Scientific paper Mass Spectrometry of Bis-quinolizidine Alkaloids: Unexpected Dimerization in Electron Ionization Mass Spectrometry Beata Jasiewicz* and Tomasz Pospieszny Faculty of Chemistry, Adam Mickiewicz University, Umultowska 98b, 61-614 Poznan, Poland * Corresponding author: E-mail: beatakoz@amu.edu.pl; Tel.: +48—61-829-1354; fax: +48-61-829-1505 Received: 01-07-2013 Abstract The electron-ionization mass spectra of dimers of bis-quinolizidine alkaloids: a-isosparteine, 2-methylsparteine, 17-hydroxylupanine, lupanine N-oxide and 2,17-dimethylsparteine are discussed and general fragmentation routes of their molecular cations are proposed. Dimers structures are also studied using PM5 methods. The recognition of the fragmentation pathways and relative abundances of the ions obtained will provide important information, useful for the identification of similar dimeric compounds. Keywords: Alkaloids dimerization, bis-quinolizidine alkaloids, EI-MS spectra, semiempirical calculations 1. Introduction The quinolizidine alkaloids have interested chemists for several decades. Interesting examples of stereochemi- cal effects on EI mass spectra of this important group of alkaloids can be found in the literature.1-4 Sparteine, the main representative of bis-quinolizidine alkaloids, is a chiral diamine and has a widespread Figure 1. Structure of bis-quinolizidine alkaloids 1-5. use as a chiral ligand in asymmetric synthesis.5'6 It appears to offer protection to plants from Leguminosae family from insects and grazing mammals.7'8 Various pharmacological and toxicological properties have been attributed by lupine alkaloids such as antiinflammatory, an-tiarrhythmic' diuretic' hypotensive' antidiabetic' respiratory, depressant and stimulant.9 As a continuation of our study of the mass fragmentation of quinolizidine alkaloids,10-12 we focused our attention on unexpected dimerization of a-isosparteine [C15H26N2, M = 234] (1), 2-methylsparteine [C16H28N2, M = 248] (2), 17-hydroxylupanine [C15H24N2O2, M = 2264] (3), lupanine N-oxide [C15H24N2O, M = 24342] (4) and 2,17-dimethylsparteine [C17H30N2, M = 262] (5) (Fig. 1) in the electron-impact ionization chamber of the mass spectrometer at inlet probe temperature 200-280 oC. Simultaneously, we found that other alkaloids such as: 2-cyano-2-methylsparteine, lupanine (2-oxosparteine), 17-oxospar-teine, 17-oxolupanine and 2-thiosparteine under the same conditions did not form dimers. There are a few reports found in the literature describing formation of bis-quinolizidine alkaloid dimers.13-15 Dimeric sparteines described in the literature have been obtained synthetically and have bonds between the carbon atom C(17) of the first molecule and the carbon atoms (C5'), C(12'), C(14') or C(17') of the second molecule. They are characterized as anti-arrhythmic agents having the basic sparteine ring structure, but with considerably improved pharmacological properties.13 The application of these alkaloids in biology requires a detailed knowledge of their structure after drug metabolism. We hope that the recognition of the fragmentation pathways and relative abundances of the ions obtained will provide important information, useful for the identification and analysis of similar dimeric compounds. 2. Experimental Low- and high-resolution electron ionization (EI) mass spectra were recorded on AMD-Intectra (Harpstedt, Germany) D-27243 model 402 double-focusing mass spectrometer of BE geometry (ionizing voltage 70 eV, accelerating voltage 8 kV, resolving power 10 000). Samples were introduced by a direct insertion probe at a source temperature of ~200 and 280 °C. The elemental compositions of the ions were determined using the same instrument by peak matching method relative to perfluorokero-sene. All masses measured agreed within ±2 ppm of those of the composition listed in Table 1. The spectra from the first field-free region were recorded using linked scans at constant