title: 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.
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 N6-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, N6-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.
authors.affiliation: Department of Medicinal Chemistry and The Cancer Center, University of Minnesota, Minneapolis, Minnesota 55455, USA.
authors.forename: Sergey
authors.initials: S
authors.lastname: Antsypovich
authors.forename: Danaè
authors.initials: D
authors.lastname: Quirk-Dorr
authors.forename: Crystal
authors.initials: C
authors.lastname: Pitts
authors.forename: Natalia
authors.initials: N
authors.lastname: Tretyakova
mesh_terms: D000241:Adenosine / Q000031:analogs & derivatives* / Q000737:chemistry; D002851:Chromatography, High Pressure Liquid; D018736:DNA Adducts / Q000737:chemistry* / Q000378:metabolism*; D003839:Deoxyadenosines / Q000737:chemistry; D004852:Epoxy Compounds / Q000737:chemistry*; D006863:Hydrogen-Ion Concentration; D009682:Magnetic Resonance Spectroscopy; D015394:Molecular Structure; D021241:Spectrometry, Mass, Electrospray Ionization
chemical_list: D018736:DNA Adducts; D003839:Deoxyadenosines; D004852:Epoxy Compounds; C521702:N6-(2-hydroxy-3,4-epoxybut-1-yl)-2'-deoxyadenosine; C007366:diepoxybutane; D000241:Adenosine