Pyrimidine radicals: Synthesis, reactivity and their applications in DNA damage study

Oxidative DNA damage has been implicated in a number of pathological conditions, including cancer, neurodegeneration, and aging. Hydroxyl radical is one of the major reactive oxygen species (ROS) formed from Fenton reaction and the ionizing radiation of water. Hydroxyl radical can react with pyrimidine bases by adding to the C5=C6 double bond or abstracting a hydrogen atom from the methyl group. The so-formed secondary radicals will further decompose or couple with its neighboring nucleobase to form stable diamagnetic products. To examine the mechanism of DNA damage and the roles of these secondary radicals played in this process, it is necessary to generate these secondary pyrimidine radicals independently through their photolabile precursors.; In this dissertation, two photolabile precursors, 5-phenylthiomethyl-2'-deoxycytidine and 5-hydroxy-6-phenylthio-5,6-dihydrothymidine, were prepared through multi-step organic synthesis and incorporated into oligodeoxyribonucleotides (ODNs) using phosphoramidite chemistry. The two reactive intermediates, cytosine-5-methyl radical and 5-hydroxy-5,6-dihydrothymidin-6-yl radical, were then generated independently by UV irradiation of their corresponding photolabile precursors. Mass spectrometry and NMR were used for structural characterization of products formed from these two radicals in both dinucleoside monophosphates and ODNs. In addition, on-line LC-MS/MS was used for the quantification of the products formed from the UV irradiation of radical precursor-containing ODNs and in gamma-irradiated DNA.; It was found that, in addition to single nucleobase decomposition products, intra-strand nucleobase cross-links were induced from the independently generated secondary pyrimidine radicals. The yield for the formation of the intra-strand cross-links was sequence dependent, with guanine on the 5' side being more abundant than with guanine on the 3' side. Moreover, this cross-link lesion could also be initiated from the methyl radical of 5-methylcytosine in the presence of molecular oxygen, suggesting that these types of intra-strand cross-link lesions might have important implications in the cytotoxic effects of ROS.