HPLC isolation and mass spectrometric characterization of two isomers of thymine glycols in oligodeoxynucleotides

Thymine glycol, or 5,6-dihydroxy-5,6-dihydrothymine, is the major oxidation product of thymine. Herein we report the isolation of both the (5S, 6R) and (5R, 6S) isomers of cis thymine glycols from several synthetic oligodeoxynucleotides (ODNs) upon oxidation with osmium tetraoxide. Our results show that tandem mass spectrometry can determine the sites of thymine glycol in ODNs by producing characteristic fragment ions, [a - 143] and its complementary w ions at the modification site. We further demonstrate that the [M + H]+ and [M + Na]+ ions of the two cis stereoisomers of thymine glycol in the dinucleotides, which are extricated from the ODNs by nuclease P1, gave distinctive product-ion spectra.