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

Abstract

The photoreactivity of 4-thiothymidine (S⁴TdR) under visible light 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 ¹H-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 (¹O₂), photogenerated by RB, and S⁴TdR, as demonstrated by the direct detection of ¹O₂ through IR luminescence spectroscopy. More specifically, a sequential reaction pathway, consisting in the generation of an electrophilic hydroxylated form of S⁴TdR and its subsequent, rapid reaction with S⁴TdR, was hypothesized to explain the presence of the S–S bridged covalent dimer of 4-thiothymidine in the reaction mixtures. The described processes make S⁴TdR an interesting candidate in the role of molecular probe for the detection of ¹O₂ under different pH conditions.