Theoretical and matrix-isolation experimental studies on 2-thiocytosine and 5-fluoro-2-thiocytosine

239

Biochimica et Biophysica Acta, 1172 (1993) 239-246 © 1993 Elsevier Science Publishers B.V. All rights reserved 0167-4781/93/$06.00

BBAEXP 92469

Theoretical and matrix-isolation experimental studies on 2-thiocytosine and 5-fluoro-2-thiocytosine Hanna Rostkowska a, Maciej J. Nowak a, Leszek Lapinski a Maria Bretner Kulikowski b, Andrzej Leg c,1 and Ludwik Adamowicz c

b

Tadeusz

a Institute of Physics, Polish Academy of Sciences, Warsaw (Poland), b Institute of Biochemistry and Biophysics, Polish Academy of Sciences, Warsaw (Poland) and c Department of Chemistry, University of Arizona, Tucson, AZ (USA) (Received 7 May 1992) (Revised manuscript received 15 September 1992)

Key words: 2-Thiocytosine;5-Fluoro-2-thiocytosine;Tautomerism; Matrix isolation; IR spectra; Ab initio calculation

2-Thiocytosine (s2fyt) and 5-fluoro-2-thiocytosine (fSs2Cyt) were studied by means of IR spectroscopy under different environmental conditions: isolated in low-temperature inert gas matrices, associated in thin amorphous and polycrystalline films. The compounds isolated in matrices were only very slightly influenced by the environment. From the analysis of the IR spectra of both compounds it appears that they exist in inert gas matrices only in the amino-thiol tautomeric form. Strong environmental effects were observed for s2Cyt and fSs2Cyt deposited in the form of thin polycrystalline films. Contrary to matrices, in polycrystalline films the amino-thione form dominates for both s2Cyt and fSs2Cyt. The experimental findings are in agreement with the ab initio quantum mechanical calculations of the relative total energies of the tautomeric forms. Those energies were calculated using the Self Consistent Field method corrected for electron correlation effects with the use of the second-order many-body perturbation theory (SCF + MBPT(2)). The theoretical calculations show that the amino-thiol tautomeric form is more stable than the amino-thione form by 38 kJ mol -~ and 48 kJ tool 1 for sZCyt and fSs2Cyt, respectively. Both molecules, s2Cyt and fSs2Cyt, may also appear in the uracil-like imino-thione tautomeric form, which is predicted to be only 8 kJ mol-1 less stable than the amino-thione form. A new method of the preparation of fSs2Cyt is reported.

Introduction 2-Thiocytosine (s2Cyt), its derivative 5-fluoro-2thiocytosine (f5 s2Cyt), and their nucleosides and nucleotides are of interest and importance because of their unusual biological properties, s2Cyt has been found in Escherichia coli t R N A ser and t R N A ~g [1]. It is located in semi-invariant position 32 in the above tRNAs and in tRNAs from several other sources [2,3]. 2-Thiocytosine nucleosides exhibit some moderate inhibitory activity against vaccina [4], human cytomegalo and Epstein-Barr [5] viruses in cell cultures. It was previously established that replacement of the hydrogen atom by fluorine at the C(5) position of the pyrimidine ring has a strong biological response [6]. For example, fluorination of uracil and 2'-deoxyuridine 1 Permanent address: Department of Chemistry, University of Warsaw, Pasteura 1, 02-093 Warsaw, Poland. Correspondence to: M.J. Nowak, Institute of Physics, Polish Academy of Sciences, AI. Lotnik6w 32/46, 02-668 Warsaw, Poland.

