The Fourier transform infrared spectra of 2`-deoxyribonucleoside — thymidine (dT) in low-temperature Ar matrices are obtained in the range 4000–1300 cm–¹. It is determined that anti-conformers of thymidine are dominant. The ribose rings of the main anti-conformers dT_a0, dT_a1 are in the C2`-endo conformation, but the ribose rings of minor anti-conformers dT_a2, dT_a3 have the C3`-endo conformation, stabilized by intramolecular hydrogen bonds O3`H…O5` and O5`H…O3`, respectively. The main syn-conformer dT_s2 is stabilized by the intramolecular hydrogen bond O5`H…O2 and has C2`-endo conformation of the ribose ring

Ivanov, A.Yu.

Krasnokutski, S.A.

Sheina, G.G.

Наукова електронна бібліотека періодичних видань НАН України (Vernadsky National Library of Ukraine)

Fizika Nizkikh Temperatur, 2003, v. 29, Nos. 9/10, p. 1065–1070Molecular structures of thymidine isomers isolated inlow-temperature inert matricesA.Yu. Ivanov1, S.A. Krasnokutski2, and G.G. Sheina11 B.Verkin Institute for Low Temperature Physics and Engineering of the National Academy ofSciences of Ukraine, 47 Lenin Ave., Kharkov 61103, UkraineE-mail: ivanov@ilt.kharkov.ua2 Max-Planck-Institut für Strömungsforschung, Bunsenstraße 10, Göttingen 37073, GermanyThe Fourier transform infrared spectra of 2’-deoxyribonucleoside — thymidine (dT) inlow-temperature Ar matrices are obtained in the range 4000–1300 ñm–1. It is determined thatanti-conformers of thymidine are dominant. The ribose rings of the main anti-conformers dT_a0,dT_a1 are in the Ñ2’-endo conformation, but the ribose rings of minor anti-conformers dT_a2,dT_a3 have the Ñ3’-endo conformation, stabilized by intramolecular hydrogen bonds O3’H…O5’and O5’H…O3’, respectively. The main syn-conformer dT_s2 is stabilized by the intramolecularhydrogen bond O5’H…O2 and has Ñ2’-endo conformation of the ribose ring.PACS: 33.15.–e, 67.80.CxThe structural components of DNA — nucleosidesand their derivatives — are important objects of inves-tigation for the modern science of life [1–11]. Themain experimental methods of investigation of nucleo-sides are NMR spectroscopy and crystallography [1].But the results of investigations by these methods de-pend strongly on the intermolecular interactions. Forexample, only one form of several possible conformerscan be stabilized in crystals [1]. Owing to competinginteractions with the solvent, NMR spectroscopy givesno way to obtain direct data about intramolecular hy-drogen bonding [4,5]. These limitations are absent inthe method of matrix isolation, where molecular iso-mers from the gas phase are trapped in low-tem-perature inert matrices [12]. We previously used theFourier transform infrared (FTIR) matrix isolationspectroscopy for the first time in the investigation ofpyrimidine nucleosides isolated in low-temperature in-ert matrices [10,11]. The evaporation of uridine anddT without thermodestruction was demonstrated, andthe intramolecular H-bond O5’H…O2 was detected[10,11]. In the present research, FTIR spectra of dTin Ar matrices were obtained by using an enhanced ex-perimental setup. The new spectral data suggest theexistence of more types of isomers with intramolecularH-bonds in the isolated pyrimidine nucleosides thanhad been considered before [7–11].Experimental and computational methodsThe basic features of the FTIR spectrometer havebeen described previously [11,13–15]. For this paperthe FTIR spectra of thymidine and the auxiliary