Tautomerism of cytosine

Cytosine is generally represented in the lactam-amine form 2 both as a free molecule and in its nucleoside or nucleotide. This is also the form considered to be involved in the base-pairing scheme in DNA. 

An overwhelming amount of experimental evidence from nearly all the available techniques of physical chemistry seems nowadays to confirm this assignment although the situation was not always clear-cut. 

Voet and A. Rich, Progr. Nucl. Acid Rea. Mol. Biol. 10,183 (1970). 

Almlof, A. Kvick, and I. Olovsson, Acta Crystallogr. Sect. B 27, 1201 (1971). 

X-ray crystal results on cytosine itself10 -13 (anhydrous and monohydrate), its complexes with different partners,15-20 cytidine,21 and cytidine 2, 3 -cyclic phosphate22 all indicate its existence in the lactam-amine form (2). In a number of crystals the cytosine ring is protonated, invariably at N-3.23-32 In cytosine 5-acetic acid33 half of the molecules 

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Carbon-13 magnetic resonance spectra of the naturally occurring cytidines have been described in several papers.79-82 The electronic structure of the compounds is reflected in the carbon-13 shifts. For instance, the observed chemical shifts for these and other pyrimidine and purine nucleosides were correlated, at least qualitatively, with the calculated charge densities (see Section VIII) and with the known reactivity of these molecules. It is difficult to draw conclusions from the carbon-13 spectra about the tautomerism of cytosine. 

Kosenkov, D. Kholod, Y. Gorb, L. Shishkin, O. Hovorun, D. M. Mons, M. Leszczynski, J. Ab initio kinetic simulation of gas-phase experiments Tautomerization of cytosine and guanine, J. Phys. Ghent. B 2009,113, 6140-6150. 

The relative stability of the tautomers of purine and pyrimidine bases is of fundamental importance to the structure and functioning of nucleic acids. The occurrence of rare tautomers was considered a factor responsible for the formation of mismatches leading to spontaneous mutations in the genetic code fl,2]. Cytosine, in particular, has been the subject of several studies, both experimental [3-5] and theoretical [5-15] which have provided a reliable picture of the relative stability of its tautomers, both in the gas phase and in solution. Tautomerization is generally the result of proton transfer (PT) reactions whose activation barriers may exert a kinetic control over the formation of some tautomers. As far as cytosine is concerned, a large majority of the studies available in the literature focus on the thermodynamic aspects of tautomerization and quite a few [16-19] are devoted to the elucidation of the kinetic aspects. The tautomerization of cytosine in the gas phase, with a special attention to the activation energy of the proton transfer reactions, has been afforded by this group in a previous paper [19]. By comparison with experimental data [4,5] it was... 

Finally, the tautomerism of cytosine has been largely studied both theoretically [118,140-142] and experimentally [143,144 and references therein] owing to its possible impact in G->A mutations and in the stabilization of d(G.C.C) triple helices at neutral or basic pH [145]. In the gas phase cytosine exists as a mixture of keto-amino and enol-amino forms, the keto-imino and enol-imino species being minoritary. In fact, experimental data... 

Podolyan, Y., Gorb, L., and Leszczynski, J. (2003) Ab initio study of the prototropic tautomerism of cytosine and guanine and their contribution to spontaneous point mutations. Int. J. Mol. 

For a discussion of tautomerism involving the carbonyl group of cytosine, see Section II,M.)... 

Uracil, thymine, and cytosine have been studied using this technique (89JA2308 and references therein). For uracil and thymine, the dioxo tautomer predominates in the case of cytosine (70), three tautomers were detected, 70a, 70b, and 70c, the last one being the least abundant. The gas-phase tautomeric equilibrium of 2-pyridone 15a and 2-hydroxypyridine 15b has been studied by MW spectroscopy (93JPC46) using both a conventional spectrometer and a jet-cooled millimeter-wave spectrometer. The relative abundances are 3 1 in favor of the hydroxy form 15b, which exists in the Z conformation shown (Scheme 23). 

Estrin, D. A., L. Paglieri, and G. Corongiu. 1994. A Density Functional Study of Tautomerism of Uracil and Cytosine. J. Phys. Chem. 98, 5653. 

In 1 JMSO-z4, or DMF-(/, the 1 1 condensation products of cytosine or iV-methylcytosine with triformylmethane, compounds 39a and 39b, showed ring-chain tautomerism with the ring-closed products 40a and 40b (see Equation (2), Section 12.04.2.4) <1996ACS1137>. 

Ring-chain tautomerism of the uracils 297 and cytosines 299 (R = H, Me) to the respective pyrimido[6,l-3][l,3]oxa-zines 298 and 300 takes place in CD3OD/DMSO-1S6 in the presence of Et3N or NaOD. Cfe-addition is predominant. 

