J. Am. Chem. SOC.1993,115, 11092-1 1098
11092
Ultraviolet Resonance Raman Spectra of Ribosyl C( 1’)-Deuterated Purine Nucleosides: Evidence of Vibrational Coupling between Purine and Ribose Rings Akira Toyama, Yoshinobu Takino,t Hideo Takeuchi,’ and Issei Haradd Contribution from the Pharmaceutical Institute, Tohoku University, Aobayama, Sendai 980, Japan Received July 19, 1993”
Abstract: Ultraviolet resonance Raman spectra of ribosyl C ( 1’)-deuterated guanosine and adenosine were measured. Most in-plane vibrations of the purine rings in the 1420-1 100-cm-1 region showed frequency upshifts upon C(1’)deuteration, while those in the 1100-700-~m-~region showed downshifts. The purine ring vibrations above 1450 cm-I were unaffected. The frequency shifts associated with the C ( 1’)-deuteration are explained by assuming couplings of the purine vibrations with a ribose C(1’)-H bending mode, which involves a hydrogen motion in the plane of N(9)C ( 1’)-H. The purine vibrations that showed upshifts in the 1420-1 100-cm-1 region are originally lowered in frequency by coupling with the ribosyl bending mode a(CH) around 1420 cm-1 and restore their intrinsic frequencies upon shifting of a(CH) to a(CD) around 1100 cm-1 in the C(1’)-D isotopomers. Some of the upshifted vibrations may be further pushed up by coupling with a(CD). On the other hand, the downshifted purine modes in the 1100-700-cm-~ region do not interact with a(CH) because of large frequency separations from a(CH) and retain their intrinsic frequencies in the C ( l’)-H species. In the C ( l’)-D species, however, a(CD) couples with these modes and pushes their frequencies downward. The observed C ( l’)-D frequency shifts provide direct evidence of vibrational coupling between the base and ribose rings, suggesting that the purine base vibrations may be affected by the ribose ring puckering and glycosidic bond orientation. Actually, most of the purine vibrations that showed significant C(1’)-D shifts are known or proved to be conformational markers of purine nucleosides and nucleotides. The conformational sensitivity of purine vibrations really arises from vibrational coupling between the base and ribose rings.
the conformation marker bands are in-plane vibrations of the base ring and their sensitivity to the ribose ring puckering and Vibrational spectroscopy is one of the powerful tools for glycosidic bond orientation has been ascribed to vibrational investigating the structures of nucleic acids and nucleic acidcoupling between base and ribose vibrations.’-3 However, no protein Hydrogen bonding,a base and direct experimental evidence has been reported for the basemetal coordinationlslz in nucleic acids can be revealed by studying ribose vibrational coupling. Since nucleic acid bases contain the Raman and infrared spectra. Further, the frequencies of several proton-donor and -acceptor sites, base vibrational freseveral Raman bands have been correlated with local conforquencies may also be affected by changes in hydrogen bonding mations of nucleic acids, namely, the ribose ring puckering and associated with changes in conformation and intermolecular glycosidic bond orientation. Such conformation marker bands interaction.’ were revealed by comparing the vibrational spectra of nucleoside In order to confirm whether conformational sensitivity really and nucleotide crystalsl3J4 or double-stranded polynucleotides~~+~9 arises from vibrational coupling between the base and ribose rings, with known crystallographic structures. Interestingly, most of we deuterated purine nucleosides at the ribosyl C ( 1’) position, which is directly connected to the base ring. If a significant * Author to whom correspondence should be addressed. vibrational coupling exists between the base and ribose rings, t Present address: Central Research Center, Ajinomoto Co. Inc., Suzukicho 1-1, Kawasaki-ku, Kawasaki 210, Japan. base vibrations would change in frequency upon C ( 1‘)-deuteration 1 Deceased August 10, 1992. as well as ribose vibrations. We employed ultraviolet resonance *Abstract published in Aduance ACS Abstracts, October 15, 1993. Raman (UVRR) spectroscopy to selectively enhance Raman (1) Nishimura, Y.; Tsuboi, M. In Spectroscopy of Biological Systems; Clark, R. T., Hester, R. E., Eds.; John Wiley & Sons: New York, 1986; pp scattering from the base in-plane ~ibrations*.20-2~ and to clearly 177-232. distinguish them from unenhanced ribose vibrations. Many (2) Thomas, G. J., Jr. In Spectroscopy of Biological Systems; Clark, R.
