Experimental and theoretical studies of molecular structure features of cytosine

Journal of Molecular Structure, 116 (1984) Elsevier Science Publishers B.V., Amsterdam

387-396 - Printed

in The Netherlands

EXPERIMENTAL AND THEORETICAL STUDIES STRUCTURE FEATURES OF CYTOSINE

E. D. RADCHENKO, Institute Kharkou

G. G. SHEINA,

for Low Temperature (U.S.S.R.)

(Received

25 October

N. A. SMORYGO

Physics

and Engineering,

OF MOLECULAR

and YU.

P. BLAGOI

UkrSSR

Academy

of Sciences,

1983)

ABSTRACT High-resolution vibrational spectra of the heterucyclic nitrogen compounds cytosine, its deutero analogue and pyrimidine, have been obtained by IR spectroscopy in an Ar matrix. On the basis of calculation results, the normal vibrations of isolated molecules are assigned and the force constants found. It is shown that an isolated molecule of cytosine exists mainly in the enol form, which agrees with theoretical predictions. INTRODUCTION

The high resolution of vibrational transitions provided by the method of matrix isolation permits investigation of fine structure features of separate molecules and molecular associates. Different tautomeric forms were predicted theoretically [l--3] for bases of nucleic acids (NA), but so far they have not been observed experimentally_ This is because most studies were carried out on solid samples [4-6], whose molecular structures were determined from their crystalline structure. Thus, investigations on solid cytosine by different methods (X-ray, IR, NMR) 16, 71 unambiguously showed the ketoamine structure. However, according to quantum-chemical calculation Cl, 21, an isolated cytosine molecule must be mainly in the aminoenol form (Fig. 1). Thus ab initio calculation shows [I] that the energy of the enol tautomer is 17.73 kcal mol-* lower than that of the ketonic cytosine. It should be noted that the question of tautomeric equilibria of NA bases has been touched upon repeatedly in connection with the possibility of spontaneous and induced genetic mutations in NA [S, 91. The experimenti and theoretical data on the vibrational structure of isolated molecules of cytosine and cytosine-D3 reported in this paper may be applied to the investigation of intermolecular NA interactions. EXPERIMENTAL

The investigation by the method of matrix isolation was performed using a standard helium cryostat (Fig. 1). The optical section and the system for OO22-2860/84/$03.00

o 1984

Elsevier Science

Publishers

B.V.

388

Fig. 1. Schematic view of experimental unit: 1, valves; 2, helium cryostat; 3, heat exchangers; 4, vacuum housing with KBr windows; 5, vacuum seal; 6, heater; 7, nitrogen screen for sample; 8, substrate holder; 9, A&a thermometer; 10, KBr substrate; 11, quartz microbalance; 12, leak; 13, container for solid Ar; 14, coil pipe; 15, Knudsen cell.

sample preparation were designed by the authors. The optical windows and substrates 10 are made of KBr plates 2 mm thick and fixed with In seals. Copper substrate block 8 is cooled with evaporated helium gas running through heat exchangers 3, preventing heat leakage from the sample to the helium bath. The helium flow is controlled with valves 1, which permits adjustment of the substrate temperature roughly in the range 100-S K without excessive consumption of coolant. The accurate temperature is maintained using thermostatic switch 6 comprising a heater and an AsGa thermometer 9. The four-substrate block can be rotated together with the cryostat within seal 5 with respect to the windows of the vacuum housing, which permits measurement of several samples in one cooling cycle. Preliminarily purified Ar is used as a matrix gas. Before filling it is cooled to liquid nitrogen temperature. The pressure above the solid Ar surface 13 is always constant being about 200 mm Hg. Such a low pressure permits smooth control of the leak-in rate with standard needle valve 12. At this stage the final distillation A is accomplished, and filled cooled matrix gas 14 suppresses heat leakage to substrate 10 and raises its accommodation coefficient_