The equilibrium between the amino and imino forms of cytosine has also been studied by Cieplak et al.9 Their results agree with those of Colominas et al. In this study, the related 2-oxopyridine and 2-oxopyrimidine molecules were also treated. In both these molecules the amine group of cytosine is not present, and in oxopyridine only one ring nitrogen is present. This enabled the keto-imino tautomerism to be studied in isolation. In both cases the imino form dominates in the gas phase, but the keto form is stabilised by solvation, and dominates in solution, in agreement with experiment. 

The Mg+ complexes of cytosine, thymine and uracil are the most complex system studied via photodissociation spectroscopy to date . A complication for these systems is that these nucleobases can exist in various tautomeric forms and that complexation of a metal can change the stability order of the tautomers. DFT calculations located four tautomeric Mg(cytosine)+ complexes, and three of these (29, 30, and 31) were suggested to be responsible for the four reactive photofragment ions 32-35 observed at a wavelength of 360 nm (Scheme 4) . Related photofragmentation reactions were observed for the Mg(thymine)+" and Mg(uracil)+" complexes . ... 

The first PMR spectra57,58 attributed the tautomeric structure (7) to the cation of cytosine, in agreement with X-ray studies and IR data. For the preferred neutral form of cytosine in solution Kokko et al.59... 

The tautomeric ratios characterizing the complex scheme of tautomeric conversions of cytosine have not been evaluated. The only quantitative results concern the relative contributions of structures 2,... 

For instance Shugar and Fox106 have shown that the spectrum of cytosine resembles that of 1-methylcytosine (form 2), not that of 2-methoxycytosine (form 1). Similarly, Kenner et al.100 have established that lactam-imine forms 6 were unimportant both for 1-methylcytosine (thus also for cytosine itself) and for its aminoacetyl derivatives. Katritzky and Waring60 have extended these studies and in particular showed that tautomers 3 and zwitterionic structures 10 were unimportant in the tautomeric equilibrium in both aqueous and dimethyl sulfoxide solutions. Although these last comparisons were made with only two partially methylated cytosines still capable of tautomerism, the reported conclusions agree with those reached later by Brown and Lyall96 on much wider evidence. 

UV spectroscopy is a sensitive tool for the elucidation of structures of molecules only if the different tautomers have distinct spectra. In some cases, cytosine or cytidine and their alkylated analogs have similar spectra with seriously overlapping bands, and thus the application of UV spectroscopy to the elucidation of the tautomeric structures of the molecules is inconclusive. In particular, the method is unreliable for the determination of tautomeric equilibria of cytosine (see below). Nevertheless the UV absorption spectra confirm the conclusions drawn from other studies as to the main form of cytosine and cytidine in aqueous solution at room temperature. 

However Morita and Nagakura104 concluded from similar studies that in aqueous solution cytosine exists as an equilibrium mixture of two forms, 2 and 6, only. According to these authors the first form predominates in trimethyl phosphate and water at room temperature, but the second prevails at high temperature. The imine form is considered to predominate also in acetonitrile. From the temperature dependence of the absorption spectrum of cytosine in aqueous solution, Morita and Nagakura estimated the tautomeric ratios Kf-e to be equal to 33, 14, and 8 at 30, 50, and 70°, respectively. The energy115 and entropy differences between the imine form 6 and the amine form 2 were evaluated as 5.5 kcal/mole and 12 cal/mole. deg., respectively. 

For comparison with the theoretical predictions, we have collected in Table II, together with the previously discussed tautomeric constants, some thermodynamic parameters characterizing the tautomeric conversion of cytosine. 

The tautomeric studies of azacytosines are not so complete as those of cytosine itself. The contribution of other tautomeric forms of azacytosines to the tautomeric equilibrium has not been evaluated (because of lack of model tautomeric compounds). It appears that 6-aza-substi-tution causes a tautomeric shift from form 3 of cytosine toward its imine form, 6. Thus, upon 6-aza-substitution the contribution of the imine tautomers to the tautomeric equilibrium of cytosine increases, while that of tautomers 3 decreases. 

The tautomeric ratio of 25 in water for 5,6-dihydrocytosine should be compared with the value of 105 for cytosine itself (see Table II). It thus appears that the hydrogenation of cytosine causes a substantial shift toward the imine form. A further shift toward the imine is observed when 5,6-dihydrocytosine is substituted at the amino group by a hydroxy group. It was shown124 by UV spectra that 1-alkyl-substituted A4-hydroxy-5,6-dihydrocytosines exist in aqueous solution in the lactam(ox)imine form 20. 

Substitution of a Hydroxy, Methoxy, or Amino Radical at the Amino Group of Cytosine Tautomeric Shift 2 — 6... 

A comparison of the tautomeric constants in the series of N4-substituted cytosines indicates an interesting trend. As seen above, in the case of cytosine itself, the ratio of the tautomers of the amine type 2 to those of the imine type 6 is about 105. In the case of jV4-amino-cytosine134 the amine form 22 predominates by a factor of about 30, while N4-hydroxy compounds have mainly the imine form 21 with tautomeric constant 10 [i.e., Kt (amine/imine) = — 10 1]. Another study136 of this constant gave the value of 25 [Kt (amine/imine) = 4 x 10-2] in favor of the imine. 

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