JOURNAL OF RAMAN SPECTROSCOPY, VOL. 25, 623-630 (1994)

Assignment of Adenine Ring In-Plane Vibrations in Adenosine on the Basis of ''N and 13C Isotopic - Frequency Shifts and UV Resonance Raman Enhancement

Akira Toyama, Naoki Hanada, Yoshiharu Abe, Hideo Takeuchi* and Issei Haradat Pharmaceutical Institute, Tohoku University, Aobayama, Sendai 980, Japan

Infrared, Raman and UV resonance Raman spectra of adenosine and its 1,3-"N2, 2 - "C, and 8 - "C isotopic analogues were measured in neutral aqueous solution (Raman and UV Raman) and in the crystalline state (infrared and Raman). The observed isotopic wavenumber shifts are useful in distinguishing adenine ring vibrations from ribose vibrations. In-plane modes of the adenine ring are selectively enhanced in UV resonance Raman spectra, which facilitates the assignment of the in-plane vibrations. In addition to the in-plane modes, a ribose vibration coupled with adenine in-plane vibrations was identified in the UV resonance Raman spectra. The fundamental wavenumbers for 22 in-plane normal modes of the 9-substituted adenine ring of adenosine in the 1700-250 cm-' region are proposed, Although the fundamental wavenumbers of adenosine correspond well with those of adenine above 1350 cm-' and below 800 cm-', the vibrations in the 1350-800 cm-' region are appreciably affected by the presence of the N - e C - 1 ' glycosidic bond and the couplings between ribose and adenine ring vibrational motions. The adenosine fundamental wavenumbers and their isotopic shifts reported here may be useful in analysing vibra- tional spectra of adenine nucleosides and nucleotides and in improving the force field of the 9-substituted adenine ring.

INTRODUCTION

There are many 9-substituted adenine derivatives of biological interest. Examples are adenosine 5'- triphosphate, cyclic adenosine 3',5'-monophosphate, nicotinamide adenine dinucleotide and adenine residues in DNA. These adenine derivatives belong to the class of nucleotides and play important roles in various phases of biological activities such as energy pro- duction, signal transmission, enzymatic reaction and genetic coding. A number of Raman spectroscopic studies have been devoted to the elucidation of the structures of adenine nucleotides in biological environ- ments. Recent advances in UV resonance Raman (UVRR) spectroscopy have further increased the appli- cability of Raman spectroscopy in the field of biological sciences. Even a single adenine nucleotide bound to a large protein can be studied by UVRR spectroscopy.'.'

In order to obtain structural information from Raman spectra, it is important to know the modes of vibrations and the structural factors that affect their wavenumbers and Raman intensities. In Raman spectra of adenine nucleotides, strong bands usually arise from vibrations of the adenine ring, and adenine has some- times been used as a model compound for adenine nucleotides. Experimental including isotopic (D, "N, I3C) sub~titutions,~.~ and theoretical studies*-" have been made to elucidate the normal

* Author to whom correspondence should be addressed. t Decreased 10 August 1992.

CCC 0377-0486/94/080623-08 0 1994 by John Wiley & Sons, Ltd.

vibrational modes of adenine. The knowledge of adenine normal vibrations, however, cannot be applied straightforwardly to detailed analyses of Raman spectra of adenine nucleotides because of substantial vibra- tional coupling of the adenine ring with ribose that is attached to N-9 of the adenine ring.''

Vibrational spectra of 9-substituted adenines such as 9-methyladenine (9-MeAde), adenosine (Ado, Fig. 1) and adenosine 5'-monophosphate have been investi- gated in order to clarify the normal modes of adenine nucleotides.' '-19 However, isotopic wavenumber data for 9-substituted adenines are limited to the 8-D and 6-ND2 analogues"-'4*'9 and even the fundamental wavenumbers of the 9-substituted adenine ring have not been established yet. Determination of the fundamental wavenumbers is a key step to full understanding of the vibrational properties of adenine nucleotides.

In this study, UVRR, Raman and infrared (IR) spectra of Ado and its isotopic analogues, [1,3-'5N,]

OH OH Figure 1. Structure of adenosine.

