1,2,3,4-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 because of 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: N⁶,N⁶-(2,3-dihydroxybutan-1,4-diyl)-2'-deoxyadenosine (N⁶,N⁶-DHB-dA), 1,N⁶-(2-hydroxy-3-hydroxymethylpropan-1,3-diyl)-2'-deoxyadenosine (1,N⁶-γ-HMHP-dA), and 1,N⁶-(1-hydroxymethyl-2-hydroxypropan-1,3-diyl)-2'-deoxyadenosine (1,N⁶-α-HMHP-dA) [Seneviratne, U., et al. (2010) Chem. Res. Toxicol. 23, 118-133]. In the work presented here, a postsynthetic methodology for preparing DNA oligomers containing stereospecific and site-specific N⁶,N⁶-DHB-dA and 1,N⁶-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 N⁶-(2-hydroxy-3,4-epoxybut-1-yl)-2'-deoxyadenosine adducts, followed by their spontaneous cyclization to 1,N⁶-γ-HMHP-dA lesions. N⁶,N⁶-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 N⁶,N⁶-DHB-dA and 1,N⁶-γ-HMHP-dA revealed that both lesions lower the thermodynamic stability of DNA. Interestingly, structurally modified DNA duplexes were more thermodynamically stable when an adenine residue was placed opposite 1,N⁶-γ-HMHP-dA instead of thymine, suggesting that these adducts may preferentially pair with dA.