Scientific paper Theoretical Considerations Regarding the Thione-thiol Tautomerism in 2-(5-mercapto-1,3,4-thiadiazol-2-ylthio)acetic Acid Raluca Pop,1* Maria Ilici,2* Mihaiela Andoni,1 Vasile N. Bercean,2 Cornelia Muntean,2 Monica M. Venter,3 and Ilie Julean2 1 University of Medicine and Pharmacy "Victor Babe§" Timisoara, Faculty of Pharmacy, Eftimie Murgu Square 2, 300041 Timisoara, Romania 2 University POLITEHNICA Timisoara, Faculty of Industrial Chemistry and Environmental Engineering, 300006 Timisoara, Romania 3 Babes-Bolyai" University, Faculty of Chemistry and Chemical Engineering, 11 Arany Janos Str., 400028 Cluj-Napoca, Romania. * Corresponding author: E-mail: ralucapop24@gmail.com; ilici2011@gmail.com Received: 24-04-2014 Abstract The acidity constants Ka1 and Ka2 of 2-(5-mercapto-1,3,4-thiadiazol-2-ylthio)acetic acid have been determined both by experimental and theoretical methods. pKa computations at B3LYP/6-311+G(d,p) level of theory were carried out for the two tautomeric forms, thiol and thione, of the above-mentioned acid. Comparisons between the experimental and theoretical values led to the establishing of the most stable tautomer of 2-(5-mercapto-1,3,4-thiadiazol-2-ylthio)acetic acid in aqueous solution. Also, a DFT study regarding the reactivity, aromaticity and population analysis of the two tau-tomers has been performed. Keywords: thiadiazole, pKa, thiol-thione tautomerism, theoretical computations, DFT. 1. Introduction Together with the other 5-membered heterocycles, thiadiazoles have gained an increased popularity during the last decades. Their importance is due to their versatility and their biologic activity as antitumor, antifungal and antiviral agents.1,2 The presence of the sulfur atom within the cyclic structure confers them, compared to the 5-mem-bered nitrogen heterocycles, an improved liposolubility and better penetration of the cellular membranes.2 Also, the 5-imino-1,2,4-thiadiazole derivatives are found to be inhibitors of glycogen synthase kinase 3 (GSK3), a protein kinase with active role in diabetes, cancer and Alzheimer's disease.3,4 In the case of heterocyclic moieties that show biologic activity and where the possibility of the co-existence of two tautomeric forms appears, it becomes very important to determine the most stable tautomer. Nowadays, computational chemistry techniques allow the prediction of the tautomeric behavior prior to the experimental stu-dies.5 There are numerous papers that where published in the last years regarding theoretical studies of the thiol-thione,6-9 keto-enol,10 formamide-formamidic acid,11 nucleic acid bases,12,13 heterocyclic rings -pyrazoles, triazo-les14-17 tautomerism. The 5-membered mercapto-aza heterocyclic derivatives (like Bismuthiol - dimercaptothiadiazole) were used with good results for the preparation of new metal com-plexes.18 Within our research group, there has been reported the obtaining of various metal complexes of the dimer-captothiadiazole derivatives.18-19 A derivative of 1,3,4-thiadiazol-2-yl-thioacetic acid, which can exist both in thiol (2-(5-mercapto-1,3,4-thiadiazol-2-ylthio)acetic acid) or thione (2-(5-thioxo-4,5-dihydro-1,3,4-thiadiazol-2-ylthio)acetic acid) form, has been used for the preparation of metal complexes with various transitional metals.19 Previous studies of our research group have highlighted two possible tautomeric forms: thiol (5SH, Ia) and thione (5S, Ib).19,20 In this regard, the pKa values for the two de-protonation stages of each tautomer may offer valuable data regarding the stability of both tautomer species in aqueous solution. The present study deals with the evaluation, both by theoretical and experimental methods, of the pKa of 2-(5-mercapto-1,3,4-thiadiazol-2-ylthio)acetic