Prototropic molecular-zwitterionic tautomerism of xanthine and hypoxanthine : unexpected biological view

The prototropic molecular-zwitterionic tautomerism of xanthine and hypoxanthine has been investigated by the semi-empirical quantum-chemical AMI method. Geometric, energetic and other physico-chemical features of their complete molecular-zwitterionic families of tautomers have been established. The influence of the environment with universal solvatation mechanism on the tautomeric equilibrium has been evaluated at Onzager approximation. The comparison of the calculations with the H NMR data favours the N7H Xan and the N9H Hyp tautomers in DM SO solutions. A possible biochemical role of tautomeric (especially zwitterionic) forms of Xan and Hyp is discussed. The stabilizing influences of both environment and specific interactions with proteins on rare tautomeric forms are considered. The biological significance of high energy tautomers is discussed.

Introduction.Biological role of xanthine (Xan), hypo xanthine (Hyp) and their nucleosides xanthosine (X) and inosine (I) is well-known.They can arise as intermediates of nucleic acid degradation, Hyp and Xan being the products of the adenine and guanine (accordingly) dearaination, that could cause a muta genic effect [1,2].The oxidation of Hyp results in Xan as well.Besides, Hyp is a substrate of metalloenzyme xanthine oxidase.At the same time they can play the role of precursors of the canonical purine bases.Anticodon triplets of some tRNAs include Hyp as a minor base [3 ].
These bases are also of interest for their pharma cological activities.Methyl derivatives of Xan (caf feine, theobromine, theophylline) are widely used due to their hypotensive and spasmolytic effects.Inosine was found to have various therapeutic effects as a component of drug complexes (see e. g. [4] and references therein).There is an information on its anti-HTLV activity [5].
Ionization states of Hyp in solution were inves tigated by UV spectroscopy [22 ].First ionization potentials of gas phase Hyp was estimated experi mentally [23 ].Conformations of crystal deoxyinosine (dl) were studied by X-ray spectroscopy [24,25].Some tautomers of Hyp isolated in a low-temperature matrix were detected by IR spectroscopy [26 ].
NMR spectroscopy was applied to the study of I and its complex inosiplex [4].The analogs of yeast alanyl tRNA, in anticodon loop of which inosine was substituted by guanine and adenine, were synthesized to elucidate its biological function [27 ].
The interactions of Xan, Hyp and a number of their methyl and glycosyl derivatives with ionized and neutral amino acid carboxylic groups were inves tigated in anhydrous dimethylsulfoxide (DMSO) by IR, UV and NMR spectroscopies [28,29].
The energetic stability, structural and electronic properties of all ketonic and enolic tautomers of Hyp were studied at the level of DFT with BP functional and the DZVP bases set [30].Energetical stabilities of seven ketonic and enolic molecular tautomers of Hyp were calculated by ab initio method at the MP2 (6-31 +G(d, p)) and DFT (B3LYP 6-31+G(d, p)) levels of the theory [31 ].
However, the prototropic tautomerism of Xan and Hyp, unlike the canonical bases, is insufficiently studied.Particularly, there is a lack of comprehensive information on energetics stability, geometric and electronic structures of Xan and Hyp, and their molecular and zwitterionic tautomers.Inasmuch as Xan, Hyp and their derivatives were shown to form often the proton transfer complexes [32 ], we suggest that their prototropic tautomers could include zwit terionic ones, which may be of great importance in biochemical reactions.
To fill the gap, in this work the complete families of molecular-zwitterionic tautomers of Xan and Hyp were investigated by the semi-empirical quantumchemical AMI method and the environmental effects (specific and nonspecific) on tautomeric equilibrium were evaluated.
Experimental.Methods.The qualitative stereo chemical analysis shows that complete molecularzwitterionic tautomeric family of Xan, the sources being three mobile (acidic) imino protons and six basic centers -acceptors of proton (two carbonyl groups and four endocyclic nitrogen atoms), com prises 53 possible structural isomers -45 molecular and 8 zwitterionic ones.
