Major Groove Orientation of the (2<i>S</i>)‑<i>N</i>⁶‑(2-Hydroxy-3-buten-1-yl)-2′-deoxyadenosine DNA Adduct Induced by 1,2-Epoxy-3-butene

Abstract

1,3-Butadiene (BD) is an environmental and occupational toxicant classified as a human carcinogen. It is oxidized by cytochrome P450 monooxygenases to 1,2-epoxy-3-butene (EB), which alkylates DNA. BD exposures lead to large numbers of mutations at A:T base pairs even though alkylation of guanines is more prevalent, suggesting that one or more adenine adducts of BD play a role in BD-mediated genotoxicity. However, the etiology of BD-mediated genotoxicity at adenine remains poorly understood. EB alkylates the <i>N</i>⁶ exocyclic nitrogen of adenine to form <i>N</i>⁶-(hydroxy-3-buten-1-yl)-2′-dA ((2<i>S</i>)-<i>N</i>⁶-HB-dA) adducts (Tretyakova, N., Lin, Y., Sangaiah, R., Upton, P. B., and Swenberg, J. A. (1997) Carcinogenesis 18, 137−147). The structure of the (2<i>S</i>)-<i>N</i>⁶-HB-dA adduct has been determined in the 5′-d­(C¹G²G³A⁴<u>C</u>^<u>5</u><u>Y</u>^<u>6</u><u>A</u>^<u>7</u>G⁸A⁹A¹⁰G¹¹)-3′:5′-d­(C¹²T¹³T¹⁴C¹⁵T¹⁶T¹⁷G¹⁸T¹⁹ C²⁰C²¹G²²)-3′ duplex [Y = (2<i>S</i>)-<i>N</i>⁶-HB-dA] containing codon 61 (underlined) of the human N-<i>ras</i> protooncogene, from NMR spectroscopy. The (2<i>S</i>)-<i>N</i>⁶-HB-dA adduct was positioned in the major groove, such that the butadiene moiety was oriented in the 3′ direction. At the C_α carbon, the methylene protons of the modified nucleobase Y⁶ faced the 5′ direction, which placed the C_β carbon in the 3′ direction. The C_β hydroxyl group faced toward the solvent, as did carbons C_γ and C_δ. The C_β hydroxyl group did not form hydrogen bonds with either T¹⁶ <i>O</i>⁴ or T¹⁷ <i>O</i>⁴. The (2<i>S</i>)-<i>N</i>⁶-HB-dA nucleoside maintained the <i>anti</i> conformation about the glycosyl bond, and the modified base retained Watson–Crick base pairing with the complementary base (T¹⁷). The adduct perturbed stacking interactions at base pairs C⁵:G¹⁸, Y⁶:T¹⁷, and A⁷:T¹⁶ such that the Y⁶ base did not stack with its 5′ neighbor C⁵, but it did with its 3′ neighbor A⁷. The complementary thymine T¹⁷ stacked well with both 5′ and 3′ neighbors T¹⁶ and G¹⁸. The presence of the (2<i>S</i>)-<i>N</i>⁶-HB-dA resulted in a 5 °C reduction in the <i>T</i>_m of the duplex, which is attributed to less favorable stacking interactions and adduct accommodation in the major groove