3253
J. Phys. Chem. 1984, 88, 3253-3260 Acknowledgment. The authors acknowledge the financial assistance of the Natural Sciences and Engineering Research Council of Canada in the form of an Operating Grant and Strategic Grant (Energy) (CAF) and Graduate Scholarships (G.C.G. and G.J.K). The support of an Imperial Oil University Research Grant is also acknowledged. The N M R spectra were obtained at the South Western Ontario High Field N M R Centre,
Manager, Dr. R. E. Lenkinski. The authors especially thank Dr. H. Robson, Exxon Research and Development, Baton Rouge, La., for his help in providing for study the very low Si/Al ratio zeolite sample whose spectrum is presented in Figure 3A, and Drs. M. Barlow and D. Stewart, B.P. Limited, Sunbury on Thames U.K., for their help in providing the highly siliceous sample whose spectrum is presented in Figures 3C and 4D.
Infrared and Raman Spectra and Vibrational Assignments for 1-Methyluracil and Isotopic Derivatives Thomas P. Lewis, H. Todd Miles,* and Edwin D. Becker National Institutes of Health, National Institute of Arthritis, Diabetes, and Digestive and Kidney Diseases, Bethesda, Maryland 20205 (Received: August 15, 1983: In Final Form: December 20, 1983)
Infrared and Raman spectra of 1-methyluracil and seven isotopic derivatives (2H and I8O) have been measured. Polarized Raman spectra of single crystals were used to distinguish in-plane and out-of-plane modes. All the fundamental vibrations except the methyl torsion have been assigned. Product rule agreement is’satisfactory. Relevance of these results to vibrational spectroscopy of polynucleotides in aqueous solution is discussed.
Introduction Infrared (IR) and Raman spectra have played a critical role in defining the structures of various synthetic and natural polynucleotides.’ The principal emphasis in this laboratory has been on infrared spectra in the C=C and C=O region since the carbonyl and ring vibrations have proved to be sensitive to helix formation and structurally most informative. DzO has been the principal solvent because of its transparency in this region, but with improvements in instruments and techniques, it is possible to use both HzO and D,O solvents to obtain useful structural information over a wide frequency range in both IR and Raman spectra.Ieg Interpretation of the polynucleotide spectra is, of course, facilitated by a detailed understanding of the spectra of individual nucleotide bases. Several surveys of IR and/or Raman spectra of nucleotides, nucleosides, and the constituent bases have been p ~ b l i s h e d . ~ -Papers ~ by Susi and ArdS and Tsuboi et a1.6 have presented detailed studies of uracil and 1-methyluracil, respectively. Several years ago, we undertook an extensive investigation of the IR and Raman spectra of 1-methyluracil (I) and seven deuterated or I80-substituted derivatives. Only one aspect of this work, the identification of the amide-I1 band, was published.’ The molecule 1-methyluracil is more representative of the base in the (1) See, for example, (a) G. B. Sutherland and M. Tsuboi, Proc. R. SOC. London, Sec. A, 239,446(1957);(b) E.R. Blout and H. Lenormant, Biochim. Biophys. Acta, 17,325 (1955);(c) H. T. Miles Proc. Natl. Acad. Sei. U.S.A., 47,791(1961);(d) T. Shimanouchi, M. Tsuboi, and Y. Kyogoku, Adv. Chem. Phys., 7 , 435 (1964); (e) H. T. Miles in “Procedures in Nucleic Acid Research”, Vol. 2, G.Cantoni and D. R. Davies, Eds., Harper and Row, New York, 1971,p 205;( f ) E. W.Small and W. L. Peticolas, Biopolymers, 10, 1377 (1971);(g) L. LaFleur, J. Rice, and G. J. Thomas, Jr., ibid., 11, 2423 (1972);(h) H. T. Miles in “Biomolecular Structure, Function, and Evolution”, R. Srinivasan, Ed., Pergamon Press, Oxford, 1980,p 251,and references cited in these papers. (2)C.L.Angell, J . Chem. Soc., 504 (1961). (3)A. Y. Kyogoku, S. Higuchi, and M. Tsuboi, Spectrochim. Acta, Part A , 23A,969 (1967). (4)R. C . Lord and G. J. Thomas, Jr., Spectrochim. Acta, Part A , 23A, 2551 (1967). (5)H. Susi and J. S . Ard, Spectrochim. Acta, Part A , 27A,1549 (1971). (6)M. Tsuboi, S . Takahashi, and I. Harada in “PhysicochemicalProperties of Nucleic Acids”, Vol.2,J. Duchesne, Ed., Academic Press, New York, 1973, pp 104-145. (7)H. T. Miles, T. P. Lewis, E. D. Becker, and J. Frazier, J . Biol. Chem., 248, 1115 (1973).
.x“;.-. H
6 ; A o I CH, I
nucleotide than uracil itself, since the heavy methyl group is expected to simulate reasonably well the ribose moiety insofar as the vibrational modes of the uracil base are concerned. Subsequent to our original publication,’ Susi and Ard8 published an assignment of the in-plane vibrations of I-methyluracil, in which some limited isotopic substitution data were employed. Nishimura et al.9 reported on the in-plane modes of the uracil residue in uridine 5’-phosphate; they included data from the molecule in which both nitrogen atoms in the uracil ring are replaced by lSN. Both of these studies, as well as that of Tsuboi et relied heavily on normal-coordinate analyses of the in-plane vibrations. Very recently, Szczesniak et a1.I0 reported the infrared spectra of uracil itself isolated in rare-gas matrices, together with an analysis of normal modes predicted by quantum-mechanical calculations. In view of the continuing interest in vibrational spectra of nucleotides and polynucleotides, we are reporting here our extensive studies on 1-methyluracil. Our results include (1) data on a number of isotopic derivatives that were not previously available, (2) single-crystal Raman polarization data, (3) comparisons of spectra in the C=O and C=C region between the solid and aqueous solution, and (4) a complete assignment of both in-plane and out-of-plane vibrations. Although our assignments are generally compatible with previous publications,6x8there are some notable differences where our more extensive isotopic and polarization data permit more reliable assignments. These data should also be of considerable value in verifying and refining normal-coordinate calculations. (8) H. Susi and J. S . Ard, Spectrochim. Acta, Part A , 30A,1843 (1974). (9)Y. Nishimura, H.Haruyama, K. Nomura, A. Y . Hirakawa, and M. Tsuboi, Bull. Chem. SOC.Jpn., 52,1310 (1979). (10) M. Szczesniak, M. J. Nowak, H. Rostkowska, K. Szczepaniak, W. B. Person, and D. Shugar, J . Am. Chem. SOC.,105, 5969 (1983).
