanu, U. Fiocco, M. Gaubitz, P. P. Geusens, T. K. Kvien, K. Pavelka, P. N. Sambrook, J. S. Smolen, J. Wa-jdula, S. Fatenejad, Ann. Rheum. Dis. 2006, 65, 1357-1362. D0I:10.1136/ard.2005.049650 Povzetek 5-Hidroksi-7-metil-3ff-tiazolo[4,5-fo]piridin-2-on smo pripravili z reakcijo med 4-iminotiazolidin-2-onom in acetoac-etatnim estrom. Nadaljnje strukturne modifikacije so vključevale uvedbo diverzitete na položajih C5 in C6. Anti-in-flamatorno učinkovanje novih tiazolo[4,5-fo]piridin-2-onskih derivatov smo ugotovili z in vivo metodo s pomočjo s karagenanom induciranega edema na podganjih tacah. Antioksidantno aktivnost sintetiziranih spojin smo določili z in vitro metodo s pomočjo radikalskega lovilca 2,2-difenil-1-pikrilhidrazilnega radikala (DPPH). Chaban et al.: Synthesis, Anti-Inflammatory and Antioxidant. DOI: 10.17344/acsi.2018.4702 Acta Chim. Slov. 2019, 66, 112-122 /^creative ^commons Scientific paper A Novel Environmentally Friendly Synergistic Mixture for Steel Corrosion Inhibition in 0.51 M NaCl Stojan Bozovic,1'* Sanja Martinez2 and Veselinka Grudic1 1 University of Montenegro, Faculty of Metallurgy and Technology, Cetinjski put, 81000 Podgorica, Montenegro 2 University of Zagreb, Faculty of Chemical Engineering and Technology, Department of Electrochemistry, ReCorr Laboratory, Savska cesta 16, HR-1000 Zagreb, Croatia * Corresponding author: E-mail: stojan.bozovic90@yahoo.com, Telephone: +38267499528 Received: 09-02-2018 Abstract The ubiquitous use of plain and low-alloy steels in neutral, chloride bearing environments presents an everyday challenge for corrosion protection professionals. This paper explores the possibility of developing a non-toxic and environmentally friendly synergistic inhibitor mixture made of propolis, tannin, sodium benzoate, PEG400 and starch that could be applied into the solution to induce the formation of a persistent protective layer on steel. Components of the mixture were chosen based on the references giving their characteristics relevant to their possible action in the solution and/or at the surface of the steel. The efficiency of the protective layer formed under the influence of the inhibitor mixture, and then measured by the LPR probe in the inhibitor-free solution, was the lowest under quiescent conditions (75%) and the highest (95%) under flow conditions. Both, LPR and EIS data indicate that the inhibitor modified layer presents a barrier for diffusion of oxygen that acts as a primary corrosion reaction depolarizer in the investigated neutral chloride solution. The demonstrated persistence of the inhibitor modified layer is of primary interest as it gives possibility batch application. Keywords: Synergistic inhibitor mixture; steel; corrosion inhibition 1. Introduction Steel is the most widely used metallic material thanks to its good mechanical properties and low price. However, when it comes into contact with aerated neutral electrolyte, especially in the presence of chloride ions, steel is subject to rapid corrosion degradation. Neutral saline aqueous environments of various origins are frequently simulated in laboratory by the use of 0.35-0.85 M NaCl solutions.1 Corrosion rates recorded on plain steel by polarization and weight loss techniques are fairly consistent and range between 0.1 and 0.5 mm per year.2,3 These rates are generally not acceptable and protective measures have to be applied. Due to the inherent characteristics of corroding systems (e.g. complex geometry or need for good thermal conductance) corrosion inhibitors frequently stand out as the most practical or the only solution. Unfortunately, protection of steel in neutral chloride media is challenging from theoretical and practical viewpoint. It can readily be observed that considerably more literature is found on inhibition of steel corrosion in acidic media than in neutral media and that the inhibitor effectiveness is generally higher. From the theoretical aspect, the reason is that in near-neutral, neutral and alkaline media, inhibitor action is complicated by formation of iron oxides and hydroxides at the surface of metal.4 In acidic media, bare iron (or steel) surface is positively charged and behaves as a soft Lewis acid, hence, soft Lewis base compounds with heteroatoms O, N, S, P and/or n electrons in their structure, are good inhibitor candidates due to the possibility of soft acid-soft base adsorption bond formation.5,6 On the other hand, when oxide is present, surface acidity and basicity depends on oxide's isoelectric point, which e.g. for Fe2O3, equals pH 8.6.7 Below this pH it may be assumed that the surface oxide is protonated, positively charged and acting as a hard Lewis acid. Hard Lewis bases would therefore be good inhibitor candidates capable of hard acid-hard base adsorptive bond formation. In the present study, tannin, sodium benzoate, propolis, starch and polyethylene glycol (PEG400) were chosen as components of the investigated inhibitor mixture. When Bozovic et al.: A Novel Environmentally Friendly Synergistic Mixture 113 Acta Chim. Slov. 2019, 66, 112-122 such a mixture rich in OH groups is used for inhibiting corrosion, in near neutral and neutral pH, part of the OH groups will be deprotonated forming anions that have electronegativity practically equal to their ionization potential, and therefore act as almost pure electron donors or hard Lewis bases.5 Adsorptive hard acid-hard base bond which is of predominantly ionic character would therefore be possible between compounds of the mixture and the oxidized steel surface. Additionally, metal cations in high oxidation state, such as ferric ions dissolved from the steel surface into the electrolyte, are hard Lewis bases, so that formation of protective deposits of hardly soluble complexes and/or chelates of ferric ions with anionic organic ligands is possible.8 Components of the inhibitor mixture investigated in the present study were chosen after careful consideration of their environmental friendliness and previously published characteristics relevant to corrosion inhibition. E.g., ethan-olic propolis extract applied to mild steel forms a very effective anticorrosion coating in neutral and near neutral aqueous 3.5% w/v NaCl solution.9 Propolis was also proven to be effective on copper in neutral chloride solution,10 but copper is more favourable for adsorption of organic compounds under such conditions.1 Adsorption of effective propolis layer on steel from the solution is probably hindered by its insufficient concentration due to the limited solubility of propolis in water. Dispersion of propolis in neutral chloride medium produces a yellowish emulsion.9 Based on literature data, PEG is used as an alternative solvent for non-ethanolic propolis extraction,11 and when used together with propolis, could beneficially influence propolis dispersibility in a water solution.12 Furthermore, it has been shown that a compact layer, with barrier effect for the diffusion of oxygen in 0.1 NaCl solution, has been deposited on mild steel from the solutions containing 1000, 2500 and 5000 g ppm of PEG after 30 min of immersion time, showing that PEG itself can act as a corrosion inhibitor.13 Formation of the protective ferric ''tannate film'' deposited from solution of tannin is also possible, and it was shown to begin at pH = 3, but relevant efficiencies are attained only at concentrations ~1000 ppm.14 Recent investigation of low carbon steel inhibition by 1-5% tannic acid solution in 3.5% NaCl shows that efficiencies up to 51% can be obtained.15 Concentrations of the same order are needed for efficient steel corrosion inhibition in chloride media by starch16 and sodium benzoate.17 In the present study, minimal quantities of individual components that would be effective, based on the above literature references, were chosen for the inhibitor mixture composition. In general, extra costs are incurred in neutral chloride media because the enhancement of inhibitor effectiveness is frequently attained through the increase in concentration up to a few thousands of ppm.18 The most explored strategy for lowering inhibitor concentration, while retaining good efficiency is application of synergistic inhibiting mixtures.1,18-22 In the present case, different chemical nature of components of the mixture used was expected to yield a combination of mechanisms of action, which is usually argued to be the basis for synergism.23 Propolis was chosen as a starting component of the mixture. It is generally accepted that propolis is composed of approximately 50% resin (polyphenolic fraction), 30% wax, 10% essential oils, 5% pollen and 5% other organic and inorganic compounds.9 Main propolis phenolic esters and flavonoids are of amphiphilic nature,24 and are expected not only to inhibit corrosion but also to increase the hydrophobicity of steel or corrosion products due to adsorption with the hydrophilic part of the molecule oriented to the surface and the hydrophobic part oriented towards the liquid phase.25 Chestnut tannin extract6 was chosen as a second component in order to increase the water-soluble inhibiting fraction of the mixture. Since polyphenols are weak organic acids known to decrease pH of neutral solutions,14 along with the inhibitive effect of the tannate layer, iron dissolution enhancing effect may be expected.15 Increased release of iron ions form steel, on the other hand, is favourable for ferric tannate formation. Hence, sodium benzoate17 has been added to the mixture for controlled iron release, until the protective surface layer is fully formed. The last two components added to the mixture were polymeric substances, PEG and starch. Besides with the intention of increasing propolis solubility, PEG was added alongside with starch, with the intention of strengthening surface layer through interaction with tannin. Tannins have an abundance of hydroxyl groups in close proximity capable of strong and specific interaction with carbohydrate polymers via hydrogen bonding and hydrophobic interactions.26 When phenolic components and PEG and starch are mixed, the hydroxyl groups of phenolic chemicals can interact with PEG oxygens27 and side chains of starch amylopectin,28 respectively. In this paper we extend the synergistic approach by hypothesising that a carefully chosen mixture of non-toxic and environmentally benign inhibitive compounds could modify the rust layer on steel. This layer would form in neutral chloride solutions, as to produce a persistent film which would lower corrosion rate considerably. The inherent advantage of the novel mixture would be environmental friendliness as well as the retention and resiliency of the inhibitor modified rust layer even in the inhibitor free solution. High concentration requirement would be counteracted by a long inhibitor free period, following batch application, during which the protective surface film would ensure acceptable corrosion rates. 2. Experimental 2. 1. Solutions Tests were performed on 0.51 M NaCl, obtained by dissolving analytical grade NaCl (Lachner, p.a.) in redistilled water. Bozovic et al.: A Novel Environmentally Friendly Synergistic Mixture ... 114 Acta Chim. Slov. 2019, 66, 112-122 Natural propolis (Naturwaren-niederrhein, GmbH, Propolis Pulver) was used at a concentration of 100 ppm in 0.51 M NaCl. 100 mg of propolis was first dissolved in 5 ml of 70% ethanol before preparing 1 dm3 of solution. Chestnut Tannin (Tanin Sevnica) and sodium benzoate (AGZ, food additive) were both used at a concentration of 2000 ppm. PEG400 (Sigma Aldrich) and corn starch (Sigma Al-drich) were used in concentrations of 200 and 400 ppm, respectively. The inhibitive mixture of 2000 ppm of tannin (T), 2000 ppm of benzoate (B) and 100 ppm of propolis (P), 200 ppm of PEG400 (PEG) and 400 ppm of starch (s) in 0.51 M NaCl is in further text denoted by abbreviation T+B+P+PEG+S, and the other combinations of single components are denoted accordingly. All the measurements were done in aerated solutions under ambient conditions. 2. 2. LPR Probe The measurements of corrosion rate were performed on the LPR (Linear Polarization Resistance) probe manufactured by RCSL with the data collector MS1500L. The corrosion rate was determined by software of the MS1500L instrument. Corrosion monitoring LPR pins made of C1010 steel had composition declared by the producer (Metal Samples) as: C (0.02-0.08), Mn (0.3-0.6), P (0-0.04), S (0.05) and Fe (bal.). LPR electrodes were, prior to immersion, treated mechanically by abrasive paper of grit 240, followed by degreasing with ethanol in an ultrasound bath and rinsing in redistilled water. Measurements were made by immersion of the electrodes in the solution of 0.51 M NaCl, as well as in solutions with the chosen concentrations of inhibitors. A protective layer at the electrode surface was formed for 24 h in quiescent solutions. In one experiment, the layer was deposited from the inhibited solution mixed at 80m RPM, in order to rate the influence of solution mixing on layer formation and its protective properties. In the non-inhibited solutions, the rust layer was formed, while in the inhibited solutions, the rust layer modified by the action of corrosion inhibitors was obtained. The layer formed without inhibitor is in the rest of the text referred to as the "rust layer", and the layer formed under the influence of the inhibitors in 0.5 M NaCl or the layer-forming solution, is in the rest of the text referred to as the" inhibitor-modified rust layer". The corrosion rate was measured after the probe was transferred into a fresh 0.51 M NaCl solution and left to stand for 24 h. The LPR measurement was then performed in a quiescent solution as well as in a solution stirred by magnetic stirrer at 90 to 150 RPM. Long-term measurements with the layer formed in the most effective inhibitor mixture as well as the pure rust layer were performed continuously for 10 days. In this way, the persisten- cy of the protective layer is tested by LPR probe, which was previously found to be particularly suitable technique for that laboratory testing of inhibitor persistence.29 2. 3. Electrochemical Impedance Spectroscopy (EIS) For EIS, the PalmSens3 device with PC Trace 5.3 software has been used. A cylindrical steel sample made of API X52 5L steel embedded in epoxy resin and having circular cross section exposed to the electrolyte, was used as a working electrode. Typical chemical composition of commercial grade API 5L used is C (0.26 max.), MN (1.40 max.), Si (0.4 max.), P (0.03 max.), S (0.03 max), Fe (bal.).30 A saturated calomel electrode was used as reference electrode and a graphite electrode was used as an auxiliary electrode. The working electrode surface was 0.2826 cm2. Prior to electrochemical testing, the surface of the working electrode was mechanically treated by abrasive paper of various grits, namely: 240, 600 and 800. Subsequently, the surface was degreased in an ultrasonic bath with ethanol and washed with redistilled water. EIS measurements were performed in quiescent solution, at the open circuit potential, at frequencies from 10 kHz to 1 mHz, with the amplitude of 10 mV. The electrodes on which a protective layer (of rust or inhibitor-modified rust) has been forming for 24 h, as in the experiment with LPR probe, were left to freely corrode for another 24 h in a fresh 0.51 M NaCl solution, in order to simulate a situation 24 h after batch inhibitor treatment. Additionally, EIS measurement has been done in the inhibited solution prior to transfer into the fresh NaCl in order to compare inhibitive effect in inhibitor bearing and inhibitor free solutions. 2. 4. Fourier Transform Infrared Spectroscopy (FTIR) FTIR spectra were recorded using a PerkinElmer spectrometer Spectrum One. Spectra were obtained in the range from 400 to 4000 cm-1, each spectrum being an average of ten scans with a resolution of 4 cm-1. Coupons used as substrate were made of previously mentioned API X52 5L material and were cut to size 30 x 40 x 3 mm, polished with abrasive paper of grit 240, 600 and 800 and degreased before exposure to inhibitor mixture solution. Inhibitor-modified rust layer has been formed for 24 hours, identically as for the EIS measurements, and then the coupons were left in 0.51 M NaCl for 10 days. 350 mg of KBr was rubbed onto the surface of steel coupons after ending the experiment and drying of the coupons. Samples bearing KBr were then hydraulically pressed into a 13 mm stainless steel die and the resulting pellets further subjected to FTIR measurement. Preparation of pellets was in conformance with the standard ASTM E1252:2007. Bozovic et al.: A Novel Environmentally Friendly Synergistic Mixture ... Acta Chim. Slov.2019, 66, 112-122 115 2. 5. Scanning Electron Microscopy (SEM) and Energy-Dispersive X-ray (EDX) Analysis SEM and EDX analysis was recorded using Tescan Vega III, SBU EasyProbe scanning electron microscope with 15 kV accelerating voltage of the electron beam at various magnifications. Measurements were done on surface of the coupons prior to FTIR experiments. 3. Results and Discussion Cathodic reaction takes place on iron or steel through two possible processes. At lower pH values of the solution, hydrogen evolution is controlled by the rate of charge transfer reaction, and at higher pH of the solution, oxygen reduction is controlled by the rate of oxygen diffusion. Lorbeer and Lorentz,31 state pH of 4.2 as critical, above which the dominant cathodic process is oxygen reduction and anodic iron dissolution is inhibited by time-dependent formation of a three-dimensional porous oxide layer on the electrode surface. In the present study, the pH of uninhibited solution was 6, while the pH of inhibited solutions was between 4.6 and 4.7 so the primary conjugate cathodic reaction of metal dissolution was reduction of oxygen. This reaction is known to be under diffusion control and satisfactory degree of corrosion inhibition requires dense blocking of the surface.23 Oxygen reduction generates OH- ions which react with the dissolved Fe2+ ions yielding Fe(OH)2 that is considered to be a precursor of other rust forming compounds.16 Formation of particular rust component depends on pH, temperature and oxygen content. The rust layer partially protects metal surface from dissolution.2 It has been found that increasing the immersion time of iron and steel in the solution enables thickening of the corrosion product layer resulting in the decrease of the corrosion rate. The decrease of the weight loss has been observed for carbon steel during the first 10 days of immersion in 3.5% NaCl, followed by a period of 20 days in which the weight lost remained low, while in the following 30 days the weight loss became more noticeable and higher.3 This observation indicates that dissolution of rust itself also occurs under the attack of chloride ions. The experiments in the present study were focused on modification of the rust layer by the inhibitors in order to obtain a persistent protective layer that lowers the corrosion rate to acceptable values. 3. 1. Measurements at the LPR Probe Figure 1 a) shows LPR corrosion rates on electrodes with the rust layer or with the inhibitor-modified rust layer formed under the influence of various inhibitors and inhibitors mixtures. The rates were measured after 24 h in layer-forming solutions, 24 h after transfer to quiescent 0.51 M NaCl solution and in in the same solution at the magnetic stirrer rate of 150 rpm. Except for tannin, single components show <30% inhibitive action both in the inhibitor solutions and quiescent 0.51 M NaCl with a formed layer. As expected,15 the solution of tannin as a chelating agent significantly increases corrosion rate of steel to 1.6 mmpy, but at the same time forms the most protective modified rust layer suppressing the corrosion rate in 0.51 M NaCl to 0.06 mmpy. Addition of benzoate to tannin solution decreases the corrosion rate to 0.11 mmpy but at the same time hinders the formation of protective tannin modified rust layer, increasing the corrosion rate in 0.51 M NaCl to 0.2 mmpy. Further addition of propolis and PEG decreased the corrosion rates in quiescent 0.51 M NaCl, while the addition of starch decreased the corrosion rate in quiescent and mixed 0.51 M NaCl. For the final inhibitor mixture, all corrosion rates were below 0.1 mmpy. a) 1.6 a. 1.4 £ E 1.2 v i (0 'Z 0.8 0.2 0 ■ SOLUTION ■ LAYER AT 0 rpm ■ LAYER AT 150 rpm b i? ,0>* J> v# j? pHzpc, then the surface charge of adsorbent is negative to attract positive ions.26 The pHzpc of the prepared materials, ACP-250 and ACP-500 have been found to be 2.66 Ibrahim et al.: Adsorptive Removal of Fluoride from Aqueous Solution ... 126 Acta Chim. Slov. 2019, 66, 123-136 and 3.06, respectively. From the results, both biosorbents may have more acidic functional groups such as carbox-ylic, phenolic groups etc., on their surfaces. It is fact that the biosorbents show different pHzpc values may be due to the presence or absences of acidic groups. These groups decrease with increasing carbonization temperature. However, during the burning of adsorbent, acidic metal oxides formed on the adsorbent surface. The pHzpc value of an aqueous solution plays an important role in the surface charge of the biosorbent. 3. 1. 2. SEM Analysis Fig. 2 (a)-(d) show the SEM images of ACP-250 and ACP-500, and reveal a rough surface with porous and crystalline structure for both biosorbents, which may be favorable for fluoride adsorption. The morphology of the two biosorbents was different, because of the thermal treatment at different temperatures. Furthermore, the adsorption of fluoride ion onto ACP-250 and ACP-500 was established by EDX analysis. Fig. 2 (e) and (f) show the EDX spectra of ACP-250 and ACP-500, which indicate Figure 2. SEM images of (a) and (b) ACP-250, (c) and (d) ACP-500; and EDX spectra of (e) ACP-250, and (f) ACP-500. Ibrahim et al.: Adsorptive Removal of Fluoride from Aqueous Solution ... Acta Chim. Slov. 2019, 66, 123-136 127 the higher percentage of carbon in ACP- 500 (57.98%), as compared to ACP-250 (50.27%). Iron was present in both materials, and Table S1 shows the weight percentage of ACP. 3. 1. 3. FTIR analysis of biosorbent The FT-IR spectra of ACP-250 and ACP-500 display numerous characteristic peaks. Figures 3 (a) and (b) show the FTIR spectra of ACP before and after fluoride adsorption, which display a number of adsorption peaks in the range (4,000-500) cm-1, which confirm the presence of different functional groups on the surface of biosorbents. The broad band peaks at (3,320.0-3,340.0) cm-1 were attributed to stretching vibrations in the hydroxyl group.27 The peaks that occur in the range (2,852.0-3,000.0) cm-1 were attributed to -C-H stretching in the hetero aromatic ring, with both alkyl and ethylene group in the side chain. Symmetric C-H stretching vibration of aliphatic acid is indicated by peaks at 2,921.9 cm-1.28 The peaks located in the range (1,690.0-1,710.0) cm-1 relate to the asymmetric stretching vibration of the ionic carboxylic group (-COO-).27 Wavenumber ranges from 1033-1500 cm-1 represents C-N streching in aromatic amines, CH2-O-O stretching in primary alcohol, OH group of carboxylic acids and CF3 group attached to benzene ring.29 The sharp peak at (500800) cm-1 was related to the C-O 30 and metal-oxygen (Fe-O) bonds 31. The presence of Fe-O band spectra before and after adsorption shows the Fe3O4 group. Fig.3(b) shows the FTIR spectra of ACP-500 and it was observed that the change in the intensity of the peaks confirms the adsorption of fluoride. 3. 1. 4. XRD Analysis Figure 3 c shows the X-Diffraction patterns of ACP-250 and ACP-500. Different diffraction peaks are observed on ACP-500, which indicate its crystalline structure; however, in the case of ACP-250, the absence of diffraction peaks is observed, which indicates its amorphous structure. (a) Wivnmnibrr (no'1) AC P-JiOBifoff idwpiloo ACP J,*(> Afttr liWplkHt t-r SM 1 (Kill (b) -ACP-500 (lie foi rAdioi pi io u) - ACP HW (Afltr Adsorption) 4000 JJW 300« ' 2WO 2W« 1500 1000 J00 U'av» auubrf t (cm (c) 3 re re 10 20 30 40 50 60 70 80 20 (degree) Figure 3. FTIR spectra of (a) and (b) before adsorption and after adsorption of ACP-250 and ACP-500, and (c) XRD spectra of ACP-250 and ACP-500. Ibrahim et al.: Adsorptive Removal of Fluoride from Aqueous Solution ... 128 Acta Chim. Slov. 2019, 66, 123-136 Consequently, the removal of fluoride from water by applying ACP-500 was found to be higher than that by ACP-250. The XRD pattern of ACP-500 reveals peaks at 20 = (24.55, 33.52, 36.04, 41.23, and 62.77o) that were assignable by their indices (220), (311), (400), (422), and (440), respectively. 32-33 All these peaks are in accord with the database in JCPDS file (82-1533), and represent the iron oxides (for example Fe3O4). The diffraction peaks at (49.88 and 54.29) corresponding to the (131) and (230) planes, respectively, represent Fe3C. Carbides of iron may be classified into two categories on the basis of the location of carbon atoms present either in trigonal-prismatic interstices (Fe3C and Fe5C2), or octahedral interstices (e.g., Fe2C, Fe2.2C). The formation of the carbides may increase the mechanical strength of the synthesized nano/micro size material.34 3. 2. Fluoride Adsorption Study 3. 2. 1. Effect of Contact Time Figures S1 (a)-(d) show the removal percentage of ACP-250 and ACP-500 toward fluoride ion with time. It was observed that in the first 30 minute of contact time, about (35 and 58.5)% fluoride adsorption was observed by ACP-250 and ACP-500, respectively; and afterward, the adsorption rate slowed down with contact time. During the early stage of the adsorption experiment, many vacant sites are available on the surface of the adsorbent for the removal of fluoride. After a certain period of time, fluoride ion is difficult to attach to the surface of the adsorbent, because of the repulsive force between the adsorbed fluoride ion on the surface, and fluoride ion in the aqueous solution. The removal of fluoride increased along with time, and then progressively reached equilibrium at 180 min. Therefore, 180 min is considered the minimum contact time to maximum removal of fluoride from the water. A similar result was also reported by Dwivedi 35 and Singh and Majumder.36 3. 2. 2. Effect of the Amount of Biosorbents The removal of fluoride ions by ACP-250 and ACP-500 was performed at different adsorbent doses i.e. (0.5, 1.0, 2.0, and 3.0) g/L, while keeping all other parameters constant, such as pH (6.6±0.5), initial fluoride concentration 10 mg/L, temperature 25 °C, with different time intervals i.e. 30, 60, 120, 180, 240, and 300 min. Figures S1 (c) and (d) indicates the removal percentage of fluoride of both of the adsorbents. The adsorption of fluoride by adsorbent was observed to be increases from 32.5 to 47% for 0.5 g/L, and the maximum removal was observed 70% in case of 1.0 g/L. While in the case of 2.0 and 3.0 g/L, the adsorption removal gradually decrease from 41 to 29.5% and 49.2 to 39%, respectively for both the bioadsorbents. The highest removal percentage was shown for both bioadsorbents at 1.0 g/L dosage. The increase of the percentage of fluoride removal with increasing biosorbent dose might be due to the avail- ability of more active site on the adsorbents surface for the binding of fluoride ions with the increased surface area,37 While further increase of the biosorbent dose, the removal percentage was not increased, because of the partial aggregation of biomass, which resulted in decrease in the active surface area of both biosorbents for the adsorption.38 3. 2. 3. The Effect of pH Values The pH of a solution is a critical aspect that affects the adsorption process, due to its close relationship with the surface charge of the biosorbents, and the ionization potential of fluoride ion; therefore fluoride adsorption on adsorbent was performed at different pH values ranging (4-10), while keeping all other parameters constant. The required pH was adjusted by adding 0.1 N NaOH or 0.1 N HCl. Fig. S1 (b) shows the effect of pH on the removal of fluoride. The result shows that the adsorption of fluoride on ACP-250 and ACP-500 observed a maximum of 76.5 and 74% respectively, at pH 4. It was perceived that the removal of fluoride is extremely sensitive to the change in pH of the solution. It was also observed that fluoride adsorption decreased approximately (44 and 26.4) % for ACP-250 and ACP-500, respectively, with increasing pH of the solution from (4 to 10). Fluoride adsorption decreases may be due to the following facts: (i) the negative charge increased on the surface of adsorbent as increased pH (> pHzpc is around 3.0 for both adsorbents) of solution, which reduces adsorption of the fluoride ions due to electrostatic repulsion; and (ii) enhancement of OH- ions reduces the adsorption of fluoride ions, as it increases the competition for adsorption sites.39 3. 2. 4. Effect of Various Fluoride Concentrations on the Adsorption Process The effects of various fluoride concentrations on its removal were studied at different concentrations in the range (1-30) mg/L with keeping all the other parameters constant, such as pH (6.6±1), temperature (25±1) °C, with different time intervals i.e. 30, 60, 120,180, 240, and 300 min, and shaking speed 100 rpm. Fig. 4 (a) and (b) show the removal percentage of fluoride. The results show that when an initial concentration of fluoride increased from (1 to 30) mg/L, the removal percentage decreased from (80 to 24) % for ACP-250, and (86 to 38.33) % for ACP-500. This might have happened because of the fixed number of active sites of the adsorbent, which might be saturated at a certain concentration. 3. 2. 5. Effect of Temperature Temperature is an essential parameter for the adsorption mechanism. Temperature determines the type of relations between the biosorbent and fluoride ions. If the removal percentage decreases with enhancing the tempera- Ibrahim et al.: Adsorptive Removal of Fluoride from Aqueous Solution ... Acta Chim. Slov. 2019, 66, 123-136 129 Time (min) Time (mill) Figure 4. Effect of various parameters (a) and (b) initial fluoride concentration, (c) and (d) temperature of ACP-250 and ACP-500 for the removal of fluoride. ture, this may represent physical adsorption or exothermic process2. In the present study, the impact of temperature on fluoride adsorption by ACP-250 and ACP-500 was studied at four different temperatures of (25, 35, 45, and 55) °C, while keeping all the other parameters constant. Fig. 4 (c) and (d) show the removal percentage of fluoride. The removal percentage of fluoride was observed to decrease with increase of the temperature of the solution. This represents that the fluoride removal from aqueous solution by both biosorbents is an exothermic process.40 Further it is proved by thermodynamic studies at section 3.5. 3. 3. Adsorption Isotherm Studies An adsorption isotherm defines the interaction between pollutant and adsorbent with adsorption properties and equilibrium information at equilibrium, a point where no further adsorption take place. To understand the nature of the interaction between the adsorbent and amount of adsorbate at a constant temperature, the adsorption isotherm is applied.21 In the present work, to understand the adsorption behavior, the Langmuir and Freundlich isotherm model were applied.41-42 3. 3. 1. Langmuir Isotherm Model The Langmuir adsorption isotherm model describes a monolayer with homogeneous adsorption, in which all sites of the adsorbent hold an equal affinity for the pollutant.43 This model also describes an equilibrium saturation point, where no further adsorption can take place.44-45 The monolayer adsorption model is given as Eq. (3). Cç__ 1_ 1 Re Qo bQ0Ce (3) where, qe is the amount of F- adsorbed per unit mass of adsorbent (mg g-1); Q0 and b are the Langmuir constant concerning the adsorption capacity (mg/g) and the binding energy constant (L/ mg), respectively. The values of Q0 and b can be obtained using linear plot between Ce/qe and 1/Ce at different fluoride concentrations (Figs. 5a and b). The Langmuir isotherm is stated as a dimensionless constant (Rl), and also denoted as a separation factor that can be calculated by applying the following equation. (4) Where, Co is the initial fluoride concentration (mg L-1), and the value of RL indicates the adsorption process i.e. if the value of RL lies between 0 and 1, then the adsorption process is favorable, RL=1 signifies linear adsorption; if RL=0, the adsorption process is irreversible; and RL > 1 represents uncomplimentary adsorption, while favorable (0 < RL < 1). Table 1 shows the RL values (0 < RL < 1) for both materials. Ibrahim et al.: Adsorptive Removal of Fluoride from Aqueous Solution ... 130 Acta Chim. Slov. 2019, 66, 123-136 3. 3. 2. Freundlich Isotherm Model The Freundlich adsorption isotherm describes the reversible, as well as non-ideal, adsorption. This model has been used only for multilayer adsorption with different binding energy spectra. The linear form of the Freundlich isotherm equation is given below: Where, Kf and n are the Freundlich isotherm constants that represent the adsorption capacity and adsorption intensity of the fluoride ions on the biosorbents. These values can be obtained from the linear plot of ln qe vs. ln Ce. The values of the Freundlich constants 3. 4. 1. Kinetics Studies Adsorption kinetics studies describe the adsorption rate and mechanism of fluoride adsorption on biosorbents. Two different types of kinetics model viz, the Lagergren first-order kinetics model and the pseudo second kinetics model were applied to understand the kinetics of fluoride adsorption. The Lagergren pseudo- first-order by Ho and McKay 46 and pseudo second kinetics model 47 are expressed as Eqs. (6) and (7), respectively. (a) (b) ♦ 5ppm HOppm Al5ppm X20ppm JOOppm (c) 120 180 t (min) 300 120 180 t (min) 300 Figure 5. Adsorption isotherm and kinetics of fluoride onto ACP-250 and ACP-500 for (a) and (b) the Langmuir isotherm, and (c) and (d) second order kinetics. Kf are (3.5886 and 4.2833) for ACP-250 and ACP-500, respectively. Table 1 shows that the values of 1/n are (5.4347 and 2.7593) for ACP- 250 and ACP-500, respectively. The results show that fluoride adsorption follows the monolayer Langmuir isotherms (R2= 0.9772 for ACP-250, and 0.9963 for ACP-500). where, k1 and k2 are the rate constants of the pseudo first order and pseudo second order kinetics, respectively; qe (mg g-1) is the adsorption capacity of the biosorbent at equilibrium, and qt (mg g1) is the amount of fluoride adsorbed on the adsorbent surface at time t (min-1). The values of k1, k2, and qe were obtained from the linear plot of log (qe-qt) vs. t and t/qt vs. t. Ibrahim et al.: Adsorptive Removal of Fluoride from Aqueous Solution ... Acta Chim. Slov. 2019, 66, 123-136 131 Table 1: Isotherms Parameters for adsorption of fluoride on the ACP-250 and ACP-500 Isotherms Parameters Values Langmuir ACP-250 ACP-500 QO(mg/g) 7.5757 12.6262 b(L/mg) 0.4404 0.4634 R, 0.6942 0.6833 Freundlich R2 0.9772 0.9963 kf(mg/g) 2.2344 3.2433 N 2.4324 1.9531 R2 0.9353 0.9513 Table 2 lists the results of the Lagergren first-order kinetics and pseudo second kinetics model (Figures 5 (c and d)) and their regression coefficient (R2). Pseudo-First-order theoretical adsorption capacity (mg/g) of fluoride was increased from 0.83 mg/g to 7.3 mg/g for ACP-250 and from 0.57 mg/g to 11.6 mg/g for ACP-500 with increasing concentration of fluoride from 1 mg/L to 30 mg/L. However, pseudo-second-order theoretical adsorption capacity (mg/g) of fluoride was also increased for both adsorbents ACP-250 and ACP-500, adsorption capacity for ACP-250 was almost similar to pseudo-first-order theoretical adsorption capacity. However, it was increased from 0.89 mg/g to 14.3 mg/g for ACP-500 with increasing concentration of fluoride from 1 mg/L to 30 mg/L. In comparison of the two models, the pseudo-second-order kinetics model fitted well with the adsorption data, as compared to the pseudo-first order kinetics model. 3. 4. 2 Intra-Particle Diffusion Model (IPDM) IPDM represents the three steps for the adsorption of contaminant on to the surface of adsorbent,48 in the Table 2: Kinetics parameters of Pseudo first, Pseudo second and Intraparticle diffusion model for fluoride adsorption on ACP-250 and ACP-500 Pseudo First Order ACP-250 ACP-500 Co(mg/L) Qe (mg g-1) ki ads (min-1) R2 Qe (mg g-1) k ads (min-1) R2 1 0.825 -0.00326 0.9091 0.5757 -0.0039 0.8007 2.5 0.825 -0.00287 0.6926 1.8513 -0.0024 0.8405 5 3.6 -0.00243 0.6608 2.5153 -0.003 0.7729 10 4.7 -0.00408 0.8914 3.8010 -0.0053 0.8425 15 5.2 -0.00126 0.3642 10.0855 -0.0034 0.9923 20 6.3 -0.00343 0.7567 9.4566 -0.0026 0.9901 30 7.3 -0.0076 0.9957 11.6064 -0.0019 0.9787 Pseudo Second Order ACP-250 ACP-500 Co(mg/L) Qe (mg g-1) K2 ads (min-1) R2 Qe (mg g-1) K2 ads (min-1) R2 1 0.825 0.3755 0.9452 0.8858 0.1288 0.9974 2.5 1.865 0.04817 0.9941 2.3889 0.0135 0.9699 5 3.6 0.07282 0.9992 4.1220 0.00473 0.9963 10 4.7 0.03008 0.9986 7.1022 0.04044 0.9996 15 5.2 0.01738 0.9952 10.2040 0.00204 0.9391 20 6.3 0.05227 0.9984 11.2612 0.00293 0.9765 30 7.3 0.02057 0.9978 14.3061 0.00095 0.8763 Intraparticle Diffusion ACP-250 ACP-500 Co(mg/L) Kid(mg g 1min -1) C(mg-1) R2 Kid(mg g1 min- 1) C(mg-1) R2 1 0.0478 0.076 0.9302 0.0456 0.2146 0.7769 2.5 0.0998 0.4898 0.7837 0.1299 0.2019 0.9157 5 0.1774 1.1369 0.6774 0.2088 1.0068 0.7863 10 0.2429 1.2854 0.7534 0.3452 2.2925 0.6642 15 0.2459 1.4985 0.6932 0.5522 0.2456 0.9445 20 0.3027 2.1699 0.6187 0.6149 0.9012 0.6645 30 0.3894 1.8909 0.7581 0.7478 0.2924 0.9547 Ibrahim et al.: Adsorptive Removal of Fluoride from Aqueous Solution ... 132 Acta Chim. Slov. 2019, 66, 123-136 first step, adsórbate is adsorb onto the surface of adsorbent through the external surface or prompt adsorption process; in the second step steady adsorption step which shows the controlled intraparticle diffusion, and in the third step, adsorbate transfers from larger pore to micropores consequently decrease in adsorption rate and finally attaining equilibrium step. Another kinetics model including IPDM has also described the mechanism of fluoride adsorption by the adsorbent. The IPDM was proposed by Weber and Morris 49 by Eq. (8): where AH is the change in enthalpy and AS° is the change in entropy. c c„ ag = -RT InK , „ AS AH In Kr —--- c R RT (9) (10) (11) qt= Kid t * + C (8) Where, Kid (mg g-1 min-1) is the Intraparticle diffusion rate constant, C (mg g-1) is the thickness of the boundary layer, and the values of Kid and C are calculated from the slope and intercept of qt vs.t1/2 plots (Figs. S2 (a) and (b)), respectively. Table 2 shows the obtained value. According to IPDM, if the adsorption of a solute is controlled by the intra-particle diffusion process, a plot of qt versus t1/2 passes through the origin. Therefore, it was concluded that intraparticle diffusion are involved in the adsorption of Fluoride onto ACP-250 and ACP-500. 3. 5. Study of Adsorption Thermodynamics The thermodynamics parameters could be responsible for comprehensive information about the essential energy and structural change after adsorption. The thermodynamics parameters were calculated as Eqs. (9)-(10): the change in Gibbs free energy (AG°) (KJ mol -1), where, Kc is the equilibrium constant, qe is the quantity (mg/g) of fluoride adsorbed on the surface of biosorbent at equilibrium, Ce represents the equilibrium concentration of fluoride in a solution (mg/L), and R denotes the universal gas constant (8.314 J/mol K), while T is the absolute temperature (K) respectively. Fluoride adsorption experiments were completed at different temperatures of (25, 35, 45, and 55) °C, and showed that the removal percentage of fluoride decreased with increased absolute temperature. The changes in enthalpy and entropy were calculated from the plot of ln Kc vs. 1/T, which are shown in Fig. S2 (c) and (d), and Table S2 gives the obtained values. The results show that the adsorption of fluoride depends on temperature. The negative value of AG° for ACP-250 represents a non-spontaneous process; however, the value of AG° for ACP-500 specifies that the adsorption process favorable at (25 and 35) °C temperature.50 The process of thermodynamics was exothermic which was confirmed by the negative value of AH° and AS° means the adsorption decreases with increase in temperature. The opportunity for favorable adsorption can be represented by the negative value of AS° for both the bioadsorbents.51 12 3 4 Reuse Cvcle Figure 6. Reusability potential of the synthesized biosorbents (ACP-250 and ACP-500). Ibrahim et al.: Adsorptive Removal of Fluoride from Aqueous Solution ... Acta Chim. Slov. 2019, 66, 123-136 133 4. Regeneration Stability Test The adsorption and desorption efficiency are the significant characteristics of an adsorbent. The reproduc-ibility of an adsorbent decreases its overall cost, hence increases its value in the continuous batch experiment. The desorption of fluoride from ACP-250 and ACP-500 for the regeneration of active site was performance in 0.1 M sodium hydroxide (NaOH) solution as a desorbing agent for two hours; the adsorbent was then washed with distilled water, until the solution pH became neutral. The regenerated adsorbents (ACP-250 and ACP-500) were tested for fluoride removal from aqueous solution. The regeneration experiment was conducted with a concentration of 5.0 mg L-1 of aqueous fluoride solution during the beginning of every cycle. Fig. 6 shows the results of reusability. For the (1st 2nd, 3rd, and 4th) cycle, the removal efficiencies of ACP-500 were (80, 76.3, 70.81, and 68.08)%; however for ACP-250, they were (80, 67.3, 60.8, and 50)%, respectively. The results clearly show that both biosorbents can be reused effectively to remove fluoride ion from water. However, ACP-500 has higher regeneration efficiency, as compared to ACP-250. 6. Comparison of the Fluoride Adsorption Capacity of ACP and other Biomass-Based Absorbents Comparisons of the maximum fluoride adsorption capacity of ACP-250 and ACP-500 with a wide variety of adsorbent have been reported. Table 3 shows that the Langmuir adsorption capacity of fluoride was (7.58 and 12.63) mg/g) using ACP-250 and ACP-500, respectively, which values are higher than those of activated alumina, wheat straw raw, original tea, Moringa indica based activated carbon, activated bagasse carbon, biomass carbon at Table 3: Comparison of the defluoridation capacities of different biomass based sorbents Adsorbent Adsorption capacity References (mg/g) Activated alumina 7.6 (Ku and Chiou, 2002)52 Wheat straw raw 1.9 (Yadav et al, 2013) 54 Original tea 3.83 (Caia et al., 2015)55 Tea-Fe 10.47 (Caia et al., 2015)55 Moringa indica based 0.23 (Karthikeyan et al., 2007)56 activated carbon Wheat straw raw 1.93 (Yadav et al., 2013)54 Activated bagasse carbon 1.15 (Yadav et al., 2013)54 Biomass carbon at 300 °C 0.52 (Sinha et al., 2003)57 Biomass carbon at 600 °C 1.54 (Sinha et al., 2003)57 Pine wood biochar 7.66 (Mohan et al., 2012)58 ACP-250 7.8247 This study ACP-500 13.0039 This study 300 °C, and biomass carbon at 600 °C based adsorbents. It requires less time (equilibrium-3hrs) to remove almost all amount of fluoride in comparison with some other stud-ies.52,53 Removal of fluoride was not much affected by pH of the aqueous solution so it can be applied for the removal of fluoride from ground water as well as from wastewater of any pH. For ACP-500 maximum 80.5% removal was observed at pH 4 and at alkaline medium i.e. pH 8, about 56% removal was obtained. While ACP-250 shows good results at pH 4. As shown in the Table 3, it was observed that the adsorbents of present study having higher adsorption capacity than other adsorbent. 7. Cost Analysis for Fluoride Removal The cost of the fluoride treatment by applying biosorbents is dependent on various factors, such as the adsorption efficiency of the adsorbent, reuse capability scale of the application, etc. The present study showed some important efforts of the regeneration and reuse of ACPs in successive cycles, which indicate that ACP-250 and ACP-500 were used to remove fluoride up to four cycles. Table S3 summarizes the cost of some of the material that is used for the removal of fluoride. As the present adsorbent is cost effective and yet it is non-commercial but can be commercialized. Citrus limetta pulp alone could not be used directly as an adsorbent for fluoride removal because it leaches in the solution as well as shows negligible adsorption. Hence, modification is done to increase the removal efficiency of the material. Material developed after modification makes adsorbent magnetic and it becomes very easy to separate the adsorbent through magnet and can be recycled many times. The cost of activated carbon prepared from pulp was calculated on the basis of raw material cost and process cost. 8. Conclusion Low-cost highly efficient magnetic or iron permeated activated carbon (ACP-250 and ACP-500) were synthesized from Citrus limetta pulp waste, and applied for the removal of fluoride ions from aqueous solution. FTIR analysis of ACP-250 and ACP-500 confirmed that fluoride-adsorbing groups were present on the surface of biosorbents. EDX results show that carbon was the dominant element in both biosorbents (Carbon content of 58% in ACP-500 and 50.3% in ACP-250. XRD results show that iron was present in the form of oxides and carbide. Slightly acidic pH and 25 °C temperature were favorable for the removal of fluoride from an aqueous solution using magnetically active resource materials. Adsorption of fluoride followed the monolayer Langmuir isotherm model and pseudo-second-order kinetics. This study confirmed that ACP-500 showed higher removal efficiency of fluo- Ibrahim et al.: Adsorptive Removal of Fluoride from Aqueous Solution ... 134 Acta Chim. Slov. 2019, 66, 123-136 ride than ACP-250. It was observed that the synthesized bioadsorbent can be used up to four cycles with effective removal of fluoride and the cost analysis proved that the synthesized biogenic iron permeated activated carbon was cost effective. Therefore, waste biomass of Citrus limetta could be applied as highly proficient material for the removal of fluoride from the water. 9. Acknowledgment The authors are grateful to the Science and Engineering Research Board, for providing financial support for this work (Department of Science and Technology (SERB-DST) Sponsored Project No. ECR/2016/001924). 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Chang, S. H. Kang, J. K. Yang, Process Saf. Environ. Prot. 2016, 99, 88-97. D01:10.1016/j.psep.2015.10.011 51. M. E. Mahmoud, G. M. Nabil, N. M. El-Mallah, H. I. Bas-siouny, S. Kumar, T. M. Abdel-Fattah, J. Ind. Eng. Chem. 2016, 37, 156-167. D0I:10.1016/j.jiec.2016.03.020 52. Y. Ku, H. Chiou, Water Air Soil Pollut. 2002, 133, 349-361. D0I:10.1023/A:1012929900113 53. H. Huang, J. Liu, P. Zhang, D. Zhang, F. Gao, Chem. Eng. J. 2017, 307, 696-706. DOI: 10.1016/j.cej.2016.08.134 54. A. K. Yadav, R. Abbassi, A. Gupta, M. Dadashzadeh, Ecol. Eng. 2013, 52, 211-218. DOI: 10.1016/j.ecoleng.2012.12.069 55. H. M Caia, G. J. Chena, C. Y. Penga, Z. Z. Zhanga, Y. Y. Donga, G. Z. Shanga, X. H. Zhua, H. J. Gaob, X. C. Wana, Appl. Surf. Sci. 2015, 328, 34-44. DOI: 10.1016/j.apsusc.2014.11.164 56. G. Karthikeyan, S. S. Llango, Iran. J. Environ. Healt. Sci. Eng. 2007, 4, 21-28. 57. S. Sinha, K. P. Pandey, D. Mohan, K. P. Singh, Ind. Eng. Chem. Res. 2003, 42, 6911-6918. DOI: 10.1021/ie030544k 58. D. Mohan, R. Sharma, V. K. Singh, P. Steele, C. U. Pittman, Ind. Eng. Chem. Res. 2012, 51, 900-914. DOI: 10.1021/ie202189 Povzetek Tekom študije smo uspešno sintetizirali biogeni aktivni ogljik iz ostankov pulpe limete (Citrus limetta) in ga uporabili za odstranjevanje fluoridnih ionov iz vodne raztopine. Za sintezo aktivnega oglja smo surovino segreli v pečici pri dveh različnih temperaturah (250 °C in 500 °C) in vzorca označili kot ACP-250 in ACP-500. Pripravljen biosorbent smo kar-akterizirali z vrstično elektronsko mikroskopijo (SEM), infrardečo spektroskopijo s Fourierjevo transformacijo (FTIR) in rentgensko difrakcijo (XRD). Šaržne študije adsorpcije smo izvedli pri različnih temperaturah, količini, pH vrednostih in začetnih pogojih. Za razumevanje adsorpcijskega mehanizma smo določili adsorpcijske izoterme in reakcijsko kinetiko. Rezultati študije so pokazali, da je bila maksimalna odstranjena količina ionov znašala za ACP-500 86 % in za ACP-250 82 %. Izoterme najbolje opišemo z Langmuirjevo izotermo, z enoslojno adsorpcijsko kapaciteto fluoridnih ionov 12.6 mg/g. Vendar pa kinetiko vezave dobro opišemo kot reakcijo psevdo-drugega reda. Sintetizirani material je bil učinkovit za odstranjevanje fluoridinih ionov iz vode pri različnih temperaturah in bil uporaben tri do štirikrat. Ibrahim et al.: Adsorptive Removal of Fluoride from Aqueous Solution ... DOI: 10.17344/acsi.2018.4726 Acta Chim. Slov. 2019, 66, 136-144 /^creative ^commons Scientific paper Fabrication of Silver-Modified Halloysite Nanotubes and their Catalytic Performance in Rhodamine 6G and Methyl Orange Reduction Maryam Akhondi and Effat Jamalizadeh* Department of Chemistry, Shahid Bahonar University of Kerman, Kerman, Iran. * Corresponding author: E-mail: jamalizadeh@uk.ac.ir, jamalizadeheffat@yahoo.com. Tel./fax: +98 34 33257433 Received: 09-12-2018 Abstract Halloysite nanotube supported Ag nanoparticles (Ag/HNT) as catalyst for reduction of Rhodamine 6G (Rh6G) and Methyl orange (MO) have been synthesized and tested. 