transforms these normal nucleobase and nucleoside into potent antitumor drugs - 5-fluorouracil and 2'-deoxy-5-fluorouridine [7]. Recently, it has been shown that introduction of fluorine at the C(5) position of 2'-deoxy-2-thiouridine yields a potent antitumor agent, 2'-deoxy-5-fluoro-2-thiouridine [8], which in the form of 5'-monophosphate, proves to be a strong, slow-binding inhibitor of thymidylate synthase [9]. Substitution of the C(5) hydrogen of 2'-deoxy-2-thiocytidine with fluorine gives potent in vitro antileukemic nucleosides aand fl-2'-deoxy-5-fluoro-2-thiocytidine [10]. s2Cyt and f5s2Cyt, like other heterocyclic compounds, may potentially exist in several tautomeric forms. The isomeric structures, for which calculations were made, are drawn in Scheme I. Two factors, the relative free energy of isolated tautomers and the free energy of their interactions with the environment, determine which isomeric form the molecule adopts under certain conditions. Tautomerism of sZCyt in different solutions was intensively investigated by Igarashi-Yamamoto et al. [11]. It was suggested that this molecule exists as an

240

H

H H \, /

H

N

N

X

i $~L.~... N / I H (II)X - H (Vl)X- F

(1) x - H

(V) X=F

H \

H H \ / N

N

H ~ N ~ X

H ~ N ~ X

I H

(m) x - H

O~ X = N

(VII) X = F

(VIII)X - F Scheme

I.

appearance of the rare tautomers with certain forms of biological activity [27]. It should be pointed out that, since in the nucleosides the thiocytosines are substituted in the N(1) position, the amino-thiol form observed for nonsubstituted s2Cyt and fSs2Cyt may not be adopted in nucleosides. In crystallographic studies [28] it was established that s2Cyt exists in crystal only as the amino-thione tautomeric form. The crystal structure of s2Cyt is stabilized by a pattern of hydrogen bonds, N H . . . N and NH • • • S. It was demonstrated that the pyrimidine ring is nearly planar in the crystal [28], with some hydrogen atoms (at C(5), C(6), N(1) and at the amino group) slightly deviating from the molecular plane. One may differentiate between two types of crystallographically nonequivalent molecules, which possess slightly different bond lengths and bond angles. The sulfur atoms are not protonated in the crystal - an indication that s2Cyt appears only in the thione tautomeric form. The existence of the amino-thione form of sZCyt in crystal was independently confirmed with the use of Raman, IR and far IR methods [29,30]. The structure of fSs2Cyt was not studied previously either in solution, or in the solid state or under isolation conditions. Materials

equilibrium mixture of the thione and thiol forms. However, it was not clarified in this work which of these two forms predominates. In the present work we address the problem of how the interaction of thiocytosines with the environment affects the tautomeric equilibrium. In a separate paper [12] we presented and interpreted the infrared absorption spectra of the observed tautomeric forms of s2Cyt and fSs2Cyt. The samples of s2Cyt and fSs2Cyt were studied in different media, i.e., isolated in inert gas low-temperature matrices (where the interactions with the environment are insignificant), in disordered amorphous layers rapidly frozen from the gas phase (at 10 K) and in polycrystalline films (where the intermolecular interactions are significant). We have chosen such different conditions for our experiments in order to test how the adopted tautomeric forms of s2Cyt and fSs2Cyt depend on the environment. In previous works [13-21] it was demonstrated that the ratio of the tautomeric forms observed in low-temperature matrices reflects the tautomeric ratio in gas phase from which the matrix was formed. It has been stated many times in the literature that the dominant tautomeric structures may be different in the gas phase (or matrix), in solutions and in the solid phase [22-26]. The environment-dependent tautomerism is an interesting phenomenon from the point of view of biological properties of the studied molecules. A hypothesis is still considered connecting

