Model Studies on the Photoreduction of the 5-Hydroxy-5,6-dihydrothymine and 5-Methyl-2-pyrimidone Moieties of (6-4) Photoproducts by Photolyase.

Photorepair mechanism of (6-4) photoproducts (6-4PP) by photolyase has been the subject of active debate over the years. The initial rationalization based on electron transfer to an oxetane or azetidine intermediate formed upon binding to the enzyme has been questioned, and there is now a more general consensus that the lesion is directly reduced from the excited flavin cofactor. However, the accepting moiety, i.e. the 5-methyl-2-pyrimidone or 5-hydroxy-5,6-dihydrothymine, has not been fully identified yet. In this work, spectroscopic experiments have been run to determine which of the 5'- or 3'-base of 6-4PP is more prone to be reduced. For this aim, the two building blocks of 6-4PP were synthesized and used as electron acceptors. Instead of the short-lived photolyase cofactor, which does not provide a time window compatible with diffusion-controlled intermolecular processes, carbazole, 2-methoxynaphthalene and phenanthrene have been selected as electron donors due to their appropriate singlet lifetimes and reduction potentials. Steady-state and time-resolved fluorescence revealed that, in solution, the pyrimidone chromophore is the most easily reduced moiety. This was confirmed by transient absorption experiments consisting of quenching of the solvated electron by the two moieties of 6-4PP.