Proc. Int. Symp. Biomol. Struct. Interactions, Suppl. J. Biosci., Vol. 8, Nos 3 & 4, August 1985, pp. 657–668. © Printed in India.

New light on tautomerism of purines and pyrimidines and its biological and genetic implications

DAVID SHUGAR and BORYS KIERDASZUK

Institute of Biochemistry and Biophysics, Academy of Sciences, 02-532 Warsaw; and Department of Biophysics, Institute of Experimental Physics, University of Warsaw, 02-089 Warsaw, Poland

Abstract. The tautomerism of the natural 1-substituted pyrimidines and 9-substituted purines found in nucleic acids has been re-examined in the light of new experimental data on various nitrogen heterocycles in solution, in the gas phase and, in part, in low-temperature inert matrices. The results are compared with those obtained by quantum chemical calculations, including improved versions of the latter. Examples are presented of natural nucleosides which exhibit appreciable tautomerism in solution, e.g. formycins A and B, isoguanosine, but are not found in DNA. Illustrations are given of synthetic promutagenic nucleosides with pronounced tautomerism in solution relevant to their role in mutagenesis, such as the N4-hydroxy- and N4-methoxy cytidines. The amino-imino tautomeric equilibria of the promutagenic N6-hydroxy- and N6 -methoxy-adenosines are highly dependent on the solvent medium, the proportion of the imino species varying from 10% in CCl4 to 90% in aqueous medium. The type of base pairing of these is dependent on the conformation of the exocyclic hydroxy or methoxy groups. At the monomer level, addition of a potentially complementary base leads to a shift in the tautomeric equilibrium in favour of the species which pairs with this base. Biological and genetical implications of the foregoing are described.

Keywords. Purines; pyrimidines; tautomerism; base pairing; mutagenesis; promutagens.

Introduction

Tautomerism of nitrogen heterocycles has long been associated in molecular biology with the natural purines and pyrimidines, and the presumed role of their rare tautomeric forms in spontaneous mutagenesis. But spontaneous mutations are not exclusively transitions, as expected from such a proposal, and include also transver- sions. Furthermore, the foregoing was formulated at a time when existence of repair enzymes, including error-prone repair systems, was not even suspected. The role of rare tautomeric species, if any, in spontaneous mutagenesis remains to be established. We briefly review here new experimental and theoretical developments leading to more reliable data on differences in binding energies between equilibrium tautomeric species, the role of the environment in such processes, and some biological implications. In the interim, tautomerism of synthetic, and some atypical natural, nucleoside analogues has assumed additional significance in relation to their antimetabolic activities, and to base analogue mutagenesis.

Tautomerism in the gas phase

Undoubtedly the most useful new experimental approach is the ability to examine tautomeric equilibria of at least some nitrogen heterocycles in the vapour phase by

657 658 Shugar and Kierdaszuk spectroscopic methods. Earlier observations by Levin et al. (1965) were extended by Beak and Fry (1973) to show that, for pyridbne-2 in the vapour phase, the ratio of the enol to the keto form is about 2:1; whereas in aqueous medium the basicity method –3 gives a KT < 10 in favour of the keto form, now known to be due to stabilization of the latter by solvation and self-association (Beak, 1977, and references cited). Furthermore, the temperature-dependence of the enol-keto equilibrium of pyridone-2 in the gas phase led to a difference in chemical binding energies between the two species of only about 1 kJ/mol, qualitatively consistent with subsequent calorimetric measure- ents in cyclo-hexane solution of about 4 kJ/mol (Cook et al., 1976). For this simplest of all possible tautomeric nitrogen heterocycle systems, various quantum mechanical calculations gave results which differed appreciably from those observed experimen- tally in the gas phase (Beak, 1977; Shugar and Szczepaniak, 1981; Schlegel et al., 1982), and raised serious doubts about the validity of such calculations for other heterocyclic systems, including the natural nucleic acid bases. On the positive side the feasibility of following tautomeric equilibria in the vapour phase has made available, for the first time, appropriate data for evaluation of the validity of the theoretical approaches, as well as extension of the latter to the role of the environment in solution studies. Concerted efforts are presently being devoted to this subject, perhaps best exemplified by the ab initio calculations of Schlegel et al. (1982) and Scanlan et al. (1983), with the use of advanced Gaussian basis sets, full geometrical optimization, and allowances for correlation energy and zero-point vibration, to obtain a value for the difference in energies between the keto and enol forms of pyridone-2 in the gas phase in excellent agreement with experimental data (Kwiatkowski et al., 1985). A number of additional heterocyclic systems, including pyrimidone-2, pyrimidone-4 and some of their analogues, have now been examined in the gas phase (Beak, 1977; Nowak et al., 1980; Shugar and Szczepaniak, 1981). The resulting KT values differ from those in solution by 3–5 orders of magnitude, further underlining the importance of environmental effects. For some heterocycles which cannot withstand the elevated temperatures required for transfer to the vapour phase, this difficulty may occasionally be circumvented by the use of low-temperature inert matrices, which possess the additional advantage of revealing the fine structure of vibrational spectra (Shugar and Szczepaniak, 1981; Szczesniak et al., 1983).