and

Methods

A commercially available sample of 2-thiocytosine supplied by Sigma was used in this study. 5-Fluoro-2-thiocytosine (fSsZCyt) [31] was prepared as follows: A 50 ml ampoule of Rasotherm glass containing 1.577 g (9.73 mmol) 2,4-dithio-5-fluoropyrimidine [9] and 20 ml concentrated aqueous NH 3 was sealed and shaken for 2 h at 37°C and then heated at 100°C for 20 h. The resulting reaction mixture was filtered, evaporated to dryness in vacuo and recrystallized from ethanol to yield 774 mg (55%) of fSs2Cyt; m.p. 218-220°C; ,~pH2 269.5 nm (e 18000); /~pH7 267 "- m a x max nm (e 19300); Amaxl pH 12 220.5 nm (e 15900), /~pHl2 262 nm (e pn 1~. max,2 12600). A.... 3 306 nm (e 4000). High resolution mass spectroscopy (EI) re~e, M+: found 145.01076; calculated 145.01096. Argon of spectral grade was purchased from VEB Technische Gase, Leipzig (Germany). The general procedure of matrix preparation was the same as that described elsewhere [32]. Matrices were deposited on the CsI window mounted on the cold finger of the continuous flow helium cryostat. The temperature of the cold window was 6-7 K and the temperature of the oven from which the sample sublimed was about 440 K for 2-thiocytosine and 410 K for 5-fluoro-2-thiocytosine. Solid films were obtained by deposition of the vapors of the compound (inert gas flux cut-off) onto the cold window maintained at 10 K. Disordered, amorphous layers, obtained in such a way,

241 TABLE I

2- Thiocytosine

Infrared spectra were measured on Perkin-Elmer 580B spectrometer.

The total and relative molecular energies for the amino-thione (I), imino-thione (III) and amino-thiol (II) tautomeric forms.

Details of calculations

I

III

II

Components of the total molecular energy, in hartrees SCF/3-21G* -711.629539 -711.628865 -711.619614 SCF/DZP -715.305444 -715.304250 -715.312363 MBPT(2)/DZP - 1.134819 - 1.133792 - 1.138198 ZPE/3-21 G * 0.105207 0.105975 0.100944 Total molecular energy, in hartrees ( S C F / D Z P + M B P T ( 2 ) / D Z P + 0.9- Z P E / 3 - 2 1 G * ) -716.345577 -716.342665 Relative tautomerization SCF/3-21G * SCF/DZP MBPT(2)/DZP 0.9. Z P E / 3 - 2 1 G * AFvib (T = 440 K)

-716.359711

energy, in kJ mol-1 0.0 1.7 0.0 3.1 0.0 2.7 0.0 1.8 0.0 2.0

26.1 - 18.2 - 8.9 - 10.1 -11.2

Cumulative free energy of tautomerization, in kJ m o l ( S C F / D Z P + M B P T ( 2 ) / D Z P + AFvib) 0.0 7.8 - 38.3 1 hartree = 2.6282.103 kJ mo1-1. Fvib = free Helmholtz vibrational energy calculated as in Ref. 19 using theoretically ( S C F / 3 - 2 1 G * ) calculated frequencies scaled down by 0.9.

The optimization of the molecular structures of different tautomeric forms of s2Cyt and fSs2Cyt were performed using the ab initio Self Consistent Field (SCF) procedure and the standard 3-21G* basis set [33]. The equilibrium geometries were found to be planar for all the tautomeric forms at this level of theory. This was confirmed by the subsequent calculations of the vibrational frequencies, which were all positive. The internal molecular energies of the tautomeric forms (for the SCF (3-21G*) optimized geometries) were evaluated by performing single point SCF calculations corrected for electron correlation effects with the use of the second-order many-body perturbation theory (MBPT(2)) [34] using the double-zeta basis set of Dunning [35] with the polarization functions of Redmon et al. [36]. Huzinaga's 5s3p [37] basis set plus an additional d (6d) polarization orbital with the exponent equal to 0.6 was used for the sulfur atom in these calculations. All of the calculations have been carried out with the use of the GAUSSIAN 86 program [38]. Results and Discussion

were heated to about 300 K and allowed to anneal overnight. Spectra of the resulting polycrystalline films were measured after cooling the layer down to 10 K.

