Spectroscopic characterization of interstrand carbinolamine cross-links formed in the 5′-CpG-3′ sequence by the acrolein-derived γ-OH-1,N2-propano-2′-deoxyguanosine DNA adduct

The interstrand (NN2)-N-2-dG DNA cross-linking chemistry of the acrolein-derived gamma-OH-1,N-2-propanocleoxyguanosine (gamma-OH-PdG) adduct in the 5'-CpG-3' sequence was monitored within a dodecamer duplex by NMR spectroscopy, in situ, using a series of site-specific C-13- and N-15-edited experiments. At equilibrium 40% of the DNA was cross-linked, with the carbinolamine form of the cross-link predominating. The cross-link existed in equilibrium with the non-crosslinked N-2-(3-oxo-propyl)-dG aldehyde and its geminal diol hydrate. The ratio of aldehyde/diol increased at higher temperatures. The 1,N-2-dG cyclic adduct was not detected. Molecular modeling suggested that the carbinolamine linkage should be capable of maintaining Watson-Crick hydrogen bonding 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 bonding at one or both of the tandem cross-linked C-G base pairs. When the gamma-OH-PdG adduct contained within the 5'-CpG-3' sequence was instead annealed into duplex DNA opposite T, a mixture of the 1,N-2-dG cyclic adduct, the aldehyde, and the diol, but no cross-link, was observed. With this mismatched duplex, reaction with the tetrapeptide KWKK formed DNA-peptide cross-links efficiently. When annealed opposite CIA, gamma-OHPdG remained as the 1,N-2-dG cyclic adduct although transient epimerization was detected by trapping with the peptide KWKK. The results provide a rationale for the stability of interstrand cross-links formed by acrolein and perhaps other a,p-unsaturated aldehydes. These sequence-specific carbinolamine cross-links are anticipated to interfere with DNA replication and contribute to acrolein-mediated genotoxicity.