Mechanistic and Kinetic Insights into Nucleobase Oxidation: Detection of Radical Intermediates in Thymine Oxidation Using SH2′ Radical Trapping

Short-lived radical intermediates, such as reactive oxygen species (ROS), play a crucial role in oxidative stress, aging, and carcinogenesis. However, understanding of these processes is impeded by limitations of existing radical characterisation techniques. Previous investigations demonstrated that the recently-developed SH2′ radical trapping technique could be used to probe biochemical oxidation mechanisms. In this study, SH2′ radical trapping and mass spectrometry (MS) techniques were used to investigate ●OH-initiated degradation of thymine, relevant to oxidative damage of DNA. Numerous substrate-derived radical intermediates were trapped and characterised, aiding elucidation of, and offering validation to, proposed mechanisms. Isotope exchange and chromatographic separations helped to distinguish between isomeric structures. Observation of thymine dimer radicals and products suggests that this methodology can be useful for studying radical cross-linking of nucleobases. Overall, SH¬2′ radical trapping was demonstrated to be a powerful technique for investigating oxidation pathways in biological systems.