Results of calculations The calculated total energies of the tautomers of s2Cyt and fSs2Cyt are presented in Tables I and II,

TABLE II

5-Fluoro-2-thiocytosine The total and relative molecular energies for the amino-thione (V), imino-thione (VII), amino-thiol (VI) and amino-thione N(3)-H (VIII) tautomeric forms. VII

VI

VIII

Components of the total molecular energy, in hartrees SCF/3-21G * - 809.942852 SCF/DZP - 814.170857 MBPT(2)/DZP - 1.309700 ZPE/3-21G * 0.096669

V

-809.942468 -814.170391 - 1.307900 0.097399

-809.936529 -814.181535 - 1.313117 0.092489

- 809.928360 -814.156409 - 1.311061 0.095773

Total molecular energy, in hartrees ( S C F / D Z P + M B P T ( 2 ) / D Z P + 0.9. Z P E / 3 - 2 1 G *) - 815.395549

- 815.390632

- 815.411412

- 815.381274

Relative tautomerization energy, in kJ m o l - 1 SCF/3-21G * 0.0 SCF/DZP 0.0 MBPT(2)/DZP 0.0 0.9.ZPE/3-21G * 0.0 AFvib (T = 410 K) 0.0

1.0 1.2 4.7 1.7 1.8

16.6 -28.0 -9.0 -9.9 -10.9

38.1 37.9 -3.6 -2.1 -2.7

Cumulative free energy of tautomerization, in kJ mol 1 ( S C F / D Z P + M B P T ( 2 ) / D Z P + AFvib) 0.0

7.7

- 47.9

31.6

1 hartree = 2.6282-103 kJ mol -x. Fvi b free Helmholtz vibrational energy calculated as in Ref. 19 using theoretically ( S C F / 3 - 2 1 G ) calculated frequencies scaled down by 0.9.

242 TABLE III

2- Thiocytosine The theoretical values of the molecular dipole moment and of the rotational constants for the amino-thione (I), imino-thione (III) and amino-thiol (II) tautomeric forms. I

III

II

Dipole moment, Debyes SCF/3-21G * 9.07 SCF/DZP 8.65

6.44 6.05

4.10 3.97

Rotational constants, GHz SCF/3-21G * 3.567 1.322 0.965

3.561 1.324 0.965

3.548 1.328 0.966

respectively. The molecular dipole moments and rotational constants are also given in Tables III and IV. Due to the lack of the experimental counterparts a direct comparison of theoretical and experimental values for the dipole moments was not possible. One may only say that the SCF dipole moments are usually overestimated, by some 10%, with respect to the experimental values [33,39,40]. The theoretical rotational constants are probably as reliable as the calculated molecular geometries, because these values are simple functions of the molecular stuctures. The calculations predicted a significant stabilization (38 and 48 kJ mol i for s2Cyt and fSsZCyt, respectively) of the amino-thiol form with respect to the amino-tione tautomer. Such a large total energy difference between the amino-thiol and the amino-thione tautomers may be, however, reduced in a polar solvent due to fairly strong interactions with the environment. In the first approximation the tautomer with higher dipole moment should be more strongly stabilized in the polar media than that with lower dipole moment [41,42]. Thus, the extraordinary stabilization of the amino-thione form in the polar environment is expected from a large dipole moment (almost 9 Debyes).

TABLE IV

5-Fluoro-2-thiocytosine The theoretical values of the molecular dipole moment and of the rotational constants for the amino-thione (V), imino-thione (VII), and amino-thiol (VI) tautomeric forms. VII

VI

Dipole moment, Debyes SCF/3-21G* 7.10 SCF/DZP 6.77

V

4.71 4.46

2.34 2.28

Rotational constants, GHz SCF/3-21G * 3.242 0.927 0.721

3.254 0.920 0.717

3.240 0.928 0.721

The energy difference between the amino-thione and imino-thione tautomeric form is estimated to be about 8 kJ mol 1, but when higher-order correlation effects are incliaded, this difference could be reduced in a similar way as it was determined for cytosine [43]. A small energetic difference between amino-thione and imino-thione tautomeric forms of thiocytosine has also been suggested in the previous quasi ab initio theoretical work of Leg and Ortega-Blake [44]. The present theoretical results may provide valuable information for biochemical investigations. As it is known, the most stable molecular structure of sZCyt and fSsZCyt, i.e., the amino-thiol form, may not be adopted in nucleosides (due to a N(1) substitution), but a strong stabilization of the amino-thiol form of s2Cyt in comparison to other tautomeric structures may explain a tendency stronger than for unmodified cytosine to form the cyclic structure (anhydro-nucleosides) that joins the C(2)-ring carbon atom via sulfur atom with either the C(2'), C(3') or C(5')-ribose carbon atoms. Observed tautomerism

