IR matrix isolation studies of nucleic acid constituents: the spectrum of monomeric thymine

Journal of Molecular Structure, 193 (1989) 35-49 Elsevier Science Publishers B.V., Amsterdam - Printed

35 in The Netherlands

IR MATRIX ISOLATION STUDIES OF NUCLEIC ACID CONSTITUENTS: THE SPECTRUM OF MONOMERIC THYMINE

MACIEJ J. NOWAK Institute of Physics, Polish Academy of Sciences, Al. Lotnikow 32/46, 02-668 Warsaw (Poland) (Received 30 November

1987)

ABSTRACT The IR matrix isolation spectra of thymine and N,,N3-dideuterated thymine in argon and nitrogen matrices are reported. The spectra are interpreted by comparison with the previously assigned spectra of uracil. The frequencies of the NH stretching and bending modes are not much changed on methyl substitution in the 5-position of the uracil ring. The most pronounced differences are observed in the frequency region of the ring vibrations.

INTRODUCTION

This work is a continuation of a series of IR spectroscopy investigations of pyrimidine derivatives of biological significance, such as uracil [ 11, N1- and N,-methylated uracils [ 21, cytosine [ 31, and their analogs such as 2-oxopyrimidine [4,5] and 4-oxopyrimidine [ 4,6,7]. Matrix isolation is a unique method which provides high quality, well resolved vibrational absorption spectra under conditions where the influence of the environment on the studied molecule is minimized. One of the advantages of such spectra is that they can be credibly assigned by the help of quantum mechanical calculations (of sufficiently high level), which generally do not consider the influence of environment on the spectra. Matrix isolation is also an excellent method for determining the structure of molecules in a weakly interacting environment. Some of the pyrimidine derivatives exhibit a strong dependence of tautomeric equilibrium constant, KT [keto/enol] , upon “reactivity” of the environment. This was found for 2oxo- and 4-oxo-pyrimidines [ 4,6]. Also in cytosine and guanine the rare tautomeric forms: enol-amino, which are absent in polar media, were detected in inert matrices [ 3,8]. The IR spectra of thymine were studied under several conditions: in Hz0 solution [ 91, solid state [ lo,11 1, gas phase [ 121 and also isolated in Ar matrix [ 131. In this paper we present IR absorption spectra in the range 4000-200 cm-l of thymine isolated in Ar and Nz matrices and of the N,,N,-dideuterated analog. Owing to the lack of quantum mechanical ab initio calculations of nor-

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0 1989 Elsevier Science Publishers

B.V.

36

ma1 modes of thymine, only an approximate assignment of the spectra is proposed. This assignment is based on comparison with the previously interpreted spectrum of uracil [ 141 and on the effect of deuteration on frequencies of the absorption bands. EXPERIMENTAL

The spectra were taken on the Perkin-Elmer 580B IR grating spectrometer working in a mode which allowed a resolution of about 2 cm-l (several cm-’ in the low frequency region). The cryostat and the method of preparing of matrices is described elsewhere [ 11. The temperature of the CsI window on which the matrices were formed was about 8 K. The matrix gases, spectral grade argon and nitrogen, were obtained from VEB Technische Gase Leipzig, G.D.R. No traces of water or other impurities were detected in the spectra. Thymine was obtained from Merck AG Darmstadt, Germany. No evidence of thermal decomposition of thymine after sublimation onto the matrix window was found. The conditions of deposition (chosen experimentally) were such that the formation of associations in a matrix was avoided. Deuterated samples were prepared by repeated recrystallization from D20. The rate of deuteration was better than 90%. Residual absorption by incompletely deuterated species was recognized by comparison with the spectra of thymine which was intentionally not completely deuterated. RESULTS AND DISCUSSION

