Factors controlling the site of protonation of the one-electron adduct of cytosine and its derivatives

Abstract

Exposure of cytosine and derivatives thereof to ⁶⁰Co γ-rays at 77 K in a range of aqueous solutions (H₂O or D₂O) at 77 K gave EPR spectra assigned to the corresponding radical anions. These spectra comprise either doublet or triplet hyperfine features due to one or two protons. Triplet features observed using H₂O media become doublets using D₂O media. Well defined triplet spectra were observed using lithium chloride glasses and methanol–water glasses. Using frozen aqueous solutions which give phase-separated solids, spectra ranging from doublets via mixtures to triplets were obtained, depending on the derivative used. It is suggested that the first formed radical anions are rapidly protonated either on the ring nitrogen [N(3)] or on the amino nitrogen. In the former, there is no extra splitting from the added proton and a major doublet is observed because of hyperfine coupling to the C(6) proton [C(6)–H]. In the latter case, the proton is thought to add along a path normal to the plane of the ring so that overlap with the SOMO is maximised, and there is a large hyperfine coupling. Coupling to the other two amino protons is too small to resolve under these conditions. The fact that the –N(CH₃)₂ derivative gives a doublet rather than a triplet supports the postulate that the –NH₂ group is indeed protonated in the triplet species. For the aqueous-methanol glasses, on annealing, this extra doublet splitting is lost irreversibly, probably because rotation of the –NH⁺₃ group sets in, making the three protons equivalent.

As has been shown by Bernhard and co-workers, certain oligomers which give C^·– centres on irradiation give rise to doublets only in H₂O glasses. Hence N(3)-protonation is favoured in polymeric systems. Reasons for these differences, and the importance of these results in relation to DNA are discussed.