Received 21 Februoiy 1994 Accepted 22 April 1994

624 A. TOYAMA ET AL.

Ado, [2-'3C] Ado and [8-I3C] Ado, were measured in neutral aqueous solution (UVRR and Raman) and in the crystalline state (Raman and IR). Vibrational bands arising from in-plane modes of the adenine ring were distinguished from those due to out-of-plane modes and ribose vibrations on the basis of resonance enhance- ment in UVRR spectra and isotopic wavenumber shifts. Some bands due to out-of-plane vibrations of the adenine ring were also identified. We propose the fun- damental wavenumbers of 22 in-plane normal modes of the 9-substituted adenine ring in the 1700-250 cm-' region and compare them with the calculated wavenum- bers reported previou~ly.~*' '

EXPERIMENTAL

Adenosine was purchased from Wako. [ 1,3-'5N,] Adenine (isotopic purity 99%), [2-'3C] adenine (90%) and [8-'3C] adenine (80%) were obtained from CEA. Labelled Ados were synthesized by coupling reactions of the labelled adenines with l-acetyl-2,3,5-tri- benzoylribofuranose (Sigma) according to the literature method." Deuteration at C-8 and C-1' was performed as described previ~usly."~'~ All samples were purified by crystallization from water. The crystals obtained were colourless needles, which were ground to a powder for the measurements of Raman and IR spectra in the crystalline state. Solution samples were prepared by dis- solving the crystalline needles in distilled, denionized water.

Raman scattering was excited with 515 nm radiation from argon ion laser (NEC GLG 3302) or the fourth (266 nm) or fifth (213 nm) harmonic of a pulsed Nd : YAG laser (Quanta-Ray DCR-3G). Raman (515 nm) and UVRR (266 nm) spectra of neutral aqueous solutions were recorded on a Jasco CT-80D double monochromator equipped with a Princeton Instruments DISIDA-700 diode-array detector." UVRR spectra excited at 213 nm were recorded on a fore-prism Raman spectrometer equipped with a charge-coupled device detector (Princeton Instruments LN/CCD-1152)." For crystalline samples, a JEOL JRS-400D Raman spec- trometer equipped with a photon-counting system was used.

IR spectra of crystalline samples in KBr discs were recorded on a Jasco FT/IR-7000 spectrometer.

RESULTS AND DISCUSSION

Assignment of Raman and IR bands

Figure 2 shows UVRR spectra of Ado and its 1,3-'5N2, 2-I3C and 8-13C analogues excited at 266 nm in aqueous solution. The UVRR spectra excited at 213 nm are shown in Fig. 3. Since the 266 and 213 nm excita- tions are in resonance with z-z* transitions of the adenine ring which are polarized in-plane but in differ- ent directions: 324 it is expected that only the vibra- tions involving in-plane atomic motions appear in the UVRR spectra and the intensity of each UVRR band

1600 1400 1200 1000 860 660 WAVENUMBER / cm-'

Figure 2. UVRR (266 nm) spectra of adenosines in neutral aqueous solution: (a) Ado; (b) [1,3-15N,] Ado; (c) [2-"C] Ado; (d) [8-13C] Ado. The concentration is 1 mM. Axes for Figs 2-7: Intensity vs. wavenumber/cm-'.

depends on the excitation ~ a v ~ e n g t h . ' ~ ~ ~ ~ Thus, the bands noticeable in the UVRR spectra are unequivo- cally assigned to in-plane vibrations of the adenine ring or vibrations strongly coupled with the in-plane modes.

Figure 4 shows Raman spectra recorded for aqueous solutions of the Ado isotopomers with 515 nm excita- tion. The 515 nm Raman and IR spectra in the crys- talline state are shown in Figs 5 and 6, respectively. In the non-resonant Raman and IR spectra, out-of-plane vibrations of the adenine ring and vibrations of ribose are expected to appear in addition to the in-plane vibra- tional bands. Table 1 summarizes the observed wave- numbers, intensities, polarization properties and isotopic wavenumber shifts.

1700-1600 em-' region. The 9-substituted adenine ring consists of 15 atoms including the substituent at N-9 and has 27 in-plane normal vibrations. Four of them are C-H and N-H stretches above 2000 cm-' and, thus, 23 normal modes occur below 2000 cm-'. The highest wavenumber mode ( V J of the Ado in-plane vibrations (below 2000 cm-') appears weakly around 1650 cm-' in the solution UVRR spectra [Figs 2(a) and 3(a)] and at 1654 cm-' in the crystalline Raman spec- trum [Fig. 5(a)]. The corresponding IR band splits into a very strong band at 1669 cm-' and a shoulder at 1656 cm-' in the crystalline state [Fig. 6(a) and Table 11. We have found that the wavenumbers and the rela- tive intensity of the two IR components depend on the