This article not subject to U S . Copyright. Published 1984 by the American Chemical Society
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Experimental Section
Syntheses. The synthetic methods developed for specific isotopic labeling at different positions of 1-methyluracil are summarized below and described in detail in the supplementary material (see paragraph at end of article). The isotopic content of all products and positions of isotopic substitution were established by mass spectroscopy or N M R spectroscopy. The mass spectrum of 1-methyluracil has the following intensity pattern: m / e 126 (M', loo), 127 (M+ f l , 7.3), 84 (4.1), 83 (46), 82 (24.4), 81 (2.4), 56 (4.1), 55 (30.1), 54 (8.1), 53 (4.9), 43 (4.1), 42 (90.2). The parent ion M + is at 126 and the peaks at 82 and 83 are presumed to arise from loss of the fragments HN-C=O and NH,-C=O, where the oxygen comes from the 2-carbonyl of 1-methyluracil. All derivatives except U-3-d were chromatographed on a silica gel column with 90% chloroform, 10% methanol as the solvent phase before the infrared spectra were measured, and samples for Raman spectra were further sublimed at least twice. All samples gave only a single spot on silica gel thin-layer chromatography. All samples melted sharply at 234-235 "C. Summary of Synthetic Methods. 1-methyluracilL MeU-4-180 (82% 4-180, 18% 4-160, no 2-180 as determined from mass spectra; reagents and conditions: (a) 98% H2-I80,concentrated H,SO,, 16 h, 120 "C) isocytosine A 1-methylisocytosine L MeU-2-180 (78% 2-180, no 4-180; reagents and conditions: (a) Me2S0,; (b) 1 N NaOH, 6 h, 120 "C) 1-methyluracil MeU-5-d (80% 5-d; position and extent of reaction confirmed by NMR; reagents and conditions: (a) 1 N D2S04 in DzO, 2 h, 100 "C) 1-methylcytosine I-methylcytosine-6-d 9 MeU-6-d (67% 6-d, no 5-d by NMR; reagents and conditions: (a) 0.2 N NaOD, 3 h, 100 "C; (b) NaNO,, HOAc, 1 h, 0 "C, then 25 "C) 1-methylcytosine MeU-5,6-d2 (first preparation, 95% isotopically pure; second preparation, dideuterated material 67%, monodeuterated 33% by mass spectroscopy; reagents and conditions: (a) 0.2 M NaOD in DzO, 2 h, 100 OC, then 2.6 N NaOD in D 2 0 , 4 h, 100 "C, neutralize and extract) dimethox pyrimidine 1-trideuteriomethyl-4-methoxypyrimidine P CD3U (83% 1-CD,, 17% 1-CH3; reagents and conditions: (a) CD31 in EtOH, 3 days, 25 "C; (b) concentrated HCl, 3 h, reflux) Spectral Methods. The 180" viewing configuration was used on a Cary Model 81 Raman spectrophotometer equipped with a Coherent Radiation Model CR-3 argon ion laser, with excitation at 514.5 nm. The frequency calibration was checked with argon atomic lines and should be reliable to f 2 cm-l. Because the 1-methyluracil molecules are oriented parallel to the ab plane of the crystal, in-plane and out-of-plane vibrational modes can be readily distinguished from Raman spectra of single crystals excited by appropriately polarized radiation. Crystals were obtained by slow evaporation from aqueous solution. Crystal faces were identified by zero-level X-ray precession photographs and by conoscopic examination. Spectral measurements were performed on a large needle elongated along the c direction. The crystal was aligned so that the direction of propagation of the incident beam was perpendicular to the c crystal axis, with the incident radiation polarized either (i) perpendicular to the c axis or (ii) parallel to the c axis and therefore perpendicular to the molecular planes. The geometry is illustrated in Figure 1 (supplementary material). The resultant Raman spectra showed clear polarization characteristics, as illustrated for typical spectra in Figures 2 and 3 (supplementary material). Infrared spectra were obtained with Perkin-Elmer Model 521, Beckman IR-7, IR-11, and Digilab FTS-14 spectrophotometers. Frequencies were calibrated by atmospheric absorption bands and should be reliable to *2 cm-'. To ascertain the polarizations of several IR bands in the 4001100-cm-l region, a sample of 1-methyluracil was melted between KBr windows and cooled slowly to promote oriented crystal growth. Conoscopic examination showed that the ab face (parallel
Lewis et al. TABLE I: Correlation of Reoresentations for 1-Methvluracil
to the molecular plane) was exposed perpendicular to the infrared beam. In-plane vibrations showed strongly, while out-of-plane modes were virtually absent. Solutions of samples in H,O or D,O were measured in 25+m CaF, cells, solvent compensated. In the 1-methyluracil crystal the methyl group for each molecule is assumed to be disposed with one hydrogen above, one below, and one in the plane of the uracil ring in order to maintain the plane of symmetry required by the crystallographic space group." In calculating moments of inertia, we assumed C H and N H distances of 1.09 A, tetrahedral angles for the methyl group, and ring C H and N H bonds bisecting the appropriate external angles. Results
Features of the Present Work. Ideally, for a vibrational assignment of the individual molecule, spectral data are obtained with the sample in the vapor phase, suspended in a solid matrix, or dissolved in a relatively noninteracting solvent. For 1methyluracil, low vapor pressure inhibited our carrying out vapor or matrix studies. (Recently, Szczesniak et al.1° were successful in obtaining matrix spectra with improved methods.) Low solubility in most solvents limited solution studies to water, DzO, and dimethylsulfoxide, each in restricted spectral ranges. The studies in H 2 0 and D 2 0 are directly relevant to solution studies of the polynucleotides and are reported in some detail. Most of our work was done, however, in the solid phase: infrared spectra with KBr pellets or mulls in hydrocarbon or fluorocarbon oils and Raman spectra with the polycrystalline solid or single crystals. Distinct differences were observed in the infrared spectra of the doublebond stretching region between mulls and KBr pellets. These differences, reported in a later section, lead us to conclude that 1-methyluracil forms a solid solution in the KBr medium, rather than merely a dispersion of small crystallites. The use of oriented single crystals and polarized exciting radiation provided Raman spectra in which in-plane and out-of-plane modes are clearly differentiated. (A limited IR study of an oriented sample was also carried out, as described previously.) 1-Methyluracil crystallizes in the orthorhombic space group Zbam (@f) with eight molecules per unit cell." The unit cell consists of two layers of cyclic hydrogen-bonded dimers with the molecular planes parallel to the crystal ab plane. The molecular symmetry, C,, is preserved in the crystal site, while the factor group, D2h, has a center of symmetry. There are 39 normal vibrations, of which 26 may be classified as A' (in-plane) and 13 as A" (outof-plane). The correlation table (Table I) shows that each of the 39 fundamental vibrations of the individual molecules is split into four modes in the crystal. However, since the major intermolecular interaction is expected to arise from hydrogen bonding within each cyclic dimer, we might anticipate that the principal observable splitting would be into two modes, one symmetric (g) and one antisymmetric (u), with respect to the center of symmetry in the dimer. Thus, the infrared and Raman frequencies should be different for all vibrations, but in somc bands the differences are smaller than the experimental uncertainties. Additional factor ~~~~
~
(1 1) (a) D. W. Green, F. S.Mathews, and A. Rich, J . Biol. Chem., 237, 3573 (1962); (b) The structure of 1.3-dimethyluracil has been shown by NMR measurements in a nematic phase to have the l-CH, group oriented with one hydrogen in the plane and pointing toward the 2-carbonyl group. See C. L. Khetrapal and A. C. Kunwar, J . Phys. Chem., 86, 4815 (1982).