Introduction
T., Hester, R. E., Eds.; John Wiley & Sons: New York, 1986; pp 233-309. (3) Peticolas, W. L.; Kubasek, W. L.; Thomas, G. A.; Tsuboi, M. In Biological Applicatiom of RamanSpectroscopy; Spiro, T.G.,Ed.; John Wiley & Sons: New York, 1987; pp 81-133. (4) Turpin, P.-Y.; Chinsky, L.; Laigle, A.; Jollls, B. J . Mol. Strucr. 1989, 214,43-70. ( 5 ) Taillandier, E.; Liquier, J.; Ghomi, M. J . Mol. Srruct. 1989,214, 185211. (6) Kyogoku, Y.; Lord, R. C.; Rich, A. J. Am. Chem. SOC.1967,89,496504. (7) Toyama, A.; Takeuchi, H.; Harada, I. J . Mol. Srrucr. 1991,242,8798. ( 8 ) Audet, P.; Simard, C.; Savoie, R. Biopolymers 1991, 31, 243-251. (9) Small, E. W.; Peticolas, W. L. Biopolymers 1971, 10, 69-88. (10) Lanir, A.; Yu, N.-T. J . Biol. Chem. 1979, 254, 5882-5887. (1 1) Uchida, K.; Toyama, A.; Tamura, Y.; Sugimura, M.; Mitsumori, F.; Furukawa, Y.; Takeuchi, H.; Harada, I. Inorg. Chem. 1989,28,2067-2073. (12) Perno, J. R.;Cwikel, D.;Spiro, T. G. Inorg. Chem. 1987,26,400-405. (13) Nishimura, Y.; Tsuboi, M.; Sato, T.; Aoki, K. J . Mol. Srrucr. 1986, 146, 123-153.
(14) Katahira, M.; Nishimura, Y.; Tsuboi, M.; Sato, T.; Mitsui. Y.; Iitaka, Y . Biochim. Biophys. Acta 1986,867, 256-267. (15) Pohl, P. M.; Ranade, A.; Stockburger, M. Biochim. Biophys. Acta 1974, 335, 85-92. (16) Thamann, T. J.; Lord, R. C.; Wang, A. H. J.; Rich, A. Nucleic Acids Res. 1981, 9, 5443-5457. (17) Benevides, J. M.; Thomas, G. J., Jr. Nucleic Acids Res. 1983, 21, 5747-5761. (18) Thomas, G. J., Jr.; Benevides, J. M. Biopolymers 1985, 24, 11011 in< ..”3 cm-1) C( 1’)-D shifts are not observed above 1420 cm-I, probably due to the same reason that the vibrations in this frequency region do not involve atomic motions around the N(9)-C( 1’) linkage. In the 1420-1 100-cm-1 region, C( 1’)-D shifts are significant and most of them are upshifts. The 1375-cm-1 shoulder of Adodo-H shows a 4-cm-1 upshift to 1379 cm-I in Ado-do-D. In the 1350-1280-~m-~region, the effect of C( 1’)-deuteration on the 266-nm Raman bands is very small (Figures 4 and 5). This is consistent with the observation that two bands of adenine corresponding to the 1337- and 1310-cm-1 bands of adenosine (Figure 4a) show very large 1,3-l5Nshifts (Table II)36 suggestive of dominant contributions of pyrimidine modes. With 21 3-nm excitation, however, the spectral pattern in the 135&1280-~m-~ region changes drastically upon C( 1’)-deuteration (Figure 6). The 1309-cm-1 band of Ado-do-H (Figure 6a) upshifts to 1326 cm-l in Ado-do-D (Figure 6b). The upshifted band is not the counterpart of the unshifted 1310-cm-1 band in the 266-nm spectrum, indicating the existence of a new vibration that is unseen with 266-nm excitation. Similarly, a new band is found at 1292 cm-l in the 213-nm spectrum of Ado-dl-H (Figure 6c), which upshifts to 1316 cm-1 in Ado-&-D. The 1307-cm-1 band of AdodS-H and the 1291-cm-’ band of Ado-ds-H are also new bands, which upshift to 1338 and 1310 cm-I, respectively, upon C(1’)(36) Hirakawa, A. Y.; Okada, H.; Sasagawa, S.;Tsuboi, M. Spectrochim. Acta, Part A 1985, 41A, 209-216.