389

The substances used can be sublimated from three separate Knudsen cells, 15, in which temperature is maintained to within better than 0.1 K. The correlation between the matrix gas and the substance (2000, 1000) is accurately measured with quartz microbalance 11 irrespective of the accommodation coefficient. Two quartz piezoelectric AT-cut resonators of 5.0 and 5.1 mHz are used as the microbalance transducers. The temperature plateau in the helium temperature region and the differential switching of the resonators fixed at the cooled substrate block provide a high measurement accuracy for the masses condensed onto the substrate. The system of working, reference generators, and the mixer being assembled using insulated-gate fieldeffect transistors, which are stable at liquid nitrogen temperatures gives the possibility of adjusting them at the nitrogen screen in immediate proximity to the resonators. Thus, a reliable thermal stability of the system is achieved and the effect of connecting leads reduced considerably. The differential frequency instability is al Hz h-i, which corresponds to 5 X lo3 g. The investigation was made on repeatedly recrystallized and dried substances produced in the U.S.S.R. and supplied by Calbiochem (U.S.A.) and Chemapol (C.S.S.R.). Amino- and imino-group deuteration was performed by recrystallization from heavy water. The sublimation temperature of 170-200°C eliminated the possibility of decompcsition and provided for precipitation of lo+ to 10’ g cm-* min-’ of the substance investigated onto the substrate. The evaporation time varied from 40 to 150 min in different experimental runs. The substrate temperature was 11 K. IR spectra were recorded with Specord-75-IR spectrophotometer, the resolution being 1 cm-’ at 1000 cm-‘. CALCULATION

TECHNIQUE

The vibrational spectra of cy’asine were calculated using the program developed by Gribov and Dementiev [lo]. Computation of the matrices of kinematic coefficients, symmetry reduction of the matrices, calculation of additional relations and derivation of secular equations were performed using the Minsk 22 computer. The geometric parameters were &ken from X-ray diffraction data [7] for an isolated molecule of cytosine in the keto form and from calculation results [l] for the enol tautomer. The molecules were assumed to be of a flat configuration and to belong to the symmet,ry group C,. The natural vibrational coordinates are shown in Fig. 2. The force field obtained in the course of calculation of the vibrational spectrum of uracil was taken as a zero approximation [ 111. The calculation results were matched to experimental IR-frequencies of isolated molecules of cytosine through varying the diagonal elements of the force field and the elements of mtho interactions. The other matrix elements were the same as in the force field matrix of uracil [ 111.

(a)

(b)

Fig. 2. Natural vibrational coordinates for (a) ketonic and (b) enol forms of cytosine. RESULTS AND DISCUSSION

Four intense bands are observed in the region of valence vibrations of NH and CH bands in the spectrum of cytosine in an Ar matrix. Two of them refer to vNHtas= 3559 cm-’ and vNHZs= 3438 cm-’ (Fig. 3a, Table 1). These are in agreement with the data for 1-methylcytosine in an Ar matrix: vNHZas= 3555 cm-‘, YNHZs= 3436 cm-’ [12]. The absorption band of cytosine Y = 3587 cm-’ coincides with the band of 2-oxypyrimidine in an A.r matrix (3587 cm-‘) [12], whose tautomeric equilibrium is almost completely shifted towards the enol form 1131. The weaker band 3468 cm-’ refers to the NIH vibration. The cytosine-D3 spectrum (Fig. 3b, Table 1) also has four intense bands at 2676, 2654, 2574, 2517 cm-‘, The correlation of maximum intensities of the cytosine absorption bands Z.‘oH= 3587 Cm-’ and vNH = 3468 cm-’ remained constant as sample evaporation temperature changed from T = 170°C to 200°C. In the low frequency region of the spectrum there is an intense band 1193 cm-‘. There is also a band in this region of the spectrum of 2-oxypyrimidine (1198 cm-‘) taken under the same conditions [11]. This band disappears in the cytosine-D3 spectrum, but a new one, 947 cm-‘, appears. The band observed is associated with the deformation vibration 60H. Thus, the comparison of cytosine spectra in the region of valence vibrations of VNH,, with those of 2-oxypyrimidine and 1-methylcytosine %H, Z’OH and 60, simulating the enol and keto forms of cytosine permits us to assume that in the gaseous phase at 2’ = 173-200°C “frozen” in an Ar matrix, cytosine and cytosine-D, are in the tautomeric equilibrium of amino-keto and aminoen01 forms [ 143.