ASSIGNMENT OF ADENINE RING IN-PLANE VIBRATIONS IN ADENOSINE 625

WAVENUMBER I cni' Figure 3. UVRR (213 nrn) spectra of adenosines in neutral aqueous solution: (a) Ado; (b) [1.3-l5N2] Ado; (c) [2-13C] Ado; (d) [8-13C] Ado. The concentration is 5 rnM.

form of crystal, suggesting that the splitting arises from intermolecular interactions in the crystal. The 1656 cm-' shoulder in the IR spectrum is related to the 1654 cm-' Raman band in the crystalline state and the very strong IR band at 1669 cm-' may be ascribed to a component of v whose wavenumber is increased by dipole-dipole interactions. Effects of dipole-dipole coupling on the wavenumbers of strong IR bands have been analysed for crystalline uracil.26 The v1 mode of Ado has been ascribed to the C-6-NH2 scissors vibra- tion because the corresponding Raman and IR bands disappear on N-deuteration.' The present Raman and IR spectra shows that the v1 mode is sensitive to both the 1,3-"N2 and 2-I3C substitutions but not to the 8-13C substitution. This is consistent with the assignment of this mode to the C-6-NH, scissors because C-6, N-1, N-3 and C-2 all belong to the six- membered (pyrimidine) ring whereas C-8 is in the five- membered (imidazole) ring (see Fig. 1).

The 1603 cm-' band in the 266 nm UVRR spectrum has counterparts at 1603 cm-' in the crystalline Raman spectrum and at 1606 cm-' in the crystalline IR spec- trum. The 1,3-15N,, 2-"C and 8-I3C shifts of the 1603 cm-' band are -2, - 4 and 0 cm-', respectively, in the 266 nm spectrum and similar isotope shifts are observed in the crystalline Raman and IR spectra. Fodor et aZ.19

observed two UVRR bands at 1648 and 1605 cm-' for 2'-deoxyadenosine 5'-monophosphate in H,O and a single band at 1626 cm-' in D20. They assigned the 1648 cm-' band to a pyrimidine ring mode and the

l * l . , . l m , . l 1600 1400 1200 1000 800 600

WAVENUMBER / cm-' Figure 4. Raman (515 nrn) spectra of adenosines in neutral aqueous solution: (a) Ado; (b) [1.3-l5N,] Ado; (c) [2-13C] Ado; (d) [8-13C] Ado. The concentration is 50 rnM.

1605 cm-' band to the NH, scissors. However, the spectral change observed on going from H,O to D,O solution can be interpreted in another way. In H20 solution, the NH, scissors mode at 1648 cm-I is coupled with the ring mode at 1626 cm-', resulting in a wavenumber decrease of the latter mode to 1605 cm-'. In D,O solution, the NH, scissors mode disappears from this wavenumber region and the vibrational coup- ling diminishes. As a result, the ring mode recovers its intrinsic wavenumber (1626 cm- '). Consistent with this interpretation is the observation that the 1600 cm-' IR band of crystalline 9-MeAde increases to 1607 cm-' on N-de~teration.'~ Hence the band around 1605 cm-' can be ascribed to a pyrimidine ring mode (v,) coupled with the NH, scissors mode around 1650 cm-'. The wavenumber of the v, band has been found to decrease with weakening of hydrogen bonding at NH,.,' This may be accounted for by the following mechanism: the NH, scissors mode decreases in wavenumber with weakening of the hydrogen bond and further pushes the v, mode to a lower wavenumber through vibrational coupling.

1600-1400 cm-' region. The Ado 266 nm UVRR spec- trum shows four bands at 1583, 1508, 1485 and 1428 cm-'. These UVRR bands undergo significant wave- number shifts on isotopic substitution except the 1508 cm-' band, whose 1,3-"N, and 2-13C shifts are zero and the 8-13C shift is not clear because of overlap with the stronger 1478 cm-' band [Fig. 2(d)]. With 213 nm

626 A. TOYAMA ET AL.

' 16b0 ' 1400 ' li00 ' 1600 ' 860 ' 660 . 460 WAVENUMBER I cm-'

Figure 5. Raman (51 5 nrn) spectra of cwstalline powers of aden- osines: (a) Ado; (b) [1,3-15N2] Ado; (c) [2-"C] Ado; (d) 18- "C] Ado.

