3520
J. Phys. Chem. 1993, 97, 3520-3524
Tautomers of 6-Thioguanine: Structures and Properties Jerzy Leszczyiiski Department of Chemistry, Jackson Stare University, 1400 Lynch Street, Jackson, Mississippi 3921 7 Received: September 15, 1992
The molecular geometries of four tautomers of 6-thioguanine (6TG) were studied using a b initio LCAO-MO method at the Hartree-Fock level. All considered species are minimum structures on the 3-21G* potential energy surface (PES) of 6TG as determined from harmonic vibrational frequencies calculations carried out at the same level. The basis set effects on the relative energies of the tautomers and their one-electron properties were studied at the HF/6-31G*, HF/6-31GS*, and HF/6-311G** levels. Our best estimation of the relative stabilities of the tautomers includes electron-correlation contributions calculated at the MP2/6-3 1G* approximation and the zero-point energy contributions scaled by 0.9. At this level the thione N(7)H tautomer 1 is the global minimum species lying on the PES 2.3 and 12.0 kJ mol-' below thiol N(9)H (3)and thione N(9)H (2) forms, respectively. The relatively large dipole moment predicted for the thione tautomer 2 indicates the computed tautomeric equilibria should be shifted toward this form in polar solvents. Calculated molecular parameters of 6TG tautomers are similar to those of the corresponding guanine forms. Thus suggest the possibility of an effortless replacement of guanine by its thioanalog in nucleotides, which would rationalize observed biological activity of 6TG.
Introduction Knowledge of the molecular structures and properties of biomolecules with potential drug activities are prerequisites for developing new active drugs. Successful synthesis and proved antitumor activity of thiopurines (e.g., 6-thioguanine (6TG) and 6-mercaptopurine)has stimulated research efforts in the chemistry and biochemistry of these species.' These compounds display a significant activity against L12 10 leukemia cells. Recent IR and UV spectral studies revealed that 6TG acts as a bidentate ligand in organomercury(I1)complexes,coordinatingthrough sulfur and deprotonation of N7.2 Milne and Townsend have synthesized and tested for biological activity 6-selenoguanosine, the next heavier analog of these bases.) Other sulfur-containing nucleosides have been proved to have chemotherapeutic and antitumor An enormous number of unusual nucleic functions a~tivities.~ acid-base (NAB) analogs, including thio derivatives, have been studied to develop a new active agent for therapy of malignant t i ~ s u e s .Some ~ of the thio bases (Zthiouridine and 6-thioguanosine) were incorporated by animal cells into their RNA, while similar thio derivatives (8-thioguanosine or 2-thiocytidine) were not metabolized.6-8 There is a continuing interest in supportingexperimentalstudies on biomolecules by accurate theoretical data. Ab inito HartreeFock (HF) calculations on molecular structures and properties of guanine tautomers employing relatively large 6-3 1G** basis set and including electron correlation effects at the second-order Moller-Plesset perturbation theory (MP2) have also been reported9-l I Theoretically predicted flat potential energy surfaces (PES) for the pyramidalization of the NH2 groups in guanine and other studied NABS suggests that adopting conformations which maximize interaction during base pairing with their counterparts should be an effortless process due to a small energy required for distortion of the amino groups.I2 Tautomeric and rotameric equilibria, vibrational frequencies, and intensities of 9-methylguaninewere studied in argon matrix by IR spectroscopic technique and by ab initio HF/3-21G level calculations.~3 It is well-known that selected substituents (e.g., S,Se, F, Br) can modify properties and structures of the nucleic acid bases. The effect of oxygen substitution by sulfur on relative energies and biological activities of uracil tautomers and their derivatives has been recently investigated by us.14-16 Our calculations predicted that the substitution of oxygens by sulfur atoms in 0022-3654/93/2097-3520504.00/0
