A Chemically Stable Interstrand Cross-Link Generated via a Reductive Amination Process Involving an Apurinic/Apyrimidinic Site and an Adenine Residue in Duplex DNA

Apurinic/apyrimidinic (AP) sites are unavoidably generated in the DNA of living organisms by the spontaneous or catalyzed loss of coding nucleobases from the deoxyribose backbone. AP sites can lead to the generation of interstrand DNA cross-links via reactions between the ring-opened AP-aldehyde residue and exocyclic NH₂ groups of nucleobases on the opposing strand of the double helix. Earlier works showed that dG-AP cross-links, which are generated in 2-5% equilibrium yields, can be converted via a reductive amination process to higher yields (15-50%) of a chemically stable alkylamine cross-link when NaBH₃CN is present in the reaction mixture. A dA-AP cross-link can be generated in equilibrium yields of 15-80%, but until now, it has been uncertain whether this cross-link could be reduced to the corresponding alkylamine cross-link by NaBH₃CN. The results presented here show that the dA-AP cross-link can indeed be reduced by NaBH₃CN to generate a chemically stable alkylamine cross-link. However, yields of the reduced dA-AP cross-link are limited by a faster, competing reduction of the AP-aldehyde to the corresponding AP-alcohol by NaBH₃CN. Similarly, faster reduction of the dG-AP cross-link in a 5'CXT/AAG sequence (X = AP), where both guanine and adenine residues compete for reaction with a single AP site, leads to a shift in the major site of the AP-derived cross-link attachment from adenine in the absence of NaBH₃CN to guanine in the presence of NaBH₃CN. The results show that two different nucleobase cross-links can coexist in equilibrium at a single AP site in duplex DNA. Overall, the reductive amination process may prove useful for detecting the dA-AP cross-link in cellular DNA using LC-MS/MS methods similar to those described here. In addition, these methods may be useful for the chemical synthesis of DNA duplexes containing chemically stable, site-specific cross-links.