Fig. 2. Canonical nucleotides react with HCHO at either exo- or endocyclic nitrogens and at different rates to form N-hydroxymethyl adducts. (A) 1H NMR time course analyses of incubations of canonical nucleotides with HCHO. Five canonical nucleotide monophosphates (dAMP, dCMP, dGMP, TMP, and UMP) were incubated with 53 eq. of HCHO in D2O at pD 7.5, and the reactions were monitored over time using 1H NMR (final nucleotide concentration = 2.4 mM). The pyrimidines TMP and UMP react via their endocyclic N-3 nitrogens to form 3hmTMP and 3hmUMP, respectively (blue). dAMP, dCMP and dGMP react via their exocyclic N-6, N-4 and N-2 nitrogens respectively to form 6hmdAMP, 4hmdCMP, and 2hmdGMP as major products (blue). Under the tested conditions, evidence for low-level formation of the dihydroxymethylated adducts 6dhmdAMP and 2dhmdGMP was also accrued (green). (B) 1H NMR time course analysis of a reaction mixture containing dAMP, dCMP, dGMP, TMP and UMP (all at 2.2 mM) and HCHO (58 eq.) in D2O at pD 7.5. The fastest adducts to form were 3hmTMP and 3hmUMP, formed via reactions of HCHO with TMP and UMP respectively. The other observed adducts (6hmdAMP, 4hmdCMP and 2hmdGMP), formed via reactions of HCHO with exocyclic nitrogens on dAMP, dCMP and dGMP respectively, were slower to form, but showed time-dependent increases in concentration. Of these three products, 4hmdCMP was fastest to form, followed by 2hmdGMP and 6hmdAMP respectively. Only the major adducts for each nucleotide are shown. (C) 1H NMR time course analyses of incubations of dAMP, dCMP and dGMP with HCHO, after two weeks reaction and subsequent dilution with D2O (25-fold). In all cases, the concentrations of the N-hydroxymethyl adducts decreased in a time-dependent manner after dilution, revealing non-immediate adjustments to new equilibrium positions. Of the three nucleotides, degradation of adducts derived from dAMP (6hmdAMP and 6dhmdAMP) were the slowest to degrade, revealing their relative stabilities compared to the other adducts. Upon dilution, 3hmTMP and 3hmUMP immediately decreased in concentration, indicating a fast dynamic equilibrium between the adducts and free nucleotides (and HCHO).
