Matrix isolation infrared studies of nucleic acid constituents

Journal of Molecular Structure, 156 (1987) 29-42 Elsevier Science Publishers B.V., Amsterdam - Printed in The Netherlands

MATRIX ISOLATION INFRARED CONSTITUENTS

STUDIES OF NUCLEIC ACID

Part 4*. Guanine and 9-methylguanine monomers and their keto-enol tautomerism

KRYSTYNA Institute (Poland)

SZCZEPANIAK**

and MARIAN SZCZESNIAK

of Physics, Polish Academy

of Sciences,

Al. Lotnikow

32/46,

02668

Warsaw

(Received 7 May 1986)

ABSTRACT Infrared absorption spectra have been studied for matrix isolated guanine and 9methylguanine, which is a formal analogue of the natural nucleoside found in DNA and RNA. Three related compounds (isocytosine, 2-amino-5-chloropyrimidine and 2-dimethylamino-6-hydroxypurine) have also been examined. The spectra provide evidence for the existence of guanine and of 9-methylguanine as mixtures of the enol-amino and ketoamino tautomers, although the keto-amino tautomer is the only tautomeric form found in solution and in solid. The estimated enol-keto equilibrium constant found in nitrogen matrices K = [E]/[K] is about 3.6 for guanine and 5.9 for 9-methylguanine. The significance of these results is evaluated in relation to the types of tautomers found in natural nucleic acids and to the concept of spontaneous and induced mutations caused by mis-pairing of the bases in the nucleic acids. INTRODUCTION

In the Watson-Crick model of DNA the bases in guanosine and cytidine must adopt the keto-amino tautomeric forms in order to form the “proper” (canonic) complementary pairs. These are not the only tautomeric forms of these bases which are, in principle, possible. Our recent studies of the IR spectra of cytosines and isocytosines isolated in inert rare gas matrices [ 1,2] indicate that while l-methylcytosine (a direct analogue of cytidine) adopts the same keto-amino form when isolated in an inert low-temperature matrix as it does in polar media, isocytosine exists predominantly in the enol-amino form when isolated in the matrix. Because the pyrimidine ring of isocytosine (IC) is the same as that in guanine (G) and in the guanosine analogue 9-methylguanine (9MG), this last observation raises the question of whether G and 9MG also adopt the enol-amino tautomeric form in the matrix. *For Parts 1, 2 and 3 see refs. 14, 17 and 18. **Present address: Department of Chemistry, University of Florida, Gainesville, FL 32611, U.S.A. 0022-2860/87/$03.50

o 1987 Elsevier Science Publishers B.V.

30

In principle G may adopt several tautomeric forms, some of which are shown. Of these, forms I, III, V and VI could also be adopted by 9MG. As I

II 0 II

012 II H,3\N,C6\c

’

HIP\ /Cz+ N14

N3

H\

/%

/C\

/N

‘i’

Y ,!;,&H”\N/C’\N,C,N// I

HI6

i

H

HIO

Ill 0

‘C-H

II NAC\CNN\

H,N,i,N,j,N/C-H

I

I

I

H

H

H

m

P

0

0

A

Ill

“-9 & /N\7 kN/kC A”\ H,~,C,~J\~/~ -’ H,N//A,N:,N/C-H

A A

ti

A

has already been briefly reported [3], the IR spectrum of G isolated in an argon matrix suggests that two keto-amino forms (I and II) are present, together with the enol-amino form (VI). Recent quantum mechanical calculations [ 4-71, in particular recent ab initio calculations [6] , have predicted that these three tautomeric forms of G are the most stable. In polar media, however, only keto-amino tautomers have been identified [8-111, in agreement with the predictions [6b] of a higher dipole moment for the keto-amino tautomer (I) (7.18 D) than for the enol-amino tautomer (3.49 D). The appearance of G and 9MG in both keto-amino and enol-amino tautomerit forms when these molecules are found in a non-polar local environment may have important biological implications, related to mutagenesis and other biological properties depending upon the integrity of the genetic code. Further studies of the tautomerism of these molecules and of the effect of the environment on their tautomeric equilibria are thus fully justified. In this report we present the IR spectra of G in argon and nitrogen matrices and of 9MG in a nitrogen matrix, all in the spectral region from 3700 to 200 cm-‘. The matrix isolation technique is ideally suited for the study of isolated monomers of these molecules, since the thermal energy needed for sublimation to prepare samples is sufficiently low that decomposition does not occur. Other studies [ 1, 13-211 have certainly verified this suitability. During the preparation of this manuscript, we became aware of a paper by Sheina et al. [22] reporting studies of the IR spectra in the OH and NH stretching regions for 9MG and some related compounds isolated in an argon matrix. Their spectra of 9MG in this region appear to be in very good agreement with our spectra, and their conclusions concerning tautomeric forms of 9MG in the argon matrix agree with ours for G and 9MG in argon and nitrogen matrices.

