Spectroscopic Characterization of Interstrand Carbinolamine Crosslinks Formed in the 5'-CpG-3' Sequence by the Acrolein-Derived γ-OH-1,N2-Propano-2'-deoxyguanosine DNA Adduct

The interstrand N 2 ,N 2 -dG DNA crosslinking chemistry of the acrolein-derived γ-OH-1,N 2 -propanodeoxyguanosine (γ-OH-PdG) adduct in the 5'-Cp G -3' sequence was monitored within a dodecamer duplex by NMR spectroscopy, in situ , using a series of site-specific 13 C- and 15 N-edited experiments. At equilibrium 40% of the DNA was crosslinked, with the carbinolamine form of the crosslink predominating. The crosslink 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 crosslink to an imine (Schiff base) crosslink, or cyclization of the latter to form a pyrimidopurinone crosslink, was predicted to require disruption of Watson-Crick hydrogen bonding at one or both of the tandem crosslinked 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,N 2 -dG cyclic adduct, the aldehyde, and the diol, but no crosslink, was observed. With this mismatched duplex, reaction with the tetrapeptide KWKK formed DNA-peptide crosslinks efficiently. When annealed opposite dA, γ-OH-PdG 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 crosslinks formed by acrolein and perhaps other α,β-unsaturated aldehydes. These sequence-specific carbinolamine crosslinks are anticipated to interfere with DNA replication and contribute to acrolein-mediated genotoxicity.