uracil may enhance the probability of its spontaneous mutations by a factor of lo3. If the biological activity of uracil and its thio analogs can be ascribed to the relative concentrations of the rare tautomers, the computational data suggest that dithiouracil has an activity similar to that of 2-thiouracil,while 4-thiouracilshould have an activity analogous to that of uracil. An experimental study, carried out on rats, suggests these compounds possess the predicted ability to interfere with the endocrine function of the thyroid gland." This paper presents ab initio studieson the molecular geometries and the relative energiesof four tautomers of 6-thioguanine.These species have not been investigated yet by ab initio calculations. The available information about the molecular structure of 6TG was derived from solid-statestudies. ThecrystallographicalX-ray analysis of 6TG showed that in the solid state it exists as tioneN(7)H tautomer 1 (a hydrogen atom is bonded to N7),'8 unlike guanine, which was found to appear as oxo N(9)H species.19 Recently reported Raman and IR spectra of 6TG supports predominant existence of 1 in a solid state.20 Since guanine and 6TG crystallize as different tautomericforms, it is not possible to determine from the crystallographical data biologically important information on specificeffects of the sulfur substituent on the molecular parameters of guanine. Furthermore, the comparison between the bond lengthsof imidazolerings of guanine and 6TG might be misleading, and several large differences observed by Bugg and Thewalt from their crystallographical structuresiscould be caused by differences of molecular parameters for different tautomeric forms rather than by the heavy substituent. Ab initio studies on various tautomeric speciescould also determine the influence of sulfur substituent on the molecular geometry of bases in question,and we shall address these problems in the paper. Method The ab initio LCAO-MO mzthod2' was used for the study of the title species. The calculations were carried out with the GAUSSIAN90 and GAUSSIAN92 series of programs.22 All geometries were optimized completelyby thegradient procedure*) at the C,symmetry. The closed-shell restricted Hartree-Fock 3-2 1G* level24was applied to find stationary pointson the potential energy surface (PES). At this level all the optimized structures were checked by analysis of harmonic vibrational frquencics 0 1993 American Chemical Society
The Journal of Physical Chemistry, Vol. 97, No. 14, 1993 3521
Tautomers of 6-Thioguanine
Q
a
P
P
b
Q Figure 1. HF/3-2IGZ optimized C, geometry of 1 (thione N(7)H tautomer of 6TG). The experimental parametersinare given in parentheses. (a) Bond lengths are in angstroms. (b) Bond anglesare in degrees.
Figure 2. HF/3-21G* optimized C,, geometry of 2 (thione N(9)H tautomer of 6TG). (a) Bond lengths are in angstroms. (b) Bond angles are in degrees.
obtained from diagonalization of force constant matrices to find the order of the stationary points. To improve the calculated energies, the extended 6-3 1G*, 6-31G**, and 6-31 1G** basis set25326 were used for single-point calculations at the optimized 3-21G* geometries. This choice of the applied basis sets allows to establish the role of d-polarization functions on the second-row elements (3-21G' basis set contains only sulfur d-polarization functions) and p-polarization functions on the hydrogen atoms, as well as thequality of thevalence triple-{ basis set on the relative energies and dipole moments of the studied tautomers. Electron-correlation contributions were determined by Maller-Plesset perturbation theory2' through single-point second-order (MP2) calculations at the 6-31G* level using the frozen-coreapproximation. Such a calculation is denoted MP2/ 6-31G*//HF/3-21G*, where // means the geometry of". The final relative energies were corrected for HF/3-21G* zeropoint energy (ZPE) differencesscaled by the recommended factor of 0.9.28
