NMR studies of an α,β-unsaturated aldehyde-derived interstrand carbinolamine DNA cross-link in the 5´-CpG-3´ sequence

Abstract

The chemistry of acrolein and crotonaldehyde-derived propano-deoxyguanosine (γ-OH-PdG and α-CH₃-γ-OH-PdG) adducts was monitored in the 5´-CpG-3´ sequence within a dodecamer duplex by NMR spectroscopy, in situ, using a series of site-specific ¹³C- and ¹⁵N-edited experiments. One striking phenomenon was the formation of an interstrand DNA cross-link, predominantly a carbinolamine. The cross-link existed in equilibrium with the non-crosslinked aldehyde and its geminal diol hydrate. The ratio of aldehyde/diol increased at higher temperatures. The effects of the pH and complementary bases were examined. Molecular modeling suggested that the carbinolamine linkage should be capable of maintaining Watson-Crick hydrogen bonds at both of the tandem C•G base pairs. In contrast, dehydration of the carbinolamine cross-link to an imine (Schiff base) cross-link, or cyclization of the latter to form a pyrimidopurinone cross-link, was predicted to require disruption of Watson-Crick hydrogen bonds at one or both of the tandem cross-linked C•G base pairs. Structural study of the <i>S</i>-crotonaldehyde-derived dG adduct ring-opened species suggested its slow generation of a cross-link was due to the configuration of the methyl group. The fully reduced <i>R</i>- and <i>S</i>-crotonaldehyde-derived cross-link studies confirm that these cross-links can be accommodated in duplex DNA. The methyl stereochemistry also affects the thermodynamic stability of the reduced cross-links in duplex DNA. The results provide a rationale for the stability of interstrand cross-links formed by acrolein and crotonaldehyde and perhaps other α,β-unsaturated aldehydes. These sequence-specific carbinolamine cross-links are anticipated to interfere with DNA replication and contribute to acrolein- and crotonaldehyde-mediated genotoxicity.