Tautomerism of thymine Thymine, like uracil, has the potential ability to exist in different tautomeric forms [ 151. Tautomeric equilibria of nucleic acid bases are the subject of continuing interest as the existence of rare enol tautomeric forms could be related to the occurrence of point mutations during DNA replication [ 161. The experimental results in the case of thymine are the same as for uracil [ 11. No traces of OH absorptions could be detected in the matrix spectra, which means that the population of the enol form (if such exists) in the gas phase, from which the matrix was formed, is below the limit of sensitivity of the spectrometer, i.e. below 1%. It is worth mentioning that recently, the UV emission (interpreted as due to the traces of the keto-enol form of uracil and thymine) was detected in a supersonic jet [ 171. This does not contradict the present result because the expected yield of fluorescence of the enol forms should be much higher than that of the keto form. Hence the small amount of the rare form (which is too low to be detected by IR) could be recognized by means of emission spectroscopy.

37

Infrared absorption spectra At present the spectra of almost all monomeric nucleic acid bases isolated in inert matrices such as those of uracil [ 14],1-methyluracil [ 21, cytosine [ 31 and guanine [ 181 are interpreted on the basis of quantum mechanical calculations. There is an evident lack of data for monomeric thymine, hence this work aims to present experimental data concerning the IR levels of thymine using matrix isolation spectroscopy. The data presented here agree, within the limit of experimental accuracy, with the results of Radchenko et al. [ 131. The IR spectra are shown in Figs. 1-4, and the frequencies and relative absorption band intensities are listed in Tables 1 and 2. Thymine has 39 fundamental modes of which 26 are in-plane modes; therefore the proper assignment of all the absorption bands without the help of high level quantum mechanical ab initio calculations is generally not possible. The assignment presented here is based mainly on that obtained for uracil, with [1,14] and without [19] the help of quantum mechanical calculations. The calculations of in-plane fundamentals scaled to the spectra of crystalline thymine of Susi and Ard [ 111 do not fit the spectra herein well. The comparison of the spectra measured in different environments (argon and nitrogen matrices) and deuteration effects studied in this work greatly helped in identification of some characteristic absorptions of thymine. This method is successful in the case of bands connected with vibrations of external groups such as N-H or C=O which are not so highly

1 0.2-

I

I I 3500 Fig. 1. The IR absorption

3LOO

I

2600

2500 cm-'

spectra in NH and ND stretching

in argon and nitrogen matrices

and N,,N,-dideuterated

frequency regions of thymine isolated

thymine isolated in Ar matrix.

B

$%,: -.._/

__I

I’ :

L--..

C

--we______---

I -

I -

L

L I

1750

1700

!I \

1650 cm-’

Fig. 2. The IR absorption spectra in carbonyl stretching frequency region oE A, uracil (Ar); B, thymine (Ar); C, thymine (N,); D, N,,N,-dideuterated thymine (Ar); isolated in low-temperature matrices.

coupled with other modes. Empirical assignment of the ring vibrations is next to impossible because of pronounced coupling between the ring stretching and bending modes with C-CH, stretching and CH3 rocking modes absent in uracil. In N-deuterated species this is even worse because ND deformations interact additionally with ring modes resulting in significant differences between spectra of deuterated thymine and uracil (Fig. 4). NH and ND stretching

IR spectra of thymine and N,,N,-dideuterated thymine in this region are illustrated in Fig. 1. Monomeric thymine in Ar matrix gave a pair of bands at 3480 cm-l and 3434 cm-‘. In nitrogen those bands are shifted towards lower frequencies: 3466 cm-’ and 3419 cm-‘. The frequencies and relative intensities are very similar to those in uracil (Table 3); hence we interpret the band

39

(a)

(b)

ii,

I

l&O0

1600

,200 cm"

1100

700

900 cm

(C)

L A

h

12-

Fig. 3. The comparison of survey IR absorption Ar matrix; C, thymine in N, matrix.

spectra oE A, uracil in Ar matrix;

B, thymine in

40

4

0.2

B 0.1

L I

I

4

900

700

500

cm“

Fig. 4. The comparison of survey IR absorption spectra OEA, N,,N,-dideuterateduracil; B, N1,N3dideuterated thymine isolated in Ar matrices.