3-glycidyloxyproyltrimethoxysilane and Triethylene tetramine were successfully utilized to modify the HNTs surface, then Ag+ ions were reduced to AgNPs on this functionalized HNT surface. The structure of AgNPs-impregnated HNT wall was characterized by FTIR, XRD, TEM, FE-SEM and EDX showing the AgNPs to be 10-15 nm in size. The catalytic activity of Ag/HNTs for Rh6G and MO reduction was investigated by UV-vis spectroscopy so as catalyzed and uncatalyzed reaction rate constants could be compared. The rate constant (k) of Rh6G and MO reduction was calculated for catalyzed reactions as 0.224 min-1 and 0.222 min-1, and for un-catalyzed reactions as 0.049 min-1 and 0.014 min-1, respectively. The Ag/HNT catalyzer was effectively recycled six times without appreciable loss of activity. Results indicate that supporting AgNPs on HNT surface functionalized with other compounds with superior performance in reducing Rh6G and MO dyes exist. Keywords: Catalyst; Ag nanoparticles; Functionalized halloysite; GPTMS; TETA 1. Introduction In recent years, great strides have been taken in use of metal nanomaterials as heterogeneous catalysts.1-3 Na-nomaterials have special properties such as the surface and interface effect which arise from the alteration of boundary between material and its surrounding, small size effect due to decrease in atom density of amorphous nanoparti-cles near the surface layer, quantum size effect arising from confinement of electrons to small regions of space in one, two, or three dimensions and also macroscopic quantum tunneling effect, leading to some new and potential applications such as catalysis, bio-engineering, medicine.4-6 Industrial dye production and use apart from forming toxic by-products and non-biodegradable wastes that affect plants, animals and fish, are suspect as potential carcino-gens.7,8 Therefore, depending on the local laws and bylaws, to prevent environmental pollution, industrial wastewa-ters retaining organic dyes need treatment to remove the residual compounds. Various techniques for removal of dye molecules exist such as membrane filtration, ion exchange, biological treatment, coagulation-flocculation, ozone flashing, Fentons reagent.9-13 The coagulation-floc- culation process is widely used, due to its simplicity and cost-effectiveness,9 however, it just transfers the dye molecules from liquid to solid form.14 Catalysis as an efficient method to treat wastewater bearing colorants has attracted much interest.15 Among different type of metal nanoparticles, noble metal nanoparticles have attracted extensive attention as chemical catalyst.16-18 Among these, Ag is particularly striking due to its remarkable catalytic activity and low cost. A significant number of studies on the catalytic reactions of Ag nanoparticles (AgNPs) have been reported, such as alcohol dehydrogenation,19 oxidation of phe-nylsilanes,20 reduction of aromatic compounds,21 and Diels-Alder cycloadditions.22 Since during a chemical reaction, the active surface atoms dislodge the Ag-NPs,22-27 various supports such as polymeric28,29 and inorganic materials, including silica,30,31 zeolite,32,33 alu-mina,34,35 ceria,36 titania,37,38 zirconia,39 activated car-bon,40,41 carbon nanotubes (CNTs),42,43 and halloysite nanotubes (HNTs),44-46 have been used to immobilize AgNPs. Halloysite silicate nanotubes of about 15 nm lumen, 50 nm external diameter and1000-2000 nm length are abundant in nature. Furthermore, HNTs are biocom- Akhondi and Jamalizadeh: Fabrication of Silver-Modified Halloysite Nanotubes and Acta Chim. Slov. 2019, 66, 136-144 137 patibile,47,48 chemically stabale,49 with high absorption capability7 and potential to settle down quickly. HNTs (Al2Si2O5(OH)4 nH2O) have two-layered (1:1) alumino-silicate with aluminol (Al-OH) group in the internal and Si-OH group on the external surfaces. Presence of hydroxyl groups allows easy surface functionaliza-tion.50-53 Existence of halloysite signicantly improves the size control of Ag nanoparticles as Ag nanoparticles without halloysite get to larger size and also aggregate heavily.54 Nanotube supported Ag nanoparticles (Ag/ HNT), are used with various precursors and for different applications such as biosensor,55-56 polymerization catalyst,44 photo catalyst,45,46 enzyme immobilization,55 dielectric material57 and antibacterial usage.58,59 In addition, the Ag/HNTs show high catalytic activity even at minute amounts towards reduction and degradation of some industrial dyes, 4-nitrophenol60-62 or methylene blue.62 The purpose of this paper is to enhance the catalytic effect of Ag nanoparticles as a heterogeneous catalyst to reduce 6G Rh6G and MO dyes by supporting them on the walls of halloysite nanotubes modified with new compounds. Such modification of the external surface of HNTs was achieved, and then in situ reduction of Ag+ to Ag nanoparticles on the functionalized HNTs surface resulted in synthetized Ag/HNTs which was then utilized as a heterogeneous catalyst in the degradation of Methyl orange and Rhodamine 6G in aqueous solution (Fig. 1). The recycla-bility of the Ag/HNTs catalyst was tested by carrying out repeated runs of the same reaction system. 2. Methods 2. 1. Materials Fig 1. Reduction of Rh6G and MO by NaBH4 catalyzed by the Ag/ HNT. The halloysite nanotube powder was provided by New Zealand China Clays Ltd., Auckland, New Zealand. The Sodium borohydride (NaBH4 > 99.99%), Sodium hydroxide (NaOH > 97.00%) and hydrochloric acid (HCl > 37.00%) were acquired from the Sigma Aldrich. The silver nitrate (AgNO3 > 99%), 3-glycidyloxyproyltrimethoxysi-lane (GPTMS > 98%), Triethylene tetramine (TETA), Eth-anol, MO and Rh6G were purchased from Merck. 2. 2. Instruments UV-vis spectra were recorded on a spectrophotometer (Varian, Cary50), with a quartz cuvette (1.0 cm path length, and 4 mL volume). Scanning electron microscope images were obtained with a field-emission scanning electron microscope (FE-SEM) KYKT, EM-3200. Transmission Electron Microscope (TEM) images were recorded from JEOL 2100F TEM. XRD patterns were obtained in reflection mode using a powder X-ray diffractometer (X'Pert Pro MPD Philips X-ray diffractometer (Ni-fil-tered Cu-Ka radiation, X = 0.154 nm). FT-IR spectra of samples as KBr discs were recorded on a FT-IR 8400-Shi-madzu in the range 400-4000 cm-1. The pH measurements were performed using a pH-meter (METROHM, Model 713). 2. 3. Functionalization of HNTs As shown in Fig 2, for functionalization of HNTs by grafting the GPTMS containing epoxy groups on the surface of HNTs via silanization; 1 mL of GPTMS was added to 0.5g of HNTs dispersed in 7 mL of ethanol and was stirred at 65 °C for 24 h and then centrifuged to collect the suspension. The precipitate (GPTMS/HNT) was washed five times with pure ethanol and dried at 60 °C for 10 h. Following that, TETA was grafted on tubes via ring opening reaction of epoxy groups by adding 6mL of pure ethanol containing TETA (0.1 mg L-1) to 0.5 g of GPTMS/ HNTs, stirred and refluxed for 24 h at 70 °C. This mixture was then centrifuged and the precipitate (TETA/HNT) was again washed with ethanol five times, dried at 70 °C for 10 h. 2. 4. Synthesis of Ag/HNTs Nanocomposite The Ag/HNT composite was prepared using AgNO3 as silver precursor, so that silver ions get reduced to silver nanoparticles on TETA/HNT's surface amine functional groups. In a typical synthesis process, 0.5 g of TETA/HNTs was added to 10 mL of deionized water under stirring for 1 h and then, 1 mL of 0.2 M AgNO3 solution was added dropwise to the mixture and left stirring for 30 min. The solution was reduced by addition of 2 mL of freshly pre- Akhondi and Jamalizadeh: Fabrication of Silver-Modified Halloysite Nanotubes and 138 Acta Chim. Slov. 2019, 66, 136-144 Fig 2. Schematic Illustration of the role of GPTMS and TETA in the formation of the TETA/HNTs. pared 0.1 M aqueous NaBH4 solution. The color of the reaction mixture turns from white to dark green, indicating formation of Ag NPs on the surface of HNTs (Ag/HNTs). The final product was washed several times with de-ionized distilled water to remove unreacted Ag+ ions and any byproducts. All experiments were performed at room temperature. 2. 5. Catalytic Reduction of Dyes by Ag/HNTs Nanocomposite Catalytic activity of Ag/HNTs nanocomposite, on decomposing Rh6G and MO dyes was investigated by comparing the rate they reacted with NaBH4 in the presence and absence of nanocomposite. The reduction of dyes was carried out in the quartz cell by admixing 1.0 mL of 1mM NaBH4 as reducing agent to a mixture of 2 mL of aqueous dye solution and 1 mg of Ag/HNTs nanocompos- ite. The process was continuously monitored by a UV-vis spectroscopy at room temperature. In order to determine its reusability after the reaction was over, the Ag/ HNT catalyst was centrifuged and washed with deionized water for two times, and dried in vacuum for 24 h at 50 °C, to be used again in dye decomposition processes. The reduction of Rh6G and MO with an excess amount of NaBH4 has been used as a model reaction to examine the catalytic performance of the Ag/HNTs. The reaction kinetics can be described as Eq. (1):40 ln(C/Co) = - kt (1) where C° is the initial concentration of dye, C is the concentration of dye at time t, k is the apparent first-order rate constant (min-1), and t is the time. Since the concentration of a solution is proportional to its adsorption, Eq. (1) can be written as follow: ln(A/A°) = - kt (2) A° and A are respectively, the initial and time t dye adsorption. Degradation efficiency (%D) of Ag/HNT is calculated according to the following Eq. (3): = 100 Ao (3) Rh6G and MO reduction was investigated under various solution pH values of 3, 5, 7, 9 and 11 using sodium hydroxide and hydrochloric acid were also performed. 3. Results and Discussion 3. 1. FTIR Spectra The FTIR spectra were used to determine the chemical structure of the HNTs during the functionalization process. As shown in Fig 3, the characteristic peaks of HNTs occurs at 3698 cm-1 and 3623 cm-1; attributable to O-H stretching of hydroxyl groups.63 Peaks observed at 1109 cm-1 and 1034 cm-1 are ascribed to in-plane stretching of Si-O bond.63 FTIR of the modified halloysite with GPTMS (GPTMS/HNT) shows new peaks at 2925 and 2854 cm-1, which is assigned to the asymmetric and symmetric stretching vibration of aliphatic -CH2 groups. This can also confirm the existence of GPTMS grafted on the HNTs.64 For GPTM/halloysite grafted by TETA (TETA/ HNT), in addition to the presence of CH2 bands at 2946 cm-1 and 2871 cm-1, there are also two new peaks at 1200 and 1600 cm-1, belonging to the C-N and NH2 groups, re-spectively.65 These results can also show the interaction between TETA and epoxide ring of GPTMS grafted on the HNTs indicating tghat nanotubes have been successfully functionalized with amine groups. In comparing these results with the FTIR spectra of pure HNTs, the positions of Akhondi and Jamalizadeh: Fabrication of Silver-Modified Halloysite Nanotubes and Acta Chim. Slov. 2019, 66, 136-144 139 Fig 3. FT-IR spectra of HNTs, GPTMS/HNTs and TETA/HNTs. most HNT peaks do not show any change, demonstrating preservation of main structure of HNT following GPTMS and TETA grafting. 3. 2. UV-vis Spectra Fig 4 shows the UV-vis optical absorption spectra of the HNTs and Ag/HNTs in the 200-800 nm spectral range. The UV-vis spectra of HNTs dispersed in water revealed no absorption band, while the Ag/HNTs sample revealed an absorption band in 427 nm, corresponding to the surface plasmon resonance (SPR) of Ag nanoparticles. 0.8 s « 0.6 a ■o 0.4 X5 < 0.2 -HNTs ---AgNPs/HNTs 700 800 200 300 400 500 600 Wavelength (nm) Fig 4. UV-vis spectra of HNTs and Ag/HNTs. 3. 3. FE-SEM and EDX Analysis The FE-SEM image of HNTs and Ag/HNTs are shown in Figs 5(a) and 5(b). The diameter of the HNTs particles are about 123 nm with random length. Fig 5 shows the halloysite nanotubes along with some non-tubolar structures. The particles of halloysite can adopt a multifariousness of morphologies, the most common of which is the elongated tube. However, short tubular, sphe- roidal and plate shapes have all been widely reported.66-68 The Ag nanoparticles are almost unobservable in Fig 5(b) but EDX analysis confirms the existence of Ag element on the surface of halloysites. a) — 4 6 8 Energy(KeV) b) 11000 5 10 Energy (KeV) 15 Fig 5. EDX spectra and FE-SEM images of (a) HNTs and (b) Ag/ HNTs. 3. 4. TEM Analysis TEM image of structure and morphology of Ag/ HNTs is shown in Fig 6. This reveals the hollow open ended tubular morphology of HNTs with the inner and outer Fig 6. TEM image of Ag/HNTs. Akhondi and Jamalizadeh: Fabrication of Silver-Modified Halloysite Nanotubes and 140 Acta Chim. Slov. 2019, 66, 136-144 diameters of about 15-40 nm and 50-140 nm, respectively. It also shows spherical single nanoparticles with average size of 10 nm dispersed evenly, indicating that amine terminated groups had adsorbed Ag+ ions from solution. 3. 5. X-ray Diffraction Analysis The X-ray diffraction (XRD) pattern of HNTs and Ag/HNTs are shown in Fig 7. The XRD patterns of the functionalized HNT reveal well defined diffraction peaks all corresponding to the HNT, confirming that modification with GPTMS and TETA does not affect the structure of the HNT. However, in the XRD pattern of the Ag/HNTs nanocomposite, four new peaks at 20 value of 37.7°, 43.5°, 64.2° and 77.4° emerge which are ascribed to (1 1 1), (2 0 0), (2 2 0) and (3 1 1) reflections of FCC structure of silver;46,69 so this further affirming successful attachment of AgNPs to the surface of the nanotubes. 35 45 55 2Teta(Dgree) Fig 7. XRD pattern of HNTs and Ag/HNTs. min = 0.34). It is observed that degradation of methyl orange is classically enhanced in presence of silver nanopar-ticles due to electron relay effect.25, 26 As presented in Table 1, the rate constant (k) of Rh6G and MO degradation has been calculated for catalyzed reactions as 0.224 min-1 and 0.222 min-1, and for un-catalyzed reactions as 0.049 min-1 and 0.014 min-1, respectively. a) b) 400 450 500 550 Wavelength (nm) 600 400 450 500 550 Wavelength (nm) 600 3. 6. Catalytic Reduction of Rh6G and MO The results of catalytic activity of synthesize Ag/ HNTs in reduction of Rhodamine 6G and methyl orange studied by UV-visible spectrophotometry are shown in Figs 8 and 9. Time dependent reduction of Rh6G and MO with NaBH4 in presence and absence of Ag/HNTs was observed by the successive decrease in the absorbance value at 516 and 467 nm, respectively. Results indicate that the reduction of Rh6G and MO in absence of catalyst at room temperature occurs which is greatly enhanced by addition of Ag/HNTs. Fig 8, exhibits a rapid Rh6G concentration decay in presence of Ag/HNTs in 12 min (AOD12 min = 0.4), compared to sluggish 30 min un-catalyzed reaction (AOD30 min = 0.4). Similarly, Fig 9, demonstrates rapid decay of MO in presence of Ag/HNTs in just 12 min (AOD12 min = 0.8), compared to its slow degradation needing 30 min when relying on un-catalyzed reaction (AOD30 Fig 8. Time-dependent UV-vis absorption spectra for the reduction of Rh6G with NaBH4 (a) in the presence and (b) absence of Ag/ HNTs at room temperature. 3. 7. Effect of Amount of Catalyst The amount of catalysts is an important factor affecting suitability of a catalyst for chemical reactions. Tables 2 and 3 present the optimization of degradation conditions for Rh6G and MO in early times. The Ag/HNTs catalyst amount was varied in 0.0002 g L-1 to 0.0025 g L-1 range at constant NaBH4 concentration of 4 mg L-1 and dye concentration of 10 mg L-1. According to the results, the rate of catalytic degradation of both dyes increased with increase in the amount of catalyst. This is due to the increase in the number of surface active sites and increased dye molecules adsorption on the catalyst sur-face.25 Akhondi and Jamalizadeh: Fabrication of Silver-Modified Halloysite Nanotubes and Acta Chim. Slov. 2019, 66, 136-144 141 300 350 400 450 500 Wavelength (nm) 550 600 b) 300 350 400 450 500 Wavelength (nm) 550 600 Fig 9. Time-dependent UV-vis absorption spectra for the reduction of MO with NaBH4 (a) in presence and (b) absence of Ag/ HNTs at room temperature. 3. 8. Effect of Amount of NaBH4 In order to evaluate the effect of NaBH4 concentration on degradation of dyes, this was varied the in the range of 1.5 to 16 g L-1, while keeping dye concentration at 10 mg L-1 and Ag/HNT concentration at 0.001 g L-1. Tables 2 and 3 show that with one exception, the increase in NaBH4 concentration, increases degradation of both dyes, due to higher number of electrons which promotes the simultaneous reduction of the Ag particles from its oxide and the reduction of dyes.25 However, as mentioned, the rate constant for the 8 g L-1 concentration of NaBH4 is observed to decrease. This is caused by excessive production of electrons from the ion leading to the saturation of electrons70 leaving AgNPs incapable of relaying the electrons. The catalytic reduction of dyes in the presence of NaBH4 on the surface of noble metal nanoparticles is considered to follow Langmuir-Hinshelwood mechanistic pathway,62,71 as Fig 10 demonstrates, these dyes are elec-trophilic with respect to Ag NPs and nucleophilic toward BH-4. Thus both dyes and BH-4 are adsorbed on the surface of AgNPs attached to the surface of HNTs. The fast nature of adsorption on the surface of nanoparticles and its reversibility has been reported.62,71,72 When NaBH4 is added to the reaction solution, the hydride from NaBH4 may be trapped by AgNPs and get adsorbed on the surface transfering its electron to the AgNPs. Following this electron exchange, a hydrogen atom is formed from BH-4 that subsequently attacks any nearby dye molecule, and a spontaneous electron transfer-induced hydrogenation of the dye occurs. In fact, a negatively charged AgNP may be re- garded as a nano-electrode at a negative potential which is trying to release its extra electrons which are finally released to dye molecule as an electron acceptor producing its reduced form.71,73 AgNPs on the surface of HNTs significantly increases the rate of catalytic reduction due to lower work function of Ag (4.26 eV) and its tendency to release electron more easily even compared to the other noble metals.62,74 Both nanoparticle's surface area and heterogeneous organic-inorganic hybrid structure supporting for AgNPs may contribute to reduction rate by increasing adsorption ability of reactant.62 Fig 10. Proposed mechanism of reduction of dyes by catalytic sample. 3. 9. Effect of pH The solution pH has significant effect on degradation of organic dyes. Fig 11 shows the reduction of MO and Rh6G at pH 3, 5, 7, 9, and 11 after 20 min. It was observed that the percentage of degradation of both dyes increased with decreasing pH from 7 to 3, and conversely, degradation fall at pH 9 and 11. The results show decreasing Rh6G solution pH from 7 to 3, increases the degradation efficiency from 78.0% to 79.7% while, pushing pH towards 11 decrease it to 73.6%. Similarly, decomposition of MO goes up from 73.9% to 77.1 % by reducing pH from 7 to 3 and solution pH of 11, cuts it to 70.6 %. In acidic media the MO molecules (pK = 3.4) undergo protonation74, 75 and the resulted proto-nated molecules are adsorbed better on negatively charged AgNPs, enhancing MO degradation. Similarly, the lower degradation rate observed for Rh6G at higher pH may attributed to the deprotonation of the Rh6G in alkaline solution since Rhodamine 6G (pK = 7.5) is a weak base and can easily lose proton under the effect of a strong base, which leads to partial neutralization of its positive charge.76 As a result, electrostatic interaction with negatively charged Ag/ HNT decreases decreasing the overall Rh6G degradation. Table 4 summaries the dye reduction studies reported in literature. The synthesized Ag/HNTs while perform- Akhondi and Jamalizadeh: Fabrication of Silver-Modified Halloysite Nanotubes and 142 Acta Chim. Slov. 2019, 66, 136-144 a) a) 100 300 400 500 600 700 Wavelength(nm) 800 b) 0.8 • 3 i "of u C IQ 0.6« Ö 0.4 < VI _Q / \ 0 min / \ -pH=3 \ -pH=5 \ -pH=7 \ -pH=9 200 300 400 500 600 700 800 Wavelength(nm) Fig 11. Time-dependent UV-vis absorption spectra for the reduction (a) Rh6G and (b) MO with NaBH4 in presence of Ag/HNTs at different pH values. ing on par with other Ag catalysts for the degradation of MO and Rh6G.25,77-79 although, the K value in this work is 0.21 min-1 which is slightly lower compared to the Ag supported on zeolite (0.30 min-1),77 it has the advantage of using much smaller amounts of Ag. In the present work, amount of used Ag/HNTs as catalyst is 2.4 times less than the AgNPs/Zeolite X, hence, the Ag/HNT catalysis would be more economical compared to the other catalysts reported in Table 4.25,78 not only because of low concentrations used but also due to the easy recovery and reusability. The halloysite as a natural mineral with high stability as support material for Ag nanoparticles, while Zeolite X77 is not natural and its preparation is costly. Also, although the Ag/tannic acid membrane79 has inner shell as natural substrate but this substrate is unstable. 3. 10. Reusability of the Synthesized Catalysts The environment impact by catalysts such as heavy metals which should include recovery and recycling is an important factor in choosing a catalyst.80 Due to the one-dimensional nature of Ag/HTNs its use as a recyclable and reusable catalysts which is easily recovered from the reaction mixture by simple filtration is very advanta- b) Fig 12. Recyclability and reusability of the synthesized Ag/HNTs for the reduction of Rh6G and MO. geous. The recycling experiments involving synthesized Ag/HNTs for reduction of MO and Rh6G are shown in Fig 12. The data show that the Ag/HNT catalyst could be repeatedly used for at least six times and still maintained degradation potency of over 85. Overall, Ag/HNT as a dye decomposing catalyst has a high operational stability with no appreciable loss in catalytic activity and good reusability. 4. Conclusion Halloysite nanotube supported Ag nanoparticles synthesized in situ by reduction of silver ions on the amine functional groups on the surface of TETA/HNTs greatly improves dispersion and stability of the Ag nanoparticles on HNT surfaces. The Ag/HNTs exhibit good catalytic activity in decomposition of MO and Rh6G by NaBH4 as the reducing agent. The catalytic efficiency was enhanced with the increasing amount of Ag/HNTs. Notably, the Ag/HNTs could be easily recycled due to the one-dimensional nano-structural property. These results signify that Ag nanopar-ticles strongly attached and immobilized on the surface of halloysite modified with GPTMS and TETA increase silver performance as Rh6G and MO dyes decomposer. 5. References 1. D. Kumar Dutta, B. Jyoti Borah and P. Pollov Sarmah, Catal Rev. 2015, 57, 257-305. Akhondi and Jamalizadeh: Fabrication of Silver-Modified Halloysite Nanotubes and Acta Chim. Slov. 2019, 66, 136-144 143 DOI: 10.1080/01614940.2014.1003504 2. T. 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DOI:10.1016/j.jallcom.2017.01.122 Povzetek Sintetizirali in okarakterizirali smo nanodelce srebra z vključenimi haloizitnimi nanocevkami (Ag/HNT) in jih uporabili kot katalizator za redukcijo rodamina 6G (Rh6G) in metiloranža (MO). 3-glicidilokiproiltrimetoksisilan in trietil-tet-raamin smo uspešno uporabili za kemijsko modifikacijo površine HNT na katero reducirani Ag+ ioni vezali v obliki nanodelcev (AgNP). Strukturo HNT prekritih z AgNP smo karakterizirali s FTIR, XRD, TEM, FE-SEM ter EDM in pokazali, da so nanodelci srebra veliki 10-15 nm. Katalitsko sposobnost reduciranja Rh6G in MO z Ag/HNT smo spremljali z UV-vis spektroskopijo, s čimer smo lahko primerjali hitrost katalizirane in nekatalizirane reakcije. Konstanta reakcijske hitrosti katalizirane reakcije je znašala za redukcijo Rh6G 0.224 min-1, za redukcijo MO pa 0.222 min-1, medtem ko je bila hitrost nekatalizirane reakcije le 0.049 min-1 oziroma 0.014 min-1. Ag/HNT katalizator smo uporabili 6 krat, ne da bi zaznali upad katalitske aktivnosti. Rezultati kažejo možnost obstoja Ag/HNT z izboljšanimi redukcijskimi sposobnostmi, v primeru njihove funkcionalizacije. Akhondi and Jamalizadeh: Fabrication of Silver-Modified Halloysite Nanotubes and DOI: 10.17344/acsi.2018.4731 Acta Chim. Slov. 2019, 66, 145-154 /^creative ^commons Scientific paper Directed Search of Biologically Active Compounds among Hydrogenated Isoindolylalkyl(alkylaryl-,aryl-)carboxylic Acids with Quinazoline Fragment that Modify the Carbohydrate Metabolism: Design, Synthesis and Modification Yulya Victorivna Martynenko,1 Oleksii Mykolayovich Antypenko,1 Oleksandr Anatolievich Brazhko,2 Irina Borisivna Labenska2 and Sergii Ivanovich Kovalenko1^ 1 Organic and Bioorganic Chemistry Department, Zaporizhzhya State Medical University, 26, Mayakovsky Ave., Zaporizhzhya, 69035, Ukraine 2 Chemistry Department, Zaporizhzhia National University, 62, Gogol st., Zaporizhzhia, 69095, Ukraine * Corresponding author: E-mail: kovalenkosergiy@gmail.com Received: 10-01-2018 Abstract An effective synthesis of (3ff-quinazoline-4-ylidene)hydrazides of N-carboxyalkyl-(arylalkyl-,aryl-)isoindoline-1,3-di-ones, using activated N-protected aminoacids and 4-hydrazinoquinazoline was proposed in the framework methodology of directed search ofhypoglycemic agents (fragment-oriented design, molecular docking). These hydrazides prepared via cyclocondensation under acid catalysis were converted to the corresponding 2-([1,2,4]triazolo[1,5-c]quinazoline-2-yl-) alkyl-(alkylaryl-,aryl-)-hydroisoindole-1,3(2H)-diones. The structure of synthesized compounds was established using IR, 'H and 13C NMR spectroscopy and LC-MS and the features of spectral pattern were discussed. The results of pharmacological screening revealed a series of compounds, that are short-acting hypoglycemic agents like prandial regulators of glucose (Mitiglinide). The SAR analysis showed, that the introduction of a hydrogenated 1,3-dioxoisoindole moiety bonded through linker groups with 4-hydrazinoquinazoline and triazolo[1,5-c]quinazoline cycles is reasonable in the context of searching for short-acting hypoglycemic agents and requires further research. Keywords: (3H-quinazoline-4-ylidene)hydrazides of N-carboxyalkyl-(arylalkyl-,aryl-)isoindoline-1,3-diones; 2-([1,2,4] triazolo[1,5-c]quinazoline-2-yl-)alkyl-(alkylaryl-,aryl-)hydroisoindole-1,3(2H)-diones; directed search; physico-chemical properties; spectral features; hypoglycemic activity 1. Introduction Quinazoline derivatives and their condensed analogues have always attracted the attention of medicinal chemists as objects of advanced research, aimed at the elaboration of new drugs. This fact is explained by the high biological activity of natural,1-3 as well as synthetic quina-zolines4-19 and, undoubtedly, their wide ability to chemical modification.10-13,18 In recent years, the interest for this heterocyclic system has increased greatly since the introduction of in silico approaches to the drug search strategy, combinatorial chemistry and high-throughput screening. These approaches have led to the discovery and creation of a number of lead-compounds and original drugs with antitumor, antihypertensive, diuretic, hypoglycemic and other biological activities.4,6,7,17 It should be noted, that information about the biological targets and fragment-oriented design based on them20,21 has played a key role in the creation of therapeutic molecule data. Using the above-mentioned search strategy we performed an attempt to one of the fragments-oriented design approaches based on the available results of hypoglycemic activity of N-carboxyalkyl-(arylalkyl-,aryl-) substituted hydrogenated isoindoline-1,3-diones,22 known hypoglycemic agents Martynenko et al.: Directed Search of Biologically Active Compounds among 146 Acta Chim. Slov. 2019, 66, 145-154 (Mitiglinide23,24 and Apabetalone25) and virtual screening. To enhance the pharmacological activity a combination in the same molecule of these compounds with a suitable pharmacophore quinazoline fragment was used (Figure 1). Thus, the aim of this work is a directed search for biologically active compounds, that modify the carbohydrate metabolism, based on unknown (3H-quina-zoline-4-ylidene)hydrazides of hydrogenated isoin-dolylalkyl(alkylaryl-,aryl-)carboxylic acids, their further modification toward triazoloquinazolines, the study of their structural peculiarities and establishment of the structure-activity relationship (SAR) for further optimization of their structure. detector Agilent LC/MSD SL (atmospheric pressure chemical ionization - APCI). Ionization mode was a concurrent scanning of positive and negative ions in the mass range 80-1000 m/z. Substances 1a-i were synthesized according to the reported procedures.22 Other starting materials and solvents were obtained from commercially available sources and used without additional purification. 2. 2. Molecular Docking Pancreatic beta-cell KATP channel (PDB ID - 6BAA) was used as the target protein to carry out molecular docking. Metglitinide and Glibenclamide were used as the reference drugs. The chemical formula of Glibenclamide was obtained from the mentioned PDB-file for calculating affinity value, and structure of Metglitinide was prepared as others ligands.26 Ligand preparation. Substances were constructed using Marvin Sketch 17.21 and saved as mol. files. Subsequently, using the Marvin Sketch 17.21 program,27 they high hypoglicemic activity according to22 pharmacophore combination derivatization Figure 1. The search strategy for hypoglycemic agents based on the fragment-oriented design (combination of isoindole and quinazoline moieties in molecule). 2. Experimental Section 2. 1. Chemistry General Methods Melting points were determined in open capillary tubes in a Stuart SMP30 apparatus and are uncorrected. The elemental analyses (C, H, N) were performed using the ELEMENTAR vario EL cube analyzer. IR spectra (4000-600 cm-1) were recorded on a Bruker ALPHA FT-IR spectrometer using a module ATR eco ZnSe. 1H NMR spectra (400 MHz) were recorded on Varian-Mercury 400 spectrometer with SiMe4 as the internal standard in DM-SO-d6 solution. LC-MS spectra were recorded using chro-matography/mass spectrometric system, which consists of a high-performance liquid chromatograph Agilent 1100 Series equipped with diode-matrix and a mass-selective were optimized with the help of the molecular-mechanical MM+ algorithm in conjunction with the semiempirical PM3 method of molecular modeling with the maximum number of cycles and the Polak-Ribiere algorithm. Molecular mechanics is used to obtain more realistic geometric values for most organic molecules, since it has a large number of parameters. The next step was to re-optimize MM+-optimized structures with using semiempirical PM3 molecular modeling method and the preservation of molecules in PDB files. Using Auto Dock Tools-1.5.6 these PDB files were converted to PDBQT while the rotational number of link options was typical. Protein preparation. PDB files have been downloaded from a data bank of proteins.26 Discovery Studio v17.2.0.16349 was used to remove water molecules and lig-ands from the file. After that, the protein was saved as PDB Martynenko et al.: Directed Search of Biologically Active Compounds among Acta Chim. Slov. 2019, 66, 145-154 147 file. Polar hydrogen atoms were added and the protein was saved as PDBQT into Auto Dock Tools-1.5.6. The search grid for docking the protein was set as following: center_x = 113, center_y = 191, center_z = 128, size_x = 22, size_y = 22, size_z = 22. Vina was used for proper docking. Discovery Studio v17.2.0.16349 was used for visualization.28 2. 3. Pharmacology 2. 3. 1. Hypoglycemia Activity Test Study of hypoglycemic activity was conducted on 150 Wistar white rats (male, weight 100-180 g, age 3.5 month) which were obtained from nursery of PE Biomodelservice (Kiev, Ukraine). Experiments on animals were done according to the bioethics principles.23 Selected after quarantine animals by random sampling were divided in groups of 6 male rats on the assumption of absence of external signs of diseases and homogeneity by weight (±15%). Experimental animals were not fed during 12 hours before injection of studied compounds. The weight of all animals was measured before experiment. Intragas-tric injection of studied compounds was conducted using atraumatic probe as water solution or finely dispersed suspension stabilized by Tween-80 in a dose of 10 mg/kg. Intact and control groups received equivalent volume of water by the same way. Hypoglycemic activity of the synthesized compounds was evaluated via changes of glucose level before and after injection of the studied substances. Measurements of glucose level were carried out 2, 4 and 6 hours after injection. Metformine (50 mg/kg) was used as a reference drug. Before the experiment the compounds were injected as an aqueous solution or 3-5% micronized water suspension, stabilized by Tween-80 in a dose of 10 mg/kg using atraumatic probe in order to identify prandial hypoglyce-mic agents. Subsequently, after 60 minutes, 40% glucose solution was injected to rats at a dose of 3000 mg/kg. Hy-poglycemic activity of the synthesized compounds was evaluated via changes of glucose level before and after injection of substances. Measurements of glucose level were carried out in 15, 30, 60 and 120 minutes after injection. Statistical analysis was done using standard software complex, namely Microsoft Office Excel 2003 and STA-TISTICA® for Windows 6.0 (StatSoft Inc., N AXXR712D-833214FAN5). For each estimated value arithmetic mean (M), and standard error of the mean (±m) were defined. During verification of statistical hypothesis, null hypothesis were declined if statistical criterion p < 0.05. General Procedure for the Synthesis of (3H-Quinazo-line -4-ylidene)hydrazides N- Carboxyalkyl-(arylalkyl-, aryl-)isoindoline-1,3-diones (3a-i) 1.62 g (0.01 mol) of N,N'-carbonyldiimidazole was added to a suspension of 0.01 mol of the corresponding 2-(isoindoline-1,3-dione)alkyl-(alkyl-,arylalkyl-,aryl-) carboxylic acids (1a-i) in 30 mL of dioxane. The mixture was kept at temperature 60-70 °C for 50-60 minutes until the carbon dioxide was completely released. After addition of 1.6 g (0.01 mol) 4-hydrazinoquinazoline (2a) the mixture was kept at a room temperature overnight or at 80 °C for 1.5 hour. Dioxane was distilled off. The water was added and the mixture was neutralized by 0.1 M aqueuos hydrochloric acid to pH 6-7. The solid product was filtered in the case of residue formation. If it was necessary, compounds were crystallized. 2-(1,3-Dioxooctahydro-2H-isoindole-2-yl)-N'-(quinazo-line-4(3H)-ylidene)propanehydrazide (3a). Yield 61.9%; m.p. 100-102 °C; IR (cm-1): 3297, 2917, 2849, 2693, 2353, 1704, 1615, 1550, 1472, 1386, 1194, 1130, 984, 759, 689; 1H NMR 8 (ppm): 11.46 (s, 1H, 3-NH), 10.16/10.03 (s, 1H, NHCO), 7.80-7.61 (m, 2H, H-2,5), 7.37 (t, J = 7.9 Hz, 1H, H-7), 7.19 (t, J = 7.9 Hz, 1H, H-6), 7.10 (d, J = 8.0 Hz, 1H, H-8), 5.15/4.75 (q, J = 7.0 Hz, 1H, CHCH3), 2.82-2.65 (m, 2H, isoindole H-3a,7a), 1.85-1.64 (m, 4H, isoindole H-4,4',7,7'), 1.58/1.51 (d, J = 7.1 Hz, 3H, CHCH3), 1.471.25 (m, 4H, isoindole H-5,5',6,6'); LC-MS: m/z = 367 [M]+; Anal. Calcd. for C19H21N5O3: C, 62.11; H, 5.76; N, 19.06. Found: C, 62.16; H, 5.82; N, 19.13. 4-((1,3-Dioxooctahydro-2H-isoindole-2-yl)methyl)-N'-(quinazoline-4(3H)-ylidene)benzohydrazide (3b). Yield: 72.6%; m.p. 263-265 °C; IR (cm-1): 3309, 3180, 3130, 2918, 2953, 2854, 2779, 2714, 1698, 1612, 1525, 1471, 1374, 1341, 1310, 1164, 1093, 906, 762, 690, 662, 646; 1H NMR 8 (ppm): 11.51 (s, 1H, 3-NH), 10.52 (s, 1H, NHCO), 9.01-6.44 (m, 9H, H-2,5,6,7,8, Ar H-2,3,5,6), 4.62 (s, 2H, -CH2-), 2.98-2.89 (m, 2H, isoindole H-3a,7a), 1.90-1.58 (m, 4H, isoindole H-4,4',7,7'), 1.50-1.29 (m, 4H, isoindole H-5,5',6,6'); LC-MS: m/z = 430 [M+1], 431 [M+2]; Anal. Calcd. for C24H23N5O3: C, 67.12; H, 5.40; N, 16.31. Found: C, 67.19; H, 5.48; N, 16.38. 4-(1,3-Dioxooctahydro-2H-isoindole-2-yl)-N'-(quina-zoline-4(3H)-ylidene)benzohydrazide (3c). Yield: 84.3%; m.p. 187-190 °C; IR (cm-1): 3421, 3280, 2993, 2913, 2855, 1698, 1626, 1537, 1493, 1475, 1385, 1167, 1129, 759, 687, 615; 1H NMR 8 (ppm): 11.60 (s, 1H, 3-NH), 10.65 (s, 1H, NHCO), 8.58-6.95 (m, 9H, H-2,5,6,7,8, Ar H-2,3,5,6), 3.15-3.08 (m, 2H, isoindole H-3a,7a), 1.97-1.70 (m, 4H, isoindole H-4,4',7,7'), 1.57-1.35 (m, 4H, isoindole H-5,5',6,6'); LC-MS: m/z = 416 [M+1], 417 [M+2]; Anal. Calcd. for C23H21N5O3: C, 66.49; H, 5.10; N, 16.86. Found: C, 66.54; H, 5.16; N, 16.93. 2-(1,3-Dioxo-1,3,3a,4,7,7a-hexahydro-2H-isoindole-2-yl)-N'-(quinazoline-4(3H)-ylidene)propanehydrazide (3d). Yield: 71.8%; m.p. 201-203 °C, IR (cm-1): 3289, 3214, 3069, 3012, 2960, 2910, 2886, 2850, 2780, 2683, 2410, 1702, 1603, 1566, 1466, 1420, 1383, 1325, 1198, 1117, 927, 776, 755, 685, 661; 1H NMR 8 (ppm): 11.49 (s, 1H, 3-NH), 10.09/10.00 (s, 1H, NHCO), 7.81-7.65 (m, 2H, Martynenko et al.: Directed Search of Biologically Active Compounds among ... 148 Acta Chim. Slov. 2019, 66, 145-154 H-2,5), 7.49 (t, J = 7.8 Hz, 1H, H-7), 7.20 (t, J = 7.8 Hz, 1H, H-6), 7.11 (d, J = 8.4 Hz, 1H, H-8), 5.89 (m, 2H, isoindole H-5,6), 5.13/4.71 (q, J = 7.9, 7.2 Hz, 1H, CHCH3), 3.052.90 (m, 2H, isoindole H-3a,7a), 2.46-2.01 (m, 4H, isoindole H-4,4',7,7'), 1.44 (d, J = 7.1 Hz, 3H, CHCH3); LC-MS: m/z = 368 [M+3]; Anal. Calcd. for C19H19N5O3: C, 62.46; H, 5.24; N, 19.17. Found: C, 62.49; H, 5.28; N, 19.23. 3-(1,3-Dioxo-1,3,3a,4,7,7a-hexahydro-2H-isoindole-2-yl)-N'-(quinazoline-4(3H)-ylidene)propanehydrazide (3e). Yield: 68.4%; m.p. 135-137 °C, IR (cm-1): 3489, 3409, 3343, 3230, 3128, 3106, 3078, 3010, 2988, 2939, 2859, 2699, 1693, 1611, 1527, 1471, 1440, 1379, 1106, 755, 687; 1H NMR 5 (ppm): 11.53 (s, 1H, 3-NH), 10.03/9.52 (s, 1H, NHCO), 7.88 (d, J = 7.2 Hz, 1H, H-5), 7.74 (s, 1H, H-2), 7.37 (t, J = 7.3 Hz, 1H, H-7), 7.20 (t, J = 7.5 Hz, 1H, H-6), 7.13 (d, J = 7.8 Hz, 1H, H-5), 5.95-5.76 (m, 2H, isoindole H-5,6), 3.67 (m, 2H, CH2CH2N), 3.24-3.08 (m, 2H, isoindole 3a,7a), 2.82 (t, J = 7.5 Hz, 2H, CH2CH2N), 2.48-2.11 (m, 4H, isoindole H-4,4',7,7'); LC-MS: m/z = 366 [M+1], 367 [M+2]; Anal. Calcd. for C19H19N5O3: C, 62.46; H, 5.24; N, 19.17. Found: C, 62.43; H, 5.18; N, 19.13. 4-(1,3-Dioxo-1,3,3a,4,7,7a-hexahydro-2H-isoindole-2-yl)-N'-(quinazoline-4(3H)-ylidene)benzohydrazide (3f). Yield: 94.3%; m.p. 261-263 °C, IR (cm-1): 3418, 3311, 3205, 3168, 3120, 3089, 3056, 3023, 2979, 2915, 2850, 1704, 1612, 1536, 1495, 1474, 1445, 1386, 1177, 845, 759, 687, 625; 1H NMR 5 (ppm): 11.58 (s, 1H, 3-NH), 10.65/9.99 (s, 1H, NHCO), 8.55-7.03 (m, 9H, H-2,5,6,7,8, Ar H-2,3,5,6), 6.15-5.81 (m, 2H, isoindole H-5,6), 3.39-3.19 (m, 2H, isoindole H-3a,7a), 2.69-2.20 (m, 4H, isoindole H-4,4',7,7); LC-MS: m/z = 414 [M+1], 415 [M+2]; Anal. Calcd. for C23H19N5O3: C, 66.82; H, 4.63; N, 16.94. Found: C, 66.89; H, 4.70; N, 17.01. 2-(1,3-Dioxo-1,3,3a,4,7,7a-hexahydro-2H-4,7-meth-anoisoindole-2-yl)-N'-(quinazoline-4(3H)-ylidene)ace-tohydrazide (3g). Yield: 71.6%; m.p. 168-170 °C, IR (cm-1): 3462, 3292, 3208, 3168, 3133, 3089, 3054, 3029, 2994, 2904, 2858, 2783, 1728, 1632, 1539, 1489, 1477, 1439, 1391, 1173, 845, 761, 683, 629; 1H NMR 5 (ppm): 11.51 (s, 1H, 3-NH), 10.27/9.66 (s, 1H, -NHCO-), 7.90 (d, J = 7.2 Hz, 1H, H-H-5), 7.80 (s, 1H, H-2), 7.40 (t, J = 7.3 Hz, 1H, H-7), 7.19 (t, J = 7.4 Hz, 1H, H-6), 7.13 (d, J = 8.0 Hz, 1H, H-8), 6.18-6.07 (m, 2H, isoindole H-5,6), 4.73/4.37 (s, 2H, CH2), 3.50-3.38 (m, 2H, isoindole H-3a,7a), 3.39-3.27 (m, 2H, isoindole H-4,7), 1.76-1.55 (m, 2H, isoindole H-8,8'); LC-MS: m/z = 364 [M+1], 365 [M+2]; Anal. Calcd. for C19H17N5O3: C, 62.80; H, 4.72; N, 19.27. Found: C, 62.87; H, 4.77; N, 19.34. 4-(1,3-Dioxo-1,3,3a,4,7,7a-hexahydro-2H-4,7-meth-anoisoindole-2-yl)methyl)-N'-(quinazoline-4-yl)benzo-hydrazide (3h). Yield: 88.1%; m.p. 265-267 °C; IR (cm-1): 3376, 3060, 2990, 2962, 2914, 2849, 2690, 1695, 1611, 1525, 1471, 1375, 1339, 1167, 1093, 907, 763, 722, 690, 659, 644; 1H NMR 6 (ppm): 11.43 (s, 1H, 3-NH), 10.55 (s, 1H, s, 1H, -NHCO), 8.18 (d, J = 7.4 Hz, 1H, H-5), 7.94-7.72 (m, 3H, H-2, Ph H-2,6), 7.55 (t, J = 7.3 Hz, 1H, H-7), 7.44-7.16 (m, 4H, H-6,8, Ph H-3,5), 6.04-5.86 (m, 2H, isoindole H-5,6), 4.46 (s, 2H, -CH2-), 3.42-3.34 (m, 2H, isoindole H-3a,7a), 3.34-3.29 (m, 2H, isoindole H-4,7), 1.74-1.48 (m, 2H, isoindole H-8,8'); LC-MS: m/z = 440 [M+1], 441 [M+2]; Anal. Calcd. for C25H21N5O5: C, 68.33; H, 4.82; N, 15.94. Found: C, 68.41; H, 4.88; N, 16.01. 4-(1,3-Dioxo-1,3,3a,4,7,7a-hexahydro-2H-4,7-meth-anoisoindole-2-yl)-N'-(quinazoline-4-yl)benzohydrazide (3i). Yield: 99.9%; m.p. 187-189 °C; IR (cm-1): 3476, 3310, 3284, 3028, 2982, 2929, 2856, 2780, 1693, 1614, 1548, 1495, 1474, 1389, 1185, 760, 744, 721, 688, 615; 1H NMR 6 (ppm): 10.63 (s, -NHCO-), 8.21 (d, J = 7.2 Hz, 1H, H-5), 8.03-7.89 (m, 3H, H-2, Ph H-2,6), 7.59 (t, J = 7.4 Hz, 1H, H-7), 7.50-7.34 (m, 2H, H-6,8), 7.26 (d, J = 7.9 Hz, 2H, Ph H-3,5), 6.31-6.19 (m, 2H, isoindole H-5,6), 3.60-3.48 (m, 2H, isoindole H-7a,3a), 3.46-3.28 (m, 2H, isoindole H-4,7), 1.82-1.54 (m, 2H, isoindole H-8,8'); LC-MS: m/z = 426 [M+1], 428 [M+3]; Anal. Calcd. for C24H19N5O3: C, 67.76; H, 4.50; N, 16.46. Found: C, 67.83; H, 4.58; N, 16.53. The synthesized compounds are yellow, soluble in DMF, DMSO, dioxane, slightly soluble in alcohol, insoluble in water. General Procedure for the Synthesis of 2-([1,2,4]Tri-azolo[1,5-c]quinazolme-2-yl-)alkyl-(alkylaryl-,aryl-) hydroisoindole-1,3(2H)-diones 4a-i 0.01 mol of the corresponding hydrazides 3a-i was dissolved in 25 mL of glacial acetic acid and was refluxed for 6 hours. The mixture was cooled. In the case of the residue formation it was filtered off. In the case of a solution, acetic acid was removed by distillation under vacuum. The resulting residue was stirred with a mixture of methanol and water and the precipitate was filtered. If necessary, the crude product can be crystallized. (2-(1-([1,2,4]Triazolo[1,5-c]quinazoline-2-yl)ethyl) hexahydro-1H-isoindole-1,3(2H)-dione (4a). Yield: 62.9%; m.p. 200-203 °C; IR (cm-1): 2922, 2843, 2690, 2359, 1705, 1381, 1342, 1195, 899, 770; 1H NMR 6 (ppm): 9.40 (s, 1H, H-5), 8.39 (d, J = 7.6 Hz, 1H, H-10), 8.03 (d, J = 7.9 Hz, 1H, H-7), 7.88 (t, J = 8.1 Hz, 1H, H-8), 7.76 (t, J = 7.3 Hz, 1H, H-9), 5.49 (q, J = 6.7 Hz, 1H, CHCH3), 3.01-2.86 (m, 2H, isoindole 3a,7a), 1.89 (d, J = 7.0 Hz, 3H, CHCH3), 1.86-1.69 (m, 4H, isoindole H-4,4',7,7'), 1.591.23 (m, 4H, isoindole H-5,5',6,6'); 13C NMR 6 (ppm): 179.41/179.29 (C-1,3 isoindole), 166.16 (C-2l), 150.74 (C-5), 142.69 (C-10b), 139.27 (C-6a), 132.80 (C-8), 129.63 (C-7), 128.99 (C-9), 123.46 (C-10), 117.76 (C-10a), 44.28 (-CHCH3), 40.35 (C-3a,7a isoindole), 23.44/23.36 (C-4,7 isoindole), 21.53/21.48 (C-5,6 isoindole), 15.82 (CH3); LC-MS: m/z = 350 [M+1], 352 [M+2]; Anal. Calcd. for Martynenko et al.: Directed Search of Biologically Active Compounds among Acta Chim. Slov. 2019, 66, 145-154 149 C19H19N5O2: C, 65.32; H, 5.48; N, 20.04. Found: C, 65.39; H, 5.52; N, 20.11. 2-(4-([1,2,4]Triazolo[1,5-c]quinazoline-2-yl)benzyl) hexahydro-1H-isoindole-1,3(2H)-dione (4b). Yield: 55.6%; m.p. 248-250 °C; IR (cm-1): 2957, 2929, 2854, 2456, 1689, 1479, 1423, 1397, 1334, 1165, 899, 775, 742; 1H NMR 5 (ppm): 9.48 (s, 1H, H-5), 8.55 (d, J = 7.7 Hz, 1H, H-10), 8.26 (d, J = 8.1 Hz, 2H, Ar H-2,6), 8.06 (d, J = 8.4 Hz, 1H, H-7), 7.90 (t, J = 7.9 Hz, 1H, H-8), 7.81 (t, J = 7.6 Hz, 1H, H-9), 7.45 (d, J = 7.9 Hz, 2H, Ar H-3,5), 4.64 (s, 2H, CH2), 2.98-2.93 (m, 2H, isoindole 3a,7a), 1.87-1.57 (m, 4H, isoindole H-4,4',7,7'), 1.54-1.26 (m, 4H, isoindole H-5,5',6,6'); LC-MS: m/z = 412 [M+1], 413 [M+2]; Anal. Calcd. for C24H21N5O2: C, 70.06; H, 5.14; N, 17.02. Found: C, 70.12; H, 5.18; N, 17.11. 2-(4-([1,2,4]Triazolo[1,5-c]quinazoline-2-yl)phenyl)hexa hydro-1H-isoindole-1,3(2H)-dione (4c). Yield: 74.6%; m.p. > 300 °C; IR (cm-1): 2989, 2924, 2863, 1701, 1514, 1477, 1445, 1357, 1164, 1122, 899, 769, 749, 713, 628; 1H NMR 5 (ppm): 9.50 (s, 1H, H-5), 8.57 (d, J = 7.9 Hz, 1H, H-10), 8.41 (d, J = 8.4 Hz, 2H, Ar H-2,6), 8.07 (d, J = 8.2 Hz, 1H, H-7), 7.96 (t, J = 7.6 Hz, 1H, H-8), 7.82 (t, J = 7.5 Hz, 1H, H-9), 7.49 (d, J = 8.4 Hz, 2H, Ar H-3,5), 3.13 (m, 2H, isoindole 3a,7a), 1.97-1.72 (m, 4H, isoindole H-4,4',7,7'), 1.57-1.39 (m, 4H, isoindole H-5,5',6,6'); LC-MS: m/z = 398 [M+1], 400 [M+2]; Anal. Calcd. for C23H19N5O2: C, 69.51; H, 4.82; N, 17.62. Found: C, 69.58; H, 4.91; N, 17.71. 2-(1-([1,2,4]Triazolo[1,5-c]quinazoline-2-yl)ethyl)-3a, 4,7,7a-tetrahydro-1H-isoindole-1,3(2H)-dione (4d). Yield: 71.2%; m.p. 180-184 °C; IR (cm-1): 2954, 2899, 1701, 1493, 1387, 1360, 1214, 900, 773, 706; 1H NMR 5 (ppm): 9.39 (s, 1H, H-5), 8.41 (d, J = 7.8 Hz, 1H, H-10), 8.02 (d, J = 8.3 Hz, 1H, H-7), 7.87 (t, J = 7.6 Hz, 1H, H-8), 7.76 (t, J = 7.5 Hz, 1H, H-9), 5.93 (m, 2H, isoindole H-5,6), 5.47 (q, J = 7.1 Hz, 1H, CHCH3), 3.38-3.09 (m, 2H, isoindole 3a,7a), 2.58-2.49 (m, 2H, isoindole H-4,7), 2.37-2.15 (m, 2H, isoindole H-4',7'), 1.83 (d, J = 7.1 Hz, 3H, CHCH3); LC-MS: m/z = 348 [M+1], 349 [M+2]; Anal. Calcd. for C19H17N5O2: C, 65.69; H, 4.93; N, 20.16. Found: C, 65.66; H, 4.98; N, 20.13. 2-(2-([1,2,4]Triazolo[1,5-c]quinazoline-2-yl)ethyl)-3a, 4,7,7a-tetrahydro-1H-isoindole-1,3(2H)-dione (4e). Yield: 40.0%; m.p. 165-167 °C; IR (cm-1): 2982, 2843, 2828, 2712, 1693, 1399, 1362, 1325, 1211, 1157, 1010, 929, 904, 770, 698; 1H NMR 5 (ppm): 9.37 (s, 1H, H-5), 8.43 (d, J = 7.6 Hz, 1H, H-10), 8.03 (d, J = 8.3 Hz, 1H, H-7), 7.88 (t, J = 7.9 Hz, 1H, H-8), 7.77 (t, J = 7.5 Hz, 1H, H-9), 5.94-5.66 (m, 2H, isoindole H-5,6), 3.85 (t, J = 7.3 Hz, 2H, CH2CH2N), 3.20-2.97 (m, 4H, CH2CH2N, isoindole 3a,7a), 2.50-2.40 (m, 2H, isoindole H-4,7), 2.32-2.06 (m, 2H, isoindole H-4',7'); LC-MS: m/z = 348 [M+1], 349 [M+2]; Anal. Calcd. for C19H17N5O2: C, 65.69; H, 4.93; N, 20.16. Found: C, 65.74; H, 5.02; N, 20.23. 2-(4-([1,2,4]Triazolo[1,5-c]quinazoline-2-yl)phenyl)-3a,4,7,7a-tetrahydro-1H-isoindole-1,3(2H)-dione (4f). Yield: 82.25%; m.p. 250-253 °C; IR (cm-1): 1703, 1514, 1478, 1446, 1360, 1315, 1177, 899, 842, 751, 717, 672; 1H NMR 5 (ppm): 9.50 (s, 1H, H-5), 8.56 (d, J = 8.3 Hz, 1H, H-10), 8.39 (d, J = 8.3 Hz, 2H, Ar H-2,6), 8.20 (d, J = 8.5 Hz, 1H, H-7), 8.06 (t, J = 8.6 Hz, 1H, H-8), 7.86 (t, J = 8.6 Hz, 1H, H-9), 7.44 (d, J = 8.4 Hz, 2H, Ar H-3,5), 6.055.92 (m, 2H, isoindole H-5,6), 3.12-2.85 (m, 2H, isoindole 3a,7a), 2.66-2.56 (m, 2H, isoindole H-4,7), 2.37-2.23 (m, 2H, isoindole H-4',7'); LC-MS: m/z = 396 [M+1], 397 [m+2]; Anal. Calcd. for C23H17N5O2: C, 69.86; H, 4.33; N, 17.71. Found: C, 69.94; H, 4.41; N, 17.80. 2-([1,2,4]Triazolo[1,5-c]quinazoline-2-ylmethyl)-3a, 4,7,7a-tetrahydro-1H-4,7-methanoisoindole-1,3 (2H)-dione (4g). Yield: 85.8%; m.p. 215-217 °C; IR (cm-1): 2994, 2863, 2812, 1721, 1514, 1475, 1446, 1368, 1323, 1183, 899, 846, 751, 721, 682; 1H NMR 5 (ppm): 9.40 (s, 1H, H-5), 8.43 (d, J = 7.6 Hz, 1H, H-10), 8.03 (d, J = 7.6 Hz, 1H, H-7), 7.89 (t, J = 7.5 Hz, 1H, H-8), 7.78 (t, J = 7.5 Hz, 1H, H-9), 6.15 (s, 2H, isoindole H-5,6), 4.73 (s, 2H, NCH2), 3.46 (s, 2H, isoindole H-4,7), 3.37 (s, 2H, isoindole H-3a,7a), 1.67 (dd, J = 20.6, 8.0 Hz, 2H, isoindole H-8,8'); LC-MS: m/z = 346 [M+1], 348 [M+3]; Anal. Calcd. for C19H15N5O2: C, 66.08; H, 4.38; N, 20.28. Found: C, 66.17; H, 4.46; N, 20.32. 2-(4-([1,2,4]Triazolo[1,5-c]quinazoline-2-yl)ben-zyl)-3a,4,7,7a-tetrahydro-1H-4,7-methanoisoin-dole-1,3(2H)-dione (4h). Yield: 33.06%; m.p. 219-221 °C; IR (cm-1): 2996, 2974, 2918, 1695, 1515, 1476, 1420, 1386, 1334, 1160, 1118, 899, 769, 756, 727, 714; 1H NMR 5 (ppm): 9.48 (s, 1H, H-5), 8.56 (d, J = 8.2 Hz, 1H, H-10), 8.24 (d, J = 7.1 Hz, 2H, 1,4-phenylene H-2,6), 8.06 (d, J = 8.2 Hz, 1H, H-7), 7.91 (t, J = 8.2 Hz, 1H, H-8), 7.81 (t, J = 8.2 Hz, 1H, H-7, H-9), 7.40 (d, J = 7.2 Hz, 1H, 1,4-phenylene H-3,4), 5.95 (s, isoindole H-5,6), 4.48 (s, 2H, -NCH2-), 3.37 (s, 2H, isoindole H-4,7), 3.32 (s, 2H, isoindole H-3a,7a), 1.63 (dd, J = 22.0, 8.5 Hz, 2H, isoindole H-8,8'); 13C NMR 5 (ppm): 177.75 (C-1,3; isoindole), 169.81 (C-2), 153.47 (C-5), 143.95 (C-10b), 137.96 (C-6a), 135.40 (C-4; Ar), 134.92 (C-5,6; isoindole), 132.76 (C-8), 131.69 (C-1a; Ar), 129.55 (c-7), 129.01 (C-9), 128.90 (C-2,6; Ar), 127.51 (C-3,5; Ar), 123.81 (C-10), 114.13 (C-10a), 52.36 (C-8; isoindole), 45.87 (C-4,7; isoindole), 44.87 (C-3a,7a; isoindole), 41.49 (-NCH2-); LC-MS: m/z = 422 [M+1], 424 [M+3]; Anal. Calcd. for C25H19N5O2: C, 71.25; H, 4.54; N, 16.62. Found: C, 71.31; H, 4.58; N, 16.68. 2-(4-([1,2,4]Triazolo[1,5-c]quinazoline-2-yl)phe-nyl)-3a,4,7,7a-tetrahydro-1H-4,7-methanoisoin-dole-1,3(2H)-dione (4i). Yield: 86.87%; m.p. 269-271 °C; Martynenko et al.: Directed Search of Biologically Active Compounds among ... 150 Acta Chim. Slov. 2019, 66, 145-154 IR (cm-1): 2982, 2932, 2861, 2812, 1703, 1477, 1447, 1357, 1186, 899, 838, 778, 744, 708, 669, 608; 1H NMR 5 (ppm): 9.50 (s, 1H, H-5), 8.57 (d, J = 7.7 Hz, 1H, H-10), 8.36 (d, J = 7.8 Hz, 2H, 1,4-phenylene H-2,6), 8.07 (d, J = 7.7 Hz, 1H, H-7), 7.91 (t, J = 7.7 Hz, 1H, H-8), 7.82 (t, J = 7.7 Hz, 1H, H-9), 7.32 (d, J = 7.7 Hz, 2H, 1,4-phenylene H-3,5), 6.27 (s, 2H, isoindole H-5,6), 3.52 (s, 2H, isoindole H-4,7), 3.43 (s, 2H, isoindole H-3a,7a), 1.59 (dd, J = 20.7, 7.9 Hz, 2H, isoindole H-8,8'); LC-MS: m/z = 408 [M+1], 409 [M+2]; Anal. Calcd. for C24H17N5O2: C, 70.75; H, 4.21; N, 17.19. Found: C, 70.82; H, 4.28; N, 17.23. The synthesized compounds are yellow, soluble in DMF, DMSO, dioxane, slightly soluble in alcohol, insoluble in water. 3. Results and Discussion 3. 1. Chemistry As it has been mentioned above, the combination in one molecule of more pharmacophore fragments, which are responsible for hypoglycemic effect, is interesting as well as offering further structural modifications and prospects to study such compounds in pharmacological screenings. Thus, following the search strategy, N-car-boxyalkyl-(arylalkyl-,aryl-)isoindoline-l,3-diones 1a-i were utilized in the reaction with N,N'-carbonyldiimi-dazole for obtaining corresponding imidazolides, in the first stage of the study (Scheme l). The last ones formed previously unknown hydrazides 3a-i in the reaction with 4-hydrazinoquinazoline (2a). The presence of anhydrous dioxane and the temperature control (not above 80 °C) is obligatory for the regioselectivity of the reaction. Reaction products 3a-i are formed with high yields and in most cas- es they do not require additional purification. The appearance in the NMR spectrum of low-field broad singlet of exocyclic NH-group proton of the quinazoline ring at the range of 11.60-11.46 ppm and NH-protons signal of the hydrazide fragment at the 10.65-9.52 ppm indicates the 3a-i compounds formation. It is important to note, that some hydrazides, like 3a, 3d-3g are characterized by doubling of NH-protons signals of the hydrazide fragment due to the prototropic tautomerism in DMSO, resulting in the appearance of aromatic and aliphatic protons in the form of multiplets or double signals. Besides, the IR spectra of hydrazides 3a-i are characterized by -C=O groups band valence vibrations of isoindole fragments at the 1728-1693 cm-1 and -C(O)NH-groups (amide-I) at the 1632-1603 cm-1, valence-deformation vibrations of the N-H and C-N (amide-II) bonds at the 1566-1525 cm-1 as well as the band of NH-groups valence vibrations at the range of the 3489-3168 and 3180-2993 cm-1. The formed hydrazides 3a-i were subjected to cyclocondensation by known methods.29 As we expected, intermediate [1,2,4]triazolo[4,3-c]quinazolines are subjected to the recyclic isomerization by the Dimroth rearrangement under acidic catalysis conditions with 2-(2-([1,2,4]tri-azolo[1,5- c]quinazoline-2-yl)-alkyl-(arylalkyl-,aryl-)-1 H-isoindole-1,3(2H)-dione formation (4a-i, Scheme 1).29,30 Signals of triazine[1,5-c]quinazoline ring protons, namely the low-field singlet of H-5 nucleus at the 9.50-9.37 ppm, doublet signals of ABCD-system at the 8.57-8.39 ppm (H-10) and 8.20-8.03 ppm (H-7) and triplets at the 8.06-7.87 ppm (H-8) and 7.86-7.76 ppm (H-9) are typical for the 1H NMR spectra of compounds 4a-i. It is important, that the aromatic proton signals of compounds 4a-i, in comparison with compounds 3a-i, undergo a significant paramagnetic shift that indicated the formation of an elec- Scheme 1. The synthesis of N-carboxyalkyl-(arylalkyl-,aryl-)isoindoline-1,3-diones (3ff-quinazoline-4-ylidene)hydrazides and hydrogenated 2-([1,2,4]triazolo[1,5-c]quinazoline-2-yl-)alkyl-(alkaryl-,aryl-)isoindole-1,3(2H)-diones. Martynenko et al.: Directed Search of Biologically Active Compounds among Acta Chim. Slov. 2019, 66, 145-154 151 tron-deficient tricyclic system. In favor of compounds 4a and 4h formation is also indicative the appearance of the characteristic signals of sp2-hybridized C-2 atoms at the 169.81-166.16 ppm and C-5 at the 153.47-150.74 ppm of triazino[1,5-c]quinazoline ring in the 13C NMR spectra.29 In addition, the 1H NMR spectra of compounds 3a-i, 4a-i are characterized by signals of the corresponding hydrogenated isoindoline moieties. Thus, a totally hydro-genated isoindole moiety of compounds 3a-3c and 4a-4c in the 1H NMR spectra is observed as a set of multiplet signals of axial and equatorial protons at the 3.15-2.65 ppm (H-3a, H-7a), at the 1.97-1.58 ppm (H-4,4',7,7') and at the 1.59-1.23 ppm (H-5,5',6,6'). The hexahydroi-soindole moiety (compounds 3d-3f, 4d-4f) is in the 1H NMR spectra characterized by multiplet signals of H-5,6 at the 6.15-5.66 ppm and a set of multiplets of axial and equatorial protons at the 3.24-2.90 ppm (H-3a, H-7a) and 2.69-2.01 ppm (H-4,4',7,7'). Whereas, the hexahy-dro-4,7-methanoisoindole moiety (compounds 3g-3i) is in the 1H NMR spectra characterized by multiplet signal of H-5,6 at the 6.31-5.86 ppm and a set of multiplet signals at the 3.60-3.34 ppm (H-3a, H-7a), 3.46-3.27 ppm (H-4,7) and 1.82-1.48 ppm (H-8,8'). The chemical shifts and multiplicity of the indicated signals in the compounds 4g-4i differs slightly: the H-5,6 signals are recorded at the 6.27-5.95 ppm, H-4,7 at the 3.52-3.37 ppm, H-3a, H-7a at the 3.43-3.32 ppm and a doublet of doublets for H-8,8' appears at the 1.67-1.59 ppm. In addition, there are signals of linker alkyl and aryl groups for which the corresponding multiplicity and chemical shift of protons are typical for the 1H NMR spectra.31 In the IR spectra of compounds 4a-i there are no bands of vibrations of exchangeable protons differing from compounds 3a-i, whereas the bands of C=O groups of isoindole fragments at the 1721-1689 cm-1 and vc=c-aromatic rings bonds at the 1515-1381 cm-1, non-flat deformation vibrations of y=C-H-bond at the 899-608 cm-1 and intensive bands of symmetric and antisymmetric valence vibrations of -CH2 groups at the 2989-2359 cm-1 are present. 