B/E, with helium as collision gas at an indicated pressure of 1.75 x 10-5 Pa with an ion source temperature of 200-280 °C, ionizing energy of 70 eV, and an accelerating voltage of 8 kV. The values of were calculated as averages of three measurements. The normal measured variation of ion intensities was 1-2% of the relative intensity. PM5 se-miempirical calculations were performed using the Win-Mopac 2003 program.16-18 3. Results and Discussion On the basis of the low-resolution EI mass spectra, exact mass determination (Table 1) and B/E linked scan spectra (Figures 2), the principal mass spectra fragmentation routes of dimers D1-D5 are interpreted as shown in Schemes 1-4. It can be concluded that the formation of C(17)-C(17') bonds for D1, D2 (elimination of H2 molecule), C(17) = C(17') bonds for D3, D4 (elimination of H2O molecule) and C(12)-C(12') for D5 (elimination of H2 molecule again) is preferred. It should be pointed out that all the fragmentation pathways have been confirmed by B/E linked scan spectra and that many of the cyclic ion structures shown in Schemes 1-4 are conjectural, similarly to those discussed previously in the literature.2-410-1219 The common feature of the mass spectral fragmentation of molecular ions of dimers D1-D5 is the cleavage of N(1)-C(10), C(6)-C(7), C(9)-C(10) (ring B); C(7)-C(17), C(9)-C(11) (ring C); C(15)-C(14), C(13)-C(12) (ring D) as well as of the C(17)-C(17') or C(12)-C(12') bonds of the skeleton of bis-quinolizidine alkaloid dimers. All suggested structures of fragment ions are consistent with the Bredt's rule, that states that a double bond cannot be placed at the bridgehead of a bridged ring system, unless the rings are large enough.20 In the mass fragmentation of dimers D1-D5 fragment ions formed from two molecules of bis-quinolizidi-ne alkaloids can be observed. The even-electron fragment ions (EE+) c, d, e, f as well as cyclic ion g are characteristic of D1, whereas in the mass fragmentation of D2, D3 and D4 the even-electron fragment ions b, c, d and e are present. It should be pointed out that for D2 the even-electron fragment ion c has been obtained from the even-electron fragment ion b by elimination of the C3H2 molecule and the even-electron fragment ion d has been obtained from the even-electron fragment ion c by elimination of the C4H7N molecule in the second and third step of the mass fragmentation of M+X respectively. The N(1)-C(2) bond cleavage of ring A of the molecular ion of D1 with elimination of the C3H6 molecule leads to the odd-electron ion (OE+^) b. In the case of molecular ions D1,D2 and D5 the charge is situated on an annular nitrogen atom N(1) or N(16) whereas in the case of molecular ions D3 and D4 the charge is probably situated on an annular nitrogen atom N(1). This fact suggests a better stabilization of the charge at N(1) of these even-electron or odd-electron fragment ions at N(16). It is probably connected with the presence of the carbonyl functional group at C(2) in the skeleton of 3 (or 4). Table 1. Elemental compositions and relative intensities of the ion peaks in the EI mass spectra of dimers D1-D5. Ion m/z Elemental Relative abundance [RA, %] composition D1 D2 D3/D4 D5 M+. a 522 C34H58N4 - - - 20 494 C32H54N4 - 10 - - 492 C30H44N4O2 - - 15/10 - 466 C30H50N4 30 - - - b 424 C27H44N4 2 - - - 410 C27H44N3 - - - 8 396 C26H42N3 - 5 - - 393 C25H37N4 - - 1/0 - c 382 C24H36N3O - - 25/4 - 368 C24H38N3 14 - - - 358 C23H40N3 - 3 - - 291 C19H35N2 - - - 3- d 340 C21H30N3O - - 7/4 - 330 C21H36N3 5 - - - 289 C19H33N2 - 4 - - 275 C18H31N2 - - - 8 e 314 C20H32N3 6 - - - 286 C17H24N3O - - 4/2 - 274 C17H28N3 - 5 - - 262 C17H30N2 - - - 75 f 286 C18H28N3 11 - - - 261 C17H29N2 - 18 - - 259 C16H23N2O - - 20/8 - 247 C16H27N2 - - - 46 g 271 C18H27N2 37 - - - 247 C16H27N2 - 65 - - 246 C15H22N2O - - 100/100 - 205 C13H21N2 - - - 28 h 245 C16H25N2 19 - - - 233 C15H25N2 - 22 - - 231 C15H23N2 - - 31/6 - 178 C11H18N2 - - - 24 i 233 C15H25N2 40 - - - 207 C13H23N2 - 18 - - 189 C11H13N2O - - 4/3 - 164 C11H18N - - - 30 J 219 C14H23N2 54 - - - 193 C12H21N2 - 8 - - 163 C10H15N2 - - 6/5 - 151 C10H17N - - - 97 k 193 C12H21N2 18 - - - 151 C10H17N - 49 - - 148 C9H10NO - - 48/50 - 136 C9H14N - - - 50 l 179 C11H19N2 9 - - - 136 C9H14N - 57 - - 134 C9H12N - - 63/16 - 124 C8H14N - - - 38 m 137 C9H15N 27 - - - 112 C7H14N - 69 14/8 100 n 98 C6H12N 100 100 7/8 33 o 84 C5H10N 25 30 10/3 30 Scheme 1. Proposed fragmentation pathway observed in the EI MS spectrum of D1. As can be seen from Schemes 2 and 4 and Table 1, the principal fragmentation pathways of D2 and D5 are similar, but show differences in the abundances of important fragment ions. In the mass spectra of 2 and 5 there are fragment ions which have been obtained by the elimination of methyl radicals from the A or/and C rings of the molecules investigated. The elimination of the methyl radical from the molecular ions of 2 and 5 can be explained by the cleavage of the C(2)-CH3 or C(17)-CH3 bond.12 Such an elimination of the methyl radical can be seen only in the mass fragmentation of the dimer D5. The even-electron fragment ions d and f of D5 have been obtained by the simple 5 cleavage of C(2)-CH3 and C(17)-CH3 bond, respectively. The common characteristic features of the EI MS fragmentation of D1-D5 are the presence of ions corresponding to the monomer part of the bis-quinolizidine alkaloids. The even-electron fragment ions i (for D1) and g (for D2) have been obtained by the cleavage of C(17)sp3-C(17')sp3 bonds. Ion i (C15H25N2, m/z 233) has only 40% of the relative intensity whereas ion g (C16H27N2 m/z 247) has 65% of the relative intensity. In both presented ions the positive charge is probably located on the nitrogen atom N(16). The odd-electron fragment ions g (for D3 or D4) and e (for D5) are obtained by the cleavage of C(17)sp2 = C(17')sp2 and C(12)sp3-C(12')sp3 bonds, respectively. The relative abundance of this ions is 100% of g (C15H22N2O m/z 246) and 75% of e (C17H30N2 m/z 262), respectively. For D2-D5 ions m formed by cleavage of ring B are located at m/z 112, whereas for compound D1 the odd-electron ion m is located at m/z 137. Although di-mers D3 and D4 have the same structure and show the same fragmentation patterns, they can be distinguished from each other based on different abundances of ions: a, c, f, h, l, o. The differences in the fragmentation of D3-D4 (molecular ions of dimers with the same elemental composition) have been expressed quantitatively by comparing the calculated values of the ^ coefficients, i.e. the abundances of selected fragment ions relative to the abundances of the odd-electron ions a. Because the values of ^ are highly dependent on the intensities of the ions, it is necessary to average three scans to obtain adequate statistical results. Table 2 presents the values of p2 and coefficients for dimers as: = % RA g / % RA a ; p2 = % RA m / % RA a ; = % RA o / % RA a The analytical ions for the calculation of the coefficients and were chosen similarly as previously for the differentiation of the stereoisomers and substituted sparteines.10-12 As can be seen from the data in Table 2, dimer D4 may be distinguished from D3 on the basis of its lower values of p2 and and higher value of p2, D3 > p2, D4 D4 > D3. Table 2. Values of fragment ions peak intensities relative to those of the corresponding molecular ions (u, see text for definitions) determined from the EI mass spectra of the dimers D3 and D4. [Intra-and inter-day repeatabilities were of the order of 1-3%.] Dimer fi f2 f3 D3 6.66 0.93 0.66 D4 10.0 0.80 0.30 Figure 2A. EI MS spectrum of the dimer D2. Scheme 3. Proposed fragmentation pathway observed in the EI MS spectra of D3 and D4. 