Behaviour of natural bases found in nucleic acids

In light of the foregoing, it appeared logical to examine the behaviour in the gas phase of nucleic acid bases, i.e. 1-substituted pyrimidines and 9-substituted purines. The nucleosides and nucleotides are not suitable for this purpose because of their thermolability. But some 1-alkylpyrimidines and 9-alkylpurines proved sufficiently thermostable. With the aid of UV and IR spectroscopy, the latter of which is by far superior, it was found that these exhibited the same predominance of keto and/or amino forms in the gas phase as in solution (Nowak et al., 1978). These interesting results prompted, in turn, additional searches for enol and imino forms in solution, but with negative results (e.g. Chevrier et al., 1980; Schwartz et al., 1983). Subsequently Beak and White (1982) described another approach to this problem, based on direct Tautomerism and its biological implications 659 determination of the energy difference between methyltropic isomers of uracil and extrapolation of the results to the corresponding uracil protomers. This led to an energy difference between the diketo form of uracil and its 4-enol of about 80 kJ/mol, as compared to an estimated value of about 20 kJ/mol in solution. The large enthalpic driving force favouring the keto species, together with the stabilizing role of hydrogen bonding, is taken to account for the striking predominance of the keto form. It is consequently not surprising that, in the course of evolution, bases such as uracil (and thymine) were selected as building blocks of nucleic acids so as to maintain fidelity of replication and transcription.

Tautomerism in the solid state

A multitude of purines and pyrimidines, their nucleosides and nucleotides, and hydrogen bonded base pairs, have been examined in the solid state, largely by X-ray diffraction (Voet and Rich, 1970), and more recently by spectroscopic methods. These are invariably in the amino and/or keto forms. An exhaustive literature search, however, revealed one striking exception, viz. 5-nitrobarbituric acid (dilituric acid). Whereas the hydrated form in the crystal is the expected tri-keto species (I in figure 1) (Craven et al., 1964), the crystalline anhydrous compound is in the 4(6)-enol form (II in figure 1), stabilized as such by intramolecular hydrogen bonding to one of the nitro oxygens (Bolton, 1963), reminiscent of the classical example of salicylic acid. This may be considered an example of "constrained" tautomerism.

Figure 1. Solid state structures of 5-nitrobarbituric acid (dilituric acid): (I) hydrated, in 2,4,6-triketo form; (II) anhydrous, in 2,6-diketo-4-enol form, hydrogen bonded to a nitro oxygen.

Constrained tautomerism

Constrainment to a given tautomeric form as a result of intermolecular interaction has hitherto received little attention. Recent examples include: (a) electrophilic trapping by 660 Shugar and Kierdaszuk benzaldehyde of one of the rare tautomers of 7-deoxydaunomycine, proposed as the biologically active form of daunomycin in its interaction with DNA (Kleyer et al., 1984); (b) binding of the histidine of the Asp-His- Ser triad of α-lytic protease via the less stable N(3)-H tautomer (Kanamori and Roberts, 1983). An additional example is furnished by the 6-arylhydrazino derivatives of uracil and isocytosine, selective inhibitors of bacterial DNA polymerase III . The corresponding 2-thiouracil analogue inhibited the polymerase by base pairing with both template thymine and cytosine, this dual specificity being ascribed to partial conversion of the thioketone to the thiol form (Wright and Brown, 1976). We shall revert to this aspect below, in relation to base pairing of the promutagenic N6-methoxyadenosine.