11096 J. Am. Chem. SOC.,Vol. 115, No. 24, 1993
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Toyama et al.
I-
e 8
I
I
L
I 1
I
I
I
1
I
1400
WAVENUMBER I cm-l Figure 6. UVRR (213 nm) spcctra of labeled adenosines in the 14001100-cm-1 region: (a) Ado-do-H, (b) Ado-do-D, (c) Ado-dl-H, (d) Adodl-D, (e) Ado-ds-H, (0 Ado-ds-D, (g) Ado-&-H, and (h) Ado-&-D. Table 11. Frequencies (cm-l) of Adenosine In-Plane Vibrations and Their Isotopic Shifts frequency . . Addo
1603 1583 1508 1485 1428 1375 1337 1310 130V 1254 1213 1174 1009 730 621
isotope shift
A-RNA" B-DNA* C(1')-D C(8)-D 8-'3CC N,GW 1,3-"NC 1603 1579 1503 1481 1422 1374 1336 1308 % 031' 1257f 121V 1174 1004 724
1605 1579 1504 1481 1422 1374 1337 1307 1342' 1254, 1224 116V lOlY 72W
-1
-2 -1 -1 -3 +4 +2 +1 +17f -5 +11 +31 -15
+2 -1
-1Y -16 -15 -7f -6 -2 -171 -14 -18 -3 +2
-6
-4
-1
-2
-318 -2,-3.6' -3.2 -7.8 -9.6 -1.5 -3.3 -2.4
4
+6 0 +I -Y +5 -4
-v
-7.7,-6.W -5.4 -6.7 -1.6 -3.6 -16.6 -11.3
4.5
-5.7
-15.0
-2.6
-3.2 -0.9
-7.4 -7.8
A-form poly(rA-rU).poly(rA-rU).* B-form poly(dA-dT)-poly(dAdT). Observed for crystalline adenine. From ref 36. (M,iP,@',@',@')deuteration. e Crystal field splitting. fobserved with 21 3-nm excitation. deuteration. These new bands are particularly enhanced with 21 3-nm excitation but are buried under stronger bands in the spectra excited at 266 nm. The 266-nm Raman spectrum of Ado-do-H shows three bands in the 1280-1 100-cm-1 region, all of which change in frequency uponC( 1')-deuteration. With 213-nm excitation (Figure6), only one of the three bands is selectively enhanced. The 1213-cm-1 band of Ado-do-H shifts to 1225 cm-I, and the 1241-cm-1 band of Ado-dl-H shifts to 1252 cm-1. For the remaining two bands, it may be reasonable to assume a correspondence in the order of frequency. Thus, the 1255- and 1174-cm-1 bands of Ado-doHshift to 1250and 1205 cm-l, respectively, upon C ( 1')-deuteration (Figure 6a,b). Similarly, the 1194- and 1171-cm-I bands shift to 1216 and 1196 cm-1 (Figure 6c,d). Among the three bands in each spectrum of Ado-do-H and Ado-dl-H, the one with the
1200 1000 800 WAVENUMBERI cm-' Figure 7. Schematic drawing of the C(1')-D frequency shifts observed for UVRR bands of guanosine and adenosine and their C(8)-D isotopomers: (a) Guo-do-H, (b) Guo-dl-H, (c) Ado-do-H,and (d) Adodl-H. Bars extending upward and downward signify upshifts and downshifts, respectively.