3. 2. Hypoglycemic Assay for Preliminary in vivo Testing The screening results of hypoglycemic activity (Table 1) showed that synthesized compounds have different effects on glucose level in the blood of experimental animals. Thus, the compounds 3a, 3f, 3h, 4a, 4c and 4f decreased the glucose level on the second hour of the experiment on 13.99%, 4.09%, 11.86%, 18.47%, 3.18% and 8.61% respectively, substantially yielded to the action of «Metformine». It is important that only compounds 3a, 3h, 4f keep this activity on the fourth hour of the experiment (reduced glucose level on 27.87%, 10.51% and 2.65% respectively). While the compounds 3b, 3c, 3e, 3i, 4b, 4e and 4i under the given experimental conditions, on the contrary, increased glucose level on the second hour of the experiment from 4.89 to 15.79% (Table 1). The insulinotropic effect of prandial glucose regulators comes very quickly unlike other oral hypoglycemic agents. Insulin secretion stimulation from ^-cells of the pancreas islands is formed in 5-7 minutes (Mitiglinide, Nateglinide) or in 10-30 minutes after using (Repaglinide) these drugs in response to food intake. This is achieved by closing ATP-dependent potassium channels in the membrane of ^-cells due to their depolarization. Molecular docking to KATP channel, PDB ID-6BAA was conducted, for a number of synthesized compounds (3a, 3f, 3h, 4a, Table 1 The synthesized compounds' influence on glucose level in blood of experimental animals (%) Comp. Initial glucose Glucose level % Glucose level % Glucose level % level in 2 h in 4 h in 6 h Intact group 6.12+0.12 6.60±0.08 +7.84 5.62±0.14 -8.17 5.85±0.13 -4.41 3a 7.93±0.83 6.82±0.69 -13.99 5.72±0.49 -27.87 8.46±0.79 +6.68 3b 6.46±0.12a 6.94±0.34 +7.43 8.08±0.79 +25.08 8.28±0.95 +28.17 3c 6.12±0.17a 6.80±0.29 +11.11 7.30±0.74 + 19.28 7.02±0.65 +14.70 3e 6.68±0.96 7.62±0.17a +14.07 8.14±0.27 +21.86 8.04±0.15a +20.36 3f 5.38±0.35 5.16±0.17a -4.09 5.80±0.29 +7.81 5.42±0.29 +0.74 3h 5.90±0.23a 5.20±0.19 -11.86 5.28±0.21 -10.51 5.20±0.19a -11.86 3i 6.56±0.05a 7.20±0.17a +9.75 7.72±0.07a +17.68 7.72±0.20 +17.68 4a 6.82±0.22 5.56±0.27 -18.47 7.34±0.45 +7.62 6.88±0.17a +0.88 4b 6.12±0.23 6.94±0.39 +13.39 7.08±0.47 +15.68 6.78±0.35 +10.78 4c 6.28±0.34 6.08±0.26 -3.18 6.56±0.33 +4.46 6.30±0.13a +0.32 4e 6.28±0.06a 7.10±0.13a +13.06 7.58±0.33 +20.70 8.36±0.41 +33.12 4f 6.04±0.20 5.52±0.33 -8.61 5.88±0.17a -2.65 5.34±0.21 -11.59 4h 5.72±0.41 6.00±0.28 +4.89 5.16±0.35 -9.79 4.98±0.37 -12.94 4i 6.46±0.07a 7.48±0.08a +15.79 8.50±0.43 +31.58 8.20±0.26 +26.93 Metformine 5.50±0.20a 3.71±0.08a -67.60 3.42±0.06a -62.30 3.64±0.06a -66.10 a differences reliable (p < 0.05) comparison to intact group of rats. Martynenko et al.: Directed Search of Biologically Active Compounds among ... 152 Acta Chim. Slov. 2019, 66, 145-154 Table 2 Types of investigated compounds' interactions with amino acid residues of the target active center according to the docking studies Investigated protein and types of interactions with amino acid residues Comp. Affinity, KATP channel, PDB ID-6BAA kcal/mol 3a -8.2 ARG1246a, ARG1300b, TYR377a, LEU434c, TYR377c, LEU592c. 3f -8.2 ARG1300a, ASP1304b, ILE381c, TRP430c, PHE433c, ARG1300c, ALA1303c. 3h -8.2 SER1238a, ARG1246a, HASN1245a, TRP430c, ILE381c. 4a -8.5 ASN1245a, ILE381c, ILE385c, MET429c, PHE433c, ILE381c. 4c -9.5 THR588a, LEU592c, PHE591c, TYR377c. TYR377c, LEU592c. 4f -9.5 THR588a, LEU592c, TYR377c, TYR377c, PHE591c. 4h -10.1 THR1242a, SER1238a, TRP430c, LEU434c, TYR377c, TRP430c, MET429c, TRP430c, TRP430c, ILE381c, LEU434c. 4i -9.0 ARG1145a, ARG1300a,b, TRP1297c, TRP1297c, TRP1297c, TRP1297c, TRP1297c, PHE591c, TRP430c, PHE433c, LEU592c. Mitiglinide -7.5 THR1242a, LEU434c, TYR377c, TRP430c, TRP430c, PHE433c. Glibenclamide* -8.4 ARG306a, ASN437a, THR1242a, ARG1246a, ASN1245a, TYR377a, ASN437a, TYR377a, MET441c, MET429c, LEU592c. a hydrogen; b electrostatic; c hydrophobic * data according to X-ray structure of protein.32 4c, 4f and 4i), taking into account the search strategy. It allowed to identify the main interaction types of the synthesized compounds, Mitiglinide and Glibenclamide with the amino acid residues with active channel centers and select compounds for further studies (Table 2).32 The visualization of the interaction of the structures with the active site of KATP channel (Figure 2) showed that compounds 3a and 4a revealed similar interaction to Glibenclamide. Such common interactions for compound 3a were: two hydrogen bonds with the amino acid residues ARG1246 (3.12Ä), TYR377 (3.96Ä) and two hydrophobic interactions with the residues LEU592 (5.44Ä), TYR377 (5.17Ä). And common interactions for compound 4a were as following: hydrogen bond with the amino acid residue ASN1245 (3.76Ä) and hydrophobic interaction with the residue MET429 (4.74Ä). This may indicate that the stated class of compounds might have ability to act in the same way as Glibenclamide does. Hypoglycemic activity studys in the shorter of the experimental periods with a previous glucose loading allowed to establish that compounds 3a, 3f, 3h, 4a, 4f reduced glucose level on 19.34%, 17.54%, 18.85%, 25.09% and 9.51%, respectively on the 15 minute of the experiment, compared to control group (Table 3). It is important that the hypoglycemic effect of these compounds is maintained throughout the experiment from 30 to 120 minutes. While compounds 4c, 4h and 4i did not affect glucose level and in some cases even increased it throughout the experiment. SAR analysis showed that the compounds with (3H-quinazoline-4-ylidene)hydrazides (3a, 3f, 3h) or [1,2,4]triazolo[1,5-c]quinazoline (4a, 4f) moeities bonded through the ethyl (3a, 4a) or phenyl (3f, 3h, 4f) linker groups with a hydrogenated isoindoline ring have hypoglycemic effect in the short term of the experiment. Modification of compounds and their further studies on hypo-glycemic activity are going to be continued. Table 3 Influence of synthesized compounds on glucose level in blood of experimental animals in the short term of the experiment Comp. Initial Glucose level Glucose level Glucose level Glucose level glucose level in 15 min in 30 min in 60 min in 120 min Control group5.84±0.05 6.10±0.21 6.60 ±0.08 5.62±0.14 5.85±0.13 3a 4.34±0.12a 4.92±0.59 4.68±0.54 4.93±1.14 3.84±0.51 3f 5.12±0.15a 5.03±0.14a 4.87±0.19a 5.23±0.12a 5.12±0.34 3h 4.48±0.23 4.95±0.21 4.92±0.19a 5.41±0.24 5.32±0.16a 4a 3.98±0.13a 4.63±0.31 4.52±0.22 4.49±0.14a 5.54±0.11a 4c 5.83±0.27 6.23±0.17a 6.93±0.26 6.08±0.19a 6.12±0.20 4f 5.34±0.22 5.52±0.23 5.48±0.22 5.52±0.15a 5.32±0.17a 4h 5.87±0.25 6.13±0.31 6.54±0.27 5.89±0.24 5.93±0.21 4i 6.10±0.21 8.50±0.58 6.78±0.85 6.34±0.49 5.98±0.38 a differences reliable (p < 0.05) comparison to control group of rats. Martynenko et al.: Directed Search of Biologically Active Compounds among Acta Chim. Slov. 2019, 66, 145-154 153 (a) (b) afteractions I vanderWaals H Conventional Hydrogen H Pi-Cation Pi-Donor Hydrogen Bond Alkyl Pi-Aikyl b) Interactions I vanderWaals I Carbon Hydrogen Bond Alkyl Pi-Alkyl asn f:1245 tyr F:377 leu F:434 c) Interactions I van der Waals I Carbon Hydrogen Bond ^ Pi-Sigma Pi-Pi T-shaped Pi-Alkyl d) Interactions Conventional Hydrogen Bond I I Carbon Hydrogen Bond I Halogen (CI, Br, I) I Pi-Donor Hydrogen Bond I Alkyl I Pi-Alkyl Figure 2. Visualization of affinity according to the docking (a) compound 3a with KATP channel; (b) compound 4a with KATP channel; (c) Mit-iglinide with KATP channel; (d) Glibenclamide with KATP channel. 4. Conclusions The synthesis of N-carboxyalkyl-(arylalkyl-,ar-yl-)-isoindoline-1,3-dione (3H-quinazoline-4-ylidene) hydrazides was carried out using activated N-pro-tected aminoacids and 4-hydrazinoquinazoline. The synthesized hydrazides were converted into the corresponding hydrogenated 2-([1,2,4]triazolo[1,5-c]quina-zoline-2-yl)-alkyl-(alkylaryl-,aryl-)isoindole-1,3(2H)-di-ones by cyclocondensation under the conditions of acidic catalysis. The structures of the synthesized compounds were established using IR, 1H and 13C NMR spectroscopy and LC-MS and the features of spectral pattern were discussed. Search strategy of hypoglycemic drugs based on the fragment-oriented design revealed a number of perspective compounds, which are short-acting drugs, like prandial glucose regulators. Conducted SAR analysis showed that the introduction of hydrogenated 1,3-diox-oisoindole moiety bonded via a linker group with 4-hy-drazynoquinazoline and triazolo[1,5-c]quinazoline cycle is reasonable in the context of searching for short-acting hypoglycemic agents and requires further research. 5. Acknowledgments The work was performed with the financial support of «Enamine Ltd» (Kyiv, Ukraine). 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Ther. 1994, 269(3), 489-495. 24. W. J. Malaisse, Expert. Opin. Pharm. 2008, 9(15), 2691-2698. DOI:10.1517/14656566.9.15.2691 25. https://www.drugbank.ca/unearth/q?c=_score&d=down& query=quinazolines&searcher=drugs. 26. Protein Data Bank, pdb. Retrieved from http://www.pdb.org. 27. MarvinSketch version: 17.21.0, ChemAxon (http://www.che-maxon.com). 28. O. Trott, A. J. Olson, J. Comput. Chem. 2010, 31, 455-461. DOI:10.1002/jcc.21334 29. S. Kovalenko, L. Antypenko, A. Bilyi, S. Kholodnyak, O. Karpenko, O. Antypenko, N. Mykhaylova, T. Los', O. Kolomoets', Sci. Pharm. 2013, 81(2), 359-392. DOI:10.3797/ scipharm.1211-08 30. El H. Sayed, El Ashrya, S. Nadeem, M. R. Shah, Ye. El Kilanyd, Adv. Heterocycl. Chem. 2010, 101, 161-228. 31. E. Breitmaier, Structure Elucidation By NMR In Organic Chemistry: A Practical Guide, John Wiley & Sons, Ltd. 2002, 258. 32. M. M. Gregory, B. Kandasamy, F. DiMaio, C. Yoshioka, S. Show-Ling, eLife 2017, 6. DOI:10.7554/elife.31054 Povzetek Opisujemo učinkovito sintezo (3ff-kinazolin-4-iliden)hidrazidov N-karboksialkil-(arilalkil-,aril-)izoindolin-1,3-dionov iz aktiviranih N-zaščitenih amino kislin in 4-hidrazinokinazolinov. Predlagana metoda temelji na neposrednem iskanju hipoglikemičnih agentov s pomočjo načrtovanja, orientiranega na fragmentih, in z molekulskim sidranjem. Željene hidrazide smo pripravili s pomočjo ciklokondenzacij pod kislimi pogoji in jih nadalje pretvorili v ustrezne 2-([1,2,4] triazolo[1,5-c]kinazolin-2-il-)alkil-(alkilaril-,aril-)-hidroizoindol-1,3(2H)-dione. Strukture pripravljenih spojin smo določili s pomočjo IR, 1H in 13C NMR spektroskopije ter LC-MS analize ter s primerjavo spektralnih vzorcev (kar je opisano v diskusiji). Rezultati farmakološkega testiranja so razkrili serijo spojin, ki delujejo kot hitri hipoglikemični agenti, podobno kot prandial regulira glukozo (Mitiglinid). SAR analiza je dodatno pokazala, da je vpeljava hidrogeniranega 1,3-dioksoindolnega fragmenta, ki je preko distančnikov vezan na 4-hidrazinokinazolinski ter triazolo[1,5-c]kinazolin-ski obroč, primerna za iskanje novih hitro delujočih hipoglikemičnih agentov ter da si zasluži nadaljnje raziskave. Martynenko et al.: Directed Search of Biologically Active Compounds among DOI: 10.17344/acsi.2018.4733 Acta Chim. Slov. 2019, 66, 155-162 /^creative ^commons Scientific paper Investigation of the Electrochemical Reduction Process of the Molybdate Ions in the Tartaric Electrolytes Vusala Asim Majidzade,^* Akif Shikhan Aliyev,1 Dunya Mahammad Babanly,1,3 Mahmoud Elrouby2 and Dilgam Babir Tagiyev1 1 Institute of Catalysis and Inorganic Chemistry named after acad.M.Nagiyev, Azerbaijan National Academy of Sciences, H. Javid ave., 113, AZ 1143, Baku, Azerbaijan 2 Chemistry Department, Faculty of Science, Sohag University, 82524 Sohag, Egypt 3 Azerbaijan State Oil and Industry University (Ufaz), Baku, Azerbaijan * Corresponding author: E-mail: vuska_80@mail.ru Received: 12-27-2018 Abstract The electrochemical reduction of molybdate ions in tartaric electrolyte has been investigated in this work. Kinetics and mechanism of the electrochemical process have been studied by cyclic and linear voltammetric polarization methods. The effect of the temperature, the rate of potential-scanning and the concentration of the molybdate ions on the electrochemical process has been also studied. By estimating the effective activation energy, it was revealed that the electrore-duction process is accompanied by mixing kinetics control. Keywords: Molybdate ions; electroreduction; tartaric acid; polarization curves; chronoamperometry 1. Introduction As known, semiconductors are one of the most important materials that differ from each other by their great manifold of the physical and electrical properties, also of the chemical composition.1-4 The need for the chalco-genide semiconductor films increases with the development of the application of the new generation of solar cells.5 Today, the using of ecologically waste free solar energy is very actual connecting with the decreasing of the natural resources of the energy. Thin films of chalcogenide can be used as photo-anodes for obtaining of the electrical energy in the solar cells. One of such photosensitive semiconductor materials is molybdenum chalcogenides. The obtaining of these films can be carried out using various methods.1,6 The most suitable method of obtaining molybdenum alloys is the electrochemical method. There are a lot of works dedicated to the electrochemical deposition of thin films of these alloys. To study well the simultaneous electrodeposition process of these compounds, at first, these components should be deposited individually. In our previous studies, we have investigated the electroreduction and co-electrodeposition of various metals together with chalcogenides.7-11 Many works were dedicated to the electrochemical reduction of molybdate ions. Some researchers12 have investigated the electrochemical reduction of the hexavalent molybdenum ions in a solution containing tartaric acid. The formed complexes of Mo-tartarate ions were examined by spectro-scopic and electrochemical methods. The resulted electrochemical peak was found to be under kinetic control at relatively high pH values or at a relatively low concentration of the tartaric ions. But, the electrochemical reaction occurred by protonation reaction. Moreover, some researchers13 studied the reduction of molybdenum (VI) on a pre-hydrogenated platinum electrode in acidic solutions. Reduction of molybdenum (VI) in a solution of 0.8-1.6 M H2SO4 occurs via two main steps. The first step is a relatively high cathodic wave attributes to the reduction of Mo (V) to Mo (III), which is accompanied with the decomposition of the solvent. The second step is a relatively low cathodic wave and attributes to the reduction of Mo (VI) to Mo (V), which takes place at +0.07 V. The second peak seems to be controlled by a diffusion and can be used for the determination of molybdenum up to 4x 10-5 M or 6 x 10-5 M. An incomplete reduction of molybdate Majidzade et al.: Investigation of the Electrochemical Reduction Process 156 Acta Chim. Slov. 2019, 66, 155-162 ions from weak acidic and alkaline solutions within the pH interval from 3.0 to 9.0 has been recorded.14 A film of the hydrated oxides of molybdenum in lower oxidation states forms on the cathode surface. Products of the incomplete reduction adsorbed at the cathode and blocked its working surface. The following up of the electroreduction process of the molybdate ions in an aqueous medium under different conditions was achieved to obtain the metallic molybde-num.15 The kinetics and the mechanism of the electroreduction process were studied by the cathodic polarization. It was observed that the rate of the electrochemical process depends on the scan rate of potential, and the temperature of the electrolyte. On the other hand, it can be concluded that the electrochemical reduction occurs in two stages. It was indicated that, during the electrochemical reduction process, the electroreduction process of the molybdate ions is accompanied by releasing of hydrogen after the potential value -0.7 V. The effect of hydrogen fluoride on the cathodic reduction of molybdate ions has been investigated.16 It was indicated that the mechanism of the process is controlled by its concentration. It was found that at concentration values less than 25 g/L, the products of the electroreduction process of the molybdate ions are black or colorful precipitations. These precipitates were formed on the cathode, and the average valent of molybdenum was determined to be 3.6-4.0. In the concentration, more than 50 g/L molybdate ions are completely reduced forming on the cathode a dense light metallic coating of thickness 3-5^m with a great adhesion to the substrate. It was concluded that the addition of hydrogen fluoric acid prevents the polymerization and aggregation of the molybdate ions. The bright, smooth metallic coating of molybdenum with 25 ^m thickness is successfully deposited from aqueous electrolyte consisting high concentration of ace-tate.17-19 The optimal conditions for the electrodeposition of metallic molybdenum have been detected to be as follow: the current density 200-450 mA/cm2 and temperature 30 °C ± 2. The obtained coatings are amorphous, but after vacuum annealing at high temperature, they converted into crystalline films. It was concluded that the current density is the major effective factor for the precipitation of a refractory metal. 19 From the published results, it has been indicated that the metallic molybdenum can be deposited through two stages through electroreduction with obtaining intermediate coating from molybdenum oxide. According to the previous studies, kinetics and mechanism of the electrochemical reduction have been investigated differently. Therefore, the main aim of our work is to study of the nature, kinetics and mechanism of the electrochemical reduction process of the molybdate ions in presence of tartaric acid as a complexing agent at different conditions, to obtain the optimal condition of obtaining a considerable coating of high quality. 2. Experimental Part The preparation of the electrolyte for the electrochemical reduction of molybdate ions was carried out as follows. The desired concentration (0.1-0.5 M) of sodium molybdate (Na2MoO4 2H2O) was dissolved in an aqueous electrolyte of 0.007 M tartaric acid. It is observed that, the pH value depends on the concentration of Na2MoO4 2H2O. The pH of the electrolyte changed in the intervals of 1.69-2.1. It is found that at the selected optimal electrolyte composition, the pH value is 2.1. The polarization experiments were performed by potentiostat of IVIUM-STAT Electrochemical Interface. A three-electrode electrochemical cell of 100 mL volume was used. Pt and Ni sheets with an area of 2.2 x 10-3 dm2 were used as working electrodes. Silver-silver chloride electrode was used as a reference electrode. Whereas, a platinum sheet with an area of 4 cm2 was used as counter electrode. The universal ultra-thermostate UTU - 4 was utilized for regulation of the temperature of the electrochemical reaction inside the cell. The detection of the nucleation mechanism of the electrodeposited Mo was carried out by taking the chron-oamperometric method (current-time), using IVIUM-STAT potentiostat. The morphology and the chemical composition of electrodeposits were studied by using scanning electron microscope (SEM) and energy-dispersive X-ray analysis (EDAX) of mark "Carl Zeiss Sigma", respectively. SEM images of the electrodeposited films of Mo on Ni substrate was captured using a JEOL T330A SEM from Japan. The prepared sample was captured at fixed temperature of 20 °C at working distance of 10 mm, at power of 20 KeV and at magnification factor of 3000 times from the real sample. 3. Results and Discussion As known, to obtain semiconductive thin films from two or more components, the first required step was to study the electrochemical reduction processes of these components individually. Hence, the electrochemical reduction behavior of molybdate ions was investigated as shown in Fig. 1. In the presented work, full data about the electrore-duction of the molybdate ions in tartaric electrolyte were given. The cyclic polarization curves were recorded by the potentiodynamic method for finding out the kinetics and the mechanism of the electroreduction process of the mo-lybdate ions. As seen from Fig. 1, the electrochemical reduction of the molybdate ions on the cathode occurs through two stages within the potential range of 0.47 -(-0.25) V. It can be expected that within interval of potential values 0.47-0.27 V the process occurs with respect to reaction (1), whereas within 0.27-0.00 V with respect to reaction (2) (Fig.1). This also corresponds to the literature data.20 Majidzade et al.: Investigation of the Electrochemical Reduction Process ... Acta Chim. Slov. 2019, 66, 155-162 157 Potential Fig. 1. Cyclic voltamperometric curves of the electroreduction process of the molybdate ions in the presence of tartaric acid on the Pt electrode. The electrolyte consists of: 0.1 M Na2MoO4 2H2O + 0.007 M C4H6O6. T = 293 K, EV = 0.02 V/s. a -the general process, b- the initial stage. MoO42- + 4H+ + 2e = MoO2 + 2H2O MoO2 + 4H+ + e = Mo3+ + 2H2O (1) (2) After that, starting from 0.0 V potential value the reduction process of Mo3+ ions to metallic Mo as (3) reaction occurred: served from Fig. 2, with increasing of temperature, the current increases due to the acceleration of the process (the peak on the polarization curve obtained near E = 0.3 V, which is shown in Fig. 1b, due to an increase in the scale of the current becomes imperceptible in both Fig. 1a and Fig. 2). Mo3+ + 3e = Mo (3) Then, starting from the potential value of -0.45 V along with electroreduction of the molybdate ions the releasing of hydrogen also occurs. Consequently, the electroreduction process was carried out up to this potential (as shown in Fig. 1 a). The linear polarization experiments were performed as a function of temperature for determination of the kinetics of the process. The effect of temperature was investigated within a temperature range of 293-353°C. As ob- Fig. 2. The effect of temperature on the electroreduction process of the molybdate ions on the Pt electrode . Electrolyte composition in (M): 0.1 Na2MoO4 2H2O + 0.007 C4H6O6. T (K): 1- 293; 2- 313; 3333; 4- 353. At a scan rate of EV = 0.02 V/s 1/T-10 ,K 0.0 -0.1 -0.2 -0.3 E V Fig. 3. (a) lgik as a function of 1/T, (b) Activation energy as a function of potential. Potential (V): 1- (-0.05); 2-(-0.1); 3- (-0.15); 4- (-0.2); 5- (-0.25); 6- (-0.3). Majidzade et al.: Investigation of the Electrochemical Reduction Process 158 Acta Chim. Slov. 2019, 66, 155-162 At a temperature of 293 K the reduction occurs at -0.094 V, but at 353 K it occurs at a potential of 0.0 V. The lgic - 1/T relation was plotted within 0.0 - (-0.3) V potential range taken from the polarization curves of Fig. 2, shown in Fig. 3 (a). The value of effective activation energy obtained by the aid of equation A3(. = -2.3 Rtga, which indicates that the electrochemical reduction of the molyb-date ions in tartaric electrolytes is accompanied by mixed kinetics mechanism. In other words, as shown in Fig. 3, at the potential values are -0.05 - (-0.2) B, the kinetics of the electroreduction process is accompanied the electrochemical, and at -0.2 - (0.3) B the concentration polarization (Fig.3 (b)). Z2 * fo ' ?2 * iu Potential v Fig. 4. The effect of concentaration of the molybdate ions to elect-roreduction process on the Pt electrode. The electrolyte consists of 0.007 C4H6O6 + (M) Na2MoO4 2H2O. M= 1- 0.1; 2- 0.2; 3- 0.3; 40.4; 5- 0.5, at T = 293 K, and EV = 0.02 V/s. The effect of concentration of the molybdate ions on the electroreduction process has been studied within the 0.1-0.5 mol/L interval (Fig. 4). At concentrations range of -0.5 0.0 0.5 v Potential Fig. 5. The effect of potential-scanning to the electroreduction process of the molybdate ions on the Pt electrode. Electrolyte (M): 0,1 Na2MoO4 2H2O + 0,007 C4H6O6. Potential-scanning (V/s): 10,005; 2- 0,01; 3- 0,03; 4- 0,06; 5- 0,09; 6- 0,12. T = 293 K. 0.1-0.3 M, the effect is regular. In more precisely, with increasing of the concentration of the ions the electrochemical reduction takes place at more positive potentials. Within these intervals (0.3-0.5 M) the displacement of reduction potential to the negative side is observed. It can be interpreted as, with increasing of the concentration of the molybdate ions their mobilities in the electrolyte decrease, and then the electroreduction process becomes hard. Furthermore, at the high concentration, the molyb-date ions form a stable complex with tartaric acid. Therefore, their subsequent dissociation becomes difficult. As a result of the investigation, the optimal conditions have been detected, then we selected the optimal concentration of the molybdate ions up to 0.2 mol/L. The effect of potential scanning onto the electrore-duction process of the molybdate ions has been studied in the potential range of -0,27- (-0,32) V as shown in Fig. 5. As seen from Fig. 5, with increasing of potential scanning, the consumed current for the electroreduction process of the moybdate ions is increased. Where, current at 0.005 V/ sec is 5.53 x 10-4 A, whereas at 0.12 V/sec is 3.97 x 10-3 A. x 102 " ip A/dm2 -0.5' -1.0' -1.5' -2.0 -2.50.1 0.2 0.3 „«v/s Fig. 6. ip as a function of u1/2 for electroreduction of the molybdate ions. Electrolyte (M): 0,1 Na2MoO4 2H2O + 0,007 C4H6O6, T = 293 K. Fig. 6 indicates that the relationship between ip and u1/2 is linear. It also demonstrates that the electroreduction process of the molybdate ions occurs under the control of concentration polarization overpotential. The chronoamperometric (CA) method can be easily used for giving more precise information about the electrochemical deposition process, at which the potential can be stopped at the deposition potential (from CV). Via CA method, the mechanisms of the nucleation and growth of the electrodeposited particles can be studied. Current-time curves were carried out at different applied potentials of 0.047; 0.03; -0.06; -0.09; and -0.1 V at room temperature as shown in Fig. 7. It seems from the shown figure that the initial regime of the current-time curve is characterized by a sudden decrease in the current under application of the Majidzade et al.: Investigation of the Electrochemical Reduction Process ... Acta Chim. Slov. 2019, 66, 155-162 159 deposition potential. This can be attributed to the presence of the double-layer between the surface of the substrate and the ions of the solution, which lead to the formation of an immediate nucleation of molybdenum in all cases as shown in Fig. 7. Fig. 7. Current-time curves of the electrodeposition of molybdenum on Pt in electrolyte consists of 0.1 M Na2MoO4 2H2O + 0.007M C4H6O6 at T = 295 K at room temperature and at different deposition potentials; 0,047; 0.03; -0.06; -0.09; and -0.1 V vs. Ag/AgCl. This sudden decrease is followed by a little increase in the resultant current. This due to an increase in the elec-troactive surface area associated with the crystal growth. Moreover, it can be noted that the current density during the electrodeposition increases in all cases by increasing the deposition potential. The mechanism of crystal nucle-ation and growth can be determined by the analysis of the resulted current-time curves. The analysis of these curves can be achieved by applying the two equations of Scharifk-er-Hills,21 comparing with the experimental calculated data as shown in Fig. 8 (a-e). The models of the theoretical transients for the instantaneous and the progressive 3D nucleation are given by equations (4) and (5), respectively as follows: (4) (5) model by which the molybdenum nucleation occurred on many active sites of Pt surface substrates. Subsequently, the deposition deviates from the instantaneous nucleation as shown in Fig. 8 (a, b, c, and d). The deviation from the ideal assumption of the Scharifker model may be attributed to that the nuclei grow under diffusion control at these conditions. In fact, through time the nuclei growth and the electrodeposition of molybdenum will be under mixed control (diffusion and charge transfer). The deviation can be also interpreted as due to the hydrogen reduction during the formation of nuclei which cause morphology change of the nucleus. But, the experimental data for Fig. 8 e are in a good agreement with the theoretical model of the progressive nucleation and growth where the deposition potential of 0.1 V. Further information for the growth mechanism can be obtained by calculating the density number of actives sites for nucleation (N0); (6) where, C the bulk concentration in mol cm-3, zf the molar charge of electrodepositing species, M and p in g cm-3 are the molecular weight and the density of the deposited material, respectively. The diffusion coefficient D of the active species in the electrolyte can be calculated via the chrono-amperometric method. According to the theoretical nu-cleation model, the D is related to the imax and the tm by the following equation; 21, 22 (7) The values of imax, tmax, D, and N0 at different deposition potentials and temperatures are shown in Table 1. From these values, it is clear that by increasing the deposition potential, the imax tmax value slightly increases. This character is typical for 3D electrochemical nucleation and growth of a new phase under the mixed diffusion and adsorption control. Moreover, the D values at these conditions of the electroactive species are very small due to the high relative density of the electrolyte and the high relative diameter of molybdate ions. It is also observed that the D value is affected by the polarization potential as seen in Table 1. Fig. 8 (a-e) shows the nondimensional I2.I2 max vs. t. tmax plots of the CA data at the different condition as in Fig. 7. The solid lines of black and red color are the theoretical transients of the instantaneous and the progressive nucleation, respectively as shown in the figure, and dotted lines for the experimental data. The nucleation and growth processes of molybdenum at these conditions can be predicted from Fig. 8 (a-e). At the early stage, the experimental curve well fits the curve of the progressive nucleation Table 1. The experimental data of the CA electrodeposition of Mo onto Pt substrate Ed, V vs. Ag/AgCl D, cm2 s1 N0, cm 2 0.047 1.89 x 10-9 25.10 x 106 0.030 3.54 x 10-9 8.51 x 106 -0.06 5.61 x 10-9 5.38 x 106 -0.09 8.24 x 10-9 1.26 x 106 -0.10 10.17 x 10-9 1.07 x 106 Majidzade et al.: Investigation of the Electrochemical Reduction Process 160 Acta Chim. Slov. 2019, 66, 155-162 Fig. 8. Comparison of theoretical non-dimensional plots of (I/Imax)2 vs (t/tmax), for instantaneous and progressive nucleation with the experimental data derived from Fig. 7. It is noted from the table that nuclei densities N0 decrease significantly with the increase of the deposition potential. This decrease with the applied deposition potential is generally understood as the decreased of the activation of the nucleation sites at higher potentials, which deviates from the classical nucleation models as confirmed by Fig. 7. This deviation may be also explained as, by increasing the deposition potential the polarization of the working electrodes increases but the diffusion of the active species is still slow because of the high density of surrounding media which hinder the diffusion of the active species. Fig. 9. shows the morphology and chemical composition of electrodeposits of the metallic Mo films investigated by SEM and EDX. Mapping SEM images and EDX Majidzade et al.: Investigation of the Electrochemical Reduction Process ... Acta Chim. Slov. 2019, 66, 155-162 161 Fig. 9. SEM (a, b) and EDAX (c) of the electrodeposited Mo from a solution consists of 0,1 M Na2MoO4 2H2O + 0.007M C4H6O6 at the potential of E = -0.4 V, at scan rate = 0.02 Vs-1 on Ni electrode at T = 295 K analysis show that the quantity of metallic molybdenum is more than that of oxygen. The presence of oxygen may be returned to that there are some adsorbed molybdate ions on the electrode surface come from the electrolyte. Both Fig. 9a and 9b refer to the molybdenum sample deposited at E = -0.4 V potential. Figure 9a presents the general SEM description including nickel electrode, molybdenum and oxygen components. That is, the area marked with red indicates the Ni electrode, blue colored dots show the oxygen in the sample, and the green dots are molybdenum. Fig. 9b represents only the molybdenum film, in order to demonstrate its deposition more noticeably. As a result of the conducted experiments, the elect-roreduction of the molybdate ions has been implemented from an electrolyte consists of 0.1 M Na2MoO4 2H2O and 0.007 M C4H6O6, at a potential scan rate of 0.02 Vs-1 and temperature of 295 K. EDX analysis shows a good quality of the electrodeposited film from the chemical composition Mo and other elements. 4. Conclusions The electroreduction process of the molybdate ions from tartaric acid solutions has been investigated on the Pt electrode. Kinetics and mechanism of the process have been studied, using the data of cyclic and linear polarization curves. The nature of polarization process in this case is accompanied by mixing kinetics. The results of all performed experiments indicate that the concentration of the molybdate ions, temperature and potential scanning have Majidzade et al.: Investigation of the Electrochemical Reduction Process 162 Acta Chim. Slov. 2019, 66, 155-162 a great effect on the electroreduction process of the molyb-date ions in the tartaric acid electrolytes. The optimal conditions and the composition of electrodeposition bath for electroreduction process of the molybdate ions was preciously detected. The electrolyte composition consists of 0.1 M Na2MoO4 2H2O + 0.007 M C4H6O6. The electrolysis conditions are detected to be as follow: temperature 295 K, scan-rate 0.02 Vs-1, electrode Pt and Ni, pH = 2.1. In addition, using galvanostatic deposition technique, where thin films of molybdenum were obtained at a current density of 2-4 mA/cm2, at electrolysis time of 360-470 minutes. The results of the EDX analysis show that 63% of metallic molybdenum in the deposited thin films. 5. Reference 1. A. Sh. Aliyev, Sh. O. Eminov, B. Sh. Sultanova, V. A. Mejidza-deh, D. A. Kuliyev, D. Jalilova, D. B. Tagiyev, Chemical Problems. 2016, 2, 139-145. 2. M. Duda, R. Kowalik, K. Mech, P. R. Zabinski, Rudy Met. Niezel. 2012, 9, 586-591. DOI:10.1016/j.mssp.2015.01.006 3. A. Sh. Aliyev, M. Elrouby, S. F. Cafarova, Mat. Sci. Semicon. Proc. 2015, 32, 31-39. 4. F. Golgovici, T. Visan, M. Buda, Chalcogenide Lett. 2013, 6, 197-207. 5. M. R. Palacin, P. Simon, J. M. Tarascon, Acta Chim. Slov. 2016, 63, 417-423. DOI:10.17344/acsi.2016.2314 6. G. M. Huseynov, N. A. Mammadova, H. A. Imanov, Chemical Problems. 2017, 3, 329-334. DOI:10.32737/2221-8688-2017-3-329-334 7. V. A. Majidzade, P. H. Guliyev, A. Sh. Aliyev, M. Elrouby, D. B. Tagiyev, /. Mol. Struct. 2017, 1136, 7-13. DOI:10.1016/j.molstruc.2017.01.082 8. V. A. Majidzade, Sh. E. Alizade, A. Sh. Aliyev, Nakhchivan State University Scientific works. 2018, 92, 3, 176-179. 9. A. Sh. Aliyev, V. A. Majidzade, N. Sh. Soltanova, D. B. Tagiyev, V. N. Fateev, Chemical Problems. 2018, 2, 178-185. DOI: 10.32737/2221-8688-2018-2-178-185 10. V. A. Majidzade, Chemical Problems. 2018, 3, 331-336. DOI: 10.32737/2221-8688-2018-3-331-336 11. V. A. Majidzade, S. F. Cafarova, A. Sh. Aliyev, D. B. Tagiyev, Azerbaijan Chemical Journal. 2018, 3, 6-10. DOI: 10.32737/0005-2531-2018-3-6-10 12. K. Ogura, Y. Enaka, T. Yosino, Electrochim. Acta. 1977, 5, 509-512. DOI:10.1016/0013-4686(77)85113-X 13. P. Zanello, G. Raspi, A. Cinquantini, Talanta. 1977, 1, 25-30. 14. V. V. Kuznetsov, D. I. Zimakov, S. A. Chepeleva, V. N. Kudryavtsev, Russ. J. Electrochem. 2004, 40, 7, 711-715. DOI:10.1023/B:RUEL.0000035253.18329.98 15. S. F. Cafarova, A. Sh. Aliyev, M. Elrouby, N. Sh. Soltanova, D. B. Tagiyev, J. Electrochem. Sci. Eng. 2015, 4, 231-235. 16. N. D. Ivanova, S. V. Ivanov, E. I. Boldyrev, O. A. Stadnik, Prot. Met. Phys. Chem. 2006, 4, 388-392. 17. S. Rajak, S. K. Ghosh, P. U. Sastry, G. Sharma, R. C. Hubli, J. K. Chakravartty, Surf. Coat. Tech. 2015, 261, 15-20. DOI:10.1016/j.surfcoat.2014.11.073 18. S. M. Deambrosis, E. Miorin, F Montagner, V. Zin, M. Fabrizio, M. Sebastiani, F. Massimi, E. Bemporad, Surf. Coat. Tech. 2015, 266, 14-21. DOI:10.1016/j.surfcoat.2015.02.006 19. T. J. Morley, L. Penner, P. Schaffer, T. J. Ruth, F. Benard, E Asselin, Electrochem. Commun. 2012, 1, 78-80. DOI:10.1016/j.elecom.2011.11.026 20. A. A. Sukhotin (Ed): Handbook of Electrochemistry, Chemistry, Leningrad, Russian, 1981, p. 488. 21. B. Scharifker, G. Hills, Electrochim. Acta. 1983, 28, 7, 879889. DOI:10.1016/0013-4686(83)85163-9 22. M. R. Khelladi, L. Mentar, A. Azizi, A. Sahari, A. Kahoul, Mater. Chem. Phys. 2009, 115, 385-390. DOI:10.1016/j.matchemphys.2008.12.017 Povzetek V tem delu smo raziskovali redukcijski potencial molibdatnih ionov v tartarski kislini kot elektrolitu. Študirali smo kine-tiko in mehanizem elektrokemijskega procesa s ciklično in linearno voltametrično polarizacijsko metodo. Študirali smo vpliv temperature, hitrosti spreminjanja potenciala in koncentracije molibdatnih ionov na elektrokemijski proces. Ocena efektivne aktivacijske energije je pokazala, da je elektrokemijski proces kontroliran s kinetiko mešanja. Majidzade et al.: Investigation of the Electrochemical Reduction Process ... DOI: 10.17344/acsi.2018.4746 Acta Chim. Slav. 2019, 66, 163-167 /^creative ^commons Scientific paper Kinetic-Spectrophotometric Determination of Thiocyanate in Human Saliva Based on Landolt Effect in Presence of Astrafloxine FF Alexander Chebotarev,1 Vitaliy Dubovyi,1 Dmytro Barbalat,1 Elena Guzenko,1 Kateryna Bevziuk1 and Denys Snigur^* 1 Department of Analytical Chemistry, Odessa 1.1. Mechnikov National University, Dvoryanskaya 2, UA-65082 Odessa, Ukraine * Corresponding author: E-mail: 270892denis@gmail.com Received: 09-26-2018 Abstract In the present study a kinetic-spectrophotometric method for thiocyanate determination is described. The suggested method for the determination of thiocyanate is based on its "Landolt effect" on the reaction of bromate with hydrobro-mic acid, which leads to the formation of only one halogen bromine. The reaction was monitored spectrophotometri-cally at the maximum wavelength of astrafloxine FF light absorption at 535 nm. The absorbance of reactants mixture decreased with an increase of the reaction time. The calibration curve for thiocyanate determination was obtained in the concentration range of 0.03-2.0 |ig mL-1 under the optimal conditions (pH 1.5; CBro3- = 7.6 x 10-4 mol L-1; Castrafloxine FF = 1 x 10-5 mol L-1). The limit of detection was 0.01 |g mL-1. The method was successfully applied to the determination of thiocyanate in human saliva samples with satisfactory results. Keywords: Thiocyanate; human saliva; Landolt effect; spectrophotometry; astrafloxine FF. 1. Introduction The toxicity of thiocyanates attracts the attention of specialists in various fields, such as medicine, ecology, food technology, etc., to the problem of developing new and improving existing methods for their determi-nation.1 In the human body, thiocyanates are formed during the metabolism of sulfur-containing tobacco substances and some vegetables of the Brassica species or fed with drinking water and food. In biological fluids thiocyanates are also formed as a result of detoxification of cyanides in the liver.2 The most common source of inorganic cyanide in the human body is tobacco smoke. Since the content of thiocyanate in body fluids, especially saliva, increases with a constant source of cyanide, which is tobacco smoke, the concentration of thiocy-anate in saliva, urine and serum is used as a biomarker to detect smokers.3,4 Various analytical methods have been proposed for the determination of thiocyanates. These include the po-tentiometry with ion-selective electrodes,5 electrophoresis,6 gas chromatography, and sequential-injection analysis or extraction-spectrophotometric methods.7-10 The property of thiocyanates to inhibit the oxidation reaction of dyes (Landolt effect) is the basis of a number of kinetic methods. Landolt reactions have frequently been used for analytical purposes because of their operational and instrumental simplicity and high sensitivity.11-16 The spectrophotometric methods for the determination of thiocyanates based on the inhibition of the oxidation of Methyl Orange,12 Methyl Red,13 Methylene Blue,14 Crystalline Violet,15 and Janus Green have been proposed.16 The main disadvantage of the existing kinetic methods for the determination of thiocyanates based on the Lan-dolt effect is the formation of a mixture of halogens in the interaction of bromate and hydrochloric acid. In this case, the oxidative destruction or halogenation of the dyes occurs at different rates with formation of several products, which leads to poor reproducibility of the measurement results. To avoid overlapping absorption spectra of the dye and its oxidation/halogenation products, a symmetric dye astrafloxine FF was chosen. The reaction was monitored spectrophotometrically at the maximum wavelength of astrafloxine FF (535 nm) while measuring the change in the absorbance over time. Chebotarev et al.: Kinetic-Spectrophotometric Determination of Thiocyanate 164 Acta Chim. Slov. 2019, 66, 163-167 In this paper, we describe the development of a new method for the determination of thiocyanate, based on its "Landolt effect" on the reaction of bromate with hydrobro-mic acid, which leads to the formation of bromine. Bromi-nation of astrafloxine FF leads to the formation of only one colorless product. 2. Experimental 2. 1. Chemicals and Reagents The analytically pure reagents were used, and all solutions were prepared using distilled water. A 0.1 mol L-1 stock solution of thiocyanate was prepared by dissolving potassium thiocyanate in water. A 0.01 mol L-1 potassium bromate solution was prepared by dissolving KBrO3 in water. A 1 x 10-3 mol L-1 solution of astrafloxine FF was prepared by dissolving dye in 5 mL of ethanol and diluting to 100 mL with water. A 2.8 mol L-1 hydrobromic acid solution was prepared by dilution of concentrated acid. Solutions with lower concentrations were prepared by the appropriate dilution of more concentrated ones. 2. 2. Instrumentation The absorbance measurements were performed using a SF-56 spectrophotometer equipped with thermo-state at 25 °C in 1 cm quartz cells. The pH measurements were made using I-160M potentiometer with a combined glass electrode. The digital weight balance Axis ANG50C was used for weighing. Centrifuge type MPW-340 was used. 2. 3. General Procedure An aliquot of the solution containing thiocyanate was transferred to a 25 ml volumetric flask with the expectation that after dilution its concentration would be in the range 0.03-2.0 ^g mL-1. Then 1.9 mL of 0.01 mol L-1 potassium bromate solution, 0.3 mL of 2.8 mol L-1 hyd-robromic acid solution, and 2.5 mL of 1 x 10-4 mol L-1 astrafloxine FF solution were added. The obtained solution was diluted to 25 mL, mixed and transferred into a quartz cell. The absorbance was recorded as a function of time at 535 nm. 2. 4. Sampling and Sample Preparation The saliva samples were collected from a smoking and a non-smoking person. These samples were centri-fuged for 5 min at 3000 rpm and analyzed according to proposed method after appropriate dilution. 3. Results and Discussion 3. 1. Reaction Chemistry In Landolt processes, a slow reaction is linked to a fast one by the reaction product of the former.11 The Landolt effect may be shown as follows: A + B P, (1) P + L C, (2) Since the second reaction is faster than the first, its product (P) can only be detected once L (the "Landolt reagent") has disappeared completely as a result of the second reaction. The reaction of bromate with the hydrobromic acid produces only bromine: BrO3- + 5Br- + 6H+ 3Br2 + 3H2O (2) A strong oxidative agents such as Br2, Cu(III), Ce(-VI), and others can cause the decolorization of the astrafloxine FF,17 and the bromination reaction mechanism probably can be described by the scheme shown in Fig. 1. Thiocyanate is a Landolt reagent in the reaction with bromate because the presence of thiocyanate in the solution causes the consumption of the produced Br2, and its reaction with thiocyanate is much faster than dye bromination. The absorption spectra of astrafloxine FF dye and a product of its decolorisation are shown in Fig. 2. As can be seen from Fig. 2, the astrafloxine FF dye has an absorbance maximum at 535 nm and its bromination reaction product is colorless and has absorbance only in UV region. 3. 2. Effects of Variables To obtain the maximum sensitivity in the determination of thicyanate, the effects of the media acidity, bromate Fig. 1. A probable scheme of astrafloxine FF interaction with bromine in aqueous solution. Chebotarev et al.: Kinetic-Spectrophotometric Determination of Thiocyanate Acta Chim. Slov. 2019, 66, 163-167 165 Fig. 2. The absorption spectra of astrafloxine FF dye and a product of its decolorisation, Castrafloxine ff = 1 x 10-5 mol L-1; CCNS- = 2 x 10-5 mol L-1; CBrO - = 7.6 x 10-4 mol L-1; l = 1 cm. is about 3 min, which is sufficient for correct registration of the change in absorbance. The effect of astrafloxine FF concentration on reaction was studied in the range of 5 x 10-6-5 x 10-4 mol L-1. The results showed that astrafloxine FF concentration does not influence the reaction. Therefore, the astrafloxine FF concentration of 1 x 10-5 mol L-1 was selected as optimal. The effect of bromate concentration was also studied. Fig. 3 shows the differential dependence of absorbance on bromate concentration (dA/dC = f(C)). As can be seen from Fig. 3, a bromate concentration equal to 7.6-10-4 mol L-1 on the differential curve is the maximum. Thus, this concentration was used in further work. The effect of ionic strength on the kinetics of the oxidation of thiocyanate was not studied in this work, because a number of studies point to the lack of or very weak influence.12-14 Despite the fact that the temperature can significantly affect the reaction rate in this study, a temperature of 25 °C was a priori chosen as the most convenient for routine work. and astrafloxine FF concentrations were studied. The effect of media acidity was studied in the interval pH 1.0-3.0 (hy-drobromic acid concentration interval was 0.0112 - 0.112 mol L-1). When the concentration of hydrobromic acid was more than 0.112 mol L-1 hydrobromic acid (pH < 1), the decolorization of astrafloxine FF was coupled almost immediately. At pH higher than 2, the reaction takes more than 1 h. Thus, the optimum media acidity was chosen as a compromise. In the case when pH is 1.5, the reaction time 3. 3. Analytical Figures of Merit and Interference Study The slope method was used to construct the calibration plot for kinetic-spectrophotometric determination of thiocyanate (Fig. 4). In the concentration range 0.03-2.0 pg mL-1 of thiocyanate, a regression equation: -tga = -5.11 x 10-3 CCNS-+ 1.14 x 10-2 with correlation coefficient of 0.99 was ob- Fig. 3.The differential dependence of absorbance on bromate concentration, Castrafloxine ff= 1 x 10-5 mol L-1; pH 1.5; Ccns- = 2 x 10-5 mol L-1; l = 1 cm. Fig. 4.The calibration plot for kinetic-spectrophotometric determination of thiocyanate by slope method, Castrafloxine ff = 1 x 10-5 mol L-1; pH 1.5; CBrO - = 7.6 x 10-4 mol L-1; l = 1 cm. Chebotarev et al.: Kinetic-Spectrophotometric Determination of Thiocyanate 166 Acta Chim. Slav. 2019, 66, 163-167 tained. Also, limit of detection LOD (3a) 0.01 ^g mL-1 was calculated. The reproducibility of the proposed method was determined on the basis of 5 parallel determinations of thiocyanate with a concentration of 1 ^g mL-1, and the obtained relative standard deviation RSD value was 2.9%. The effect of some interfering ions on the determination of the 2 x 10-5 mol L-1 CNS- by the suggested method was studied and it was established that ions of alkaline metals, alkaline earth metals and a number of other ions did not interfere at the following molar ratios: 1:1500 (Na+, K+, Ca2+, Cl-); 1:1000 (CO32-, F-, PO43-, Cu2+, Mg2+, Al3+); 1:500 (Co2+, Fe2+, Fe3+); 1:100 (SO32-, NO2-, C2O42-). 3. 4. The Analytical Applications of the Method To demonstrate the analytical applications of the proposed method, the non-smoker and smoker human saliva samples were analyzed according to the suggested procedure. The received results are summarized in Table 1. The accuracy and reliability of the proposed method were investigated by applying it to determination of thiocyanate using the standard addition method. High (98.8-99.6%) recoveries (R) indicated that the suggested kinetic-spec-trophotometric procedure was accurate, reliable and can be used for the thiocyanate quantification. 3. 4. Comparison with Literature Studies A comparison of the proposed kinetic-spectropho-tometric method with other spectrophotometric methods which were reported in literature for thiocyanate determination is given in Table 2. The spectrophotometric method based on formation of Fe(III)-thiocyanate complex is poorly sensitive.18 The increased sensitivity of the determination of thiocyanate by kinetic-spectrophotometric methods should be noted.12-16,19,20 Methods for the determination of thiocyanate based on the formation and extraction of its ion pair with astrafloxine FF are also of low sensitivity.9,10 Zaruba with coauthors have proposed an original two-in-one device for online monitoring of direct immersion single-drop microextraction for precon-centration and determination of thiocyanate.21 This is the first time an optical probe was proposed as the microdrop holder and measuring cell. However, with the simplicity and convenience of this approach, its disadvantages are Table 1 .Determination of thiocyanate in human saliva by the proposed method (n = 5, P = 0.95) Sample range1 Spiked, ^g mL 1 Found, ^g mL 1 RSD, % R, % 'Thiocyanate concentration Non-smoker saliva "*M 20.0 20.0 86.25±2.46 105.8±3.28 2.3 2.5 99.6 78.54±3.02 99.12±3.57 3.1 2.9 100.6 51.98-155 Smoker saliva M 20.0 20.0 251.4±8.74 268.2±8.99 2.8 2.7 98.8 235.7±8.49 254.1±9.47 2.9 3.0 99.4 170.23-265 *The interval of thiocyanate concentration (|ig mL 1) in human saliva according to the literature data; **Male; ***Female; R - recovery. Table 2. Comparison of the developed method with some other methods of thiocyanate determination with spectrophotometric detection F F Method Reagent (\, nm) Linearity, ^g mL 1 LOD, ^g mL1 Ref. Spectrophotometry Fe(NO3)3 (447) 2-100 - 18 Kinetic-spectrophotometry Methyl Orange (525) 2 x 10-7 - 4 x 10-5 * 7 x 10-8 * 12 Kinetic-spectrophotometry Methyl Red (520) 0.05-1.1 0.0025 11 Kinetic-spectrophotometry Ferroine/IO4-(510) 0.02-0.2 0.016 19 Kinetic-spectrophotometry 4-amino-N,N- diethylanilinesulphate/Fe3+ (515) 0.05-0.5 0.012 20 DLLME* Astrafloxine FF (555) 3.1-28.2 0.11 9 SIA** Astrafloxine FF (555) 2.9-29 1.16 10 DI-SDME** Astrafloxine FF (555) 0.29-4.35 0.12 21 Kinetic-spectrophotometry*** Astrafloxine FF (535) 0.03 - 2.0 0.01 This work DI-SDME - direct immersion single drop microextraction; DLLME - dispersive liquid-liquid microextraction; SIA - sequential injection analysis; * concentration given in mol L-1; **ion pair formation between thiocyanate and astrafloxine FF and its extraction; ***decolorization of astrafloxine FF Chebotarev et al.: Kinetic-Spectrophotometric Determination of Thiocyanate ... Acta Chim. Slov. 2019, 66, 163- 167 167 low sensitivity and the need for optical probe with a special design. Also, the micropumping multicommutation flow system was successfully applied to determine thiocy-anate in human saliva samples.22 In general, the proposed kinetic-spectrophotometric method for the determination of thiocyanate based on the decolorization of astrafloxine FF can be attributed among the most sensitive methods. At the same time, the proposed method lacks the disadvantages of other kinetic methods, namely, poor reproduc-ibility. 