sub-stance 1-methyl-thymine were obtained in the ranges4000–1300 cm–1 with a CaF2 beamsplitter at anapodized resolution of 0.4 cm–1. The matrix isolation© A.Yu. Ivanov, S.A. Krasnokutski, and G.G. Sheina, 20031234567He8Fig. 1. The general scheme of low-temperature setupbased on liquid He cryostat: rotating vacuum seal (1),cryogenic block with cold mirrors and QCM (2), rotatingnitrogen shield (3), flange with indium seal (4), Knudsencell (5), electric heater of Knudsen cell (6), Ar flowthrough Knudsen cell (7), outside Ar flow (8).setup was based on a liquid helium cryostat with a ni-trogen shield (Fig. 1). Two low-temperature differen-tial quartz crystal microbalances (QCM) and twometal mirrors were placed on the copper holder in thevacuum chamber (Fig. 1) and had a working tempera-ture in the range 5–40 K. The QCM was used for themeasurements of the absolute intensity of the molecu-lar beams and the matrix-to-sample ratio (M/S) [13].Owing to QCM, we have the capability of workingnot only with an Ar flux passing through the Knudsencell but with an outside flux of cold Ar gas also(Fig. 1).All nucleosides are very thermally labile molecules,and for their evaporation a special Knudsen cell withreduced molecular beam losses was constructed. As isshown in Fig. 2, the geometry of the Knudsen cell andits disposition to low-temperature mirror are very im-portant for effective operation. The data in Fig. 2were obtained by the statistical Monte Carlo method,which is very useful for the simulation of complicatedvacuum systems [16]. This evaporation cell is cha-racterized by a working vapor pressure of around10–5 Torr and Knudsen number of over 100. This evap-oration cell is more effective by a factor of more than500–1000 over one of the cells used in our previous ex-periments with simple compounds [13–15]. In com-parison with our previous work [11], for the presentexperiments the distance L (Fig. 2) was decreasedfrom 4.5R to 2.5R. The typical intensities of molecu-lar beams of thymidine were about 40–70 ng/(s·cm2)at evaporation temperatures 410–430 K without anythermodestruction. Thymidine (commercial substancefrom Sigma) was used without additional purifica-tion. The auxiliary substance 1-methyl-thymine wassynthesized at the Kharkov National University(Kharkov, Ukraine). All substances were annealed toremove impurities such as sorbed H2O, CO2, and N2 inthe initial phase of evaporation. The inert gas Ar wasmore than 99.99% pure and deposited on the mirrors at11 K. To improve the optical characteristics of the Armatrices, before the deposition of matrix samples athin layer of pure Ar was deposited on the mirrors overa temperature range of 35–20 K [15]. The quan-tum-chemical ab initio calculations of the relative en-ergies and vibrational spectra of thymidine conformerswere performed by the program PC GAMESS version6.0 [17] of the GAMESS (US) QC package [18].Results and discussionThe peculiarities of vibrational spectra of dT con-formers. As is evident from Fig. 3, in the region of thestretching vibrations (OÍ), (NÍ), the frequency ofthe (N3H) stretching vibration of dT coincides wellwith (N3H) of 1-methyl-thymine. Because of this,four absorption bands in this region (Fig. 3) can be-long to two OH groups of 2’-deoxyribose — O3’H,O5’H only (Fig. 3). The magnification of the numberof bands can be explained by intramolecular hydrogenbonds in the conformational structures, which