of higher frequency as due to N,-H stretching and the second one to N3-H stretching. The NH absorptions in gas phase reported by Ferro et al. [ 121 have lower frequencies (3434 cm-’ and 3407 cm-‘) which is highly unusual since the matrix environment lowers NH stretching frequencies. In deuterated thymine the ND absorptions are split into several bands, with the three stronger bands dominating: 2588 cm-‘, 2567 cm-l and 2538 cm-‘. In pyrimidines the NH frequency shift after deuteration is usually constant v (NH ) / v (ND ) = 1.35 [1,2]. This factor was obtained for the bands at 3480 and 2588 cm-’ interpreted here as due to NiH, N,D (respectively). We suppose t,heother two bands of deuterated species are connected with the N,D vibration at approximate frequency 2552 cm-’ (assuming the same shift on deuteration) which is split

41 TABLE 1 Observed frequencies ( v ) , relative absorptions in maxima (I), relative integrated absorptions (A ) for infrared spectrum of thymine in argon and nitrogen matrices” Nitrogen matrix

Argon matrix Z rel.

A rel.

v (cm-‘)

43 40 2 2 3

111 69 7 7 8

117 83 61

124 120 89

57 151

69 191

20 15

29 17

3465 3419 2995 2968 2940 1771 sh 1770 1751 sh 1747 1737 vw 1730 1710 1700 vw 1684 1669 1600 vw 1554 1550 1538 VW 1518 1478 1456 VW

ycrn-‘) 3480 3434 2992 2971 2940 1769 1752 1747 1731 VW 1725 1712 1701 VW 1683 1668

1510 1472 1456 1452 1437 1433 1406 1395 1389 1365 vw 1357 1347 1315 1308 vw 1297 1221 1208 vw 1198 1184

7 55 3 2 2 5 44

7 60 3 2 2 4 39

3 7

3 6

4 2 4

4 3 5

2 2

4 1

3 88

3 92

Z rel.

Assignment A rel.

35 27 1 2 2

143 93 6 8 9

54

123

90

239

22 141

93 294

29 6

76 25

2 2

2 2

4 30

6 58

3

5

1417 1412

15 12

29 22

1390

4

5

1358 1345

2 1

5 3

1192 1188 sh

v(CH,), u(CHs)s v(C,H)

1 u(C,=O) or

1435

1223 1212 VW

uOJ,H) VP&H)

2

4

v(C,=O)

i 1 ,

lJ(C=C) Overtone Overtone Overtone Overtone Overtone BP&H) v(ring)

1

P(CH,)

Wng)

I

B(CW) v(ring)

/3(C-H) u(ring) Overtone u(ring) /3(C-H) B(CH,) Overtone 1

P(CH,)

v(ring)

P(CeH) v(ring) v(ring) u(C-CH,)

36 21

60 34

I

P(N,H) B(N,H) B(C,H)

42

TABLE 1 (continued) Nitrogen matrix

Argon matrix I rel.

A rel.

ycrn-‘) 1143 1140 1087 1005 960 936 890 800 785 vw 764 754 727 662 660 sh 545 545 455 391

2 6 4 9 3 9 3

1 2 9 4 8 5 15 4

19 21 3 39 24 30

21 19 3 29 14 43

10

13

1

16

26

v (cm-‘)

Z rel.

Assignment A rel.

T(CH,) (?I 1008 957

2 5

2 6

901 805 795 769 758 729 686

3 1 2 23 17 2 10

11 2 4 23 17 2 53

585 542 457 400 sh

8 6 10

44 7 15

_

392

11

14

.

