Mechanistic Insights into the Borane-Mediated Conversion of 5-Carboxylcytosine to Dihydrouracil in DNA Enable an Efficient Labeling Method Using Alkoxyamines

TET-assisted pyridine borane sequencing (TAPS) is a powerful method for mapping cytosine modifications, yet the underlying mechanism of conversion of 5-carboxylcytosine (5caC) to dihydrouracil (DHU) remains poorly understood. Here, through both experimental and computational studies, we show that the reaction proceeds via reduction and decarboxylation to give 5,6-dihydrocytidine as the intermediate, which is then slowly hydrolyzed to DHU as the rate-limiting step. We also found that in double-stranded DNA, the reaction can be catalyzed by an adjacent 5caC with high efficiency. The mechanistic discovery of the slow hydrolysis step by water in the conventional TAPS guided us to use more nucleophilic alkoxyamines to improve this protocol, achieving complete conversion within 1 h. This approach, when coupled with a click-chemistry pull-down, enables up to a 47-fold enrichment of 5caC-containing DNA. Our work thus provides fundamental mechanistic insights into a key epigenetic sequencing reaction and translates this knowledge into a new chemical biology tool for the selective labeling of DNA modifications.