comparison, due to a large experimental uncertainty in their positions (the experimental error is equal 0.05 A and 2O for hydrogen bond lengths and angles, respectively), the average difference between experimental and theoretical bond distances is 0.0166 A, while the mean deviation of the calculated bond angles from their experimental counterparts is equal to 0 . 8 8 O . This degree of agreement suggestsa reliability of the theoretically predicted geometries as well as only a minor distortion of the molecular structure of 6TG by crystal and packing forces. The calculated geometrical parameters of the 6TG tautomers are very similar to those of the corresponding guanine species.I0 The observed differences are primary in the C6-NI and C b C 5 bond lengths. The calculated bond distances are always shorter for the thio species, by 0.023 and 0,013 A, respectively, when comparing 1 with the analogous N(7)H tautomer of guanine. The corresponding differences between bond distances of other studied oxo and thio tautomers are even smaller. Calculated HF, MP2, and ZPE energies (Table I) and computed relative energies (Table I1 and Figure 5 ) of 6TG tautomers allow for prediction of the tautomeric equilibrium for the isolated species. Apparently, only positive vibrational frequencies indicated that all studied species are minimum structures at the HF/3-21G* PES. Clearly, 1 is the global minimum tautomer at all applied levels of theory, being stabilized relative to the second thione species (2); (thioneN(9)H tautomer) byadditionofthed- (5.7 kJ mol-') and p-polarization functions (0.05 kJ mol-') and by inclusion of the electron-correlation effects (2.3 kJ mol-'). Improvement of
Results and Discussion An important question which can be answered on the basis of the results of theoretical calculations is the consequence of oxygen substitution by sulfur on molecular parameters of the studied NABS. Figures l a s h o w molecular parametersof 6TG calculated at the HF/3-21G* level. In the case of the N(7)H tautomer 1 the experimental X-ray crystallographicalparameters'* are listed. Good overall agreement between ab initio and crystallographical data was found for 1. If one excludes hydrogens from the
Leszczyfiski
3522 The Journal of Physical Chemistry, Vol. 97,No. 14, 1993
b
Figure 3. H F/3-2 1G* optimized C, geometry of 3 (thiol N(9)H tautomer of 6TG). (a) Bond lengths are in angstroms. (b) Bond angles are in degrees.
the basis-set-quality (HF/6-3 1 1G** level) stabilized tautomer 2 by 0.7 kJ mol-' relatively to the HF/6-31G** level and ZPE contributions decreased the relative energy of 2. This effect was negligible (0.24 kJ mol-'). Relative energies of the thiol tautomers (N(9)H 3 and N(7)H 4) depended noticeably on the basis set applied. The inclusion of the d-polarization functions on the second-row elements (6-3 1G* vs 3-2 lG* basis sets) significantly stabilized these species (by 23.2 and 27.0 kJ mol-' for 3 and 4, respectively). The effect of the ppolarization functions on hydrogens (6-31G* vs 6-31G** basis sets) was insignificant, contributing less than 0.5 kJ mol-' toward increasing of their relative energies. At the HF/6-31G* level the order of relative stabilities of 2 and 3 reverses and the thiol N(9)H tautomer 3 becomes the second most stable species. The electron correlation energy contributions introduced through a second-order perturbation theory at the MP2/6-31G* approximation provide additional stability to the thiol N(7)H tautomer but slightly destabilize 3. ZPE contributions play a signficant role in stabilizationof the both thiol tautomers, lowering their relative energies by approximately 10 kJ mol-'. At the MP2/6-3 lG* + 0.9 ZPE level relative energy of 3 amounts only to 2.3 kJ mol-', while 2 is the third most stable tautomer with the relative energy of 12.0 kJ mol-'. The contribution of the p-polarization functions on hydrogens to the relative energiesof tautomers deserves additional comments. Comparing available theoretical results from the studies on oxohydroxy tautomeric equilibria in I ~raci1,~6.2~ pyri-
b
Figure4. HF/3-21G* optimized C,$geometryof4(thiol N(7)H tautomer of 6TG). (a) Bond lengths are in angstroms. (b) Bond angles are in degrees.
