Synthesis of the diastereomers of thymidine glycol, determination of concentrations and rates of interconversion of theircis-transepimers at equilibrium and demonstration of differential alkali lability within DNA

Abstract

5,6-dihydroxy-5,6-dihydrothymidine (thymidine glycol) is a major product of the reaction of thymidine with reactive oxygen species, including those generated by ionizing radiation. Thymidine glycol exists as 2 diastereomeric pairs by virtue of the chirality of the C(5) and C(6) atoms. A simple procedure is described for synthesizing and purifying each of the diastereomeric pairs separately. After brominating thymidine, the two trans 5-bromo-6-hydroxy-5,6-dihydrothymidine (thymidine bromohydrin) C(5) diastereomers were easily separated by High Performance Liquid Chromatography. Each thymidine bromohydrin was quantitatively converted to the corresponding diastereomeric thymidine glycol pair by reflux in aqueous solution. The concentrations at equilibrium of the cis (5S,6R),(5R,6S) and trans (5S,6S),(5R,6R) forms of the thymidine glycol diastereomers were determined and were 80% cis and 20% trans for the 5S pair and 87% cis and 13% trans for the 5R pair. At equilibrium, the rate of cis-trans epimerization of the two sets of diastereomers was essentially identical. The 5S diastereomeric pair was significantly more alkali labile than the 5R pair due to the higher concentration of the 5S trans epimer at equilibrium. This differential alkali lability was also manifest when the thymine glycol moiety was present in chemically oxidized poly(dA-dT).poly(dA-dT) indicating that the chemical differences between the diastereomeric pairs are preserved in DNA. These chemical differences may affect the biological properties of this important oxidative derivative of thymine in DNA.

Human DNA Polymerase κ Bypasses and Extends beyond Thymine Glycols during Translesion Synthesis in Vitro, Preferentially Incorporating Correct Nucleotides

“…Both procedures result in a mixture of the four possible stereoisomers of Tg in different relative proportions (Fig. 1B) (32,33). We show by both qualitative and quantitative steady-state kinetic analysis that pol supports TLS across both of these substrates.…”

“…Additionally, the stereochemical composition of this sample was assayed by digestion of the deoxynucleoside triphosphate with alkaline phosphatase and separation of the deoxynucleosides by HPLC according to a previously published method (33). This assay returned three peaks: 1) trans (5S,6S), 2) trans (5R,6R), and 3) the cis isomers, which elute together ((5S,6R) and (5R,6S)) with nearly identical retention times and peak area ratios, as had previously been reported for oxidation of thymidine deoxynucleoside (data not shown).…”

“…One substrate (Tg Br2 ) was generated by incorporation of Tg from thymidine glycol-5Ј-triphosphate prepared by bromination and oxidation as previously described (33). This procedure has been shown to yield a stereochemical mixture containing 64.3% of the two 5R stereoisomers, and 35.7% of the 5S stereoisomers (33).…”

“…One substrate (Tg Br2 ) was generated by incorporation of Tg from thymidine glycol-5Ј-triphosphate prepared by bromination and oxidation as previously described (33). This procedure has been shown to yield a stereochemical mixture containing 64.3% of the two 5R stereoisomers, and 35.7% of the 5S stereoisomers (33). Following oxidation, the stereochemistry at C-5 is fixed, but epimerization occurs about the C-6 center to yield 87% cis, 13% trans for the 5R stereoisomer and 80% cis, 20% trans for the 5S stereoisomer (33).…”

“…Following oxidation, the stereochemistry at C-5 is fixed, but epimerization occurs about the C-6 center to yield 87% cis, 13% trans for the 5R stereoisomer and 80% cis, 20% trans for the 5S stereoisomer (33). Epimerization equilibrates at room temperature within a few hours (33), yielding an equilibrium distribution of ϳ55.9% (5R,6S), 8.4% (5R,6R), 28.6% (5S,6S), and 7.1% (5S,6R) (Fig. 1B).…”