acid. This way, an estimation of the pKa values by theoretical means -using both thermodynamic cycles21-24 and isodesmic reac-tions25 - may also result in clarifying the issues regarding the dominant tautomer in aqueous solution. Scheme 1. Tautomeric structures of 2-(5-mercapto-1,3,4-thiadia-zol-2-ylthio)acetic acid I (C4H4S3N2O2) (Ia - thiol form; Ib - thione form). In order to estimate the theoretical pKa value of the 2-(5-mercapto-1,3,4-thiadiazol-2-ylthio)acetic acid, two thermodynamic cycles and one isodesmic reaction scheme have been employed. Method 1: Thermodynamic cycle 1 TDzCOOH (gas) .^gp.ii^ TDzCOO' (gas) + H+ (gas) TDzCOOH (aq) TDzCOO" (aq) + H* (aq) Scheme 2. Thermodynamic cycle (1) used for the pKa computation. tively. Within the present study, the values of -265.9 kcal/mol (solvation) and -6.28 kcal/mol (gas-phase) of the free energy of H+ were considered. Also, the correction to the gas-phase free energy of the deprotonating reaction (RTln24.46, representing 1.895 kcal/mol) was added. The correction factor is required for transforming the gas-phase free energy of the deprotonation reaction, computed to a reference state of 1 atm, in a value computed for a reference state of 1M (specific to the aqueous phase computations). Method 2: Thermodymanic cycle 2 The second thermodynamic cycle is based on the reaction with a water molecule, when the corresponding anion of the thiadiazole derivative, along with the hydro-nium ion, is obtained. This way, the source of errors consisting in the thermodynamic parameters of H+ is avoided. Within the following computations, the accepted values of the AGsolH3O+ and AGsolH2O were considered -110.3 kcal/mol and -6.32 kcal/mol, respectively. TDzCOOH (gas) + H20 (gas) aG0. - TDzCOO' (gas) л Gsol (TDzCOOH) TDzCOOH (aq) AGsoHHjO) H20 (aq) де0 ;1GsoI(TDzCOQ"> TDzCOO (aq) Scheme 3. Thermodynamic cycle (2) used for the pKa computation. The equations that were used for the computation of pKa are the following: (1), where: The value of pKa was calculated by means of the thermodynamic cycle presented in Figure 1. According to literature data, one of the main challenges in pKa computations consists in determining the appropriate value of the free energy of solvation of H+, as it is well known that an error of 1.36 kcal/mol leads to an 1 unit of pKa error.22,24 The experimental data range within -254 and -265.9 kcal/mol,21-24 but two of the most often used values are -264.0 kcal/mol24 and -265.9 kcal/mol,22 respec- AG = AG + AAG , ciij gas sol AAGw = ДGw ( products) - AGMll ( reagents ) (2) (3) (4) Method 3: Isodesmic reaction scheme with thioglycolate The isodesmic reactions are characterized by the conservation of the type of broken/formed bonds. Nama-zian and Heidary25 proposed an isodesmic reaction sche- TDzCOOH + HSCHzCOO" -TDzCOO + HSCH2COOH HSCH2COOH + H20 --HSCH2COO" + H30+ TDzCOOH + H20 -TDzCOO" + H30+ Scheme 4. Isodesmic reactions used for pKa computations me with acetate/acetic acid for the computation of pKa for a number of carboxylic acids with similar structure. In this regard, the thioglycolate ion and the thioglycolic acid were chosen as references for the pKa computation of the 2-mercapto-1,3,4-thiadiazol-5-yl thioacetic acid. 2. Experimental the experimental determination of Ka values, conductome-tric and potentiometric titrations have been performed using a MM 41 Crison multimeter, fitted with a conductivity cell and a glass electrode. For the determination of the equivalence points of potentiometric titration and for the calculation of Ka1 and Ka2 values, software applications developed by the authors (I. Julean and C. Muntean) have been used. The geometric optimization of compounds Ia and Ib and their