31 EXPERIMENTAL

The IR spectra were studied with a Perkin Elmer 580B spectrophotometer. The matrix was formed on a CsI window in a continuous flow liquid helium cryostat. The details of the experiment were the same as those previously described [ 141. The sample of G was obtained from Reanal (Hungary) and Sigma while the 9MG was synthesized by Dr. L. Dudycz (IF, PAN, Warsaw) by a method described in ref. 23. Both G and 9MG were purified by vacuum sublimation during the process of deposition of the matrix sample. The IC sample was prepared as described [ 241 by Dr. B. Kierdaszuk (Warsaw University). To prove that our compounds did not decompose during sublimation to form the matrix isolated sample we compared the spectrum of a solid film formed on sublimation from the same furnace temperature as for the matrix study with that from an unheated solid sample in a KBr pellet. The frequencies of the bands in both spectra were found to be identical, and agree with those reported [ 81. RESULTS

AND DISCUSSION

IR survey spectra of G and 9MG isolated in nitrogen matrices are shown in Fig. 1. Figure 2 compares the spectrum of G in an argon matrix with that of the polycrystalline solid and also with that predicted in a recent ab initio molecular orbital calculation [6]. The latter includes predictions of both frequencies and intensities for the in-plane modes and out-of-plane modes, for G in the keto-amino tautomeric form (I). Table 1 summarizes the frequencies and relative intensities observed for matrix isolated samples of G and 9MG, and those for crystalline samples of G. These experimental values are compared in the table with the frequencies and intensities predicted by the ab initio calculation [6] for the keto-amino tautomer (I) of the G monomer. The assignment of the experimental spectra shown in Table 1 is based upon this comparison, as well as upon the characteristic group frequencies for the NH, OH and CHJ groups. This assignment has to be considered to be only preliminary, since the calculations with a 3-21G basis are still not very accurate, particularly for the out-of-plane modes, since calculations for 9MG have not yet been made, and since calculations have not been made for other tautomeric forms. The first striking indication that more than one tautomer is present in the matrix sample is that the number of bands observed in the spectrum of G isolated in the matrix is much larger than the number of bands observed in the spectrum of the crystalline solid (where G is known to exist in the ketoamino form [8-lo]) and also larger than the number of bands predicted by the ab initio calculation for the keto-amino tautomer (I) of G [6, 71. The most straightforward information about the tautomeric forms present in the matrix is usually obtained from the examination of the spectral regions in which the characteristic absorption is expected for the groups that are

32

3600

3300

3000

1700

1600

1500

1400

1300

[WiiJ

Fig. 1. The IR spectrum in different regions (a-d) (---) both isolated in N, matrices at about 10 K.

of guanine

(-)

and 9-methylguanine

directly involved in the tautomeric transitions: namely, those of NH, NH,, OH and CO stretching modes. However, the interpretation of the absorption found in these spectral regions is not straightforward for matrix isolated G and 9MG. Figures 3 and 4 show the spectra in these regions on an expanded scale. For the keto-amino form (I) of G four bands are expected in the NH stretching region (vBNH2, v,NH2, vN~H, and vN,H); only the first three of these are expected for the keto-amino form of 9MG. In contrast with this simple expectation, absorption from at least eight bands can easily be recognized for G in argon (Fig. 3a) and an even more complicated pattern is observed both for G and for 9MG in nitrogen matrices (Fig. 3b, c). Comparison

3600

3400

3200

3000

,-

2800

-1800

1700

.f!fi’,,, ,,!

