DNA Oligomers Containing Site-Specific and Stereospecific Exocyclic Deoxyadenosine Adducts of 1,2,3,4-Diepoxybutane: Synthesis, Characterization, and Effects on DNA Structure

Abstract

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.

Base Excision Repair of N6-Deoxyadenosine Adducts of 1,3-Butadiene

“…28,29 DNA strands containing ( R,R )- N 6 -(2-hydroxy-3,4-epoxybutan-1-yl)-dA were converted to the corresponding ( R,S )-1, N 6 -HMHP-dA containing strands via intramolecular nucleophilic substitution in an aqueous solution. 28,29 These reactions were conducted on solid support in order to maximize the yield of the final product. All synthetic oligodeoxynucleotides were purified by HPLC to achieve > 98% purity and characterized by liquid chromatography-mass spectrometry (Table 1, Supplement S-1).…”

“…63,64 Circular dichroism (CD) experiments indicated that DNA duplexes containing N 6 -HB-dA, 1, N 6 -HMHP-dA, and N 6 , N 6 -DHB-dA adducts maintain a B-type DNA conformation. 28 Solution NMR structures of ( R , R ) and ( S , S )- N 6 , N 6 -DHB-dA and ( S )- N 6 -HB-dA containing DNA duplexes show that they are readily accommodated in the major groove of DNA. 65 Only small perturbations in base stacking and hydrogen bonding interactions were observed for ( S )- N 6 -HB-dA, while the presence of N 6 , N 6 -DHB-dA prevented its hydrogen bonding with thymidine in the opposite strand and disrupted stacking interactions of the adducted base with neighboring nucleotides.…”

“…28–30 Briefly, ( R,S )-1, N 6 -HMHP-dA adducted DNA strands were prepared by coupling ( R,R )- N -Fmoc-1-amino-2-hydroxy-3,4-epoxybutane with the oligomers containing site-specific 6-chloropurine at position X . The resulting ( R,R )- N 6 -(2-hydroxy-3,4-epoxybut-1-yl)-adenine ( R,R -HEB-dA) containing oligodeoxynucleotides were isolated by high performance liquid chromatography (HPLC) and subjected to cyclization in water to afford the corresponding ( R,S )-1, N 6 -HMHP-dA strands.…”

1,3-Butadiene-Induced Adenine DNA Adducts Are Genotoxic but Only Weakly Mutagenic When Replicated in Escherichia coli of Various Repair and Replication Backgrounds

“…Even though two studies have found that both isomers reduce the melting point of duplex DNA to a similar extent, it was proposed that the different orientations of the hydroxyl groups on the DHB adducts could interact with DNA polymerases differently during TLS, thus leading to different outcomes. 44, 46 Investigation of the three stereoisomers of the DEB metabolites also found that the S,S -isomer is more cytotoxic and mutagenic than the meso - and R,R -isomers. 47 Our results here support the hypothesis that lesion stereospecificity modulates mutagenicity and genotoxicity, which has also been previously observed with the stereoisomers of three other BD adducts.…”

“…44 This decrease in stability was reflected in a similar decrease in melting points for both the R,R - and S,S -duplexes. 44, 46 Previously, we showed that all four bases can be inserted opposite R , R - N 6 , N 6 -DHB-dA when bypassed by TLS polymerases in vitro . 26 Here, we found that both N 6 , N 6 -DHB-dA lesions might induce low levels of A→G (~1%) and A→T (<1%) mutations.…”