Rose Bengal-photosensitized oxidation of 4-thiothymidine in aqueous medium: evidence for the reaction of the nucleoside with singlet state oxygen

The photoreactivity of 4-thiothymidine (S(4)TdR) under visible Eight in the presence of Rose Bengal (RB), acting as a photosensitizer, was investigated in aqueous solutions at pH 7 and 12, using UV-vis, FTIR-ATR and H-1-NMR spectroscopic techniques, time resolved absorption spectroscopy and electrospray ionization mass spectrometry (ESI-MS). Evidence for the generation of thymidine (TdR) as the main product, after one hour of irradiation, was obtained from UV-Vis data, that suggested 4-thiothymidine photodegradation to be faster at basic pH, and confirmed by FTIR-ATR and 1H-NMR data. Clues for the presence of a further product, likely corresponding to a dimeric form of S4TdR, were obtained from the latter techniques. Besides indicating the presence of thymidine, the ESI-MS and MS/MS spectra of the reaction mixtures enabled the identification of the additional product as a S-S bridged covalent dimer of 4-thiothymidine. The concentration of the dimeric species could be estimated with the aid of 1H-NMR data and was found to be lower than that of thymidine in pH 7 reaction mixtures and almost negligible in the pH 12 ones. From a mechanistic point of view, time-resolved absorption spectroscopy measurements provided direct evidence that the formation of the two products cannot be ascribed to a photoinduced electron transfer involving S4TdR and the excited triplet state of RB. Rather, their generation can be interpreted as the result of a bimolecular reaction occurring between singlet state oxygen (102), photogenerated by RB, and S4TdR, as demonstrated by the direct detection of 102 through IR luminescence spectroscopy. More specifically, a sequential reaction pathway, consisting in the generation of an electrophilic hydroxylated form of S(4)TdR and its subsequent, rapid reaction with S(4)TdR, was hypothesized to explain the presence of the S-S bridged covalent dimer of 4-thiothymidine in the reaction mixtures. The described processes make S(4)TdR an interesting candidate in the roEe of molecular probe for the detection of O-1(2) under different pH conditions.