Base analogue mutagenesis

The mutagenicity of various base analogues is still frequently ascribed to mispairing resulting from tautomerism. Relevant are only those consisting of 1-substituted pyrimidines or 9-substituted purines present in DNA; e.g. 3-methylcytosine exhibits a –3 significant proportion of the imino form in solution KT =2·5×10 (Dreyfus et al., 1976), but 3-methylcytidine is obviously in the fixed imino form. Isocytosine in aqueous medium is a mixture of the N(l)-H and N(3)-H protomers; while all purines are mixtures of N(7)-H and N(9)-H protomers, clearly absent in the nucleosides.

5-Bromouracil

Most widely employed is the thymidine analogue, 5-bromodeoxyuridine, presumed to exhibit enhanced propensity for existence as the 4-enol. Evidence for this is based on use of the basicity method to purportedly show that l-methyl-5-bromouracil displays a higher proportion of the 4-enol than 1-methyluracil, the KT values being 103·3 and 4·0 10 , respectively (Katritzky and Waring, 1962). Apart from some questionable assumptions in procedure, the experimentally measured pKa values (as acids) for 5- bromouracil and l-methyl-5-bromouracil were erroneous (cf. Berens and Shugar, 1963). Furthermore, since 5-bromouracil replaces thymine in DNA, and not uracil in RNA, the comparison should have been made between the 1-methyl congeners of 5- bromouracil and thymine, and this would still be worth while doing. For the corresponding cytidines and their 5-halogeno analogues, where the validity of the basicity method was checked by showing that the conjugate cations of the amino and fixed imino species share a common structure, the calculated KT values for cytidine, 5-fluorocytidine and 5-bromocytidine did not differ significantly within the limits of experimental error (Kulikowski and Shugar, 1979). Furthermore there is now considerable evidence that in vivo 5-bromouracil-induced mutagenesis is due to factors other than mispairing by the enol form (Pietrzykowska et al., 1983, and references cited).

2 -Aminopurine

Mutagenesis by this base analogue has likewise been attributed to mispairing due to amino-imino tautomerism. But, both in the solid state, and in synthetic polynucleotide Tautomerism and its biological implications 661 systems in solution, the 2-aminopurine residues in the amino form base pair with poly(U), but not with poly(C) (Janion and Shugar, 1973). More recently Goodman and Ratliff (1983), with the aid of UV spectroscopy, showed that in polydeoxynucleotides containing 2-aminopurine residues, the latter pair with cytosine residues via hydrogen bonding at the N(l) of 2-aminopurine. While this may indeed be construed as constrainment to the imino form, it may also be interpreted in terms of imposed protonation of N(l). This ambiguity could undoubtedly be resolved with the aid of NMR spectroscopy.

Tautomerism of some atypical natural nucleosides

Several nucleosides of natural origin do indeed exhibit appreciable tautomerism in solution. Typical examples are the nucleoside antibiotics formycin A (figure 2) and its deamination product, formycin B, structural analogues of adenosine and inosine, respectively. Another is isoguanosine (figure 3), originally isolated from croton seeds,

Figure 2. Tautomeric equilibrium forms of formycin A. In aqueoues medium the propor- tion of the N(2)-H form is about 15%. R = ribose.

