Formation of a N 2-dG: N 2-dG carbinolamine DNA cross-link by the trans -4-hydroxynonenal-derived (6 S,8 R,11 S) 1, N 2-dG adduct

Abstract

Michael addition of trans-4-hydroxynonenal (HNE) to deoxyguanosine yields diastereomeric 1,N2-dG adducts in DNA. When placed opposite dC in the 5′-CpG-3′ sequence, the (6S,8R,11S) diastereomer forms a N 2-dG:N2-dG interstrand cross-link [Wang, H.; Kozekov, I. D.; Harris, T. M.; Rizzo, C. J. J. Am. Chem. Soc.2003, 125, 5687-5700]. We refined its structure in 5′-d(G1C2T 3A4G5C6X7A 8G9T10C11C12)- 3′•5′-d(G13G14A15C 16T17C18Y19C20T 21A22G23C24)-3′ [X7 is the dG adjacent to the C6 carbon of the cross-link or the α-carbon of the (6S,8R,11S) 1,N2-dG adduct, and Y19 is the dG adjacent to the C8 carbon of the cross-link or the γ-carbon of the HNE-derived (6S,8R,11S) 1,N2-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 (6S,8R,11S) 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 Y19N2H and X7 N1H protons, respectively. A strong H8→H11 NOE and no 3J(13C→ H) coupling for the 13C8-O-C11-H11 eliminated the tetrahydrofuran species derived from the (6S,8R,11S) 1,N2-dG adduct. The (6S,8R,11S) carbinolamine linkage and the HNE side chain were located in the minor groove. The X7N2 and Y19N2 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 C20O2 of the 5′-neighbor base pair G5•C20 and O11H with C18O 2 of X7•C18. These may, in part, explain the stability of this cross-link and the stereochemical preference for the (6S,8R,11S) configuration. © 2011 American Chemical Society.