oJy=$~~~ H Keto Enol equilibrium of cytosine

.gyjyj I CH3

1-CH,cytosine

2-Oxypyrimidine

Pyrimidine

391

Fig. 3. IR spectra of (a) cytosin e and (b) cytosine-D,

in Ar matrix at 11 I(. Ar:M

= 2000.

The presence of two tautomeric forms of cytosine was proved by applying the calculation technique to vibrational spectra, which had been used to detect the ketonic and enol forms of barbituric acid [ 15]_ The calculated frequencies and shapes of in-plane normal vibrations of the ketonic and enol forms of cytosine are given in Tables 1 and 2. The ketonic vibrational spectra were calculated for two molecular states: the crystal and isolated in a rare gas. Vibrational spectra of crystalline cytosine were calculated by Susi et al. [5]. Their interpretation and force constants agree with our data in principle, though there are some discrepancies (Table 2). Thus, ref. 5 does not mention th frequency referring to vDN_ However, this is one of the characteristic frequencies in the spectrum of cytosine. According to our calculation, the vczN frequency is 1595 cm-‘. Deformational vibrations of N and H occur at Y = 1331 cm-‘. Some discrepancies are observed while comparing the frequencies and the interpretation of results below 1000 cm-‘. These arise from the complexity of interpretation of valence-deformation vibrational spectra of the pyrimidine ring. The force constants of the cytosine ring valence bonds obtained in ref. 5 are in good agreement with ours (Table 3). Since these are the first results

vc=c,vy

1663 1622 1608 1694

1669 0.17 1636 0.09 1490 0.09 1473 0.29 1438 0.97 1436 1426 0.82 1379 sh 1376 0.23 I 1372 sh 11336 0.14

rC=O 6aclaNH,

1712 1672

1426 1382

vc,o,vr

1476 1444

UC,-0, vy’,6C,-H,

vr, 6 C,H

ur, 6 C,H, 6 C,H

vr

1664

vN,=C:I,vr vNI=C1,UN+, vN,=C,

NH,

6 sds.

1743

0.17 0.07 0.66 0.17 0.19 0.61 1.00 1.00 0.26 811

1749 1729 1714 1670 1666 1666 1620 I1617 1696 1687

vOH *m NH, uN,H vs NH,

3688 3668 3468 3438

V

Type of vibrntion

0.14 0.12 0,07 0,20

I

Calcw Iation

3687 3669 3468 3438

u

Experiment

Cytosine

6 C,--H

E E

E K

K

K E E K

1423 1384 1379 1370

1489 1434

1660 1644 1610 I1604 1591 1677 1667 1620

vr, 6C,H vr, 6C,H

sh 1419 0.16 1389 0.24 0.34

ur

vc=c, vr v&N, vr uC=N,vr vC=N, vr

vC=O

vNID voBND, uOD “5 ND,

vr I+, 6C,H, 6C,H

1643

1662 1644 1600 1689

2G76 2672 2647 2630

Type of vibration

0.66 1477 0.97 1445

0.24 0.66 0.99 0.67 0.17 0.23 0.18 0.10

0.43 0.60 0.78 1701

1737 1714 1706

K K E

0.09 0.16 0.27 0.26

2674 2676 2664 2617

E K,E K K,E

V

I

Calculation

v

Tnutomer Experiment

Deuterocytosine

E E

E K

K E E K

K

K K,E E K,E

Tautomer

1419 1400 1386

0.1 1.0 0.2

0.16

0.9 0.8

1670 1667

1464

0.19 0.14 0.15

0.21 0.33

I

1624 1611 1607

3062 3042

V

Experiment

Pyrimidine

Calculatedand observed (T = 11 K Ar matrix) frequencies of in-plane normal vibrations of cytosine and deuterocytosine (u cm-‘)*