corresponding anions has been performed at B3LYP/6-311+G(d,p) level of theory. Vibrational analysis, carried out at the same level of theory, showed that no imaginary frequency was obtained. In order to quantify the effects of the aqueous environment, two solvation models -conductor-like polarizable continuum model (CPCM) and integral equation formalism polarizable continuum model (IEFPCM - with the solute characterized by the electronic density) - have been employed.26 The geometric optimization of the two tautomers (and of the corresponding anions) in aqueous environment have been also performed at B3LYP/6-311+G(d,p) level of theory, using both CPCM and IEFPCM solvation models. Geometry optimizations, frequency computations, thermochemistry data and reactivity indices (the condensed Fukui functions27) have been performed using the Gaussian 09W package.28 Also, for the NICS(0) and NICS(1) computations (NICS - Nucleus-Independent Chemical Shift), the GIAO (Gauge Independent Atomic Orbitals) approach within Gaussian 09W program has been employed. For 3. Results and Discussions 3. 1. Theoretical Calculation of pKa First ionization stage of 2-(5-mercapto-1,3,4-thia-diazol-2-ylthio)acetic acid consists in the removal of the H atom from the carboxyl group (reactions (1) and (3), respectively). The reaction is very similar for both tautomeric forms, leading to the obtaining of the thioaceta-te anion. Instead, significant differences appear during the second ionization stage, that involves H+ removal from the -SH group (tautomer Ia) and the H+ removal of the -NH group, a (tautomer Ib). One can approximate that a more "acidic" character is expected for the proton removal from the -SH group, which is directly bonded to the aromatic skeleton of 1,3,4-thiadiazole. In the case of the second ionization stage of the thione tauto-mer Ib, the hydrogen atom that has to be removed belongs to a secondary amino group (also, the cyclic delo-calization of the electrons within the heterocyclic ring is affected). (1) (2) Scheme 5. The heterocyclic species involved in the first and second ionization of the thiol tautomer (Ia) of the 2-(5-mercapto-1,3,4-thiadiazol-2-ylthio)acetic acid. (3) (4) Scheme 6. The heterocyclic species involved in the first and second ionization of the thione tautomer (Ib) of the 2-(5-mercapto-1,3,4-thiadiazol-2-ylthio)acetic acid. The experimental results (see detailed information in Appendix) are presented in Table 1 and show a 3rd order difference between the Ka values (and, corresponding, 3 p-Ka units) that were obtained for the two stages of the ioni-zation process. Table 1. Experimental values of Ka and pKa, determined for the first and second ionization stages of compound I Compound Ka1(PKa1) Ka2 (PKa2) I (C4H4S3N2O2) 8.72 • 10-3 (2.06) 8.632 • 10-6 (5.06) The theoretical computations of pKa values allow the evaluation of the acid character of both tautomeric forms. Similarities with the experimental results will be used for the prediction of the dominant tautomer in aqueous solution). Several conclusions can be drawn regarding the results presented in Table 2 and Table 3; pKa1 values are very similar, regardless of the chosen method of computation. As expected, loosing the H atom of the carboxyl group (namely, the pKa1 value) is only slightly influenced from the thiol-thione tautomerism. The computed values show a difference of 0.65 pKa units between the two tautomers, the more acidic being the thione form (Ib). Significant differences appear between the pKa2 values (almost 7 pKa units is the difference between the thiol and thione tautomers). These results lead to the conclusion that, in aqueous solution, the dominant tauto-mer is Ia (the thiol form of the 