Y(C-H) v(C-CH,) v(C-CH,)

v(ring) v(ring)

r(W) Y(W) y(CH) r(CO) YWH,) y(N,H)

B(ring) P(G=O)

B(G=O)

P(C,=C) P(C,=O)

“I (absorptions in the maxima of the bands) and A (integrated absorptions of the bands) were normalized in such a way that the sum of the intensities of the two bands in the region 764-752 cm -’ was constant. Abbreviations: VW,very weak; sh, shoulder; v, stretching; j?, bending in-plane; y, bending out-of-plane; T, torsion.

into the doublet 2567,2538 cm-l by Fermi resonance with the combination of the following bands: 1461 cm-‘+ 1093 cm-‘= 2554 cm-‘.

CH stretching region

In the spectra of pyrimidines the C-H stretches usually have such low intensity that it is difficult to distinguish them from the background. In the case of thymine, where a methyl group is substituted at C5 of the ring, the CHBstretches were also weak, but sufficiently strong for identification. The observed frequencies and intensities are given in Tables 1 and 2.

43 TABLE 2 Observed frequencies ( v ) , relative absorptions in maxima (I), relative integrated absorptions (A ) for infrared spectrum of N,,N,-dideuterated thymine in argon matrix* v (cm-‘)

3479 * 3434 * 2993 2972 2941 2600 vw 2594 sh 2588 2581 2567 2553 2542 sh 2538 2527 2516 1776 1768 l 1762 * 1756 * 1744 1735 1714 * sh 1707 1700 1689 1679 1670 1665 VW 1633 vw 1625 VW 1620 VW 1613 VW 1526 1506 1483 1480 1471* vw sh 1461 1446 1436

Z rel.

A rel.

20 8 11 3

39

10 4 1 8 14 5 11 141 35

15 4 1 28 26 7 22 345 85

184 46 27 22 22

552 110 65 66 52

Assignment

v(N,D)

9

17 4

1

vND ) Overtone Overtone

v(C,=O) or/and v(C,=O)

I

v(C=C) Overtone

Overtones 2 7 2

2 9 3

38 41 57

43 48 83

v (ring) or overtone

B(NID) vbing)

44

TABLE II (continued) v (cm-‘) 1415 1404 * 1389 1363 1353 1323 1303 * 1269 * 1238 1230 VW 1216 * 1207 1196 vw 1164 * 1156 l 1093 1085 1045 1031 1008 927 890 858 777 768 763 753 702 660 l 553 * 528 505 451 413 387 378 l 370 280

I rel.

A rel.

Assignment

3

6

P(CH,) Wng)

8 4 15 65

9

6 19 76

/3(C-H) v(ring) u(ring) j?(C-H) P(CHs) B(N,D) P(ND) v(ring)

3

3

vbing) P(CH)

8

11

10 15 1 7 1 2 9 22 2 2 18 22 3

12 1 10 1 2 17 22 2 2 16 24 3

4 20 11 5 13

5 28 18 8 19

21 2

35 3

9

v(ring)

v(ring)

Y(C-CHB) P(ND)

B(ring) POW) T(CH,) (?) /I(ring) v(ring) v(C5-CHs) (?) p(ring) u(ring) v(C5-CHa) (?) /3(ring) v(ring) v(C&-CHa) (?) y(C,H) P(ring) K&H) v(ring) P(CH,) B(ND) (7)

Y(C*=O) Y(c,=o) u(ring)

Bring) P(C,=O) P(G=O)

YW,D) P(C,=O)

;cc2=0, YPJD) 5

“I (absorptions in the maxima of the bands) and A (integrated absorptions of the bands) were normalized in such a way that the sum of intensities of the two bands in the region 764-752 cm-’ was the same as for undeuterated species. Abbreviations: *, bands due to a small amount of partially deuterated species; VW,very weak; sh, shoulder; v, stretching; p, bending in-plane; y, bending out-of-plane; ‘5,torsion.