4. Conclusions In this study, a novel, accurate and sensitive kinet-ic-spectrophotometric procedure for determination of thiocyanate trace concentrations has been reported. The method for the determination of thiocyanate is based on its "Landolt effect" on the reaction of bromate with hydro-bromic acid, which leads to the formation of only one halogen bromine and promotes decolorization of astrafloxine FF. The reaction was monitored spectrophotometrically at the maximum wavelength of astrafloxine FF at 535 nm. The limit of detection was 0.01 ^g mL-1.The method was successfully applied to the determination of thiocyanate in human saliva samples with satisfactory results. 5. References 1. Z. Glatz, P. Bouchal,O. Janiczek, M. Mandl, P. Ceskova, J. Chromat. A. 1999, 838, 139-148. 2. Z. Glatz, S. Novakova, H. Sterbova, J. Chromat. A. 2001, 916, 273-277. 3. G. F. Wang, M.G. Li, Y.C. Gao, B. Fang, Sensors, 2004, 4, 147-155. DOI:10.3390/s40900147 4. S. Narkowicz, E. Jaszczak, Z. Polkowska, B. Kielbratowska, A. Kotlowska, J. Namiesnik, Biomed. Chromatogr. 2018, 32, e4111. DOI: 10.1002/bmc.4111 5. N. Giorgio, Anal. Chem. 1975, 47, 763-764. DOI: 10.1021/ac60354a002 6. C. Bjergegaard, P. Muller, H. Sorensen, J. Chromat. A. 1995, 717, 409-414. 7. Y. Tanaka, N. Naruishi, H. Fukuya, J. Sakata, K. Saito, S. Wakida, J. Chromatogr. A. 2004, 1051, 193-197. DOI:10.1016/S0021-9673(04)00979-3 8. S. Kage, T. Nagata, K. Kudo, J. Chromatogr. B. 1996, 675, 2732. DOI: 10.1016/0378-4347(95)00344-4 9. V. Andruch, C. C. Acebal, J. Skrliková, H. Sklenárová, P. Solich, I. S. Balogh, F. Billes and L. Kocurová, Microchem J. 2012, 100, 77-82. DOI:10.1016/j.microc.2011.09.006 10. C. C. Acebal, H. Sklenárová, J. Skrliková, I. Síamková, V. Andruch, I.S. Balogh and P. Solich, Talanta, 2012, 96, 107-112. DOI:10.1016/j.talanta.2012.01.021 11. D. Perez-Bendito and M. Silva, Kinetic Methods in Analytical Chemistry, 1st ed. Ellis Horwood, Chichester, 1988, 330. 12. T. Madrakian, A. Esmaeili, A. Abdolmaleki, J. Anal. Chem. 2004, 59, 28-32. DOI:10.1023/B:JANC.0000011664.22353.e3 13. J. Ghasemi, R. Amini, A. Afkhami, Anal. Sci. 2001, 17, 435437. DOI: 10.2116/analsci.17.435 14. M. R. Shishehbore, N. Nasirizadeh, A.A. Kerdegari, Anal. Sci. 2005, 21, 1213-1216. DOI:10.2116/analsci.21.1213 15. G. Bagherian, M.A. Chamjangali, Z. Berenji, Eurasian J. Anal. Chem. 2008, 3, 307-317. 16. M. Keyvanfard, K. Alizad, P. Elahian, J. Chem. 2013, 2013, 1-5. 17. I. S. Balogh, M. Ruschak, V. Andruch, Y. Bazel, Talanta. 2008, 76, 111-115. DOI:10.1016/j.talanta.2008.02.011 18. J. Hovinen, M. Lahti, J. Vilpo, J. Chem. Educ. 1999, 76, 12811282. DOI:10.1021/ed076p1281 19. A. Afkhami, H. Bahrami, Anal. Letters. 1995, 28, 1785-1791. 20. V. R. Igov, R. M. Simonovic, R. P. Igov, Facta Univ. Ser. Phys., Chem. Technol. Univ. Nis. 2002, 2, 209-213. DOI: 10.1080/00032719508000356 21. S. Zaruba, A. Vishnikin, J. Skrliková, A. Diuzheva, I. Ozimanicová, K.Gavazov, V. Andruch, RSC Advances. 2017, 7, 29421-29427. DOI:10.1039/C7RA02326J 22. J. J. Silva Júnior, M. A. Farias, V. L. Silva, M. C. B. S. M. Montenegro, A. N. Araújo, A. F. Lavorante, A. P. S. Paim, Spec-trosc. Lett. 2010, 43, 213-219. DOI: 10.1080/00387010903287094 Povzetek V predstavljeni študiji opisujemo kinetično spektrofotometrično metodo za določanje tiocianata. Predlagana metoda za določanje tiocianata je osnovana na njegovem "Landoltovem učinku" na reakcijo med bromatom in bromovodikovo kislino, ki vodi do nastanka ene molekule broma. Reakcijo spremljamo spektrofotometrično pri absorpcijskem maksimumu astrafloksina FF pri 535 nm. Absorbanca reakcijske mešanice se zmanjšuje z naraščanjem reakcijskega časa. Umeritveno krivuljo za določitev tiocianata smo pridobili v koncentracijskem območju 0,03-2,0 |ig mL-1 pod optimalnimi pogoji (pH 1,5; CBro3- = 7,6 x 10-4 mol L-1; Castrafloksin FF = 1 x 10-5 mol L-1). Meja zaznave je bila 0,01 |g mL-1. Metodo smo uspešno uporabili za določitev tiocianata v vzorcih človeške sline z zadovoljivimi rezultati. Chebotarev et al.: Kinetic-Spectrophotometric Determination of Thiocyanate DOI: 10.17344/acsi.2018.4749 Acta Chim. Slov. 2019, 66, 168-172 /^creative ^commons Scientific paper Synthesis and Crystal Structure of a Polymeric Copper(II) Complex Derived from 2-Hydroxy-5-methylbenzaldehyde Oxime with Antibacterial Activities Ya-Li Sang* and Xue-Song Lin Department of Chemistry and Chemical Engineering, Chifeng University, Chifeng 024001, P. R. China * Corresponding author: E-mail: sangyali0814@126.com Received: 09-28-2018 Abstract A centrosymmetric O-bridged polynuclear copper(II) complex, [CuL2]n, where L is the deprotonated form of the Schiff base ligand 2-hydroxy-5-methylbenzaldehyde oxime, has been prepared and characterized by IR, UV and single-crystal X-ray determination. There is a crystallographic inversion center in the complex. The Cu atom in the complex is coordinated by the phenolate oxygen, imino nitrogen and hydroxyl oxygen atoms from two Schiff base ligands, forming octahedral geometry. The complex was tested in vitro for its antibacterial activity. Keywords: Schiff base; copper complex; synthesis; crystal structure; antibacterial activity 1. Introduction Schiff bases are readily synthesized by the condensation reaction of carbonyl compounds with primary amines.1 Schiff bases have been widely investigated for their biological activities, such as antibacterial and antitumor activities,2 biomimetic catalytic properties, etc.3 Metal complexes of Schiff bases have also been received much attention. These complexes not only play an important role in the development of coordination chemistry related to catalysis and enzymatic reactions, magnetism and molecular architectures,4 but also exhibit interesting biological activities.5 In recent years, a number of Schiff bases derived from salicylaldehyde and its derivatives with various amines, and their complexes have been reported.6 Most of the compounds show versatile biological properties especially antibacterial activities.7 In addition, copper complexes show effective biological activities.8 In the present work, we choose 2-hydroxy-5-methylbenzaldehyde oxime (HL) as ligand, to prepare a novel polymeric copper complex [CuL2]n. To our knowledge, there have been no complexes derived from HL reported so far. The antibacterial activities against Bacillus subtilis, Staphylococcus aureus, Escherichia coli, and Pseudomonas fluorescens, were evaluated for the Schiff base and the copper complex. Scheme 1. The Schiff base HL and the copper complex 2. Experimental 2. 1. Materials and Measurements 2-Hydroxy-5-methylbenzaldehyde, hydroxylamine hydrochloride, and copper perchlorate hexahydrate with AR grade were obtained from Aldrich and were used as received. Elemental analyses were performed using a Per-kin-Elmer 240C analytical instrument. Infrared spectra were recorded on a Nicolet 5DX FT-IR spectrophotometer Sang and Lin: Synthesis and Crystal Structure of a Polymeric Copper(II) Acta Chim. Slov. 2019, 66, 168-172 169 with KBr pellets. NMR spectrum was recorded on a Bruker instrument at 300 MHz. UV-Vis spectra were recorded on a JASCO V-570 spectrophotometer. Molar conductance was measured with a Shanghai DDS-11A con-ductometer. Caution! Although no problems were encountered in our work, perchlorate salts are potentially explosive. Therefore, only a small amount of copper perchlorate should be used at a time and handled with proper care. 2. 2. Synthesis of HL Hydroxylamine hydrochloride (0.70 g, 0.010 mol) and NaOH (0.40 g, 0.010 mol) were reacted in absolute ethanol (30 mL). The solid was removed by filtration. To the filtrate an ethanol solution of 2-hydroxy-5-methyl-benzaldehyde (1.36 g, 0.01 mol) was added with stirring. The mixture was stirred at room temperature for 30 min to give yellow solution. The solution was evaporated to give yellow powder, which was recrystallized from methanol and dried in air. Yield: 87%. Anal. Calcd. for C8H9NO2 (%): C, 63.56; H, 6.00; N, 9.27. Found (%): C, 63.45; H, 6.12; N, 9.15. 1H NMR (d6-DMSO): 5: 2.32 (s, 6H, CH3), 6.87 (d, 1H, ArH), 7.14 (d, 1H, ArH), 7.53 (s, 1H, ArH), 8.28 (s, 1H, CH=N), 11.71 (s, 1H, OH), 12.23 (s, 1H, OH). 13C NMR (d6-DMSO): 5: 21.2, 116.7, 118.1, 130.5, 131.3, 133.0, 146.7, 157.2. 2. 3. Synthesis of [CuL2]n To an ethanolic solution (15 mL) of HL (15.1 mg, 0.10 mmol) an ethanolic solution (10 mL) of copper per-chlorate hexahydrate (37.1 mg, 0.10 mmol) was added with stirring. The mixture was stirred for half an hour and filtered. The filtrate was kept undisturbed at room temperature to slowly evaporate for a few days, generating blue crystals suitable for X-ray diffraction. Crystals were isolated by filtration and dried in air. Yield 37% with respect to HL. Anal. Calcd. for C16H16CuN2O4 (%):C, 52.82; H, 4.43; N, 7.70. Found (%):C, 52.63; H, 4.55; N, 7.81. 2. 4. X-ray Crystallography A suitable single crystal with high quality of the complex was selected and mounted on a Bruker Smart 1000 CCD area-detector diffractometer with graphite monochromatized Mo-Ka radiation (X = 0.71073 A). Diffraction data for the complex were collected by « scan mode at 298(2) K. Data reduction and cell refinement were performed by the SMART and SAINT programs.9 Empirical absorption correction was applied by using SADABS.10 The structure was solved by direct methods and refined with the full-matrix least-squares technique using the SHELXL97 package.11 The non-H atoms in the structure were subjected to refined anisotropic refinement. Hydrogen atoms were located in geometrically and treated with the riding mode. Crystallographic data and experimental details for the complex are summarized in Table 1. Selected bond lengths and angles for the complex are listed in Table 2. Table 1. Crystallographic and experimental data for the copper complex Parameter Value Chemical formula C16H16CuN2O4 Formula weight 363.85 T (K) 298(2) Crystal system orthorhombic Space group Pbca a (A) 7.5170(5) b (A) 6.4796(4) c (A) 32.076(2) V (A3) 1562.3(2) Z 4 p (g/cm3) 1.547 |i(Mo-Ka) (mm-1) 1.419 F(000) 748 No. of measured reflections 12412 No. of unique reflections 1440 No. of observed reflections 1269 Parameters/ restraints 107/0 Rint 0.0234 Goodness of fit on F2 1.300 R1, wR2 [I > 2ff(I)]a 0.0397, 0.0859 R1, wR2 (all data)a 0.0450, 0.0884 Apmax/Apmin, e A-3 0.269, -0.254 R = ZjFoi - |Fc|/Z|Fo|, wR2 = [Iw(Fo2 - F/m^i-,2)2]1'2. Table 2. Selected bond lengths (Ä) and angles (o) for the copper complex Bond d, A Bond d, A Cu(1)-O(1) 1.921(2) Cu(1)-N(1) 1.934(2) Cu(1)-O(2A) 2.571(3) Angle w, ° Angle w, ° O(1)-Cu(1)-O(1A) 180 O(1)-Cu(1)-N(1A) 88.36(9) O(1)-Cu(1)-N(1) 91.64(9) N(1)-Cu(1)-N(1A) 180 O(1)-Cu(1)-O(2A) 98.85(9) N(1)-Cu(1)-O(2A) 84.28(9) O(1)-Cu(1)-O(2B) 81.15(9) N(1)-Cu(1)-O(2B) 95.72(9) Symmetry codes: A: 1 - x, 1 - y, - z; B: /+ x, V - y, -z. 2. 5. Antibacterial test Antibacterial activities of the Schiff base and the complex were tested in vitro against Bacillus subtilis, Staphylococcus aureus, Escherichia coli, and Pseudomonas fluo-rescens using MH medium (Mueller-Hinton medium: casein hydrolysate 17.5 g, soluble starch 1.5 g, beef extract 1000 mL). The minimum inhibitory concentrations (MIC) Sang and Lin: Synthesis and Crystal Structure of a Polymeric Copper(II) 170 Acta Chim. Slov. 2019, 66, 168-172 of the test compounds were determined by a colorimetric method using the dye MTT (3-(4,5-dimethylthi-azol-2-yl)-2,5-diphenyltetrazolium bromide).12 A solution of the compounds (50 ^g mL-1) in DMSO were prepared and graded quantities of the test compounds were incorporated in specified quantity of sterilized liquid MH medium. A specified quantity of the medium containing the compound was poured into microtitration plates. Suspension of the microorganism was prepared to contain about 105 colony forming units cfu mL-1 and applied to microtitration plates with serially diluted compounds in DMSO to be tested and incubated at 37 oC for 24 h. After the MICs were visually determined on each of the microtitration plates, 50 ^L of PBS (Phosphate Buffered Saline 0.01 mol-L-1, pH 7.4: Na2HPO4-12H2O 2.9 g, KH2PO4 0.2 g, NaCl 8.0 g, KCl 0.2 g, distilled water 1000 mL) containing 2 mg of MTT was added to each well. Incubation was continued at room temperature for 4-5 h. The content of each well was removed, and 100 ^L of isopropyl alcohol containing 5% 1.0 mol L-1 HCl was added to extract the dye. After 12 h of incubation at room temperature, the optical density (OD) was measured with a microplate reader at 550 nm. The observed MICs are presented in Table 3. Table 3. Antibacterial activities of the compounds MIC (|ig ml1) Bacillus Esche Pseudo- Staphylo- subtilis richia monas coccus coli fluorescens aureus HL > 100 12.5 > 100 25 [CuL2]n 3.12 1.56 12.5 1.56 Penicillin B 1.3 > 100 > 100 2.1 Kanamycin G 0.78 3.12 3.12 0.78 3. Results and Discussion The Schiff base HL was readily prepared by the reaction of 2-hydroxy-5-methylbenzaldehyde with hydroxyl-amine in ethanol. The copper complex was prepared by reaction of equimolar quantities of the Schiff base ligand and copper perchlorate hexahydrate in ethanol. The molar conductivities of the copper complex measured in acetoni-trile at concentration of 10-3 and 10-4 M are 25 and 7.0 O-1 cm2 mol-1, indicating the dissociation of the polymeric complex to form single [CuL2] units in such solution.13 3. 1. Crystal structure Description Figure 1 gives the perspective view of the copper complex. The molecule of the complex possesses crystallo-graphic inversion center symmetry, with the inversion center located at the Cu atom. The smallest repeat unit of the complex contains [CuL2] unit. The adjacent [CuL2] units are bridged by hydroxyl groups, with Cu—Cu separation of 4.962(2) A. The Schiff base ligand forms one six-membered chelate ring with bite angle of 91.64(9)°. The Cu atom is in an octahedral coordination, with the two phenolate oxygen and two imino nitrogen donor atoms from two Schiff base ligands defining the equatorial plane, and with two hydroxyl oxygen donor atoms from symmetry related Schiff base ligands occupying the axial positions. The axial coordinate bonds are much longer than those in the equatorial plane, which is caused by the Jahn-Teller distortion. The cis bond angles are in the range 88.36(9)-91.64(9)°. The Cu-O and Cu-N bond lengths in the complex are comparable to the values observed in Schiff base copper(II) complexes.14 The molecules are linked by hydroxyl groups to form two-dimensional network (Figure 2). Figure 1. Molecular structure of the copper complex. Displacement is drawn at the 30% probability level. Atoms labeled with the suffix A are at the symmetry position 1 - x, 1 - y, - z. Hydrogen atoms of the carbon atoms were omitted for clarity. Figure 2. Molecular packing diagram of the copper complex, viewed along the c axis. Hydrogen atoms of the carbon atoms were omitted for clarity. Sang and Lin: Synthesis and Crystal Structure of a Polymeric Copper(II) Acta Chim. Slov. 2019, 66, 168-172 171 3. 2. IR and UV-Vis Spectra The IR spectra of the free Schiff base and the copper complex provide information about the metal-ligand bonding. The weak and broad absorptions at 3350-3500 cm-1 are assigned to the stretching vibrations of the phenolic O-H group of the compounds. Several bands in the range of 2900-3150 cm-1 are assigned to the characteristic absorption of CH groups. The phenolic v(C-O) in the spectrum of the Schiff base is observed as a medium band at 1278 cm-1. However, in the spectrum of the complex, the absorption appears at 1287 cm-1. The strong band at 1639 cm-1 of the free Schiff base is assigned to the azome-thine group, vC=N,15 which is observed at low wave number of 1627 cm-1 for the complex, indicating the coordination of the imino nitrogen atom to the Cu atom. The newly observed bands in the region 450-650 cm-1 can be assigned to the Cu-O and Cu-N bonds. The electronic spectra of the compounds in ace-tonitrile were recorded in the range of 200-800 nm. The intense absorption band at 270 nm may be assigned to intra-ligand n-n transitions in the complex. The weak absorptions centered at 350 nm may be assigned to the phenolate of Schiff base ligand to Cu center charge transfer band (LMCT). Unlike the spectrum of the free ligand, much weaker and less well-defined broad band found in the spectrum of the complex at 605 nm which is assigned to the d-d transition. The transition is typical for square-planar copper(II) com-plexes,16 which indicates that the complex dissociated to form [CuL2] units with square planar geometry in such solution. 3. 3. Antibacterial Activity The Schiff base HL and the complex were screened in vitro for antibacterial activities against Bacillus subtilis, Staphylococcus aureus, Escherichia coli, and Pseudomonas fluorescens by the MTT method. The MICs of the compounds against the bacteria are presented in Table 3. Penicillin B was used as a reference. The Schiff base HL shows medium antibacterial activities against Escherichia coli and Staphylococcus aureus, and no activity against Bacillus subtilis and Pseudomonas fluorescens. In general, the copper complex has stronger activities against the bacteria than the free Schiff base. The complex has strong activity against Bacillus subtilis, Escherichia coli and Staphylococcus aureus, and medium activity against Pseudomonas fluo-rescens. The present complex has stronger activities against Bacillus subtilis and Escherichia coli than the copper complex we reported recently.17 It is quite interesting that the complex has better activity against Staphylococcus aureus than Penicillin B, and better activity against Escherichia coli than Kanamycin G, which deserve further study and to explore new antibacterial drugs. 4. Conclusion A new centrosymmetric O-bridged polynuclear cop-per(II) complex derived from 2-hydroxy-5-methylbenzal-dehyde oxime has been prepared and characterized. The Cu atom in the complex is in octahedral coordination. The antibacterial activities of the Schiff base and the complex were assayed. The results indicated that the complex is a potential antibacterial material. 5. Supplementary Material CCDC reference number 1445981 for the copper complex contain the supplementary crystallographic data for this paper. These data can be obtained free of charge at www.ccdc.cam.ac.uk, or from Cambridge Crystallographic Data Center, 12, Union Road, Cambridge CB2 1EZ, UK; Fax: +44 1223 336 033; e-mail: deposit@ccdc.cam.ac.uk. Acknowlegements The authors gratefully acknowledge the financial support by the Research Program of Universities of Inner Mongolia Autonomous Region (NJ10245). 6. References 1. (a) J. Valentova, S. Varenyi, P. Herich, P. Baran, A. Bilkova, J. Kozisek, L. Habala, Inorg. Chim. Acta 2018, 480, 16-26; D01:10.1016/j.ica.2018.04.058 (b) P. Nithya, R. Rajamanikandan, J. Simpson, M. Ilanchelian, S. 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Hosseinzadeh, F. Mohsenzadeh, K. Harms, Polyhedron 2017, 129, 189-198. D0I:10.1016/j.poly.2017.03.035 9. SMART and SAINT. Area Detector Control and Integration Software, Siemens Analytical X-Ray Systems, Inc., Madison, Wisconsin, USA, 1996. 10. G. M. Sheldrick. SADABS. Program for Empirical Absorption Correction of Area Detector Data, University of Göttingen, Germany, 1996. 11. G. M. Sheldrick. SHELXTL V5.1 Software Reference Manual, Bruker AXS, Inc., Madison, Wisconsin, USA, 1997. 12. J. Meletiadis, J.F. Meis, J.W. Mouton, J.P. Donnelly, P.E. Ver-weij, J. Clin. Microbiol. 2000, 38, 2949-2955. 13. J. W. Geary, Coord. Chem. Rev. 1971, 7, 81-122. D0I:10.1016/S0010-8545(00)80009-0 14. (a) F. Niu, K.-X. Yan, L.-H. Pang, D. Qu, X. Zhao, Z. You, Inorg. Chim. Acta 2015, 435, 299-304; D0I:10.1016/j.ica.2015.07.014 (b) Z. You, M. Liu, C. Wang, G. Sheng, X. Zhao, D. Qu, F. Niu, RSC Advances 2016, 6, 16679-16690. DOI: 10.1039/C6RA00500D 15. R. Golbedaghi, M. Rezaeivala, M. Khalili, B. Notash, J. Karimi, J. Mol. Struct. 2016, 1125, 144-148. D0I:10.1016/j.molstruc.2016.05.096 16. A. B. P. Lever. Inorganic Electronic Spectroscopy, 2nd ed., Elsevier, Amsterdam, 1984, p. 553. 17. Y.-L. Sang, X.-S. Lin, W.-D. Sun, Acta Chim. Slov. 2016, 63, 856-863. D0I:10.17344/acsi.2016.2795 Povzetek Sintetizirali smo centrosimetrični O-premosteni polimerni bakrov(II) kompleks, [CuL2]n, kjer je L deprotonirana oblika Schiffove baze 2-hidroksi-5-metilbenzaldehid oksim, ter ga okarakterizirali z IR, UV in monokristalno rentgensko di-frakcijo. Kompleks vsebuje kristalografski center inverzije. V kompleksu je Cu atom koordiniran s fenolatnih kisikovim atomom, imino dušikovim atomom in hidroksilnim kisikovim atomom dveh ligandov Schiffove baze in ima oktaedrično geometrijo. Kompleks smo testirali in vitro za antibakterijsko aktivnost. Sang and Lin: Synthesis and Crystal Structure of a Polymeric Copper(II) DOI: 10.17344/acsi.2018.4762 Acta Chim. Slov. 2019, 66, 173-181 /^creative ^commons Scientific paper Density Functional Approach: To Study Copper Sulfide Nanoalloy Clusters Prabhat Ranjan1 and Tanmoy Chakraborty^* 1 Department of Mechatronics Engineering, Manipal University Jaipur, Dehmi-Kalan, Jaipur-303007, India 2 Department of Chemistry, Manipal University Jaipur, Dehmi-Kalan, Jaipur-303007, India * Corresponding author: E-mail: tanmoychem@gmail.com; tanmoy. chakraborty@jaipur. manipal.edu Received: 10-20-2018 Abstract Nano clusters of Copper sulfides (CuS) have paramount importance due to its significant adsorption property and non-toxic behaviour. In this report, nanoalloy clusters of (CuS)n; (n = 1-8) have been theoretically analyzed in terms of Conceptual Density Functional Theory (CDFT) based descriptors, aiming to explore its electronic and other properties. Global DFT based descriptors have been computed for ground state configurations and low-lying isomers of (CuS)n clusters. Computed HOMO-LUMO gaps, lying in the range of 1.25-3.53 eV, indicate that (CuS)n clusters may be utilized as renewable energy sources specially in photocatalysis and solar cell applications. A statistical correlation has been established between electronic and photo-catalytic properties of copper-sulfide clusters with their computational counterparts. The close agreement between experimental and computed data supports our analytical approach. Keywords: Density Functional Theory; Copper-Sulfide Cluster; Descriptors; Optoelectronic Property; Regression Analysis 1. Introduction A search of alternative energy resources is one of the most popular topics of the scientific research. Recently, transition metal chalcogenide clusters have gained considerable importance due to their potential applications in the field of energy conversion, storage and optoelectronics.1-6 Among them, the compound formed between Cu and S is well known semiconducting material that has been extensively used in technological and strategic industries, including thermoelectric cooling materials, solar cells, clean-energy sectors, nonlinear optical materials, lithium ion batteries, gas sensors, nanoscale switches, photocata-lysts, supercapacitors, petrochemicals, pharmaceuticals etc.7-21 In addition, transition metal clusters particularly Cu, Ag and Au have huge applications in diverse technological domains due to its superior catalytic, magnetic, electronic and optical properties.16,22-24 In similar line, advancement of methodologies and characterization techniques have a strong dependence on utilization of different compositions of nanoalloys.16,25-26 A deep insight of core-shell structure of nano compounds has paramount importance since its properties may be regulated through proper control of other structural and chemical parameters. In view of regulating power of band gap on electronic properties of any materials, analysis of band gap of semiconductor materials has emerged as a major research domain in science and engineering. Band gap signifies energy difference between the top of the valence band and bottom of the conduction band. The highest occupied energy band represents the valence band and the lowest unoccupied energy band is known as conduction band.27 The nature of interaction of light absorbed by semiconducting material can be explained in terms of atomic structure of material. This complex interaction generates the free charge carriers i.e. electrons and holes, which are responsible factors for energy transformation. In order to free an electron from a covalent bond, the light particles, photons, must have to carry at least the energy equal or greater than the band gap of a semiconductor material.28 Band gap of semiconductor electrode in the range of 1.63-3.27 eV (760-380 nm), facilitates generation of free charge carriers in the visible light. It is already reported that zero consumption of energy (no external voltage) for hydrogen production with the use of UV solar light can be achieved utilizing TiO2 electrodes in Photo Electro Chemical (PEC) cells sunlight-absorbing semiconductor.29 A large number of theoretical Ranjan and Chakraborty et al.: Density Functional Approach: To Study Copper Sulfide 174 Acta Chim. Slov. 2019, 66, 173-181 and experimental studies based on catalytic materials for water splitting reaction have also been reported, which generate the new results on bulk semiconductor photocat-alysts.30 Recently, heterogeneous nanomaterials have also confirmed their highest catalytic performance to be developed as novel semiconductor electrodes in PEC cells.31 Copper Sulfides (CuS) have become popular in view of their three major properties namely high adsorption, non-toxic behaviour and the most importantly abundance in nature (i.e cheap material).32 By customizing the structure and concentration ratio of copper or sulfide, their respective energy band gap can be regulated. A number of reported data reveals the band gap of stoichiometric composition of copper sulfides, chalcocite (Cu2S), djurleite (Cu197S), digenite (Cu18S) and covellite (CuS) as 1.22, 1.40, 1.55 and 2.0 eV respectively.33-36 Keeping in view the data, it can be assumed that there is inverse relationship energy between band gap and the number of copper atoms in the clusters. The energy band gap for copper sulfide clusters, ranging from 1.2 eV to 2.5 eV, supports behavior of cluster as a leading p-type semiconducting material and suitable candidate for solar cells, nonvolatile memory devices, optoelectronics and non-liner optical devices.1,8,11-13,37-38 Recently, Li et al. have reported copper and sulfur doped copper clusters, describing the growth pattern of the Cun-1S clusters.39 In this work, authors have computed the optimized geometry, HOMO-LUMO gap, molecular orbital energy and density of states of Cun-1S clusters. The computed HOMO-LUMO gaps are in the range of 1.42-2.96 eV, which make CunS clusters suitable for renewable energy sources. The cluster Cu2S (Planar structure, C2v) has been identified as the most stable geometry with the highest HOMO-LUMO gap (2.96 eV). Sánchez et al. have also reported the structure and electronic properties of (CuS)N clusters (N = 1-6) in terms of DFT calculations.15 The authors also claimed the renewable energy property of the clusters on basis of computed HOMO-LUMO gaps, ranging between 1.3-3.3 eV. Due to high theoretical capacity, flat discharge curve and very good specific charge capacities, copper sulfide has been proven an excellent cathode material for lithium ion bat-teries.40-42 The inclusion of copper nanocrystals into sulfur electrode helps to intensify the cyclability of Li-S battery by alteration of sulfur to more electrochemically stable CuS.43,44 All these properties make copper sulfide a suitable candidate for capacity-contributing conductive filler for the sulfur electrode.45 Xie et al. have studied the compositional transformations of CuS nanocrystals when exposed to divalent cations, as Cd2+ and Hg2+ at room temperature in organic solvents.46 The authors claimed that the feature and mechanism of this analysis may be helpful in study of nanoscale chemical transformations. Karikalan et al. have studied the sonochemical route to synthesis of sulfur doped reduced graphene oxide and CuS nanocom-posite.47 The authors have validated the practicability of the developed glucose sensor in real biological samples. Selvi et al. have synthesized CuS and CdS nanoparticles capped with CTAB using hydrothermal technique.48 The result obtained from this study indicate that CuS and CdS with band gap of 1.8 eV and 2.3 eV respectively can be suitable for optoelectronic devices and photovoltaic applications. However, it is felt that deep insight in terms of experimental and theoretical analysis is required to further explore the intrinsic properties of these clusters.49-50 Density Functional Theory has gained importance due its computational friendly behavior. DFT is a widely accepted method to study the electronic structure of many-body systems. In the domain of material science research, particularly in super conductivity of metal based alloys,51 magnetic properties of nano-alloy clusters,52 quantum fluid dynamics, molecular dynamics,53 nuclear physics,54 DFT has become popular technique. The study of density functional theory spreads over three major areas viz. theoretical, conceptual, and computational.55-57 Conceptual density functional theory is recognized as an important tool to study the chemical reactivity of materi-als.58-60 The conceptual density functional theory is highlighted following Parr's dictum "Accurate calculation is not synonymous with useful interpretation. To calculate a molecule is not to understand it".61 We have been extensively applying conceptual density functional based global and local descriptors to study physico-chemical properties of nano-engineering materials and drug designing pro-cess.62-68 Computational study invoking DFT on CuS clusters is very limited. In this venture, we have analyzed electronic and photo-catalytic properties of (CuS)n nanoalloy clusters as a function of their size; n = 1-8. Conceptual DFT based descriptors namely electronegativity, hardness, HO-MO-LUMO gap, softness, electrophilicity index and dipole moment have been computed to correlate physico-chemical properties of the compounds. It also helps to analyze the stability and the semiconducting behavior of the clusters. A comparative analysis has been done between available experimental reports with our computed data. Considering paucity of DFT based study on these compounds, it is assumed that our analysis may help to bring a new insight in the domain of renewable energy sources. 2. Computational Details In this report, we have done computational analysis of the nanoalloy clusters of (CuS)n (n = 1-8) in terms of Density Functional Theory based global descriptors. 3d modeling and structural optimization of all the compounds have been performed using Gaussian 0369 within Density Functional Theory framework. Hybrid functional Becke, three parameter, Lee-Yang-Parr (B3LYP) exchange correlation with basis set LanL2dz has been adopted for geometry optimization. No restriction is imposed on mo- Ranjan and Chakraborty et al.: Density Functional Approach: To Study Copper Sulfide 175 Acta Chim. Slov. 2019, 66, 173-181 lecular spin during energy minimization process. Z-axis is considered as spin polarization axis. In order to ensure or-thogonalization on the (frozen) Core Orbitals (COs), Symmetrized Fragment Orbitals (SFOs) have been combined with auxiliary Core Functions (CFs). Invoking Koopmans' approximation,58 we have computed Ionization Energy (I) and Electron Affinity (A) for all the nano alloys using the following ansatz- I = ■ £homo a - - £lumo (1) (2) Thereafter, using I and A, the conceptual DFT based global molecular descriptors viz. electronegativity (x), global hardness (r|), molecular softness (S) and electro-philicity index («) have been computed. The equations used for such calculations are as follows- (3) Where, ^ represents the chemical potential of the system. (4) (5) (6) 3. Results and Discussion 3.1. Equilibrium Geometries In this section the structural aspects of low lying isomers of (CuS)n (n = 1-8), which were obtained during the search of ground state geometries, are discussed. The results for the most stable structure and low-lying structures of CuS clusters are presented in Figure-1. The linear structure is obtained for diatomic CuS cluster. The structure with C„v symmetry has a bond length of 2.18 A, which is nicely correlated with the data reported by Sánchez et al.15 We have listed five low-lying isomers for n = 2. It is observed that the optimized ground state geometry of (CuS)2, 2-a, with D2h symmetry is energetically more favorable than the other isomers, because rhombus structure of this cluster forces Cu-Cu bond distances to be shorter. In the lowest energy structure of (CuS)2, the bond length of Cu-Cu and CuS is 2.30 and 2.12 A respectively. The structure 2-b and 2-c with symmetry of C2 and C1 respectively, have an angle of 109.47°. The structure 2-b is energetically higher than the lowest energy structure of 2-a by 0.3 eV but at the same time it is lower in energy than structure 2-c. Magnitude of energy difference between the structures 2-d and 2-e is very small. Six low lying isomers of (CuS)3 clusters are identified. The cluster 3-a, having a 3D geometry and symmetry Cj, is more stable than the planar structures. The 3-a cluster arises from the coordination of one copper and one sulfur atom from top and bottom of rectangular cluster. The bond lengths of Cu-Cu, Cu-S and S-S in 3-a cluster are 2.31, 2.19 and 2.06 A respectively. The second most low lying isomer is 3-b which is 0.82 eV lower than the most stable structure of 3-a. Four more low-lying isomers, namely 3-c, 3-d, 3-e and 3-f, are also found in the range of 2.77 eV. A close agreement of energy value is observed for the structures 3-c and 3-d. Cluster 3-d is energetically higher than 3-c by 0.16 eV. A number of low-lying isomers are found out for the (CuS)4 cluster. Our computed result indicates that first three low-lying three dimensional structures (4-a, 4-b and 4-c) are lower in energy than the planar structure. The most stable structure 4-a has a triangle in between the cluster, surrounded by sulfur atoms, forming triangle with copper. The bond lengths of Cu-Cu, Cu-S and S-S in cluster 4-a are 2.35, 2.27 and 1.94 A respectively. The clusters 4-d and 4-e are found to be very close in energy and 4-e is only 0.08 eV higher than 4-d. The most stable structure (4-a), with C1 symmetry and 3D geometry, is energetically lower than the planar structure (4-d) by 1.00 eV. The planar structures of three isomers (4-d, 4-f and 4-g), formed by placing Cu atoms on all sites of structures, are within an energy range of 2.72 eV. It is observed that for cluster (CuS)5, structure 5-a, having symmetry group C1, is the most stable isomer. The structure corresponds to an interaction of two four atoms cluster of Cu2S2 connected back to back, while one sulfur and copper atom are coordinated to an edge of the same Cu2S2 cluster. In the structure 5-a, the bond lengths between Cu-Cu, Cu-S and S-S are 2.25, 2.17 and 1.89 A respectively. The second most stable isomer, 5-b is found to be 1.02 eV higher in energy than 5-a. Four more low-lying structures are found in the range of 4.61 eV. The structure 5-e is also formed through the interaction of two Cu2S2 clusters connected back to back, though Cu and S atoms arranged in a different way compared to structure 5-a. The isomer 5-e is 4.61 eV higher in energy than the most stable structure 5-a. Five low-lying isomers are identified for (CuS)6 cluster. The cluster 6-a with symmetry C1 is found to be the most stable structure. It arises from the coordination of four sulfur atoms on the edge forming triangles with six copper atoms, while remaining two sulfur atoms interacting with the face of the cluster. The bond lengths between Cu-Cu, Cu-S and S-S in the lowest energy structure of (CuS)6 are 2.28, 2.19 and 2.13 A respectively. The second most stable structure 6-b is a star like structure with interaction of two six atoms double layered cluster. The second and third most stable structures, 6-b and 6-c are energeti- Ranjan and Chakraborty et al.: Density Functional Approach: To Study Copper Sulfide 176 Acta Chim. Slov. 2019, 66, 173-181 ^ Jj 1-a C„v, AE= 0 eV (AE= 0 kcal/mol) V * / /' J ^ J , / 2-a 2-b 2-c 2-d 2-e D2h, AE= 0 eV (AE= 0 kcal/mol) C2, AE= 0.3 eV (AE= 6.91 kcal/mol) Ci, AE= 0.5 eV (AE= 11.53 kcal/mol) Cs, AE= 0.8 eV (AE=18.44 kcal/mol) Cs, AE= 0.82 eV (AE=18.91 kcal/mol) ik 3-a 3-b 3-c 3-d 3-e 3-f Ci, AE= 0 eV (AE=0 kcal/mol) Cs, AE= 0.81 eV (AE= 18.67 kcal/mol) Cs, AE=1.36 eV (AE=31.36 kcal/mol) Ci, AE= 1.52 eV (AE=35.05 kcal/mol) Ci, AE=2.01 eV (AE= 46.35 kcal/mol) C2v, AE= 2.77 eV (AE= 63.87 kcal/mol) A * * A T&T * * * J * * j - * 4-a 4-b 4-c 4-d 4-e 4-f Ci, AE= 0 eV (AE= 0 kcal/mol) C5, AE= 0.27 eV (AE= 6.22 kcal/mol) Ci, AE= 0.68 eV (AE= 15.68 kcal/mol) C2v. AE= 1.00 eV (AE= 23.06 kcal/mol) Ci, AE- 1.08 eV (AE= 24.90 kcal/mol) C2v, AE= 2.06 eV (AE=47.50 kcal/mol) __ C2v, AE= 2.72 eV (AE- 62.72 kcal/mol) M V/.v 5-a 5-b 5-c 5-d 5-e Ci, AE OeV (AE= 0 kcal/mol) Ci, AE= 1.02 eV (AE= 23.52 kcal/mol) Cs, AE= 2.70 eV (AE= 62.26 kcal/mol) C,, AE=3.8eV (AE= 87.62 kcal/mol) Ci, AE=4.61 eV (AE= 106.30 kcal/mol) Äi ¡X 'A- M 6-a 6-b 6-c 6-d 6-e Ci, AE=0 eV (AE= 0 kcal/mol) Ci, AE- 0.70 eV (AE= 16.14 kcal/mol) Ci, AE- 0.76 eV (AE= 17.52 kcal/mol) C2, AE-1.08 eV (AE= 24.90 kcal/mol) Ci, AE- 1.44 eV (AE= 33.20 kcal/mol) Figure 1. Ranjan and Chakraborty et al.: Density Functional Approach: To Study Copper Sulfide Acta Chim. Slov. 2019, 66, 173-181 177 I lût SÊÊ " - " * * * 7-a 7-b 7-c 7-d Ci, AE=0 eV (AE- 0 kcal/mol) Ci, AE=0.38 eV (AE- 8.76 kcal/mol) Ci, AE=0.76 eV (AE- 17.52 kcal/mol) Ci, AE=2.80 eV (AE- 64.56 kcal/mol) M 1% 8-a 8-b 8-c 8-d Ci, AE=0 eV (AE= 0 kcal/mol) Ci, AE=0.73 eV (AE= 16.83 kcal/mol) Ci, AE= 1.46 eV (AE= 33.66 kcal/mol) Ci, AE=3.78 eV (AE= 87.16 kcal/mol) Figure 1. Structures of low-energy isomers for (CuS)n clusters (n = 1-8). Relative energies and symmetry group are given at B3LYP/ LanL2dz level. Red spheres represent Cu atoms and yellow ones represent S atoms. cally less stable than 6-a by 0.70 and 0.76 eV respectively. Among four low-lying isomers of (CuS)7 cluster, the structure 7-a, having symmetry group Q , is the most stable cluster. In the structure 7-a, the bond lengths between Cu-Cu, Cu-S and S-S are 2.24, 2.14 and 2.13 A respectively. The second most stable cluster is energetically higher than 7-a by 0.38 eV. Two more low-lying isomers are found to be within 2.80 eV energy range. Four low-lying isomers, within the range of 3.78 eV, are identified for (CuS)8 cluster. The 8-a structure arises from the coordination of double layer Cu4S4, C1 cluster. The bond lengths between Cu-Cu, Cu-S and S-S in the lowest energy structure of (CuS)8 are 2.32, 2.29 and 2.15 A respectively. The second and third most stable isomers, 8-b and 8-c are 0.73 and 1.46 eV less stable than 8-a, respectively. Other isomers are also found but they are of high energy. The computed bond lengths between Cu-Cu, Cu-S and S-S of lowest energy structure reveals that the bond lengths in (CuS)n clusters follow the order Cu-Cu > Cu-S > S-S. The calculated bond lengths are consistent with the results obtained by Sanchez et al. [15]. 3. 2. The HOMO-LUMO Gaps and DFT Based Global Descriptors In this paper, we have reported computational analysis invoking electronic structure theory of copper sulfide nanoalloy clusters. The orbital energies as HOMO (Highest Occupied Molecular Orbital)-LUMO (Lowest Unoccupied Molecular Orbital) gap along with computed DFT based global descriptors for copper sulfide nanoalloy clusters have been presented in the Table-1. The molecular dipole moment of the compounds in Debye unit is also reported in the Table-1. Our computed HOMO-LUMO gap for the copper sulfide clusters (CuS)n, n = 1-8 has range from 1.25 to 3.53 eV. This is in close agreement with the desirable band gap of semiconducting nano materials, suitable for photo-catalytic processes and renewable energy application.15,32,39 The HOMO-LUMO gap of these clusters is running parallel with the reported data.15 From Table-1, a direct relationship is revealed between HO-MO-LUMO gaps of the nano-clusters and their evaluated global hardness. Frontier orbital energy gap and computed global hardness run hand in hand. This trend is expected considering experimental analysis. The molecule possessing the highest HOMO-LUMO gap will be the least prone to response against any external perturbation. It validates that clusters, having larger HOMO-LUMO gap, require significant amount of energy to promote electrons from the occupied to the unoccupied molecular orbitals. Our computed data reveals that (CuS)3 restricts itself to exhibit any response against the external perturbation whereas (CuS)4 exhibits the maximum reactivity under similar condition. In absence of quantitative benchmark pertaining to optical properties of aforesaid clusters, it is tacitly assumed that there must be a direct qualitative relationship between optical properties (specifically photo-catalytic) of CuS nano-clusters with their computed HO-MO-LUMO gap. The assumption has the basis that optical properties of materials are governed by flow of electrons within the systems, which in turn depends on the energy difference between valence and conduction band. A linear relationship between HOMO-LUMO gap with the difference in the energy of valence-conduction band is already reported.70 In view of that, it can be concluded that optical properties of the nano-clusters will be enhanced with an increase in their hardness values. Similarly, the softness data exhibits an inverse relationship towards the experimental optical properties. Table-1 also signifies that computed electronegativity and electrophilicity index have an Ranjan and Chakraborty et al.: Density Functional Approach: To Study Copper Sulfide 178 Acta Chim. Slov. 2019, 66, 173-181 inverse relationship with HOMO-LUMO gap of CuS na-no-clusters. Parr et al. have already reported that electro-philicity index measures the energy lowering of a ligand due to maximal electron flow between donor and acceptor and it depends on conjoint action of ionization potential and electron affinity.71 The linear correlation between HO-MO-LUMO gap along with their evaluated electrophilicity index is depicted in the Figure 2. The high value of regression coefficient (R2 = 0.902) supports our prediction. The shell model of metal clusters arises on the concept of full delocalization of molecular orbitals for the cluster electrons, in which the valence electrons of the cluster atoms are placed in shells of s, p and d character of the overall system.72-75 The analysis of the (CuS)3 clusters in terms of orbitals i.e., HOMO, HOMO-1, LUMO and LUMO+1, exhibiting the highest HOMO-LUMO gap among the mentioned nano-clusters, is plotted in the Figure 3. A* Figure 2. A Correlation Plot between Electrophilicity Index (eV) Vs HOMO-LUMO Gap (eV) HOMO HOMO-1 LUMO Figure 3. Orbitals of (CuS)3 Nanoalloy Cluster A comparative analysis has also been done in the Ta-ble-2, between experimental bond length and frequency with our computed data of the species namely Cu2, S2 and CuS. The result reveals that bond lengths follow the order as- Cu-Cu > Cu-S > S-S, which is in same line as previously reported.15 The close agreement between the experiment data and our computed data signifies the theoretical analysis. 4. Conclusion Study on copper-sulfide nanoalloy clusters has become important considering its wide field applications. In this paper, computational analysis of the structure, electronic and optical/ photo-catalytic properties of (CuS)n, [n = 1-8] nanoalloy clusters in terms of conceptual DFT based descriptors, are reported. The ground state configurations and low-lying isomers of (CuS)n clusters have been analyzed invoking electronic structure theory. The result exhibits an appearance of 3D structure of (CuS)3 with C1 Table 1. Computed DFT Based Global Descriptors of (CuS)n nanoalloy clusters (n = 1-8) Species HOMO- Electronegativity Global Global Electrophilicity Dipole LUMO Gap (eV) (eV) Hardness (eV) Softness (eV) Index (eV) Moment (Debye) CuS 2.394 5.142 1.197 0.417 11.045 4.440 (CuS)2 1.278 6.054 0.639 0.781 28.661 0.658 (CuS)3 3.537 4.870 1.768 0.282 6.706 0.817 (CuS)4 1.251 6.013 0.625 0.798 28.890 3.986 (CuS)5 2.041 6.135 1.020 0.490 18.448 1.533 (CuS)6 1.877 5.701 0.938 0.532 17.308 4.814 (CuS)7 2.829 5.442 1.414 0.353 10.465 2.142 (CuS)8 2.503 5.686 1.251 0.399 12.910 1.744 Table 2. A comparative analysis between computed bond lengths (A) and frequency (cm-1) with their experimental counterparts for the species Cu2, S2 and CuS.76-79 Species Computed Experimental Computed Experimental bond length bond length Frequency Frequency Cu2 2.25 2.21 257 265 S2 2.07 1.89 662 726 CuS 2.18 2.05 372 415 symmetry, which is more stable than the planar structure. The computed HOMO-LUMO gap, ranging from 1.25 to 3.53 eV, makes these clusters as potential candidates for renewable energy sources. Our computational data identifies the most reactive and least reactive species. The computed data describes that optical property of the instant clusters and global hardness run hand in hand, which is very much expected considering experimental facts. The high value of regression coefficient between electrophilici- Ranjan and Chakraborty et al.: Density Functional Approach: To Study Copper Sulfide Acta Chim. 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Slov. 2019, 66, 173- 181 181 Povzetek Nanoklastri bakrovih sulfidov (CuS) so zaradi njihovih adsorpcijskih lastnosti in netoksičnosti izjemnega pomena. V prispevku smo nanoklastre (CuS)n (n = 1-8) analizirali s pomočjo računske kemije na podlagi teorije gostotnega potenciala (conceptual DFT (CDFT)), z namenom dobiti vpogled v njihove elektronske in druge lastnosti. S pomočjo DFT kalkulacij smo izračunali nekatere parametre (»global descriptors«) za konfiguracije osnovnega stanja in nizko ležeče izomere (CuS)n klastrov. Izračunane HOMO-LUMO vrzeli, ki ležijo v območju od 1,25 do 3,53 eV kažejo, da bi lahko (CuS)n klastre uporabljali v procesu pridobivanja energije iz obnovljivih virov, zlasti pri fotokatalizi in kot material v sončnih celicah. Prikazali smo statistično korelacijo med elektronskimi in fotokatalitičnimi lastnostmi nanoklastrov na osnovi bakrovih sulfidov. Takšen pristop računske kemije podpira tudi ujemanje med eksperimentalnimi in izračunanimi podatki. Ranjan and Chakraborty et al.: Density Functional Approach: To Study Copper Sulfide DOI: 10.17344/acsi.2018.4767 Acta Chim. Slov. 2019, 66, 182-189 /^creative ^commons Scientific paper Voltammetric Investigation of Inclusion Complexes of the Selected Succinimides with ß-Cyclodextrin and (2-Hydroxypropyl)-ß-Cyclodextrin Jelena Lovič,1'* Milka Avramov Ivič,1 Bojan Božič,2 Jelena Ladarevič3 and Dušan Mijin3 1 Institute of Electrochemistry, University of Belgrade, Belgrade, Serbia 2 Faculty of Biology, University of Belgrade, Belgrade, Serbia 3 Department of Organic Chemistry, Faculty of Technology and Metallurgy, University of Belgrade, Belgrade, Serbia * Corresponding author: E-mail: jlovic@tmf.bg.ac.rs, jelena.lovic@ihtm.bg.ac.rs Received: 12-08-2018 Abstract The inclusion complexes of the selected, potentially biologically active, succinimides with p-cyclodextrin (pCD) and (2-hydroxypropyl)-p-cyclodextrin (HPpCD) were prepared. The formation of the inclusion complexes of the investigated monophenyl and diphenyl succinimide derivatives was confirmed using attenuated total reflection (ATR) study. Their electrochemical behavior was examined by cyclic voltammetry (CV) and square wave voltammetry (SWV) in 0.05 M NaHCO3 on a gold electrode. The stability constants for compound 1 were determined by cyclic voltammetry and calculated as KpCD = 350.87 M-1 and KHPpCD = 250.67 M-1. The SWV measurements reveal well defined peak at potential Ep = ~ 60 mV and the higher currents at Ep for both inclusion complex of the succinimides compared to the free compounds. The impact of chlorine atom in the phenyl moiety of succinimide derivatives on the activity in electrooxidation reaction is presented. Among the studied succinimides, according to SWV measurements, the most active is the monophenyl succinimide derivative (compound 1) in complex with pCD. The difference of peak current of compound 1 + pCD compared to compound 1 + HPpCD and free compound is 6.3 and 35.2 |A cm-2, respectively. Keywords: Diphenylsuccinimide; threephenylsuccinimide; ATR analysis; inclusion complexes; voltammetry. 1. Introduction Succinimides are derivatives of pyrrolidine-2,5-di-one which can be modified by the introduction of various alkyl and/or aryl groups on nitrogen or carbon atoms. Some of the modifications can be illustrated by the structures presented in Fig. 1. Succinimide ring represents a renowned pharmacophore in the drug discovery since its derivatives possess anticonvulsant activity.1-3 Substituted succinimides show anticholinesterase and antioxidant po-tentials,4 posses antimicrobial,5,6 antitumor,7 analgesic8 and antispasmodic9 activity. Apart from their biological importance, they also find application in liquid crystal displays (LCD)10 and in the production of water soluble reactive copolymers and polymers.11,12 They are appropriate scaffolds for organic synthesis,13 and in some cases, they are used as effective and recyclable catalysts.14 Evaluation of pharmacokinetic properties and in vitro cytotoxic activ- ity of N-phenyl substituted succinimides showed that all compounds were predicted for good permeability and solubility, oral absorption rate and moderate volume of distribution even for blood brain permeation, followed by acceptable observed toxicity.15,16 Furthermore, quantum mechanical and experimental studies of N-phenyl succin-imides were performed and detailed interpretation of spectral data were reported.17,18 For 1-aryl-3-methylsuc-cinimides, it is shown that interactions with hydrogen bond donor solvents and non-specific interactions with solvents play an important role in the solvatochromic behavior of these compounds. Moreover, MEP (Molecular electrostatic potential) data revealed regions of possible intermolecular interactions of N-phenyl succinimides and according to the analysis, oxygen atoms are suitable sites for electrophilic attack as well as for hydrogen bonding interactions with environment. Lovič et al.: Voltammetric Investigation of Inclusion Complexes of the Acta Chim. Slov. 2019, 66, 182-189 183 Figure 1. The structure of the investigated succinimides: (1) R1=H, R2=Ph, X=H; (2) R1=Ph, R2=Ph, X=H; (3) R1=H, R2=Ph, X=4-Cl; (4) R1=Ph, R2=Ph, X=4-Cl; (5) R1=H, R2=Ph, X=3-Cl; (6) R1=Ph, R2=Ph, X=3-Cl. The synthesis and physico-chemical properties of the succinimides given in Fig. 1, classify as monophenyl succinimide derivatives (1, 3, 5) and 3,3-diphenyl succin-imide derivatives (2, 4, 6) have been reported earlier.19,20 Recently, the results obtained from cyclic and square wave voltammetry, on the gold electrode, combined with computational studies, revealed that these compounds are electrochemically active and oxidized by the mechanism involving the conversion of COCHPh or -CH2- group in free radical by the loss of one proton in one electron pro- 21 cess.21 Succinimide electrochemistry has received modest attention so far. Previous electrochemical studies involve the electrochemical reduction of succinimides.22,23 Voltammetric studies on Pt cathode in aqueous media reveal that the N-bromosuccinimide is a two-electron reduction process and succinimide anion is an intermediate in this reaction while succinimide and N-alkylated succinimide are the observed products.22 The electrochemical reduction of succinimide examined on Pt cathode by cyclic voltammetry in aprotic media (acetonitrile) shows a single irreversible reduction peak. The product of these reaction is the succinimide anion in good yield and it was shown that this anion is stable in electrolysis medium.23 The aim of this work is to compare the already observed electrochemical activity of six selected succinim-ides with their activity in the form of inclusion complexes with cyclodextrines. Cyclodextrines (CD) are widely applied as non toxic encapsulated materials for a variety of guest molecules in order to improve their physico-chemical properties, such as aqueous solubility, physical chemical stability, and bioavailability of drug.24-29 Due to the fact that the investigated succinimides are slightly soluble in water, their inclusion complexes are prepared in order to enhance their water solubility. Among the host molecules, P-cyclodextrins (P-cyclo dextrin (PCD) and (2-hydroxy-propyl)-^-cyclodextrin (HP^CD)), built up from seven glucopyranose units, are chosen since they are suitable for complexing molecules bearing phenyl groups such as investigated succinimides. A number of nitrogen containing compounds have been electrochemicaly investigated, but to the best of our knowledge, the activity of succinimides (heterocyclic "pyrrolidine-2,5-dione" derivatives) in the form of inclusion complexes with cyclodextrines have not been subjected to the electrochemical studies. Since the impact of certain functional groups on the activity has been neglected in the literature, in this manuscript we provide the explanation about the influence of hydroxypropyl groups, chlorine atom or phenyl ring on the reactivity of investigated complexated compounds. 