2-(5-mercapto-1,3,4-thia-diazol-2-ylthio)acetic acid). The results presented in Table 2 show a ApKa value around 3, in good correlation with the experimental one. The results seem to illustrate the Gustafson's paradox: the most acidic tautomer is also the less abundant.5 As regards the accuracy of the results, it is obvious that the theoretical calculations of pKa, regardless the chosen method, overestimate the pKa values. A possible ex- planation for the lower values of experimental pKa (when compared to the calculated ones) can be attributed to the undervaluing of the energies of the compounds when carrying PCM computations. Among the three variants that were used for the estimation of pKa, differences appear only for the pKa2 values computed by means of the first thermodynamic cycle. As the pKa1 calculations (by means of all of the three methods) led to insignificant differences among the obtained values, it may be assumed that the underestimation of the pKa2 value, when using the first ther-modynamic cycle, cannot be caused by the free energy of solvation of H+ (considered -265.9 kcal/mol within the present study). 3. 2. Similarities and Differences Between the Thiol-thione Tautomers Ia and Ib 3. 2. 1. Aromatic Character of the Thiol- and Thione- Derivatives of the 2-(5-mercapto-1,3,4-thiadiazol-2-yl-thio)acetic acid The magnetic criterion was chosen in order to evaluate the aromaticity of the two tautomers. In this regard, NICS(0) and NICS(1) computations were performed; the results are presented in Table 4. Table 4. NICS(0) and NICS(1) values, computed at B3LYP/6-311+G(d,p) level of theory C4H4S3N2O2 NICS(0) (ppm) NICS(1) (ppm) Tautomer Ia -10.156 -8.994 Tautomer Ib -7.310 -5.194 The results confirm that the thiol form of the 2-(5-mercapto-1,3,4-thiadiazol-2-ylthio)acetic acid is more aromatic than the thione one; given that in the case of the thione tautomer, formation of the exocyclic C=S bond disrupts the n-electron conjugation and leads to a decrease of the aromatic character. Table 2. Theoretical pKa values calculated for the two ionization stages of the thiol tautomer (Ia) C4H4S3N2O2 PKa1 PKa2 APKa = pKaQ-pKaa) Ia IEFPCM CPCM IEFPCM CPCM IEFPCM CPCM Method 1 4.63 4.63 6.08 6.12 1.45 1.49 Method 2 4.64 4.64 7.82 7.80 3.18 3.16 Method 3 4.63 4.56 7.82 7.62 3.26 3.06 Table 3. Theoretical pKa values calculated for the two ionization stages of the thiol tautomer (Ib) C4H4S3N2O2 PKa1 PKa2 APKa = PKa(2)-PKa(1) Ib IEFPCM CPCM IEFPCM CPCM IEFPCM CPCM Method 1 3.98 3.97 12.30 12.28 8.32 8.31 Method 2 3.98 3.99 14.00 14.05 10.02 10.06 Method 3 3.94 3.86 13.99 13.80 10.05 9.94 In addition to the magnetic index of aromaticity (NICS), the global stability of the six species involved in the ionization process (the two tautomers Ia and Ib and their corresponding monoanions and dianions) was evaluated. As well as in the case of other aromatic compounds, HOMO-LUMO gap computation represents an useful tool for comparisons among the stability of various species.29 Table 5. HOMO-LUMO gap of the Ia-IIIa and Ib-IIIb species (B3LYP/6311+G(d,p)) HOMO-LUMO gap (eV) Ia 5.11 Ib 4.59 IIa 3.38 IIb 3.21 IIIa 4.26 IIIb 4.26 According to the results presented in Table 5, the most stable specie is the tautomer Ia, whereas at the opposite side the monoanions IIa and IIb are found. The identic value obtained for the dianionic species IIIa and IIIb suggests that a delocalization of both the negative charge and the double bond appears among the N-C-S atoms, leading to the same specie (see Scheme 7). 3. 2. 