45 TABLE 3 Comparison

of the absorption

band frequencies

N,H stretch N,H bend, in-plane N,H bend, in-plane N-H bend, in-plane (C,H bending) N,H bend, out-of-plane N,H bend, out-of-plane

NH modes for thymine

and uracil”

v (cm-‘)

NH mode

NIH stretch

of different

Thymine Uracil Thymine Uracil Thymine Uracil Thymine Uracil Thymine Uracil Thymine Uracil Thymine Uracil

Ar matrix

Nz matrix

3480 3482 3434 3433 1472 1473 1406 1401 1184 1186 662 664 545 551

3465 3470 3419 3423 1478 1477 1417,1412 1405 1192 1192 686 685 585 592

aData for uracil from ref. 1.

Double bond stretching region In the region 1800-1600 cm-’ the Cz=O, Cd=0 and C=C stretching vibrations are expected. As usual in the spectra of two-keto pyrimidines [ 1,2,19], many more than three bands are observed in this region. Figure 2 shows seven bands of different intensity in Ar matrix. The strong dependence of the pattern and relative intensities upon the change of matrix from argon to nitrogen, and upon deuteration, indicates that the Fermi resonance is a cause of the composed multiplet structure in this region. The proper assignment of all bands in this region is difficult. We propose to divide the observed bands into three groups originating from three fundamentals: (1769,1753,1747 cm-‘) the first one, (1724,1712 cm-‘) the second one, and (1684,1669 cm-‘) the third one. From theoretical calculations for uracil it is known that two C=O and C=C vibrations are strongly coupled together. Calculations done for uracil [ 1,14,20] assigned the highest frequency band as due mainly to C2=0 (1760 cm-‘), the second to C,=O (1746 cm-‘) and the third to C=C (1645 cm-l) stretches. The substitution of the methyl group, manifested by the increase of the frequency of the third set of bands, changed the conditions of the coupling. Calculations of transition moments, carried out with a fixed partial charge model [21], showed that the coupling between double bonded stretches in thymine is quite different from that in uracil and suggest that the highest frequency band originates primarily from the Cd=0 stretch, while the other two bands have an

46

almost equal share of C&=0 and C=C vibrations. Usually the C=O absorption bands have a very high absorption coefficient. Such intense bands are placed in our spectrum above 1700 cm-l. The absorptions 1684 and 1669 cm-’ are relatively weak, hence it is supposed that the coupling between C=O and C=C is not so strong and these bands are mainly due to the C=C stretch. NH deformation modes Table 3 presents the influence of 5-methyl substitution in the uracil molecule on the NH stretching and deformation frequencies. It can be seen that the differences between thymine and uracil are not significant and are of the order ofafewcm-‘. The NH bands are considerably easier to determine since they disappear (shift towards lower frequencies) on deuteration. It should be stressed here that the bending modes are usually highly coupled with other vibrations, mainly the ring vibrations (stretching or deformation), hence they are not as characteristic as the stretching modes. The frequencies of all NH modes, not only those of the stretching vibrations, are sensitive to intermolecular interactions with the environment. Frequencies of the stretching modes shift towards lower frequencies, while those of the bending modes towards higher ones. The change of matrix from argon to nitrogen caused sufficiently large changes in frequencies to help with the assignment of the NH deformation bands. We want to mention that the deformation out-of-plane modes as shown in Fig. 3 and Table 3 are extremely sensitive to the environment. Such great sensitivity of NH out-of-plane bands in uracil derivatives to the strengths of hydrogen bonds was observed by Bandekar and Zundel [ 221. The band at 1184 cm-’ in thymine corresponding to the one at 1186 cm-l in uracil is characteristic of all keto-pyrimidines. In the case of uracil this was interpreted as the C&-H bend [ 141, but this band is very sensitive to deuteration. Hence it is deduced that NH bending motions strongly contribute to this absorption which is in accordance with assignment proposed by Barnes et al. [ 191. The potential energy distribution calculated for uracil [20] also confirmed the contribution from N,H and NBH bending to that normal mode. C=O bending modes The carbonyl Cz=O, C4=0 deformation modes in uracil are strongly coupled with each other and with the ring deformation modes [ 201. The same behaviour is expected for thymine. Experimentally it was not possible to determine the contribution from other modes in these bands. Our assignment is based mainly on the observed small shift of the bands after deuteration, on a weak shift towards higher frequencies in nitrogen matrix in relation to the argon matrix and the comparison with the frequencies of uracil. The frequencies connected with carbonyls are collected in Table 4. The band of uracil at 769 cm-’