largest C(1')-D shift (at 1174 and 1171 cm-I, respectively) is assigned to a coupled mode of the N(9)-C(1') stretch and a C(1')-H motion by analogy with the 1208-cm-l band ofguanosine. In D2O solutions, 1204-cm-1 band of Ado-dS-H and the 1212cm-I band of Ado-ds-H show large C(1')-D upshifts (Figure 6e-h). These bands may correspond to the 1174- and 1 171-cm-1 bands in H20 solution. A band around the 1185-cm-1 region has been assigned to the ND2 scissors,22 and it does not show a significant C ( 1')-D shift. Below 1100 cm-l (Figure 4), an Ado-do-H band at 1009 cm-1 (101 1 cm-l for Ado-dl-H) shows a large downshift (-15 cm-1) upon C(1')-deuteration. The 730-cm-I band of Ado-do-H, which is one of the strongest bands in the visible Raman spectra and has been assigned to the ring-breathing mode,35 shows a 6-cm-1 downshift, in contrast to no C ( 1')-D shift of the guanine ringbreathing mode at 680 cm-I. Discussion
Mechanism of C( 1')-D Shifts of Purine Vibrations. The effects of C ( 1')-deuteration on the guanosine and adenosine vibrational frequencies are depicted schematically in Figure 7. Most bands in the 1420-1 100-cm-1regionshift upwardand thosein the 1100700-cm-1 region shift downward for both nucleosides, though the shifts are smaller for adenosine. If the isotopic substitution simply increases the effective mass of vibration, the vibrational frequency would decrease. The observation of frequency upshifts in the 1420-1 100-cm-l region cannot, therefore, be explained by the simple mass effect and requires more complex mechanisms of vibrational coupling. Significant coupling of two vibrations occurs if the directions of vibrational displacement and the locations of vibrating atomic groups as well as the frequencies of vibrations are close to each other. The frequencies of the two vibrations are pushed apart by the coupling, the magnitude of the frequency
Spectra of Ribosyl C(1‘)-Deuterated Guo and Ado
Figure 8. Definitions of two C( 1’)-H bending modes: a(CH) = aAa b(A& + A&) and j3(CH) = (Aj31 - A,32)/21/2.For the tetrahedral geometry, the coefficients, u and b, in the definition of a(CH) are 2/5Il2 and 1 / W ,respectively.
change being dependent on both the strength of interaction and the separation of their intrinsic frequencies. In any case, the low-frequency component becomes lower and the high-frequency one becomes higher. As described above, several purine vibrations show large frequency upshifts upon C ( 1’)-deuteration, indicating a strong vibrational coupling between the purine base and ribose. Any purine vibration that showed a significant C ( 1’)-D frequency shift must be close to a ribose vibration in direction, location, and frequency. Further, such a ribose vibration must involve an appreciable C( 1’)-H motion. If these conditions are fulfilled, the C ( 1’)-deuteration decreases the frequency of the ribose vibration and concomitantly the purine vibrational frequency will upshift or downshift as a result of changes in vibrational coupling. The largest C ( 1’)-D shift of purinevibrations is 60 cm-I, implying that the ribosevibration that couples with purinevibrations should undergo a much larger C ( 1’)-D shift. Most likely candidates for such a ribose vibration are the. two C ( 1’)-H bending vibrations, a(CH) and @(CH),defined in Figure 8. In a(CH), the hydrogen atom moves in the plane of N(9)C(1’)-H and the atomic displacements of a(CH) and the N(9)C(1’) stretch are in the same plane, while the hydrogen motion in @(CH) is perpendicular to the plane and to the atomic displacements of the N(9)-C(1’) stretch. Thus the N(9)-C( 1’) stretchcancouple witha(CH) but not with@(CH). Thestrength of vibrational coupling between the C ( 1’)-H bends and purine ring modes depends on the glycosidic bond orientation defined by the C(4)-N(9)-C( 1’)-0(4’) torsional angle (x). Usually the xangleisaround 55O (syn) or-125’ (anti).37 In eitherorientation, the C(1’)-H hydrogen atom lies close to the purine ring plane and a(CH) can mix with purine ring in-plane vibrations. In order for @(CH)to mix with purine in-planevibrations, the N(9)C ( 1’)-H plane should be nearly perpendicular to the purine ring plane with a x angle around -35O or 145’. This perpendicular orientation is, however, rarely found for nucleosides and their derivatives.3’ Hence, only a(CH) is geometrically able to couple with purine ring in-plane vibrations as well as with the N(9)C ( 1’) stretch. The a(CH) mode is a bending vibration of an isolated C-H bond, and its frequency is expected to be around 1400 cm-I. For instance, the corresponding C-H bending mode in cyclopentanol, a five-membered-ring compound like ribose, has a frequency of 1382 cm-I and downshifts to 1027 cm-I upon deuteration of the C-H group [vibrations corresponding to @(CH)and @(CD)were found at 1074 and 8 12 cm-I, respectively] .38 We have attempted to find the a(CH) and a(CD) bands of adenosine and guanosine in the UVRR, visible Raman, and infrared spectra. However, (37) Saenger, W. Principles of. Nucleic Acid Structure; Springer-Verlag: . . New York, 1983. (38) Durig, J. R.; Karriker, J. M.; Harris, W. C. Spectrochim. Acta, Part A 1971, 27A, 1955-1971.