II a

Ih

1600 1400 1200 1000 800 600 400 WAVENUMBER / cm-'

Figure 6. IR spectra of adenosines in KBr discs: (a) Ado; (b) [1. 3-''N,] Ado; (c) [2-13C] Ado; (d) [8-''C] Ado.

excitation, however, the 1478 cm- ' band becomes much weaker and a shoulder is clearly seen at 1500 cm-', indicating an 8-I3C shift of -8 cm-' [Fig. 3(d)]. In the visible Raman spectra in solution and crystal, the corre- sponding band also shifts by -5 to -10 cm-' on 8-13C substitution. Hence the 1508 cm-' band is ascribed to an imidazole ring vibration (vJ. The 1428 cm-' band is also assigned to another imidazole ring vibration (v6) on the basis of its particularly large 8-"C shift (- 10 cm-'). Actually, deuteration of C-8 causes large (ca. - 15 cm- ') wavenumber decreases of the 1508 and 1428 cm-' On the other hand, the 1,3- "Nz shift is larger than the other isotopic shifts for the 1583 cm-' band. The N-1-C-6 stretch and/or N-3-C-4 stretch may contribute to this pyrimidine ring mode (VJ. The isotopic wavenumber shift of the 1485 m-' band is generally large, irrespective of the site of substi- tution. This band is assigned to a mode involving both the pyrimidine and imidazole rings ( v ~ ) . In addition to the four 266 nm UVRR bands, a band in the visible Raman spectrum is observed at 1458 cm- in solution (1466 cm-' in crystal), which does not show any isotope shift and is ascribable to a C-H bend of ribose as proposed previou~ly.'~

The 1429 cm-' band observed with 213 nm excita- tion [Fig. 3(a)] is very close in wavenumber to the 1428 cm- ' band (v6) in the 266 nm spectrum. However, the isotopic wavenumber shifts differ significantly between the two bands (Table 1). An analogous but larger differ- ence in isotopic wavenumber shift is observed on deu- teration at (2-8. In the 266 nm UVRR spectra, the 1428 cm-' band has been found to shift to 1413 cm-' on C-8-deuteration,' ' whereas the same substitution does not affect the 1429 cm-' band significantly and a peak at 1427 cm- ' with a shoulder at 1412 cm- ' is seen in the 213 nm spectrum of [8-D] Ado [Fig. 7(a)]. Since the 1427 cm-' band of [S-D] Ado shifts to 1026 cm-' on C-1'-deuteration [see Fig. 7(b)], this band (and the 1429 cm-' band of Ado) is ascribed to a ribose vibration. Th wavenumber ofthe ribose vibration is very close to

W .+ m

I ' I - I - I . , . I 1600 1400 1200 1000 800 600

WAVENUMBER / cm"

Figure 7. UVRR (213 nrn) spectra of (a) [8-D] Ado and (b) [8, l '-D2] Ado. The concentration is 5 rnM.

ASSIGNMENT OF ADENINE RING IN-PLANE VIBRATIONS IN ADENOSINE 627

Table 1. Observed Roman and IR wavenumbers (cm-') and their isotope shifts of adenosioe

Aqueous solution Crystrltine powder

UVRR (266 or 213 nmpa Raman (515 nm)'.= Raman (515 nm)*.c

(1 650.

1603 1563 1508 1485

1429' 1 428

1376

1337 1310 1309'

1253

1213' 1199 1176

[-5. -5. -21)

[-2. -4. 01 [-8. -2. -21 [O. 0. -8'1 [-5, -11, -71

[O. -1. -41 [-4. -2. -101

[-3, -3, -41

[-lo, -6. -21 [-16, -17, -21 [-lo. -10. -61

[-5. -3. -11

[-1. -1.01 [-2, -4, -91 [-4, -2. -91

1009 1-6. -4. -31

916. [-19, -5. -11

851. [-2, -3, 01

730 [-7, -6, -41 (701 [+1. -. + 2 ] )

6361620 [-51-6. 01-2. -21-21

534 [-4. -1, -11

1582 I, p 1510s. p 1485 s, p 1458 vw, p

1428 w. P

1377 s, p

1337 m. p 1307 6, p

1254 m, p

1213m. p

1176 w, p

1128w. p

1086 w, p

(1044 w. P) 1007 m. p

[-7, -1, -21 (-1.0. -101

[O. 0, 01

[O. -1. -101

[O. -1. -21

[-9. -5, -21

[-5. -9, -71

[-14, -14, -11

[-5. -5, -11

[-2. -2. -11

[-3. -4, -91

[O. +1. -11

[O. 0. -11

[-5, -4, -31

(916 w, p (899 w, p 867 m, p

[+3. +3. -11) [-2. 0. 0)) [O. 0. -11

828 vw. dp 799 vw, dp

[-3. -3. -21 [-3. -2, -11

730 vs. P 701 vw, p

[-7, -6. -41

638161 8

534

(16Wvw. b

(1603 vw. b 1573 s 1506m 1474 m

(1 466 rh)