47 TABLE 4 Comparison between thymine and uracil”

the frequencies

of the absorption

bands of different

Thymineb

C,=O) bend, out-of-plane (C,-C, stretch”) C,=O, Cd=0 bend, in-plane Ring bendd C,=O bend, in-plane (Ring bend’) C,=O bend, in-plane

Thymine Uracil Thymine Uracil Thymine Uracil Thymine Uracil Thymine Uracil

“Data for uracil from ref. 1. bOnly the most intensive from ref. 14. dAssignment for uracil from ref. 20.

Ar matrix

Nz matrix

1769

1770 1747 1710 1761 1736 1704 769 811 758 762 542 538 457 518 392 394

1752 1712 1762 1733 1707 764 806 754 769 545 537 455 516 391 393

Uracilb

C,=O bend, out-of-plane

modes for

v (cm-‘)

C=O mode

C,=O stretch and C,=O stretch (Fermi resonance)

carbonyl

absorptions

listed. ‘Assignment

for uracil

was interpreted as C,-C, stretch, and the band at 516 cm-l as in-plane ring deformation [ 141. We suppose that the corresponding bands in thymine (754, 454 cm-’ respectively) behave as bands in which carbonyl bends dominate. As seen from Table 4,5-substitution of the methyl group in thymine disturbs the C4=0 vibrations more than the &=O. Ring, CH,, and CH deformation modes The ring stretching and ring bending modes are usually coupled together, and with C-H, N-H and C=O modes [ZO]. The substitution of the methyl group to the uracil ring could change the coupling conditions in some cases. The assignment concerning ring and CH, vibrations in Tables 1 and 2 should be treated as an approximate one. This was based in part on the comparison with frequencies found for uracil and in part on the predictions of in-plane vibrations calculated by Susi and Ard [ 111. Some absorptions found in the spectrum of thymine correspond quite well with those in the uracil spectrum (Fig. 3). The 1389, 1357 cm-’ bands correspond to the v,, mode in ref. 14, 1315 cm-l was assigned as the band 1314 cm-’ in ref. 1, the band 1219 cm-l

48

corresponds in ref. 14.

to v12, 960 cm-l was interpreted

Spectrum of NI,N,-dideuterated

as mode v22 and 727 cm-l

as v25

thymine

A comparison between the spectra of N,,N,-dideuterated thymine and uracil is presented in Fig. 4. It can easily be seen that in this case the spectra differ more than for non deuterated species. The most pronounced differences concern the ring modes. The ND deformation modes which shift considerably to lower frequencies with respect to NH frequencies in thymine are assigned in Table 2. If we compare these frequencies with the absorption frequencies of deuterated uracil in refs. 1 or 19 we can find the bands whose frequencies are very close to the ND deformations in thymine. How far these absorptions are really connected with the ND movements we do not know. Calculations of potential energy distribution done for various deuterated uracil species [ 201, showed that the change of a proton mass leads to considerable changes in the relative contributions from symmetry coordinates to these normal modes. Hence the assignment presented in Table 2 should be treated as approximate. The carbonyl deformation frequencies show weak dependence on deuteration. The differences between deuterated uracil and thymine concern only the C4=0 modes which are disturbed by the methyl group (as in the undeuterated species). ACKNOWLEDGMENTS

These investigations were conducted following a suggestion by Dr. L. Harsanyi from the Hungarian Academy of Sciences; he also assisted in some of the experiments. This work was financed by the Polish research programs: CPBR 11.5 and CPBP. 01.12.

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18 19 20 21 22

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