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these bending bands were too weak to be identified with confidence. Although the exact frequencies of a(CH) and a(CD) are unknown, they may not be much different from those in cyclopentanol. It is interesting to note that the highest frequency of the upshifted bands is close to the expected frequency of a(CH) and the boundary of upshift and downshift regions is close to the frequency expected for a(CD). This coincidence may not be accidental but suggests that the observed C ( 1’)-D shifts are caused byvibrationalcoupling witha(CH) anda(CD), which arelocated around 1420 and 1100 cm-1, respectively. By assuming such vibrational coupling, theobserved C ( 1’)-D shifts can be explained as follows. TheN(9)-C( 1’) stretching mode of Guo-do-H showed the largest (+60 cm-I) C(1’)-D shift from 1208 to 1268 cm-1. Geometrically, the N(9)-C( 1’) stretch can couple with a(CH) as described above. The only remaining factor that will affect the coupling scheme is their intrinsic frequencies. Since the N(9)C(1’) stretch is lower in frequency than a(CH), the coupling between them pushes the N(9)-C( 1’) stretching frequency downward. On the other hand, coupling with a(CD) in the C ( 1’)-D species pushes the N(9)-C( 1’) frequency upward because the order of frequency is reversed in the C ( 1’)-D species. The frequency lowering in the C ( 1’)-H species and the raising in the C(1’)-D species result in an upshift of 60 cm-I upon C(1’)deuteration. In this case, both the couplings with a(CH) and a(CD) may contribute comparably to the C ( 1’)-D shift. On the other hand, the contribution from the coupling with a(CH) may be dominant for higher-frequency vibrations such as the 1414and 1321-cm-I modes of Guo-do-H because they are close to a(CH) in frequency but far away from a(CD). These vibrations are pushed down by coupling with a(CH) in the C ( 1’)-H species but restore their intrinsic frequencies in the C ( 1’)-D species. Conversely, coupling with a(CH) is negligible for the modes below 1100 cm-1 and their C(1’)-D downshifts are ascribed solely to the frequency lowering by coupling with a(CD). The modes above 1420 cm-1 lack such coupling because they are vibrations far away from the N(9)-C( 1’) moiety as described in the Results. Similar explanations apply to the C(1’)-D shifts in the other isotopomers of guanosine as well as in adenosine. Relation between C( 1’)-DShift and Conformational Sensitivity. The purine vibrations that shift upon C ( 1’)-deuteration are coupled with a(CH) and/or a(CD) as described above? It is probable that such purine vibrations are also coupled with other ribose vibrations. Changes in ribose ring puckering and glycosidic bond orientation will affect the modes and frequencies of ribose vibrations including a(CH), resulting in changes in vibrational coupling with the purine vibrations. Thus, the purine vibrations sensitive to C ( 1’)-deuteration are expected to serve as conformation markers of purine nucleosides and nucleotides. 