Scheme 2. Acid Trapping of the Proposed Intermediates in the Reaction with Deuterium-Labeled Flavin (FADD₂).

Formation of 5-HM-dUMP in (a) requires oxidation of the reduced intermediates at C6, i.e., loss of a hydron (H⁺ or D⁺) and two electrons. Due to an isotope effect on this nonenzymatic oxidation, the majority of 5-HM-dUMP is expected to be deuterated. Molecular oxygen has been proposed as the oxidant,¹¹ since quenched reactions are exposed to oxygen during quenching.