1419 m 1386 m 1371 s

1332 vs 1302 r

1271 w 1246 m

(1210 w, b

1178m

( 1 1 3 2 ~ ) ( 1 1 1 3 ~ )

(1 070 vw)

1035 m 1011 m 978 w

911 w

(114ovw)

(1088 vw)

859 vw 843 6

821 vw 794 vw 762 m 720 s 701 vw Wvw

[-1. -4.01)

[-5. -4.01) [-7, -1. -11 [-1.O. (-5)l [O. -5. -41

[-1.0. -111 [O. 0. -11 [O. -1. -11

[-11. -6, +1] [-11. -11, +1]

[+1.0. +1] [-2,O. +1]

[+1.0. -11)

[-3, -2. -81

[O. 0. 01 [-5, -2. -11 [+1, +l. -11

[-14. -4, 01

[-1.0. -81 [+1, +1.0] [+1, +1.0] [-2. -1.01 [-1.O. -11 [-4, -2, -21 [O. 0. 01 [O, +I. +l ]

639 m [-4, 0. 01

587 w 571 w 551 w 537 w 525 w 412 w 382 vw 350 vw 319 s 305 sh 285 w 271 w

1-2. -1.01 [O. 0, 01 [-4. -3.01 [(-6). -2. 01 [+1. +1.0] [O. 0-01 [O. 0, 01 [O. 0. +1] [O. 0. 01

10. 0. 01 [O. 0. -11

1669 m

(1656 sh 18066 1574 in 1507 w 1475 s

1428 w 1415m 1388 m 1372 m 1353 w 1334 S 1303

1252 m 1243 m 1224 m 1211 s

1179m 1143 m 1127 m 1109 8 1092 w 1072 s 1057 s 1038 S

1012 m 978 in 965 w

904m

859 w 8 4 4 W 824 m 795 w 769 m 723 m 706 w

(573 m. b (645 sh)

639 m 593 m

548vw 539 w 525 w 412 vw

[-2. -2, 01

[-7. -1.01) [ -3. -5.01 [-7, -7, -11 [-1, 0. -61 [-3, -8. -6

[-3, -1. -31 [-1.0. -101 [-1. -1, -21 [O. -2. -21 [O. 0.01

[-12, -11, -11 [-13. -8, -11

[O. 0. 01 [ -5. -4. 01 [O. 0.0] [-2. -1, -21

[-4, -3, -91 [O. 0. 01 [O. 0. 01 [-1, -1, -11 [O. 0.01 [-I, 0. -11 [-2. -1, -21 [+I. +1.0]

[O. 0. 01 [O. -10.01

[-6, -3. -21

[+1.0.0]

[+1.0. -81 [O. 0. -11 [-1, -1, -11 [-2. -1.01 [-1. -1, -21

[-1. -1, +1]

[-1. -1. -31)

[-5, -3, -41

[O. 0. -31 [-1, -2, -21

N-5). -3.01 [-6. -2.01 [-1. -1,Ol [-, -1, -21

a Wavenumber shifts on 1,3-'5N, 2-'% and 8-'% substitutions are listed in this order in brackets. Data in parentheses are less certain because of broad bandwidths and/or weak intensities. Wavenumbers marked with asterisks were observed with 213 nm excitation and the others were observed with 266 nm excitation.

"Abbreviations: vs, very strong; s. strong; m. medium; w, weak; vw, very weak; sh, shoulder; b, broad; p, polarized; dp, depolarized.

that expected for a C-l'-H bending vibration, a-CH, from the analysis of UVRR spectra of a series of C-1'- deuterated Ados." The wavenumber of the a-CD mode in C-1'-deuterated Ados is also close to that expected previously." It has also been proved that a-CH is strongly coupled with adenine ring vibrations." Such vibrational coupling is likely to lend UVRR intensity to the a-CH band. The 1428 cm-' IR band of crystalline

Ado may also be assigned to a-CH. This mode prob- ably corresponds to a conformational marker band of polynucleotide secondary structure around 1420 cm- (mode 4 in the numbering scheme of Ghomi et al.").