Figures. Plot of the relativeenergiesversustheoretical levels. Allenergies arerelative tothat of 1. TOTaretheMP2/6-31G*//HF/3-2lGS+0.9 ZPE(HF/3-2lG*) relative energies.
done,30and pyrazolo[4,3-d]pyrimidine-S,7-(4H,6H)-dione ( T ) , ~ ' one concludes that relative energies of hydroxy vs oxo forms calculated at the HF/6-31GZ* level are lower by about 6.5 kJ mol-' incomparison totheHF/6-31G*results. Thiseffect,being almost independent of the considered system, is also additive. The dihydroxy tautomer of T is stabilized by inclusion of the
Tautomers of 6-Thioguanine
The Journal of Physical Chemistry, Vol. 97, No. 14, 1993 3523
TABLE I: Total Energies (-au) of 6TC Tautomers HF/3-21G* HF/6-31G* HF/6-31G** HF/6-311G** MP2/6-31G* ZPEU
1
2
3
4
857.582 37 862.032 29 862.050 43 862.165 90 863.580 02 -0.124 71
857.580 75 862.028 50 862.046 64 862.162 36 863.575 36 -0.124 61
857.569 99 862.028 75 862.046 74 862.162 86 863.575 6 4 -0.120 83
857.558 54 862.018 71 862.036 80 862.152 61 863.568 34 4 . 1 2 0 21
Zero-point energies (uncorrected) at the HF/3-21G* level.
Conclusions
TABLE 11: Relative Energies (kJ mol-') of 6TC Tautomers HF/3-21G* HF/6-31G* HF/6-3 1G** HF/6-31 IG** MP2/6-3 1G* TOT"
1
2
3
4
0 0 0 0 0 0
4.2 9.9 10.0 9.3 12.2 12.0
32.5 9.3 9.7 8.0 11.5 2.3
62.6 35.6 35.8 34.9 30.7 20.0
MP2/6-31Gz//HF/3-21G* energies corrected for 0.9 scaled ZPE(HF/3-21G*).
TABLE 111: Dipole Moments (debyes) of 6TG Tautomers H F/ 3-2 1G* HF/6-3 1G* HF/6-3 1G** HF/6-31 IG**
studied species calculated a t different levels. The calculated dipole moments show little sensitivity to the applied basis set. At all levels the calculated dipole moment of 2 is more than 2.5 times larger than those of the other species. This strongly suggests that this tautomer is stabilized by the polar solutions. We conclude that the calculated dipole moment magnitude and small relative energy of 2 might result in increasing of its relative concentration in the polar environment.
1
2
3
4
2.25 2.41 2.40 2.43
8.50 8.64 8.64 8.69
3.59 3.59 3.59 3.59
3.51 3.27 3.27 3.26
p-polarization functions by 13.2 kJ mol-' while its monohydroxy tautomers are stabilized only by 6.5 kJ m01-I.~' Interestingly, our results for 6TG as well as those obtained from the study of the basis set effects on the relative energies of 2,4-dithiouracil3* indicate destabilization of the thiol vs thione forms upon inclusion of the p-polarization hydrogen functions. This effect is on order of the one-tenth (0.5 kJ mol-l) of that noticed for the oxo counterparts. Closer examination of the calculated bond lengths shows that the sulfur-thiol's hydrogen (thiol's hydrogen-N1) distances are longer by 0.4(0.2) A, in 6TG than corresponding distances to the hydroxy hydrogen in guanine. Consequently, the interaction of the thiol's hydrogen with electronegative atoms S and N is minimized, resulting in a drastic reduction of the stabilization effect of the hydrogen p-polarization functions in the studied thiol derivatives of NABS. Thecomputed relativestabilitiesof 6TG andguanine tautomers deserve some comments. For both studied systems N(7)H forms are the global minimum species. However, the relative energy of 1 at the HF/3-21G//HF/3-21G PES of guanine amounts to 10.5 kJ mol-', and its stabilization at the MP2/6-31G**//HF/ 6-31G level is a consequence of both an increase in basis set size and inclusion of the electron-correlation contributions. In contrast, the corresponding tautomer of 6TG is the lowest energy species at all applied levels of calculations. Interestingly, the important contributions stabilizing rare tautomers of 6TG are ZPEs. These contributions