The IR spectra of sZCyt and fSsZCyt obtained for monomers isolated in the Ar matrix and for the associated compound in cold (10 K) amorphous and polycrystalline films are shown in Figs. 1-4. The detailed analysis of these spectra and assignment of the absorption bands to the theoretically predicted normal modes of sZCyt and f5sZCyt tautomers has been published elsewhere [12]. Isolated monomers

The tautomeric forms present in the matrix may be determined by analyzing the range of the IR spectrum, in which the absorption bands due to vibrations of the functional groups characteristic for a particular tautomer are expected. Such groups are: NH 2 for the amino forms, NH for the thione forms and SH for the thiol forms. In the higher frequency range (3600-2000 c m - l) the IR spectra of s2Cyt and fSsZCyt are very similar (Figs. 1A and 3A). In both spectra the bands due to the NH 2 antisymmetric and symmetric stretching vibrations (at 3562, 3444 cm -1 for sZCyt and 3563, 3446 cm - t for fSsZCyt) and the weak band due to the SH stretching vibration (2617 cm -1 for s2Cyt, 2619 cm -1 for fSsZCyt) were recognized. The very low absolute intensity is characteristic for the band due to the SH stretching vibration. That is why it is so difficult to detect this band in the spectrum. The weak SH stretching bands were found at similar spectral positions as in the spectra of thiol tautomers of other pyrimidine-derivatives isolated in matrices [15,45,46]. The appearance of the NH 2 and SH stretching bands indicates that the amino-thiol tautomeric form (II and VI) is present in the matrix. Lack of the bands due to the N(1)H stretch-

243 ing vibration, which are expected to appear, within few c m - 1 , a t 3400 c m - 1 [15,46], shows that the thione N(1)H form is not populated to a detectable extent. We do not think that the band due to stretching of the N(1)H group may be hidden underneath the band of the NH 2 symmetric vibration which is placed about 40 cm-1 above the expected frequency of N(1)H stretching. The possibility of the existence of certain amounts of thiocytosines in the thione N(3)H form was rejected because the theoretical calculations places the thione N(3)H tautomer significantly higher in energy than the amino-thiol form. In the IR spectra of previously studied thione derivatives of heterocycles [15,18,46] the stretching vibration

0.5 ~

~

L.~ 0 Z

~,-~O.5 O Or)

0

0

. 0

5 i

~ b

i

i

i

i

i

i

0 c m

--1

Fig. 2. Infrared absorption spectra of 2-thiocytosine (s2Cyt) in the 1700-400 cm -1 region. (A) Compound isolated in Ar matrix (10 K). (B) Amorphous layer (10 K). (C) polycrystalline film (10 K), the sample (B) annealed.

0.2 I

2640 0.1

j~

0

I

I

of the C=S group was usually coupled with ring deformations giving rise to several bands, one of which was particularly intense. This band was observed at: 1148 cm -1 for 2-thiouracil [18], 1142 cm -1 for 4-thiouracil [18], 1183 cm -1 for 3(2H)-pyridazinethione [15], 1140 cm -1 for 4(3H)-pyrimidinethione [15] and 1144 cm -1 for 2(1H)-pyridinethione [46]. In the IR spectrum of matrix isolated s2Cyt no bands were observed in the region between 1216 and 1098 cm-1 [12] (see Fig. 2A). That might be regarded as a further indication of the absence of the thione forms of this compound. Unfortunately, the same argument may not be applied to fSs2Cyt since the band due to the ring stretching of the thiol form [12] is present in the spectrum at 1182 cm-1 (At matrix) (see Fig. 4A). The detailed analysis of It~ spectra of Ar matrix isolated s2Cyt and fSs2Cyt ha~ been presented in Ref. 12. The general agreement between observed spectra and the spectra theoretically predicted (at the SCF/6-31G** level) for the aminothiol forms of both compounds gives further support to the conclusion of the present work. The high dominance of the amino-thiol (II and VI) tautomeric form in matrices i.e., under the conditions where the intermolecular interactions are negligible, is in agreement with the results of quantum mechanical calculations (Tables I and II) predicting the amino-thiol form to be the most stable.

