Continuous-flow and spin-trapping EPR studies on the reactions of cytidine induced by the sulfate radical-anion in aqueous solution. Evidence for an intermediate radical-cation

Abstract

Radicals generated by reaction of the sulfate radical-anion, SO₄˙⁻, with cytidine in aqueous solution were characterized by EPR spectroscopy. Two different methods were employed. First, radicals were generated in situ in a continuous-flow system by photolysis of solutions containing the substrates and K₂S₂O₈. Secondly, SO₄˙⁻ was generated either photolytically or by means of the redox couple Ti(III)/S₂O₈²⁻ in presence of the spin trap 2-methyl-2-nitrosopropane (MNP) and EPR spectra were taken from the ensuing persistent spin-adducts. In the continuous-flow experiments the signals of the 2′-oxo-1′-yl sugar radical 1 decreased in intensity upon addition of increasing amounts of phosphate dianions (HPO₄²⁻, pH > 7.2) and the complex spectrum of base radical 2 appeared. The radicals detected in the spin-trapping experiments were not identical with those observed in the flow-system: in the absence of phosphate the open-chain C(1′) radical 4 was trapped at pH 6–9. It was replaced by spin-trapped 5-yl and 6-yl base radicals 5a/5b in solutions containing phosphate dianions (pH > 7.2). The results from the in situ experiments as well as from the spin-trapping studies are explained by rapid reaction of the intermediate base radical-cation with HPO₄²⁻ competing efficiently with transfer of the radical site to the sugar. From the failure to generate the base radicals 2 and 5 with phosphate monoanions (pH < 7.2) a reaction pathway via the negatively charged cytidyl sulfate-adduct radical is excluded.