Nitrosation of 1,2-Phenylenediamine by Peroxynitrite/CO₂:  Evidence for a Free Radical Mechanism

Abstract

Nitric oxide and S-nitrosothiols function as critical signaling species. 1 S-Nitrosothiols have a longer half-life 2 than does • NO, and typical concentrations of S-nitrosothiols in blood plasma are 3-4 orders of magnitude higher than those of • NO. 3 Thus, nitrosation can lead to the formation of S-nitrosothiols, which can serve as carriers of • NO; however, since • NO itself does not nitrosate, 4 the mechanism by which nitrosation occurs in vivo is still unclear. 5 We have recently shown that peroxynitrite (PN), 6 an oxidant formed in the down-regulation of • NO, 7 can nitrosate phenol 8 in a CO 2 -dependent pathway. The nitrosating species was suggested to be a nitrosonium ion (NO + ) carrier X-NdO (where X can be -ONO, -ONO 2 , -O 2 NO 2 , or -OCO 2 -) or the free radicals CO 3 •-and • NO, all 8 of which can be formed from the reactions of PN 9 and its adduct with CO 2 , ONOOCO 2 -10 (Scheme 1). We here present unequivocal evidence for nitrosation of nucleophiles by PN/CO 2 based on the oxidation of 1,2-phenylenediamine (4), which gives up to 20 mol % yield of 1,2,3-benzotriazole (9). The inhibitory effects of azide support a free radical mechanism for the reaction.A characteristic probe for nitrosation involves the reaction of a vicinal diamine such as 4 or 2,3-diaminonaphthalene 11 to give a triazole (9), formed as a result of an intramolecular nucleophilic displacement on the diazo hydroxide (8) by the neighboring amino group (Scheme 2). Reactions of this type give quantitative yields of nitrosation under mildly acidic, neutral, or even somewhat alkaline conditions. Figure 1A shows the typical product profile for the reaction of PN with 4 in the presence of trace amounts of CO 2 (curve a). One of the major products of this reaction, 12 which elutes with a retention time of 8.0 min, has been identified as 9 based on coelution with authentic 1,2,3-benzotriazole ( Figure 1A, curve b) as well as GC/MS/EI analysis giving ions at m/z 119, 91, and 64, corresponding to M + (C 6 H 5 N 3 •+ ), and fragmentation to C 6 H 5 N •+ (loss of N 2 ) and C 5 H 4 •+ (further loss of HCN).The pH profile of the yields of 9 in the PN/CO 2 /4 system parallels the formation of NO 2 -(in the absence of 4) ( Figure 1B), confirming 8-10 that the nitrosation reaction is mechanistically related to the pathways that produce NO 2 -(Scheme 1, eqs e-h). Like most oxidation 13 and nitration 7,14 reactions mediated by PN/ CO 2 , the nitrosation reaction levels off with a maximum yield that is only about 0.2 mol/mol of PN used ( Figure 1B). 15 This confirms the existence of competing steps 8,10,13,14 in which PN Lippton, H.;Edwards, J. C.;Baricos, W. H.;Hyman, A. L.;Kadowitz, P. J.; Greutter, C. A. J. Pharmacol. Exp. Ther. 1981, 218, 739. (3) Stamler, J. S.; Jaraki, O.; Osborne, J.; Simon, D. I.; Keaney, J.; Vita, J.; Singel, D.; Valeri, C. R.; Loscalzo, J. . van der Vliet, A.; Hoen, P. A. C.; Wong, P. S.-Y.; Bast, A.; Cross, C. E. J. Biol. Chem. 1998, 273, 30255.(6) The term PN refers to the sum of PN anion (ONOO -) and its acid, ONOOH (...

Biological chemistry of reactive oxygen and nitrogen and radiation-induced signal transduction mechanisms

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Performance of diamino fluorophores for the localization of sources and targets of nitric oxide

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“…These new findings add to the growing body of knowledge on how to best optimize their use [9,10] and avoid potential pitfalls [62][63][64][65][66], which will ultimately help investigators to use this relatively new class of fluorophores more judiciously for the investigation of NO-related events.…”

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“…33 These data can justify also the strong protection effect, almost against free radicals, of MLT on cell membranes, that have been reported. [4][5][6][7][8] Infrared spectra on the solid complex demonstrated that in the formation of inclusion complexes an important contribution is due to polar bonds formation-modification, thus evidencing once more 33 that MLT is preferentially localised in interfacial nano-districts, between polar and apolar domains. In these interfacial regions MLT can strongly act as protective agent.…”

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