Formation of a <i>N</i>²-dG:<i>N</i>²-dG Carbinolamine DNA Cross-link by the <i>trans</i>-4-Hydroxynonenal-Derived (6<i>S</i>,8<i>R</i>,11<i>S</i>) 1,<i>N</i>²-dG Adduct

Abstract

Michael addition of <i>trans</i>-4-hydroxynonenal (HNE) to deoxyguanosine yields diastereomeric 1,<i>N</i>²-dG adducts in DNA. When placed opposite dC in the 5′-CpG-3′ sequence, the (6<i>S</i>,8<i>R</i>,11<i>S</i>) diastereomer forms a <i>N</i>²-dG:<i>N</i>²-dG interstrand cross-link [Wang, H.; Kozekov, I. D.; Harris, T. M.; Rizzo, C. J. <i>J. Am. Chem. Soc.</i> <b>2003</b>, <i>125</i>, 5687–5700]. We refined its structure in 5′-d(G¹C²T³A⁴G⁵C⁶<u>X</u>⁷A⁸G⁹T¹⁰C¹¹C¹²)-3′·5′-d(G¹³G¹⁴A¹⁵C¹⁶T¹⁷C¹⁸<u>Y</u>¹⁹C²⁰T²¹A²²G²³C²⁴)-3′ [X⁷ is the dG adjacent to the C6 carbon of the cross-link or the α-carbon of the (6<i>S</i>,8<i>R</i>,11<i>S</i>) 1,<i>N</i>²-dG adduct, and Y¹⁹ is the dG adjacent to the C8 carbon of the cross-link or the γ-carbon of the HNE-derived (6<i>S</i>,8<i>R</i>,11<i>S</i>) 1,<i>N</i>²-dG adduct; the cross-link is in the 5′-CpG-3′ sequence]. Introduction of ¹³C at the C8 carbon of the cross-link revealed one ¹³C8→H8 correlation, indicating that the cross-link existed predominantly as a carbinolamine linkage. The H8 proton exhibited NOEs to Y¹⁹ H1′, C²⁰ H1′, and C²⁰ H4′, orienting it toward the complementary strand, consistent with the (6<i>S</i>,8<i>R</i>,11<i>S</i>) configuration. An NOE was also observed between the HNE H11 proton and Y¹⁹ H1′, orienting the former toward the complementary strand. Imine and pyrimidopurinone linkages were excluded by observation of the Y¹⁹ <i>N</i>²H and X⁷ N1H protons, respectively. A strong H8→H11 NOE and no ³<i>J</i>(¹³C→H) coupling for the ¹³C8–O–C11–H11 eliminated the tetrahydrofuran species derived from the (6<i>S</i>,8<i>R</i>,11<i>S</i>) 1,<i>N</i>²-dG adduct. The (6<i>S</i>,8<i>R</i>,11<i>S</i>) carbinolamine linkage and the HNE side chain were located in the minor groove. The X⁷ <i>N</i>² and Y¹⁹ <i>N</i>² atoms were in the gauche conformation with respect to the linkage, maintaining Watson–Crick hydrogen bonds at the cross-linked base pairs. A solvated molecular dynamics simulation indicated that the anti conformation of the hydroxyl group with respect to C6 of the tether minimized steric interaction and predicted hydrogen bonds involving O8H with C²⁰ <i>O</i>² of the 5′-neighbor base pair G⁵·C²⁰ and O11H with C¹⁸ <i>O</i>² of X⁷·C¹⁸. These may, in part, explain the stability of this cross-link and the stereochemical preference for the (6<i>S</i>,8<i>R</i>,11<i>S</i>) configuration