Synthesis of dihydrothymidine and thymidine glycol 5'-triphosphates and their ability to serve as substrates for Escherichia coli DNA polymerase I

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Abstract
5,6-Dihydrothymidine 5,6-Dihydrothymidine 5''-triphosphate (DHdTTP) was by catalytic hydrogenation of thymidine 5''-triphosphate (dTTP). Thymidine glycol 5''-triphosphate (dTTP-GLY) was prepared by bromination dTTP followed by treatment with Ag2O. The modified nucleotides were extensively purified by anion-exchange high-performance liquid chromatography (HPLC). Alkaline phosphatase digestion of DHdTTP and dTTP-GLY gave the expected products (5,6-dihydrothymidine and cis-thymidine glycol), the identifies of which were confirmed by reverse-phase HPLC using authentic markers. HPLC analysis of the alkaline phosphatase digested DHdTTP revealed that DHdTTP was a mixture of C5 diastereoisomers [(5S)- and (5R)-DHdTTP]. Despite the significant distortion of the pyrimidine ring in DHdTTP, it was incorporated in place of dTTP during primer elongation catalyzedby Escherichia coli DNA polymerase I Klenow fragment. The rate of incorporation of DHdTTP was abotu 10-25-fold lower than that of dTTP. On the other hand, dTTP-GLY, which also has a distorted pyrimidine ring, did not replace dTTP, and no elongation of the primer was observed. In order to study the preference of incorporation of the diastereoisomers of DHdTTP into DNA, salmon testes DNA, activiated by exonuclease III, was used as a template for DNA polymerase I Klenow fragment in the presence of [3H]DHdTTP (S and R mixture) and normal nucleotides. After enzymatic digestion of the DNA to nucleosides, the products were analyzed by HPLC. The ratio of the isomers incorporated into DNA (S:R = 73:27) was virtually the same as that of the [3H]DHdTTP substrates (S:R = 79:21). This result suggests that Escherichia coli DNA polymerase I uses both isomers of DHdTTP as substrates and that the overall efficiency of incorporation is primarily determined by the concentration of the isomers in the nucleotide pool.