TABLE 1

w 2

0.23 0.12 0.05 0.09 0.09 0.33 0.10

ur,60H,6 C,H

1201 1118 1156

0.17 0.03 0.06 0.03 0.05 0.15 0.07 0.06 0.05 0.32 0.14 0.15 0.08

v,6, u,hr

u,hr u,6r ~,6~

6, hC=O,b,

bC=O,sCN

6CN cc=0

767 732

633 590 572

534 519

483

363 270

K E

E

K K

K E E

E

E

E

K K E K E K

478

0.05 484 436 340 252

522

763 731 700 686 613 579 563

828

0.25 0.17 0.08 0.04 0.05 0.02

809 783 769 753 709 665

947 913

0.091016 0.06 989 985 0.40 941 0.09 846

1167 0.041129

0.301306 1261 0.151212 1212 1206

1053 987

1116

1243 1239

$309

ND, 6(7-i 6ND,

K E IC E

6 C=O,6 CN

ac=o 6 C=O,6 CN 6C=0

K 6 c=o

6poudul.NDz K E V,6r E 6pendul. ND, K vr, 6N,D K “9 6r E ‘jr E I’, 6,

K

u,,aOD

6N,D

E

"r *r V,6r 6OD

K E K E K E

bC,H "I

6sds. ND2

vr,

“r,

6 sti.

“i-16C,H

I(, ltcto and E, en01 l’orms.

"I,relative intensit.y;sh, bandshoulder; {,mergedbantls;r, ring;v,stretching mode;b,bending mode; as, nntisymmetric;~,

780 767 749 709 635 613 574 567 535 519 607 498 441

v,6r ur V,~P dpcndul.NH, 6pcndul. NH, 6 pcndul.NHz, "r

vt6r

6C,H

E E K

K E

ur,6C,H,6 N,H “r, 6C5H

1230 1221

ur, 6 OH

E

vr,sC,--N,C,H

1310

0.05 0.08 1092 0.15 1068 0.03 992 971 926 913 847 817 0.04 807 0,21

1318 1256 1242 1223 1209 1193 1123 1107 1090 1082 980 -

0.1 0.1 0.9 0.08 0.26

symmetric;

767 749 719 678 621

803 0.12

1074 0.08 1034 0.05 990 0.1

1157

1223 0.28

E cu

394

TABLE 2 Calculated frequeucies of in-plane normal vibrations the isolated state (Xr) and a crystalline sampiea Crystalline

Isolated 3559 3468 3438

oNH+S

1743 1712 1653 1594

SW, vc=o vc=c, vC=N

1554 1444 1317

“r 6 CH, ur IJ=,vC-N,

123O 1156

Yr, 6CH, 6CH

1092 1068

v, 6, *, 6, v, 6,

uNH VNH,S

971 913 767 633 534

“I 61. v-6, 6,

519 363

6C0,6, 6CN

“v, valence;

of the ketonic

vy

6 NH 6NH

PNH,

6, deformation;

form

Crystalline

of cytosine

samp!e

[5 ]

vNH vCHs vCHas 6NH, vc=o vc=c, Y1 vC=N, V~

3356 3240 3190 3121 3114 1689 1650 1616 1530

vNH,as vNH,s VNH vCHs vCHas 6 NH, lJc=o vc=c 6NH, LJr

1508 1458 1362

vr 6 CH, vr urr UC-N,

1496 1450 1344

vr., 6NH 6 CH, LJC-N UC-N, 6 CH

1283 1224

vn b C&H

3380 3266 3164 3108 3095 1698 1664 1624 1549

vNH,as

1107 1044 957 844 752 534 525 534 366 P, torsional

VNH,S

6 NH

yr Y, 6r “r ur vr. P NH, y* sr 6r sco, 6C-N 6r 6CN vibrations;

