A covalent thymine-tyrosine adduct involved in DNA-protein crosslinks: Synthesis, characterization, and quantification

A thymine-tyrosine adduct, (3-[(1,3-dihydro-2,4-dioxopyrimidin-5-yl)methyl] -L-tyrosine), was synthesized using a simple, single-step condensation betw een 5-(hydroxymethyl)uracil and L-tyrosine. This approach provides access t o useful quantities (mg-g) of analytically pure reference material, and wit h minor modification, to stable isotope-labeled analogues (isotopomers). Wi th reference material and a suitable internal standard available, isotope-d ilution liquid chromatography-electrospray ionization-tandem mass spectrome try (LC/MS/MS) was used to assay the adduct in a model system purged of oxy gen, i.e., a gamma-irradiated N2O-saturated aqueous solution of thymine and tyrosine. The convenient synthetic route to standards and the method for q uantification reported here will prove useful in assessing the significance of the adduct in biological systems. These studies also highlight the pote ntial for artefactual adduct formation if the appropriate substrates are pr esent under acidic conditions. (C) 1999 Elsevier Science Inc.