Formation of a Methide Derivative upon Photolysis of Thymidine Bromohydrins Thierry Douki,* Guillaume Vadesne-Bauer, and Jean Cadet* Laboratoire Le´ sions des Acides Nucle´ iques, Service de Chimie Inorganique et Biologique, UMR 5046, CEA/DSM/De´ partement de Recherche Fondamentale sur la Matie` re Condense´ e, CEA-Grenoble, 38054 Grenoble Cedex 9, France
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[email protected] Received October 25, 2002
Reaction of bromine with thymidine in aqueous solution produces, in high yield, the corresponding 5-bromo-6-hydroxy-5,6-dihydroderivative (thymidine bromohydrins). UVC photolysis of thymidine bromohydrins gives rise to a reactive intermediate that is converted into 5-(hydroxymethyl)-2′deoxyuridine upon incubation in water. When the former compound is left in methanol, ethanol, or propanol, the corresponding 5-alkoxymethyl derivatives are produced. The proposed structure for the primary photolysis product of thymidine bromohydrins is a methide derivative of the thymine ring. This compound could be an interesting intermediate in the synthesis of methyl-substituted thymidine. Introduction
Results
Modification of the chemical structure of DNA bases is associated with deleterious cellular processes including lethality and mutagenicity. In that respect, radicalinduced degradation of nucleobases has been extensively investigated on model systems such as nucleosides and short oligonucleotides.1 Photochemical generation of nucleobase radicals is a powerful tool for the understanding of the degradation pathways.2 Recently, we used thymine bromohydrins, previously used as synthetic precursors of pyrimidine hydroperoxides,3 to photochemically trigger the formation of tandem lesions in a dinucleoside monophosphate carrying a vicinal guanine moiety.4 In the latter experiment, products only modified on their thymine moiety were also obtained in high yield. We presently report a more thorough study of the photolysis of thymine bromohydrins at the nucleoside level. The final stable products were characterized, on the basis of 1H NMR and mass spectrometry analyses, as 5-(hydroxymethyl)-uracil nucleosides. Their formation involved an intermediate that was partially identified by 1 H NMR and mass spectrometry. A methide structure, in agreement with the reactivity in water and alcohols, was proposed for this transient bromohydrins photolysis product.
Identification of the Final Stable Products of Photolysis of Thymidine Bromohydrins. (5R,6S)and (5S,6R)-5-Bromo-6-hydroxy-5,6-dihydrothymidine (trans bromohydrins of thymidine, 2) were synthesized in high yield as previously reported by addition of bromine to an aqueous solution of thymidine (1).5 The reaction mixture was injected on a reverse-phase HPLC column, and a fraction containing the two bromohydrins was collected. The resulting solution was then exposed to the UVC light emitted by a germicidal lamp. Thymidine bromohydrins are very reactive under these conditions, since, after 30 min of irradiation, HPLC analysis shows that 2 is completely converted. Instead, the HPLC-UV chromatogram exhibits two main peaks corresponding to closely eluting photoproducts 3 and 3′ (yield ca. 95%) at shorter retention times than those obtained for 2 (Figure 1). Thymidine glycols that may arise from either the hydrolytic conversion of bromohydrins or the fate of the 6-hydroxy-5-yl radical are also obtained in low yield (