1400-1150 cm-' region. Seven UVRR bands are seen in the 1400-1150 cm-' region of the Ado 266-nm spec- trum CFig. 2(a)]. The isotope shifts of the 1376 cm-'

628 A. TOYAMA ET AL.

band are all small, indicative of a highly delocalized character of this vibration (v,). The 1337 and 1310 cm-' bands show large wavenumber decreases on both 1,3-I5N2 and 2-I3C substitutions but not on 8-I3C sub- stitution. They are assigned to pyrimidine ring vibra- tions (v8 and vg). The 1309 cm-' band in the 213 nm spectrum of Ado is distinguished from the 1310 cm-' band in the 266 nm spectrum by its 1,3-I5N, 2-I3C, and 8-13C shifts. The distinction between the 1309 and 1310 cm-' bands has been made more clearly by the 8-D and 1'-D shifts: - 17 and + 17 cm-', respectively, for the 1309 cm-' band in the 213 nm spectrum and -2 and +1 cm-' for the 1310 cm-' band in the 266 nm spectrum." Since the 1309 cm-' band shows a compa- rable (-10 to -6 cm-') shift on each of the 1,3-I5N, 2-13C and 8-I3C substitutions, this band seems to arise from a vibration involving both the pyrimidine and imidazole rings (vl0). The 1253 cm-' band is sensitive to the 1,3-"Nz and 2-13C substitutions and is assigned to a mode with contributions from pyrimidine vibra- tions (vl '). However, contributions from imidazole vibrations cannot be neglected for the v l l mode because it also shows a large 8-D shift (-14 cm-').'I The 1213 cm-' band in the 266 nm UVRR spectrum of Ado has a shoulder at 1199 cm-' [Fig. 2(a)]. The 1213 cm- ' band is virtually insensitive to the isotopic substi- tutions, whereas the 1199 cm-' shoulder shows a -9 cm-' shift on 8-13C substitution and becomes more separated from the 1213 cm-' band in the spectrum of [8-13C] Ado [see Fig. 2(d)]. The 1199 cm-' band is therefore assigned to an imidazole ring mode (v13). Although neither of the 1,3-I5N2, 2-I3C and 8-I3C sub- stitutions affects the 1213 cm-' band, deuteration at C-8 causes a very large decrease (-248 cm-') from 1213 to 965 cm-' [compare Figs 3(a) and 7(a)], indica- tive of the predominant contribution from C-8-H bend to the 1213 cm-' mode (v12). For the 1176 cm-' band, the 8-I3C shift (-9 cm-') is much larger than the 1,3- 15N2 and 2-I3C shifts. This band has been found to shift by +31 cm-' on C-1'-deuteration and assigned to an imidazole ring mode ( ~ 1 4 ) coupled with the N-9-(2-1' stretch". In the Raman spectrum of crystalline Ado, additional bands are seen at 1386, 1271 and 1246 cm-', which are insensitive to the isotopic substitutions. The IR bands at 1388, 1353, 1252 and 1224 cm-' are also insensitive. These additional bands are ascribed to ribose vibrations.

Below 1150 em-'. The Ado 266 nm Raman spectrum shows seven bands in the 1150-500 cm- ' region [Fig. 2(a)]. The 1,3-15N, 2-"C and 8-13C shifts of the 1009 cm-' band are comparable to one another and this band is ascribed to a delocalized mode (v J. This is also the case of the 730 cm-' band (v18). Although the iso- topic wavenumber shifts for the very weak band at 916 cm-' are not clear in the 266 nm UVRR spectra, the band becomes stronger with 213 nm excitation (Fig, 3) and a large 1,3-I5N2 shift (- 19 cm-') is observed. Its 2-I3C and 8-13C shifts are much smaller than the 1,3- "N2 shift. Parallel isotope shifts have been observed for the 911 cm-' band in the visible Raman spectrum of crystalline Ado and these two bands have the same origin, a pyrimidine ring vibration (v16). A broad band with two peaks at 636 and 620 cm-' is observed in the 266 nm UVRR spectrum of Ado solution and the shape

of this band is conserved in the spectra of 1,3-15N-, 2-"C- and 8-'3C-substituted Ados with parallel shifts of the peaks (see Fig. 2). In contrast to the double- peaked UVRR band, a single band appears in the visible Raman spectra of crystals (at 639 cm-' for Ado, Fig. 5). The double-peaked UVRR band is probably due to Fermi resonance of an in-plane fundamental with the overtone of a low-wavenumber mode, possibly the 319 cm-' mode observed strongly in the Raman spectra of crystalline Ados. The 1,3-"N2 shift of the 636/620 cm-' band is -5/-6 ax-' but the 2-"C and 8-I3C shifts are no more than -2 cm-'. This band may arise from a pyrimidine ring deformation (v19). The 534 cm-' band bears resemblance with the 636/620 cm-' band in its isotopic wavenumber shift and may be due to another pyrimidine ring deformation (vgl). A band at 851 cm-' is too weak to be detected with 266 nm exci- tation but it is clearly seen in the 213 nm UVRR spec- trum. This band corresponds to the 843 cm-' band in the crystalline Raman spectrum (844 cm-' in IR) and is not sensitive to the 1,3-15N2, 2-I3C and 8-I3C substi- tutions. However, the 8-D substitution causes a shift of - 14 cm-' and therefore the 851 cm-' band is assigned to an in-plane adenine ring mode, possibly an imidazole ring mode (v16).