2600

h

~-

0j

t.d 7°0.8

< m nO m <

0.4

J 600'

' '5200 ' ' ' '2800 ' ' ' '2400 ' ' '

'2000 '

Amorphous layers -1

cm Fig. 1. Infrared absorption spectra of 2-thiocytosine (s2Cyt) in 36002000 cm-1 region. (A) Compound isolated in Ar matrix (10 K); the cumulated spectrum (10 scans) of the band due to the SH stretching vibration is shown in the box. (B) Amorphous layer (10 K). (C) polycrystalline film (10 K), the sample (B) annealed.

Contrary to rare gas matrices, in which the guest molecules may be treated as non-interacting, in amorphous layers the molecules do interact with each other. The very low temperature during deposition causes rapid loss of energy of the molecules condensing from the vapor phase to the amorphous layer. That is why

244 midine [47]. After relaxation at room temperature, when the layer achieved thermodynamical equilibrium, the band in the region near 2400 cm -1 disappeared while the massive band near 3000 cm-1 became much more intense (Figs. 1C and 3C). The same effect, reflecting the change of the tautomeric form (thiol --* thione) during annealing, was previously observed for other mercaptocompounds [46,47]. For both compounds, the broad absorption bands similar to those observed in the spectra of crystals are present in the region 3500-2700 cm-1. Those bands are due to vibrations of the NH 2 and NH groups, testifying to the presence of the amino-thione tautomers. Because the NH 2 and NH groups are directly involved in the hydrogen bondings, those bands are broad and shifted to lower frequencies (with respect to the usual positions of the NH 2 and NH stretching bands in the matrix spectra). The additional confirmation of the existence of the amino-thione and amino-thiol tautomers in amorphous films is provided by the analysis of the lower frequency range shown in Figs. 2B and 4B. The same bands as observed in the spectra of the matrix isolated aminothiol tautomers may be recognized in the spectra of the amorphous layer. However, in the later case the bands are broader and somewhat shifted. In addition to those absorptions, other bands could be recognized at frequencies close to that of the bands observed in the spectra of crystalline samples. Those bands were assigned to the amino-thione tautomeric form.

0.04 0.4 0.02 , , 2640 2600 0.2

A

0 0.2

Ld

0 Z

~0.4 o m

0.2

I

3600

I

I

I

I

3200

I

I

2800

I

[

I

I

I

2400

cm

I

I

2000 -1

Fig. 3. Infrared absorption spectra of 5-fluoro-2-thiocytosine (fSs2Cyt) in 3600-2000 cm-l region. (A) Compound isolated in Ar matrix (10 K); the cumulated spectrum (10 scans) of the band due to the SH stretching vibration is shown in the box. (B) Amorphous layer (10 K). (C) polycrystalline film (10 K), the sample (B) annealed.

the complexes in such a layer may not achieve their most stable form. The IR spectra indicate that in the amorphous layers the hydrogen-bonded complexes composed of molecules in both thiol and thione forms are present. In the spectra of s2Cyt and fSsZCyt (Figs. 1B and 3B) we find weak and very broad bands of the hydrogen bonded SH group in the 2580-2250 cm-1 region. The IR absorption bands due to the stretching of the SH group engaged in the hydrogen bond are usually weak (because of very low absolute intensity of the SH stretching band) and shifted down in comparison to the frequency of the unperturbed SH stretching vibration. Spectra of similar shape were reported for amorphous layers of 2-mercaptopyridine [46] and 2-mercaptopyri-

Polycrystalline films During annealing the structure of amorphous layer was transformed into the structure of polycrystal, as it was demonstrated in the case of layers of 2-pyrimidinones [47]. The IR spectra of sZCytand fSseCyt in

0 Z

,..~ O. 4 .,-w0 {../3 CD

~:

0

0

.