1295 1222 1126 1036 1007 955 790 592 560 533 386

in

6 CHas “I vr, P NH, vr v, 6, “r, P NHz v,s, 6, 6 CO, 6 C-Ns 6, .Sc=o,

6CNas

r, ring.

on IR spectra for isolated molecules of cytosine in an Ar matrix at a dilution of 1:2000, i.e. under conditions when no intermolecular H bonds are formed, it is interesting to trace variations of frequencies corresponding to the same types of cytosine vibrations during transition from the crystalline to the isolated state. The frequencies VNH,~ and vm.rSnn are increased significantly: Av = 300 cm-‘. In the spectrum of a crystalline sample the vibrations of vNH occur only within one band 3380 cm-‘; in an isolated sample the band is split into u,, = 3559 cm-’ and vS = 3438 cm-‘. The deformational vibration frequencies are increased by 46 cm-’ for aNH and 52 cm-’ for vca. Also, the frequencies of the ring valence vibratiok are increased, which are in the region of 1700-1400 cm-‘. These are the frequencies which are mainly contributed by valence vibrations of C=O, C=N and C=C bonds. In this case the force constants of the bonds also increase (Table 3), which suggests redistribution of electron density in cytosine due to the fact that the free electron pairs O4 and N3 are no longer involved in the formation of H bonds and hence increase the electron density in the ring. The frequencies of valencedeformation vibrations of the ring vary much less.

395

TABLE

3

Force constants K

Kq1 *qa Kq3 Kq4 Kqs Kqa Kq7 Kqs Kq9 KPIO KS11 Kqrz Kqm

of cytosine

Keto

En01

Solid

Ax

Matrix

9.0 16.3 9.0 11.5 11.0 10.2 10.2 8.5 8.8 13.0 8.8 9.0 9.3

9.0 17.8 9.0 10.0 10.0 11.3 11.3 8.5 8.8 14.4 8.8 10.6 11.1

12.2 8.0 9.0 12.5 12.5 11.3 11.3 7.5 8.8 14.0 8.8 9.0 12.05

( lo6 cm*) K

*PI KPa Ka, Kg. Kos

KP6 PC@,

Kpa Kb

EllC.1

Keto Solid

Ax

Matrix

2.0 ;.i .

2.0

2.0 f:",

2.0 ;$' 0.8 0.15 0.8 ;.;

2.0 0.8 0.15 0.8 0.92 0.92

Kp,,

017

E-z

Kg,, KLh Kpt3

0.' 0.71 0.71

0175 0.75

H

Keto Solid

H q1qo

Hwq3

;*g -

Hqrqs 2.0 0.8 0.15 0.8 0.8 0.8 0.67 0.67 1.1 -

H qsq10

Hqeqlz Hqroq,z

;.z * 0.4 2-O

En01 AZ

Matrix

1.8 0.1 1.5 2.0 0.4 2.0

0.8 0.1 4.0 1.0 0.4 3.5

The analysis of the force field of the enol form of cytosine shows that in the pyrimidine ring there is a stronger delocalization of n-electrons as compared to the keto form. This is due to increasing force constants of muItipIe bonds Fc,=,> and Fc,=N, = Fc,--N,, a slight decrease in Fc+ and a change in the coefficients of bond-ring interactions. The force field of the enol form of cytosine becomes close to that of aromatic molecules. Indeed, the majority of vibrational modes of the enol form of cytosine, in which the greatest contribution arises from vibrations of the ring bonds, are close to the appropriate frequencies of pyrimidine oscillations (Table 1). The region of 1400-1000 cm-’ embraces the frequencies corresponding to the ring vibrations greatly contributed by deformation vibrations of C-H bonds for both the tautomeric forms of cytosine. They are smaller for the enol form than for the ketonic one, but the differences are not large: 10 to 20 cm-‘. In the same spectral region are the frequencies characteristic of vibrations of the fragment vC,-O-H, vCZ-O = 1379 cm-‘, 60H = 1193 -I. The appropriate bands are also observed in the cytosine-D3 spectrum. ZZlow 1000 cm-’ a number of characteristic frequencies of valence-deformation vibrations of the pyrimidine ring are seen, which are practically insensitive to deuteration or substitution effects. These are 980, 807, 767 and 749 cm-’ frequencies. it should be noted in conclusion that the results obtained in this work must be taken into account in experiments involving isolated molecules of cytosine, for example in mass-spectrometry, ion cyclotron resonance, etc. Besides, the results would permit a correct interpretation of IR spectra of molecular complexes containing the molecules studied. CONCLUSIONS