The complete molecular-zwitterionic family of Hyp tautomers, which is generated by three mobile (acidic) protons and five proton acceptor atoms (one exocyclic oxygen and four endocyclic nitrogens), consists of 12 molecular and 2 zwitterionic tautomers.The topological properties of multidimensional surface of potential energy for each of 53 possible tautomers of Xan and of 14 tautomeric forms of Hyp were studied by AMI method, which is the most proper one among semi-empirical quantum-chemical methods for tasks and objects of this kind.
The calculations were performed in a wide range of initial geometries, which were formed by various disturbances of primarily optimized tautomeric struc tures with full optimization of all the structural parameters at gradient norm below 0.01.
The room temperature concentrations of isolated tautomers of Xan and Hyp were evaluated according to Boltzmann distribution.*H NMR spectra were obtained on a Gemini-200 spectrometer (Varian) in 5 mm tubes; the chemical shifts of proton resonances are presented in parts per million (ppm) against TMS as internal standard. Materials.
Results and Discussion.Xanthine.
The molecu lar-zwitterionic family of Xan tautomers, covering the range of relative energies about 43 kcal/mol, includes 32 structural isomers -24 molecular and 8 zwit terionic ones (fig. 1 and tables 1-4).The rest of 53 possible isomers are plane symmetrical transitional states of hydroxylic group rotation of some enolic tautomers which correspond to saddle points on the multidimensional surface of potential energy.The most molecular-zwitterionic tautomers of Xan, with exceptions of the XVIII and XX molecular tautomers (symmetry C,) and their XIX and XXI enantiomers respectively, are planar symmetrical dipole-stable structures (symmetry C $ ).At that the atom deviations from planarity are far smaller than amplitudes of vibrations.In the case of non-planar tautomers and their enantiomers there are significant atom de viations from planarity.So, e. g. for enantiomeric pairs of tautomers XVIII, XIX and XX, XXI dihedral angles I C6C5N7H | are 53,5° and 30,3° respectively.Non-planarity of the XVIII-XXI molecular tau tomers is probably determined by steric factor, in particular, the repulsion between adjacent hydrogen atoms, one of which is hydroxyl one.
As it could be seen from the table 1, the Xan molecular tautomers in free state occupy the range of relative energies about 33 kcal/mol, their dipole moments are within the limits from 1.03 (IX) up to 9.36 D (XXII) and the first potentials of ionization change from 8.91 (XX, XXI) up to 9.34 eV (I).The all tautomers-zwitterions except the main tautomerzwitterion XXVI are the structures with higher energy compared to molecular tautomers and they occupy the range of relative energies about 25 kcal/mol, having much greater dipole moments within the limits from 9.36 (XXVII) up to 14.81 D (XXXII) and slightly smaller ionization potentials, which change from 8.36 (XXXI) up to 9.03 eV (XXXII) (table 1).It should be noted that tautomer-enantiomers (their numbers are given via commas in table 1) have the identical scalar physical and chemical characteristics and differ only in spatial orientations of dipole moments.
The X, XI, XIII, XIV, XX, XXI, XXIII mo lecular tautomers and the XXVIII and XXX zwitterion-tautomers are the high energy rotamers of the VI, IX, XV, XVIII, XIX, XXII molecular tautomers as well as the XXVII and XXIX zwitterion-tautomers, respectively (fig. 1 and 2).They are formed from the low energy tautomer-prototypes through rotation of hydroxy lie groups by 180° round the ordinary bonds C-O.The barriers of these rotations are within the range about 4.5-7 kcal/mol; as it has been shown by calculation of rotamerization reaction coordinates, the saddle points correspond to the pairs of mirrorsymmetrical transitional states of rotamerization with orthogonal orientation of hydroxylic group relative to the ring plane (symmetry C,) and considerably nonplanar geometry of the latter.In the case of other Xan tautomers with one or two hydroxylic groups the transitional state of rotamerization, as evidenced by the calculation of reaction coordinates, is the planar symmetrical one (symmetry C s ).It is realized through the rotation of hydroxylic group on 180° around the ordinary bonds C-O, the barriers of these rotations being within the range 5-9 kcal/mol.