1-Methyluracil and Isotopic Derivatives TABLE 11: Summary of Spectral Data Tabulations table IR Raman Raman no. compd spectra state spectra state polarizn KBr pellet crystal 3 MeU" KBr pellet crystal 4 MeU-2-I80 crystal KBr pellet 5 MeU-4-I8O 6 MeU-3-d crystal mull, pellet 7 MeU-5-d crystal KBr pellet KBr pellet 8 MeU-6-d crystal 9 MeU-J,6-d2 crystal KBr pellet crystal KBr pellet 10 CD3U 14 MeU, MeU-2-I80, H2O H2O MeU-6-d, MeU-4-180, MeU-54, CD3U 15 MeU-3-d, D2O D2O MeU-3,6-d2, MeU-3-d-2-I80, MeU-3-d-4-I80
" MeU = I-methyluracil. group splitting into doublets in the infrared or Raman frequencies may occur. Recent papers by Bandekar and ZundelI2 have focused on the C=O transition dipole-dipole coupling in uracil crystals as the source of splittings in vibrational bands. Such interactions in the closely related 1-methyluracil would account for as many as four bands (two active in IR, two in Raman) for each fundamental mode. We observe a number of splittings in both infrared and Raman spectra of all derivatives, some of which may be. attributable to dipolar coupling. However, it is equally plausible to interpret these splittings as Fermi resonance, which would be expected to be significant in a molecule of this complexity, and we indicate possible combinations or overtones that might participate in such an interaction. A sizable g-u splitting may have important ramifications for the observation of Fermi resonance, as a combination or overtone level with appropriate symmetry can sometimes lie close enough to one of the two components, g or u, to interact while the other may be relatively unaffected. Thus, a further splitting of a fundamental by Fermi resonance might occur in the infrared but not for the same fundamental in the Raman, or vice versa. In order to test various aspects of the vibrational assignment, we have synthesized and studied complete infrared and Raman spectra of a large number of isotopic derivatives the two mono-I80 species (2-180, and 4-lS0), the three monodeuterio species (3-d, 5-d,and 6-d), a dideuterio species (5,6-d2), and deuteriomethyl derivative (1-CD3). In addition, several of the species (2-180, 4-180, and 6-d) were studied over a limited range in D 2 0 solution, where the 3-hydrogen was also replaced by deuterium. Although we did not prepare 15Nderivatives of 1-methyluracil, we did study uracil-I5N2and report those data where pertinent. Vibrational Assignments. Infrared and Raman spectral data are presented in a number of tables, many of them appearing in the supplementary material. As a guide to the observed data, Table I1 summarizes the various data tabulations. Table I11 lists the observed infrared and Raman frequencies of 1-methyluracil (hereafter abbreviated as MeU), the results of the polarized Raman spectra of the single crystal, approximate intensities of all bands, and the assignments, as described in the following sections. Tables IV-X (supplementary material) provide similar data for the seven isotopic derivatives. Our assignments of the 39 fundamentals of MeU and seven isotopic derivatives are given in Table XI-XIII. We have found little interaction between the nine internal vibrations of the methyl group and the vibrational modes of the rest of the molecule. Consequently, we discuss the internal methyl modes (v3,-vj9) separately. The remaining fundamentals divide into 21 in-plane (ip) and nine out-of-plane (op) modes (uI-vZI and ~ 2 2 - ~ 3 0 ,respectively). (12) J. Bandekar and G. Zundel, Spectrochim. Acta, Part A , 39A, 337, 343 (1983).
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The Journal of Physical Chemistry, Vol. 88, No. 15, I984 TABLE III: Observed IR and Raman Bands of IR Raman freq, cm-' freq, cm-' mode 3147 w 3128 w, ipb u2 3087 w 3077 w, ip y3 3023 w, ip 3015 m Vl 2980 vw 2980 w, op u3 1 2948" w, ip 2950 w u32 2935" w, ip 2875 vw 2835 w 2840 w w , ip u33 2802 w w , ip 1986 w 1965 vw 1964 w w 1789 vw 1794 w w 1768 vvw 1763 w 1763 w w 1718" vw, ip 1695' vs y4 1707" vw, ip 1660' vw 1652 vs, ip y5 1623'm 1622 w, ip u6 1526' vw 1527 vw, ip 1486 m 1488 w, ip y7 1462 w 1468 w, ip y34 1442 w, op 1438 m y35 1432 m 1432 m, ip v36 1428 m 1423 s 1418 w, ip V8 1379 s 1384 s, ip v9 1331 s 1327 w, ip VI0 1247" m, ip 1224 w 1228" vs, ip v11 1201 w 1200" w, ip 1161 w 1162 vw, ip VI2 1146 m 1154 w, ip y13 1135 w w , op y37 1046 vw, ip 1048 m U38 1040 w (sh), ip 995 w 995 w, ip v14 990 w 983 w, op y12 869 m, op u23 808 m, ip 804 w, ip VI5 804 w (sh) 761 w 773 vs, ip u16 757 m, op y24 724 w, op 723 vvw, op y25 630 w 628 vw 626 w, ip u17 563 m, ip 547 m, ip VI8 482 m, ip 448 vw
426 m 356 vw 257 w 195 w
478 m, ip 446 s, op 394 vw, ip 353 w w
y19 v26 v20
v2 1
267 vw, op
u28
177 w, op
u30
v29
1-Methyluracil approx descripnc 5 C-H str 6 C-H str
+ u l o = 3026
u4
N-H
str
deg Me str (op) deg Me str (ip) 2X ~3= 4 2936 v4 + vi2 = 2879 sym Me str ~5 + ~ l =j 2806 2X ~ 1= 4 1990 UT + ~ 1 = 9 1966 ~9 + ~ 2 0= 1805
+ ~ 1 =8 1775
+ +
v i 6 = 1768 2 C=O str ~ 1 2 ~ 1 = 8 1709 vi4
4 C=O str C=C str 2 X V i 6 = 1546 ring str deg Me bend (ip) deg Me bend (op) sym Me bend VIS ~ 1 = 7 1436
+
N-H
bend
ring str ring str
+ V I 9 = 1251 VIS + ~ 2 = 0 1198 VI6
ring str
C-H bend N-Me str Me rock (op) Me rock (ip) Vi8 + ~ 1 9= 1041 C-H bend (ip) C-H bend (op) N-H bend (op) ring str
vi7 + ~ 3 = 0 803 ring breathing ring bend (op) C-H bend (op) 826 u29 = 641 ring bend (ip) C=O and ring bend (ip) C=O bend (ip) ring bend (op) C=O bend (ip) N-Me bend (ip) N-Me bend (op) ring bend (op)
+
ring and C=O bend (op)
150 w, op
lattice mode or
121 m, op 97 m, ip 85 s, op 79 w (sh), ip 57 s, op 48 w (sh), ip 30 vs, op
lattice mode lattice mode
v ~ I- ~ 2 = 9
98
78
152
lattice mode lattice mode lattice mode
lattice mode lattice mode
'
"Probable Fermi resonance. In Nujol mull. cQualitative description of probable dominant contribution to fundamentals; probable assignment of combinations and overtones. In-Plane Vibrations Hydrogen Stretching Modes. The two C-H stretches, u2 and v3, are readily identified at 3147 cm-' (IR), 3128 cm-' (R) and at 3087 cm-' (IR), 3077 cm-' (R), respectively. Infrared spectra were not measured for most of the isotopic derivatives in the hydrogen stretching region, but the Raman data (Table XI) show
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The Journal of Physical Chemistry, Vol. 88, No. 15, 1984
Lewis et al.