,,I,

)!;,,!;

2

L

o.l,

W?

A

b

1300 1200 1100 1000 900

1500

1400

,i,i;,

800

1300 1200 1100 1000 900

[

1600

F [Cm-q

C [W-q

h

,a,

800

700 .

o,~~~~~,

1

600

500

400

300

200

Fig. 2. The IR spectrum in different regions (a-d) of guanine isolated in an Ar matrix at about 10 K, (-), as the polycrystalline solid (at about 300 K) in KBr pellet (---) and as predicted by ab initio quantum mechanical calculations (in-plane vibrations, vertical solid lines; out-of-plane vibrations, vertical broken lines). Vertical arrows indicate the position predicted for very weak bands.

of the spectra of G and 9MG (both in nitrogen matrices) shows that the absorption near 3500-3480 cm-’ is present only in the spectrum of G, but not in 9MG. This observation suggests that this absorption is due to the NgH stretching mode. Such an assignment agrees with those for other purines [ 261 and with the theoretical prediction [ 6, 71. Because the structure of this absorption suggests that more than one band absorbs in this region, it seems that some contribution from the NTH stretching mode from tautomer II may also appear here. This suggestion seems to be confirmed by the presence of two strong bands in the carbonyl region for G in both argon and nitrogen

1

47

35

3540

3526

31

270

330

3428

1749

1736

2855 2770

vr

1610

1653

1773

480

315

635

NH +c

NH pc

vc=o

LJC,H

YN ,H

3570

1641

1660

1696

2900

2927

2962

3000

3422

3435

3442

3454

3534

3560

1547

1484

1506

1531

1548

m

“r

1523

166

1525

1545

1558

1555

v, 51 48 I 100

458

1553

1570

1587

1597 1592

1570 vr

NH,sc,

NH,sc

vC=O

z

WH ,

vaNH

(cm”)

va

1564 98

235

760

635

1

3164

mole’)

Assignmentc

1565

1635

1675

1695

47

147

127

82

(km

I

3437

3454

3505

3563

(cm“)

va

1577

s

m

m

NH+

NH.+

vc=o

u,NH,(K)

usNH,(E)

vN,H(2) uC,H

VN,H

vsNH, vN,H

VP%

AssignmentC

1583

1580

ur

1592

140

1599

1588

vr

1593

49

s

m

s

s s

s

s

sh

vN,H

2700

. 4390

1e

1603

1602

1629

1654

1716

1731 1725

1745

3423

3437

m

s

3473

3455

s

3482

vN,H

s

v,NH,(K)

w

3488

3521

2920

w

3090

3528

.

3115

3170

3340

w

vOH

v,NH,(E)

3539

m

m

m

(cm”)

va

Calculatedf

1606

NH+

NH+

vc=o

v,NH,(K)

vN,H

@H,(E)

vN,H

vN,H

V>H,(K)

3553

3560

3571

AssignmentS

(in KBr)

1626)

375

210

vOH

, %NH,(E)

(cm-‘)

vd

Solid

1629 1620

1654

1692

30

64

3439

1705

29

3455

3485

109

74

3493

sh

3578

3570

AsignmentC

matrix

1

\

22

38

136

392

526

68’

17

108

94

256

rb

matrix

9-Methylguanine

Nitrogen Nitrogen

matrix

Guanine

VI

vr

t+

Yr

NH+

vN,H

QNH,(E)

v,NH,(K)

u,NH,(E)

vOH.