2. Experimental 2. 1. Material and Methods All investigated compounds have been previously synthesized.20 All used chemicals were p.a. grade (sodium hydrogen carbonate and sulphuric acid) obtained commercially from Sigma. 18 MO cm deionised water was obtained from a Milipore Waters Milli-Q purification unit. ATR spectra of the investigated succinimides were collected on a Nicolet™ iS™ 10 FT-IR Spectrometer (Thermo Fisher SCIENTIFIC) with Smart iTR™ attenuated total reflectance sampling accessories. 2. 2. Preparation of Inclusion Complexes The inclusion complexes of the investigated succinimides were prepared by mixing of the succinimides solution (0.15 mmol in 1 cm3 of methanol and then added to 100 cm3 of 0.05 M NaHCO3) with cyclodextrines solution (0.15 mmol in 50 cm3 of 0.05 M NaHCO3) to obtain 150 cm3 of1:1 succinimide:cyclodextrine complex in 0.05 M NaHCO3. The resulting mixture was shaken thoroughly and allowed to equilibrate at room temperature for 30 min before the addition to the electrochemical cell for electrochemical analysis. 2. 3. CV and SWV Measurements Electrochemical studies were performed using three-electrode cell with an Au working electrode (surface area 7 mm2), an Au wire auxiliary electrode and a calomel reference electrode. The Au working electrode was polished with diamond paste, cleaned with a mixture of 18 MO water and sulfuric acid and further cleaned with 18 MO deionized water in an ultrasonic bath. Prior to each experiment working electrode was checked by cycling the potential scan between -0.40 and 1.1 V in basic solution (0.05 M NaHCO3; pH = 8.4) at the scan rate of 50 mV s-1 until the unchanged CV characteristics for Au electrode were obtained. After that the electrode was transfer to the electrochemical cell containing succinimides or complex-ated succinimides and CVs were performed. For SWV measurements the accumulation of the succinimides and their inclusion complexes at the Au elec- Lovic et al.: Voltammetric Investigation of Inclusion Complexes of the 184 Acta Chim. Slov. 2019, 66, 182-189 trode was carried out for 0.2 s at - 0.45 V. After that a square-wave voltammetric stripping initiated in the positive potential direction was performed. The following instrumental parameters were used to record the square-wave voltammograms: step size 5 mV, pulse size 75 mV, frequency 10 Hz, scan rate 50 mV s-1. CV and SWV was carried out using PGZ 402 Volta Lab (Radiometer Analytical, Lyon, France). 3. Results and Discussion In the first part of the work, inclusion complexes were prepared by dissolving corresponding succinimide and CD in 0.05 M NaHCO3. The solutions were shaken for 15 min, and then evaporated under vacuum and dried. In order to study the formation of inclusion complexes, ATR analysis was performed. For illustration, Fig. 2A depicts ATR spectra of the relevant samples for the study of the inclusion complex of the compound 1 with HP^CD. The spectrum of HP^CD (b) displays characteristic bands at 3280 and 2926 cm-1 originating from O-H and C-H stretching vibrations, respectively and 1659 cm-1 as a result of H-O-H bending vibrations. The most prominent peaks at 1080 and 1021 cm-1 are ascribed to composition of the valence vibrations C-O-C, C-C-O and C-C-C. ATR spectra of 1 (c) show characteristic vibrations of car-bonyl groups at 1775 and 1701 cm-1. The spectrum of the physical mixture (d) represents the simple sum of the spectra of the succinimide molecule and HP^CD implying that this mixture do not provide inclusion of the guest molecule into the host molecule cavity. On the other hand, spectrum of inclusion complex (e) significantly differ from all of the spectra (a-d). Bands at 1425 and 878 cm-1 of the inclusion complex appear from NaHCO3, since the complexes are prepared in bicarbonate solution. The disappearance of the some corresponding peaks originating from succinimide ring and HP^CD is observed in this spectrum due to the restricted vibrations of the groups after the insertion of succinimde into HP^CD cavity. The decrease of the intensity and the shift from 1701 to 1685 cm-1 of the vibrations of the carbonyl group is observed in the spectrum of the inclusion complex as a result of the intermolecular hydrogen bonds between host and guest molecules. Furthermore, a broad band at 3280 cm-1 of HPpCD significantly decreased intensity and moved to 3458 cm-1 in complex clearly indicating the formation of inclusion complex. The same conclusions are derived for the inclusion complex of 1 with ^CD (Fig. 2B). In order to confirm the stoichiometry of the complex 1:1, CVs were recorded for different concentrations of sucinimide 1 while the HP^CD concentration remains unchanged, as it was proposed by 30. From Fig. 3 it is clear that the oxidation peak corresponding to the succinimides is growing proportional to the amount added suggesting that the molar ratio is 1:1. In inset of Fig. 3 it is presented linearity of current density vs. concentration dependency recorded at 0.7 V with excellent regression coefficient R = 0.998. For compound 1, with the most pronounced electrochemical activity 21, the stability constants were determined by cyclic voltammetry and calculated by "electrochemical current method" for both inclusion complexes.30,31 With the constant concentration of compound 1 and with different concentrations of both complexes the CVs were recorded as is presented in Fig. 4 and using A) B) Figure 2. ATR spectra of the corresponding compounds in the formation of inclusion complex of 1 with HP|CD (A) and |CD (B): (a) NaHCO3; (b) corresponding CD; (c) compound 1; (d) physical mixture of the compound 1 and corresponding CD and (e) the inclusion complex of 1 with corresponding CD. Lovic et al.: Voltammetric Investigation of Inclusion Complexes of the ... Acta Chim. Slov. 2019, 66, 182-189 185 Figure 3. CVs of Au electrode in 0.05 M NaHCO3 in the presence of compound 1 complexated with HP|CD (c = 1 mM). Concentrations of compound 1 are 0.2; 0.4; 0.6; 0.8 and 1 mM. v = 100 mV s-1. Inset: current density vs. concentration dependency at 0.7 V. equation from 30,31, the obtained stability constants are: KpcD = 350.87 M-1 and KHP(3CD = 250. 67 M-1. Those calculated values are in accordance with the determined constants of cyclodextrin inclusion complexes for aromatic carbonyl compounds 31 and inclusion complexes with ascorbic acid, uric acid, acetaminophen and some xantine alkaloids.30 The electrochemical investigation consisted of voltammetric and kinetic studies of selected succinimides performed by cyclic voltammetry is already published.21 The cyclic voltammetry of the all examined inclusion complexes of succinimides show the same response as free compounds, with the higher current in the region of the oxide formation. In 21 is shown and pointed out that the anodic currents of investigated succinimides, appeared only in the region of the oxide formation at gold electrode, are more pronounced when SWV technique is applied. With the same succinimides inclusion complexes with 250- 200- E ° 150-< =L 100- 50- compound 1 + |SCD -0.6 mM -------------0.8 mM -1 mM ..........1.2 mM 0.6 0.7 0.8 0.9 1.0 E / V (SCE) 1.1 250- 200- < =L 100- 50- 0- compound 1 + HPpCD 7 y — 0.6 mM 0.8 mM — 1 mM 1.2 mM 0.6 0.7 0.8 0.9 1.0 E /V (SCE) 1.1 Figure 4. CVs of Au electrode in 0.05 M NaHCO3 in the presence of compound 1 (c = 1 mM) complexated with |CD (A) and complexated with HP|CD (B). Concentrations of CDs are 0.6, 0.8, 1 and 1.2 mM. v = 100 mV s-1. Lovic et al.: Voltammetric Investigation of Inclusion Complexes of the 186 Acta Chim. Slov. 2019, 66, 182-189 A) 50- 40 fc o J 30 20 10 B) 90 80 CM E 70 o 60 < i 50 —, 40 30 20 10 C) 80 70 (Ni 60 E o 50 J 40 —, 30 20 10 A) -compound 1 — compound 2 ■•■-- compound 3 ------compound 4 compound 5 -♦— compound 6 -0.4 -0.2 0.0 E/V (SCE) 0.2 -compound 1 + pCD -----compound 2 + [3CD • compound 3 + pCD -------compound 4 + pCD —o— compound 5 + pCD // \ \\ —»— compound 6 + pCD T -0.4 ~i-1-r -0.2 0.0 E/V (SCE) 0.2 -compound 1 + HPpCD ----compound 2 + HPpCD — -compound 3 + HPpCD ------compound 4 + HPpCD -o—compound 5 + HPpCD -♦— compound 6 + HPpCD/'' i— -0.4 -0.2 0.0 E/V (SCE) 0.2 Figure 5. SWVs of investigated compounds (A); of the inclusion complexes of the investigated compounds with |CD (1:1) (B) and of the inclusion complexes of the investigated compounds with HP|CD (1:1) (C), obtained on Au electrode using 0.05 M NaHCO3. Accumulation time: 0.2 s at E = - 0.45 V; step size 5 mV, pulse size 75 mV, frequency 10 Hz, scan rate 50 mV s-1. P-cyclodextrin and (2-hydroxypropyl)-P-cyclodextrin are prepared and examined in this work by SWV. Figure 5A shows the SWVs of the selected succinim-ides recorded at the Au electrode in 0.05 M NaHCO3. Their 1:1 inclusion complexes with P-cyclodextrin are presented in Fig. 5B and their 1:1 inclusion complexes with (2-hy-droxypropyl)-P-cyclodextrin are presented in Fig. 5C. The SWV measurements reveal well defined peak at potential Ep = ~ 60 mV (Fig. 5) and the higher currents at Ep for both inclusion complex of the succinimides (Fig. 5B and 5C) comparing to succinimides presented in Fig. 5A. It indicates that the complexation of succinimides improves their electrooxidation ability. E o < 40 B) 60- 70- E o "5. 40 V///À free compound compound + ßCD KX2 al free compound compound + HPßCD Figure 6. Bar plots of the peak current densities obtained by the oxidation of the free succinimides and their inclusion complexes with |CD (A) and HP|CD (B) derived from Fig. 5 and their difference (network grid bar). According to Fig. 5A higher currents of the SWV signal for the compounds 1 and 5 in the region of Ep are observed. The highest peak current is observed for compound 1 complexated with 6CD as is presented in Fig. 5B. Concerning the inclusion complexes with HP6CD, the highest peak current is observed for compound 5 according to Fig. 5C. The difference between the peak current of free compound and inclusion complex (Djp), presented in Fig. 6 is a characteristic of improved electrooxidation ability. Re- Lovic et al.: Voltammetric Investigation of Inclusion Complexes of the ... Acta Chim. Slov. 2019, 66, 182-189 187 garding the inclusion complexes of succinimides with PCD, the highest Djp is noticed for compound 1, while for the inclusion complexes of succinimides with HPpCD the highest Djp is observed for compound 6. According to the computational analysis for the free compounds reported earlier,21 the obtained improved electrooxidation ability of the investigated succinimides was ascribed to the influence of the structural parameters. Namely, the monophe-nyl succinimide derivatives (1, 3, 5) were characterized by the formation of radical and anion at position C3, while 3,3-diphenyl succinimide derivatives (2, 4, 6) can form only one species of radical and anion as consequences of deprotonation at position C4.21 The complexing pattern likely involves hydrophobic N-phenyl ring bound in the hydrophobic cavity by van der Waals interactions. The structure is additionally stabilized through the formation of hydrogen bonds between car-bonyl groups of succinimide and hydroxyl groups outside the cavity (Fig. 7). Thusly, the part of the molecule responsible for electrochemical activity is preserved and available for oxidation process in the complex. The stabilization of the formed anion/radical through the hydrogen bonds and non specific interactions in CD:succinimide complex may be the reason for improved electrochemical activity with regard to corresponding succinimide. Proposed complex-ing mode is in accordance with complex formed between cyclodextrin and maleimides reported earlier.32 showed higher electrochemical activity, inclusion complexes with HPpCD were more electrochemically active. The presence of hydroxypropyl groups additionally stabilize complex since this cyclodextrin provides more hydrophobic microenvironment convenient for N-phenyl ring and thus interactions in greater extent than in the case of PCD. The prominent activity of 1 with PCD is observed, while with HPpCD the highest activity is observed for 5. From Fig. 6, it can be observed that introduction of phenyl ring in the position 3 of the succinimide ring has a different impact on the electrochemical activity depending on the nature of the substitutent in N-phenyl ring. Namely, unsubstituted (1) and meta-Cl substituted (5) diphenyl-succinimide: CD complexes show higher activity than corresponding threephenylsuccinimides:CD complexes (2 and 6, respectively), while for 3 and 4, the opposite trend is observed. For both CD, the lowest electrochemical activity is observed for compound 3 which could be attributed to the weak mesomeric effect of chlorine atom affecting accumulation of electron density and hindering the formation of radicals and anions during the electrooxidation of molecule. On the basis of the obtained electrochemical activity, it can be concluded that introduction of phenyl ring in 3, noticeable increases the current of complexes of 4 indicating that phenyl group contributes in stabilization of this particular anion/radical forms. For other compounds (1 and 5), by insertion of phenyl ring (2 and 6) the lower electrochemical activity is observed due to destabilization of complex caused both by conformational changes of the molecule and weakening of the interactions between host and guest molecules. Different behavior of different positioned chlorine atoms is related to diverse electronic effect in meta- and para- positions. As mentioned before, chlorine in para- position (3, 4) exerts weak mesomeric positive effect, while meta-chlorine (5, 6) exhibit strong negative inductive effect causing withdrawal of electron density and stabilization of the obtained anion/radical. The overall conclusion is that the effects of different groups and different cyclodextrins on the electrochemical activity of the complexes is complicated and involves syn-ergetic interplay of each effect both through the electronic nature of the substituents and the strength of the interactions between guest and host molecules. Figure 7. Schematic illustration of the succinimide:cyclodextrin complex. For consideration of complex activity, three structural factors should be taken into account: structure of the employed CD, electronic nature of substituent at N-phenyl ring and the presence/absence of phenyl group at the position 3 of succinimide ring. As can be seen from the data given in Fig. 6, except for compound 1, where inclusion complex with PCD 4. Conclusions The complex formation between selected succinimides and ^-cyclodextrin and (2-hydroxypropyl)-^-cyclo-dextrin was confirmed with ATR spectral analysis. The stability constants for compound 1 were determined by cyclic voltammetry and calculated as KpCD = 350.87 M-1 and KHPpCD = 250. 67 M-1. The SWV measurements reveal well defined peak at potential Ep = ~ 60 mV and the higher currents at Ep for both inclusion complex of the succinimides comparing to the free compounds. It indicates that the Lovic et al.: Voltammetric Investigation of Inclusion Complexes of the 188 Acta Chim. Slov. 2019, 66, 182-189 complexation of succinimides improves their electrooxi-dation ability. The highest peak current in this study is observed for compound 1 complexated with 6CD. Inclusion complexes with HP^CD of succinimide derivatives (2, 3, 4, 5, 6) were more electrochemically active in regard to the inclusion complexes with ^CD since the presence of hy-droxypropyl groups additionally stabilize complex through interaction with the succinimide molecule. The complex-ing mode is suggested and involves entering of hydrophobic N-phenyl ring into the hydrophobic cavity of cyclodex-trins which is additionally strengthened by hydrogen bonds between carbonyl groups of succinimide and hy-droxyl groups outside the cavity. Different electrochemical activity of the complexes is caused by synergetic interplay of the substituent effects at N-phenyl ring, absence/presence of the phenyl group at the position 3 of succinimide ring and the structure of the employed CD on the stability of the formed radical/anion and its interactions with corresponding CD. Acknowledgment The work was supported by the Ministry of Education, Science and Technological Development of the Republic of Serbia under the research projects: ON172013 and ON172060. 5. References 1. M. K. Hargreaves, J. G. Pritchard, H. R. Dave, Chem. Rev. 1970, 70, 439-469. DOI:10.1021/cr60266a001 2. K. Kaminski, J. Obniska, Bioorg. Med. Chem. 2008, 16, 49214931. DOI:10.1016/j.bmc.2008.03.037 3. K. Kaminski, S. Rzepka, J. Obniska, Bioorg. Med. Chem. Lett. 2011, 21, 5800-5803. DOI:10.1016/j.bmcl.2011.07.118 4. A. Sadiq, F. Mahmood, F. Ullah, M. Ayaz, S. Ahmad, F. U. Haq, G. Khan, M. S. Jan, Chem. Cent. J. 2015, 9, 31-39. DOI:10.1186/s13065-015-0107-2 5. N. P. Shetgiri, B. K. Nayak, Indian J. Chem. B 2005, 44B, 19331936. 6. T. N. Bansode, J. V Shelke, V. G. Dongre, Eur. J. Med. Chem. 2009, 44, 5094-5098. DOI:10.1016/j.ejmech.2009.07.006 7. S. H. Han, S. Kim, U. De, N. K. Mishra, J. Park, S. Sharma, J. H. Kwak, S. Han, H. S. Kim, I. S. Kim, J. Org. Chem. 2016, 81, 12416-12425. DOI:10.1021/acs.joc.6b02577 8. O. N. R. Fhid, T. H. Zeglam, S. E. A. Saad, M. S. Shlaka, M. A. Dao, World Pharm. Pharm. Sci. 2015, 4, 24-34. 9. V. C. Filho, R. J. Nunes, J. B. Calixto, R. A. Yunes, Pharm. Pharmacol. Comm. 1995, 1, 399-401. 10. K. Kishikawa, Y. Furukawa, T. Watanabe, M. Kohri, T. Tani-guchi, S. Kohmoto, Liq. Cryst. 2017, 44, 1332-1339. DOI: 10.1080/02678292.2017.1295477 11. H. Peng, M. Kather, K. Rübsam, F. Jakob, U. Schwaneberg, A. Pich, Macromolecules 2015, 48, 4256-4268. 12. K. M. Doll, R. L. Shogrent, R. A. Holser, J. L. Willett, G. Swift, Lett. Org. Chem. 2005, 2, 687-689. DOI: 10.2174/157017805774717553 13. P. Chauhan, J. Kaur, S.S. Chimni, Chem. Asian. J. 2013, 8, 328-346. DOI:10.1002/asia.201200684 14. F. Shirini, N.G. Khaligh, Dyes Pigm. 2012, 95, 789-794. DOI:10.1016/j.dyepig.2012.06.022 15. N. U. Perisic-Janjic, R. Kaliszan, N. P. Milosevic, G.S. Uscum-lic, N.R. Banjac, J. Pharm. Biomed. Anal. 2013, 72, 65-73. DOI:10.1016/j.jpba.2012.09.006 16. N. P. Milosevic, V. Kojic, J. Curcic, D. Jakimov, N. Milic, N. Banjac, G. Uscumlic, R. Kaliszan, J. Pharmaceut. Biomed. 2017, 137, 252-257. DOI:10.1016/j.jpba.2017.01.042 17. V. D. Vitnik, Ž. J. Vitnik, N. R. Banjac, N. V. Valentic, G. S. Ušcumlic, I. O. Juranic, Spectrochim. Acta A 2014, 117, 4253. DOI:10.1016/j.saa.2013.07.099 18. N. R. Banjac, B. D. Božic, J. M. Mirkovic, V. D. Vitnik, Ž. J. Vitnik, N. V. Valentic, G. S. Ušcumlic, J. Mol. Struct. 2017, 1129, 271-282. DOI:10.1016/j.molstruc.2016.09.086 19. N. U. Perisic-Janjic, R. Kaliszan, P. Wiczling, N. P. Milosevic, G. S . Uscumlic, N. R. Banjac, Mol. Pharm. 2011, 8, 555-583 DOI:10.1021/mp100373d 20. N. Banjac, N. Trisovic, Z. Vitnik, V. Vitnik , N. Valentic, G. Uscumlic, I. Juranic, Monatsh. Chem. 2013, 144, 1525-1535. DOI:10.1007/s00706-013-1052-1 21. B. Božic, J. Lovic, N. Banjac, Ž. Vitnik, V. Vitnik, D. Mijin, G. Ušcumlic, M. Avramov Ivic, Int. J. Electrochem. Sci. 2018, 13, 4285-4297. DOI:10.20964/2018.05.54 22. J. E. Barry, M. Finkelstein, W. M. Moore, S. D. Ross, J. Org. Chem. 1982, 47, 1292-1298. DOI:10.1021/jo00346a026 23. W. M. Moore, M. Finkelstein, S. D. Ross, Tetrahedron 1980, 36, 727-730. DOI:10.1016/S0040-4020(01)93685-3 24. Z. Z. Stoiljkovic, V. M. Jovanovic, D. Ž. Mijin, V. Nikolic, Lj. Nikolic, S. D. Petrovic, M. L. Avramov Ivic, Int. J. Electro-chem. Sci. 2013, 8, 9543-9557. 25. I. V Terekhova, N. A. Obukhova, Mendeleev Commun. 2005, 15, 38-40. DOI:10.1070/MC2005v015n01ABEH001901 26. I. V. Terekhova, T. V. Volkova, G. L. Perlovich, Mendeleev Commun. 2007, 17, 244-246. DOI: 10.1016/j.mencom.2007.06.022 27. I. V. Terekhova, Mendeleev Commun. 2009, 19, 110-112. DOI:10.1016/j.mencom.2009.03.021 28. A. Yu. Ziganshina, S. V. Kharlamov, E. Kh. Kazakova, S. K. Latypov, A. I. Konovalov, Mendeleev Commun. 2007, 17, 145-147. DOI:10.1016/j.mencom.2007.05.004 29. S. S. Jambhekar, P. Breen, Drug Discov. Today 2016, 21, 363368. DOI:10.1016/j.drudis.2015.11.016 30. L. Fritea, M. Tertis, T. L. Topala, R. Sandulescu, Farmacia 2013, 61, 1054-1068. 31. M. Vilar, M. Navarro, Electrochim. Acta 2010, 56, 305-313. DOI:10.1016/j.electacta.2010.08.079 32. S. Chaudhuri, T. Phelan, M. Levine, Tetrahedron Lett. 2015, 56, 1619-1623. DOI:10.1016/j.tetlet.2015.01.185 Lovic et al.: Voltammetric Investigation of Inclusion Complexes of the ... Acta Chim. Slov. 2019, 66, 182- 189 189 Povzetek Z ATR spektralno analizo smo potrdili tvorbo kompleksa med izbranimi sukcinimidi in p-ciklodekstrinom ter (2-hi-droksipropil)-p-ciklodekstrinom. S ciklično voltametrijo smo določili stabilnostne konstante za spojino 1 in izračunali vrednosti konstant KpCD in KHPpCD kot KpCD = 350.87 M-1 in KHPpCD = 250.67 M-1. SWV meritve so pokazale dva dobro definirana vrhova pri potencialu Ep = ~ 60 mV in, v primerjavi s prostimi spojinami, višje tokove pri Ep za oba vkl-jučitvena kompleksa s sukcinimidom. To kaže, da vključevanje sukcinimidov v ciklodekstrine izboljša njihovo elektro-oksidativno sposobnost. Najvišji tok pri vrhu smo v tej študiji opazili za spojino 1 kompleksirano z 6CD. Vključitveni kompleks derivatov (2, 3, 4, 5, 6) sukcinimida z HPpCD so bili bolj elektrokemijsko aktivni v primerjavi z vključitvenim kompleksom z pCD, ker prisotnost hidroksipropilnih skupin dodatno stabilizira kompleks preko interakcij z molekulo sukcinimida. Predlagali smo način tvorbe kompleksa, ki vključuje vstop hidrofobnih N-fenilnih obroče v hidrofobno votlino ciklodekstrina, kar je dodatno okrepljeno še z vodikovimi vezmi med karbonilnimi skupinami sukcinimida in hidroksilnimi skupinami izven votline. Različno elektrokemijsko aktivnost kompleksov povzroči sinergistična interakcija med substituentami na N-fenilnem obroču, odsotnost/prisotnost fenilnih skupin na položaju 3 v sukcinimidnem obroču in struktura uporabljenega CD s svojim vplivom na stabilnost nastalega radikala/aniona ter njegove interakcije z ustreznim CD. Lovic et al.: Voltammetric Investigation of Inclusion Complexes of the DOI: 10.17344/acsi.2018.4773 Acta Chim. Slov. 2019, 66, 190-195 /^creative ^commons Scientific paper Synthesis, Crystal Structures, and Antimicrobial Activity of Zinc(II) and Manganese(II) Complexes Derived from N,-(1-(Pyridin-2-yl)Ethylidene)Isonicotinohydrazide Ling-Wei Xue,* Hui-Jie Zhang and Pan-Pan Wang College of Chemistry and Chemical Engineering, Pingdingshan University, Pingdingshan Henan 467000, P.R. China * Corresponding author: E-mail: pdsuchemistry@163.com Received: 10-08-2018 Abstract Two mononuclear zinc(II) and manganese(II) compounds, [ZnL2] (1) and [MnL(HL)]ClO4 (2), where L is the mono-anionic form of N'-(1-(pyridin-2-yl)ethylidene)isonicotinohydrazide (HL), and HL is the zwitterionic form of HL, have been prepared and characterized by elemental analyses, IR and UV-Vis spectroscopy, and single-crystal X-ray crystallo-graphic determination. Compound 1 contains a neutral [ZnL2] complex molecule. Compound 2 contains a [MnL(HL)] + complex cation and one perchlorate anion. The metal atoms in the complexes are in octahedral coordination. The hydrazone ligands coordinate to the metal atoms through the pyridine N, imine N, and enolate O atoms. The compounds were investigated for their antimicrobial activity against Staphylococcus aureus, Escherichia coli, and Candida albicans. Keywords: Hydrazone, Zinc complex, Manganese complex, Crystal structure, Antimicrobial activity 1. Introduction Schiff bases bearing characteristic C=N bonds are a kind of versatile ligands in coordination chemistry.1 In recent years, metal complexes of Schiff bases have attracted considerable attention due to their interesting biological activity, such as antifungal, antibacterial and antitumor.2 Schiff base complexes derived from salicylaldehyde and its derivatives with primary amines, bearing the N2O, N2S, NO2 or NSO donor sets, have particular biological activi-ties.3 Hydrazone is a kind of special Schiff base compound, which possess interesting biological activities.4 Metal complexes usually show stronger biological activities than the hydrazone ligands.5 Chang and coworkers have reported that some transition metal complexes derived from N'-(1-(pyridin-2-yl)ethylidene)isonicotinohydrazide (HL; Scheme 1) have interesting antitumor and antioxidant ac-tivities.6 However, no report on the antimicrobial activity of complexes derived from HL has been reported so far. As an extension of our work on Schiff base complexes,7 a new H li O Scheme 1. The ligand HL zinc(II) compound, [ZnL2] (1), and a new manganese(II) compound, [MnL(HL)]ClO4 (2), are reported. 2. Experimental 2. 1. Material and Methods 2-Acetylpyridine and pyridine-4-carbohydrazide were purchased from Fluka. Other reagents and solvents were analytical grade and used without further purification. Elemental (C, H, and N) analyses were made on a Perkin-Elmer Model 240B automatic analyzer. Infrared (IR) spectra were recorded on an IR-408 Shimadzu 568 spectrophotometer. UV-Vis spectra were recorded on a Lambda 35 spectrometer. X-ray diffraction was carried out on a Bruker SMART 1000 CCD area diffractometer. The ligand HL was prepared according to the literature method.8 Caution! Perchlorate salts are potentially explosive. Only a small amount of material should be prepared, and they should be handled with great care. 2. 2. Synthesis of Compound 1 The hydrazone (48.0 mg, 0.20 mmol) was dissolved by methanol (10 mL) and then a methanol solution (10 mL) of Zn(ClO4) 6H2O (37.2 mg, 0.10 mmol) was added with stir- Xue et al.: Synthesis, Crystal Structures, and Antimicrobial Activity Acta Chim. Slov. 2019, 66, 190-195 191 ring. The mixture was stirred for 1 h at ambient temperature to give a colorless solution. Colorless block-shaped single crystals suitable for X-ray diffraction were formed by slow evaporation of the solution in air for several days. The yield is 45% (based on Zn). IR data (KBr, cm-1): 1595 (C=N), Table 1. Crystallographic data and refinement parameters for complexes 1 and 2 Parameters 1 2 Habit, color Block, colorless Block, brown Molecular formula C26H22N8O2Zn C26H23ClMn- N8O6 Formula weight 543.89 633.91 Crystal system Orthorhombic Monoclinic Space group Aba2 P2Jn Unit cell dimensions: a, Â 12.363(2) 11.755(2) b, Â 20.251(2) 9.437(2) c, Â 9.595(2) 26.198(2) a, ° 90 90 P, ° 90 98.197(2) ^ ° 90 90 V, Â3 2402.2(7) 2876.5(8) Z 4 4 Pcalcd, g cm-3 1.504 1.464 F(000) 1120 1300 Absorption coefficient, mm-1 1.065 0.607 Reflections collected 6959 16410 Independent reflections 1732 5322 Data/parameters 1353/169 3869/381 Restraints 1 0 R1, wR2 [I > 2ff(I)] 0.0333, 0.0634 0.0879, 0.2408 R1, wR2 (all data) 0.0538, 0.0718 0.1133, 0.2583 Goodness-of-fit on F2 1.020 1.070 1498, 1451, 1363, 1160, 1072, 947, 854, 543, 522. UV-vis data in methanol [Amax (nm), £ (L mol-1 cm-1)]: 277, 6450; 362, 15510. Anal. Calcd. (%) for C26H22N8O2Zn: C, 57.42; H, 4.08; N, 20.60. Found (%): C, 57.61; H, 4.17; N, 20.45. 2. 3. Synthesis of Compound 2 The hydrazone (48.0 mg, 0.20 mmol) was dissolved by methanol (10 mL) and then a methanol solution (10 mL) of Mn(ClO4)2 6H2O (36.2 mg, 0.10 mmol) was added with stirring. The mixture was stirred for 1 h at ambient temperature to give a brown solution. Brown block-shaped single crystals suitable for X-ray diffraction were formed by slow evaporation of the solution in air for several days. The yield is 33% (based on Mn). IR data (KBr, cm-1): 1596 (C=N), 1499, 1450, 1365, 1160, 1079 (ClO4), 945, 855, 756, 696, 622, 546, 518. UV-vis data in methanol [Amax (nm), £ (L mol-1 cm-1)]: 271, 7270; 366, 16480. Anal. Calcd. (%) for C26H22ClMnN8O6: C, 49.34; H, 3.50; N, 17.70. Found (%): C, 49.16; H, 3.62; N, 17.83. 2. 4. X-ray Structure Determination Data were collected from selected crystals mounted on glass fibres. The data were collected with Mo^ radiation (0.71073 A) at 298(2) K with a Bruker SMART 1000 CCD area diffractometer. The data for the two complexes were processed with SAINT9 and corrected for absorption using SADABS.10 Multi-scan absorption corrections were applied with ^-scans.11 The structures were solved by direct methods using the program SHELXS-97 and were refined by full-matrix least-squares techniques on F2 using anisotropic displacement parameters.12 All hydrogen atoms were placed at the calculated positions. Idealized H atoms Table 2. Selected bond lengths (Â) and bond angles (°) for complexes 1 and 2 1 Zn(1)-N(3) 2.069(3) Zn(1)-O(1) 2.149(3) Zn(1)-N(4) 2.193(4) N(3)-Zn(1)-N(3A) 173.3(3) N(3)-Zn(1)-O(1A) 101.34(13) N(3)-Zn(1)-O(1) 74.22(13) O(1)-Zn(1)-O(1A) 100.30(17) N(3)-Zn(1)-N(4) 75.14(14) N(3)-Zn(1)-N(4A) 109.70(15) O(1)-Zn(1)-N(4A) 91.47(13) O(1)-Zn(1)-N(4) 148.77(11) N(4)-Zn(1)-N(4A) 93.19(19) 2 Mn(1)-O(1) 2.144(4) Mn(1)-O(2) 2.151(4) Mn(1)-N(7) 2.191(4) Mn(1)-N(3) 2.193(4) Mn(1)-N(4) 2.314(5) Mn(1)-N(8) 2.326(4) O(1)-Mn(1)-O(2) 107.34(16) O(1)-Mn(1)-N(7) 122.05(15) O(2)-Mn(1)-N(7) 72.27(15) O(1)-Mn(1)-N(3) 72.01(14) O(2)-Mn(1)-N(3) 125.51(15) N(7)-Mn(1)-N(3) 155.58(17) O(1)-Mn(1)-N(4) 142.00(15) O(2)-Mn(1)-N(4) 90.07(16) N(7)-Mn(1)-N(4) 95.15(16) N(3)-Mn(1)-N(4) 70.41(15) O(1)-Mn(1)-N(8) 90.69(16) O(2)-Mn(1)-N(8) 142.58(14) N(7)-Mn(1)-N(8) 70.38(15) N(3)-Mn(1)-N(8) 90.90(15) N(4)-Mn(1)-N(8) 95.35(16) Symmetry code for A: -x, -y, z. Xue et al.: Synthesis, Crystal Structures, and Antimicrobial Activity 192 Acta Chim. Slov. 2019, 66, 190-195 were refined with isotropic displacement parameters set to 1.2 (1.5 for methyl groups) times the equivalent isotropic U values of the parent atoms. The large average Ueq of residue including Cl1 (0.335) in complex is caused by the disorder of the perchlorate anion. The crystallographic data for the complexes are listed in Table 1, selected bond lengths and bond angles for compounds 1 and 2 are given in Table 2. 2. 5. Antimicrobial Assay Qualitative determination of antimicrobial activity was done using the disk diffusion method as described in the literature.13 Suspensions in sterile peptone water from 24-h cultures of microorganisms were adjusted to 0.5 McFarland. Muller-Hinton Petri dishes of 90 mm were inoculated using these suspensions. Paper disks (6 mm in diameter) containing 10 ^L of the substance to be tested (at a concentration of 2048 ^g/mL in DMSO) were placed in a circular pattern in each inoculated plate. Incubation of the plates was done at 37 °C for 18-24 h. Reading of the results was done by measuring the diameters of the inhibition zones generated by the test substance. Tetracycline was used as a reference substance. Determination of MIC was done using the serial dilutions in liquid broth method. The materials used were 96-well plates, suspensions of microorganism (0.5 McFarland), Muller-Hinton broth (Merck) and stock solutions of each substance to be tested (2048 ^g/mL in DMSO). The following concentrations of the substances to be tested were obtained in the 96-well plates: 1024, 512, 256, 128, 64, 32, 16, 8, 4 and 2 ^g/mL. After incubation at 37 °C for 18-24 h, the MIC for each tested substance was determined by microscopic observation of microbial growth. It corresponds to the well with the lowest concentration of the tested substance where microbial growth was clearly inhibited. 3. Results and Discussion 3. 1. Chemistry The hydrazone compound HL was prepared by the condensation of equimolar quantities of 2-acetylpyridine with pyridine-4-carbohydrazide in methanol at ambient temperature. The hydrazone compound prepared in this way was formed in nearly quantitative yield and of high purity. The compounds 1 and 2 were readily synthesized by reaction of the hydrazone compound HL with zinc perchlorate and manganese perchlorate in methanol at ambient temperature. All the compounds are very stable at room temperature in the solid state, and soluble in common organic solvents, such as methanol, ethanol, and acetonitrile. The results of the elemental analyses are in accord with the composition suggested for the hydrazone and the complexes. 3. 2. IR and UV-Vis Spectra The typical band indicative of the azomethine group in the ligand of the complexes was observed at 1595 cm-1 for compound 1 and 1596 cm-1 for compound 2. The strong absorption at 1079 cm-1 in the spectrum of compound 2 is assigned to the stretching vibration of the perchlorate anion. UV-visible spectra of HL and the complexes were carried out in methanol. In the hydrazone, the band at 345 nm is attributed to the azomethine chromophore n-n* transition. The band at higher energy (265 nm) is associated with the benzene n-n* transition. In the spectra of the complexes, however, the azomethine chromophore n-n* transition is shifted to 362-366 nm, indicating that the im-ino nitrogen is involved in coordination to the metal ion. The absorption frequencies ascribed to the benzene n-n* transition (271-277 nm) are slightly changed. 3. 3. Structure Description of Compound 1 The molecular structure of compound 1 is shown in Fig. 1. Selected bond distances and angles are listed in Table 2. The Zn atom is coordinated by two pyridine N, two imine N, and two enolate O atoms from two deprotonated forms of hydrazone ligands, forming an octahedral geometry. The hydrazone acts as a tridentate ligand, forming two five-membered chelate rings with the Zn atom. The bond distances subtended at the Zn atom are comparable to those observed in the similar zinc(II) complexes with Schiff bases.14 The cis and trans coordinate bond angles are Table 3 Distances (Â) and angles (°) involving hydrogen bonding of the complexes D-H-A d(D-H) d(H-A) d(D-A) Angle(D-H-A) 1 C(9)-H(9)—0(1)#' 0.93 2.36(5) 3.180(5) 148(6) C(10)-H(10)—N(1)#2 0.93 2.54(5) 3.455(5) 170(6) C(13)-H(13A)—N(2) 0.96 2.43(5) 2.811(5) 103(6) 2 N(1)-H(1)—N(5)#3 0.93 2.39(3) 3.284(4) 161(7) C(10)-H(10)—0(5)#4 0.93 2.39(3) 3.284(4) 161(7) C(13)-H(13C)—N(2) 0.96 2.40(3) 2.778(4) 103(5) C(23)-H(23)—0(6)#5 0.93 2.48(4) 3.258(5) 142(6) C(26)-H(26A)—N(6) 0.96 2.37(3) 2.788(4) 106(5) Symmetry codes: #1: V + x, - y, -V + z; #2: V - x, -V + y, -1 + z; #3: V + x, -V - y, -V + z; #4: V - x, V + y, V - z; #5: -x, 1 - y, -z. Xue et al.: Synthesis, Crystal Structures, and Antimicrobial Activity Acta Chim. Slov. 2019, 66, 190-195 193 Fig. 1. Perspective view of complex 1 with 30% probability thermal ellipsoids. Atoms labeled with the suffix A are related to the symmetry operation -x, -y, z. Fig. 2. Molecular packing structure of complex 1, with hydrogen bonds drawn as dashed lines. range from 74.22(13) to 109.70(15)° and from 148.77(11) to 173.3(3)°, respectively, indicating the distortion of the octahedral coordination from ideal geometry. In the crystal structure of the compound, the complex molecules are linked through hydrogen bonds (Table 3), to form a three-dimensional network (Fig. 2). 3. 4. Structure Description of Compound 2 The molecular structure of compound 2 is shown in Fig. 3. Selected bond distances and angles are listed in Table 2. The compound contains a [MnL(HL)]+ complex cation and a perchlorate anion. The Mn atom in the [Mn-L(HL)]+ complex cation is coordinated by two pyridine N, two imine N, and two enolate O atoms from one deproto-nated form of the hydrazone ligand and one zwitterionic form of the hydrazone ligand, forming an octahedral geometry. The hydrazones act as tridentate ligands, forming two five-membered chelate rings with the Mn atom. The bond distances subtended at the Mn atom are comparable to those observed in the similar manganese(II) complexes with Schiff bases.15 The cis and trans coordinate bond angles are range from 70.38(15) to 125.52(15)° and from 141.99(14) to 155.58(17)°, respectively, indicating the distortion of the octahedral coordination from ideal geometry. In the crystal structure of the compound, the [MnL(HL)]+ complex cations and the perchlorate anions are linked through hydrogen bonds (Table 3), to form a three-dimensional network (Fig. 4). Fig. 3. Perspective view of complex 2 with 30% probability thermal ellipsoids. y Fig. 4. Molecular packing structure of complex 2, with hydrogen bonds drawn as dashed lines. 3. 5. Antimicrobial Activity The results are summarized in Table 4. A comparative study of minimum inhibitory concentration (MIC) values of the hydrazone and the metal complexes indicates that the complexes have better activity than the free hy- Xue et al.: Synthesis, Crystal Structures, and Antimicrobial Activity 194 Acta Chim. Slov. 2019, 66, 190-195 drazone. Generally, this is caused by the greater lipophilic nature of the complexes than the ligand. Such increased activity of the metal chelates can be explained on the basis of chelating theory.16 On chelating, the polarity of the metal atoms will be reduced to a greater extent due to the overlap of the ligand orbital and partial sharing of positive charge of the metal atoms with donor atoms. Further, it increases the delocalization of p-electrons over the whole chelate ring and enhances the lipophilicity of the complexes. This increased lipophilicity enhances the penetration of the complexes into lipid membrane and blocks the metal binding sites on enzymes of microorganisms. From Table 4, it is obvious that the two complexes have higher antibacterial and antifungi activities against Staphylococcus aureus, Escherichia coli, and Candida albicans when compared to the free hydrazone. Complex 1 has strong activity against Escherichia coli, with MIC value of 0.20 ^g/mL, which is comparable to Tetracycline. Complex 1 has medium activity against Staphylococcus aureus and weak activity against Candida albicans. Complex 2 has effective activity against Escherichia coli, and weak activity against Staphylococcus aureus and Candida albicans. In general, the zinc complex has stronger activity against Staphylococcus aureus, Escherichia coli, and Candida albicans than the manganese complex. So, the metal type of the complexes can influence the antimicrobial activity. Table 4. MIC values (^g/mL) for the antimicrobial activities of the tested compounds Compounds Staphylococcus Escherichia Candida aureus coli albicans The hydrazone 64 32 > 512 1 8.0 2.0 64 2 16.0 8.0 128 Tetracycline 0.32 2.12 > 1024 4. Supplementary Mateiral CCDC 1871743 for 1, and 1871744 for 2 contain the supplementary crystallographic data for this paper. These data can be obtained free of charge at http://www.ccdc. cam.ac.uk/const/retrieving.html or from the Cambridge Crystallographic Data Centre, 12 Union Road, Cambridge CB2 1EZ, UK; fax: +44(0)1223-336033 or email: deposit@ ccdc.cam.ac.uk. 5. Acknowledgments This research was supported by the Top-class foundation of Pingdingshan University (no. PXY-BSQD-2018006 and PXY-PYJJ-2018002). 6. References 1. (a) K. J. Davis, N. M. O. Assadawi, S. Q. T. Pham, M. L. Bir-rento, C. Richardson, J. L. Beck, A. C. Willis, S. F. Ralph, Dal-ton Trans. 2018, 47, 13573-13591; DOI: 10.1039/C8DT02727G (b) S. Banerjee, R. Patra, P. Ghorai, P. Brandao, S. G. Chow-dhury, P. Karmakar, A. Saha, New J. Chem. 2018, 42, 1657116582; DOI: 10.1039/C8NJ02235F (c) S. Asadizadeh, M. Amirnasr, F. F. Tirani, A. Mansouri, K. Schenk, Inorg. Chim. Acta 2018, 483, 310-320. DOI:10.1016/j.ica.2018.08.037 2. (a) Y. Salman, F. B. Barlas, M. Yavuz, K. Kaya, S. Timur, F. C. Telli, Inorg. Chim. Acta 2018, 483, 98-105; DOI:10.1016/j.ica.2018.08.010 (b) A. S. Burlov, V. G. Vlasenko, Y. V. Koshchienko, N. I. Makarova, A. A. Zubenko, Y. D. Drobin, L. N. Fetisov, A. A. Kolodina, Y. V. Zubavichus, A. L. Trigub, S. I. Levchenkov, D. A. Garnovskii, Polyhedron 2018, 154, 65-76; DOI:10.1016/j.poly.2018.07.034 (c) S. Alyar, C. H. Sen, H. Alyar, S. Adem, A. Kalkanci, U. O. Özdemir, J. Mol. Struct. 2018, 1171, 214-222. DOI: 10.1016/j.molstruc.2018.06.004 3. (a) A. K. Ghosh, C. S. Purohit, R. Ghosh, Polyhedron 2018, 155, 194-201; DOI:10.1016/j.poly.2018.08.021 (b) Y.-T. Li, J.-W. Dong, Y. Lu, Y.-T. Gu, C.-N. Shang, F.-Y. Li, Y. Xin, C.-L. Jing, Z.-L. You, Chinese J. Inorg. 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Liu, G.-D. Huang, Z.-W. Li, P. Yu, Y. Gou, F. Yang, Eur. J. Med. Chem. 2018, 156, 368-380; DOI:10.1016/j.ejmech.2018.07.022 (d) R. Fekri, M. Salehi, A. Asadi, M. Kubicki, Inorg. Chim. Acta 2019, 484, 245-254; DOI:10.1016/j.ica.2018.09.022 (e) Z.-Y. Hao, Q.-W. Liu, J. Xu, L. Jia, S.-B. Li, Chem. Pharm. Bull. 2010, 58, 1306-1312. DOI:10.1248/cpb.58.1306 6. H.-Q. Chang, L. Jia, J. Xu, W.-N. Wu, T.-F. Zhu, R.-H. Chen, T.-L. Ma, Y. Wang, Z.-Q. Xu, Transition Met. Chem. 2015, 40, 485-491. Xue et al.: Synthesis, Crystal Structures, and Antimicrobial Activity Acta Chim. Slov. 2019, 66, 190- 195 195 7. (a) G.-P. Cheng, L.-W. Xue, C.-X. Zhang, Acta Chim. Slov. 2017, 64, 261-265; D01:10.17344/acsi.2016.3036 (b) L. Wang, Y.-J. Han, Q.-B. Li, L.-W. Xue, Acta Chim. Slov. 201 6, 63, 822-826; D0I:10.17344/acsi.2016.2699 (c) Q.-B. Li, Y.-J. Han, G.-Q. Zhao, L.-W. Xue, Acta Chim. Slov. 2017, 64, 500-505. D0I:10.17344/acsi.2017.3416 8. M. R. Maurya, S. Khurana, W. Zhang, D. Rehder, Dalton Trans. 2002, 3015-3023. D0I:10.1039/b202852m 9. SMART and SAINT. Area Detector Control and Integration Software, Madison (WI, USA): Bruker Analytical X-ray Instruments Inc., 1997. 10. G. M. Sheldrick, SADABS, Program for Empirical Absorption Correction of Area Detector Data, Göttingen (Germany): Univ. of Göttingen, 1997. 11. A. C. T. North, D. C. Phillips, F. S. Mathews, Acta Crystallogr. A 1968, 24, 351-359. D0I:10.1107/S0567739468000707 12. G. M. Sheldrick, SHELXL-97, Program for the Refinement of Crystal Structures, Göttingen (Germany): Univ. of Göttingen, 1997. 13. (a) A. Barry, Procedures and Theoretical Considerations for Testing Antimicrobial Agents in Agar Media, In: Antibiotics in Laboratory Medicine, Lorian, V. (Ed.), Baltimore: Williams and Wilkins, 1991; (b) T. Rosu, M. Negoiu, S. Pasculescu, E. Pahontu, D. Poirier, A. Gulea, Eur. J. Med. Chem. 2010, 45, 774-781. D01:10.1016/j.ejmech.2009.10.034 14. (a) S. Konar, A. Jana, K. Das, S. Ray, S. Chatterjee, S. K. Kar, Polyhedron 2012, 47, 143-150; D0I:10.1016/j.poly.2012.07.080 (b) Z. He, C. He, Z.-M. Wang, E.-Q. Gao, Y. Liu, C.-H. Yan, Dalton Trans. 2004, 502-504; D0I:10.1039/b315281b (c) C.-F. Chow, S. Fujii, J.-M. Lehn, Chem. Asian. J. 2008, 3, 1324-1335. D0I:10.1002/asia.200800101 15. (a) C.-F. Wang, Z.-J. Chen, K.-D. Zhao, X.-F. Chen, C.-Y. Zhang, G.-H. Sheng, H.-L. Zhu, J. Coord. Chem. 2016, 69, 2656-2665; D0I:10.1080/00958972.2016.1213819 (b) N. A. Mangalam, S. R. Sheeja, M. R. P. Kurup, Polyhedron 2010, 29, 3318-3323; D0I:10.1016/j.poly.2010.09.007 (c) S. Banerjee, A. Ray, S. Sen, S. Mitra, D. L. Hughes, R. J. Butcher, S. R. Batten, D. R. Turner, Inorg. Chim. Acta 2008, 361, 2692-2700. D0I:10.1016/j.ica.2008.01.019 16. J. W. Searl, R. C. Smith, S. Wyard, J. Proc. Phys. Soc. 1961, 78, 1174-1181. D01:10.1088/0370-1328/78/6/311 Povzetek Sintetizirali smo dve enojedrni cinkovi(II) in manganovi(II) spojini, [ZnL2] (1) in [MnL(HL)]ClO4 (2), kjer je L mon-oanionska oblika N'-(1-(piridin-2-il)etiliden)izonikotinohidrazida (HL) in HL ion dvojček HL, ter ju okarakterizirali z elementno analizo, IR in UV-Vis spektroskopijo ter monokristalno rentgensko strukturno analizo. Spojina 1 je nevtralen [ZnL2] kompleks. Spojina 2 je zgrajena iz [MnL(HL)] + kompleksnega iona in enega perkloratnega aniona. Kovinski ioni v obeh kompleksih so oktaedrično koordinirani. Hidrazonski ligand se koordinira na kovinski ion preko piridinskega N, iminskega N in enolatnega O atoma. Spojinama smo določili antimikrobno aktivnost proti Staphylococcus aureus, Escherichii coli in Candidi albicans. Xue et al.: Synthesis, Crystal Structures, and Antimicrobial Activity DOI: 10.17344/acsi.2018.4783 Acta Chim. Slov. 2019, 66, 196-207 /^creative ^commons Scientific paper Evaluation of DNA/BSA Binding and Chemical Nuclease Activity of L-Tyrosine-Based Mn(III) and Fe(III) Metallo-Intercalators Biju Bennie Rajaretnam,1 Joel Chellappa,1 Daniel Abraham Solomon,2 Iyyam Pillai Subramanian3 and Theodore David Manickam Selvanayagam4,* 1 Postgraduate Department of Chemistry, St. Johns College, Tirunelveli-627002, Tamil Nadu, India. 2 Department of Chemistry, Madras Christian College, Chennai-600059, Tamil Nadu, India. 3 Postgraduate and Research Department of Chemistry, Pachaiyappas College, Chennai-600030, Tamil Nadu, India. 4 Department of Applied Chemistry, PSN College of Engineering and Technology, Tirunelveli-627152, Tamil Nadu, India. * Corresponding author: E-mail: s.theodore.david@gmail.com Mobile: +91 9366705118 Received: 10-11-2018 Abstract A novel class of Mn(III) and Fe(III) complexes of L-tyrosine-based ligand has been synthesized and characterized through various analytical and spectroscopic techniques. These complexes were found to exhibit efficient binding properties with the biomolecules viz. calf thymus DNA and BSA. The ability of complexes to bind with such biomolecules has been explored through absorption, emission and viscosity measurements. Based on spectroscopic techniques we can conclude that the complexes could bind to DNA via intercalation. It was observed that these complexes can cleave pBR322 DNA in gel-electrophoresis technique through oxidative mechanism. The BSA was quenched by the complexes around 340 nm adopting a mechanism of static mode. The binding constants, thermodynamic parameters and the donor to acceptor distance were calculated. Besides, molecular docking simulations were carried out for the complexes with human DNA topoisomerase and BSA protein. The docked poses are visualized to provide supportive evidence to the interaction of the synthesized complexes with DNA/BSA. Keywords: BSA; CT DNA; Coordination complex; L-Tyrosine; Molecular docking 1. Introduction Biomolecules such as proteins or nucleic acids are particularly interesting as the target since they act as the key molecules in the metabolic pathways associated with a specific disease.1 A drug is a molecule designed in order to obstruct the target function in the particular disease modifying pathway. The transition metal complexes containing multidentate aromatic ligands play important role in different biological processes which eventually helps to develop different structural and more importantly functional model systems.2-4 DNA is known to be an important cellular receptor. Interactions of small metal complexes with DNA and proteins are the key research areas of current years as there are enough potentials of development of new therapeutic agent particularly showing anti- tumor properties and possibility of the transportation of these molecules throughout the physiological system via protein binding.5-8 The metal complexes-nucleic acid interaction has gained the attention of researchers due to their excellent biological activity. The activity of the complexes is described based on their mode and affinity of binding with DNA.9-11 Generally, DNA has been considered as a target molecule for a wide range of disease therapies such as anticancer and antiviral. Any disorder in gene expression may cause diseases and plays a secondary role in the outcome and severity of human diseases.12 Therefore, various metal-based drugs have been opted with improved pharmacological properties and aimed at different targets. Proteins play fundamental roles in sustaining life and are an integral part of origin, evolution, Manickam Selvanayagamet al.: Evaluation of DNA/BSA Binding and Chemical Acta Chim. Slov. 2019, 66, 196-197 207 and metabolism. Being one of the most abundant proteins in blood constituent, serum albumin aids in the transportation of various molecules (ligands) including fatty acids, steroids and metal ions.13 Hence the binding of these molecules to serum albumins is taken under consideration in various fields.14 The homology of 76% in structures between the bovine serum albumin (BSA) and the human serum albumin (HSA) makes it a unique one for the researchers to be studied more comprehensively.15 Serum albumins are model globular proteins found abundant in plasma. Serum albumins exhibit a character of reversible binding with various molecules through hydrophobic, hy-drophilic and electrostatic interactions. The binding of metal complexes with BSA can be detected by the decrease in intensity of the fluorescence emitted by one of the amino acid residues called tryptophan 212, which is exposed outwards. It is expected that the ligand could bind into the two binding sites of BSA marked as site I and site II located in the hydrophobic cavities of subdomains IIA and IIIA, respectively. The binding of drug molecules to BSA on a particular site can be probed through various selective site markers.16 Serum albumin also binds metal ions, predominantly soft/intermediate metal ions at one or more of at least four proposed specific sites. Albumin binds hard metals in chelate form. For example, albumin scavenges hemin, the ferric state of heme, to avoid the formation of reactive oxygen species. Albumin binding affects the pharmacokinetics, trafficking and efficacy of metal-based therapeutics.17 Amino acids are small molecules capable of metal complexation via amino and car-boxylate groups.18 It has been reported that transition metal complexes of amino acids could possess better fungicidal, anti-bacterial, antiviral and anti-tubercular activities.19 Hence, we herein report the synthesis of Mn(III) and Fe(III) complexes of L-tyrosine-based ligand and their interaction with CT-DNA/BSA using various spectral methods. 