wereconsidered in this paper (Fig. 4). It is known that theconformational-flexible ribose rings of nucleosides hasa puckered structure and C2’-endo, C3’-endo are the1066 Fizika Nizkikh Temperatur, 2003, v. 29, Nos. 9/10A.Yu. Ivanov, S.A. Krasnokutski, and G.G. Sheina0 4 800.40.8L(R)IH2RNozzleH = 2RFig. 2. The relationship of the efficiency of evaporationcell (I) as function of the distances L between cell andcold mirror. L is in units R, R is the radius of outlet noz-zle of Knudsen cell.3600 3500 34000,40,8D ,  ñm–121O5'HO5'Hsyn-conf.O3'HN3HFig. 3. The FTIR spectra of thymidine (1) and1-methylthymine (2) isolated in Ar matrices (T = 12 K,M/S = 700) in the O–H, N–H stretching region(3690–3390 cm–1).main equilibrium conformations of the ribose ring insolutions [1]. As is indicated in Fig. 4, the transitionC3’-endo  C2’-endo has no effect on the structure ofthe H-bonds in the syn-conformers dT_s1, dT_s2(Fig. 4). In the anti-conformers dT_a2, dT_a3 thetransition C2’-endo  C3’-endo leads to the formationof the H-bonds O3’H…O5’, O5’H…O3’ (Fig. 4). Thechoice of the structure dT_a1 with the orientation ofhydroxymethyl group gauche+ and torsion angle (< C3’–C4’–C5’O5’) = + 50o are close to the struc-tures with hydrogen bonds C6H…O5’ in the anti-con-formers of uridine and cytosine [7,8]. The structuredT_a0 with the orientation of hydroxymethyl groupgauche— (<   – 65o) and the lack of any classical lin-ear H-bonds (Fig. 4) were considered also. The stabi-lization of the position of the pyrimidine ring in thedT_a0, dT_a2, dT_a3 conformation can be effected bythe electrostatic interaction between atoms: C6H O4’ and C2O  H1’. The orientation of the methylgroup with respect to the carbonyl group C2O areclose to the structure A from [19], where two hydro-gen atoms positioned above and below N1–C2–Oplane (Fig. 4). All conformers from Fig. 4 may be con-sidered as local minima, since they were stable at theHF/3–21G(p), HF/6–31G(d,p), and MP2/6–31G(d,p)levels of calculation and have no imaginary frequen-cies in the calculated spectra.The experimental and calculated spectra are com-pared in the region of OÍ), NH), CH) (Ta-ble 1). From this table we notice that the calculatedfrequencies of the free O5’H, O3’H, and N3H groupsare in good agreement with the experimental frequen-cies for all conformers. The band 3482 cm–1 (Fig. 3,Table 1) can be assigned to the H-bonded vibrationO5’H…O2 in the syn-conformations. According to ourcalculations, the vibration (hb_O5’H) in conformerdT_s2 has a frequency about 40 cm–1 lower than in thedT_s1 conformer (Table 1). We can see in our experi-mental spectra that the band at 3482 cm–1 does notMolecular structures of thymidine isomers isolated in low-temperature inert matricesFizika Nizkikh Temperatur, 2003, v. 29, Nos. 9/10 1067OHHHOHNHRO4NHO2HHHOHHHOHHHOHNHR ON HOHHHOHHOH HHOHNHRONHOHHHOHOHHHOHNHRONHOHHHOHHHOH HHOHNHR ON HOHHHOHOHHHOHNHRONHOHHHOHHdT_a1dT_s1dT_a2dT_s2dT_a3dT_a05' 1'3'432165Fig. 4. The conformational structures of thymidinewhich are stable at the different level of calcu-lation (HF/3–21G(p), HF/6–31G(d,p) andMP2/6–31G(d,p)). Intramolecular H-bonds are repre-sented with dashed lines. The symbol R – represents aCH3 group.Table 1The parameters of experimental FTIR spectra in Ar matrices and calculated by the method 6–31G(d,p) spectral bands ofthymidine conformers in the 3700–3000 cm–1 regionConformermodeExperiment dT_a0 dT_a1 dT_a2 dT_a3 dT_s1 dT_s2, ñm–1 