Abstract1,2,3,4-Diepoxybutane (DEB)1 is considered the ultimate carcinogenic metabolite of 1,3-butadiene, an important industrial chemical and environmental pollutant present in urban air. Although it preferentially modifies guanine within DNA, DEB induces a large number of A → T transversions, suggesting that it forms strongly mispairing lesions at adenine nucleobases. We now report the discovery of three potentially mispairing exocyclic adenine lesions of DEB:. The structures and stereochemistry of the novel DEB-dA adducts were determined by a combination of UV and NMR spectroscopy, tandem mass spectrometry, and independent synthesis. We found that synthetic N 6 -(2-hydroxy-3,4-epoxybut-1-yl)-2′-deoxyadenosine (compound 1) representing the product of N 6 -adenine alkylation by DEB spontaneously cyclizes to form 3 under aqueous conditions or 2 under anhydrous conditions in the presence of organic base. Compound 3 can be interconverted with 4 by a reversible unimolecular pericyclic reaction favoring 4 as a more thermodynamically stable product. Both 3 and 4 are present in double stranded DNA treated with DEB in vitro and in liver DNA of laboratory mice exposed to 1,3-butadiene by inhalation. We propose that in DNA under physiological conditions, DEB alkylates the N-1 position of adenine in DNA to form N1- (2-hydroxy-3,4-epoxybut-1-yl)adenine adducts, which undergo an S N 2-type intramolecular nucleophilic substitution and rearrangement to give 3 (minor) and 4 (major). Formation of exocyclic DEB-adenine lesions following exposure to 1,3-butadiene provides a possible mechanism of mutagenesis at the A:T base pairs.

Abstract1,2,3,4-Diepoxybutane (DEB) is a carcinogenic metabolite of 1,3-butadiene (BD), an important industrial and environmental chemical present in urban air and in cigarette smoke. DEB is considered the ultimate carcinogenic species of BD due to its potent genotoxicity and mutagenicity attributed to its ability to form DNA-DNA cross-links and exocyclic nucleoside adducts. Mutagenesis studies suggest that DEB adducts formed at adenine bases may be critically important, as it induces large numbers of A → T transversions. We have recently identified three types of exocyclic DEB-dA lesions: N 6 ,N 6 -(2,3-dihydroxybutan-1,4-diyl)-2′-deoxyadenosine (N 6 ,N 6 -DHB-dA), 1,N 6 -(2-hydroxy-3-hydroxymethylpropan-1,3-diyl)-2′-deoxyadenosine (1,N 6 -γ-HMHP-dA), and 1,N 6 -(1-hydroxymethyl-2-hydroxypropan-1,3-diyl)-2′-deoxyadenosine (1,N 6 -α-HMHP-dA) (Seneviratne et al Chem. Res. Toxicol. 2010, 23, 118-133). In the present work, a post-synthetic methodology for preparing DNA oligomers containing stereo-and site-specific N 6 ,N 6 -DHB-dA and 1,N 6 -γ-HMHP-dA adducts was developed. DNA oligomers containing site specific 6-chloropurine were coupled with optically pure 1-amino-2-hydroxy-3,4-epoxybutanes to generate oligomers containing N 6 -(2-hydroxy-3,4-epoxybut-1-yl)adenine adducts, followed by their spontaneous cyclization to 1,N 6 -γ-HMHP-dA lesions. N 6 ,N 6 -DHB-dA containing strands were prepared analogously by coupling 6-chloropurine containing DNA with 3S,4S or 3R,4R pyrrolidine-3,4-diols. Oligodeoxynucleotide structures were confirmed by ESI-MS, exonuclease ladder sequencing, and HPLC-MS/MS of enzymatic digests. UV melting and CD spectroscopy studies of DNA duplexes containing N 6 ,N 6 -DHB-dA and 1,N 6 -γ-HMHP-dA revealed that both lesions lower the thermodynamic stability of DNA. Interestingly, structurally modified DNA duplexes were more thermodynamically stable when adenine residue was placed opposite 1,N 6 -γ-HMHP-dA instead of thymine, suggesting that these adducts may preferentially pair with dA.

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.

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