In most cases of Xan molecular tautomers the energetically preferable orientation of hydroxylic gro up is the czs-position, when it shades the adjacent double bond C=N or C=C (fig.1); the same situation is observed for the molecular tautomers of the canonic nucleotide bases [6,7 ] with some exceptions.Appa rently, the interaction of hydroxylic groups with лг-electron system of the adjacent double bond is

Table 1 Some calculated physico-chemical characteristics of the xanthine molecular and zwitterionic tautomers*
•Total energy of the I molecular tautomer with fixed nuclei is equal to 49875.9 kcal/mol; **ab initio data.
decisive for its as-orientation.In the separate cases of fra/ts-orientation of OH group the main role is probably played by an intramolecular hydrogen bond 0-H...N.
It should be emphasized that exactly the cisorientation of hydroxylic group about the adjacent double bond C2=N3 is typical for the molecular tautomer III (fig.1), which coincides with the results [21 J. So, the explanation of elementary mechanisms of point mutations (caused by guanine deamination) based on molecular configuration of Xan III with hydroxyl ґгяля-огіепіаііоп [2 J, which is the high energy transitional state of tautomer HI rotame rization (its energy is over 5.95 kcal/mol than mole cular tautomer III), is incompetent.
Molecular and zwitterionic tautomers of Xan are quasi-rigid structures, which is evidenced by the existence of low frequency (v < 150 cm" 1 ) fundamental vibrations in their calculated spectra.For example, the frequencies of non-planar vibrations of «butterfly» type (bend of purine ring along the C4=C5 bond) for the energetically preferable molecular tautomers I and II, as well as for the main tautomer-zwitterion XXVI are equal to 98, 91 and 110 cm -1 , accordingly (the non-scaled values of calculated frequencies are given).
The obtained results evidence (fig. 1 and table 1) that prototropic tautomerism of Xan in free state is caused, as a rule, by tautomerism of its imidazole ring.The main and practically single tautomer of Xan in isolated state (~ 99.9 %) is molecular diketo form

*In brackets are given calculated energies of deprotonation for the N7H (I) and N9H (II) Xan tautomers (E NH7 /E N9H ) from [34J
I with the localization of imidazole ring imino proton at the N7 atom (tables 2, 3), which agrees with the results of the work [21 1, where four N7H and N9H tautomers of Xan were investigated by the ab initio LCAO-MO method at high approximations.We note that the concentration of the main tautomer-zwitterion XXVI (the most energetically preferable one from the family of Xan tautomer-zwitterions) with imino pro ton localization at the N1, N3 and N9 atoms is practically zero (~ 3.4-10~' 5 %) in free state at room temperature.
It is obvious that the main molecular tautomer I as compared to tautomer II with higher energy has weaker both proton donor and proton acceptor pro perties of imidazole ring (table 3).The transition of Xan from the isolated state to solution with universal mechanism of solvatation («inert» solvent) doesn't practically change the char acter of tautomer equilibrium.In fact, even under є > > 1 the energy gap between tautomers II (N9H) and I (N7H) in solution, the value of which in free state with regard to zero vibrational energy (66.28 and 66.02 kcal/mol for tautomers I and II, correspon dingly) measures AE = E u -E ] = 3.94 kcal/mol, dec reases, as estimation indicates accordingly to Onzager model by the following formula: (E -dielectrical permeability of the solvent; a" a nradii of Onzager's spherical cavities for tautomers I and II, correspondingly (a 7 ~ a u = 4.7 A); /*" /u ucorresponding dipole moments of tautomers I and II (table D), compared to free state only by Ai? = == 1.96 kcal/mol.This fact as well as results [211 evidences insignificant population of tautomeric state II of molecular Xan in «inert» solvent at room temperature.The concentration of tautomer-zwitterion XXVI under the same conditions is too low (~ 1.6* 10~1 0 %) to be taken into consideration.The energy gap E xxyi -E \ under transition from gase phase into the «inert» solvent decreases with the same formula (a XXVI = = 4.7 A) by Ais=6.36 kcal/mol and reaches 16.02 kcal/mol with regard to the zero vibrational energy (66.15 kcal/mol).
