9738
J. Am. Chem. Soc. 1999, 121, 9738-9739
Scheme 1
Nitrosation of 1,2-Phenylenediamine by Peroxynitrite/
CO2: Evidence for a Free Radical Mechanism
Rao M. Uppu* and William A. Pryor
The Biodynamics Institute, 711 Choppin Hall
Louisiana State UniVersity
Baton Rouge, Louisiana 70803
ReceiVed June 22, 1999
Nitric oxide and S-nitrosothiols function as critical signaling
•
species.1 S-Nitrosothiols have a longer half-life2 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
phenol8 in a CO2-dependent pathway. The nitrosating species was
suggested to be a nitrosonium ion (NO+) carrier X-NdO (where
X can be -ONO, -ONO2, -O2NO2, or -OCO2-) or the free
Scheme 2
•-
•
radicals CO3 and NO, all8 of which can be formed from the
reactions of PN9 and its adduct with CO2, ONOOCO2-10 (Scheme
1). We here present unequivocal evidence for nitrosation of
nucleophiles by PN/CO2 based on the oxidation of 1,2-phenylene-
diamine (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-diaminonaphthalene11 to give
a triazole (9), formed as a result of an intramolecular nucleophilic
displacement on the diazo hydroxide (8) by the neighboring amino
* To whom correspondence should be addressed. Telephone: (225) 388-
(1) Ignarro, J. L. Annu. ReV. Pharmacol. Toxicol. 1990, 30, 535. Moncada,
S.; Palmer, R. M. J.; Higgs, E. A. Pharmacol. ReV. 1991, 43, 109. Bredt, D.
S.; Snyder, S. H. Neuron 1992, 8, 3. Jia, L.; Bonaventura, C.; Bonaventura,
J.; Stamler, J. S. Nature 1996, 380, 221.
(2) Ignarro, L. J.; 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. Proc. Natl. Acad. Sci. U.S.A. 1992,
89, 7674.
(4) Pryor, W. A.; Church, D. F.; Govindan, C. K.; Crank, G. J. Org. Chem.
1982, 47, 156.
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
CO2 (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+ (C6H5N3•+), and fragmentation
(5) Wink, D. A.; Darbyshire, J. F.; Nims, R. W.; Saavedra, J. E.; Ford, P.
C. Chem. Res. Toxicol. 1993, 6, 23. Lewis, R. S.; Tannenbaum, S. R.; Deen,
W. M. J. Am. Chem. Soc. 1995, 117, 3933. Goldstein, S.; Czapski, G. J. Am.
Chem. Soc. 1996, 118, 3419. 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 (pKa 6.8).
to C6H5N•+ (loss of N2) and C5H4 (further loss of HCN).
•+
The pH profile of the yields of 9 in the PN/CO2/4 system
parallels the formation of NO2- (in the absence of 4) (Figure 1B),
confirming8-10 that the nitrosation reaction is mechanistically
related to the pathways that produce NO2- (Scheme 1, eqs e-h).
Like most oxidation13 and nitration7,14 reactions mediated by PN/
CO2, 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 steps8,10,13,14 in which PN
(7) •NO decomposes in vivo primarily through the reactions with O2
•-
and oxyhemoglobin, both reactions being extremely rapid and giving PN,
which is then trapped by the reaction with CO2. For example, see: Beckman,
J. S.; Koppenol, W. H. Am. J. Physiol. 1996, 271, C1424. Radi, R. Chem.
Res. Toxicol. 1996, 9, 828. Lymar, S. V.; Hurst, J. K. J. Am. Chem. Soc.
1995, 117, 8867. Uppu, R. M.; Squadrito, G. L.; Pryor, W. A. Arch. Biochem.
Biophys. 1996, 327, 335. Houk, K. N.; Condroski, K. R.; Pryor, W. A. J. Am.
Chem. Soc. 1996, 118, 13002. Squadrito, G. L.; Pryor, W. A. Free Radical
Biol. Med. 1998, 11, 718.
(8) Uppu, R. M.; Lemercier, J.-N.; Squadrito, G. L.; Zhang, H.; Bolzan,
R. M.; Pryor, W. A. Arch. Biochem. Biophys. 1998, 358, 1.
(9) Mere´nyi, G.; Lind, J. Chem. Res. Toxicol. 1998, 11, 243. Pfeiffer, S.;
Gorren, A. C. F.; Schmidt, K.; Werner, E. R.; Hansert, B.; Bohle, D. S.; Mayer,
B. J. Biol. Chem. 1997, 272, 3465. Coddington, J. W.; Hurst, J. K.; Lymar,
S. V. J. Am. Chem. Soc. 1999, 121, 2438.
(10) Goldstein, S.; Czapski, G. J. Am. Chem. Soc. 1998, 120, 3458. Bonini,
M. G.; Radi, R.; Ferrer-Sueta, G.; Ferreira, A. M. D. C.; Augusto, O. J. Biol.
Chem. 1999, 274, 10802.
(11) Misko, T. P.; Schilling, R. J.,; Salvemini, D.; Moore, W. M.; Currie,
M. G. Anal. Biochem. 1993, 214, 11.
(12) The other product, 11 (Figure 1A), coelutes with authentic 2,3-
diaminophenazine.
(13) Goldstein, S.; Czapski, G. Inorg. Chem. 1997, 36, 5113. Lymar, S.
V.; Hurst, J. K. Inorg. Chem. 1998, 37, 294. Hodges, G. R.; Ingold, K. U.,
personal communication.
(14) Uppu. R. M.; Pryor, W. A. Biochem. Biophys. Res. Commun. 1996,
229, 764. Lymar, S. V.; Jiang, Q.; Hurst, J. K. Biochemistry 1996, 35, 7855.
Lemercier, J.-N.; Padmaja, S.; Cueto, R.; Squadrito, G. L.; Uppu, R. M.; Pryor,
W. A. Arch. Biochem. Biophys. 1997, 345, 160. Zhang, H.; Squadrito, G. L.;
Pryor, W. A. Nitric Oxide: Biol. Chem. 1997, 1, 301.
10.1021/ja9921165 CCC: $18.00 © 1999 American Chemical Society
Published on Web 10/01/1999