Assignme&

and nitrogen matrices, in a nitrogen matrix

Argon

Frequencies (v) integrated intensities (I) and assignment of the bands of the IR spectrum of guanine in argon the crystalline solid in KBr pellets, and calculated by ab initio methods (3-21G basis); and for 9-methylguanine

TABLE in

r;:

16 73 21

1375 1361 1353 1329

752' 738 721 709 693 670

v,,GNH

v,.tTOH

11 6 13 10 10 I 5 TNH

11 v,,vco 40 1 v,,SCsH 40 24 v,,6C=O 10 10 vr.NH,ro 10 30 &OH,", 20 4 Yr,?C=O 6 5 iv=O 5 T'CH 14 14 2‘ 11 g:v: 15

69

23 20 vr.6C,H 14' +,&OH 11 v,,60H

44 31 68 29

1443 1432 1418 1405

1276 1271 1260 1210 1195 1 &l.& 1179 1158 1140 1131 1104 1063 1052 ,1049 1018 1010 987 975 932 925 855 827 821 794

46

1472

Y~.+H, -rNH YNH

706~

668w

Yr

6,,vr

YCO,Y, 6,,yCH

60H,v,

705 690

781 778sh 730

44

885 Jzv&) 840 yNH

6,

Yr,yC=O

v,,NH,ro

3

28

Yr

-/NH

37 i FVr r

43

950

43

669

722

835 808 796

953 950 909

1043

1045

1134

1306 1263

1346

1353

1401

1486

v,NH,ro

ur.6C,H

SC,H,+

VI

u,.6C,H

v,,tiNH

vr

+,sC,H. +GNH

1103

37 5 61

43

1215

1175 1150 1120

52

sh 415 sh

1390 1375 1360

1265

28

195

1417

1475 1465

6 c=o

VI.

",.&OH v,.vco

vr,sC,H

k&H

20,

730w

825~ 797w 7831-n

965w 930w

1183m 1161~

1377m 1368~ 1356111 1331w 1316 w 1311w 1281~ 1260~

1473m 1458~ 1446~ 1439w 1419 m 1405m

38

197

105 9 123

152 13 70

15

22

32

24 43

2

148

148

23

6 c=o

yc=o

VI

-rN,H, yN,H

Yrv yNH

Yr

i:.

-YC,H

6,

Y*,

vr,NH,ro

I+,

vi-. 6C,H

vi=,6N,H +r 6C,H

672 662 644

1148 1136 1083 1060 1053 1047 1016 1000 993

1276 1227 1217 1205 1197 1192 1183

1295

1372

1431

1488 1485 1465

J

34

v,,60H v,,60H

v,.6CH

v,,SC,H

SCH,. I+,N*H,ro

20 1 yNH 2 yNH,yC=O 26 S,,&C=O

14 60H,v, 32 sh ' YpW=O

80

6' v,.6C=O

32

80 20 16 8 sh

24

28

46

1041

m

W

matrix

Solid

(in KBr) AssignmentC

Calculatedf

86

&NH,, X=0,6,

w

m

307

VW

346

367

VW

m

542

373

m

564

tNH,,

m

617

w

w

625

485

w

663

(cm-‘)

vd

Yr

yNH

AssignmentC

6r 348

I mol-‘)

AssignmentC

15 217

Yr

6C=O,

&NH,, 3

4

Yr

6, tNH,.

4

1

1

TNH,

r br

6C=O,

6,

SC=0

yNH

6,

YC=O. V’J,H. -or

75

253

11

23

6

(km

305

332

361

356

6NH,, lsc=o,

473 390

523

400

495

505

516

542

tNH,.

yNH

651

658

561

6,

6C=O.6,

560

l2

(cm-‘)

va

606

1e

608

640

@&

(cm”)

ua

Yr

WH,

-rNH, ^/NH,

6,

6C=O, I br

yC=O

yNH,

AstignmentC

268 250

317

337

358

368

378

538

552

585

602

625

635

(cm-‘)

va

26

6

matrix lb

yNH,.

rbr

@H

Assignmentc

aThe more intense band is underlined. bIntegrated intensities in arbitrary units, chosen so that the sum of intensities of all bands within the whole spectrum is approximately the same for argon and nitrogen matrix and for calculated spectra. In these units intensities of the corresponding bands in matrix and in calculated spectra can be compared. CAbbreviations: K, keto form; E, enol form; V, stretching; a, asymmetric; s, symmetric; SC, scissors; r, ring; 6, bending in-plane; ro, rocking; y, bending out-of-plane; t, twisting; r br, ring breathing. dInstead of intensities only approximate characteristics in terms of: s, strong; m, medium; w, weak; VW very weak are given. “Integrated intensities in arbitrary units; the carbonyl stretching absorption equals that of matrix isolated molecules. In these units intensities of the corresponding bands of solid films could be compared with those of matrix isolated molecules, because absorption intensities of carbonyl stretching vibrations are not strongly affected by hydrogen bonding in crystals. fFrom ref. 5.