Ado shows many visible Raman and IR bands below 1150 cm-' that have no counterparts in the UVRR spectra. Most of these bands are insensitive to isotopic substitutions in the adenine ring and are assignable to ribose vibrations. Exceptionally, the 55 1 cm- ' Raman band (548 cm-' in IR) shows -4 (1,3-I5N,) and -3 cm-' (2-'3C) shifts. These small but significant shifts indicate that the 551 cm-' band arises from a vibration of the adenine ring (vzo). A strong Raman band at 319 cm-' in crystalline Ado shifts to 298 cm-' in [6-ND2] Ado (spectrum not shown) and is assigned to the C-6- NH, bend (v2J. The same mode has been identified at 331 cm-' for Thomas and Livramento14 assigned the 1135 and 1045 cm-' bands in the visible Raman spectrum of adenosine 5'-monophosphate to ribose vibrations. The corresponding bands at 1128 and 1044 cm-' in the Raman spectrum of Ado solution do not show any significant isotope shift, supporting the previous assignments.

Out-of-plane vibrations. The 9-substituted adenine ring is expected to have 12 out-of-plane vibrations. These vibrations would be absent from UVRR spectra but might be observed in visible Raman or IR spectra. The 965 cm-' IR band of Ado shows a shift (- 10 an-') on 2-' 3C substitution only and is unambiguously assigned to the out-of-plane wagging mode of C-2-H. On the other hand, the 859 cm-' IR band is affected onIy by the 8-I3C substitution (-8 cm-' shift) and ascribed to the C-8-H out-of-plane wagging vibration. The present assignments of the C-2-H and C-8-H wagging modes are consistent with the results of a neutron inelastic scattering study on crystalline adenine' but are different from the previous assignments proposed by Raman and IR s t u d i e ~ . ~ . ~ . ' ~ Kyogoku et al." studied polarized IR spectra of single-crystal 9-MeAde and identified five out-of-plane IR bands below 800 cm-' on the basis of their polarization. The wavenumbers of the out-of-plane IR bands of 9-MeAde are 795, 690, 525, 358 and 210 cm-'. Among them, the 690 cm-' band shifted to 489

ASSIGNMENT OF ADENINE RING IN-PLANE VIBRATIONS IN ADENOSINE 629

cm-' on N-deuteration and was assigned to the NH, wagging mode. The corresponding band of Ado is seen at 673 cm-' in the crystalline IR spectrum. Other out-of-plane modes are seen at 795 (Raman, IR), 525 (Raman, IR) and 350 cm-' (Raman). The lowest wave- number out-of-plane mode corresponding to the 210 cm-' mode of 9-MeAde is outside the wavenumber range of the present measurements.

Fundamental wavenumbers of in-plane vibrations

In the UVRR (266 and 213 nm) spectra, we have identi- fied the fundamental bands of 20 normal modes (v1-v19 and vzl) in the 1700-500 cm-' region as described above. In addition, the 551 cm-' visible Raman band (548 cm-' in IR) observed for crystalline Ado has been assigned to an in-plane fundamental (v,,,) on the basis of significant 1,3-I5N, and shifts. Below 500 cm-', two in-plane bending modes of exocyclic C-6- NH, and N-9-C-1' bonds are expected. The C-6-NH2 bending mode (v2J has been found at 319 cm-' in the crystalline Raman spectrum. On the other hand, the location of the N-9-(2-1' bend (v2J is not clear in the spectra reported here. Presumably, this bending mode has a wavenumber below 250 cm-'. Table 2 shows the in-plane fundamental wavenumbers of the 9-substituted adenine ring together with their 1,3-I5N,, 2-13C and

8-I3C wavenumber shifts. The 8-D and 1'-D shifts deter- mined previously" or in this work (spectra not shown) are also included in Table 2.

