Cross-linked thymine-purine base tandem lesions: synthesis, characterization, and measurement in gamma-irradiated isolated DNA.

5-(Phenylthiomethyl)-2'-deoxyuridine has been recently shown to be a specific photolabile precursor of 5-(2'-deoxyuridilyl)methyl radical that is involved in the formation of tandem base lesion with vicinal guanine in oxygen-free aqueous solution. The thionucleoside was incorporated by either liquid or solid-phase phosphoramidite synthesis into dinucleoside monophosphates with a 2'-deoxyadenosine residue as the vicinal nucleoside located either at the 3' or 5'-extremity. UV-C irradiation of the modified dinucleoside monophosphate under anaerobic conditions gives rise to cross-linked thymine(CH2-C8)adenine tandem base lesions which were isolated and characterized by (1)H NMR and mass spectrometry analyses. The formation of the latter tandem lesions involved an intramolecular addition of the 5-(2'-deoxyuridilyl)methyl radical to the C8 of the adenine moiety. A sensitive and specific assay aimed at monitoring the formation of the four thymine(CH2-C8)purine adducts, namely d(T Delta G), d(G Delta T), d(T Delta A), d(A Delta T), within DNA, was designed. This was based on a liquid chromatography analysis coupled to tandem mass spectrometry (HPLC-MS/MS) detection of the dinucleoside monophosphates which were quantitatively released from gamma-irradiated DNA and oligodeoxyribonucleotides by enzymatic hydrolysis. The four lesions were detected in both single-stranded oligodeoxyribonucleotide and isolated DNA upon exposure to gamma-radiation in oxygen-free aqueous solution. It was found that the tandem guanine-thymine lesions were produced more efficiently than the adenine-thymine cross-links. Moreover, a significant sequence effect was observed. Thus, the yield of formation of the tandem lesions is higher when the purine base is located at the 5' position of the 5-(2'-deoxyuridilyl)methyl radical.