TABLE XI: In-Plane Fundamental Frequencies (cm-I) of 1-Methyluracil (MeU) and Isotopic Derivatives
VI
v2 y3 v4 y5
v6 y7 "8
V¶ VI0
mode IR R IR R IR R IR R IR R IR R IR R IR R IR R IR
R YlI VI 2 y13 VI4 y15
u16
u17 VI8
VI9 y20
y11
IR R IR R IR R IR R IR R IR R
IR R IR R IR R IR R IR R
product ruleb
MeU 3015
MeU-2-I80 (3015)I
MeU4-180 (3015)
MeU-3-d MeU-5-d 2235 (3015) 2227
MeU-6-d (3015)
MeU5,64 (3015)
CDIU (3015)
approx descripn" NHstr
3147 3128 3087 3077 1695 1718 1660 1652 1623 1622 1486 1488 1423 1418 1379 1384 1331 1327 1224 1228 1161 1162 1146 1154 995 995 808 804 761 773 628 626 563 547 482 478 426 394 356 353
3128
3128
3129
2336
3127
2338
3128
5 C-H
3075 1675 1699 1660 1646 1624 1623 1483 1488 1422 1416 1378 1383 1331 1327 1224 1228 1161 1162 1146 1156 995 993 807 803 (755) 769 623 620 560 542 480 478 419 387 349 350
3078 1693 1717 1647 1647 1618 1624 1484 1488 1417 1409 1378 1384 1331 1327 1223 1228 1160 1160 1145 1156 993 995 807 803 (758) 772 626 620 561 546 472 473 418 389 354 353
3085 1691 1711 1638 1643 1620 1607 1486 1488 1119 1131 1378 1382 1336 1335
2297 1700 1718 1660 1652 1600 1600 1485 1489 1420 1415 1379 1383 1330 1329 1219 1223 973 1149 1152 864 863 808 803 763 770 619 618 561 546 480 479 425
2298 1704 1717 1673 1648 1601 1598 1484 1488 1418 1416 1349 1354 1329 1329 1221 1223 834 833 1157 1155 764 767 800 (797) 748 760 608 608 543 534 478 478 422
3076 1690 1716 1659 1653 1624 1623 1465 1470 1427 1420 1387 1394 1317 1312 1223 1226 1151 1162 1120 1123 995 99 5 808 803
6 C-H str 2 C=O str
1148 1156 914 922 794 794 (752) 760 624 62 1 556 542 480 477 422 392 355 353
3079 1692 1717 1662 1650 1615 1616 1485 1488 1421 1416 1358 1362 1330 1326 1221 1222 1088 1092 1151 1156 773 (770) 807 803 757 770 614 613 547 535 480 478 423 395 353 352
353 353
350 354
obsd calcd
1.07 1.096
1.08 1.090
1.92 1.974
1.99 1.968
1.94 1.970
3.82 3.880
1271 1164
str
4 C=O str and C=C str C=C str and 4 C=O str ring str N H bend ring str ring str ring str C H bend NCH3 str C H bend ring str ring breathing
754 617 613 564 547 472 467 422 392 313 315
ring bend ring bend ring, 4 C=O bend ring, C=O bend NCH, bend
1.30
Qualitative description of probable dominant contributions; not based on normal-coordinate calculations; character may change with isotopic substitution. bApplied to ratio of MeU/derivative frequencies.
TABLE XII: Out-of-Plane Fundamental Frequencies (cni') of 1-Methyluracil (MeU) and Isotopic Derivatives MeU-4mode MeU MeU-2-I80 MeU-3-d MeU-5-d MeU-6-d MeU-5,6-d2 y22 IR 990 990 99 1 975 854 800 R 983 983 983 983 968 853 797 ??3 IR 869 865 868 624 864 (864) 869 R v24 IR 757 755 758 752 754 757 755 R b 5 IR 724 723 723 725 582 717 583 R 723 723 725 578 716 579 y26 IR R 446 446 446 446 446 443 442 y17 IR (430) 430 430 (430) 430 392 39 1 R 40 1 403 y28 IR 257 256 259 R 267 266 266 264 267 266 264 y29 IR 195 (195) 195 (195) (195) (195) (195)
CD,U
approx descripn' 6 C-H bend
983 869
N H bend
749
ring bend
724 725
5 C-H
443 43 1
ring bend ring bend
247 25 1 195
NCH3 bend ring bend C=O bend
bend
R u30
product ruleb
IR R
177 obsd calcd
173
171
177
174
174
171
170
1.04 1.027
1.04 1.029
1.42 1.388
1.30 1.381
1.32 1.378
1.79 1.905
1.12
Qualitative description of probable dominant contribution; not based on normal-coordinate calculations; character may change with isotopic substitution. bApplied to ratio of MeU/derivative frequencies.
