Carbon dioxide but not bicarbonate inhibits N-nitrosation of secondary amines. Evidence for amine carbamates as protecting entities

Hydrogen carbonate (bicarbonate, HCO3-) has been proposed to accelerate the decomposition of N2O3 because N-nitrosation of morpholine via a nitric oxi de/oxygen mixture ((NO)-N-./O-2) was inhibited by the addition of HCO3- at pH 8.9 [Caulfield, J. L., Singh, S. P., Wishnok, J. S., Deen, W. M., and Ta nnenbaum, S. R. (1996) J. Biol. Chem. 271, 25859-25863]. In the study prese nted here, it is shown that carbon dioxide (CO2) is responsible for this ki nd of protective effect because of formation of amine carbamates, whereas a n inhibitory function of HCO3- is excluded. N-Nitrosation of morpholine (1- 10 mM) at pH 7.4-7.5 by the (NO)-N-.-donor compounds PAPA NONOate and MAMA NONOate (0.5 mM each) was not affected by the presence of large amounts of HCO3- (up to 100 mM) in aerated aqueous solution. Similar results were obta ined by replacing the (NO)-N-.-donor compounds with authentic (NO)-N-. (900 mu M). In agreement with data from the study cited above, (NO)-N-./O-2-med iated formation of N-nitrosomorpholine (NO-Mor) was indeed inhibited by abo ut 45% in the presence of 50 mM HCO3- at, pH 8.9. However, 500 MHz C-13 NMR analysis with C-13-enriched bicarbonate revealed that significant amounts of morpholine carbamate are formed from reaction of equilibrated CO2 with m orpholine (1-100 mM) at pH 8.9, but only to a minor extent at pH 7.5. The p rotective effect of morpholine carbamate formation is explained by a signif icantly reduced charge density at nitrogen. This view is supported by the r esults of density functional theory/natural population analysis, i.e., quan tumchemical calculations for morpholine and morpholine carbamate. In agreem ent with its lower pK(a), another secondary amine, piperazine, had already produced significant amounts of piperazine carbamate at pH 7.4 as shown by C-13 NMR spectrometry. Consequently, and in contrast to morpholine, N-nitro sation of piperazine (2 mM) by both (NO)-N-./O-2 (PAPA NONOate, 0.5 mM) and the (NO)-N-./O-2(-.)-releasing compound SIN-1 (1 mM) was inhibited by abou t 66% in the presence of 200 mM HCO3-.