2. Experimental All the chemicals utilized in this research work are of AR grade and also 99% pure solvents are used. The ligands employed in our present investigation, viz. 9,10-phenan-threnequinone (Aldrich) and L-tyrosine (Loba Chemie) were purchased in pure form and used as such. CT-DNA and pBR322 DNA was purchased from Genei, Bengaluru, India. The protein bovine serum albumin (BSA) used in this experiment was purchased from Sigma whereas Tris buffer from HIMEDIA. Carlo Erba 1108 elemental analyzer was involved in analysing the elemental content (C, H, N) of the ligand as well as its Mn(III) and Fe(III) complexes. The percentage of metal ions in the complexes were estimated as per the procedure given in the book.20 Nuclear magnetic study was performed on Bruker Advance DRX 300 FT-NMR spectrometer in CDCl3 solvent and the refer- ence material taken was TMS. JEOL DX-303 EI mass spectrometer and Bruker-Daltonics micro TOF-Q II mass spectrometer were used to record EI mass spectrum of the ligand and electrospray ionization mass spectra (ESI) of the complexes respectively. Vibrational spectral analysis of the ligand and its manganese and iron complexes were carried out on JASCO FT-IR/4100 Spectrometer, involving KBr disc method. The UV-vis spectra of both the synthe-sised complexes were taken in 200-800 nm wavelength range on a Perkin Elmer Lambda 35 spectrophotometer using the solvent DMSO. Hertz SG8-5HJ model Gouy magnetic balance was employed to study the magnetic susceptibility measurements by taking CuSO4 • 5H2O as the calibrant. The molar conductivities of MnL and FeL were measured using Elico model SX 80 conductivity-bridge taking DMSO as solvent. 2. 1. Synthesis of Ligand About 2.08 g of 9,10-phenanthrenequinone (10.0 mmol) dissolved in 50 mL of ethanol was taken in a two-neck flat bottomed flask. To this, 3.62 g of L-tyrosine (20.0 mmol) dissolved in 20 mL of 1 M sodium hydroxide was added. The reaction mixture was subjected to stirring for 10 hours to give a bright yellow colored precipitate of the Schiff base ligand (L). After washing the precipitate repeatedly with water and diethyl ether, it was then dried in vacuum over anhydrous calcium chloride. The dried product obtained (Scheme 1) was then characterized using IR, EI-MS and 13C NMR spectroscopic tools. Yield: 75%, m.p.: 178 °C, Anal. Found (%): C, 71.84; H, 4.88; N, 5.20; Calc.: C, 71.90; H, 4.91; N, 5.22; EI-MS: m/z, 534.46; IR (KBr, cm-1): v(C=N) 1673, vasy(COOH), 1589, vSy(COOH) 1450, V(oh) 3205; 13C NMR (5, ppm in CDO3): 163.19 (C=N), 180.47 (COOH). 2. 2. Synthesis of Metal Complexes 1.0 mmol (0.58 g) of finely powdered Schiff base li-gand was dissolved in 20 mL ethanol. To this, 20 mL etha-nolic solution of 1.0 mmol metal salts (manganese(III) acetate 0.27g / iron(III) chloride 0.40 g) were added drop wise (Scheme 2). Then the mixture was subjected to stirring as well as refluxing at 50 °C for 12 hours. The resulting homogenous solution was subjected to slow evaporation at room temperature and the product obtained was washed with ethanol and dried and stored in air tight container for further analysis. MnL: Yield: 57%; Brown color; Molecular weight: 623.51; Molecular formula C32H28MnN2O8; Anal. Found (%): C, 61.57; H, 4.46; N, 4.82 and Mn, 8.76. Calc.: C, 61.6; H, 4.5; N, 4.9 and Mn, 8.81, ESI-MS: m/z, 624.25; IR (KBr, cm-1): V(C=N) 1598, VaSy(COO-) 1512, v^coo-) 1435, V(m-o) 547, v(M-N) 423; Am(S mol-1 cm2) 51.78; ^eff (BM) 4.89; UV-Vis (DMSO, nm, transition): 280 (LMCT), 530 and 720 (d-d). Manickam Selvanayagamet al.: Evaluation of DNA/BSA Binding and Chemical ... 198 Acta Chim. Slov. 2019, 66, 196-207 FeL: Yield: 52%; Green color; Molecular weight: 624.42; Molecular formula C32H28FeN2O8; Anal. Found (%): C, 61.46; H, 4.44; N, 4.47 and Fe, 8.87. Calc.: C, 61.5; H, 4.5; N, 4.5 and Fe, 8.94, ESI-MS: m/z, 625.91; IR (KBr, cm-1): V(c=n) 1611, VaSy(Coo-) 1513, vsy(Coo") 1402, V(m_o) 528, V(M-N), 435; Am(S mol-1 cm2) 51.56; ^eff (BM) 5.93; UV-Vis (DMSo, nm, transition): 275 (LMCT), 525 and 670 (d-d). 3. Results and Discussion 3. 1. Scheme The schematic representation of the Schiff Base li-gand synthesized from the effective condensation of 9,10-phenanthrenequinone and L-tyrosine is shown in Scheme 1 and Scheme 2 represents the complexation of the ligand with Mn(III)/Fe(III) ions. 3. 2. FTIR Spectra The FTIR spectrum of the ligand L (Fig. S1a) exhibit the imine stretching v(-C=N) vibration at 1673 cm-1. The peaks due to (CooH) asymmetric stretching and (CooH) symmetric stretching vibration are observed in the regions of 1589 cm-1 and 1450 cm-1 respectively. The less intense and weak band at 3570 cm-1 corresponds to phenolic -oH stretching. In the vibrational spectra of complexes (Fig S1b and S1c), the imino (C=N) stretching frequency has been shifted to lower frequency regions 1598 and 1611 cm-1, respectively, when compared to the imino stretching frequency of ligand (1673 cm-1). This indicates the coordination of nitrogens in the imino group to the metal ions.21 Moreover, the asymmetric stretching and the symmetric stretching vibrations of Coo- group has been shifted down to 1512 cm-1, 1513 cm-1 and 1435 cm-1, 1402 cm-1, respectively, which confirms the bonding of the metal ions by the carboxylato oxygen atom.22 This is further support- nh2 Scheme 1: Synthesis of Schiff base ligand Scheme 2: Synthesis of Mn(III)/Fe(III) complexes Manickam Selvanayagamet al.: Evaluation of DNA/BSA Binding and Chemical ... Acta Chim. Slov. 2019, 66, 196-199 207 ed by the presence of Mn-N, Fe-N and Mn-O, Fe-O bands at 423 cm-1, 435 cm-1 and 547 cm-1, 528 cm-1, respectively.23 The -OH stretching vibrations exhibits broad band at 3423 cm-1 and 3200 cm-1 for the MnL and FeL complexes, respectively. The peaks around 750 cm-1 and 650 cm-1 correspond to pr(H2O) and pw(H2O) of coordinated water molecules, respectively. 3. 3. 13C NMR Spectra The 13C NMR spectrum of the ligand L (Fig S2) shows a signal at 8 155.98 assigned to two -OH attached carbons. The signal at 8 36.13 is assigned to -CH2 carbons present in between azomethine groups. The signal for the methane carbon of L-tyrosine appears at 8 67.10. The signals in the range 8 114.12-136.16 are due to aromatic car-bon24 atoms of L-tyrosine as well as 9,10-phenanthrenequi-none. The signal at 8 163.19 corresponds to the two azomethine carbons.25 The signal at 8 180.47 is due to the two carboxylate carbons. 3. 4. Mass Spectra The EI mass spectrum of ligand L (Fig S3a) posses a molecular ion (M+) peak at m/z = 534 which is meant to the molecular weight of the ligand. The peaks at m/z = 485, 440, 383, 305, 253, 236, 204, 181, 154, 136, 108, 78 and 58 corresponds to various fragments C31H23N2O4, C26H2qN2O5, C2qH18N2O6, C18H13N2O3, C^H^NO,, C15H9NO2, C14H8N2, C12H9NO, C12H10, C7H6NO2, C7H7O, C6H4 and C2H2O2, respectively, which confirms the structure of the ligand. The structure of the complexes was corroborated with ESI-MS studies. The ESI mass spectra of the complexes exhibit the molecular ion peak which is in correct accordance with their molecular weights. The various other peaks in the spectra correspond to different fragments of the complexes. In the spectrum of MnL (Fig S3b), the molecular ion peak is observed at m/z = 624.25 and for the complex FeL (Fig S3c), the molecular ion peak is obtained at m/z = 625.91 which are in correct coherency with the molecular weight of the complexes. Therefore, the mass spectral analysis substantiates well with the proposed chemical structure of the complexes MnL and FeL. 3. 5. Electronic Spectra, Molar Conductance, and Magnetic Measurements The UV-Vis spectrum of MnL (Fig 1a) exhibit ligand field absorption at 280 nm and two d-d bands at 530 nm and 720 nm corresponding to 5B1g^5Eg and 5B1g^5B2g transitions, respectively. Normally these two weak transitions are expected for tetragonally distorted octahedral MnL complex.26 The electronic spectra of FeL (Fig 1b) complex show three absorption maxima at 670 nm, 525 nm, and 275 nm. The transitions corresponding to the aforesaid absorption bands are 6A1g(S)^4T1g(G), 6A1g(S)^ 4T2g(G) and 6A1g(S)^4Eg respectively, suggesting the octahedral geometry for FeL complex.27 The magnetic moment values of 4.86 BM and 5.91 BM for MnL and FeL, respectively, further confirms the octahedral geometry.28 The values obtained for molar conductance (51.78 and 51.56) of MnL and FeL complexes, respectively, reveals that the complexes are 1:1 electrolytes.29 Wavelength (nm) Wavelength (nm) Fig. 1 (a) Electronic spectra of MnL complex (b) FeL complex 3. 6. DNA Binding Studies 3. 6. 1. Absorption Spectral Studies The binding ability of the MnL and FeL complexes in DMSO solutions with CT-DNA were studied by measuring their effects on the absorption spectral method. In this method, the titrations were done at fixed complex concentration against different concentrations of DNA. If only could the complexes bind intercalatively to DNA helix, it shows hypochromism and also bathochromism. Since there exists a stacking interaction of an aromatic chromophore between the base pairs of nucleic acid, hy- Manickam Selvanayagamet al.: Evaluation of DNA/BSA Binding and Chemical ... 200 Acta Chim. Slov. 2019, 66, 196-207 pochromism arises30 whereas the bathochromic shift is a characteristic of coupling of n* orbital of the complexes with the n orbital of the DNA nucleotide pairs which in turn causes decrease of the n^n* transition energy.31 Hy-pochromism is generally a measure of the extend of inter-calative mode of binding.32 An aqueous solution of the complexes showed intra-ligand charge transition bands at 276 nm (MnL) and 264 nm (FeL) which resulted in hy-pochromism on addition of increasing concentration of CT-DNA (Fig S4a and S4b) from which the binding constants Kb of the complexes were evaluated. The intrinsic equilibrium binding constant Kb was found to be 1.22 x 105 M-1 and 1.56 x 105 M-1 for MnL and FeL complexes, respectively, which is in compliance with the observed trend in hypochromism. These experimentations conclude that both the complexes could interact through intercalation with DNA. 3. 6. 2. EB Fluorescence Displacement Assay A competitive binding titrations using ethidium bromide have been performed to substantiate the above results. Being a non-fluorescent compound in Tris-buffer, ethidium bromide (EB) shows fluorescence when combined with DNA, because of its strong intercalation between the nucleotides of DNA.33 It is reported that the emission of DNA pretreated with EB is being quenched by some complexes resulting in the reduction of emission intensity. On addition of the complexes MnL and FeL to EB-DNA, the fluorescence intensity at 615 nm was decreased with increasing the concentration of the complexes without any shift in the position of emission maxima34 as seen in (Fig 2a and 2b). From the classical Stern-Volmer equation, the apparent binding constant values (Kapp) for the complexes MnL and FeL were calculated and it is found to be 4.8 x 106 M-1 and 12.3 x 106 M-1. From the observed Wavelength (nm) Wavelength (nm) Fig. 2: Emission spectrum of EB bound to DNA in the presence of complexes ([EB] = 3.3 |iM, [DNA] = 40 |iM, [complex] = 0-30 |iM, \ex = 430 nm). Inset shows the plots of emission intensity Io/I vs [DNA] / [complex]. (a) Emission spectra of MnL complex (b) Emission spectra of FeL complex Manickam Selvanayagamet al.: Evaluation of DNA/BSA Binding and Chemical ... results, it may be concluded that both the complexes interact with CT-DNA via intercalative mode. 3. 6. 3 Viscosity Experiments Viscosity experiments provide additional information regarding the intercalative nature of complexes between the nucleotide pairs of CT-DNA (Fig 3). The increase in complex concentration enhances the DNA viscosity as the base pairs of DNA get separated at its intercalation sites causing increase in the overall DNA length.35 The viscosity of the DNA increases gradually with increase in complex concentration. These results indicate that both the complexes under study can intercalate the adjacent DNA base pairs, causing an expansion in the helix, and thus enhance the viscosity of DNA. Thus, the intercalative binding of the Fig. 3: Effect of increasing amounts of MnL and FeL on the relative viscosities of CT-DNA at room temperature in 5 mM Tris-HCl buffer Acta Chim. Slov. 2019, 66, 196-201 207 complexes to CT-DNA is determined as established through absorption and fluorescence measurements. 3. 7. DNA Cleavage Analysis The cleavage efficiency of the complexes was studied on pBR322 plasmid DNA using agarose gel electrophoresis so as to identify the conformational changes of the DNA. When electrophoresis is performed on circular plasmid pBR322 DNA (Fig. 4), there occurs a rapid migration for the supercoiled SC form (Form I). The super coiled form of the DNA was relaxed to slow moving open circular OC form (Form II) in the presence of oxidant H2O2 which indicates that one strand is being cleaved.36 Both the MnL and FeL complexes possess the tendency to cleave the DNA from SC form to OC form on various concentrations (10, 20 and 30 ^g). From the Figure 4 it is observed that no cleavage occurred in the case of control DNA (lane 1) and control DNA/ H2O2 (lane 2). The complex MnL shows no cleavage at 10 ^g (lane 3) but partial cleavage is observed at 20 ^g (lane 4) and 30 ^g (lane 5) from SC form (Form I) to OC (Form II) in the presence of H2O2. Similarly, the complex FeL does not cleave the DNA at 10 ^g (lane 6) but in the presence of the oxidant it exhibits partial cleavage from SC form (Form I) to OC (Form II) at 20 ^g (lane 7) and 30 ^g (lane 8). An effective cleavage is visualized as the concentration of the complexes increases. Both the MnL and FeL complexes could cleave DNA more effectively in the presence of an oxidant (H2O2) which may be attributed to the involvement of hydroxyl free radicals. In oxidative cleavage, the hydroxyl free radicals formed oxidize the deoxyribose moiety and further hydro-lytic cleavage of sugar phosphate back bone occurs.37 Fig. 4: DNA cleavage analysis. 1st well- Control only pBR322; 2nd well- pBR322+H2O2 and Buffer; 3rd well- Sample (MnL)-10 |ig; 4th well- Sample (MnL)-20 |ig; 5th well-sample (MnL)-30 |ig; 6th wellsample (FeL)-10 |ig; 7th well- sample (FeL)-20 |ig; 8th well- sample (FeL)-30 ^g 3. 8. BSA Binding Studies 3. 8. 1 Fluorescence Spectroscopy The intrinsic BSA fluorescence at 340 nm due to tryptophan moiety38 is quenched on addition of drug mol- ecules. The mechanistic process involved in quenching is categorized to be static or dynamic. The molecular collisions results in dynamic quenching while static quenching arises as a result of complex formation in groud-state between the protein and the quencher. Dynamic quenching values are found to increase along with temperature whereas the static quenching constants decrease with tem-perature.39 This quenching mechanism can be explored from Stern-Volmer equation.40 ■=1 i A"sv[Q] = 11 A"qro[Q] (1) The emission maxima in-absentia and presence of quencher molecules are denoted as F0 and F, respectively. Ksv being Stern-Volmer quenching constant and the quencher concentration is designated as [Q] with t0, the mean life time of protein and for BSA it is found to be 10- 1 Kq is a) 500- 400 - wavelength (nm) b) 600 500- 300 - wavelength (nm) Fig. 5: Emission spectra of BSA in the presence of various concentrations of metal complexes in DMSO (T = 298 K), c(BSA)=2.0 x 10-6 mol L-1, c(Metal complex) = 2, 4, 6, 8, 10 x 10-6 mol L-1. Insert: Stern-Volmer plot for quenching of BSA by metal complexes (a) Emission spectra of BSA with MnL complex (b) Emission spectra of BSA with FeL complex Manickam Selvanayagamet al.: Evaluation of DNA/BSA Binding and Chemical ... 202 Acta Chim. Slov. 2019, 66, 196-207 the quenching rate constant of the protein and it is equal to Ksv/t0. The Ksv values are obtained from the slope of linear fitting plots F0/F vs [Q]. The Stern-Volmer plots for MnL and FeL complexes at three different temperatures 298 K, 304 K and 310 K are provided in Figs. 5, S5 and S6 and their Kq values are presented in Table 1. The results conclude that Stern-Volmer quenching constants (Ksv) decreases with increasing temperature. Also, it was found that the Kq values are greater than 2.0 x 1010 L mol-1 s-1 and this value is attributed to the maximum scattering collision quenching constant of various quenchers for dynamic quenching.42 This exposes that the BSA quenching by MnL and FeL is due to ground-state complex formation i.e., static and not dynamic quenching. Table 1: Ksv and K„ values of MnL and FeL complexes Complexes T (K) Ksv Kq (x 104 L mol-1) (x 1012 L mol-1 s-1) MnL 298 2.10 2.10 304 1.39 1.39 310 1.07 1.07 FeL 298 3.53 3.53 304 2.69 2.69 310 2.29 2.29 3. 8. 2. Absorption Spectroscopy Ground state interaction of MnL and FeL with BSA is further proved by UV-vis absorption spectra. The absorption spectra were recorded both for the BSA and BSA-MnL/FeL system in the range of 200-350 nm (Fig. 6). A bathochromic shift occurs at the absorption maxima compared to that of BSA which confirms the static quenching mechanism.43 3. 8. 3. Analysis of Binding Constants and Binding Sites From the binding of complexes MnL and FeL with BSA, the binding constants (.Kb) and the binding sites (n) can be calculated from the following equation.44 rF0 - F\ log log Kb + nlog[Ç] (2) The values of Kb and n are obtained (Table 2) from the intercepts and slopes of the linear fitting plots of log F0 - F/F vs log [Q] at 298 K are displayed in Fig. 7 for MnL and FeL complexes, respectively. Similarly, the plots for BSA-MnL complex at 304 K and 310 K and for BSA-FeL complex at 304 K and 310 K are shown in Figs. S7 and S8, respectively. The binding site values approximated to 1 reveals the existence of only one binding site in BSA where the complexes could bind. The BSA molecule possess two tryptophan residues, ie., Trp-134 in sub-domain IA and Trp-212 in hydrophobic sub-domain IIA. Any molecules which bind to sub-domain IIA can result in conformation-al changes.45 These results confirm that the complexes could fit into the hydrophobic pocket in sub-domain IIA. Table 2: Binding constant (Kb) and Binding site (n) values of MnL and FeL complexes Complexes T (K) Binding constant Binding (104 L mol-1) site MnL 298 5.72 1.31 304 4.95 1.16 310 4.37 1.06 FeL 298 5.17 1.12 304 4.71 1.05 310 4.66 1.02 Wavelength (nm) Wavelength (nm) Fig. 6: UV-Vis absorption spectra of BSA and BSA-Metal complex solutions: c(BSA) = c(Metal complex) = 2 x 10-6 mol L-1. (a) BSA-MnL solution (b) BSA-FeL solutions Manickam Selvanayagamet al.: Evaluation of DNA/BSA Binding and Chemical ... Acta Chim. Slov. 2019, 66, 196-203 207 log [Q] log [Q] Fig. 7: Double logarithm plot for BSA-metal complexes at 298 K (a) Double logarithm plot for BSA-MnL (b) Double logarithm plot for BSA-FeL 3. 8. 4 Thermodynamic Parameters The interactions between the complexes and BSA could be hydrophobic, hydrogen bonds, van der Waals forces and electrostatic interactions.46 It can be found out from the thermodynamic parameters such as enthalpy (AH) and entropy (AS). The thermodynamic parameters were investigated at 298 K, 303 K and 310 K and the result shows no structural degradation was observed in BSA. The values for AH and AS can be obtained from the following van't Hoff equation. (3) The free energy changes can be obtained from, AG = -RT\nK (4) The AH and AS values can be obtained from the slopes and ordinates of linear fitting plots of lnK vs 1/T (Fig. 8). The positive values of AH and AS are attributed to hydrophobic interactions. The AH and AS values being negative shows the existence of hydrogen bonding and van der Waals forces, very low positive or negative AH value and positive AS values are attributed to electrostatic interactions.47 From the thermodynamic parameters (Table 3), the negative enthalpy (AH) and positive entropy (AS) values ascertains that electrostatic interactions played an important role between BSA and MnL/FeL. The negative AG values calculated, proves the spontaneous binding process. 3. 8. 5 Energy Transfer Between Metal Complexes and BSA The binding of metal complexes to BSA becomes effective only when there is considerable transfer of energy between the donor and the acceptor which can be calculated from Forster's resonance energy transfer theory.48 For an effective energy transfer, the following conditions must be 1000/T(K ') 1000/T (K 1) Fig. 8: van't Hoff plot for the interaction of BSA with (a) MnL complex (b) FeL complex Manickam Selvanayagamet al.: Evaluation of DNA/BSA Binding and Chemical ... 204 Acta Chim. Slov. 2019, 66, 196-207 Table 3: Thermodynamic parameters of the binding interaction of metal complexes with BSA Complexes T (K) AH (kJ mol1) AS (J mol1 K1) AG (kJ mol1) MnL 298 -17.23 33.22 -27.14 304 -27.33 310 -27.54 FeL 298 -6.68 67.72 -26.89 304 -27.20 310 -27.71 satisfied: a) the donor must be a fluorescent in nature b) there must be a spectral overlap between the emission of donor and the absorption of acceptor; c) the donor-acceptor distance should be less than 8 nm. From the following equation, the efficiency of energy transfer can be calculated.49 F E= 1 = fi (L Fn fin+ r6 (5) Where r is the donor-acceptor distance, R0 is the critical distance at which 50% energy transfer occurs and is given by,50 (6) K2 the orientation factor whose value is found to be 2/3 for random alignment in fluid medium; n being the average refractive index of the medium in the wavelength region of significant spectral overlap (n = 1.336); O is denoted for the quantum efficiency for emission of donor and for BSA it is 0.15;51 J is found to be the overlap integral amid the emission and absorbance spectra of donor/acceptor which is calculated as follows 7 = I e(A) F (A);I4 AA (7) 2 F(X) AA F(X) is represented for corrected donor fluorescence intensity in wavelength range A, e(X) is the acceptor's ab- sorptivity at wavelength X. The spectral overlap between the emission of BSA and absorption of MnL and FeL are shown in Fig. 9 along with the energy transfer parameters given in Table 4. The donor-acceptor distance (r) is calculated to be lesser than 8 nm,52 ascribing the occurrence of transfer energy from BSA to metal complexes with high possibility. Table 4: Energy transfer parameters for the interaction of metal complexes with BSA Complexes J (cm3 L mol1) Ro (nm) E r (nm) MnL 6.47 x 10-14 3.44 0.037 5.91 FeL 2.27 x 10-15 1.96 0.051 3.20 3. 9. Molecular Docking Studies of Human DNA Topoisomerase I and BSA The molecular docking system can add normal medication outline by putting a little particle into the coupling site of the DNA/BSA targets particularly in a non-covalent mode. The most suitable binding mode, binding site and possible interactions of the complexes with DNA/BSA could be probed using this study. By using X-ray structural pattern of the human DNA topoisomerase I and BSA a) b) 350 375 wavelength (nm) 0.004 0.000 wavelength (nm) Fig 9: Spectral overlap of UV-Vis absorption spectrum of metal complexes with the fluorescence emission spectrum of BSA. c(BSA) = c(Metal complex) = 2 x 10-6 mol L-1, T = 298 K (a) Spectral overlap of MnL (b) Spectral overlap of FeL Manickam Selvanayagamet al.: Evaluation of DNA/BSA Binding and Chemical ... Acta Chim. Slov. 2019, 66, 196-205 207 a) b) c) d) Fig. 10: (a) Molecular docked model of MnL with DNA. (b) specific interaction of MnL with DNA(c) Molecular docked model of MnL with BSA. (d) specific interaction of MnL with BSA (downloaded from RCSB PDB)53 the molecular docking of the complexes were carried out. Interactions of the complexes with the receptors DNA-topoisomerase I and BSA along with most preferable binding sites are given in Fig. 10 for MnL and Fig. 11 for FeL. The binding energies of the MnL and FeL complexes with DNA were found to be -11.8 and -11.7 kcal mol-1, respectively. Also it was observed that the binding energies of the MnL and FeL complexes with BSA were -10.9 and -10.8 kcal mol-1, respectively. The possible binding modes, bond distances and binding sites for both the complexes are provided in Tables S1-S4. 4. Conclusion A tetradentate Schiff base ligand involving L-tyros-ine and 9,10-phenanthrenequinone along with its Mn(I-II)/Fe(III) complexes has been synthesized and characterized using various analytical and spectroscopic tools. The absorption and emission spectra and viscosity measurement provides information on intercalative mode of interaction of the metal complexes with CT-DNA. The interaction of the complexes with BSA was studied at three varying temperatures and observed that the fluorescence quenching process may be accomplished as static quenching. The changes in enthalpy and entropy are ascertained to electrostatic interaction between the Mn(III)/Fe(III) complexes and BSA. The distance between the BSA and MnL and FeL was found to be 5.91 and 3.20, respectively, which indicates that the energy transfer occurs with high possibility. These results reveal that the synthesised Mn(I-II)/Fe(III) complexes could be promising compounds as target-based drugs. 5. Supporting Information Summary The experimental details of DNA/BSA binding and chemical nuclease activity, spectral techniques used such as FTIR, 13C NMR, Mass, absorption and emission titrations and tables for binding energy values of this work can be found in supplementary information. 6. Acknowledgement The authors thank SAIF, Indian Institute of Technology, Madras for providing EI-mass spectra. The authors Manickam Selvanayagamet al.: Evaluation of DNA/BSA Binding and Chemical ... 206 Acta Chim. Slov. 2019, 66, 196-207 a) c) At99.0%), sodium phosphate monobasic (>99.0%), sodium phosphate dibasic (>99.0%), albumin from bovine serum (BSA) (>98.0%), malt extract, agar, D-(+)-glucose, sodium acetate, Sigmacell, glucose assay reagent, Casein, Hammarsten bovine, trichloroacetic acid (TCA), D-(-)-Salicin (> 99.0%), starch azure and sodium chloride were supplied by Sigma (Schnelldorf, Germany). 2. 2. Preparation of Halophilic Fungi Suspension The microbial strains used in this study were H. werneckii, P. triangularis, T. salinum, and W. ichthyophaga. H. werneckii, P. triangularis and T. salinum were grown on solid malt extract agar (MEA) for 5-7 days at room temperature, while cells of W. ichthyophaga were grown on MEA with 17% NaCl under the same conditions. Black yeast cells were suspended in sterile saline solutions, incubated at 25 °C and stirred at 300 rpm for 10 min to achieve homogeneity of the cell suspension. All cultures were freshly prepared by the same procedure. 2. 3. Disintegration of Halophilic Fungi Cells Rotor-stator homogenizers consist of two parts: a fast-spinning inner rotor and a stator (a stationary outer sheath. Their function is to homogenize samples based on the action of mechanical tearing, shear fluid forces, and/or cavitation (the rapid forming and collapsing of bubbles). Rotor-stator homogenizers are broadly capable of homogenizing a wide variety of tissues or cells. The experiments were conducted in a 100 mL glass centrifuge tube. The sterile centrifuge tube was filled with a fresh cell suspension of selected black yeasts, mixed and then placed into a water bath at a fixed temperature of 35 °C. For black yeast cell disruption, a rotor-stator homogenizer (Homogenizer, Polytron Pt1200, Kinematica AG, Switzerland) for volumes from 1 mL to 1 L was used. The cell suspension was homogenized from 10 to 100 min at 25,000 rpm, and at a predetermined time, samples were taken for subsequent analysis. The experiments were repeated three times. 2. 4. Survivor Colony Count The number of colonies of H. werneckii, T. salinum and P. triangularis was determined before and after the defined homogenization time. The initial concentration (No) was around 105 CFU mL-1 (colony forming units mL-1) for each fungus. Parameters for determination of CFU mL-1 were volume of sample 100 ^L, an incubation temperature of 25 °C and an incubation time of 5 to 7 days. 2. 5. Bradford Assay A Bradford assay is a highly sensitive colorimet-ric method, with perceived linearity, and it presents high Primozic et al.: Release of Halophilic Extremozymes by ... 220 Acta Chim. Slov. 2019, 66, 217-228 speed of analysis.32 35 Bovine serum albumin (BSA) was used as the standard protein. 2. 6. Determination of Enzymatic Activity The optical density of halophilic fungi cell suspensions was determined at 600 nm by the UV-Vis spectrophotometer and were between 0.47 and 0.54 before homogenization. Samples of halophilic fungi cell suspensions after homogenization were centrifuged for 2 min, and the supernatant without cells and debris was analyzed spectrophotomet-rically. The amount of total proteins was determined with the Bradford method.32 Activities of the cellular enzymes of halophilic fungi, a-amylase, cellulase, ß-glucosidase and protease, which are the most commonly used enzymes in applied biocatalysis were determined by the UV-Vis spectrophotometer at wavelengths of 595 nm, 340 nm and 280 nm, before and after homogenization of the black yeast culture. 2. 6. 1. Assay of Cellulase Enzyme Activity Cellulase activity was defined at 340 nm (25 °C) with a UV-Vis spectrophotometer using the method with a Sig-macell solution as a substrate.36 2. 6. 2. Assay of a-amylase Enzyme Activity a-Amylase activity was defined spectrophotometri-cally at 595 nm, using starch azure as a substrate. The activity was expressed as the change in absorbance at 595 nm per minute per mL of cell suspension.36 2. 6. 3. Assay of Protease Enzyme Activity Protease enzyme activity was determined at 280 nm on a UV-Vis spectrophotometer, using the TCA method.36 3. Results and Discussion 3. 1. Viability of Halophilic Fungi Exposed to Mechanical Forces Structural damage or physiological dysfunction of the cell are often associated with the reason for its death. Other effects that influence or lead to cell death are disruption of the envelopes, DNA conformational changes, ribosome alterations or protein aggregation, as well as physiological disorders (membrane selective permeability alteration or loss of key enzyme function).37 In addition, elevated temperatures can also affect the cellular structure; therefore, it is very difficult to determine which parameter contributes most to cell death. For that reason, in our study a low temperature (35 °C) was chosen for exposure of the fungi to mechanical forces. The influence of mechanical forces on the viability of H. werneckii, P. triangularis, T. salinum and W. ichthyophaga cells in the cell suspension was a subject of interest (Figure 1). Cell division, growth and morphogenesis are phases during which the fungal cell structure dynamic is changing. No similarity in the composition and mechanical properties of the cell wall is expected either between genera, or even within closely related species. Moreover, growth conditions and genetic modifications could affect the fungal cell structure.38,39 Therefore, it was interesting to study the influence of homogenization on cell death of these four halophilic fungi. Before homogenization of the halophilic fungi, the number of viable cells was cca. 105 CFU mL-1 for H. werneckii, T. salinum and W. ichthyophaga, and 102 CFU mL-1 for P. triangularis. The viability of all four treated halophilic fungi after 10 minutes of homogenization was relatively high, from 2. 6. 4. Assay of ^-glucosidase Enzyme Activity ^-glucosidase enzyme activity was determined at 340 nm with a UV-Vis spectrophotometer, using salicin as a substrate.36 2. 6. 5. Statistical Method For the statistical evaluation of the deviation of individual measurements, the standard deviation method was used, and for regression analysis, the coefficient of determination (R2) for linear regression was determined. For the calculation of standard deviation, the following equation was used: (1) Symbols: x - each value in the population; x - the mean value of the sample; n-1 - the number of values in the sample minus 1. Figure 1: Survivor lines for H. werneckii, P. triangularis, T. salinum, and W. ichthyophaga after homogenization of cell suspension at 35 °C in relation to homogenization time. Experiments were repeated three times. Coefficients of determination (R2) for linear regression are 0.985 (for P. triangularis); 0.942 (for H. werneckii); 0.987 (for T. salinum); and 0.974 (for W. ichthyophaga). Primožič et al.: Release of Halophilic Extremozymes by ... Acta Chim. Slov. 2019, 66, 217-228 221 88% (corresponding to the 68-105 CFU mL-1 for H. wer-neckii), to 99% (corresponding to the 79-105 CFU mL-1 for W. ichthyophaga). With further increases in homogeniza-tion, a rapid decline in the viability of T. salinum and P. triangularis cells was observed. In comparison to T. salinum and P. triangularis, W. ichthyophaga and H. werneck-ii cells are more resistant to the influence of mechanical forces. After 100 minutes of homogenization of the W. ichthyophaga cell suspension, 1% of the cells (corresponding to the 1-105 CFU mL-1 for W. ichthyophaga) had survived, and for the same time of treatment, 13% (corresponding to the 10-105 CFU mL-1 for H. werneckii) of the H. werneckii cells had survived. In our earlier studies, it was also found that the black yeasts W. ichthyophaga and H. werncekii were highly resistant to high pressure.36,40 On the basis of the survivor lines constructed for these halophilic fungi, decimal reduction times (D-values) were determined. The times required to kill 90% of tested microorganisms exposed to mechanical forces at 35 °C, expressed as D-val-ues. The greatest resistance to mechanical forces was observed for the H. werneckii cells (D-value = 115.0 min), while the least resistant were the T. salinum cells (D-value = 17.0 min). D-values for W. ichthyophaga and P. triangularis were following; 51.5 min and 31.7 min. After treatment of the halophilic fungi cell suspensions by mechanical forces, the shape of the cells was examined under an environmental scanning electron microscope (ESEM). Kralj Kuncic et al.41 reported that at low salinity (15% NaCl), the cell walls of W. ichthyophaga cells were threefold thicker than the cell walls of the hyphae of both W muriae and W. sebi (both belong also to the halophilic fungal genus Wallemia). This could be the reason for the very high D-values for W. ichthyophaga and H. werneckii that were obtained in our study. Figure 2 shows the ESEM figures for these fungi before (Figures 2 a, c, e, g) and after (Figures 2 b, d, f, h) exposure to mechanical forces, using the homogenization method. As can be seen from Figure 2, untreated halo-philic fungi cells had a smooth surface and typical shape. Homogenized halophilic fungi cells were damaged and deformed. Slightly wrinkled cell walls of dead halophilic fungi cells were detected, especially among the treated cells of T. salinum (Figure 2d). Ruptures and broken pieces of the cell walls were also detected. All these morphological changes indicate damage to the cell walls of the treated fungi. Therefore, the release of intracellular proteins could be predicted. 3. 2. Residual Protein Concentration Besides the cell types, the appropriate time of exposure of the biological material to homogenization is also a critical parameter influencing the activity of the intra-cellular compounds. The residual protein concentration in the selected halophilic fungi suspensions increased linearly with the increase in homogenization time (Figure 3). The highest residual protein concentration (330%), which is equivalent to 8.7780 mgprotdns mL-1 of cell suspension, was detected after 100 min of homogenization in the P. trian-gularis cell suspension. The lowest protein concentration after 100 min of homogenization was observed in the H. werneckii cell suspension. These results coincide with those obtained by determination of the survivor curves in selected halophilic fungi. The lowest D-value was obtained for P. triangularis, Figure 2: Halophilic fungi cells observed under an environmental scanning electron microscope (ESEM). H. werneckii cells a) before (magnification: 3000 X) and b) after 100 min of homogenization, T. salinum cells (magnification: 1000 X) c) before (magnification: 4000 X) and d) after 100 min of homogenization, P. triangularis cells (magnification: 4000 X) e) before (magnification: 1000 X) and f) after 100 min of homogenization and W. ichthyophaga (magnification: 2000 X) g) before (magnification: 3500 X) and h) after 100 min of homogenization (magnification: 3500 X). Primozic et al.: Release of Halophilic Extremozymes by ... 222 Acta Chim. Slov. 2019, 66, 217-228 where the highest protein concentration in the cell suspension was also detected. In contrast, the highest D-value and greatest viability was observed for H. werneckii, where the residual concentration of proteins in the cell suspension was the lowest. Obviously, intracellular proteins were extracted from the cells and consequently, the total protein concentration in selected halophilic fungi cell suspensions was increased. The strength of the fungal cell wall is defined by its structure, which comprises glycoproteins and polysaccharides, mainly glucan and chitin. Several studies suggest that chitin is a primary effector for melanin deposition within the fungal cell wall.42 It has an enormous tensile strength and significantly contributes to the overall integrity of the cell wall. However, chitin microfibrils account for only 1-2% of the yeast cell walls by dry weight.43 This could be the reason for the very high level of proteins released under variable conditions during the homogeniza-tion disruption procedure. have absorbance maxima at 260 and 280 nm, respectively. The ratio of absorbances at 260 nm and at 280 nm could be used as a measure of purity in both nucleic acid and protein extractions. A 260/280 ratio is generally accepted as "pure" for DNA; a ratio of ~2.0 is generally accepted as "pure" for RNA. A ratio lower than ~1.8 may indicate the presence of protein, phenol or other compounds, which are characterized by an absorption maximum at 280 nm.44 The absorbic ratio of the UV-absorb-ing materials was measured after the mechanical treatment of the halophilic fungi cell suspensions. Figure 4 shows the absorbic ratio of the absorbance 260 nm and 280 nm. The highest absorbic ratio was determined for T. salinum cell suspension after 100 min of homogeniza-tion (A 260/280 = 1.9). Obviously, during the homogeni-zation of the T. salinum cell suspension, pure DNA was also extracted. Absorbic ratios A260/280 for the other halophilic fungi (P. triangularis, W. ichthyophaga and H. werneckii) were lower than 1.8. Regarding the trend Figure 3: Residual total protein concentration in halophilic fungi cell suspensions in relation to homogenization time at 35 °C and a ho-mogenization speed of 25,000 rpm. Experiments were repeated three times. Standard deviation for all measurements was less than ± 3%. Figure 4: Absorbic ratio (A 260/280) after homogenization of halophilic fungi cell suspensions at 35 °C and a homogenization speed of 25,000 rpm. Experiments were repeated three times. Standard deviation for all measurements was less than ± 0.004. Initial concentration of the proteins in the halophilic fungi cell suspensions before homogenization are presented in Table 1 and were set to a value of 100%. curves for other studied halophilic fungi, it is expected that with prolongation of homogenization time, pure DNA will also be extracted from other halophilic fungi cells. Table 1: Initial concentration of proteins in halophilic fungi cell suspensions before homogenization and standard deviation of measurements (SD). Halophilic c (mgproteins SD (mgproteins fungi mL of cell suspension) mL of cell suspension) T. salinum 0.0341 ± 0.0010 H. werneckii 0.0472 ± 0.0014 P. triangularis 0.0266 ± 0.0007 W. ichthyophaga 0.0114 ± 0.0003 Nucleic acids and their related compounds, such as pyrimidines and purines, are known to absorb UV light at a wavelength of 260 nm. Nucleic acids and proteins 3. 3. Influence of Homogenization Time on Residual Activity of Different Enzymes from the T. Salinum Cell Suspension T. salinum is a melanized meristematic fungus that was isolated from saltern water along the Adriatic coast.25 The influence of homogenization time on the residual activity of a range of extracellular and intracellular enzymes, a-amylase, ^-glucosidase, protease and cellulose, from the T. salinum cell suspension was studied. As can be seen from Figure 5, the highest residual enzyme activity (440%) in the T. salinum cell suspension, which is equivalent to the enzyme activity of 1.6720 U mL-1 of cell suspension, was determined for a-amyl- Primožič et al.: Release of Halophilic Extremozymes by ... Acta Chim. Slov. 2019, 66, 217-228 223 ase after 60 min of homogenization time. a-Amylase belongs to the group of endoamylases that catalyse initial hydrolysis of starch into shorter oligosaccharides through the cleavage of interval a-D-(1-4) glycosidic bonds, resulting in a-anomeric product.45,46 Therefore, it is a very important enzyme in industrial processes such as starch saccharification, the textile, paper and food industries and pharmaceuticals.47-49 Since a-amylase from T. Salinum is an intracellular enzyme 50, an increase in its residual activity was expected after the defined time of homogenization. By prolonging the exposure of the T. salinum cell suspension to mechanical forces, the cell walls were damaged and intracellular enzymes were released in the cell suspension. A similar trend was detected for protease (maximal residual activity was determined after 30 min of homogenization), which is also an intracellular enzyme of T. salinum.50 Prolongation of homogenization time results in a decrease of a-amylase and protease activity. Probably, the overly long exposure to shear forces caused disruption in the enzyme tertiary structure and, consequently, its deactivation. Figure 5: Residual activity of various enzymes in the T. salinum cell suspension in relation to homogenization time at 35 °C and a homogenization speed of 25,000 rpm. Experiments were repeated three times. Standard deviation for all measurements was less than ± 3%. Initial activity of the studied enzymes in the T. sali-num cell suspension before homogenization are presented in Table 2 and were set to the value of 100%. Table 2: Initial activity of the studied enzymes in the T. salinum cell suspension before homogenization and standard deviation of measurements (SD). Enzyme Enzyme activity (U mL 0f cell suspension) SD (U mL of cell suspension) ß-glucosidase 0.0035 ± 0.0001 protease 0.0108 ± 0.0003 cellulase 0.0350 ± 0.0010 a-amylase 0.0038 ± 0.0001 ^-Glucosidase and cellulase are extracellular enzymes of T. salinum. The residual activity of ^-glucosidase in the T. salinum cell suspension after 10 min of homoge-nization was detected to be 150% (the enzyme activity was 0.5250 U mL-1 of cen suspension), and it remained unchanged until 40 min of homogenization time, when by prolonging the homogenization time, a decrease in ^-glucosidase activity was detected. A similar trend was observed for cellu-lase. Cellulases are of great interest in the field of organic acids and biotechnology industries. They have remarkable applications in areas such as alternate energy, detergent, textile, food, the pharmaceutical industry, nutrition and the agriculture industry.51 The reason for a decline in the residual activity of ^-glucosidase and cellulase after a longer homogenization time could lie in the denaturation of both enzymes as a result of longer exposure to shear forces. 3. 4. Influence of Homogenization Time on Residual Activity of Various Enzymes from the W Ichthyophaga Cell Suspension Wallemia belongs to the cosmopolitan xerophilic fungi and can be found in habitats with low aw and high concentrations of toxic inorganic ions.50 When a fungal cell is exposed to a high salinity environment, it uses common molecular mechanisms as well as specific molecular mechanisms to react rapidly to the consequent loss of water. In W. ichthyophaga and H. werneckii, this is achieved by the synthesis of glycerol.52 The W. ichthyophaga cell suspension was homogenized from 10 to 100 min, and the residual activity of a-amylase, ^-glucosidase, protease and cellulase vs. the time of homogenization was studied (Figure 6). Initial activity of the studied enzymes in W. ichthyophaga cell suspension before homogenization are presented in Table 3 and were set to the value of 100%. Figure 6: Residual activity of various enzymes in the W. ichthyophaga cell suspension in relation to homogenization time at 35 °C and a homogenization speed of 25,000 rpm. Experiments were repeated three times. Standard deviation for all measurements was less than ± 3%. Primozic et al.: Release of Halophilic Extremozymes by ... 224 Acta Chim. Slov. 2019, 66, 217-228 Table 3: Initial activity of various enzymes in the W. ichthyophaga cell suspension before homogenization and standard deviation of measurements (SD). Enzyme Enzyme activity (U mL of cell suspension) SD (U mL of cell suspension) ß-glucosidase 0.0279 ± 0.0008 protease 0.0082 ± 0.0002 cellulase 1.2200 ± 0.0317 a-amylase 0.0074 ± 0.0002 With the increase in homogenization time, the activity of the intracellular enzymes a-amylase, cellulase and protease also increased. The highest residual activity of a-amylase (610%), which is equivalent to an enzyme ac- tivity of 4.5140 U mL-1 of ceii suspension, was detected after 60 min of homogenization, and with a further increase in homogenization time, its residual activity decreased. A slight increase in the activity of protease from 0 to 40 min homogenization time and cellulase from 0 to 20 min was also observed. W ichthyophaga cells are obviously rich in a-amylase, while concentrations of the other enzymes were much lower. An increase in homogenization time up to 100 min caused a rapid decrease in all enzyme activity. Longer exposure to shear forces resulted in conformation changes to the proteins' molecular structure, which led to their activity loss. 3. 5. Influence of Homogenization Time on Residual Activity of Various Enzymes from the H. Werneckii Cell Suspension The living environments of H. werneckii are seawa-ter and natural or man-made saltpans with reduced water activity53, but it can also be present in house dust.54 It is a halophilic species with the capacity to survive in environments with high salt concentrations (3-30% NaCl). This microorganism has been used as a model for conditions of extremotolerance studies (e.g. oxidative stress, osmotic ad- aptation and melanisation).55-57 The influence of homog-enization time on residual activity of selected enzymes in the H. werneckii cell suspension was studied (Figure 7). Initial activity of the enzymes in the H. werneckii cell suspension before homogenization are presented in Table 4 and were set to the value of 100%. Table 4: Initial activity of various enzymes in the H. werneckii cell suspension before homogenization and standard deviation of measurements (SD). Enzyme Enzyme activity (U mL of cell suspension) SD (U mL of cell suspension) ß-glucosidase 0.0042 ± 0.0001 protease 0.0164 ± 0.0004 cellulase 0.0320 ± 0.0008 a-amylase 0.0029 ± 0.0001 Figure 7: Residual activity of various enzymes in the H. werneckii cell suspension in relation to homogenization time at 35 °C and a homogenization speed of 25,000 rpm. Experiments were repeated three times. Standard deviation for all measurements was less than ± 3%. An immense increase in residual activity of protease was detected in the H. werneckii cell suspension (800%), which is equivalent to the enzyme activity of 13.1200 U mL-1 of cell suspension, after 60 min of homogenization. As expected, with the increase in homogenization time, the viability of H. werneckii cells decreased, and intracellular enzymes were released in the cell suspension. Among the selected enzymes, H. werneckii cells contain mostly protease (intracellular enzyme).50 Proteases are among the three largest groups of industrial enzymes and represent about 60% of the total worldwide sale of enzymes, where approximately 40% of sales comprise microbial proteases.58 A slight increase in the residual activity of a-amylase in the H. werneckii cell suspension in relation to homoge-nization time was also detected. The highest residual activity of a-amylase was observed after 90 min of homogenization, when the viability of the H. werneckii cell was also low (less than 25%, corresponding to the 20 ■ 105 CFU mL-1). Since a-amylase is an intracellular enzyme,50 sufficient destruction of the cell walls was needed to release a-amylase in the cell suspension and, consequently, a higher residual activity was reached after 90 min of homogenization. Obviously, enzymes released in the suspension (a-amylase, as well as protease) were less resistant to mechanical forces, since a rapid decrease in residual activity was determined after only an additional 10 min of homogenization. The highest residual activity of ^-glucosidase and cellulase was observed after 10 min and 30 min of homogenization, and with an additional increase in homogenization time, a decrease in their residual activity was also determined. 3. 6. Influence of Homogenization Time on Residual Activity of Various Enzymes from the P. Triangularis Cell Suspension P. triangularis is an obligate halophile meristemat-ic black yeast that can live in humidifiers.59 Meristematic growth of the P. triangularis cell is slow, and it possesses Primožič et al.: Release of Halophilic Extremozymes by ... Acta Chim. Slov. 2019, 66, 217-228 225 very thick, darkly pigmented cell walls.30 The P. triangularis cell suspension was treated with a rotor-stator homog-enizer to disintegrate the halophilic fungus cells. Residual activity of various enzymes from P. triangularis was studied in relation to homogenization time (Figure 8). Figure 8: Residual activity of various enzymes in the P. triangularis cell suspension in relation to homogenization time at 35 °C and a homogenization speed of 25,000 rpm. Experiments were repeated three times. Standard deviation for all measurements was less than ± 3%. Initial activity of the enzymes in the P triangularis cell suspension before homogenization is presented in Table 5 and was set to the value of 100%. Table 5: Initial activity of various enzymes in the P. triangularis cell suspension before homogenization and standard deviation of measurements (SD). Enzyme Enzyme activity (U mL of cell suspension) SD (U mL of cell suspension) ß-glucosidase 0.0040 ± 0.0001 protease 0.0648 ± 0.0019 cellulase 0.0237 ± 0.0007 a-amylase 0.0064 ± 0.0002 As can be seen from Figure 10, the highest residual activity was detected for ^-glucosidase. By prolonging the homogenization time from 10 min to 60 min, an increase in ^-glucosidase residual activity was observed, while additional prolongation of the homogenization yielded a decrease in its residual activity. Many bacteria, yeasts and filamentous fungi can produce ^-glucosidas,60 which is an essential component of the cellulose complex, since it relieves the glucanases from product inhibition by hydrolys-ing cellobiose to glucose and thus is crucial for rapid and efficient saccharification of cellulose.61 High residual activity of a-amylase, which is an intracellular enzyme of P. triangularis, after only a short time of homogenization indicates that P. triangularis has low resistance to mechanical forces (D-value P. triangularis was 31.7 min). The cell walls were damaged, resulting in the release of intracellular enzymes into the cell suspension. The influence of mechanical forces on the released enzymes in the suspension was high, since with an increase in homog-enization time, a rapid decrease in their residual activity was detected, except for cellulose, where a slight increase in its residual activity with increase in homogenization time was observed. No significant increase was detected in the residual activity for protease (an extracellular enzyme) after 60 min of homogenization. Too long exposure of enzymes in the cell suspension to mechanical force may result in enzyme inactivation. 