I , ñm–1 Ia , ñm–1 Ia , ñm–1 Ia , ñm–1 Ia , ñm–1 Ia , ñm–1 I aO5’H 3665 3.1 3673 76 3367 72 3673 84O3’H 3641 1.8 3654 64 3659 54 3659 65 3681 76 3660 54 hb_O3’H 3620* 0.8* 3638 107 hb_O5’H 3597* 0.5* 3639 86 hb_O5’H 3482 6.2 3626 161 3589 351N3H 3428 9.2 3439 106 3441 103 3438 106 3438 106 3435 103 3436 106C6H 3075 0.5 3059 4 3043 14 3071 7 3070 8 3022 6 3026 5C o m m e n t: I — relative integral intensities. Ia — absolute integral intensities (km/mol). hb_OH — bands ofgroups involved in the intramolecular H-bonds. * — after deconvolution of the wide band 3598 cm–1 on the Gaussiancontours.have a high-frequency shoulder in Ar matrices (Fig. 3)and the occupancy of conformer dT_s1 can be ne-glected. The calculated frequencies and intensities ofthe characteristic bands of the conformers dT_a2 anddT_a3 are in close agreement (Table 1). This result issupported by the experiment in Ar matrices, where wecan see only the one wide band 3598 cm–1 with ahigh-frequency shoulder (Fig. 3). Contrary to the cal-culated parameters of C6H (Table 1), only one bandof C6H vibration was detected in the experimentalspectrum (Fig. 5). The influence of the intramolecularhydrogen bonds C6H…O5’ in conformer dT_a1 on theC6H parameters was not detected experimentally, asthe free C6H group in 1-methyl-thymine has Fermiresonance splitting in the spectra (Fig. 5). Conse-quently, it may be considered that the anti-conformersdT_a0 and dT_a1 are practically indistinguishable inthe investigated region.The populations of dT conformers in the low-tem-perature matrices. It is known that the conformers’occupancies in matrices may differ widely from thosein the gas phase. The effect of interconversion is ob-served at low barriers between conformational isomers[12]. We tested the interconversion by using the an-nealing of matrix samples. The annealing of matrixsamples at 30 K has no influence on theconformational equilibrium. It follows that the barrierheights of dT conformers > 2.5–3 kcal/mole and theirconformational equilibrium in the gas phase at theevaporation temperature must be close to equilibriumin Ar matrices at 12 K. For comparison with the realexperimental data the relative free Gibbs energy Gwas estimated by the standard method [20]:    G T E ZPE CdT T S TABT( ) ( ) 	 	 0.The relative electronic energy E was estimated atthe MP2/6–31G(d,p) level of ab initio calculation,and the relative zero-point vibrational energy ZPEand temperature-dependent contributions of rotationand vibration were estimated at the HF/6–31G(d,p)level for temperatures of 298 and 420 K (Table 2).The association of G and the experimental spec-tral data can be expressed by the standard equation:G T RT K RT /AB AB A B( ) ln ln ( )      ,where KA,B is the equilibrium constant of conformersA, B, and A, B are the populations of these confor-mers.To define the equilibrium constants by using theexperimental spectra it is necessary to know the molarextinction coefficients of the characteristic spectralbands or their ratios. The ratios of molar extinctioncoefficients of characteristic bands may be determinedthrough the redistribution of the intensities of thecharacteristic bands under the influence of changingevaporation temperature or through the UV irradia-tion of matrix samples. If the corresponding experi-ments are difficult, the ab initio calculations of the in-tensities of the characteristic spectral bands may beused. Ab initio calculations