Based on the results of UV study [33] the conclusion was made that in water solutions Xan exists mainly as the N7H tautomer.The analysis of chemical shifts values of the N1H, N3H, N9H/N7H and C8H protons in 'H NMR spectra of Xan, m 9 Xan and m 3 Xan and the comparison them with calculated [34 ] values of the deprotonation energy of the relative Xan protons for the N7H and N9H tautomers (table 4) give evidence in favour of the N7H tautomeric form of Xan and m 3 Xan in DMSO.But it should be taken into consideration that interactions with amino acid residues in active sites of enzymes may induce transition to the N9H tautomeric form.For example, deprotonated carboxylic groups of aspartic and glu tamic acids can obviously form complex with Xan via two cooperative hydrogen bonds involving adjacent protons at the N3 and N9 nitrogen atoms [29,30 ].
The analysis of electronic characteristics of Xan molecular-zwitterionic tautomers (tables 1, 3) points to the essential distinctions in their reactivity, par ticularly their proton donor and acceptor properties.The most considerable changes in charges at atoms and appropriate electron densities, as well the first potentials of ionization are therewith observed during transition from molecular to zwitterion form.The character of these changes indicates the greater reactvity of zwitterionic forms of Xan than molecular ones.
Among the zwitterionic tautomers the most atten tion should be paid to the ylidic form in which imino protons are localized at the N1, N3, N7 and N9 atoms and the C8H proton is absent.The molecularkinetic mechanism of hydrogen-tritium exchange of C8H group of Xan with water at acidic and neutral pH values is considered to be associated with the ylidic form [35 J. Our results may be regarded as the conformation of the ylidic mechanisms of H<*T ex change.In fact, the XXV Xan ylidic form in free state is the main one, i. e. energetically the most favorable form among the family of ylidic tautomers, and it is characterized by high proton affinity of the C8 atom (226.9 kcal/mol), exceeding its affinity in Xan mo lecular form II with proton of the N7 atom.The energy barrier of the XXV ylidic form formation from the molecular one in vacuum with participation of free proton (18.2 kcal/mol) agrees with experimental ene rgy of activation of hydrogen-tritium exchange in the C8H group of Xan (21 ±1 kcal/mol [331) in water solution at acidic and neutral pHs.In essence, the main ylidic form XXV is properly the transitional state of H««»T exchange reaction in the C8H group of isolated Xan.
Hypoxanthine.By calculation results, the tauto meric molecular-zwitterionic family of Hyp, occuping the energy range about 26 kcal/mol, consists of 12 structural isomers -10 molecular (I-VII, IX-XI) and 2 zwitterions (XII, XIII) (fig. 2 and table 5).The other part of the total of 14 possible ones (E7t and Elc from [30]) are planar symmetrical transitional states of hydroxyl group rotation around the ordinary C-O bond in the V and XI enolic molecular tautomers with the barriers of internal rotation ~ 9.6 kcal/mol.All without exception tautomers of Hyp are the planar symmetrical (symmetry C 4 ) and dipole stable struc tures: atom deviations from planarity (<7.5 10~4 A) are much less than amplitude of out-of-plane vibra tions.This coincides with the data [30].