4. Conclusion The influence of a mechanical cell-disruption method (use of a rotor-stator homogenizer) on the viability of the halophilic fungi T. salinum, W. ichthyophaga, H. werneckii and P. triangularis and residual activity of selected enzymes were studied. The homogenization method was successfully used for the destruction of selected halophilic fungi cell walls. The greatest resistance to mechanical forces was observed for the H. werneckii cells (D-value = 115.0 min), while the least resistant were the T. salinum cells (D-value = 17.0 min). Due to the damage of cell walls, the residual concentration of proteins and residual activity of the intra-cellular enzymes from extremophilic fungi were increased. McMillan et al.11 reported about 92.95% efficiency of algae cell disruption using mechanical solid shear, while ultrasonication was less efficient. Recovery of intracellular enzymes using homog-enization is a challenging problem because the released enzymes can be inactivated in the homogenizer. Another challenge is to keep the activity of extracellular enzymes unchanged during the homogenization method, while at the same time obtaining a high level of active intracellular enzymes. Figure 9: The highest residual activity of a-amylase, ^-glucosidase, cellulase and protease obtained in various halophilic fungi cell suspensions vs. optimal homogenization time. Primozic et al.: Release of Halophilic Extremozymes by ... 226 Acta Chim. Slov. 2019, 66, 217-228 The highest residual activity among the selected enzymes was detected for protease in the H. werneckii cell suspension (800%), with 13.1200 U mL-1 of cen suspension, after 60 min of homogenisation time (Figure 9). Furthermore, the highest residual activity of ^-glu-cosidase in the P. triangularis cell suspension (509%) of 2.0360 U mL-1 of cen suspension, and a-amylase in W. ichthy-ophaga cell suspension (610%) of 4.5140 U mL-1 of cen suspension, was also detected after 60 min of cell suspension treatment with the rotor-stator homogenizer. T. salinum cells are rich in cellulase, since after 20 min of homogeni-zation, the highest residual activity of cellulose among the tested halophilic fungi was detected in the T. salinum cell suspension. The level of proteins released under variable conditions and with these specific disruption method is usually higher in the case of yeast cells than fungi. Different strengths and structures of the fungal cell walls, which comprise glycoproteins and polysaccharides, mainly glu-can and chitin, are the reason for the lower levels of protein released from fungi in comparison to those from yeast. Due to the diversity of environmental sources of isolation and the cultivation conditions for these individual halophilic fungi, the content of individual enzymes may vary. Therefore, the results of our study did not turn out as expected. Moreover, no standard trend of enzyme release from the cells that would apply to all microbial cells could be determined. The appropriate time of exposure of the biological material to certain forces causing cell lysis is a critical parameter that could influence the activity of the intracellular compounds. Klimek-Ochab et al.5 reported that the prolongation of sonication cycles from 11 to 30s admittedly resulted in a decrease in the amount of proteins released from P. citrinum, but allowed a higher level of specific activity of G6P dehydrogenase. In contrast, for the A. fumig-atus cells, the short period of sonication enables the release of a high amount of protein, and a high specific activity of G6P dehydrogenase was detected. Table 6 shows the highest residual activity of the enzymes related to viability of the halophilic fungi and residual protein concentration obtained in the cell suspensions. While the a-amylase in these four halophilic fungi is an intracellular enzyme, whose residual activity increased after exposure to mechanical forces, regardless of the treated fungi, ^-glucosidase belongs to the group of extracellular enzymes not exhibiting the expected significant increase in its residual activity after homogenization. Nevertheless, its residual activity significantly increased when the cells from P. triangularis were disrupted by the homogenization method. Most likely, the ^-glucosidase in this fungus is located on the inner edge of the cell wall, and only after exposure to shear forces is released from the cell wall, which is reflected in the increase in its residual activity. Consequently, no uniform estimate of the content and activity of each enzyme can be given, and results thus did not turn out as mostly expected. An essential first step in the enzyme extraction process from a microbial cell is its rupture. Homogenization is an important and widespread method for acquiring intra-cellular enzymes and organelles from microbial, plant and animal sources. It enables extraction of intracellular substances without the presence of solvents or other chemicals to break down the cell wall. Although some studies of cell wall disintegration and extraction of intracellular components privilege the use of non-mechanical methods and advise the use of a homoge-nizer for fungus cell disruption, it has been demonstrated that even using mechanical methods such as homogeniza-tion, many intracellular enzymes from the halophilic fungal cells can be released while their activity is maintained. Extremophiles such as T. salinum, W. ichthyophaga, H. werneckii and P. triangularis present a source of chemically diverse and often novel metabolites and proteins (e.g. enzymes such as protease, a-amylase, ^-glucosidase and cellulase) which are interesting for food and pharmaceutical industry and offer new catalytic alternatives for industrial applications and represent the basis for the development of environmentally friendly, efficient, sustainable and cleaner industrial technologies. Therefore, obtained results represent significant new findings, which are of broad biotechnological importance and are especially interesting for industrial application and useful also for a wider scientific sphere working in bio- Table 6: Correlation between the viability of halophilic fungi, residual protein concentration and residual enzyme activity for selected enzymes (a-amylase, ^-glucosidase, cellulase and protease) in the halophilic fungi cell suspensions. Halophilic a-amylase jS-glucosidase cellulase protease fungi RPC VHP REA RPC VHP REA RPC VHP REA RPC VHP REA (%) (%) (%) (%) (%) (%) (%) (%) (%) (%) (%) (%) T. salinum 245 0.15 440 185 2 158 150 44 290 178 7 240 W. ichityophaga 230 8 610 100 100 100 150 47 120 220 13 150 H. werneckii 226 25 213 175 87 143 206 61 156 210 47 800 P. triangularis 280 14 348 296 4 509 330 0.1 220 296 4 116 Legend: RPC (%) - Residual protein concentration (%); VHP (%) - Viability of halophilic fungi (%); REA (%) - Residual enzyme activity (%). Red color - intracellular enzyme; Blue color - extracellular enzyme Primožič et al.: Release of Halophilic Extremozymes by ... Acta Chim. Slov. 2019, 66, 217-228 227 technology on protein purification. Based on the obtained results, studied halophilic fungi contain industrial important enzymes with high catalytic activity which can be released from the cells with a cheap and simple method and are suitable for different industrial applications as batch or continuous operations e.g. reduction of mixed wastes, leading in reduction of environmental impacts. 5. Acknowledgments The authors acknowledge the financial support of the Slovenian Research Agency (research core funding No. P2-0046 - "Separation Processes and Product Design"). Special thanks goes to Prof. Dr. Nina Gunde-Cimerman, University of Ljubljana, Biotechnical Faculty, Slovenia for the donation of halophilic fungi. The authors would like to thank Kaja Grobelnik, Laura Kolednik, Jadranka Svigelj and Marko Kresnik for technical support. 6. References 1. R. C. Kuhad, A. Singh, K. K. Tripathi, R. K. Saxena, K. E. Eriksson, Nutr. Rev. 1997, 55, 65-75. DOI:10.1111/j.1753-4887.1997.tb01599.x 2. Y. Chisti, M. 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Reichelt (Eds.): Industrial enzymology: the application of enzymes in industry, Macmillan, London, 1983. 59. G. S. de Hoog, H. Beguin, W. H. B. de Vegte, Antonie Van Leeuwenhoek 1997, 71, 289-295. DOI:10.1023/A:1000156820793 60. A. Abdella, T. E.-S. Mazeed, A. F. El-Baz, S.-T. Yang, Process Biochem. 2016, 51, 1331-1337. DOI:10.1016/j.procbio.2016.07.004 61. S. Chamoli, P. Kumar, N. K. Navani, A. K. Verma, Int. J. Biol. Macromol. 2016, 85, 425-433. DOI:10.1016/j.ijbiomac.2016.01.001 Povzetek Trimatostroma salinum, Wallemia ichthyophaga, Hortaea werneckii in Phaeotheca triangularis so halofilne glive, ki lahko uspevajo v širokem razponu slanosti. Predstavljajo vir dragocenih bioaktivnih spojin, encimov in beljakovin, ki so zanimivi za živilsko in farmacevtsko industrijo. Za ločevanje encimov iz celic halofilnih gliv smo uporabili mehansko metodo. Dobljeni rezultati in nova spoznanja so pomembna z biotehnološkega vidika, saj je ločevanje encimov v obliki koktajla iz halofilnih gliv zanimivo za industrijsko uporabo, zlasti za kaskadne reakcije. Encimi iz ekstremofilov imajo namreč izboljšane lastnosti in se lahko uporabljajo v težkih pogojih, pri katerih se lahko neekstremofilni encimi deak-tivirajo. Primožič et al.: Release of Halophilic Extremozymes by ... DOI: 10.17344/acsi.2017.4815 Acta Chim. Slov. 2019, 66, 229-238 /^creative ^commons Scientific paper Characterization and Atmospheric Implication of Hydrotrioxy Radical-Water-Methylamine-Formic Acid-Sulphuric Acid Complexes Matjaž Dlouhy and Antonija Lesar* Department of Physical and Organic Chemistry, Jožef Stefan Institute, Jamova c. 39, SI-1000 Ljubljana, Slovenia * Corresponding author: E-mail: antonija.lesar@ijs.si Received: 10-26-2018 Presented at the 24th Annual Meeting of the Slovenian Chemical Society Abstract New particle formation is an important source of atmospheric aerosols, but the nucleation phenomena is still poorly understood. Here the formation of bimolecular complexes of HOOO^ radical with H2O, CH3NH2, HCOOH and H2SO4 has been investigated by quantum chemical methods. The stabilising effect of methylamine is found to be close to that of sulphuric acid, both complexes are formed spontaneously at studied atmospheric conditions. In addition, the hydration of the four most stable 1:1 complexes forming the 1:1:1 complexes have been considered. Water stabilises a HOOOV. .H2SO4 complex, the most effectively. Complexation process is spontaneous as well. Complex formations induced significant red-shift and enhancement of the IR intensity for the OH stretching vibration relative to that of the free radical. Equilibrium constants have been examined and the relative abundance of complexes in the troposphere is discussed. Keywords: Hydrotrioxy radical; atmospheric acids; amine; water; nucleation precursors; quantum chemistry 1. Introduction Hydrotrioxy (HOOO^) radical, or hydridotrioxygen according to IUPAC nomenclature,1 belongs to the group of polyoxides with general molecular formula, ROnR, where R stands for hydrogen, any other atom or chemical group and n > 3.2,3 It is believed that such polyoxides, especially the HOOO^ radical, play an important role in atmospheric processes in Earth's troposphere and environmental reactions as well as in biological oxidation reactions.4 Hence the understanding of its structure, stability and reactivity with other atmospheric species is a matter of the utmost importance. The HOOO^ radical can be regarded as an adduct of HO^ and O2, and represents a temporary reservoir of OH radicals.5 Many theoretical and experimental data suggest that the formation of HOOO^ radical in the atmosphere can be described by the following reactions5,6: HOO^ + O HOOO^ (1) H + O3 HOOO^ (2) HO^ + O2 HOOO^ (3) Reaction 3 is exothermic with the activation energy of around 3 kcal mol-1 implying that a certain amount of the radical is indeed formed.6 The first mention of the hydrotrioxy radical dates back to the 1970's and ever since it has been a subject of pomum Eridis.7 Despite its long postulated existence, the HOOO^ radical was inadvertently predicted in 1996 by Speranza8 and was first detected three years later by Ca-cace et al.9 in the gas phase experiments based on neutral-ization-reionization/collisionally activated dissociation mass spectrometry. At the turn of the millennium, Nel-ander et al.10, investigated and detected HOOO^ radical in argon matrices using infrared spectroscopy. A few years later, in 2005 Suma et al.11 reported the rotational spectrum and structure of the radical. Their experimental study was the first to confirm the planar geometry of the radical, suggesting trans-HOOO' conformation with inner O-O bond length of 1.688 A. Numerous theoretical studies on the structure and stability of the radical have been published, but there is still no consensus on its planar structure (cis-HOOO^ or trans-HOOO^) as the quantum chemical calculations are strongly dependent on the level of theoretical method and basis sets used in the calculations.1,4,12,13,14 Dlouhy and Lesar: Characterization and Atmospheric Implication 230 Acta Chim. Slov. 2019, 66, 229-238 HOOO^ is a reactive molecule and can form complexes by interaction with other species present in the atmosphere. Recent works of Kim et al.15 show that the hydrated radical may be 20 to 500 times more stable compared to the isolated HOOO^ structure. Furthermore, Cannon et al.16 have reported that the radical forms a stable binary and ternary complexes with water and sulphuric acid, which are up to 800 times more stable than the isolated radical. Water is the most abundant species in the atmosphere, its concentration varies between 1-4 %17, and can form strong hydrogen bonds with other molecules and radical species. Such interactions can lead to the formation of new atmospheric particles that serve as a predecessors of secondary aerosols.18,19 In addition to the water, sulphuric acid is the main molecular species in new particle forma-tion.19 Many recent studies reported the importance of gaseous amines (especially methyl-, dimethyl- and trimethyl-amine) in nucleation processes.16 Nucleation is enhanced also by organic compounds, formic acid is an important trace constituent of the troposphere.18 The abundance of these species varies, but experimental data proposes that the concentration of sulphuric acid and methylamine is hovering around 1 x 107 molecule cm-3 18,20, while a concentration of formic acid is higher, around 2 x 1011 molecule cm-3 18. Their concentration can significantly increase in urban and industrial areas (up to a few orders of magnitude) and varies pretty much by daytime and season.20 In this paper we have performed a study on electronic structure calculations of possible conformations of the hydrotrioxy radical and its complexes with water, sulphuric acid, methylamine and formic acid molecules. Further, the hydration of the most stable 1:1 complexes were investigated. Based on these calculations, we reported valuable properties of complexes, such as the equilibrium structure, stability, the nature of interaction between each component and the IR spectral features. Thermodynamic properties of the clusters were calculated at four temperatures relevant to the atmospheric conditions. The computed Gibbs free energies were used to estimate the relative concentration of the formed complexes. 2. Computational Methods All calculations were performed out with the GAUSSIAN 16 software package21 using default convergence criteria. Equilibrium geometry of monomers and complexes were optimised using the Becke three-parameter non-local exchange functional with the non-local correlation of Lee, Yang, and Parr (B3LYP)22,23 and the People-type 6-311++G(3df,3pd)24 basis set. The harmonic and anharmonic frequencies of all species were computed at the same level of theory in order to confirm the nature of the stationary points. The final energies of the complexes were improved by a multi-step composite method using the CBS-QB3 level of theory.25 The selection of above methods was based on the comparison of test calculations for hydrotrioxy radical, and available experimental structural, spectroscopic and binding parameters. These calculations included B3LYP, M06-2X26 density functional methods and CCSD(T) wave function method27 in combination with the 6-311++G(2df,2pd), 6-311++G(3df,3pd) and aug-cc-pVDZ basis sets. The results of these computations are summarised in the next section. Binding energy (D0) of the complexes has been estimated as a negative difference between the zero-point corrected energy of the complexes (E0, complex) and monomers (E0, monoi). Specifically, for three-body complex the equation is written as: Do = — (E0, complex — E0, mono1 — E0, mono2 — E0, mono3). (4) Thermochemical parameters, i.e. Gibbs free energies, are calculated at four atmospherically relevant conditions: at the surface, 298.15 K and 278.15 K, 1atm; at altitude 5km, 255.15 K, 0.533 atm and at altitude 8 km, 235.15 K, 0.351 atm. Further, the equilibrium constants, Kc, of the complex formations were calculated according to equation 5: KJT =a-e rt . cV ' "a (5) In this equation a is the symmetry number, R is the universal gas constant, T is the temperature, NA is the Avogadro's number and AGT is the complex formation Gibbs free energy change. 3. Results and Discussion Let us first consider the HOOO^ radical, the optimised structures of cis and trans conformations denoted as c-HTO and t-HTO, respectively, are presented in Figure 1 with the atom enumeration. Our calculations confirm that both conformations exhibit planar geometry. Due to a very low isomerization energy barrier the rotation around inner O2-O3 bond is nearly spontaneous. Figure 1: Optimised structures of cis-HOOO^ (c-HTO) and trans-HOOO^ (t-HTO) configuration of radical. Structural parameters obtained at different levels of theory are summarised in Table 1. Calculated values for bond lengths and angles are in good agreement with available experimental data also presented in the Table. In general, c-HTO has longer bond lengths and narrower angles in Dlouhy and Lesar: Characterization and Atmospheric Implication ... Acta Chim. Slov. 2019, 66, 229-238 231 Table 1: Optimised geometry parameters for c-HTO and t-HTO. Bond distances are given in A and angles in degrees. For comparison, experimental data are included. Method/basis set: d(H'O2) d(O2O3) c-HTO d(O3O4) a(H'O2O3) a(O2O3C M06-2X/ aug-cc-pVTZ 0.973 1.434 1.251 100.8 111.6 B3LYP/aug-cc-pVTZ 0.974 1.505 1.250 99.9 112.7 B3LYP/6-311++G(2df,2pd) 0.973 1.503 1.249 100.0 112.7 B3LYP/6-311++G(3df,3pd) 0.973 1.501 1.247 99.9 112.7 CCSD(T)/ aug-cc-pVTZ 0.973 1.500 1.241 99.8 111.9 t-HTO M06-2X/aug-cc-pVTZ 0.969 1.458 1.240 100.0 109.4 B3LYP/aug-cc-pVTZ 0.971 1.546 1.233 98.5 110.2 B3LYP/6-311++G(2df,2pd) 0.970 1.454 1.232 98.6 110.2 B3LYP/6-311++G(3df,3pd) 0.970 1.544 1.230 98.6 110.2 CCSD(T)/ aug-cc-pVTZ 0.970 1.542 1.231 100.2 109.1 experiment" 0.913 1.684 1.235 92.4 110.7 experimentb 0.972 1.688 1.225 90.0 111.0 experiment 0.970 - 1.208 - - a McCathy et al., FTMW spectrometry1, b Suma et al., FTMW spectrometry u, c Huber et al., Photoionization mass spectrometry28 comparison to the same parameters in t-HTO. Interestingly, for inner O2-O3 bond length we detected largest differences between calculated and experimental values, absolute deviation is between 0.14 A and 0.25 A. Calculations at B3LYP/ 6-311++G(3df,3pd) show good agreement with available experimental data for t-HTO, even for the most variable inner bond length, with a discrepancy of around 8 %. Calculated binding energies for both stable conformations of the radical are represented in Table 2. The energy difference between them is rather small, but D0 is strongly dependent on the method and basis set used for calculations. Results computed at M06-2X/aug-cc-pVTZ level show no correlation between experimental and computed value. Pople's B3LYP method, CCSD(T) method and CBS-QB3 composite method show a stronger correlation with the experiment, with the latter being the most reliable. At the CBS-QB3 level of calculation t-HTO is slightly more stable, by 0.12 kcal mol-1, than the cis conformation. The predicted energy difference is at the limit of computational accuracy, and for this reason, we decided to take both stable conformations into account in our study. Table 2: Binding energies (D0; eq. 4) in kcal mol 1 for c-HTO and t-HTO. Method/basis set: c-HTO t-HTO M06-2X/aug-cc-pVTZ 1.23 1.36 B3LYP/aug-cc-pVTZ 3.07 3.20 B3LYP/6-311++G(2df,2pd) 2.82 2.94 B3LYP/6-311++G(3df,3pd) 2.80 2.88 CCSD(T)/ aug-cc-pVTZ 2.71 2.83 CBS-QB3 2.69 2.81 experimenta - 2.94 a Picard et al., CRESU experiment29 Harmonic and anharmonic frequencies for both configurations of the radical along with the IR absorption intensities at the B3LYP/6-311++G(3df,3pd) level of theory are presented in Table 3 and graphically illustrated in Figures S1 and S2 of Supplementary Material. The radical has six different vibrational modes. The comparison between calculated harmonic and anharmonic vibrational frequencies shows that OH stretching absorption peaks for t-HTO and c-HTO differ for 160 cm-1 and 166 cm-1, respectively; while all the others are shifted to higher energies on average by 24 cm-1 in t-HTO and 31 cm-1 in c-HTO. Calculated anharmonic frequencies for t-HTO and c-HTO show reasonable agreement with observed gas-phase spectra. Predicted HO stretching peak in t-HTO appears 13 cm-1 lower than experimentally observed; implying that the calculated OH stretching mode for the complexes should be adequate. The differences between anharmonic and experimental values for other vibrations are slightly more significant. 3.1 Structure, Intramolecular Interaction and Binding Energy Binary complexes A systematic sampling was used to search for global minima of the complexes. Nearly a hundred initial guess structures were generated where all rational interaction of radical with water (W), methylamine (MA), formic acid (FA) and sulphuric acid (SA) molecules were considered. After full geometry optimization and on the basis of the Boltzmann probability distributions two stable low-energy structures for the HTO-W, two for HTO-MA, six for HTO-FA and three for HTO-SA complexes were identified. All these structures are shown in Supplementary Material, Figure S3, while the most stable structure for each type of com- Dlouhy and Lesar: Characterization and Atmospheric Implication .. 232 Acta Chim. Slov. 2019, 66, 229-238 Table 3: B3LYP/6-311++G(3df,3pd) calculated harmonic and anharmonic frequencies (u) in cm 1 and IR absorption intensities (I) in km mol-1 of c-HTO and t-HTO. Available experimental values are given. harmonic anharmonic experimenta Mode: u I u I u t-HTO H'O2 srt. 3742 70 3582 59 3569 O3O4 str. 1389 22 1353 36 - H'O2O3 bend. 1239 136 1199 113 1202 O2O3O4 bend. 659 64 636 53 482 O2O3 str. 460 76 445 74 244 H'O2O3O4 tor. 183 109 193 98 129 c-HTO H'O2 srt. 3687 49 3521 42 - O3O4 str. 1413 72 1362 72 - H1O2O3 bend. 1240 12 1219 8 - O2O3O4 bend. 715 150 691 138 - O2O3 str. 463 19 450 22 - H1O2O3O4 tor. 230 106 163 93 148 a Murray et al., values for isolated radical in gas-phase30 plex is illustrated in Figure 2. The binding energies D0, are given in the figure to present the complex's stability. c-HTO-W complex. After complexation a planar structure of radical moiety in the complex is preserved. The inner O-O bond in the radical unit is shortened by 0.03 A compared to that in the isolated radical. The hydrogen atom from the radical and the electron rich oxygen atom from the water molecule form one strong H-bond with the length of 1.782 A. Our calculated binding energy for this structure is 4.80 kcal mol-1, which is in good agreement with the previously reported value of 4.86 kcal mol-1 by Kim et al.15 c-HTO-MA complex. The formation of a binary complex between c-HTO and MA forced the radical out of a planar geometry. The inner O-O bond in the c-HTO-MA complex is shortened by 0.04 A in comparison to the isolated radical. The hydrogen bond, 1.725 A in length, is formed between the hydrogen atom from the radical and the nitrogen atom of the methylamine. Binding energy of this complex is 9.85 kcal mol-1. c-HTO-FA complex. Due to the interaction between c-HTO and FA, the radical moiety undergoes a structural change, its geometry in the complex is no longer planar and the inner bond is shortened by 0.05 A. The complex is found to be of a cyclic structure with two H-bonds. A shorter hydrogen bond, 1.734 A, is formed between the carbonyl oxygen atom of the formic acid and the hydrogen atom of the radical, but the radical can also act as a proton acceptor, resulting in a second H-bond, 1.885 A, with the acid proton. The calculated binding energy for this complex is 8.14 kcal mol-1. t-HTO-SA complex. When t-HTO interacts with SA, the inner O-O bond in the HOOO moiety of the complex is by 0.03 A shorter than that in the free radical. Two H-bonds with the length of 1.790 A and 2.080 A are formed resulting in a cyclic structure with a planar t-HTO unit in the complex. In the shorter H-bond SA acts as a proton donor while in the second, rather longer H-bond, the hy-droxyl oxygen atom from the radical interacts with a lone oxygen electron pair of SA. Our calculated binding energy for this structure is 10.09 kcal mol-1, which is in good agreement with the previously reported value of 10.00 kcal mol-1 by Cannon et al.16 Ternary complexes In addition, we carried out an extensive search of a possible structure of the ternary complexes formed by hy- Figure 2: Optimised structures of the most stable c-HTO-W, c-HTO-MA, c-HTO-FA and t-HTO-SA complexes with the B3LYP/6-311++G(3d-f,3pd) bond lengths and the CBS-QB3 binding energies. Dotted lines represent intermolecularly formed H-bonds. Dlouhy and Lesar: Characterization and Atmospheric Implication ... Acta Chim. Slov. 2019, 66, 229-238 233 Figure 3: Optimised structures of the most stable t-HTO-W-W, c-HTO-MA-W, t-HTO-FA-W and c-HTO-SA-W complexes with the B3LY-P/6-311++G(3df,3pd) bond lengths and the CBS-QB3 binding energies. Dotted lines represent intermolecularly formed H-bonds. dration of the four stable binary complexes described above. Around two hundred initial guess structures were full geometry optimised and three, five, three and six stable low-energy structures of the HTO-W, HTO-MA, HTO-FA and HTO-SA complexes with water, respectively, were selected. The identified most stable structures of the HTO-W-W, HTO-MA-W, HTO-FA-W and HTO-SA-W are shown in Figure 3, whereas all structures are found in Figure S4 of the Supplementary Material. The binding energies D0 and D0,W displayed in the figure are related to the monomer interaction process and the process of hydration of corresponding binary complex, respectively. t-HTO-W-W complex. In the most stable ternary complex the conformation of the hydrotrioxy radical unit is changed to the trans arrangement compared to that in the most stable binary structure being cis (c-HTO-W). The inner O-O bond of the radical subunit is approximately 0.03 A shorter than that in the isolated radical. Three H-bonds are formed by the association of the radical with two water molecules. The shortest H-bond with a length of 1.697 A is formed between two water molecules. Next, the 1.771 A long H-bond is a result of an interaction between the radical proton and the lone electron pair of oxygen from the water molecule. The longest H-bond at 2.142 A results from an interaction of the hydroxyl radical oxygen atom being an electron acceptor and water hydrogen being a proton donor. t-HTO-W-W is quite stable with a binding energy of 11.21 kcal mol-1. When comparing energies of the ternary complex and the corresponding binary one, we observe that the additional water molecule contributes around 6.55 kcal mol-1 to the stability of the t-HTO-W-W complex. c-HTO-MA-W complex. Cis configuration of the radical moiety is retained in the hydrogenation of the HTO-MA complex. The radical inner O-O bond is again shortened by 0.05 A. The c-HTO-MA-W complex is a cyclic structure with three H-bonds. The shortest intermolecular H-bond at 1.631 A results from an interaction between the radical hydrogen and the electron rich amino nitrogen atom. The water molecule acts as an H-donor to the terminal radical oxygen atom, and as an H-acceptor from the amine group, the formed H-bonds are 2.021 and 2.070 A long, respectively. Further, calculated binding energy for this complex related to the monomer interaction process is 14.87 kcal mol-1, whereas the additional water contributes 5.02 kcal mol-1 to the stability of the complex. t-HTO-FA-W complex. The hydrotrioxy radical moiety in the complex changes the orientation of atoms when the water molecule is added to the binary HTO-FA complex. The inner O-O bond is shortened by 0.02 A. Three stable intermolecular bonds are formed, resulting in an eight-membered ring structure. The water molecule acts as a proton donor and proton acceptor and forms two 1.887 A and 1.668 A long H-bonds, respectively. The lone electron pair on the carbonyl oxygen atom participates in the 1.720 A long H-bond with the hydrogen atom from the HOOO subunit. The binding energy is predicted to be 16.96 kcal mol-1. Additional water stabilises the most stable HTO-FA complex even further, by 9.06 kcal mol-1. c-HTO-SA-W complex. Similar to the t-HTO-W-W and t-HTO-FA-W complexes, the radical moiety changes the orientation in comparison to the corresponding binary complex. As in all the other cases, the inner O-O bond in the HTO unit is shortened, in this case by 0.03 A. A strong H-bond, 1.574 A long, is formed between the hydrogen atom of SA and the lone electron pair on the water oxygen atom, whereas the H-bond with the acid being a proton acceptor is 1.755 A long. The third intermolecular H-bond is longer, 1.956 A, where water acts as the proton donor and terminal oxygen contributes a lone electron pair to the bond. The c-HTO-SA-W complex with the binding energy of 20.79 kcal mol-1 is predicted to be the most stable ternary structure reported in this study. Water stabilises the HTO-SA significantly, as it contributes 11.62 kcal mol-1 to the stability of the c-HTO-SA-W complex. 3. 2. Vibrational Spectra The complete set of harmonic and anharmonic frequencies with IR absorption intensities calculated at Dlouhy and Lesar: Characterization and Atmospheric Implication .. 234 Acta Chim. Slov. 2019, 66, 229-238 B3LYP/6-311++G(3df,3pd) level of theory for the both HOOO^ conformers and the precursor molecules is collected in Table S1 of the Supplementary Material, where available experimental data for gas-phase molecules of precursors are given for comparison. Figures 4 and 5 show harmonic vibrational spectra for the four most stable binary and the four most stable ternary complexes, respectively. The intermolecular interactions in complex formation induced new degrees of freedom and consequently new intermolecular vibrational modes which are not present in monomer spectra. An examination of the calculated data shows that the frequencies and IR absorption intensities of H-bonded OH stretching regions are the most significantly affected by complexation. As expected, a large frequency red-shift is mainly observed for vibration of OH bonds involved in H-bond formation. Binary complexes c-HTO-W complex. The complex has 15 fundamental vibrational modes, 6 of them are related to the free radical and three are related to modes in an isolated water molecule. The remaining six are present due to intermolecular interactions. The most noticeable change in the IR spectrum is a red-shift of 314 cm-1 related to the OH stretching of the HOOO^ radical subunit. This stretching vibration is decreased to 3207 cm-1 in the complex relative to the 3521 cm-1 in the isolated c-HTO, while the intensity of the transition increases to the maximum in the spectra of the complex. c-HTO-MA complex. This particular complex has even more vibrational modes due to more participating atoms and bonds in the complex formation. When the OH group from the radical subunit in the complex is compared to the isolated HOOO% it is seen that frequency is red-shifted by 472 cm-1 and is accompanied by a substantial increase in IR absorption. c-HTO-FA complex. When the anharmonic vibra-tional frequencies of the OH symmetric stretch mode of cyclic c-HTO-FA is considered, a shift of 454 cm-1 to the red region of the spectra with increased intensity is observed. The OH vibration of the formic acid subunit is also shifted to the higher energy by 229 cm-1. t-HTO-SA complex. In this complex, the OH stretching vibration of the enol HTO and sulphuric acid subunits are also red-shifted compared to the isolated monomers, the shifts are 150 cm-1 and 537 cm-1, respectively. Figure 4: B3LYP/6-3++1G(3df,3pd) calculated IR absorption spectra for the most stable binary complexes. Graphs represent the harmonic absorption spectra, while in the insterted tables the relevant harmonic and anharmonic absorption peaks as well as calculated shifts of anharmonic frequences in cm-1 are summarised. Dlouhy and Lesar: Characterization and Atmospheric Implication ... Acta Chim. Slov. 2019, 66, 229-238 235 Ternary complexes In general, ternary complexes have even more vibrational modes due to the three body molecules interactions forming stable complexes. t-HTO-W-W complex. The radical and both water OH stretching frequencies are moderately red-shifted; the shifts are 419 cm-1, 198 cm-1 and 87 cm-1 compared to the OH symmetric stretching motions of the radical and water subunits. The vibrational modes are all accompanied by increased intensity. c-HTO-MA-W complex. The most pronounced change in the vibrational spectrum of the complex with regard to the isolated species is the 419 cm-1 red-shift of the OH anharmonic stretching frequency in the HOOO moiety. The OH and NH stretching of the water and amine moieties are decreased only by 87 cm-1 and 48 cm-1, respectively, implying the less strong participation of water and amino groups in the bonding. t-HTO-FA-Wcomplex. When the OH-stretching an-harmonic frequencies of the OH-stretching in the t-HTO-FA-W complex are considered and compared to the isolated species, the red-shifts of 671 cm-1, 511 cm-1 and 237 cm-1 are observed for the OH modes of the radical, formic acid and water moiety, respectively, all involved in the H-bond formation. c-HTO-SA-W complex. In this specific complex, shifts in three OH vibrational modes are observed due to participation in the H-bond formation. The OH stretching in HOOO moiety is shifted by 304 cm-1. Sulphuric acid OH bond related vibration is decreased by 30 cm-1, and symmetric OH vibrational mode of water moiety is red-shifted by 180 cm-1. Evaluated data provides strong evidence that the complex formation of the hydrotrioxy radical with water, methylamine, formic acid and sulphuric acid introduces new intermolecular vibrations. The vibration frequencies of those OH bonds that participate in H-bonding interaction are generally shifted to lower frequencies, while their intensity absorption bands strongly increase. The other modes in the complexes are similar to that in the isolated monomers. 3. 3. Atmospheric Implication Binding energies are important parameters related to the stability of the complexes formed, and the vibrational Figure 5: B3LYP/6-311++G(3df,3pd) calculated IR absorption spectra for the most stable ternary complexes. Graphs represent harmonic absorption spectra, while in the inseted tables the relevant harmonic and anharmonic absorption peaks as well as calculated shifts for anharmonic frequences in cm-1 are summarised. Dlouhy and Lesar: Characterization and Atmospheric Implication .. 236 Acta Chim. Slov. 2019, 66, 229-238 Table 4: Thermodynamic properties (AH, TAS and AG) in kcal mol 1 and calculated Kc values in cm3 molecules 1 at different altitude (h) [km], temperature (T) [K] and pressure (p) [atm]. Complex h T p AH TAS AG Kc Binary Complexes c-HTO-W 0 298.15 1 -5.42 -8.40 2.98 2.8 x 10-22 0 278.15 1 -5.44 -7.84 2.40 5.1 x 10-22 5 255.15 0.533 -5.46 -7.53 2.07 6.1 x 10-22 8 236.15 0.351 -5.47 -7.18 1.71 8.8 x 10-22 c-HTO-MA 0 298.15 1 -9.94 -9.24 -0.70 1.4 x 10-19 0 278.15 1 -9.97 -8.64 -1.33 4.4 x 10-19 5 255.15 0.533 -9.99 -8.25 -1.74 1.1 x 10-18 8 236.15 0.351 -10.01 -7.84 -2.16 3.4 x 10-18 c-HTO-FA 0 298.15 1 -8.37 -10.29 1.92 1.7 x 10-21 0 278.15 1 -8.40 -9.63 1.23 4.3 x 10-21 5 255.15 0.533 -8.44 -9.19 0.75 8.3 x 10-21 8 236.15 0.351 -8.47 -8.72 0.26 1.9 x 10-20 t-HTO-SA 0 298.15 1 -10.02 -9.45 -0.56 1.1 x 10-19 0 278.15 1 -10.06 -8.86 -1.20 3.5 x 10-19 5 255.15 0.533 -10.10 -8.49 -1.61 8.8 x 10-19 8 236.15 0.351 -10.13 -8.08 -2.05 2.7 x 10-18 Ternary Complexes t-HTO-W-W 0 298.15 1 -12.66 -18.04 5.38 4.8 x 10-24 0 278.15 1 -12.70 -16.86 4.16 2.1 x 10-23 5 255.15 0.533 -12.72 -16.13 3.41 4.4 x 10-23 8 236.15 0.351 -12.74 -15.33 2.59 1.3 x 10-22 c-HTO-MA-W 0 298.15 1 -15.59 -18.29 2.70 4.5 x 10-22 0 278.15 1 -15.65 -17.12 1.47 2.8 x 10-21 5 255.15 0.533 -15.71 -16.40 0.69 9.3 x 10-21 8 236.15 0.351 -15.75 -15.61 -0.14 4.5 x 10-20 t-HTO-FA-W 0 298.15 1 -17.94 -19.69 1.75 2.2 x 10-21 0 278.15 1 -17.99 -18.42 0.43 1.8 x 10-20 5 255.15 0.533 -18.04 -17.58 -0.46 8.9 x 10-20 8 236.15 0.351 -18.07 -19.69 -1.38 6.3 x 10-19 c-HTO-SA-W 0 298.15 1 -21.45 -18.89 -2.56 3.2 x 10-18 0 278.15 1 -21.50 -17.67 -3.83 4.1 x 10-17 5 255.15 0.533 -21.55 -16.69 -4.66 3.6 x 10-16 8 236.15 0.351 -21.58 -16.06 -5.53 4.4 x 10-15 spectra provide valuable data for laboratory and atmospheric identification. To provide insightful information into the atmospheric relevance of the complexes the ther-mochemical properties have also to be evaluated. The enthalpy (AH), entropy (TAS) and Gibbs free energy (AG) changes for global minimum structures of binary and ternary complexes were calculated as a difference between the values for the complex and infinity separated constituents. These quantities at different atmospheric conditions as noted in the introduction section together with calculated Kc values according to the equation 5 are gathered in Table 4. An inspection of the table illustrates that both the binary and ternary complex formations are exothermic processes at all studied conditions. The AH value decreases with increasing atmospheric height, but according to the AG value, not all processes are spontaneous. The c-HTO-MA and t-HTO-SA binary complexes, are spontaneously formed at all conditions with AG236.is = -2.16 kcal mol-1 and -2.05 kcal mol-1, respectively. Only the c-HTO-SA-W ternary complex is formed spontaneously at all studied altitudes with AG236 15 = -5.53 kcal mol-1, while c-HTO-MA-W and t-HTO-FA-W have a negative Gibbs free energy value at higher levels of the troposphere. Kc values of the complexes increase with height, probably due to lower pressure and lower concentration of the reactive species. From the equilibrium constants and the gas-phase concentration of molecular species, we have evaluated relative abundance of the particular binary complexes. The reported atmospheric concentrations at 298 K are as fol- Dlouhy and Lesar: Characterization and Atmospheric Implication ... Acta Chim. Slov. 2019, 66, 229-238 237 lows: [H2O] = 8 x 1017 molecules cm-3 17, [CH3NH2] = 1 x 107 molecules cm-3 20, [HCOOH] = 2 x 1011 molecules cm-3 18 and [H2SO4] = 1 x 107 molecules cm-3 18. For example, 0.002 % of the HOOO^ radical is bounded into the c-HTO-W complex at the Earth's surface, while only 4 x 10-4 % of the radical forms a binary complex with water at an altitude of 8 km and 100 % relative humidity. For other complexes the values of relative abundance can go as far as ten decades lower than those calculated for water. Figure 6 illustrates the computed equilibrium constants at the different temperatures at the ground level and different altitudes of the Earth's atmosphere for the all spontaneous processes. The equilibrium constants for the formation of the bimolecular t-HTO-SA and c-HTO-MA complexes are comparable. A formation of the ternary c-HTO-SA-W complex from monomes appears to be more favourable than the hydration of binay c-HTO-SA complex. The equilibrium constant for the hydration of binary complexes is one decade lower than for the monomer interaction process resulting in the same ternary c-HTO-SA-W complex formation. Thermodynamic quantities for the hydration process of binary complexes are listed in the Table S2 of the Supplementary Material. drotrioxy radical with lengths of around 1.75 A, while the complexes with both acids have a cyclic structure with two H-bonds, the second H-bonds are somewhat longer. The binding energies of complexes with methylamine and sulphuric acid are comparable, they approach to 10 kcal mol-1. The addition of water to the most stable dimer complexes resulted in multiple H-bonded cyclic structures with H-bonds of lengths between 1.6 A and 2.1 A and stabilised the most effectively the complex with sulphuric acid, with binding energy of 20.8 kcal mol-1. The present calculations confirmed that large spectral red-shift and enhancement of IR intensities could be used for spectroscopic identification in the atmosphere. In particular, the most substantial red-shift of 671 cm-1 is observed for the H-bonded OH stretching vibrational mode of HOOO moiety in the hydrated complex involved formic acid. From the calculated thermodynamic quantities, it should be concluded that a binary complex with methylamine and sulphuric acid, and a ternary complex with sulphuric acid are all formed spontaneously at the Earth's surface to the height of 8 km in the troposphere. Equilibrium constants of the complexes increase with increasing altitude. Figure 6: Calculated equilibrium constants, Kc, as a function of the altitude of Earth's atmosphere for the formation of selected processes indicated along each curve on the figure above. The dashed line represents a variation of temperature. 4. Conclusion The hydrogen-bonded complexes of hydrotrioxy radical with water, methylamine, formic acid and sulphuric acid were studied using the B3LYP/6-311++G(3df,3pd) and CBS-QB3 methods. Our results indicate that due to the negligible energy difference, 0.1 kcal mol-1, either the cis or trans isomer is involved in the complexation. Water and methylamine formed one strong H-bond with the hy- 5. Acknowledgment This research was funded by the Slovene Research Agency, Program grant number P2-0393 and, partly, PR-08976. M. D. is grateful to prof. Tomaž Urbič for his contribution to the master's work. Dlouhy and Lesar: Characterization and Atmospheric Implication .. 238 Acta Chim. Slov. 2019, 66, 229-238 6. References 1. M. C. McCarthy et al., J. Chem. Phys. 2012, 136, 034303. DOI: 10.1063/1.3673875 2. D. G. Semes'ko, S.L. Khursan, Russ. J. Phys. Chem. A 2008, 82, 1277-1282. DOI: 10.1134/S0036024408080074 3. K. Bhattacharjee, P. K. Shukla, Struct. Chem. 2018, 29, 11091118. DOI:10.1007/s11224-018-1095-3 4. S. Chalmet, M. F. Ruiz-Lopez, Chem. Phys. Chem. 2006, 7, 463-467. DOI: 10.1002/cphc.200500326 5. S. L. Khursan, PATAI'S Chem. of Func. Group. 2014, 1, 1-72. 6. C. 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Tuttle, J, Koller, B. Plesnicar, Comput. Theor. Chem. 2013, 1010, 19-24. DOI:10.1016/j.comptc.2013.01.009 17. M. B. McElroy: The atmospheric environment: effects of human activity. Princeton University Press, Princeton, United States, 2002, pp. 205-211. 18. R. Zhang et al., Science 2004, 304, 1487-1490. DOI:10.1126/science.1095139 19. L. Yao et al., Science 2018, 361, 278-281. DOI: 10.1126/science.aao4839 20. F. Yu, G. Luo, Atmos. Chem. Phys. 2014, 114, 12455-12464. DOI:10.5194/acp-14-12455-2014 21. M. J. Frisch et al., Gaussian 16, Revision B.01. Gaussian, Inc., Wallingford CT, 2016. 22. C. Lee, W. Yang, R. G. Parr, Phys. Rev. B 1988, 37, 785-789. DOI: 10.1103/PhysRevB.37.785 23. B. Miehlich, A. Savin, H. Stoll, H. Preuss, Chem. Phys. Lett. 1989, 157, 200-206. DOI:10.1016/0009-2614(89)87234-3 24. . Krishnan, J. S. Binkley, R. Seeger, J. A. Pople, J. Chem. Phys. 1980, 7, 650-654. DOI:10.1063/1.438955 25. J. A. Montgomery, M. J. Frisch, J. W. Ochterski, G. A. Peters-son, J. Chem. Phys. 1999, 110, 2822-2827. DOI: 10.1063/1.477924 26. Y. Zhao, D. G. Truhlar, Acc. Chem. Res. 2008, 41, 157. DOI:10.1021/ar700111a 27. K. Raghavachari, G. W. Trucks, J. A. Pople, M. Head-Gordon, Chem. Phys. Lett. 1989, 157, 479-483. DOI:10.1016/S0009-2614(89)87395-6 28. K. P. Huber, G. Herzberg: Molecular Spectra and Molecular Structure, Van Nostrand Reinhold Company, New York, United States, 1979, pp. 463. DOI: 10.1007/978-1-4757-0961-2 29. S. D. Le Picard et al., Science 2010, 328, 1258-1262. DOI: 10.1126/science.1184459 30. C. Murray, E. L. Derro, T. D. Sechler, M. I. Lester, J. Phys. Chem. A 2007, 111, 11592. DOI:10.1021/jp071473w Povzetek Delo povzema študij kompleksov hidrotrioksi radikala z vodo, metilaminom, mravljinčno kislino in žveplovo kislino z metodama B3LYP/6-311++G(3df, 3pd) in CBS-QB3. Rezultati kažejo, da je razlika energije med cis in trans izomero radikala le 0,1 kcal mol-1, zato smo obe izomeri vključili v preučevanje kompleksov. Voda in metilamin sta v interakciji z radikalom tvorili eno močno H-vez z dolžino okoli 1,75 A, medtem ko imata kompleksa z obema kislinama ciklično strukturo z dvema H-vezema, dolžini druge H-vezi v kompleksih pa sta nekoliko daljši. Vezavni energiji kompleksov z metilaminom in žveplovo kislino sta med seboj primerljivi, približujta se 10 kcal mol-1. Dodana molekula vode najbolj stabilnim dimernim kompleksom vodi do tvorbe cikličnih struktur z več H-vezmi z dolžinami med 1,6 in 2,1 A. Najbolj se stabilizirana kompleks z žveplovo kislino, njegova vezavna energija je 20,8 kcal mol-1. Z izračuni anharmonskih frekvenc smo pokazali, da je komplekse v ozračju mogoče identificirati z IR spektroskopijo. Zlasti velik premik v rdeči del spektra, pomik 671 cm-1, se pojavi pri OH-nazteznem nihanju v hidratiranem kompleksu z mravljično kislino. Na osnovi izračunananih termodinamskih količin zaključimo, da se binarna kompleksa z metila-minom in žveplovo kislino ter kompleks z žveplovo kislino in vodo tvorijo spontano od površja zemlje do višine 8 km v troposferi. Ravnotežne konstante kompleksov naraščajo z večanjem nadmorske višine. Dlouhy and Lesar: Characterization and Atmospheric Implication ... DOI: 10.17344/acsi.2018.4848 Acta Chim. Slov. 2019, 66, 239-246 /^creative ^commons Scientific paper Determination of Dissolved Iron Redox Species in Freshwater Sediment using DGT Technique Coupled to BDS Hanna Budasheva,1^ Aleksander Kravos,2 Dorota Korte,1 Arne Bratkič,3,4 Yue Gao3 and Mladen Franko1 1 University of Nova Gorica, Laboratory for Environmental and Life Sciences, Vipavska 13, SI-5000 Nova Gorica, Slovenia 2 University of Ljubljana, Faculty of Chemistry and Chemical Technology, Večna pot 113, SI-1000 Ljubljana, Slovenia 3 Vrije Universiteit Brussel, Analytical, Environmental and Geo-Chemistry, Boulevard de la Plaine 2, 1050 Brussels, Belgium 4 Université de Liège, Chemical Oceanography Unit, Allée du 6 Août 17, 4000 Liège, Belgium * Corresponding author: E-mail: hanna.budasheva@ung.si +386 70 718 204 Received: 11-13-2018 Abstract In this work we have developed a novel method for determination of iron redox species by the use of diffusive gradients in thin-film (DGT) technique coupled to photothermal beam deflection spectroscopy (BDS). The combination of both methods achieved low limit of detection (LOD) of 0.14 |M for Fe (II) ions. The total Fe concentration determined in the Vrtojbica river sediment (Slovenia, Rožna Dolina, 5000 Nova Gorica) was 49.3 |gL-1. The Fe (II) and Fe (III) concentration amounted to 12.8 |igL-1 and 39.9 |gL-1, respectively. Such an approach opens new opportunities for monitoring the content of iron species in natural waters and sediments and provides highly sensitive chemical analysis and an accurate qualitative and quantitative characteristic of the materials under study. Keywords: Beam deflection spectroscopy; diffusive gradients in thin-film technique; iron redox species; photothermal techniques; sediment 1. Introduction Metals in trace amounts are natural components of the environment, but at high concentrations they can become toxic to living organisms since they act as conservative pollutants. All trace elements (including iron, Fe) that are essential for supporting various life processes have a fairly narrow "concentration window" between their biogenic and toxic levels. Iron is a vital constituent of plant life since it is essential for photosynthetic and respiratory electron transport, nitrate reduction, chlorophyll synthesis, and detoxification of reactive oxygen species.1 At low concentrations, Fe plays an important role in metabolic and fermentation processes, as an enzyme activator, stabilizer and functional component of proteins, and may be limiting for growth of organisms. Its redox state will also have influence on being available for the uptake. Human populations in areas contaminated by iron and other heavy metals could be significantly exposed to these contaminants due to their bioaccumulation properties. They can accumulate in bone, hair and in some soft tissues, such as the liver, kidney and lungs. Prolonged exposure and high concentration levels can lead to heart disease, the development of cancer, as well as other complications such as arthritis, diabetes or liver disease.2 As a result of these health concerns various methods have been developed for determination of iron concentration in the environmental samples, including UV-Vis spectrophotometry,3 atomic absorption spectrometry,4 ion chromatography5 and high-performance liquid chromatography.6 Unfortunately, the information about the bio-available fraction content of its redox species is very difficult to measure and is in most cases lacking, although it is very important for understanding Fe toxicity.7,8 This is partly due to complex Fe geochemistry; either of the two redox states (Fe(II), Fe(III)) may be present in various Budasheva et al.: Determination of Dissolved Iron Redox Species 240 Acta Chim. Slov. 2019, 66, 239-246 complexes and size fractions (e.g. as truly dissolved, in soluble coordination complexes with inorganic ligands and organic ligands, or in a variety of colloidal and/or particulate forms). Investigation of the fractions accessible to biota (bioavailable) is often hampered by their extremely low environmental concentrations, which requires the use of contamination-prone detection methods (e.g. voltamme-try and potentiometry).9-11 Studying Fe cycling in the environment is further complicated also because the distribution of its chemical species often changes during sampling and storage. Since the above methods are not sensitive enough to satisfy the requirements associated with detection of ultra-trace amounts of Fe, thus, there is need to develop new sensitive techniques that provide reliable measurement of Fe redox species in natural environments. Diffusive gradients in thin-film (DGT) technique has been increasingly used for monitoring of environmental pollution due to its robustness, versatility, precision and capacity of pre-concentrating trace-level metal pollutants. In the uptake process, metals diffuse from natural waters through the diffusive layer to the binding layer (commonly Chelex-100 resin), which is selective to transition metals and their species,12,13 such as Fe(II) and Fe(III). It is important to point out that DGT technique advantage is capability of pre-concentrating Fe species from the dissolved phase. It samples labile fraction passively, which is without external pressures, sample manipulation, transport, derivations, etc. It provides also a time-average of environmental species concentrations during the deployment time. In contrast to the above enumerated methods, opto-thermal methods provide high-sensitivity measurements for spectroscopic characterization and detection of low-absorption transparent samples.14-17 The high sensitivity of the optothermal methods has already been repeatedly improved by combining with other methods.18-22 In this work the detection of iron species strongly bound in the resin gel was performed by the photothermal beam deflection spectroscopy (BDS). In the theoretical approach to the coupled DGT-DBS method, an intensity modulated beam of light illuminates (excitation beam EB) the absorbing sample with iron species. As a result of nonradiative deexcitation processes, thermal waves are generated. They diffuse into the sample and the adjacent medium inducing the thermal oscillations (TOs) called the temperature field. Causing the intensity change of another light beam (probe beam PB) passing through the samples adjacent medium and grazing its surface.23,24 Intensity changes are correlated to the iron concentration bonded in the examined gel.25 Presumably, the BDS technique would provide a highly sensitive chemical analysis, will be non-invasive and will retain optical and structural characteristics of the sample, thus, offering new possibilities for determination of iron species in natural water environments. The goal of this work was therefore to couple DGT and BDS methods, and to determine dissolved Fe redox species concentration as well as the amount of dissolved total Fe in the river sediments. 