O

i

1700

4

~

1400

~

i

i

1 100

8QO

500

200 c m

--1

Fig. 4. Infrared absorption spectra of 5-fluoro-2-thiocytosine (fSsZCyt) in the 1700-200 cm -1 region. (A) Compound isolated in Ar matrix (10 K). (B) Amorphous layer (10 K). (C) polycrystalline film (10 K), the sample (B) annealed.

245 matrices and in crystalline films differ considerably. Such a substantial change in the spectral patterns must reflect the change of the tautomeric form. They are too pronounced to result only from intermolecular interactions in the crystalline state. The effect of the change of the tautomeric form is well demonstrated in the spectra of amorphous and crystalline layers in the SH and NH stretching region (Figs. 1B and 1C, 3B and 3C). After annealing the relatively weak band of the SH stretching at approx. 2450 cm-1 in the spectrum of amorphous s2Cyt vanished, whereas the strong absorption of the NH stretching increased. The more detailed analysis of the IR spectra of polycrystalline layers of s2Cyt and fSs2Cyt is reported in a separate paper [12]. From X-ray crystallographic studies [28] it is known that the crystal of sZCyt consists of molecules in the amino-thione (I) form. This was confirmed by Yadav et al. [29], who studied sZCyt in KBr and polyethylene pellets by means of the IR spectroscopy. Our results are in full agreement with their conclusions. We found in the spectra of crystalline layers the bands due to the vibrations of the hydrogen-bonded NH and NH 2 groups of the form (I), but no absorption of the SH group (characteristic to the thiol form). The similarities between the spectral patterns of crystalline s2Cyt and fSs2Cyt indicate that also in the case of crystalline 5-fluoro-2-thiocytosine the amiflo-thione (V) form dominates. The imino-thione forms were predicted by theory as being 8 kJ mo1-1 less stable than the amino-thione tautomers. We cannot exclude the existence of small amounts of those forms in the crystals. The spectra of the crystals are composed of much broader bands than the spectra of the matrices and therefore, due to their overlapping, it is not possible to detect absorption of small amounts of rare tautomeric forms. Conclusions In the quantum-chemical calculations of internal energies of 2-thiocytosine and 5-fluoro-2-thiocytosine in different tautomeric forms (performed at the ab initio (SCF(DZP) + MBPT(2)(DZP) + ZPE(3-21G*)) level) the amino-thiol form was predicted to be the most stable. Large internal energy differences between the amino-thiol forrn and other tautomers strongly suggest that, in both s2Cyt and fSs2Cyt, only the amino-thiol tautomer should be observed in the gas phase as well as in the Ar matrices. In the matrix isolation studies of 2-thiocytosine and 5-fluoro-2-thiocytosine monomers only the molecules in the amino-thiol form were detected. This observation is in qualitative agreement with the theoretical results. In the crystalline state, contrary to low-temperature matrices, the dominance of the amino-thione tautomers of s2Cyt and f5s2Cyt was found.

The theoretical calculations indicated that substitution of 2-thiocytosine at C(5) with fluorine caused additional stabilization of the amino-thiol tautomeric form. The similar effect was observed for cytosine where fluorination (also at C(5) position) shifted the tautomeric equilibrium in low-temperature matrix towards high predominance of the amino-hydroxy form [48]. The tautomeric equilibria of s2Cyt and fSs2Cyt exhibit strong dependence on the interactions with the environment and are significantly different in low-temperature matrices (this study), in solutions [11] and in the solid state.

Acknowledgements The present work was supported in part by a grant from the National Science Foundation (INT-9100935). Ludwik Adamowicz was supported by the American Cancer Society in the form of the Junior Faculty Research Award.

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