The IV and X enolic molecular tautomers are the high energy rotamers of the III and IX molecular tautomers, correspondingly: they are formed from the low energy tautomers on account of rotation of hydroxylic group by 180° around the ordinary bond C-O.In this case the saddle points correspond to the pairs of mirror-symmetrical transitional states with orthogonal orientation of hydroxylic group against the purine ring «ріапе» (symmetry C,) and significantly non-planar geometry of the latter.It takes notice of the fact that the energetically favourable orientation of hydroxylic group is c/s-position, when it «shades» the adjacent double bond C6=N1 or C5=C6 (fig. 2 and table 6).The same situation takes place in the case of molecular tautomers of canonical nucleotide bases 16, 7 ] with rare exceptions.Obviously, it is the general case that in enolic molecular tautomers of nucleotide bases the interaction of hydroxylic group with я-electron system of the adjacent double bond is decisive for its as-orientation.
The Hyp tautomers are quasi-rigid structures, which is evidenced by the presence of low frequency (< 150 cm" 1 ) fundamental vibrations in their cal culated spectra.Thus, for example, the frequencies of the lowest energy planar vibrations of «butterfly» type (bend of purine ring along the СФ=С5 bond) for the most energetically favourable tautomers I (N9H) and II (N7H) measure 134 and 145 cm" 1 , correspondingly (the non-scaled values of calculated frequencies are given).
In accordance with data [30,31 ], results obta ined (fig.2, table 5) give evidence that prototropic tautomerism of Hyp, as in the case of Xan is mainly caused by the tautomerism of its imidazolic ring.The stability order of Hyp tautomers agrees with the ab initio data [31 ], except the most stable I and II Hyp tautomers.The AMI method overestimates the I tautomer stabiliity.The ab initio calculations predict a slight energetics advantage (0.8-0.9 kcal/mol) of the II tautomer.However, tautomer relative energy deviations between our results and ab initio calcu lation [31 ] do not exceed such deviations between the [30] and [31 ] data.Furthermore, our dipole moment values for Hyp tautomers and analogical values for six most stable Hyp tautomers from [31 ] are rather close.The latter is also true for a first ionizations potentials which should be regarded as upper limits of expe rimental values [23 ].It is noteworthy that the ground state tautomer of Hyp is the least polar (its dipole moment is equal to 1,72 D (table 5)).
In isolated state at room temperature two ener getically favourable ketonic tautomers N7H (II) and N9H (I) coexist, less polar form N7H being the main one (80 %).Under the same conditions the concen tration of the main enolic tautomer (III) measures only 0Л5 %.This fact quantitatively confirms the conclusion about the character of Hyp tautomeric equilibrium in vacuum made on the bases of expe rimental investigation of its IR spectra in low tempe- •Lengths of the valence bonds are given in A and valence angles -in degrees.
rature matrix isolation [261.It should be noted that in order to interpret the weak vibrational bands observed at significant accumulation of signals at the Hyp evaporation temperature 262 °С and assigned to enolic tautomers, in the first turn its rotamer IV (along with the main enolic tautomer III of Hyp) should be taken into consideration, but not the high energy rotomer V (fig.2, table 5), as done in [26].
The analysis of electron characteristics of Hyp molecular-zwitterionic tautomers (tables 5, 7) points to the significant difference in their reaction ability, particularly proton donor and proton acceptor proper ties.The greatest changes of charges at atoms and correspondent electron densities, as well the most considerable deviations in the first potentials of ionization are observed under the transition from molecular to zwitterionic form.The character of these changes shows the greater reactivity of zwitterionic form of Hyp as compared to molecular ones.
The Hyp transition from the isolated state to

Compound N9H/N7H C2H
solvent with the universal mechanism of solvatation shifts completely the N7H«»N9H tautomeric equi librium to the more polar tautomer I (80 % at the room temperature).Really, the energy gap between the I and II tautomers in the solvent with e > 1 increases (valued as in the case of Xan; a x ~ a n = 4.5 k f*\> -as * n table 6) compared to vacuum by AE = 1.63 kcal/mol and reaches 0,83 kcal/mol.The concentrations of all other tautomers with except of the II tautomer (14.1 %) under the same conditions are too small for consideration.