1. Guanosine. Seven vibrations of guanosine showed C ( 1’)-D shifts larger than 3 cm-1 (Table I). Among them, three bands at 1414, 1081, and 869 cm-1 are known to be conformationsensitive. The guanineribose conformation in double-stranded poly(dG-dC) changes from C2’-endo/anti to C3’-endo/syn on going from the right-handed B form to the left-handed Z form. Concomitantly, UVRR bands of the guanine residue at 1419, 1086, and 862 cm-I shift to 1406, 1074, and 842 cm-1, respectivelyz3(see Table I). In addition, a single band at 1321 cm-I of guanosine, which showed a +lO-cm-l C(1’)-D shift, becomes a doublet at 1335 and 1319 cm-I in B-form poly(dGdC) and reverts to a singlet a t 1318cm-I in the Z form. Therefore, this vibration is also sensitive to the conformation. The 1208cm-1 band showed the largest (+60 cm-I) C(1’)-D shift. The frequency of this band varies with conformation in crystalline guanine nucleosides and nucleotides.’3 Guanosine-2‘-monophosphate bound to ribonuclease T139or FIMtakes the C3’-endo/syn conformation and gives a characteristic band at 1218 cm-1. These (39) Takeuchi, H.; Satoh, Y.; Harada, I. J . Mol. Strucr. 1991,242,49-59. (40) Takeuchi, H.; Harada, I.; Yoshida, H. Biochim. Biophys. Acta 1991, 1078. 307-3 12.
Toyama et al.
11098 J. Am. Chem. SOC.,Vol. 115, NO.24, 1993
endo/anti in theB f ~ r m . I - ~ ,With ~ ’ 266-nm excitation (Figure 9a,c), the A-RNA gives two Raman bands at 1336 and 1308 cm-1, which are practically identical to those of the B-DNA. This is because the expected conformation-sensitive mode at 1309cm-I is not resonance enhanced by 266-nm excitation and only the two modes insensitive to the conformation appear in the spectra. On the other hand, the 2 13-nmspectra of A-RNA and B-DNA largely differ from each other (Figure 9b,d). A band at 1308 cm-I, which corresponds to the 1309-cm-’ band of adenosine, is dominant in the 213-nm spectrum of A-RNA. The intensity maximum shifts to 1343 cm-’ for the B-DNA, where no peak was observed with 266-nm excitation. This large spectral change is accounted for by a shift of the conformation-sensitive vibration from 1308 (C3’-endo/anti) to 1343cm-I (C2’-endo/anti). In visible Raman spectra, a band around 1335-1340 cm-l has been proposed to be a conformationmarker of adenine residues in polyn~cleotides.lJ.~~ The band shifted from 1335 (C3’-endo/anti) to 1340 cm-1 (C2’-endolanti). This observation can be explained as follows. The actual conformation marker band at 1308 cm-I in the C3’endo/anti conformation is weak with visible excitation and is overlapped by a conformation-insensitiveband at 1310cm-I. Thus, a stronger band around 1335 cm-I, which is also conformationinsensitive, was regarded as a marker of that conformation. In the C2’-endo/anti conformation, on the other hand, the conformation-sensitive band at 1343cm-’ becomes stronger and overlaps the insensitive band around 1335 cm-I. Accordingly, the 1340cm-1 band looked as if it shifted from 1335 cm-I in the visible Raman spectrum. Three bands of adenosine at 1209,1177, and 1009 cm-l showed large C(1’)-D shifts, suggesting a possibility of their being conformation-sensitive. Actually, these bands are observed at 1219, 1176, and 1006 cm-I in A-form poly(rA-rU).poly(rA-rU) (Figure 9b) and shift to 1226, 1169, and 1013 cm-’ in B-form poly(dA-dT).poly(dA-dT) (Figure 9d). The adenine ringbreathing mode at 730 cm-1 also showed a C(1’)-D shift of -6 cm-I . This shift is attributable to a frequency lowering by coupling witha(CD) andnot tocouplingwitha(CH). Ifthering-breathing mode does not couple with any other C( 1’)-H ribose vibrations either, it will not be sensitive to the conformation. Actually, the ring-breathing mode of adenosine is not regarded as a conformation marker, indicating a lack of such coupling.