Figure 3. Keto–enol tautomeric equilibrium of isoguanosine. The proportion of the enol

form varies from 10% in CCl4 to 90% in aqueous medium. R = ribose. 662 Shugar and Kierdaszuk now known to be widespread amongst marine organisms. For the formycins, the proportions of the N(2)-H species may attain 15 % (Chenon et al., 1976; Wierzchowski et al., 1982), and their antimetabolic activities may be manifested through one or the other of these. The difference in KT between formycin A and its 5'-phosphate is accompanied by modifications in conformation about the glycosidic bond, with marked changes in emission spectra; this has been exploited to develop a sensitive continuous fluorimetric assay of 5'-nucleotidase (Wierzchowski et al., 1984). Isoguanosine (2-oxoadenosine) is even more unusual in that its neutral form exists to an appreciable extent as the enol in solution, the proportion varying from 5% in aqueous medium to 80% in an aprotic solvent (Sepiol et al., 1976). In the solid state the cation of 9-methylisoguanine is C(2)-keto, N6-amino, with N(3) protonated (Banerjee et al., 1978), but attempts to obtain the enol form in the solid state have been unsuccesful. Bearing in mind the foregoing, it is not surprising that these nucleosides are not found in DNA. In some systems, e.g. parasitic protozoa, the formycins may be incorporated into RNA, leading to disruption of translation. Extensive searches for isoguanosine in the RNA of marine organisms have been negative, but the potent pharmacological activities of isoguanosine and 1-methylisoguanosine (Kim et al.,1981) are of interest in relation to their tautomerism.

Xanthosine

Xanthine, its N-methyl congeners, and its nucleoside and nucleotides are involved in numerous metabolic processes, but xanthine is not a constituent of nucleic acids.

Treatment of the latter with the mutagen HNO2 leads to partial deamination of guanine residues to xanthine, but the genetic effects of this, if any, have not been clarified. The neutral form of xanthosine, and xanthine, is 2,6-diketo (Roy and Miles, 1983; Psoda and Shugar, in preparation), also observed in the solid state (Lesyng et al., 1984). Acid dissociation leads to loss of the proton from N(3), with the formation of only one tautomeric species, the 6-keto-2-enolate anion, pKa = 5·6, as for the anion of xanthine in the solid state (Mizuno et al., 1969). The synthetic homopolymer, poly(X), differs from all other homopolymers in its versatility to complex with a variety of other potentially complementary homopoly- ucleotides (Michelson and Monny, 1966; Fikus and Shugar, 1969). Of the various multi-stranded structures proposed for these, the one for which there is reasonably good evidence is the double-stranded poly(X) · poly(U) via the diketo form of the xanthine residues which are thus prevented from ionizing. The structure of poly(X) itself has been proposed to consist of from 3 (Fikus and Shugar, 1969) to 4 or 6 (Roy et al., 1979) strands. The latter authors demonstrated, with the aid of IR spectroscopy, that only the diketo forms of xanthine residues are involved in these structures. In sharp contrast, diffraction studies on poly(X) fibres have been interpreted in terms of two different duplex forms, similar to the A forms of RNA and DNA, respectively, with base pair hydrogen bonding via the 6-enol species (Arnott et al., 1981). Further attempts to resolve the foregoing discrepancies should be based, in part, on the use of hydrodynamic methods in solution to determine the number of strands involved in a given structure. Tautomerism and its biological implications 663

Hydroxylamine (methoxyamine) mutagenesis

We now turn to what are undoubtedly the most interesting of promutagenic base analogues, the N4-hydroxy- and N4-methoxycytidines (figure 4), and the N6-hydroxy- and N6-methoxyadenosines (figure 5), the products of reaction of cytidine and adenosine with the potent mutagens NH2OH and NH2OCH3, respectively. In vivo these analogues lead to point transition mutations, C → T(U) and A → G, respectively (Budowsky, 1976; Marfey and Robinson, 1981). In in vitro transcription they exhibit dual functional activity, viz. N4-hydroxycytidine (OH4C, 4a in figure 4) behaves like U(T) or C (Budowsky et al., 1971; Flavell et al., 1974; Singer et al., 1984), whereas N4- methoxy(deoxy)cytidine (OMe4C, 4b in figure 4) behaves like T(U) (Singer et al., 1984). Similarly N6-methoxyadenosine (OMe6A, 5b in figure 5) mimics A or G (Singer and Spengler, 1982). The foregoing are strikingly reflected in the tautomerism of these promutagenic analogues and their base pairing properties, which we now describe.