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The Journal of Physical Chemistry, Vol. 88, No. 15, 1984 3257
1-Methyluracil and Isotopic Derivatives
TABLE XIII: Fundamental Freauencies (cm-') of the Methvl Grow in 1-Methvluracil (MeU) and Isotopic Derivatives ~
MeU-4mode y31 y32
y33 y34
u35 y36
y37
V38
v39
product rule
IR R IR R IR R IR R IR R IR R IR R IR R IR R
MeU
MeU-2-180
2980
2980
2948
2947
2840 1462 1468 1438 1442 1432 1432
1459 1466 1437 1442 1425 1431
1135 1048 1046
1048 1045
approx descripna
MeU-3-d
MeU-Sd
MeU-6-d
MeU-5,6-d2
CD3U
2980
2919
2980
2980
2987
2241
op C H str
2947
2936
2945
2947
2945
2173
ip CH str
2842 1461 1467
2841
2833 1459 1464
2838 1458 1465 1443 1436 1432
2123 1084 1082 1044 1050 1060 1060
ip CH str ip CH bend
1442 1430 1429
2834 1457 1465 1437 1444 (1437) 1431
1134 1049 1046
1044 1043
893 783 786
OD
1045
180
1443 1437 1437
1456 1448 1443 1427 1434
1132 1048 1046
1045 1041
op C H bend ip C H bend
~-wag ip wag
torsion
C C
obsd calcd
21.0b 2016
r? Qualitative description of probable dominant contribution; not based on normal-coordinate calculations; character may change with isotopic substitution. bApplied to ratio of all 38 assigned frequencies (Tables XI-XIII), with a factor of 1.41 applied for the unobserved q g .'Not observed.
that these frequencies remain constant except on deuterium substitution at C-5 or C-6. In MeU-5-d v2 shifts to 2336 cm-l (a frequency ratio of 1.34), while u3 remains unaltered. In MeU-6-d v 3 shifts to 2297 cm-I (also a ratio of 1.34), and v2 is unchanged. The frequency assigned to v 2 is unusually high for an aromatic or olefinic C-H stretch. It is considerably higher than assigned by several other authors for uracil and other nucleotides4-6,8but at essentially the same frequency found for uracil in a matrix.l0 The deuteration shifts are very clear in the Raman spectra, and the assignment is unambiguous. It is somewhat surprising that the stretching vibrations of these two vicinal hydrogen atoms are completely uncoupled. In the related molecule uracil, the two C-H stretching modes were characterized as "in-phase" and "out-of-pha~e".~Again, the evidence of the monodeuterio derivatives of MeU is clear, however, that the two C-H stretches are not coupled. The N-H stretch, v l , is expected to be the highest frequency in the isolated (non-hydrogen bonded) molecule. Szczesniak et al.1° recently assigned the N H stretch at 3415 cm-I in the IR spectrum of 1-methyluracil in a nitrogen matrix and the N3H stretch in uracil at 3423 cm-', with the latter shifting to 2549 cm-I in uracil-1,3-d2. In the solid, hydrogen bonding is expected to lower the N H frequency. Other authors have assigned it in the vicinity of 3130-3160 cm-' in solid uracil and 1-methyluracil. Our IR spectrum of MeU (Table 111) shows, in addition to the bands assigned to v 2 and v3, only the band of medium intensity at 301 5 cm-I as a reasonable candidate for vl. In MeU-3-d the weak bands at 2235 cm-I (IR), 2227 cm-I (R) can probably be assigned to the N D stretch. If similar isotopic shift factors apply in solid MeU and in the isolated uracil molecule, this implies that v 1 should be about 3000 crn-'. Thus, the IR band at 3015 cm-I is in the correct range and is assigned as vl. Note that the Raman band at 3023 cm-I in MeU cannot be assigned to v, since it occurs also in MeU-3-d (Table VI). On the basis of its behavior with isotopic substitution, especially deuteration of the methyl group (Tables IV-X), it is assigned to a combination, v4 + vl0. C=O and C=C Stretching Modes. MeU has three bands between 1600 and 1730 cm-' (v4, v5, and v6) which correspond primarily to the stretching vibrations of the C=O bonds and a C=C bond. v4 at 1695 cm-I (IR), 1718 cm-' (R) undergoes a shift of -20 cm-' in MeU-2-I80 but exhibits little or no change in MeU-4-I80, MeU-5-d, MeU-6-d, or CD,U. v4 corresponds primarily to the Cz=O stretch, with smaller contributions from other modes. v5 a t 1660 cm-' (IR), 1652 cm-' (R) has a major contribution from the C4=0 stretch but is mixed to a significant extent with other modes. v6 at 1623 cm-' (IR), 1622 cm-' (R) exhibits a large shift in MeU-6-d and MeU-5,6-d2 (-22 cm-l)
and a smaller shift in MeU-5-d. There is no shift in MeU-2-ls0 and only a small one in MeU-4-180. These data support a major C = C stretching character of the vibration, with greater movement of C-6 than of C-5 and probably a small contribution of C4=0 stretch. We note the contrast between MeU-3-H and MeU-3-d in v5 and v6 in which the C=O and C=C character of the two modes is strikingly changed (vide infra). The infrared frequencies of MeU bands in KBr pellets differ in the double-bond region from those of Nujol mulls but are in other regions quite similar. For example, the double-bond vibrations are observed at 1701, 1678, and 1620 cm-' in a KBr pellet but at 1695, 1660, and 1623 cm-I in a Nujol mull. It appears that the molecules are dispersed as a solid solution in KBr rather than as crystallites and that the double-bond frequencies, with their C=O character, are most strongly affected. The pattern of frequency shifts on isotopic substitution is essentially the same for mulls and for KBr pellets. These data lead us to the same assignments as those presented above for the mulls. The infrared spectrum of polyuridylic acid has been measured in H 2 0 in the carbonyl region (Table XIV) by computer subtraction of the water spectrum (the solubility of MeU was too low for convenient measurement, but the spectra in this region are quite similar). In this case, as well as in the KBr pellets, only a single broad band ( A q p = 60 cm-I) was observed above 1650 cm-' with v, at 1695 cm- and a possible shoulder at about 1670 cm-I. In contrast to the Raman, where v4 is not resolved, it is v5 which is not resolved in the infrared spectrum in H 2 0 solution. We include an explicit discussion of MeU-3-d in the doublebond region primarily because of its relevance to the extensive polynucleotide studies in D 2 0 referred to in the Introduction. Spectra of several of the isotopic derivatives of MeU measured in D 2 0 solution are summarized in Table XV. v4 (1684 cm-l, IR; 1683 cm-', R) is primarily a C2=0 stretching vibration as in MeU itself) but also involves small contributions from motions of the C4 oxygen and N , methyl group. v5 is assigned at 1652 cm-' (IR), 1654 cm-' (R) but is more difficult to describe since l S 0 and 6-d substitution produce only small shifts. In contrast, v5 of MeU itself has major C4=o stretching character. v6 is assigned at 1618 cm-' (IR), 1620 cm-' (R) and exhibits large shifts on 4-180 and 6-d substitution. We conclude that this mode is predominantly a coupled vibration of the C=C double bond and the 4-carbonyl group with minor mixing of C2=0 motion. Again, the contrast with v6 of MeU-3-H is striking. N-H Bending Mode. This fundamental, vx, is readily identified in spectra of aqueous solutions of 1-methyluracil. Both infrared and Raman spectra show in H 2 0a band at 1416 cm-I which is absent in D,O.' Polarized Raman single-crystal experiments show
3258 The Journal of Physical Chemistry, Vol. 88, No. 15, 1984
Lewis et al.