61

309

42

41’

93

129

Ib

346

371

382

482

494

500

505

514

528 520

(cm-‘)

va

9-Methylguanine

Nitrogen

Nitrogen

matrix

GWUline

1 (continued)

Argon

TABLE

37

J ,,,,,,,,, \:

:1;f

[C/N*

n

3550

3500

3650

3600

Fig. 3. The IR spectrum in the NH and OH stretching region of (a, b) guanine, (c) 9methylguanine, (d) 2-dimethylamino-6-hydroxypurine, (e, f) isocytosine and (g) L-amino5-chloropyrimidine isolated in Ar and/or N, matrices at about 10 K (20 K in d).

b

G/N2

e

KIN,

d

2DMAP/

Ar

.1-

:.-::

F4

a

.l-

GlAr

,A,, c 9MGI

Nz

_J

I

1750

8

J.._

I

1700

1750

1700

Fig. 4. The IR spectrum in the C=O stretching region of (a, b) guanine, (c) 9-methylguanine, (d) 2-dimethylamino-6-hydroxypurine and (e) isocytosine in Ar and/or N, matrices at about 10 K, (20 K in d).

38

matrices (Figs. 4a, b) while only one band is present in this region for 9MG in a nitrogen matrix (Fig. 4~). The interpretation of the remaining bands in the spectra of G and 9MG shown in Fig. 3a-c is more difficult. In order to understand these spectra we may compare them with the spectrum of matrix isolated IC shown in Fig. 3e, and of 2-dimethylamino-6-hydroxypurine (2DMAP) [ 251 in Fig. 3d. In this last model compound of guanine no v,NH~ band can be present near 3560-3570 cm-‘, and the only absorption possible in this range would be uOH, if the molecule adopts the enol form. As mentioned above, the pyrimi dine ring of G and 9MG, is the same as that of IC which exists in the matrix in the enol-amino form. We may also compare these spectra with the spectrum of matrix isolated 2-amino-5-chloropyrimidine (2AP) in Fig. 3g, which provides us with a spectrum typical of an NH2 group attached to the Cz carbon atom in the pyrimidine ring [ 1, 21. On examination of Fig. 3a-c we see that it is difficult, on first glance, to find in the spectra of G or 9MG the characteristic doublet absorption for the asymmetric V, and symmetric V, stretching vibrations of the NH2 group similar to that observed for the NH, group in 2AP, with the typical frequency difference (v, - V, from 113 to 120 cm-‘) and intensity ratio (1,/I, of about 0.7) [l, 2, 25-271. Such an “unusual” ratio of 1,/I, is also observed in the spectrum of IC in an argon matrix (Fig. 3e), and it can be explained as being caused by the overlap of the NH2 V, vibration by the vOH absorption from the enol tautomer. The IC molecule exists predominantly in the enol-amino form, as proved by the absence of any significant absorption in the carbonyl region (see Fig. 4e), so absorption is expected from both V, of the NH* group and from the OH group, giving rise to the overlapped absorption in the IC spectrum near 37503560 cm-’ [ 13, 211. When IC is isolated in a nitrogen matrix, the absorption in this region clearly splits into several components (Fig. 3f). If we assign the less intense, high frequency component to v,, we see that it exhibits the “usual” frequency separation and intensity ratio for IJI, (with v,NH, near 3455 cm-‘). We believe that G in argon has a similar overlap between v, and vOH in this region. For G and for 9MG in nitrogen matrices much broader absorption with several sub-bands is observed in the region where we expect va, vOH, and V, (Fig. 3b, c). We make our assignment of the bands of G and 9MG using as a guide the v,, V, bands of 2AP, IC and also 7-methylguanine and 1,7_dimethylguanine (both only in the keto-amino form in the matrix) and BDMAP [ 251, looking for a pair of bands with the same frequency separation for V, - V, as observed for these compounds and approximately the same ratio for I,/I,. The appearance in the experimental spectrum of more than one pair of bands that may be assigned to the v, and V, modes of NH2 may possibly be due to slightly different frequencies for these vibrations for the keto-amino than for the enol-amino tautomers of G and of 9MG. Another explanation for at least some of the observed subbands may be matrix splitting [14, 281. The presence in the matrix of the enol-amino tautomers of G and 9MG with characteristic vOH absorption near 3570 cm-’ is also strongly supported