decrease the relative energies of the thione tautomers by as much as 10 kJ mol-l; the same effect noticed for the hydroxy tautomers of guanine amounts only to 1 kJ mol-'. Similar trends have been observed in HF calculations on uracil,I6 thiouracil^,^^ and dithioura~il'~ and in MP2 level calculations for the series of seleno and thio analogs of phosphine oxide and phosphinous acids. Though ZPE contributions at the MP2/TZP approximation stabilize hydroxy vs oxo forms of phosphorous compounds by 0.1 kJ mol-!, the analogous contributions for thioj3 and ~ e l e n forms o ~ ~ amounts to 5.4 and 6.5 kJ mol- 1, respectively. The predicted tautomeric equilibrium pertain only to isolated tautomers and might be changed by biological environment. Quantitatively, the interaction between different tautomers and polar environment can be correlated to the magnitude of the solute dipole moment. Table 111 shows dipole moments of the
The important conclusions from the present study can be summarized as follows: All studied tautomers are minimum structures on the HF/ 3-21G* potential energy surface. Thione N(7)H tautomer 1 is the global minimum species with an energy difference of 2.3 and 12.0 kJ mol-' (at the MP2/6-31G* ZPE level) with the thiol N(9)H and thione N(9)H forms, respectively. There is an overall good agreement between a b initio HF/321G* and experimental bond distances and angles. Compared to the parent hydroxy compounds, relatively small changes in the relative energies of thiol forms are found upon inclusion p-type polarization functions on hydrogens. However ZPE contributions significantly stabilize thiol tautomers. Due to a large difference in dipole moments, 2 is predicted to be stabilized in the polar solvents. Calculated molecular parameters of 6TG tautomers are very similar to those of the corresponding guanine forms. This similarity together with the different relative stabilities calculated for their tautomers has important biological consequences. Our calculations indicate that due to comparable molecular structures 6TG could easy replace guanine in nucleotides. Such replacement followed by interaction with polar biological environment might result in stabilization of the thione form 2. However due to its higher relative energy (12.0 kJ mol-') than that of the corresponding guanine tautomer (4.6 kJ mol-'), the relative concentration of 2 is predicted to be smaller than would be for guanine.
+
Acknowledgment. This study was supported by Grant # 332090 from the NIH foundation and by the DOE/LBL/JSU/AGMEF consortium. The Mississippi Center for Supercomputing Research is acknowledged for generous allotment of computer time. The author isgrateful to Drs.J. S.Kwiatkowski and J. Zubkowski for helpful discussions. References and Notes ( I ) Bennett Jr., L. L.; Montgomery, J.A. InMerhodsinCancerResearch; Busch, H., Ed.; Academic Press: New York, 1967; Vol. 111. (2) Ahluwalia,V. K.;Kaur,J.;Ahuja,B.S.;Sodhi,G.S.J. Inorg. Biochem. 1991, 42, 147. (3) Milne, G. H.; Townsend, L. B. J. Hererocyd. Chem. 1971, 8, 379. ( 4 ) Calabresi, P.; Parks, R. E. In The Pharmacological Basis of Therupeurics; Goodman, L. S., Gilman, A,, Eds.; Macmillan: New York. 1970. (5) Yamada, 0.; Larrson, B. S.; Amilcar, R.; Dencker, L.; Ullberg, S. J. Inuesr. Dermarol. 1988, 88, 873 and references therein. (6) Yu, M. W.; Sedlack, J.; Lindsay, R. H. Arch. Biochem. Biophys. 1973, 1955, 1 1 I . (7) Ono, M.; Kawakami, M. J. Biochem. 1977,81, 1247. (8) Melvin, W. T.; Milne, H . 8.;Slater, A. A.; Allen, H. J.; Keir, H. M. Eur. J. Biochem. 1978, 92, 373.
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