According to the results of UV spectroscopic investigations 133], neutral molecule of Hyp exists mainly as the N9H tautomer in water solution.Closed values of chemical shifts of the N1H, C2H and C8H protons in the !H NMR spectra of the Hyp and dl solutions in DMSO points out to prevalence of the N9H tautomer in DMSO as well (table 8).
Thus, in conditions similiar to physiological ones the content of Hyp enolic form compared to content of guanine enolic form [6,7,15] may be neglected as the value of Hyp enolic form is smaller than guanine enolic form by one and half order.So, the suggestion [26 ] on exactly this reason, favouring the decrease of possibility of formation mismatched pairs Hyp.Ura and thereby possible mistakes of codon-anticodon PROTOTROPIC MOLECUIAR-ZW1TTERIONIC TAUTOMERISM recognition, of substitution of guanine to Hyp in anticodon triplets of tRNAs is absolutely reasonable.
As in the case of Xan, exactly the VIII ylidic form in free state is the main one, i. e. the most energetically favorable among the zwitterionic tau tomers family.It is characterized by the high proton affinity of the C8 atom (232.77kcal/mol), the energy barrier of reaction of ylidic form VIII formation from molecular tautomer I in vacuum with the free proton participation (14.48 kcal/mol) conforming to expe rimental energy of hydrogen-tritium exchange activa tion in the C8H group of Hyp (21 ±1 kcal/mol 133]) in water solution under acidic and neutral pHs.The main ylidic form VIII is in fact the transition state of reaction H«*T exchange in the C8H group of Hyp in isolated state.
Concluding remarks.All the conceivable mole cular and zwitterionic tautomers of the minor nucleo tide bases Xan and Hyp were investigated by the semi-empirical quantum-chemical AMI method with full optimization of all the parameters.Geometric and electronic structures and energetic features of their complete molecular-zwitterionic families of tautomers were determined.It should be emphasized that the Xan and Hyp prototropic tautomerism comprises all their protons including so-called carboprotons, i. e. the protons bound to the carbon atoms.
In this case the AMI method appears to be quite relevant for estimation of tautomer physico-chemical features which are biologically significant.
One of the consequences of tautomer formation may be significant non-planarity of some of them.So, in spite of planarity of the molecule of Xan in the ground state and most of its tautomers, some enolic tautomers of Xan were proved to be essentially non-planar because of the hindrance of hydroxyl groups with two adjacent imino groups.In the case of one neighboring imino group, due to steric conflict between hydroxyl and imino protons, enolic tauto mers of Xan and Hyp with as-orientation of hydroxyl group to adjacent imino group can not be considered as stable structures but as transition states of hydro xyl group internal rotation.Exactly because of that reason energetic disadvantage of the E7t and Elc enolic «tautomers» from [30] relative to the E7c and Elt tautomers are as high as 8.83 and 10.65 kcal/mol respectively, which are close to the barriers of hydro xyl rotations (~ 9.6 kcal/mol).
So then, it is a steric hindrance in enolic tau tomers that reduces the numbers of molecular-zwit terionic tautomers in comparison with prediction of qualitative stereochemical analysis.Apropos, ener getically favourable orientation of hydroxylic group is a еде-position to the adjacent double bound.It seems to be typical for oxopurine tautomers of nucleotide bases.Thus, prototropic tautomerism of Xan and Hyp is mainly attached to the imidazolic part of purine ring.This type of tautomerism in purinic bases is essentially affected by the nature of pyrimidinic rest.The N9H*>N7H tautomerism prevails in Hyp [30,31 ], Pur [9,10], Ade Xan [18,19,21 ], but in the case of Gua main tautomeric events take place in pyrimidinic ring [6,7,11,13,15,26].