Normal coordinate calculations of the 9-substituted adenine ring were reported by MajoubeI7 and Wiorkiewicz-Kuczera and Karplus,' who employed empirical and ab initio force fields, respectively. The normal wavenumbers calculated in the two studies differ widely from each other. For example, Majoube predicted seven modes in the 1700-1400 region whereas Wiokiewicz-Kuczera and Karplus predicted six modes. Experimentally we have found six fundamentals in that region. In Table 2, we list the calculated wavenumbers of Wiorkiewicz-Kuczera and Karplus, which are in better agreement with the experimental wavenumbers. However, very large discrepancies of ca. 50-130 cm-' are still seen between the calculated and experimental wavenumbers for v l 0 , v14 , v16 and ~ 1 9 . The present data on fundamental wavenumbers and isotopic wave- number shifts may help one to improve the ab initio force field.

Hirakawa et aL4 examined the Raman and IR spectra of adenine and its 1,3-I5N,, 2-I3C and 8-13C analogues. The reported fundamental wavenumbers of adenine cor- respond well with those of the 9-substituted adenine ring studied here in the 1700-1350 cm-' region and below 800 cm-'. However, in the 1350-800 cm-' region, the wavenumbers of adenine vibrations cannot

~~ ~ ~ ~

Table 2. Fundamental wavenumbers (cm-I) and their isotope shifts for in-plane modes of the 9- substituted adenine ring.

V1

v 2

v3

v4

v5

v 7

V 8

V 9

v 1 0

v 1 1

"1 2

'6

'1 3

'1 4

v 1 5

'1 6

'1 7

V 1 8

V 1 9

vzo v 2 1

v22

'23

Observed

1650 1603 1583 1508 1485 1428 1376 1337 1310 1309 1253 1213 1199 1176 1009 91 6

730

551' 534 31 9'

a51

6288

h -

1,3-"N2

-2' -2

0 -5 -4 -3

-10 -16 -10 -5 -1 -2 -4 -6

-19 -2 -7 - 6g - 4' -4 0'

-a

2-'3c

-2' -4 -2

0 -1 1 -2 -3 -6

-1 7 -10 -3 -1 -4 -2 -4 -6 -3 -6 -18 - 3' -1 0'

Isotope shih

8-"C

0" 0

-2

-7 -1 0 -4 -2 -2 -6 -1

0 -9 -9 -3 -1

0 -4 -20

0' -1

0'

-a

8-Db

-1 = +2 -1

-1 3 -16 -1 5 -7 -6 -2

-17 -14

-5 -3 +2

-27 -14 -4 - 2'

-1 3' -2 -1 '

-248

l '-Db

-1 -2 -1 -1 -3 +4 +2 +1

+17 -5

+11

+31 -1 5 -4

-24' -6 -1'

0' 0'

- 2f

+I a

Calculated'

1639 1590

1512 1474 1414 1363 1319 1301 1263 1242 1195 1177 1043 974

729 700 568 529 528 305 208

1568

aai

Assignmentd

N H , scissors Pyr + N H , scissors PY Im Pyr + Im Im Pyr + Im PY r PY r Pyr + Im Pyr + Im C-8-H bend Im Im + N-94-1' stretch Pyr + Im PY r Im Pyr + Im PY r Pyr + Im PYr C-6-N H a bend N - 9 4 - 1 ' bend

a Observed wavenumbers and isotope shifts in UVRR spectra of aqueous solutions. Ref. 11 and this work. Ref. 9. Ab inirio calculations on O-methyladenine. Approximate description based on the isotopic wavenumber shifts. Pyr, pyrimidine ring vibration; Im,

imidazole ring vibration. ' Data from IR spectra of crystals. ' Data from Raman spectra of crystals. gAverage value for the doublet.

Expected below 250 cm-'.

630 A. TOYAMA ET AL.

be simply correlated with those of the 9-substituted and its isotopic analogues. The only missing fundamen- adenine ring. Replacement of the N-9-H bond in tal is that due to the N-9-C-1' in-plane bend, which is adenine by the N-9-C-1' bond in adenosine causes expected below 250 cm-'. In addition to the in-plane large changes in fundamental wavenumbers and normal fundamentals, we have identified a ribose vibration modes in the 1350-800 cm - region. coupled with adenine in-plane modes and several

out-of-plane vibrations. The present data may help in interpreting the Raman and IR spectra of adenine nucleosides and nucleotides and in improving the force field of the 9-substituted adenine ring. CONCLUSION

Acknowledgement

This work was supported by a grant from the Japan Society for the Promotion of Science.

We have determined the fundamental wavenumbers for 22 in-plane vibrations of the 9-substituted adenine ring by analysing the UVRR, Raman and IR spectra of Ado

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