39

by the spectrum of BDMAP in an argon matrix, shown in Fig. 3d. In this molecule both hydrogen atoms of the amino group are substituted by methyl groups; hence, no absorption bands from the NH2 group appear in the spectrum. In spite of this fact, strong absorption near 3560 cm-’ is observed for this compound in the matrix spectrum. This band is interpreted to be vOH, for the molecules in the enol form, and a second band in the spectrum at 3440 cm-’ is interpreted to be vNIH for the molecules which are present in the keto form. The origin of the relatively weak bands near 3540 and 3520 cm-’ observed for G in argon and nitrogen matrices is quite mysterious (Fig. 3a-c). Only the first of them appears to be present in the spectrum of 9MG in a nitrogen matrix. One of the possible interpretations of these bands is that one of them corresponds to the V, absorption of the NH2 group from the keto forms of G. The corresponding symmetric modes are located near 34403430 cm-‘. In this last case the separation between V, and V, for the amino groups is only about 100 cm-‘, much less than usually observed. Such an “unusual” spacing between V, and v, might possibly occur if these vibrations for the keto form are coupled with vNIH (see ref. 25). Such coupling is predicted by the normal coordinates from the ab initio calculation [29]. This coupling is expected to increase the frequency of V, for the NH2 group in G relative to that in 2AP, and thus to decrease the separation between v, and vs. Another possible explanation for the band near 3540 cm-’ is that it may be due to vOH from molecules in which the OH group is directed toward the N, atom, in an internal hydrogen bonding interaction, instead of towards the N1 atom [ 301. Ab initio calculations made with an STO-3G basis set and with geometry optimization indicate that molecules with the OH groups toward the N1 atom are only slightly more stable (2.1 kcal mol-‘) than are molecules with the OH group directed toward the N, atom [ 311. An alternative interpretation of the band near 3520 cm-’ which appears in the spectrum of G but not in the spectrum of 9MG, is that it may be due to vOH for the enol-amino tautomer of G with the imidazole hydrogen attached to the N, atom. The relatively low frequency of this band (compared to 3570 cm-l for vOH in the “normal” enol tautomer) would then be explained by a weak interaction between the lone pair on the oxygen atom with the hydrogen atom from the N,H group. The spectra observed for G and for 9MG in the carbonyl region (Fig. 4a, b) provide further support for the postulated presence of the enol-amino forms. although the absorption in this region observed in the spectrum of G and 9MG (Fig. 4a, b) is quite strong compared with that observed for IC (Fig. 4d), the ratio of the integrated absorbance in the carbonyl region to that due to the NH2 and N,H stretching modes (3600-3400 cm-‘, excluding the bands due to N9H and N7H stretching) for G and for 9MG is much lower than that observed for 1-methylcytosine (lMC), which is known to exist in the matrix only in the keto-amino form [ 1, 21, and is also much lower than the ratio

