Independent generation and characterization of 5,6-dihydro-2'-deoxyuridin-6-yl

Abstract

Design of a photolabile precursor (122) enabled the independent generation of 5,6-dihydro-2'-deoxyuridin-6-yl (123), a model for nucleobase radicals (2, 15) produced in DNA by ionizing radiation. Prior to generation in DNA, the reactivity of monomeric radical 123 was characterized. Using competitive kinetic methods, rate constants for the reaction of 123 with thiol and 2-deoxyribose sugar models were estimated. Under aerobic conditions, 2'-deoxyuridine C6-hydrate (140) is the major product of 5,6-dihydro-2'-deoxyuridin-6-yl. 2-Deoxyribonolactone (133) is produced in modest yield, the product of an intramolecular hydrogen atom abstraction reaction by the nucleobase peroxyl radical (145). Incorporation of the radical precursor (122) into synthetic oligonucleotides allowed investigation of the role of a 5,6-dihydropyrimidin-6-yl in DNA damage. Generation of 5,6-dihydro-2'-deoxyuridin-6-yl (123) in DNA resulted in oxygen-dependent alkali-labile lesion formation at the site of the radical and at adjacent nucleotides. The mechanism of the DNA damage amplification process was elucidated by deuterium isotope effect measurements and examining the effects of modified nucleotides upon transfer of damage. It was determined that the nucleobase peroxyl radical (158) results in alkali-labile lesion formation at adjacent nucleotides by three distinct mechanisms: Cl' hydrogen atom abstraction, C5 methyl hydrogen abstraction, and addition to the nucleobase. The yield of tandem alkali-labile lesions was estimated to be ~ 65 % in duplex DNA, suggesting that the significance of this class of damage in the ionizing radiation mediated damage of nucleic acids may be vastly underestimated.