2. NBO (Natural Bond Orbitals) Analysis The evaluation of the donor-acceptor interactions and their energetic contributions to the stability of the compounds is performed by means of the 2nd order perturbation theory within NBO analysis. The stabilization energy E2, as difference between the localized-delocalized structures, is reported in tables below: The results presented in Table 6 and Table 7 emphasize the differences among the thione and thiol form (namely between the neutral and monoanionic species, Ia-Ib and IIa-IIb). The lone pair of the nitrogen atom N4 has an exclusive p character in the thione form, leading to stronger stabilization interactions. Regarding the S6 atom hybridization, it results that the content in s and p orbitals is changing when transition to the thiol to the thione form occurs. The dianionic species III-a-IIIb are characterized by the same hybridization -as obtained for the thiol tautomer- for N4, while S6 has the same character as in the case of the thione tautomer, leading to the structure of the mesomeric form III. The specific donor-acceptor interactions of the 5SH- and 5S- tautomers are preserved within the dianionic form III. s-—^SCHjCOO IIIb S ^SCHjCOO" IIIa JHL s III SCH,COO" Scheme 7. Mesomeric species IIIa and IIIb. Table 6. NBO analysis of the 6SH tautomer Thiol tautomer LP N3 hybridization Donor-acceptor interactions E2 (kJ/mol) Ia IIa IIIa Ia IIa IIIa 36.34% s, 63.54% p 35.76% s, 64.13% p 36.43% s, 63.52% p LP S6 hybridization 68.91% s, 31.07% p 68.25% s, 31.73% p 81.17% s, 18.82% p LP N4 ^ o* C5-S6 LP N4 ^ o* C5-S6 LP N4 ^ o* C5-S6 LP S6 ^ n* C5-N4 LP S6 ^ n* C5-N4 LP S6 ^ n* C5-N4 14.58 14.54 14.79 24.52 21.56 39.22 Table 7. NBO analysis of the 6S tautomer Thione tautomer LP N4 hybridization Donor-acceptor interactions E2 (kJ/mol) Ib 99.99% p LP N4 ^ o* C5-S6 74.37 LP N4 ^ n* C2-N3 32.36 IIb 99.99% p LP N4 ^ o* C5-S6 80.22 LP N4 ^ n* C2-N3 28.47 IIIb 36.40% s, 63.50% p LP N4 ^ o* C5-S6 14.91 LP S6 hybridization Ib 81.39% s, 18.59% p LP S6 ^ n* C5-N4 11.94 IIb 81.02% s, 18.95% p LP S6 ^ n* C5-N4 11.62 IIIb 81.17% s, 18.81% p LP S6 ^ n* C5-N4 39.80 3. 2. 3. Reactivity Indices Condensed Fukui functions have been computed for the N4, N3 and S6 heteroatoms, in order to assess their reactivity during a reaction with an electrophile.26 Table 8. Condensed Fukui functions computed for the 6SH tauto-mer (B3LYP/6-311+G(d,p)) Atom f (Ia) f (IIa) Г (IIIa) N4 0.064 0.111 0.091 N3 0.068 0.072 0.034 S6 0.171 0.028 0.356 Table 9. Condensed Fukui functions computed for the 6S tautomer (B3LYP/6-311+G(d,p)) Atom f-(Ib) f (IIb) Г (IIIb) N4 0.109 0.048 0.091 N3 0.056 0.028 0.034 S6 0.335 0.021 0.353 The condensed Fukui functions computed for the neutral forms Ia and Ib show that the S6 atom has the largest electrophilic character (higher for the thione tauto-mer). The two N atoms of the thiadiazole heterocycle have similar reactivity in the case of the tautomer Ia (see Table 8); the reactivity difference between the two N atoms of the tautomer Ib is outlined by the almost double value obtained for the N4 atom. The S6 atoms within the monoa-nionic species present the lowest reactivity; the graphical presentation of the HOMO orbitals of the thioglycolate anions clearly shows that the highest electrophilic character appears at the thyoglycolate residue, not within the hete-rocyclic ring (see Appendix). The dianion III shows a similar reactivity of the S6 atom as in the case of the thione form, whereas the reactivity of the N4 atom is between (almost the mean value) the values of Fukui function Г of the N4 atom in the thiol and thione tautomers. 4. Conclusions Theoretical evaluation of the pKa for the two ionization stages of 2-(5-mercapto-1,3,4-thiadiazol-2-ylthio) acetic acid (both tautomeric forms) have been performed, in order to predict the more stable tautomer in aqueous solution. Two solvation models, IEFPCM and CPCM, have led to similar results for the solvation free energies of all the species involved in the ionization process. Two ther-modynamic cycles and one isodesmic reaction scheme have been used for the theoretical computation of pKa. 