SITE OF PROTONATION OF ONE-ELECTRON-REDUCED CYTOSINE AND ITS DERIVATIVES IN AQUEOUS-METHANOL GLASSES

Electron paramagnetic resonance spectra of one-electron-reduced cytosi ne, cytidine, 2'-deoxycytidine, 5'-dCMP, and 3'-dCMP produced by Co-60 gamma-irradiation in aqueous lithium chloride glasses (LiCl/H2O) and aqueous methanol glasses (MeOH/H2O) at 77 K exhibited a triplet which has been interpreted in terms of the radical anion protonating on the exocyclic amino group of the base C(N4+H)(.) (II), The exchangeable na ture of the extra proton coupling is demonstrated by the collapse of t he triplet into a doublet in LiCl/D2O and CD3OD/D2O glasses. The EPR s pectrum of cytidine-5,6-d(2) in either LiCl glasses or aqueous methano l led to a doublet in each case, consistent with the loss of the coupl ing to the hydrogen on C6, with the remaining coupling due to the prot onation on the exocyclic amino group. Comparable experiments in LiCl/D 2O or CD3OD/D2O glasses led directly to a singlet. The results in aque ous methanol glasses parallel closely those in aqueous LiCl glasses an d provide clear evidence for protonation on the exocyclic amino group even in the absence of metal ion coordination to N3. Hence, N4 protona tion of the cytosine radical anion is not a result of metal ion coordi nation to N3 as has been suggested by others. We suggest that N4 proto nation is kinetically controlled, possibly brought about by solvation effects, whereas the N3 protonation would occur under thermodynamic co ntrol.