usually overestimate theabsolute infrared intensity of vibrational bands[21,22], but the ratio of the experimental and calcu-lated intensities I coincides much better. The equilib-rium constant of isomers a and b has been presented as[23,24]:KIIIIABaabb(exp)( )( )(exp)calccalc . (1)Unlike some studies [23,24], we have used Eq. (1)with the intensities of the OH), NH), and CH)stretching vibrations only. With our data the bestagreement between the calculated and experimentalresults is observed for the ratio of the intensities ofstretching vibrations. For determination of KAB dif-ferent combinations of the experimental and calcu-lated intensities from Table 1 were used. As discussedabove, the anti-conformers in the pairs: dT_a0 anddT_a1, dT_a2 and dT_a3 are practically indistin-guishable in the matrix spectra. Therefore, for thesake of simplicity, the conformer dT_a0 was ignored.According to the relative electronic energycalculations, the syn-conformer dT_s2 with the1068 Fizika Nizkikh Temperatur, 2003, v. 29, Nos. 9/10A.Yu. Ivanov, S.A. Krasnokutski, and G.G. Sheina3120 3080 304000.04D ,  ñm–121 C H6Fig. 5. The FTIR spectra of thymidine (1) and1-methylthymine (2) isolated in Ar matrices (T = 12 K,M/S = 700) in the C5–H, C6–H stretching region(3140–3040 cm–1).intramolecular hydrogen bond O5’H…O2 and confor-mation of the sugar ring — C2’-endo has the minimalenergy among all conformers at all levels of optimiza-tion (Table 2). The data in Table 2 demonstrate dis-agreement between the experimental and calculatedoccupancies of the dT_a1 and dT_s2 conformers, butthe experimental and calculated data coincide well forthe minor anti-conformers dT_a2 and dT_a3 (Ta-ble 2). The influence of the vibrational-rotational con-tribution to the free energy reduces the difference ofconformers’ energies significantly, and this is espe-cially noticeable with increasing evaporation tempera-ture (Table 2). Because of this, conformers dT_s2 mayhave an essential effect on biological processes at therelatively low physiological temperatures.ConclusionsIt was shown that FTIR matrix isolation spectros-copy is a helpful method for the investigation of themolecular structure of nucleoside conformational iso-mers. It was established that at evaporation tempera-tures of up to 430 K thymidine may be evaporated foran appreciable length of time and trapped in inert ma-trices without any thermodestruction. Anti-conform-ers of thymidine are dominant in the isolated state.The main anti-conformer dT_a1, has the Ñ2’-endo con-formation of the ribose ring. The minor anti-conform-ers dT_a2, dT_a3 have the Ñ3’-endo conformation ofthe ribose ring, stabilized by intramolecular hydrogenbonds O3’H…O5’ and O5’H…O3’ respectively.The intramolecular hydrogen bonds O3’H…O5’, andO5’H…O3’ may be regarded as an indicator of thetransition Ñ2’-endo  Ñ3’-endo between conforma-tions of ribose ring in the anti-conformers ofthymidine. The thymidine dT_s2 syn-conformer is sta-bilized by the intramolecular hydrogen bondO5’H…O2 and the dominant conformation of ribosering is Ñ2’-endo.AcknowledgementThis investigation was supported by theUkrainian Academy of Sciences and in part bythe INTAS-International Association under grantNo. INTAS 00–00911.1. W. Saenger, Principles of Nucleic Acid Structure,Springer-Verlag, New York (1984).2. G.A. Jeffrey and W. Saenger, Hydrogen Bonding inBiological Structures, Springer-Verlag, Berlin (1991).3. S.N. Rao, Nucleosides and Nucleotides 14, 1179(1995).4. F. Seela, H. Debelak, H. Reuter, G. Kastner, and I.A.Mikhailopulo, Tetrahedron 55, 1295 (1999).5. J.J. Barchi Jr., L.