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Mennucci, R. Cammi, Chem. Rev. 2005, 105, 2999-3093. http://dx.doi.org/10.1021/cr9904009 27. R. R. Contreras, P. Fuentealba, M. Gaban, P. Perez, Chem. Phys. Lett. 1999, 304, 405. http://dx.doi.org/10.1016/S0009-2614(99)00325-5 28. Gaussian 09, Revision D.01, M. J. Frisch, G. W. Trucks, H. B. Schlegel, G. E. Scuseria, M. A. Robb, J. R. Cheeseman, G. Scalmani, V. Barone, B. Mennucci, G. A. Petersson, H. Nakatsuji, M. Caricato, X. Li, H. P. Hratchian, A. F. Izmay-lov, J. Bloino, G. Zheng, J. L. Sonnenberg, M. Hada, M. Ehara, K. Toyota, R. Fukuda, J. Hasegawa, M. Ishida, T. Na-kajima, Y. Honda, O. Kitao, H. Nakai, T. Vreven, J. A. Montgomery, Jr., J. E. Peralta, F. Ogliaro, M. Bearpark, J. J. Heyd, E. Brothers, K. N. Kudin, V. N. Staroverov, T. Keith, R. Ko-bayashi, J. Normand, K. Raghavachari, A. Rendell, J. C. Bu-rant, S. S. Iyengar, J. Tomasi, M. Cossi, N. Rega, J. M. Mil-lam, M. Klene, J. E. Knox, J. B. Cross, V. Bakken, C. Adamo, J. Jaramillo, R. Gomperts, R. E. Stratmann, O. Yazyev, A. J. Austin, R. Cammi, C. Pomelli, J. W. Ochterski, R. L. Martin, K. Morokuma, V. G. Zakrzewski, G. A. Voth, P. Salvador, J. J. Dannenberg, S. Dapprich, A. D. Daniels, O. Far-kas, J. B. Foresman, J. V. Ortiz, J. Cioslowski, and D. J. Fox, Gaussian, Inc., Wallingford CT, 2013. 29. J. Aihara, J. Phys. Chem. A 1999, 103 (37), 7487-7495. http://dx.doi.org/10.1021/jp990092i Povzetek Za 2-(5-merkapto-1,3,4-tiadiazol-2-iltio)ocetno kislino smo določili eksperimentalne in teoretične vrednosti konstant Ka1 in Ka2. Izračune pKa smo za dve tavtomerni obliki izvedli na B3LYP/6-311+G(d,p) nivoju. S primerjavo eksperimentalnih in izračunanih vrednosti smo določili najbolj stabilno tavtomero preiskovane kisline v vodni raztopini. Dodatno smo izvedli smo še DFT analize reaktivnosti, aromatskega značaja in populacije obeh tavtomer. Theoretical considerations regarding the thione-thiol tautomerism in 2-mercapto-1,3,4-thiadiazol-5-yl thioacetic acid Supplementary data 1. Computational part Я 0 и 9 vi Г.И w л -■ • ■■ • a- Ia (HOMO orbitals) Ib (HOMO orbitals) " . - -, jJ -'" Ila (HOMO orbitals) IIb (HOMO orbitals) W i i 'С € ->ч rv': r; - £ jf III (HOMO orbitals) Figure 1. Graphical representation of HOMO orbitals Free energies of solvation, computed at B3LYP/6-311+G(d,p), are presented in table below: AGsolv ( ical/mol) Compound IEFPCM CPCM Ia -9.194 -9.270 IIa -58.119 -58.192 IIIa -158.131 -158.158 Ib -9.740 -9.810 IIb -55.228 -55.300 IIIb -158.143 -158.159 Thioglycolic acid -5.962 -6.021 Thioglycolate -58.523 -58.420 Table 1. AGsolv (kcal/mol) (B3LYP/6-311+G(d,p) level of theory) 2. Experimental part The acid-base titration data that have been used to determine the equivalence volume and pH and to calculate the acidity constants Ka1 and Ka2 by means of software applications (Visual Basic) developed by the authors (I. Julean and C. Muntean): C2HN2S3CH2COOH V(mL) Figure 2. Conductometric ( ) and acid-base titration (*) of 0.01M C4H4S3N2O2 with 1M NaOH Theoretical considerations regarding the thione-thiol tautomerism in 2-(5-mercapto-1,3,4-thiadiazol-2-ylthio)acetic acid Supplementary data 1. Computational part ЪйИ la (HOMO orbitals) Ib (HOMO orbitals) H Ila (HOMO orbitals) IIb (HOMO orbitals) Bf III (HOMO orbitals) Figure 1. Graphical representation of HOMO orbitals Free energies of solvation, computed at B3LYP/6-311+G(d,p), are presented in table below: AGsolv (kcal/mol) Compound IEFPCM CPCM Ia -9.194 -9.270 IIa -58.119 -58.192 IIIa -158.131 -158.158 Ib -9.740 -9.810 IIb -55.228 -55.300 IIIb -158.143 -158.159 Thioglycolic acid -5.962 -6.021 Thioglycolate -58.523 -58.420 Table 1. AGsolv (kcal/mol) (B3LYP/6-311+G(d,p) level of theory) 2. Experimental part In order to evaluate the acidity constants of 2-(5-mercapto-1,3,4-thiadiazol-2-ylthio)acetic acid, both potentiometric and conductometric titration were performed. Five identical samples of acid were employed, for obtaining the averaged value for each of the two acidity constants. For each of the five samples, the pH at the equivalence point and the volume at the second equivalence point were determined by using the experimental data obtained during the potentiometric titration. The experimental data have been used for determining the acidity constants Ka1 and Ka2 by means of software applications (Visual Basic) developed by the authors (I. Julean and C. Muntean). Figure 2 depicts the titration curves of the 2-(5-mercapto-1,3,4-thiadiazol-2-ylthio)acetic acid. It can be observed the inflection of the curve at the first equivalence point, while the second equivalence point is characterized by a larger jump of pH. This behavior is usual for diprotic acids with similar values of the two acidity constants (Ka1/Ka2<104). V(mL) t 4 -- 3.5 -- 3 E о 2.5« 2 > > 1.5o з 1 1 о о -- 0.5 Figure 2. Conductometric ( ) and acid-base titration (*) of 0.01M 2-mercapto-1,3,4-thiadiazol-5-yl thioacetic acid with 1M NaOH Synthesis of 2-(5-thioxo-4,5-dihydro-1,3,4-thiadiazol-2-ylthio)acetic acid 2-(5-Thioxo-4,5-dihydro-1,3,4-thiadiazol-2-ylthio)acetic acid (I) was prepared according to Scheme 1. The alkylation of 2,5-dimercapto-thiadiazole (IV) was carried out with ethylchloroacetate in chloroform in the presence of N,N-dimethylaniline, followed by the hydrolysis of the raw ethyl 2-(5-thioxo-4,5-dihydro-1,3,4-thiadiazol-2-ylthio) acetate (V) with aqueous NaOH, which afforded the sodium salt (VI) of 2-(5-thioxo-4,5-dihydro-1,3,4-thiadiazol-2-ylthio)acetic acid (I). Compound VI was transformed in the free acid (I) with diluted HCl. HH n-N C|CH2COOC2H5^ NaOH / H20 4|N—N H^SSH N'N"DMA ' CHC^3 ^^S-CH2C00C2H5 ^^S-CH2C00Na IV V VI H4 H 4N-N HC| ^-N S-CH2C00Na S-CH2C00H VI I Scheme 1. Synthesis of 3#-2-thioxo-1,3,4-thiadiazole-5-il) thioacetic acid (I). Method: To a suspension of 0.04 moles of 2.5-dimercapto-thiadiazole and 0.044 moles of N,N-dimethylaniline in 25 mL CHCl3 was added, without cooling, a solution of 0.041 moles ethylchloroacetate in 10 mL CHCl3. After refluxing 90 min. and repeated washings at room temperature with water, diluted HCl, the solution was treated with anhydrous Na2SO4. After the solvent removal, the row product (V) was suspended at reflux in a solution of 0.044 mol NaOH in 30 mL water. The obtained solution was washed out by active charcoal, hot filtered and then cooled. After water recrystallization, the obtained product (VI) has a m.p.=268-270°C. Compound VI was characterized by FT-IR-Raman spectroscopy and by single-crystal X-ray diffraction which prove its molecular structure as hydrate, [Na^H^SsC^COOX^O^b^O. A water solution of sodium salt (VI) was treated with diluted HCl, and the precipitated acid (I) was recrystallized from water. The product was characterized by m.p.=160-162°C (lit1 164-166°C, FT-IR and Raman spectroscopy2, 1H-NMR and 13C-NMR spectra recorded on a Bruker Avance 300MHz Spectrometer. 1H-NMR: [DMSO-Jć, 5(ppm)]: 4.01 (s, 2H, CH2) 13C-NMR [DMSO-dć, S(ppm)]: 188.24 (C=S), 169.40(C-S), 157.88 (C=O), 35.43 (CH2) References 1. CarloErba SA, Dérivés thiéadiazolyliques d'acide 7-acylamido-3-cépheme-4-carboxylique et procédés de leur préparation, FR Patent Number 2,335,230, date of patent July 15, 1977. 2. M. M. Venter, S. Cinta Pinzaru, V. Bercean, I. Haiduc, Studia Univ. Babes-Bolyai, Physica 2004, 3(XLIX), 285-288.