2. Experimental 2. 1. Solutions and Reagents The solution of 3 mM 1.10-phenanthroline (PHN) was prepared by adding 2.61 g of PHN (Merck) to 5.0 mL of 6 M HCl and dissolving both in 500 mL of double-deion-ized water (18 MO m-1, NANOPURE), then diluted 10-times in 100 mL flasks. While 6 M of hydrochloric acid (HCl) solution was prepared by dissolving 5.9 mL of 32% pure HCl (Sigma-Aldrich) in 10 mL of double-deionized water. The solution of 5.1 mM of L-ascorbic acid (Sig-ma-Aldrich) was prepared in 100 mL flask by dissolution of 9 mg of solid L-ascorbic acid in 0.1 M acetic acid. While 0.1 M acetic acid solution was prepared by dissolving 0.6 mL of 99.8% pure acetic acid (Merck) in 100 mL flask and diluted with double-deionized water. Working solutions of Fe(II) and Fe(III) to construct the calibration curves were prepared using concentrations of 4, 8, 12, 16 and 20 ^mol L-1 in 25 mL flasks by proper dilution of stock solution in the double-deionized water (18 MO m-1, NANOPURE). The Fe(II) stock solution was prepared by dissolving 695 mg of ferrous sulphate heptahydrate (FeSO4 7H2O) reagent (Merck) in 250 mL of 0.1 M HCl solutions (Sig-ma-Aldrich). The Fe(II) concentration of stock solution was than 10 mmol L-1. While the 0.1 M of HCl prepared by dissolving 4.9 mL of 32% HCl in 500 mL of double-deion-ized H2O. The Fe(III) stock solution was prepared by dissolving 677.6 mg of Iron (III) chloride hexahydrate (FeCl3 6H2O) reagent (Riedel de Haen) in 250 mL of 0.1 M HCl solutions. The Fe(III) concentration of stock solution was than 10 mmol L-1. All reagents and solvents were used as purchased without further purification. 2. 2. Preparation of DGT The procedure for DGT probe preparation is in depth described elsewhere.26 Briefly, the probe base was overlaid in the following order with 1) ground Chelex-100 resin gel, 2) polyacrylamide diffusive gel (APA, 0.8 mm final thickness) and 3) acid-precleaned 0.45 ^m HVLP filter (Millipore). The layers were secured by the probe cover with a fixed-area window for diffusion (Figure 1). To avoid contamination of the samplers, all used equipment, as well as DGT sediment probes were pre-cleaned in 5% HNO3. Before assembling DGT sampler, its parts were thoroughly washed with double-deionized wa- Budasheva et al.: Determination of Dissolved Iron Redox Species Acta Chim. Slov. 2019, 66, 239-246 241 Figure 1. The components and assembled DGT probe for sediment deployment. ter (18 MO m-1, NANOPURE) to protect from acid coming into contact with the gels. Both polyacrylamide gel and Chelex-100 resin were stored in closed plastic vials in dou-ble-deionized water before use to prevent them from drying. The gels were cut with Teflon-covered blade to fit the sediment probe. One day before field work, the assembled DGT probe was inserted into a bottle filled with double-deion-ized water and purged with nitrogen to expel oxygen, which could affect redox speciation. Immediately before sampling, the bottle was tightly closed and transferred to the sampling site, where the sampler was inserted into the sediment. 2. 3. Field Work DGT sediment probes were used for accumulation and pre-concentration of the Fe redox species in situ in the Vrtojbica River sediment. Vrtojbica flows through an an-thropogenically-impacted environment of the city of Nova Gorica and its sedimentary Fe content is expected to be sufficiently high for reliable analysis. Two assembled DGT sediment probes were placed back-to-back in the river sediment for 5 days (from 18.07.2018 to 23.07.2018), reaching approximately 7.5 cm deep. The water temperature recorded at the beginning and the end of the experiment ranged between 23.5 °C and 24.5 °C. After sampling, the DGT probes were carefully recuperated from the sediment, rinsed with a double-deion-ized water, inserted into the plastic bag and transferred to the laboratory. 2. 4. Laboratory Analysis In the laboratory the diffusive layer and filter were discarded, and resin layer was transferred into clean vial with double-deionized water until analysis. One probe gel was used to determine the dissolved Fe(II) concentration, whereas the second one to determine the total dissolved amount of iron. Fe(III) was calculated as the difference between dissolved total and Fe(II) values. The procedure for total Fe determination was the same as described below (see 2.3). After determination of Fe redox values in the gel, DGT equation was applied to calculate the concentration of Fe species (C) in the sediment pore waters: c = M Ad D At' (1) where M is the mass of accumulated Fe species, t is the time of exposure, A is the area of exposed surface (A = 0.15 x 10-3 m2), Ad is the diffusive layer thickness and D is the diffusive coefficient of the labile Fe species (D = 5.9 x 10-6 m2/s). The determination of Fe redox species is based on colorimetric reaction of the Fe(II) and PHN, accompanied by the formation of a stable orange complex, named Fer-roin ([Fe(phen)3]2+), with high absorptivity at 508 nm (Figure 2). For the total iron content, the Fe(III) was reduced to Fe(II) with L-ascorbic acid, followed by determination of total Fe as Fe(II). Figure 2. The Ferroin complex absorption spectrum. For a comprehensive understanding of how Fe ions are distributed in the aqueous phase of the sediments using of the sampler described above, the binding gel was cut into smaller pieces vertically and horizontally by Teflon-covered blade. They were then separately, piece by piece, immersed directly in the 3 mM solution of PHN for the formation of a coloured complex. Before immersing in the PHN solution the pieces of gel from the second sampler were enriched by 5.1 mM L-ascorbic acid for reduction reaction of Fe(III) to Fe(II) for determination of the total Fe content. After 24 hours of soaking in the PHN solution, the gels were dried between clean glass layers for another 24 hours before performing the BDS measurements. Budasheva et al.: Determination of Dissolved Iron Redox Species 242 Acta Chim. Slov. 2019, 66, 239-246 2. 5. Experimental Setup for BDS Method For determination of Fe concentration, the dried gels on glass support was placed on the sample's holder in BDS system (Figure 3, 4). Figure 3. Experimental setup of BDS system. Figure 4. The scheme of BDS system with view from above, where: 1 - glass layer with a sample; 2 - 3D translation stage; 3 - detector; M - mirror; IF- interference filter; L - lens. As EB (excitation beam) was chosen a solid-state laser at 532 nm output wavelength because the absorption maximum of Ferroin complex is close to it (508 nm), and 30 mW output power (CST-H-532nm-1000 MW). He-Ne laser (Uniphase, Model 1103P) was used as PB (probe beam) source at 633 nm output wavelength and 3 mW output power since this wavelength is not absorbed by Fer-roin complex. Both beams were focused by a set of lenses (Bi-Convex, AR Coated: 350-700 nm, EDMUND OPTICS). A variable-speed mechanical chopper (SCIENTIC INSTRUMENTS, Control unit model 300C, chopping head model 300CD, chopping disks model 300H) at frequency of 3.0 Hz was used to modulate the EB. The used frequency range was chosen to ensure the TOs penetration only within the sample (the thickness of the dried gel is 0.04 mm) to get the information only from it without the influence of the support. The sensitivity of the BDS system was improved by using additional mirrors (400-750 nm, Thorlabs) that directed PB through the TOs to increase its intensity change and thus enhance the BDS signal. The intensity change of PB was measured by a quadrant photodiode (RBM-R. Braumann GmbH, Model C30846E) equipped with an interference filter (633 nm, Edmund Optics) and connected to the lock-in amplifier (Stanford research instruments, Model SR830 DSP). The examined sample was placed on a 3D translation stage (CVI, Model 2480M/2488) to vary its position in x, y and z direction and optimize the experimental configuration. 3. Results and Discussion 3. 1. Determination of Fe(II) Content The calibration curve obtained for the Chelex-100 resin spiked with different concentration of Fe(II) including best fit equation is shown on the Figure 5. After immersing the gels in the Fe(II) solution for 5 days, the gels were transferred to the PHN solution for 1 day to form a coloured Ferroin complex, then transferred to the glass layers for drying. The achieved limit of detection was 0.14 ^mol L-1. 12 10 8 6 4 2 > CO Q m y = 0,4651x + 0,3158 R2 = 0,9948 12 C, fjM 16 20 24 Figure 5. Calibration curve for Fe(II) determination. A linear relationship between Fe concentration and BDS signal was obtained between 0 and 20 ^M of Fe(II). All our samples fit in this concentration range. To determine the 2D distribution of Fe redox species in the gels, the binding gel was cut into 4 parts horizontally and into 3 parts vertically. In each part, Fe(II) concentration was determined. The gel concentrations from Vrtojbica River sediment are presented in Table 1 and Figure 7a. The lower horizontal part was damaged during the deployment, so the data for Fe (II) (respectively for Fe (III) also) in this layer at the depth 7.5 cm is not available. Budasheva et al.: Determination of Dissolved Iron Redox Species Acta Chim. Slov. 2019, 66, 239-246 243 Table 1. Concentrations of the Fe(II) ions in the Chelex-100 resin. (The "-" sign indicates data lower than the LOD.) Horizontal position in the sample, cm 0.6 1.2 1.8 Vertical position in Molar concentration Fe(II), Average, the sample, cm .mol L-1 .mol L-1 0 2.1±0.2 1.9±0.4 1.9±0.4 2.0±0.3 -2.5 2.3±0.2 - - 2.3±0.2 -5.0 2.5±0.4 1.5±0.4 - 2.0±0.3 Average, ^mol L-1 2.3±0.3 1.7±0.4 1.9±0.4 2.0±0.3 The obtained data indicate that the concentrations of Fe(II) do not vary much in the sediment pore waters. Generally, the absence of Fe(II) indicates oxidative environment and the presence implies reductive conditions. There is an increase on the left side of the investigated area, which could imply more reductive localized condition during the time of the sampling. Considering that DGT binds the dissolved and labile fractions of total Fe(II), our data suggest there was a constant amount of Fe(II) available for geochemical transformations and as well for organisms. Surprisingly, no decrease in Fe(II) concentrations were observed at the sediment-water interface (SWI), suggesting that there might be a loss of this species to the water. Also interestingly and somewhat contrary to general behaviour of reduced metals species, the values around -2.5 and -5 cm were below LOD on the right side of the gel. Usually, in the sediments the anoxia begins somewhere below SWI and extends in the interior of the sediment, where the reduced species domi-nate26. The Vrtojbica River however, is a quickly flowing stream of water, hence the absence of Fe(II) in the part of the sediment of might represent a well aerated sediment. 3. 2. Determination of Total Fe Content Dissolved total Fe was determined by conversion of the Fe (III) to Fe (II) with L-ascorbic acid as a reducing agent, using this method previously described in the literature for photothermal techniques.18-19 The calibration curve obtained for the Chelex-100 resin spiked with different concentration of Fe(III) re- duced into Fe(II) including best fit equation is shown in the Figure 6. The achieved limit of detection in this case was 0.21 ^mol L-1. Figure 6. Calibration curve for total Fe determination. Using a linear equation of the calibration curve the total Fe concentrations in river water and sediment were calculated. The results are presented in Table 2 and its distribution in the gels in Figure 7b. The distribution of the total dissolved Fe in the sediment of the Vrtojbica River is quite uniform. This suggests stable conditions during the deployment, and also a stable pool of labile, dissolved Fe species that was continuously present in the sediment during the time of the sampling. Table 2. Concentrations of total Fe in the Chelex-100 resin. Horizontal position in the sample, cm 0.6 1.2 1.8 Vertical position in Molar concentration total Fe, Average, the sample, cm .mol L 1 .mol L-1 0 3.0±0.4 8.4±0.3 2.3±0.0 4.6±0.2 -2.5 10.1±0.0 10.8±0.2 7.8±0.1 9.6±0.1 -5.0 9.8±0.3 8.3±0.3 8.4±0.2 8.8±0.3 -7.5 8.5±0.1 6.9±0.1 9.7±0.2 8.3±0.1 Average, ^mol L-1 7.9±0.2 8.6±0.2 7.0±0.2 7.8±0.2 Budasheva et al.: Determination of Dissolved Iron Redox Species 244 Acta Chim. Slov. 2019, 66, 239-246 At the SWI, the concentration of total dissolved Fe species is lower than inside of the sediments, clearly indicating general loss to the water of both redox species. The increase of dissolved species in the sediment interior is associated with dissolved Fe(III) increase (see 3.3). 3. 3. Determination of Fe(III) Content One of the goals of the study was to determine the concentration of Fe(III) ions in the sediment since it is one of the species in which iron as essential metal appears in the nature. Numerous factors contribute to the environmental ratios of Fe(II) and Fe(III), e.g. pH, temperature and reductive-oxidative environmental conditions, presence of sulphide ions, ammonia and oxygen. Furthermore, the ratio is also dependant on geological features of the river sediment. The content of Fe(III) was calculated as a difference between the total Fe content (Table 2) and Fe (II) (Table 1). The results are given in the Table 3 and presented in the Figure 7c. Generally, the distribution of the Fe(III) in the sediment follows the distribution of total dissolved Fe. There is an increase of Fe(III) below the SWI at the depth of ap- proximately 2.5 - 5 cm in the centre of the gel. This might indicate a geological source dissolving and releasing Fe(I-II) into the pore waters, or a local oxidation hotspot that would oxidize any Fe(II) to Fe(III). The oxidation source might be geochemical or microbial. Very likely this feature indicates sediment heterogeneity, which we were able to observe as a result of the newly developed method. The coupling of DGT and BDS methods enables the determination of the distribution of Fe redox species in two dimensions. While Fe(III) is present over all investigated area and occurs simultaneously with Fe(II), Fe(III) is exclusively present on the right side of the gel. Combined with the Fe(II) results, this part of the sediment appears to be fully oxygenated and/or excludes formation of Fe(II), at least during the sampling period. As the DGT technique reports time-average values, this indicates very stable conditions in the time of the sampling. The DGT-BDS method does not require intensive manipulation after the sampling, which renders the possibility of transport or storage artefacts that could influence Fe speciation less likely and increases the reliability of the obtained results. Therefore, the observed patterns of Fe re-dox species likely accurately represent the sedimentary conditions. To summarize the data obtained in Figure 7 Table 3. Concentrations of the Fe (III) ions in the Chelex-100 resin. Horizontal position in the sample, cm 0.6 1.2 1.8 Vertical position in the sample, cm Molar concentration Fe(III), .mol L-1 Average, .mol L-1 0 1.0±0.3 6.5±0.3 0.4±0.2 2.6±0.3 -2.5 7.8±0.1 10.8±0.3 7.8±0.2 8.8±0.2 -5.0 7.3±0.4 6.9±0.4 8.4±0.2 7.5±0.3 Average, .mol L-1 5.3±0.3 8.1±0.3 5.5±0.2 6.3±0.3 Figure 7. Graphical presentation of Fe redox species distribution in the Chelex-100 resin. Budasheva et al.: Determination of Dissolved Iron Redox Species Acta Chim. Slov. 2019, 66, 239-246 245 presents the Fe (II), total Fe and Fe (III) distribution in the Chelex-100 resin, respectively. Although our preliminary results are not accompanied with a suite of other geochemical parameters, they clearly demonstrate the potential and applicability of the newly developed method to be used for two-dimensional imaging of dissolved, bioavailable Fe redox species in natural environments. 3. 4. Concentrations of the Fe Redox Species in the River Sediment Using the equation (1) we calculated the concentration of Fe species in the sediment pore waters (Table 4). Although not much data exist for comparison of DGT-derived Fe redox species concentrations, our data fit in the range of the published results for pristine environments.18 Total concentrations obtained from polluted or strongly impacted river sediments are higher for factor of 10 or 100.28-30 Nonetheless, the distribution of dissolved, labile and potentially bioavailable fraction of Fe redox species in the Vrtojbica River indicates a dynamic sediment system. As observed before, the low values at SWI indicate sediments are a source of both Fe redox species to the river water. The observed increases in dissolved Fe(II) and dissolved total Fe in the sediment interior could be attributable either to geochemical and microbial interactions with Fe-rich minerals, or both. Since these are the first data on iron speciation in this system, it cannot be said with cer- tainty if the concentrations are representative of this particular environment. However, despite its city location, Vrtojbica river sediment is far from polluted and not likely to increase river water Fe concentration to above WHO guidelines on Fe in drinking water (0.3 mg/L). Together with low SWI concentration our data suggests that anthropogenic activity might have not affected the river and that the sediment does not act as a sink for Fe. Simultaneously, however, it is also yet unclear which factors have highest influence on the redox state of dissolved Fe in the sediment. 4. Conclusions We report for the first time the dissolved Fe redox species distribution in freshwater sediments measured by coupled DGT technique and BDS method. The average total iron concentration in the Vrtojbica River river sediment was found to be 49.3 ^gL-1. The average amount of Fe(III) was 3 times higher than the average Fe(II) concentration and reached the value of 39.9 ^gL-1 and 12.8 ^gL-1, respectively. The obtained results show the potential of using DGT method coupled to BDS technique for monitoring biologically relevant Fe species at environmental concentrations in natural waters and sediments. The information received from the newly coupled method will advance our understanding of the basic biogeochemical processes gov- Table 4. Concentrations of the Fe redox species in the Vrtojbica sediment. Horizontal position in the sample, cm 0.6 1.2 1.8 Vertical position in the sample, cm Fe(II) in pore waters, ^g/L Average, ^g/L 0 13.2±1.3 12.0±2.5 12.0±2.5 12.4±2.1 -2.5 14.5±1.3 14.5±1.3 -5.0 15.7±2.5 9.4±2.5 12.6±2.5 Average 14.5±1.7 10.7±2.5 12.0±2.5 12.8±2.1 Vertical position in the sample, cm Total Fe in pore waters, ^g/L Average, ^g/L 0 18.9±2.5 52.9±1.9 14.5±0.0 28.7±1.5 -2.5 63.6±0.0 68.0±1.3 49.1±0.6 60.2±0.6 -5.0 61.7±1.9 52.2±1.9 52.9±1.3 55.6±1.7 -7.5 53.5±0.6 43.4±0.6 61.0±1.3 52.7±0.8 Average 49.4±1.3 54.1±1.4 44.4±0.8 49.3±2.0 Vertical position in the sample, cm Fe(III) in pore waters, ^g/L Average, ^g/L 0 6.3±1.9 40.9±1.9 2.5±1.3 16.6±1.7 -2.5 49.1±0.6 68.6±1.9 49.1±1.3 55.6±1.3 -5.0 45.9±2.5 43.4±1.9 52.9±1.3 47.4±1.9 Average, ^g/L 33.8±1.7 51.0±1.9 34.8±1.3 39.9±2.2 Budasheva et al.: Determination of Dissolved Iron Redox Species 246 Acta Chim. Slov. 2019, 66, 239-246 erning trace metal behaviour in pristine and anthropogen-ically-impacted environments. Its results may be well incorporated in the existing mathematical models, which are currently based primarily on one-dimensional profiles, thereby increasing reliability of the model predictions. Further development, application and testing of the DGT-BDS is warranted, since it is reliable, precise, resistant to contamination, inexpensive, and time-saving analytical method. 5. Acknowledgements This work was supported by Slovenian Research Agency within the research program P2-0393; Advanced materials for low-carbon and sustainable society. 6. References 1. W. G. Sunda, S. A. Huntsman, Mar. Chem. 1995, 50, 189-206. 2. S. Tautkus, L. Steponeniene, R. Kazlauskas, J. Serb. Chem. Soc. 2004, 69(5), 393-402. DOI:10.1016/0304-4203(95)00035-P 3. A. T. Pilipeko, V. G. Safronova, L. V. Zakrevskaya, Zavod. Lab. 1998, 1, 54. 4. M. Feng, Y. Yang, P. He, Y. Fang, Spectrochim. Acta A 2000, 56(3), 581-587. DOI:10.1016/S1386-1425(99)00157-2 5. B. Divjak, M. Franko, M. Novic, J. Chromatogr. 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Cambridge University Press, Cambridge, UK, 2016, pp. 66-92. DOI:10.1017/CBO9781316442654.005 13. W. Li, C. Li, J. Zhao, R. J. Cornett, Anal. Chim. Acta 2007, 592(1), 106-113. DOI:10.1016/j.aca.2007.04.012 14. M. Pawlak, S. Pal, A. Ludwig, A. D. Wieck, J. Appl. Phys. 2017, 122(22), 135109-1-135109-7. DOI:10.1063/1.5013305 15. D. Korte, M. Franko, J. Opt. Soc. Am. A. Opt. 2015, 32(1), 61-74. DOI:10.1364/J0SAA.32.000061 16. M. Pawlak, S. Pal, S. Scholz, A. Ludwig, A. D. Wieck, Thermo-chim. Acta 2018, 662, 69-74. DOI:10.1016/j.tca.2018.02.009 17. L. Chrobak, M. Malinski, J. Zakrzewski, Thermochim. Acta 2018, 606, 84-89. DOI:10.1016/j.tca.2015.03.014 18. L. Gojat, M. Martelanc, V. Vinatier, A. Delort, M. Franko, 23th YISAC. 2016, 44. 19. L. Gojat, M. A. Proskurnin, I. V. Mikheev, M. Franko, Euroa-nalysis XIX. 2017, 203. 20. H. Cabrera, D. Korte, M. Franko, Int. J. Thermophys. 2015, 36(9), 2434-2440. DOI:10.1007/s10765-015-1933-0 21. E. Cedeño, H. Cabrera, A. E. Delgadillo-López, O. Delgado-Vasallo, A. M. Mansanares, A. Calderón, and E. Marín, Talanta. 2017, 170, 260-265. DOI:10.1016/j.talanta.2017.04.008 22. H. Cabrera, J. Akbar, D. Korte, E. E. Ramírez-Miquet, E. Marín, J. Niemela, Z. Ebrahimpour, K. Mannatunga, and M. Franko,, Talanta. 2018, 183, 158-163. DOI:10.1016/j.talanta.2018.02.073 23. M. Malinski, L. Chrobak, L. Bychto, Solid State Commun. 2010, 150(9-10), 424-427. DOI:10.1016/j.ssc.2009.12.002 24. M. Malinski, L. Chrobak, Arch. Acoust. 2009, 34(4), 727-734. 25. M. Malinski, L. Chrobak, M. Pawlak, Appl. Phys. A-Mater. 2015, 118(3), 1009-1014. 26. Y. Gao, S. van de Velde, P. N. Williams, W. Baeyens, H. Zhang, TrAC. 2015, 66, 63-71. 27. S. V. Pakhomova et al., Mar. Chem. 2007, 107, 319-331. DOI:10.1016/j.marchem.2007.06.001 28. F. Leermakers, J. C. Eriksson, H. Lyklema, Fundamentals of Interface and Colloid Science 2005, 5, 1-123. DOI:10.1016/S1874-5679(05)80014-5 29. F. Gao et al., J. Exp. Bot. 2006, 57, 3259-3270. DOI:10.1093/jxb/erl090 30. C. Montalvo et al., Environ. Pollut. 2014, 3. Budasheva et al.: Determination of Dissolved Iron Redox Species DOI: 10.17344/acsi.2018.4914 Acta Chim. Slov. 2019, 66, 247-254 /^creative ^commons Scientific paper Development of a Dispersive Liquid-Liquid Microextraction Followed by LC-MS/MS for Determination of Benzotriazoles in Environmental Waters Ida Krasevec* and Helena Prosen Faculty of Chemistry and Chemical Technology, University of Ljubljana, Vecna pot 113, Ljubljana, Slovenia * Corresponding author: E-mail: ida.kmsevec@fk.kt.uni-lj.si Received: 12-18-2018 Abstract Emerging environmental pollutants are becoming a global concern, since the acceptable concentrations are currently not set by legislation in EU or elsewhere. Benzotriazoles are an important group of emerging pollutants found in low |ig/L concentrations, entering the environment through wastewater treatment facilities due to their insufficient removal, and through industrial and other use. Two new dispersive liquid-liquid microextraction (DLLME) methods were developed for the extraction of hydrophilic and hydrophobic benzotriazoles from environmental waters. Liquid chromatographic method coupled to tandem mass spectrometric detection (LC-MS/MS) was developed and validated for surface water. Validation parameters were satisfactory and the overall DLLME-LC-MS/MS method was found to be applicable to analysis of the chosen analytes in environmental waters. It was used to determine benzotriazoles in surface water and wastewater from a municipal wastewater treatment plant. In surface waters, concentration was below the limit of detection, while concentrations determined in wastewater were estimated between 2.7 and 12.0 |ig/L. Keywords: Benzotriazoles; dispersive liquid-liquid microextraction; liquid chromatography-mass spectrometry; natural water; wastewater 1. Introduction Benzotriazole and its derivatives are heterocyclic compounds used as anti-corrosive agents in industrial fluids, household dishwasher detergents, de-icing liquids, cooling systems, and hydraulic fluids, while less polar derivatives are used as UV stabilizers in plastics and cosmet-ics.1 Their widespread use has led to a ubiquitous presence in the environment: surface fresh and sea water,2-4 groundwater and drinking water,5-7 river sediments,8 soils,9 sewage sludge,10 indoor dust,11 and air.12 Their main point of entry to environmental waters is through the wastewater treatment plants (WWTP) effluents where they are insufficiently removed.13-14 A survey from European Union in 201315 has shown their presence in 97-100% of effluent wastewaters (90 WWTPs with various sources) with concentrations in low |g/L range, while concentrations in untreated wastewaters were up to ten times higher.14 Nevertheless, no environmental limit concentrations are set for benzotriazoles in EU yet. Benzotriazoles are classified as emerging pollutants with low acute toxicity, but their chronic effects are less well known: possible endocrine-disrupting activity,16 toxicity for plants and some aquatic organisms,17 and suspected human carcinogenesis.18 Measurable concentrations of benzotriazoles have been found in human urine and amniotic fluid.19-21 The concentrations of benzotriazoles in environmental matrices are in the ng/L to ^g/L range, therefore it is necessary to perform some form of extraction and pre-concentration before the analysis. The most frequently used method is solid-phase extraction (SPE).3,6,21-25 Stir-bar sorptive extraction (SBSE)26 and solid-phase microextraction (SPME)21-27 have also been applied. More recently, liquid-phase microextractions were introduced as an alternative to sorbent-based extraction, but with a very low solvent consumption. One of the most popular is dispersive liquid-liquid microextraction (DLLME), which has to date been applied to the extraction of benzotriazoles only in few instances,28-29 in both cases with the use of lighter-than-water solvents, which requires the use of special glassware to collect the solvent. Air-assisted liquid-liquid microextraction, a variant of DLLME, with lighter-than-water solvent, has also been developed for benzotriazoles in water samples.30 Krasevec and Prosen: Development of a Dispersive Liquid-Liquid 248 Acta Chim. Slov. 2019, 66, 247-254 Following the extraction, benzotriazoles are usually determined by chromatographic techniques. HPLC coupled to MS or MS/MS detection is the most frequently applied technique,2,3,7,10,19,22,23,25-27 with LODs in the range 0.2-200 ng/L (depending also on the extraction method), occasionally also with UV detection,28,30 which is usually leading to higher LODs. GC-MS or GC-MS/MS methods, either with or without derivatization of analytes, are less frequently encountered,6,13,21,25,29 their LODs tend to be comparable or a bit higher than with LC-MS/MS. In the present work, two DLLME methods were developed and optimized for the extraction of six hydrophilic and two hydrophobic benzotriazoles from aqueous samples. The optimization of DLLME for extraction from water samples with heavier-than-water solvents was done stepwise and with a two-level fractional factorial experimental design. The extracts were analysed with LC-MS/ MS. The method for hydrophilic benzotriazoles was evaluated in terms of the analytical parameters and the efficiency to determine analytes in the environmental aqueous samples. The validated method was applied to the determination of selected benzotriazoles in Slovenian environmental waters. 2. Experimental 2. 1. Materials Solid standards of the analytes in this study (Table 1) were purchased from Sigma-Aldrich, USA (OHBZ, ClBZ, BTZ, TBZMF, and BZPF), from Fluka, Switzerland (4MBZ, 5MBZ, and DMBZ), or from Santa Cruz Biotechnology, USA (BTZ-d4 as internal standard - IS). Ultrapure water (MQ) was prepared by Milli-Q water system (Millipore, USA). HPLC grade solvents acetonitrile (Fisher Chemical, UK), methanol (J. T. Baker, UK), acetone (Honeywell, USA), and isopropanol (Sigma-Aldrich, USA) were used. Other chemicals were of p.a. or higher purity from various producers: formic acid and CS2 from Sigma-Aldrich (USA), 25% NH3 from Gram-mol (Croatia), HCl, ethanol, and CCl3 from Honeywell (USA), NaOH and CCl4 from Merck (USA), NaCl from Scharlau (Spain), chlorobenzene from Fluka (USA), and 1,1,1-trichloroetane from Codex (Italy). 2. 2. DLLME Extraction Extensive optimization of DLLME procedure with heavier-than-water solvents was performed, partially with a two-level fractional factorial experimental design and partially stepwise. The final conditions for DLLME of hydrophilic and hydrophobic benzotriazoles were: pH of an aqueous sample (3 mL) in a conical test tube was adjusted to 3.5 with HCl, and solid NaCl was added up to 8% w/v. A mixture of 80 ^L CHCl3, 20 ^L CCl4, and 700 ^L acetonitrile was quickly injected into the sample. After shaking for 5 s, the mixture was centrifuged at 3000 rpm for 5 min. The extract was collected from the bottom of the tube with a syringe and transferred to a vial, dried under nitrogen and re-dissolved in 50 ^L of MQ:ACN (3:7). The final conditions for DLLME of hydrophilic ben-zotriazoles were: pH of an aqueous sample (5 mL) in a conical test tube was adjusted to 4.0 with HCl, and solid NaCl was added up to 10 % w/v. A mixture of 160 ^L CHCl3 and 800 ^L acetonitrile was quickly injected into the sample. After shaking for 5 s, the mixture was centrifuged at 3000 rpm for 5 min. Extract was collected from the bottom of the tube with a syringe and transferred to a vial, dried under nitrogen and re-dissolved in 25 ^L of MQ. 2. 3. HPLC-DAD and LC-MS/MS Analysis DLLME conditions were optimized using an HPLC-DAD method with the following parameters: an Agilent 1100 Series HPLC-DAD instrument (Agilent, USA) equipped with autosampler was used. LC separation was performed on a Kinetex XB-C18 column (Phenomenex, USA, 150 x 4.6 mm, 5 ^m) at room temperature and flow rate of 0.7 mL/min. The mobile phase was composed of acetonitrile (A) and 0.1% HCOOH in MQ (B), with the following gradient profile: 5% A, increased to 50% A in 5.0 min, then to 100% A in 9.0 min and retained at 100% A for 11.0 min. The injection volume was 20 ^L. UV spectra were recorded in 200-400 nm range, and detection wavelengths for quantification were set at 260, 304, and 350 nm. LC-MS/MS method for hydrophilic benzotriazoles: a PerkinElmer LC system, coupled with TurboSpray ESI ionization and 3200 QTRAP mass analyser (Sciex, USA) Table 1: Analyte abbreviations, molecular weights, logKow, and pKa. Abbrev. Name M (g/mol) logKow pKa OHBZ 4-hydroxy-1H-benzotriazole 135.12 0.80 7.25 BTZ 1H-benzotriazole 119.12 1.44 8.38 4MBZ 4-methyl-1H-benzotriazole 133.15 1.82 8.74 5MBZ 5-methyl-1H-benzotriazole 133.15 1.98 8.74 ClBZ 5-chloro-1H-benzotriazole 153.57 2.13 7.46 DMBZ 5,6-dimethyl -1H-benzotriazole 147.18 2.28 8.92 TBZMF 2-fert-butyl-6-(5-chloro-2H-benzotriazol-2-yl)-4-methylphenol 315.80 6.81 9.31 BZPF 2-(2H-benzotriazol-2-yl)-4,6-di-tert-pentylphenol 351.49 7.87 8.85 Kraševec and Prosen: Development of a Dispersive Liquid-Liquid ... Acta Chim. Slov. 2019, 66, 247-254 249 was used. LC separation was performed on the same column and with same parameters as for HPLC-DAD, except for the mobile phase gradient. which was as follows: 5% A, increased to 15% A in 0.5 min, then to 35% A in 12.5 min, then to 100% A in next 7 min. The injection volume was 10 ^L. Ionization was performed with the electrospray in positive mode with the curtain gas pressure at 30 psi, ion spray voltage of 4 kV, drying gas temperature at 450 °C, sheath gas 1 at 50 psi and sheath gas 2 at 50 psi. Nitrogen, supplied by Messer (Germany), was used both as drying and collision cell gas. The mass spectrometer was operated in selected reaction monitoring (SRM) mode; the declus-tering potential, entrance potential and collision cell exit potential were fixed at 40 V, 10 V, and 3 V, respectively. Transition parameters were optimized for each analyte separately by injecting separate solutions directly into the ion source by flow-injection; two fragment ions were monitored for each compound (Table 2). Quantification was performed as analyte/IS signal ratio, using the first transition for each analyte and BTZ-d4 as the IS, while the second transition and the fragment ion ratio were used for identity confirmation. During sample analysis, the calibration standard at 0.1 mg/L level and a MQ blank were injected every 12 samples to check for drift in response and carryover effect. 2. 4. Samples Grab samples of surface water were taken from the Glinscica (46,050972 °N, 14,468536 °E), Soca (46,152460 °N, 13,739944 °E), and Idrijca (46,04803 °N, 14,02272 °E) rivers (Slovenia) and two groundwater samples were taken at two different drinking water sources (Roje, PIS) of Ljubljana, Slovenia. Samples were stored in the dark at 4 °C. Influent and effluent municipal wastewater samples in the form of 24 h composite samples were taken at Central wastewater treatment plant in Ljubljana, Slovenia, and stored at -20 °C. Due to high matrix effect, which was ob- servable with the use of the internal standard, the influent wastewater sample was diluted 5 times before extraction procedure. 3. Results and Discussion 3. 1. Optimization of HPLC-DAD and LC-MS/MS Method Two different reverse-phase columns were initially tested to separate polar and hydrophobic analytes in the same run: Kinetex XB-C18 column (Phenomenex, USA, 150 x 4.6 mm, 5 ^m, 100 A) and Gemini C18 column (Phenomenex, USA, 150 x 4.6 mm, 3 ^m, 110 A). The Ki-netex column provided better resolution of the peaks, which was expected since the sorbent is prepared with core-shell technology, and was selected for further use. The optimization of mobile phase gradient was challenging due to very different polarities of the analytes (Table 1). Therefore, the final gradient started at low acetonitrile percentage (5%), which was then steeply increased to 50% and then to 100% to reach the conditions that were favourable for the elution of hydrophobic benzotriazoles (TBZMF, BZPF). Still, the separation of methyl isomers 4MBZ and 5MBZ could not be achieved. These conditions were chosen as final since HPLC-DAD was used only for the optimization of DLLME extraction parameters and the later use of LC-MS/MS in SRM mode enabled us to tolerate the less than satisfactory separation. Wavelengths for quantification were set at 260, 304, and 350 nm due to different UV spectra of analytes. Under these conditions, the calibration curves for the selected benzotriazoles were linear in the 0.5-50.0 mg/L range with R2 > 0.99. An interesting finding was that the standard solutions had to be prepared in a mixture with high percentage of organic solvent to achieve repeatable peak areas for hydrophobic analytes (Figure 1), possibly due to their low solubility in water and adsorption to the walls of the glassware. For this reason all Figure 1: Peak area of different analytes at various compositions of solvent (MQ and acetonitrile) for 50 mg/L standard solution. Krasevec and Prosen: Development of a Dispersive Liquid-Liquid 250 Acta Chim. Slov. 2019, 66, 247-254 solutions were prepared in 70% ACN. A chromatogram of standard solution is given in Supplementary Information SI 1. Although it would be possible to determine all selected analytes with LC-MS/MS and it was also possible to achieve favourable DLLME extraction conditions for all of them, we decided to omit the two most hydrophobic analytes (TBZMF, BZPF) from further method development since it was highly unlikely that they would be present in aqueous samples due to their hydrophobicity. Therefore, the LC-MS/MS method was optimized only for the hydrophilic benzotriazoles. At this point, a deu-terated internal standard (IS; 1H-benzotriazole-d4, BTZ-d4) was also introduced. The same chromatograph-ic column was used and the elution gradient started with very low concentration of organic solvent with the slow increase in its percentage (35% ACN in 12.5 min) to achieve the best separation of methyl isomers 4MBZ and 5MBZ. Even so, their peaks were not baseline separated (see Table 2). A chromatogram of standard solution under final conditions is given in Supplementary Information SI 2. For MS parameters, electrospray conditions were optimized by flow-injection of analyte solution. Positive ESI ionization was chosen because the signals were of a much higher intensity than with negative ionization, which is in agreement with other published methods.7,22,23 The optimized SRM transition conditions are presented in Table 2. The first transition was used for quantification, while the second transition and the fragment ion ratio were used for the identification of analytes. Table 2: Retention times (iR), observed SRM transitions, their collision energies (CE) and fragment ion ratios. analyte tR SRM1 CE1 SRM2 CE2 Ion (min) (V) (V) ratio OHBZ 6.96 136>80 29 136>90 27 9.74 BTZ 8.63 120>65 30 120>92 25 2.74 4MBZ 11.3 134>77 34 134>79 25 1.46 5MBZ 11.5 134>77 34 134>79 25 1.40 ClBZ 13.7 154>99 32 154>73 46 2.05 DMBZ 14.0 148>77 37 148>91 31 1.75 IS 8.55 124>69 33 124>96 24 Instrumental limits of detection (ILOD) and quantification (ILOD) were calculated from calibration curves in standard solutions with added IS, using formulae ILOD = 3.3 x s(res)/k and ILOD = 10 x s(res)/k, where s(res) is the residual sum of squares and k the slope of the calibration curve. ILOD were at 5-30 ^g/L, while ILOQ were determined in the range 16-91 ^g/L. Acceptable linearity of the calibration curve was observed in the range from ILOQ to 10 mg/L. 3. 2. Optimization of DLLME Extraction DLLME extraction was studied in MQ water with added analytes at 1 mg/L level. Two sets of conditions were optimized: for both hydrophilic and hydrophobic ben-zotriazoles; and for hydrophilic analytes only. The following parameters were changed: choice and volume of extraction solvent; choice and volume of dispersive solvent; volume, pH and ionic strength of aqueous sample; time and speed of centrifugation. All of the tested extraction solvents were denser than water: dichloromethane, 1,1,1-trichloroethane, CHCl3, CS2, CCl4, and chloroben-zene. Solvents with lower density than water (light-er-than-water) can also be applied,28-30 but after centrifu-gation, a thin layer of solvent with extracted compounds is formed on the surface of aqueous phase, which is impossible to collect. For that reason, special glassware with narrow neck should be used to allow for formation of a thicker upper layer of solvent.28-30 To avoid the need for special equipment, heavier-than-water solvents were used to form an easy-to-collect drop at the bottom of the vessel after centrifugation. For dispersive solvent, methanol, acetoni-trile, acetone, isopropanol, and ethanol were tested. The extract was dried and re-dissolved in MQ or MQ/ACN because of the incompatibility of the chlorinated solvents with the LC column. Out of all tested extraction solvents, the best recoveries were achieved with CHCl3 for hydrophilic and CCl4 for hydrophobic analytes. A mixture of these solvents was used, the ratios determined in further steps. Acetoni-trile proved to be the best dispersive solvent for all ana-lytes. Optimization of the previously listed parameters was first performed by two-level fractional factorial experimental design, the values of parameters investigated are presented in Table 3. 8 different experiments with parameters set at these levels (with 4 repetitions) were performed to estimate the significance of influences of the parameters. For most analytes the only significant parameter seemed to be the pH value (Figure 2), which could be due to its broad range investigated (4-10). As it is well known that other parameters can also influence the extraction efficiency, we decided to further perform a stepwise optimization. Table 3: Values of parameters, investigated with fractional factorial design. Parameter Level - Level + Sample volume (mL) 2 5 CHCl3 volume (^L) 20 100 CCl4 volume (^L) 20 100 ACN volume (mL) 0,3 1 Sample pH 4 10 NaCl (% w/v) 0 10 rpm 1500 3000 Kraševec and Prosen: Development of a Dispersive Liquid-Liquid ... Acta Chim. Slov. 2019, 66, 247-254 251 Figure 2: Influence of investigated parameters on 4MBZ extraction efficiency, the line denotes significant influence. In the stepwise optimization it turned out, that sample pH below 6 was necessary for efficient extraction of hydrophilic compounds and that the addition of salt up to 10 % w/v improved the extraction for the hydrophilic an-alytes, while the hydrophobic analytes remained unaffected. Variations in centrifugation speed and time had almost no effect on extraction efficiency, while smaller sample volumes and larger solvent volumes increased the efficiency for all analytes. Due to aiming for higher enrichment factors (not recoveries), the ratio between sample and extract volume was adjusted further, with sample volume set at 3 mL and final solvent volumes 80 ^L CHCl3, 20 ^L CCl4, and 700 ^L acetonitrile, which resulted in 200-220 ^L of extract before drying. With the parameters set as described in Experimental, the enrichment factors ranged between 9.5 and 28.7. Further experimental data on this optimization is given in Supplementary Information (SI3-5). For hydrophilic analytes, only CHCl3 was used as the extraction solvent, as the addition of CCl4 was unnecessary. The best enrichment factors in general were obtained with the CHCl3/acetonitrile ratio 1:5 (Figure 3) and the highest available sample volume (5 mL). Relatively high volumes of solvents contributed to higher enrichment factors due to the additional concentrating step (drying and re-dissolving). Again, the acidic pH and salt addition in- Figure 3: The effect of solvent ratios (CHCl3:ACN) on enrichment factors for hydrophilic analytes. creased the efficiency, while the centrifugation speed and time had negligible effect. While the addition of 15 % w/v of NaCl further increased the efficiency, the density of the sample increased to such amount, that the extract floated to the surface, and the repeatability dropped drastically. With the final parameters, the obtained enrichment factors ranged from 9.5 to 84.7. 3. 3. Validation of the Method in Environmental Samples DLLME method for hydrophilic analytes in surface water in combination with LC-MS/MS analytical method was evaluated for enrichment factor, linearity, repeatability, limit of detection, and limit of quantification, using matrix matched calibration. River water was spiked with analytes in the range 0.01-50 ^g/L and 1 ^g/L of IS, and then extracted by the optimized DLLME procedure. The repeatability was estimated at two concentration levels (0.5 and 10 ^g/L) in at least 3 replicates, and blank river water extracts were also prepared. All signals were calculated as the ratio of peak areas of analytes and IS. The validation results are shown in Table 4. High linearity was observed from LOQ to 10 ^g/L for all analytes (R2 0.99010.9985, except for OHBZ 0.9827). Moreover, the matrix effect (ME) was estimated by comparing the signal of the blank extract, spiked with the analytes and IS (post-extraction spiking), and the signal of the standard solutions in MQ water at the same theoretical concentration. Negative values of ME indicate ionization suppression in ESI, while positive ME values indicate ionization enhancement. Interestingly, ME is in the range +15% to +31%, except for BTZ (-19%). Matrix effect is commonly explained by the co-elution of compounds from the sample matrix that are still present in the sample extract and compete with analytes during the ESI ionization process. Positive ME values are in fact favourable because lower LODs can be achieved, but nevertheless, matrix-matched calibration should be used for the quantitative determination of analytes in real samples. As seen from Table 4, LODs and LOQs are sufficiently low to cover the expected range of concentrations in wastewater,14,15 while using only 5 mL of sample, which is much lower compared to sample volumes usually required for SPE.3,6,21-25 Extraction efficiency, given as enrichment factor (EF), is calculated by comparing concentration in the extract and initial concentration in sample. It is obvious that EFs for the most polar analytes (OHBZ, BTZ) are lower than EFs for the other analytes, which results from their lower solubility in the non-polar extraction solvent. Repeatability is acceptable at higher spiking level, but it is obvious that DLLME is a manual technique requiring a highly skilled operator. On the lower spiking level, the repeatability decreases, even with the use of an internal standard, but this is understandable, since this level is close to or below LOQs. Krasevec and Prosen: Development of a Dispersive Liquid-Liquid 252 Acta Chim. Slov. 2019, 66, 247-254 Table 4: Method validation parameters in surface water. OHBZ BTZ 4MBZ 5MBZ ClBZ DMBZ LOD (|ig/L) 0.07 0.14 0.75 0.19 0.06 0.04 LOQ (|ig/L) 0.23 0.47 2.51 0.62 0.19 0.14 %RSD 19.4 4.4 8.8 10.7 12.1 16.2 (10 re/L) %RSD 44.0 42.4 35.2 24.4 16.8 14.3 (0.5 ^g/L) EFa 10 34 75 66 81 70 aEF - enrichment factors, determined at spiking concentration 5 |g/L 3. 4. Comparison to Other DLLME Methods for Benzotriazoles Three other DLLME or similar methods were found in the literature for the determination of polar benzotriazoles. The comparison of methods from the analytical standpoint is presented in Table 5. Pena et al.28 used 100 ^L of the ionic liquid tri-butyl-phosphate as the extraction solvent, but due to the high viscosity of the ionic liquid, the extract had to be diluted 10-times before injection into LC-Flu-UV. In our method, the recoveries are lower, but no ionic liquids are used, so it is not necessary to dilute the extract and further preconcentration is gained with the drying step and the LOQs achieved are quite similar. Casado et al.29 used 60 ^L of toluene as the extraction solvent with 100 ^L of acetic anhydride added for simultaneous acetylation of the analytes, which were then analysed by GC-MS. With this method, they obtained very low LOQs, but the recoveries are comparable to those obtained with our method, with the exception of the most polar analyte (OHBZ) which was not analysed in their study. Lu et al.30 used 80 ^L of 1-hexanol extraction solvent, which was dispersed with the assistance of air instead of a dispersive solvent. The extracts were analysed by HPLC-UV, which also contributes to the higher LOQs in comparison to our method's. In general, our method is comparable to those from the literature. For all of these methods, the extracts were lighter-than-water, which demanded either special glassware or a twofold transferring of the extract for exact separation of the phases. In this work, we managed to avoid these disadvantages connected to the use of heavier-than-water solvents. In comparison to conventional SPE methods, for which the reported LOQs range from 1 to 100 ng/L,3,6,22,23,25 the LOQs obtained with all of these DLLME methods are quite higher. This is a direct consequence of lower sample volumes, which reduces the possible preconcentration factors. 3. 5. Determination of Benzotriazoles in Environmental Samples The developed and validated method was applied to the determination of analytes in environmental water samples. The quantification was performed with matrix-matched calibration. In groundwater and rivers Glinscica, Idrijca, and Soca, all analytes were below LOD of the method, while BTZ, 4MBZ, and 5MBZ were found in influent and effluent wastewaters from the Central wastewater treatment plant in Ljubljana. Estimated concentrations in effluent wastewater ranged from 4.8 to 7.3 ^g/L. In influent wastewater, the same analytes were present in estimated concentrations 2.7 to 12.0 ^g/L. Influent water was diluted 1:5 with MQ water before extraction due to enhanced matrix effect compared to surface water. However, for accurate quantification, matrix-matched calibration for wastewater should be applied. The concentrations for wastewater are in good agreement with those usually determined in European waste-waters.14,15 In a recent study on micropollutants in surface and groundwater from the area of Maribor, Slovenia, where SPE extraction was performed (LOQs 3-7 ng/L), two benzotriazoles (2-methyl-2H-benzotriazole, 2,4-di-methyl-2H-benzotriazole) were found in concentrations 1.4 to 273.3 ng/L in groundwater, and in concentrations 5.8 to 27.9 ng/L in surface water.31 These two compounds were not considered in our method. 4. Conclusions In this study, two DLLME microextraction methods were developed for the determination of either hydrophilic benzotriazoles, or hydrophilic and hydrophobic benzotriazoles together. Especially the hydrophilic benzotriazoles are emerging pollutants present in the aquatic system. Microextraction methods for benzotriazoles are rarely encountered in literature, and to our best knowledge, this is Table 5: Comparison of analytical performance in pure water with DLLME methods from literature (EF - enrichment factor, RSD - repeatability). Analytes EF Absolute recovery RSD (%) LOQ Ref. (%) (at spiking level) OHBZ, BTZ, 4MBZ, 5MBZ, ClBZ, DMBZ ND 67-86 5.0-7.8 (18 |ig/L) 0.1-7.3 |ig/L 28 BTZ, 4MBZ, 5MBZ, ClBZ, DMBZ 93-172 24-46 2-8 (0.2 |g/L) 7-80 ng/L 29 BTZ, 5MBZ, ClBZ 43-87 ND 3.0-4.3 (50 |g/L) 2.9-4.8 |g/L 30 OHBZ, BTZ, 4MBZ, 5MBZ, ClBZ, DMBZ 10-85 5-42 4.7-13 (50 |g/L) 0.1-2.5 |g/La This work ND - no data available; a In surface water Kraševec and Prosen: Development of a Dispersive Liquid-Liquid ... Acta Chim. Slov. 2019, 66, 247-254 253 the first time a DLLME method with denser-than-water extracts for benzotriazoles has been developed. The developed methods use smaller volumes of samples and solvents than classical methods and in this aspect contribute to a more environmentally friendly chemistry. The DLLME method for hydrophilic benzotriazoles was validated in surface water. Matrix effect was compensated for in sample analysis with the usage of matrix matched calibration. The method was applied for determination of ana-lytes in groundwater, surface water and wastewater samples. However, in wastewater samples the matrix effect was more pronounced than in surface water. Therefore, matrix-matched calibration using wastewater matrix should be applied for accurate quantification. This study was also one of the very few investigating the presence of benzotriazoles in Slovenian water environment. We confirmed their presence in the wastewater at ^g/L levels, comparable to other European samples, but the analytes were g vitamina Bz enkrat nadan Ji* (Agí 20 tfrečk po ?rlggranulata z; <č*KRKI\ .jr s«3& www.magnezijkrka.si ® Okus po pomaranči in limeti. ® Brez konzervansov. ® Brez umetnih barvil, arom in sladil. ® Ena vrečka na dan. Prehransko dopolnilo ni nadomestilo za uravnoteženo in raznovrstno prehrano. Skrbite tudi za zdrav življenjski slog. 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