The evaluation of polar environmental influence carried out in the framework of classical Onzager model shows the shift of the N9H«*N7H equilibrium to the more polar N9H tautomer for both bases.For isolated and dissolved in anhydrous DMSO Xan the N9H<*N7H equilibrium is strongly shifted to the diketonic -N7H tautomer.Unlike isolated state [30,31 ], in anhydrous DMSO solution Hyp was shown to exist mostly as the keto-N9H tautomer with noti ceable amount of the keto-N7H tautomer.It is noteworthy to mention that the N7H tautomers of purine nucleotide bases play a key role in synthesis of nucleic acids ( [36] and refs.therein).
So, tautomeric equilibrium of Xan and Hyp may be disturbed by environment both via non-specific interactions determined by tautomer dipole moment and dielectric permeability of solvent at the first approximation and by local specific interactions (e. g., with amino acid residues in the active sites of enzymes) derived from geometric and electronic com plementarity of the partners.The authors believe that the latter is the leading factor in real biological systems.Molecular-zwitterionic families of tautomers are situated in the energetic range which is com mensurable with the energy of a few H-bonds.High energy tautomers can be realized through expendient hydrogen bonding with amino acid residues of pro teins, for example.
We mean that biological significance of nucleotide base tautomers could not be confined by their direct physico-chemical detection under thermodynamic equilibrium and homogenic environment.In other words, from biological point of view the most ener getically favourable tautomers are merely «a top of iceberg*.Such approach to the problem of tauto merism in nucleotide bases, in general, may cast a new light on the mechanisms of enzymatic catalysis, interconversions of the bases, their metabolism etc., though traditionaly the interest in tautomerism of nucleotide bases was confined only to its possible mutagenic effects.
Perceptibly greater polarity of zwitterionic tauto mers determines their greater ability of participating in chemical reaction.In spite of their energetic disadvantage they may be formed in essentially non-equilibrium environment of living cell in the course of biochemical reactions.Besides, it is known that metal ions are able to stabilize them [371, and in the work [7] the stabilizing role of their inter actions with proteins is discussed.
The most favourable ylidic tautomers formed with the participation of the C8H carboprotons of tau tomers among the zwitterionic families may be rea lized as an intermediates in the course of the H*>T exchange between the C8H proton of purine bases and water, which gives a support to the suggestion of its ylidic mechanism [35 J.This mechanism should be taken into account under investigation of lethal and mutagenic effects of tritium incorporated into the purine 8 positions of DNA [38 ].
It should be emphasized that investigation of all the possible tautomeric forms is not «purely academic question*, as it may seem at the first view.To understand the possible mechanisms of biochemical reactions and the participation of nucleotide bases in nucleic acid functioning, it is necessary to take into consideration not only the most favourable tautomers.
It is quite clear that all the tautomers with the excess of formation energy (with respect to the most favourable tautomer), which does not exceed the activation energy of biochemical reactions (20-30 kcal/mol), should be regarded as possible partici pants in them.The authors believe that there is a large field to be investigated in this direction.
Finally, we would like to emphasize the growing significance of quantum-chemical investigation on nu cleotide bases' prototropic tautomerism because their biologically important patterns are far not in every case experimentally detectable.
The complete numerical information is available in database of Department of Molecular Biophysics at the Institute of Molecular Biology and Genetics of the Ukrainian National Academy of Sciences.The work was sponsored by the Ukrainian State Committee on Science and Technology (Contract 5.4/77).

•
Lengths of the valence bonds are given in A and valence angles -in degrees.

Table 5
Some calculated physico-chemical characteristics of the hypoxanthine molecular and zwitterionic tautomers* •Total energy of the I molecular tautomer with fixed nuclei is equal to 42475.1 kcal/mol; **ab initio data; ***DFT data.