Spectroscopic Characterization of Interstrand Carbinolamine Cross-Links Formed in the 5‘-CpG-3‘ Sequence by the Acrolein-Derived γ-OH-1,N 2-Propano-2‘-deoxyguanosine DNA Adduct

Abstract

The interstrand N²,N²-dG DNA cross-linking chemistry of the acrolein-derived γ-OH-1,N²-propanodeoxyguanosine (γ-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 ¹³C- and ¹⁵N-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 N²-(3-oxo-propyl)-dG aldehyde and its geminal diol hydrate. The ratio of aldehyde/diol increased at higher temperatures. The 1,N²-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 γ-OH-PdG adduct contained within the 5‘-CpG-3‘ sequence was instead annealed into duplex DNA opposite T, a mixture of the 1,N²-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 dA, γ-OH-PdG remained as the 1,N²-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 α,β-unsaturated aldehydes. These sequence-specific carbinolamine cross-links are anticipated to interfere with DNA replication and contribute to acrolein-mediated genotoxicity.