Site specific N6-(2-hydroxy-3,4-epoxybut-1-yl)adenine oligodeoxynucleotide adducts of 1,2,3,4-diepoxybutane: Synthesis and stability at physiological pH

Sergey Antsypovich, Danaè Quirk-Dorr, Crystal Pitts, Natalia Tretyakova

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Abstract

1,2,3,4-Diepoxybutane (DEB) is an important metabolite of 1,3-butadiene, a high volume industrial chemical classified as a human and animal carcinogen. DEB is a bifunctional alkylating agent that exhibits both mutagenic and cytotoxic activity, presumably a result of its ability to form bifunctional DNA adducts. Initial reactions of DEB with DNA produce 2-hydroxy-3,4-epoxybut-1-yl (HEB) lesions at guanine and adenine nucleobases. The epoxy group of the monoadduct is inherently reactive and can then undergo further reactions, for example, hydrolysis to the corresponding 2,3,4-trihydroxybutyl adducts and/or second alkylation to yield 2,3-butanediol cross-links. In the present work, synthetic DNA 16-mers containing structurally defined racemic N6-(2-hydroxy-3, 4-epoxybut-1-yl)-2′-deoxyadenosine (N6-HEB-dA) adducts (5′-AATTATGTXACGGTAG-3′, where X = N6-HEB-dA) were prepared by coupling 6-chloropurine-containing oligodeoxynucleotides with 1-amino-2-hydroxy-3,4-epoxybutane. The latter was generated in situ from the corresponding Fmoc-protected amino epoxide. The N6-HEB-dA-containing DNA oligomer was isolated by reverse-phase HPLC, and the presence of N 6-HEB-dA in its structure was confirmed by molecular weight determination and by HPLC-UV-ESI+-MS/MS analyses of enzymatic digests. An independently prepared N6-HEB-dA nucleoside served as an authentic standard. The fate of N6-HEB-dA within DNA at physiological conditions in the presence of various nucleophiles (e.g., cysteine, dG, and the complementary DNA strand) was investigated. Under all conditions tested, N 6-HEB-dA rapidly cyclized to produce previously unidentified exocyclic dA lesions (t1/2 < 2 h at physiological conditions). Only trace amounts of hydrolyzed and cross-linked products were detected, suggesting that the rate of cyclization was much greater than the rates of other reactions at the epoxide ring. These results indicate that DEB-induced alkylation of N6-adenine in DNA is unlikely to lead to DNA-DNA cross-linking but instead can result in the formation of exocyclic dA adducts.

Original languageEnglish (US)
Pages (from-to)641-649
Number of pages9
JournalChemical research in toxicology
Volume20
Issue number4
DOIs
StatePublished - Apr 2007

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