TABLE X I V Infrared and Raman Bands in H20Solution
1-MeU
MeU-4-ls0 IR
R
IR 1695' s 1636O w 1485 m 1467 w 1437 w 1416 m 1389 m -1370 w, sh 1332 m
1676 vs 1630 m 1488 vs 1465 vw 1438 w 1417 vw 1389 m
R
IR
MeU-6-d R
IR
1668 vs -1625 sh
-
,1481 m 1467 w 1438 w 1407 m 1389 m 1332 m
1333 w 1242 vs 1195 vw 1162 w 996 vw 800 w 796 vs 628 vw 600 w 557 w 479 w
MeU-2-I80
1438 w 1407111 1390111 1333 w 1244 vs 1195 vw 1162 w 997 vw
1483 m 1465 w 1436 w 1411.5 vw 1389 m *,1370 w 1331 m
768 vs 600 w 556 w
1483 m -1460 w 1412 m -1389w 1372 m 1332111
MeU-5-d R
1675 vs 1630 m 1488vw 1438 w 1412vw 1389111 1330w 1243 vs 1195 vw 1162 w 993 vw 797 m 766 vs -630 vw 600 w 550 w
IR
1483 m -1460 w 1438 vw 1415 m 1390 vw 1365 w 1331 m 1225 w 1175 vw
-
CD,U IR R
R
1482 m 1467 w 1415 m 1320 m 1237 vw 1197 w 1131 vw
uMeasured with polyuridylic acid for increased solubility (cf. ref lh). vI4 all include contributions from the N D bend.
TABLE X V Infrared and Raman Bands in D20Solution
MeU IR 1684 1652 1618 1486 1459' 1437 1390
R 1683 1654 1620 1488 1460 1437 1391 1272 1213 1170 1120 1070
MeU-2-I8O IR R
1667 sh -1671 1679 1652 1650 1648 1604 ,1615 1613 1484 1488 1479 1460 1458 1459 1437 -1437 sh 1435 1391 1389 1389 -1370 1270 1213 1170 1120 1065
-
925 788 752 620 543 483
925 79 1 753 622 551 483
'6(HOD)
-
MeU-4-180 IR R 1677 br 1652 1605 1488 1460 1437 1390
MeU-64 IR 1684 1646 1601 1484 1451 ,1435 , . 1390 1372
1270 1213 1170 1120 1081 1065 920 782 753 621 549
1457 cm-'.
the band (1418 cm-I) to be in-plane polarized (Table 111). There is a relatively large isotopic shift (-9 cm-I) on 4-180 substitution but only a -2-cm-l shift on 2-180 substitution. The N-H bending is thus strongly coupled to C4=0 stretching and less so to the Cz=O vibration. We have also observed the related molecule, uracil, and its I5N derivative, uracil-1,3-I5N2. There are two N-H vibrations at 1509 and 1418 cm-' in the normal molecule, assigned to 6(Nl-H) and 6(N3-H), respectively, by Susi and ArdS5In ura~il-1,3-'~N~, these bands shift, -16 and -4 cm-I, respectively, indicating major motion of N l in 6(Nl-H) and significant motion of N 3 in 6(N3-H). We assume that the result with 6(N3-H) in uracil indicates a qualitatively similar contribution of N 3 motion of v8 of 1-methyluracil. Possibly, this contribution may be described as a C4-N3 stretch coupled to the bending vibration. Assignment of v8 in MeU-3-d is not straightforward. Rather than a single band shifting to about 1000 cm-I, three bands change frequency, indicating a marked redistribution of the coupling in the normal modes. A redistribution is also generally consistent with the normal-coordinate calculation of Tsuboi et a1.,6 which indicated contributions of N-D bending to modes calculated at 1258, 1091, and 904 cm-'. We assign the bands at 1119 cm-l (IR), 1131 cm-' (R) to v8 but note that in MeU-3-d v8, vI1, and
Ring Stretching Modes. In addition to the C=C stretch, discussed previously, there should be five vibrations, v,, v9, vl0, vI1, and ~ 1 5 that , can be described approximately as ring stretching modes. The strong infrared band in MeU at 1486 cm-' (1488 cm-', R) is assigned to q. Its frequency is virtually constant with isotopic substitution except for approximately a 20-cm-' shift on deuteriomethylation. The strong band at 1379 cm-I (IR), 1384 cm-' (R) in MeU is assigned to v9. It is invariant to I8O, 3-d, and I-CD, substitution. The deuteration shifts indicate significant localization of the motion of C-5 and C-6, possibly accompanied by a very small amount of mixing with the hydrogen bending vibrations. Tsuboi et aL6 calculate the frequency of this mode about 100 cm-' higher than our assignment, and Susi and Ards assign the vibration at 994 cm-I, a frequency we assign to a C-H bend (vide infra). The band in MeU at 1331 cm-I (IR), 1327 cm-' (R) is largely unaffected by isotopic substitution and is assigned to vlo. It decreases by 15 cm-' in CD3U and increases by 5 cm-' in MeU3-d. The strong Raman band in MeU at 1228 cm-I, with its weak infrared counterpart at 1224 cm-', is assigned to v l l . It too is almost unaltered by isotopic substitution except in MeU-3-d, where it appears at 1271 cm-I. In MeU-3-d the entire frequency distribution in this region is different from the other isotopic species, as discussed previously in the assignment of the N-D bending mode. The final ring stretching mode, vI5, is assigned at 808 cm-' (IR, ip), 804 cm-' (R, ip), a frequency that is almost invariant to isotopic substitution. C-H Bending Modes. There are no prominent infrared or Raman bands with appropriate frequency shifts on deuteration that are obviously attributable to the two in-plane C-H bends, vlZ and ~ 1 4 .We assign to vl2 the weak bands at 1161 cm-' (IR), 1162 cm-' (R) in MeU, 1088 cm-' (IR), 1092 cm-' (R) in MeU-J-d, and 834 cm-' (IR), 833 cm-' (R, ip) in MeU-5,6-dZ. In MeU-6-d the assignment is not certain, but we choose a weak Raman band at 973 cm-', which has no infrared counterpart. Another possible choice is 1014 cm-' (IR), 1026 cm-I (R), but the frequency shift between MeU and MeU-6-d would then be too small to fit the product rule. For v14 we assign 995 cm-' (IR, R) in MeU, 794 cm-' (IR), 796 cm-' (R, ip) in U-5-d, and 718 cm-' (IR, R) in MeU-5,6-d2. In MeU-6-d we assign as v14 the Raman in-plane polarized band at 864 cm-'. Its infrared counterpart is obscured by the broad band at 864 cm-I, which we shall later confidently assign to ~23,the out-of-plane N-H bend. (A possible alternative choice for v i 4 in MeU-6-d is 897 cm-' (IR), 896 cm-' (R, ip), but we think this less likely on the basis of the product rule.) Overall, these assignments seem quite reasonable but are not absolutely firm.