-S. Jeong, M.A. Siddiqui, V.E.Marquez, and J. Biochem, Biophys. Methods 34, 11(1997).6. J.M. Gavira, M. Campos, G. Diaz, A. Hernanz, andR. Navarro, Vibrational Spectroscopy 15, 1 (1997).7. N. Leulliot, M. Ghomi, G. Scalmani, and G. Berthier,J. Phys. Chem. A103, 8716 (1999).8. N. Leulliot, M. Ghomi, H. Jobic, O. Bouloussa, V.Baumruk, and C. Coulombeau, J. Phys. Chem. B103,10934 (1999).9. O.V. Shishkin, A. Pelmenschikov, D.M. Hovorun,and J. Leszczynski, J. Mol. Struct. 526, 329 (2000).10. S.A. Krasnokutski, A.Yu. Ivanov, V. Izvekov, G.G.Sheina, and Yu.P. Blagoi, XXIV European CongressMolecular structures of thymidine isomers isolated in low-temperature inert matricesFizika Nizkikh Temperatur, 2003, v. 29, Nos. 9/10 1069Table 2Calculated and experimental relative energies (kcal/mole) of the thymidine conformers in the isolated stateConformermethoddT_a0 dT_a1 dT_a2 dT_a3 dT_s1 dT_s2HF/3–21G(p) 5.4 2.4 2.6 4.6 3.3 0(–865.3452)*HF/6–31G(d,p) 2.9 1.6 1.8 3.4 1.5 0(–870.1091)*MP2/6–31G(d,p) 5.7 3.0 3.8 4.9 2.6 0(–872.6776)*G (298 K)**MP2/6–31G(d,p)3.3 1.8 2.1 3.4 1.6 0G (420 K)**MP2/6–31G(d,p)2.4 1.4 1.6 2.9 1.2 0G (420 K)Experiment— 0 1.8 2.2 — 1.1* — absolute energies in a.u. are indicate in brackets. ** — vibrational-rotational contribution was estimated atHF/6–31G(d,p) level.on Molecular Spectroscopy, Prague, Czech Repablic,470 (1998).11. S.A. Krasnokutski, A.Yu. Ivanov, V. Izvekov, G.G.Sheina, and Yu.P. Blagoi, J. Mol. Struct. 482–483,249 (1998).12. A.J. Barnes, J. Mol. Struct. 113, 161 (1984).13. A.Yu. Ivanov, A.M. Plokhotnichenko, E.D. Radchen-ko, G.G. Sheina, and Yu.P. Blagoi, J. Mol. Struct.372, 91 (1995).14. A.Yu. Ivanov, A.M. Plokhotnichenko, V. Izvekov,G.G. Sheina, and Yu.P. Blagoi, J. Mol. Struct.408–409, 459 (1997).15. A.Yu. Ivanov, G. Sheina, and Yu.P. Blagoi, Spectro-chim. Acta A55, 219 (1999).16. G.L. Saksagansky, Molecular Beams in the CompositeVacuum Structures, Atomizdat, Moscow (1980) (inRussian ).17. Alex.A. Granovsky, http://classic.chem.msu.su/gran/gamess/index.html.18. M.W. Schmidt, K.K. Baldridge, J.A. Boatz, S.T. El-bert, M.S. Gordon, J.J. Jensen, S. Koseki, N. Matsu-naga, K.A. Nguyen, S. Su, T.L. Windus, M. Dupuis,and J.A. Montgomery, J. Comput. Chem. 14, 1347(1993).19. M.J. Nowak, L. Lapinski, D.C. Bienko, and D. Mi-chalska, Spectrochim. Acta A53, 855 (1997).20. J.S. Kwiatkowski and J. Leszczynsky, Chem. Phys.Lett. 204, 430 (1993).21. M.J. Nowak, L. Les, and L. Adamowicz, Trends Phys.Chem. 4, 137 (1994).22. Y. Yamaguchi, M. Frisch, J. Gaw, H.F. Schaefer III,and J.S. Binkley, J. Chem. Phys. 84, 2262 (1986).23. M.J. Nowak, L. Lapinski, and J. Fulara, Spectrochim.Acta 45A, 229 (1989).24. H. Vranken, J. Smets, G. Maes, L. Lapinski, M.J.Nowak, and L. Adamovicz, Spectrochim. Acta 50A,875 (1994).1070 Fizika Nizkikh Temperatur, 2003, v. 29, Nos. 9/10A.Yu. Ivanov, S.A. Krasnokutski, and G.G. Sheina

Molecular structures of thymidine isomers isolated in low-temperature inert matrices

http://dspace.nbuv.gov.ua/xmlui/bitstream/123456789/128932/1/27-Ivanov.pdf

Molecular structures of thymidine isomers isolated in low-temperature inert matrices

Abstract

Similar works

Full text

Available Versions

Наукова електронна бібліотека періодичних видань НАН України (Vernadsky National Library of Ukraine)