40

predicted in the ab initio calculation [6]. This intensity ratio (11,50--1670/ 13600_-3400) was found to have the following values: 1.1 for G in argon; 1.0 for G in nitrogen; 0.6 for 9MG in nitrogen; 3.6 for 1MC in nitrogen [Z] ; and 2.3 from the ab initio calculation. These values suggest that an appreciable fraction of the G and 9MG molecules isolated in the matrix exist in the enolamino form, which does not absorb in the carbonyl region, but which does contribute to the absorption in the 3600-3400 cm-’ region. In the latter region we may expect that the intensities for vOH and for vN~H, in the enol and keto forms, respectively, are nearly the same [ 12, 131. The much lower value observed for this intensity ratio for 9MG indicates that the keto-enol equilibrium is shifted more toward the enol form for that molecule. This postulated increase in the enol-amino form over the keto-amino form for 9MG, as compared with G, is also consistent with the observation that the absorption assigned to V, for the NH2 group in the enol-amino tautomer near 3455 cm-’ is much stronger in the spectrum of 9MG in the nitrogen matrix than is the corresponding band for G in either argon or nitrogen matrices (see Figs. 3a-c) . The enol-keto equilibrium constant, K, ([E]/[K] ) estimated from the IR spectrum and the integrated molar absorption coefficients’ ratio for C = 0 and NH absorptions transferred from 1MC [2] is 3.6 for G and 5.9 for 9MG (both in Nz matrices). CONCLUSIONS

The results presented here suggest strongly that monomeric molecules of both G and 9MG isolated in inert matrices (argon or nitrogen) exist in both the enol-amino and keto-amino tautomeric forms, even though both apparently exist only in the keto-amino forms in polar media. Hence, guanine and 9-methylguanine (the analogue of guanosine) are the only biologically significant nucleic acid bases that have been observed to show such drastic changes in the tautomeric equilibria when the environment is changed from a polar medium to a non-polar one. All the nucleic base monomers of biological significance previously studied in the vapor phase or isolated in inert matrices, as well as those studied in polar media, have been found to exist in only one tautomeric form, regardless of the nature of the medium [l, 2, 14-16, 18, 19,26, 271. This behavior has been in sharp contrast with the dependence of the tautomeric forms on the environment observed for a number of other pyrimidine bases [ 1, 2, 13, 20, 211. A number of uncertainties still remain in the interpretation of the IR spectra of G and of 9MG, and more work remains to be done to verify the assignment of the bands in the spectra to different vibrations and to different tautomers and to estimate quantitative tautomeric equilibria. Such studies are in progress in our laboratories.

41 ACKNOWLEDGEMENTS

We wish to dedicate this paper to Professor David Shugar (Warsaw) on the occasion of his 70th birthday and to express our gratitude to him for introducing us to tautomerism of nucleic acid bases as well as to acknowledge our valuable and stimulating discussions with him. The authors are grateful to Professor Willis B. Person (Gainesville) for helpful and stimulating discussion, for help with revising this manuscript, and also for providing us with results from the ab initio calculations of the IR spectrum of guanine prior to their publication. We want also to thank Professor J. S. Kwiatkowski (Torun) and Dr. M. J. Nowak (Warsaw) for their suggestions and lengthy discussion. We are grateful to Mr. A. Orlowski for his instrumental and technical assistance. This investigation was supported by the Polish National Cancer Research Program PR-6, C.P.B.R.-11.5 and Research Program of the Polish Academy of Sciences C.P.B.P.Ol.12. Finally, we are grateful to the Department of Chemistry of the University of Florida, Gainesville, for hospitality while writing the final version of this manuscript, and to NIH Research Grant No. GM-32988 for partial support. REFERENCES 1 M. Szczesniak, M. J. Nowak and K. Szczepaniak, J. Mol. Struct., 115 (1984) 221. 2 M. Szczesniak, Ph.D. Thesis, Warsaw, 1985. 3 K. Szczepaniak, Z. Latajka, K. Morokuma and W. B. Person, Spectroscopic des Especes Isolees en Matrice, geme Colloque International, ass. au CNRS Abbaye de Fontevraude, France, 1985, p. 86. 4 A. Sygula and A. Buda, J. Mol. Struct. (Theochem), 92 (1983) 267. 5 A. Sygula and A. Buda, J. Mol. Struct. (Theocbem), 121 (1985) 133. 6 (a) Z. Latajka, W. B. Person and K. Morokuma, J. Mol. Struct. (Theochem), 135 (1986) 253. (b) Z. Latajka, W. B. Person and K. Morokuma, unpublished results. 7 Y. Nishimura, M. Tsuboi and K. Morokuma, Bull. Chem. Sot. Jpn., 58 (1985) 8 9 10 11 12 13 14 15 16 17 18 19

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