1-Methyluracil and Isotopic Derivatives Susi and Ard8 show the C-H bends as a component of several modes but assign the C-H bends principally at 1434 cm-' (IR), 1444 cm-' (R) and a t 1202 cm-' (IR, R). The former Raman band, 1442 cm-' (Table 111), is clearly an out-of-plane mode that cannot be v I 2 . (We assign it later to a CH3 deformation.) The band a t 1202 cm-' does not show any appreciable isotopic shift; hence, it cannot be a C-H bend. (We assign it to the overtone of v I 7 ,interacting via Fermi resonance with v l l , which gives strong Raman bands in all isotopic derivatives.) N-Methyl Stretching Mode. One vibration, vI3,might be described approximately as a stretching of the entire methyl group relative to the nitrogen atom to which it is attached. The band at 1146 cm-I (IR), 1154 cm-' (R) in MeU, which shifts to 1120 cm-' (IR), 1123 cm-' (R) in CD3U, has appropriate behavior for such a vibration and is assigned to ~ 1 3 . The shift on deuteriomethylation is smaller than might be expected, suggesting that this mode is mixed. We have seen that vl, vl0, and v12all undergo small shifts (10-20 cm-I) on CD3 substitution. Ring Bending and C=O Bending Modes. Of the five fundamentals, V16-v2O3 three might best be described as in-plane distortions or bending of the uracil ring and two as C=O bends, but there is probably some mixing. The very intense band at 773 cm-I in the Raman spectrum of MeU (761 cm-I, IR) is confidently assigned to It shows only very minor alterations in frequency with isotopic substitution. The second ring bending mode, ~ 1 7 , is assigned at 628 cm-' (IR), 630 cm-' (R, ip) in MeU, with small shifts in the other isotopic species. v18is assigned at 563 cm-' (IR), 547 cm-l (R, ip) and is also relatively invariant to isotopic substitution, with the largest change, about 15 cm-I, occurring in MeU-5-d. The 5-cm-' shift in MeU-2-I80 may indicate some C=O bending character. The fourth vibration, v19, is assigned a t 482 cm-' (IR), 478 cm-I (R, ip) in MeU. It shifts about 15 cm-' on deuteriomethylation and about 5 cm-' on 4-180 substitution, with smaller shifts on other isotopic substitutions. For v2,,, the final one of the five modes, the leading candidates are the IR band of medium intensity at 426 cm-' in MeU and the weak Raman band at 394 cm-' in MeU. Both are in-plane polarized. We believe that both of these bands represent urn,with a very large g-u splitting. Both bands have essentially the same shifts of 5-8 cm-' on both 2-180 and 4-lS0 substitution. The large g-u splitting may be explained by large dipolar and hydrogen-bond interactions in the dimer. N-Methyl Bend. The lowest frequency in-plane vibration, vZ1, is assigned at 356 cm-I (IR), 353 cm-' (R) in MeU and at 318 cm-I (IR), 315 cm-' (R, ip) in CD3U. This mode is primarily a bending of the entire methyl group. Isotopic substitutions other than deuteriomethylation are generally small, but the shift of about 5 cm-' on 2-180 substitution indicates some participation of 2carbonyl bending in this mode.
Out-of-Plane Vibrations Excluding the internal methyl vibrations, there are nine outof-plane fundamentals, v21-v30,which can be described approximately as an N-H and two C-H bends, two C=O bends, a bend of the entire CH, group relative to the uracil ring, and three ring distortions (essentially twisting motions about the bonds in the ring). Actually, considerable mixing occurs among many of these modes. The N-H bend ~ 2 is3 readily assigned as the broad band at 869 cm-I in the IR spectrum of MeU, which shifts to 630 cm-' in MeU-3-d (a frequency ratio of 1.38). One of the C-H bending modes, v22, is readily assigned at 990 cm-' (IR), 893 cm-' (R, op). In MeU-5-d, MeU-6-d, and MeU-5,6-d2, the corresponding out-of-plane polarized Raman bands are easily located at 968, 853, and 790 cm-I, respectively. The deuteration shifts show that this mode is largely a bending of the 6-hydrogen. The other C-H 5 associated with the 5-hydrogen) is found at 724 bend ~ 2 (largely cm-I (IR), 723 cm-' (R, op), with its counterparts at 582 cm-l (R, op) in MeU-6-d and 583 cm-I (IR), 579 cm-' (R, op) in MeU-5,6-d2. The IR band of medium intensity at 757 cm-I in MeU is clearly an out-of-plane mode, as shown by the IR spectrum of an oriented
The Journal of Physical Chemistry, Vol. 88, No. 15, 1984 3259 sample. Its insensitivity to isotopic substitutions (Table XII) marks it as a ring mode; we assign it as the fundamental ~ 2 4 . No Raman counterpart is observed (except possibly in MeU-6-d and MeU5,6-d,), but that is not surprising in view of its proximity to the extremely intense in-plane band at 773 cm-I. The weak but distinctly out-of-plane polarized Raman band at 267 cm-' in MeU (IR counterpart probably at 257 cm-I) is assigned with confidence to the methyl bending mode, v28. This band undergoes only a 1-3-cm-I shift on deuterium or I8O substitution in the ring but shifts 16 cm-' on substitution of CH3 by CD,. The 6.5% shift is quite reasonable for a motion that is primarily a bend of the entire methyl group. The medium-intensity out-of-plane polarized Raman band at 446 cm-I in MeU shows virtually no shift on any isotopic substitution. This is almost certainly a fundamental, v26, probably a ring deformation centered primarily in a C N bond. We observe no IR counterpart for this band. In MeU-6-d and MeU-5,6-d2, where the Raman frequencies of v26 are 443 and 442 cm-', respectively, an additional intense out-of-plane polarized line appears at 401 and 403 cm-I, respectively. A Raman band of this intensity is very probably a fundamental, v2,. No counterparts are found in the Raman spectra of any of the other species. However, the IR counterparts of the Raman bands at 401 and 403 cm-I and weak IR bands in the other isotopic derivatives at 430 cm-I may be due to ~ 2 7 . We have no explanation for the reduction in intensity of the Raman band so as to render it unobservable in these isotopic derivatives except to suggest that the character of the normal mode varies considerably with substitution and that there is an accidental cancelation of polarizability change in those species. There are a number of bands observed in the Raman spectra at frequencies below 200 cm-I, one or two of which may be fundamentals. Harada and Lord,I3 in their study of the lowfrequency vibrational spectra of 1-methyluracil and 1-methylthymine, assigned the bands in MeU at or-below 121 cm;' to lattice modes. Their assignment for 1-methylthymine was supported by a normal-coordinate calculation for the lattice modes that is compatible with lattice modes at 119 cm-I and below. (Later calculations by E r i n et al. supported their concl~sions.'~)Harada and Lord thus relegated the observed Raman bands in MeU at 177 and 150 cm-I to intramolecular fundamentals. For the isotopic derivatives we find only small frequency shifts, which might be compatible with either intermolecular (lattice) vibrations or with intramolecular modes (excluding the methyl torsion). The Raman band in MeU at 177 cm-' certainly seems too high for a lattice mode, and we assign it with reasonable confidence to the fundamental ~ 3 0 . In order to exclude lattice modes, we attempted to obtain a Raman spectrum in the low-frequency region of a saturated solution of MeU in dimethyl-d, sulfoxide. Because of low solubility, the quality of the spectrum was poor, but a shoulder on a sloping background appeared at approximately 185 cm-'. We take this as support for the assignment of a band in this region to ~ 3 0 . We were unable to discern a band in the vicinity of 150 cm-' and tentatively ascribe the band at 150 cm-I to a lattice mode or possible a difference band, rather than a fundamental. We examined the far-infrared spectra of MeU, MeU-4-I80, and CD3U and found bands of moderate intensity at 195, 98, and 78 cm-I, essentially unchanged in frequency among the three derivatives. The last two bands are almost certainly lattice modes, but the band at 195 cm-I is assigned to an out-of-plane fundamental, vZ9. An alternative possibility, that this band is due to the methyl torsion, can, of course, be excluded by the lack of frequency shift on CD, substitution. We also considered and rejected the possibility that the Raman band at 177 cm-' is the g counterpart of the infrared fundamental at 195 cm-I, since the frequency shifts on '*Oand CD3 substitution are quite different. (13) I. Harada and R. C. Lord, Spectrochim. Acta, Part A , 26A, 2305 (1970). (14) D. Kirin, L. Colombo, K. Furic, and W. Meier, Spectochim. Acta, Part A , 31A, 1721 (1975).
