Formation of a fairly stable diazoate intermediate of 5-methyl-2'-deoxycytidine by HNO2 and NO, and its implication to a novel mutation mechanism in CpG site

The intermediate produced from 5-methyl-2'-deoxycytidine ((5me)dCyd) by HNO2 and NO treatments was isolated and characterized. When 10mM (5me)dCyd was incubated with 100mM NaNO2 at pH 3.7 and 37 degrees C, a previously unidentified product was formed. The product was identified as a diazoate derivative of (5me)dCyd, 1-(beta-D-2'-deoxyribofuranosyl)-5-methyl-2-oxopyrimidine-4-diazoate ((5me)dCyd-diazoate), on the bases of several measurements including LC/MS. The time course of the concentration change of the diazoate showed a characteristic profile of a reaction intermediate, and the steady state concentration was 2.3 microM (0.023% yield). When an aqueous solution of 10mM (5me)dCyd (10 mL) was bubbled by NO at 37 degrees C under aerobic conditions holding the pH around 7.4, the diazoate was also generated. The yield of the diazoate was 0.041 micromol (0.041% yield) at 20 mmol of NO absorption. At physiological pH and temperature (pH 7.4, 37 degrees C), the diazoate was converted to dThd exclusively with a first order rate constant k=9.1x10(-6) x s(-1) (t(1/2)=21 h). These results show that the diazoate is generated as a relatively stable intermediate in the reactions of (5me)dCyd with HNO2 and NO and further suggest that the diazoate can be formed in cellular DNA with biologically relevant doses of HNO2 and NO.