Figure 4. Amino imino tautomeric equilibrium for the syn rotamers of N4- 4 hydroxycytidine (OH4C, 4a), N4-methoxycytidine (OMe4C, 4b), l-methyl-N -hydroxy- 4 4 1 4 cytosine (Me1OH Cyt, 4c), l-methyl-N -methoxycytosine (Me OMe Cyt, 4d). Note: In the solid state, the syn rotamer of the imino tautomer is further stabilized by intramolecular hydrogen bonding, shown as a dashed line. 664 Shugar and Kierdaszuk

Figure 5. Amino'⇌imino tautomeric equilibrium for the syn rotamers of N6- 6 6 6 hydroxyadenosine (OH A, 5a), N6-methoxyadenosine (OMe A, 5b), 2',3',5'-tri-O-methyl-N - 6 methoxyadenosine (OMe A(Me)3, 5c). The sugar methylated derivative is used because of its higher solubility in low polar solvents. Note: In the solid state, the syn rotamer of the imino tautomer is further stabilized by intramolecular hydrogen bonding, shown as a dashed line.

N4-hydroxy- and N4-methoxy- cytidines

4 Both OH C (4a) and OMe4C (4b) exhibit two tautomeric forms, amino⇌imino (figure 4), the equilibrium being dependent on the solvent. The imino form of 4 Me1OMe C (4c) predominates in aqueous medium (Brown et al., 1968; Sverdlov et al., 1971; Janion, 1972). The KT for 4c in aqueous medium, determined by the basicity method, is about 10 (Brown et al., 1968), and for OH4C about 25 (Sverdlov et al., 1971), in favour of the imino form. Transfer to a less polar medium further shifts the equilibrium towards the imino species. Spectroscopic methods, including IR, and 1Η, 13 C and 15N NMR (Psoda et al., 1981; Kierdaszuk and Shugar, 1983; Kierdaszuk et al., 1983a) point to predominance, and probably exclusively, of the imino species in weakly polar solvents such as CHC13, as well as in the gas phase (Kulinska et al., 1980). In the solid state (Shugar et al., 1976; Birnbaum et al., 1979) the imino form is stabilized by an intramolecular hydrogen bond, N(3)–H · · · O–N4 (figure 4).

N6-hydroxy- and N6-methoxy- adenosines

OH6A (5a) and OMe6A (5b) each exhibits an amino ⇌imino equilibrium (figure 5) (Morozov et al., 1982), with marked dependence of KT on the solvent medium Tautomerism and its biological implications 665

(Kierdaszuk et al., 1983b; Kierdaszuk et al.,1984; Stolarski et al., 1984). The proportion of the imino form varies from about 10% in a non-polar solvent like CCI4 to about 90% in aqueous medium (table 1). In the solid state the imino form is stabilized by intramolecular hydrogen bonding, N(l)–H · · · O–N6 (see figure 5) (Birnbaum et al., 1984). An exceptionally interesting feature of the solid state structure is the observed base pairing of two molecules via N(l)–H · · · N(7) and C(2)-H · · · N6, the first reported example of base pairing involving a C-H as donor.

6 Table 1. Populations of the amino and imino tautomers of OMe A(Me)3 in various solvents, at two concentrations and diferent temperatures, and the changes in populations on addition of an equimolar amount of the potentially complementary a U(Me)3 or C(Me)3.

a 6 6 OMe A(Me)3, U(Me)3 and C(Me)3 are the 2',3',5'-tri-O-methyI derivatives of N - methoxyadenosine (O Me6A), uridine (U) and cytidine (C), employed because of their better solubility in solvents of low polarity. b Estimated accuracy ± 2 %. c Based on IR spectroscopy, accuracy ± 10 % (Kierdaszuk et al., 1984). d From spectra run on a JEOL JNM 4H-100 cw instrument, hence somewhat less accurate, ± 10 % .