3260 The Journal of Physical Chemistry, Vol. 88, No. IS, 1984 Overall then, we regard the assignment of the out-of-plane modes as reasonably satisfactory, with six fundamentals very firmly assigned and with considerable (though not conclusive) evidence supporting the other three assignments. The product ratios, as given in Table XII, indicate that the assignments are compatible with the product rule.
Methyl Vibrations There are nine vibrational modes which can be attributed to internal methyl group motions. There is little interaction between these modes and those of the uracil ring. Methyl motions may appear with in-plane or out-of-plane polarization, depending on the particular normal mode, and the polarization may be utilized as an aid for the assignment. The carbon-hydrogen stretching modes of the methyl group occur below 3000 cm-'. The asymmetric methyl stretch (doubly degenerate for an isolated methyl group) is observed as an outof-plane polarized Raman band at 2980 cm-', ~31,and an in-plane doublet at 2948 and 2935 cm-', v32,one member of which presumably arises from Fermi resonance with the overtone of the in-plane degenerate deformation of 1468 cm-'. The symmetric methyl stretch, v33,occurring with only in-plane polarization, is assigned at 2840 cm-I. These bands shift down to the 21002250-cm-I region in CD3U. The asymmetric (degenerate) methyl deformations, v34 and v35, are assigned at 1462 (IR), 1468 (R, ip) and at 1437 (IR), 1442 (R, op). The bands are, of course, greatly shifted on deuteriomethylation, and the in-plane vibration appears at 1084 cm-I (IR), 1082 cm-' (R) and the out-of-plane band at 1044 cm-' (IR), 1050 cm-' (R) in CD3U. It is interesting that other isotopic substitution produces little change in these bands, indicating little mixture of the internal methyl and uracil ring motions. The symmetric methyl deformation, v36, is in-plane polarized and is assigned at 1432 cm-' (IR and R). In CD3U, this vibration is assigned at 1060 cm-I (IR and R). Susi and Ard8 assign the symmetric CH3 deformation at 1378 cm-l, a frequency characteristic of this vibration in hydrocarbons. However, for the CH3N moiety, the frequency is expected to be higher (1418-1488 ~ r n - ~ ) . ' ~ , ' ~ (15) L. J. Bellamy in "The Infrared Spectra of Complex Molecules", Vol. 1, 3rd ed., Chapman and Hall, London, 1975, p 27. (16) C. N. R. Rao in "Chemical Applications of Infrared Spectroscopy", Academic Press, New York, 1963, p 139.
Lewis et al. The methyl rocking mode has in-plane and out-of-plane components. The latter, v37,probably appears as a very weak outof-plane polarized transition in MeU at 1135 cm-'. There is no visible IR counterpart, and an analogous weak out-of-plane band appears for CD3U at 893 cm-I. The in-plane methyl rock, v38, is assigned at 1048 cm-I (IR), 1046 cm-' (R, ip). The band shifts to 783 cm-' (IR), 786 cm-' (R, ip) in CD3U. The only methyl motion not accounted for is the methyl group torsion or internal rotation, v39. This is expected in the 200300-cm-' region but has not been observed.
Consistency with the Product Rule On the assumption that the nine internal methyl vibrational modes are completely separable-an assumption that is verified to a high degree of approximation by the data, as indicated in previous sections-each isotopic derivative provides independent product rule checks of in-plane and out-of-plane frequencies. The experimental values given in the last lines of Tables XI and XI1 show generally acceptable agreement with the theoretical values. For the l 8 0 species, the shifts are so small that experimental uncertainties in the frequencies largely determine the exact numbers obtained. For the deuterated species anharmonic effects are expected to produce experimental ratios that are 2-3% too small. However, in the present work other discrepancies can arise from crystal field coupling effects, as discussed previously, and from the effects of Fermi resonance. Some of the values for out-of-plane modes show discrepancies of more than 5%, which suggests that some of the low frequencies that were assumed invariant in lieu of direct measurement may be in error. Acknowledgment. We thank Drs. I. W. Levin and R. Pearce for their investigation of the normal coordinates of 1-methyluracil and discussion of these results. We are also indebted to Dr. Henry Fales for his aid in measuring and interpreting the mass spectra. We wish to thank Mary Lou Miller for her excellent help in preparation of the manuscript. Registry No. I, 615-77-0; I-2-I80,57362-90-0; 1-4-'*0, 90742-77-1; I-3-d, 18372-36-6; I-5-d, 90742-78-2; I-6-d, 90742-79-3; 1-5,6-d2, 90742-80-6; I-a,a,a-d3, 90742-8 1-7.
Supplementary Material Available: Details of isotopic synthesis and characterization of products, tables of infrared and Raman frequencies of isotopic derivatives (Tables IV-X), and figures illustrating Raman spectra (Figures 1-3) (3 1 pages). Ordering information is given on any current masthead page.