The method of matrix isolation is employed to obtain high-resolution IR spectra of cytosine and cytosine-I&. As the analysis of characteriitic frequencies of these compounds shows, the isolated molecules of cytosine

396

are characterized and amino-enol.

by an equilibrium of two tautomeric forms: amino-keto

For the first time the measurement technique for matrix dilution of complex molecules in a solid rare gas is realized using a quartz differentidl low-temperature microbalance whose sensitivity is 5 X lO+ g. Frequencies and shapes of flat normal vibrations are calculated for the ketonic form of crystalline cytosine and keto and enol forms of cytosine and cytosine-II3 isolated in an Ar matrix. The vibrational frequencies and force fields of crystalline and Ar-isolated cy-tosine molecules are shown to be different_ The difference is due to the fact that perturbing environmental factors causing frequency and force field variations are practically absent in an Ar matrix. The force fieId and the frequencies of in-plane normal vibrations of the ketonic and enol forms differ significantly. The ring vibration frequencies of the enol form, which is more aromatic, are lower than the appropriate frequencies of the ketonic form, being closer to the frequencies of pyrimidine. REFERENCES 1 2 3 4 5 6 7 8 9 10

11 12 13 14 15

J. D. Goddard, P. G. Mezey and I. G. Csizmadia, Theor. Chim. Acta, 39 (1975) 1. D. L. Breen and R. L. Flurry, Theor. Chim. Acta, 23 (1971) 138. B. Ya. Simkin, Khim. Geterotsiki. Soedin. (1978) 94. C. L. Angell, J. Chem. Sot., (1961) 504. H. Susi, J. S. Ard and J. M. Purcell, Spectmchim. Acta, Part A, 29 (1973) 725. H. Susi and J. S. Ard, Spectrochim. Acta, Part A, 27 (1971) 1549. D. Barker and R. E. Narch, Acta CrystaUogr., 17 (1964) 1581. B. I. Sukhorukov, A. S. Gukovskaya, L. V. Sukhoruchkina and G. I. Lavrentieva, Biofizika, 17 (1972) 5. B. I. Sukhorukov and V. I. Poltev, Biofizika, 9 (1964) 148. L. A. Gribov and V. A. Dementiev, Metody i Algoritmy vychislenii v teorii Kolebatelnykh Spektrov Molekul (Methods and Algorithms of Computation in the Theory of Vibrational Spectra of Molecules), Nauka, Moscow, 1981. N. A. Smorygo and B. A. Ivin, Khii. Geterotsiki. Soedin., 1 (1975) 98. E. D. Radchenko, A. M. Plokhotnichenko, G. G. Sheina and Yu. P. Blagoi, Biofizika, 28 (1983) 559; Stud. Biophys., 87 (1982) 251. M. J. Nowak, K. Szczepaniak, A. Barski and D. Shugar, J. Mol. Struct., 62 (1980) 47. G. Sheina, E. Radchenko, A. M. Plochotnichenko and Yu. Blagoi, Biofiiika, 27 (1982) 933. N. A. Smorygo and B. A. Ivin. Khim. Geterotsikl. Soedin., 10 (1975) 1402.