From the temperature-dependence of KT for 5c in (CD3)2SO, the lifetimes of the tautomers were evaluated as τ(amino) = 1/7·5 s and τ(amino) = 1/15·5 s; and the free enthalpy for the energy barrier between them at 80°C as ∆G# = 80 ± 5 kJ·mol–1.The difference in free enthalpy between the energy minima for the two species is ∆G° = 2·2 kJ · mol–1. The high energy barrier between the two tautomers leads to two 666 Shugar and Kierdaszuk indpendent sets of 1H and 13 C signals (Stolarski et al., 1984) and two series of UV and IR bands (Kierdaszuk et al., 1984). This facilitated studies on the solvent-dependence of KT, as well as dependence of the latter on base pairing with the potentially complementary uridine and cytidine (see below).

Role of tautomeric equilibrium and exocyclic group conformation

The imino form of OH4C (or OMe4C) resembles U (or T) in that it possesses a proton donor, N(3)–H, a proton acceptor, C(2)=O, and a potential acceptor, C(4)=N4. The acceptor properties of the latter are, however, dependent on the conformation of the 4 4 4 N -OH (or N -OCH3) aboutthe C(4)=N bond. At the monomer level in solution, the 4 N - OCH3 is oriented syn to the ring Ν (3), as shown in figure 4 (Kierdaszuk and Shugar, 1983), thus precluding Watson-Crick base pairing, and leading to inverse Watson-Crick pairing with C(2)=O as acceptor (Kierdaszuk et al., 1983a). At the homopolymer level it provokes destabilization of secondary structure (Janion and Shugar, 1968, 1969; Singer and Spengler, 1981; Spengler and Singer, 1981), as also for polymers with Me6A residues (Engel and von Hippel, 1978). Nonetheless, in vitro replication with copolymer templates containing such residues led to AMP incorporation opposite OMe4C (Singer and Spengler, 1981) or OMe4dC (Singer et al., 1984) residues. By contrast, OH4CTP simulated both UTP and CTP in replication and transcription (Budowsky et al., 1971; Flavell et al., 1974). Mis- incorporation due to mispairing is, consequently, dependent on the marked influence of the polymerase. On the other hand, the recent finding that OH4dCMP is a competitive inhibitor of thymidylate synthetase (Goldstein et al., 1984) was interpreted implicitly as involving the amino form, without consideration of the conformation of the exocyclic N4-OH group. The amino form of OMe6A formally resembles adenosine, and the imino form inosine, theoretically permitting of base pairing with uridine and cytidine, respectively, depending on the conformation of the exocyclic OCH 3 group (Stolarski et al., 1984). The tautomeric equilibrium of this compound, and its specific planar association in solution, are reflected in its dual functional activity in an RNA polymerase system, with incorporation of UMP and CMP in a 10:1 ratio (Singer and Spengler, 1982).

Influence of base pairing on tautomeric equilibrium

Formation of hydrogen-bonded planar autoassociates of OMe6A, and preferential heteroassociation of the amino form with U and the imino form with C, in CHCI3, was found to be accompanied by shifts in tautomeric equilibrium (see table 1). An increase in concentration and/or a decrease in temperature, which favour atuoassociation, led to a singificant shift in the equilibrium towards the amino form. Furthermore, addition of 6 an equimolar amount of U to a solution of OMe A in CHCI3 shifted the tautomeric equilibrium of the latter towards the amino form (8–9 % at 30°C, with an equal decrease in population of the imino form, see table 1). By contrast, addition of an equimolar amount of C shifted the equilibrium of OMe6 A in the direction of the imino species Tautomerism and its biological implications 667

(22 % at 0·2 M, and 13 % at 0·04 M, at – 30°C, accompanied by an equal decrease in population of the amino form, see table 1. The foregoing is, to our knowledge, the first example of a shift in tautomeric equilibrium of a base analogue as a result of pairing with a potentially complementary residue. In the case of OH6A (or OMe6A), this phenomenon undoubtedly plays a role in the mechanism of A → G transitions provoked by these promutagenic analogues.

Acknowledgements

We would like to acknowledge support by the Polish Academy of Sciences (09.7.1), the Polish Cancer Research Program (PR-6), and the Ministry of Science and Higher Education (MR.I.5).

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