N (6)-Hydroperoxymethyladenosine: a new intermediate of chemical oxidation of N(6)-methyladenosine mediated by bicarbonate-activated hydrogen peroxide

N (6)-Methyladenosine (m(6)A) represents a relatively abundant modification in eukaryotic RNA. Because m(6)A has similar properties to adenosine and a low reactivity, limited research has been focused on this nucleoside. In this study, we revealed an important intermediate in the oxidation of m(6)A through the bicarbonate-activated peroxide system. Over the course of oxidation, we found a new mechanism in which N(6)-hydroxymethyladenosine (hm(6)A), N(6)-formyladenosine (f(6)A) and N(6)-hydroperoxymethyladenosine (oxm(6)A) were intermediate products, and adenosine was the final product. In this study, oxm(6)A was isolated using HPLC and characterized by mass spectrometry, NMR and diphenyl-1-pyrenylphosphine (DPPP) fluorescence detection. This study provides a new modified nucleoside and demonstrates oxidative demethylation of m(6)A by reactive oxygen species at the nucleobase level and in RNA strands.