15000000 20000000 25000000 30000000 35000000 40000000 45000000 Figure 2B. B/E mass spectrum of the [M+' a] ion at m/z 494 of D2. Scheme 4. Proposed fragmentation pathway observed in the EI MS spectrum of D5. Table 3. Heats of formation of alkaloids and its dimers D1-D5 calculated by PM5 semiempirical method. Compound Heat of formation [kcal/mol] 1 -26.3335 2 -31.4112 3 -107.4384 4 -69.3802 5 -36.3871 D1 -35.9377 D2 -45.3540 D3/D4 -86.9464 D5 -63.8692 lated structure of dimers D1-D5 are presented in Figure 3. Calculation shows that heat of formation (HOF) of a-isos-parteine, 2-methylsparteine, 2,17-dimethylsparteine and lupanine N-oxide is higher than that of corresponding dimers, whereas the HOF of 17-hydroxylupanine is lower than that of D3 (Table 3). The latter may be explained by the fact that the water loss is much more favorable in the monomer 3 than 4.21 The highest heat of formation observed for a-isosparteine and its dimer is probably related to the trans-trans all chair conformation of rigid molecule. 4. Conclusion Thus, PM5 semiempirical method is reliable method for visualization of the structures in the gas phase. Calcu- In the mass fragmentation of dimers D1-D5 fragment ions formed from two molecules of bis-quinolizidi- Figure 3. The optimized structures of the bis-quinolizidine dimers D1-D5. ne alkaloids as well as the presence of ions corresponding to the monomer part of the alkaloids can be observed. The results obtained have proved that it is possible to differentiate between dimers obtained. On the basis of the EI mass spectra, differentiation of the dimers D3 and D4 can be achieved using the values of coefficients 4. 1. Dedication This paper is dedicated to the memory of Prof. E. Wyrzykiewicz. 5. References 1. S. P. Sastry, in: G. R. Waller (Ed.): Biochemical Application of Mass Spectrometry, Wiley-Interscience, New York, 1972, p.655. 2. E. Wyrzykiewicz, W. Wysocka, Org. Mass Spectrom. 1990, 25, 453-456. 3. E. Wyrzykiewicz, W. Wysocka, M. Wiewiorowski, Org. Mass Spectrom. 1988, 25, 700-704. 4. U. Majchrzak-Kuczynska, M. Wiewiorowski, E. Wyrzykiewicz, Org. Mass Spectrom. 1984,19, 600-604. 5. J. L. Stymiest, G. Dutheuil, A. Mahmood, V. K. Aggarwal, Angew. Chem. Int. Ed. 2007, 46, 7491-7494. 6. I. Coldham, J. J. Patel, S. Raimbault, D. T. E. Whittaker, H. Adams, G. Y. Fang, V. K. Aggarwal, Org. Lett. 2008,10, 141-143. 7. M. Wink, in: E. A. Bernays (Ed.): Insect-plant interaction, CRC-Press, Boca Raton, 1992, p.136. 8. M. Wink, in: G. A. Cordell (Ed.): The Alkaloids: Chemistry and Biology, San Diego, 1993. 9. T. Schmeller, M. Sauerwein, F. Sporer, M. Wink, W. E. Müller, J. Nat. Products 1994, 57, 1316-1319. 10. E. Wyrzykiewicz, W. Boczon, B. Koziol, J. Mass Spectrom. 1998, 33, 971-975. 11. B. Jasiewicz, W. Boczon, E. Wyrzykiewicz, J. Mass Spectrom. 2004, 39, 541-547. 12. E. Wyrzykiewicz, W. Boczon, B. Koziol, J. Mass Spectrom. 2000, 35, 1271- 1277. 13. B. Hachmeister, W. Kehrbach, U. Kuehl, G. Buschmann, Bis-Sparteine Derivatives and Method of Using Same In Therapy, U.S. Pat. No. 4,415,577. 14. H. G. Bolt, Achievements in Alkaloid Chemistry Up to 1960, Akademie Verlag, Berlin, 1961, p. 197. 15. M. Hesse, H. O. Bernhard, in H. Budzikiewicz (Ed.): Alkaloide, Verlag Chemie GmbH, Weinheim, 1975, p. 264. 16. CAChe 5.04, UserGuide, Fujitsu. 2003 17. J. J. P. Stewart, J. Comput. Chem. 1998, 10, 209-220. 18. J. J. P. Stewart, J. Comput. Chem. 1998, 10, 221-264. 19. J. Tamas, in J. S. Splitter, F. Turecek (Eds.): Applications of Mass Spectrometry to Organic Stereochemistry, New York, 1994, pp. 625-626. 20. G. L. Buchanan, Chem. Soc. Rev. 1974, 3, 41-63. 21. B. Jasiewicz, E. Wyrzykiewicz, Int. J. Anal. Chem. 2011, Article ID 652589. Povzetek Razpravljamo o masnih spektrih z elektronsko ionizacijo za dimere bis-kvinolizidinskih alkaloidov: a-izospartein, 2-metilspartein, 17-hidroksilupanin, lupanin N-oksid in 2,17-dimetilspartein. Predlagamo tudi splošne fragmentacijske poti njihovih molekulskih kationov. S PM5 metodami preučujemo strukture dimerov. Prepoznavanje fragmentacijskih poti in relativnih intenzitet opaženih ionov daje pomembne informacije, koristne pri identifikaciji podobnih dimernih spojin.