-
1250
1000
1250
WAVENUMBER I cm-’ Figure 9. UVRR spectra of double-stranded polynucleotides: (a) and (b) poly(rA-rU).poly(rA-rU),(c) and (d) poly(dA-dT).poly(dA-dT).The excitation wavelength was 266 nm (a and c) or 213 nm (b and d).
-
observations indicate that the 1208-cm-’ band also bears conformational sensitivity. In double-stranded poly- and oligonucleotides, however, stronger Raman bands due to the cytosine residue overlap the guanosine 1208-cm-l band and the guanosine conformation cannot be deduced from this vibration. The remaining two bands at 1026 and 825 cm-I are weak and are sometimes overlapped by other bands in nucleotides. Although the conformational sensitivities of these bands are not known, they may potentially be conformation markers as are the other guanosine vibrations discussed above. The guanine ring-breathing mode at 680 cm-l is well characterized as a marker of the ribose ring puckering and the glycosidic bond 0rientation.’-~*3~*35 The frequency change associated with the conformational change is very large as shown in Table I (682 cm-l for C2’-endo/anti and 624 cm-I for C3’endo/syn). However, this vibration did not show any C( 1’)-D shift. The large frequency separation from a(CH) and a(CD) may be responsible for the lack of C(1’)-D shift. It is highly Conclusions probable that the ring-breathing vibration is strongly coupled with a ring deformation vibration of ribose around 650 cm-I, Deuteration at the ribose C( 1’) position has provided experwhich makes the breathing mode conformation-~ensitive.~’ The imental evidence of vibrational coupling between the purine and 1361-cm-I band of guanosine is also regarded as a conformation ribose rings. Generally, purine in-plane vibrations in the 1420marker, though its frequency is insensitive to C( 1’)-deuteration. 1100-cm-I region couple with a C(1’)-H bend, (r(CH), around This mode does not involve a significantN(9) motion, as described 1420 cm-I, leading to frequency upshifts upon C( 1’)-deuteration. in the Results, and its direct coupling with ribose vibrations is On the other hand, a(CD) in the C( 1’)-D speciespushes purine unlikely to happen. Possibly, this mode couples with the 1321v i b r a t i o n a l frequencies d o w n w a r d in t h e 1100cm-’ one, which is a conformation marker involving a large N(9) 700-cm-’ region and, possibly, upward in the 1300-1200-cm-~ motion, and gains conformational sensitivity through coupling region through vibrationalcoupling. The observed C( 1’)-D shifts with the 1321-cm-l mode. are well explained by the coupling scheme described above. Such 2. Adenosine. As revealed by the 213-nm spectra, adenosine vibrational coupling is absent for the purin!: vibrations above has three vibrations in the 1340-1300-~m-~region. Among the 1420 cm-l for the geometrical reason that their vibrational modes three, only the 1309-cm-l mode showed a large C( 1’)-D shift do not involve atomic motions in the N(9)-C(1’) moiety. Most (+17 cm-I) and theremaining twomodesat 1337 and 1310cm-I of the purine UVRR bands that showed significant C( 1’)-D shifts were unaffected. This observation suggests that the 1309-cm-I are conformation markers that have been established already or mode is conformation-sensitive while the others are not. Figure proved to be so in this work. It is concluded that the confor9 compares UVRR spectra of A-form p~ly(rA-rU).poly(rA-rU)~~ mational sensitivity of purine in-plane vibrations really arises and B-form poly(dA-dT)-poly(dA-dT).I4 The adenine residue from vibrational coupling with ribose. takes the CY-endo/anti conformation in the A form and C2’Acknowledgment. This work was supported by research grants (41) Benevides, J. M.; Lemeur, D.; Thomas, G. J., Jr. Biopolymers 1984, (Nos. 62430004 and 0277 1680) from the Ministry of Education, 23, 101 1-1024. Science, and Culture of Japan and by a grant from the Japan (42) Morikawa, K.;Tsuboi, M.; Takahashi, S.; Kyogoku,Y.; Mitsui, Y.; Society for the Promotion of Science. Iitaka, Y.;Thomas, G. J., Jr. Biopolymers 1973, 23, 1011-1024.
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