Nitration and hydroxylation of phenolic compounds by peroxynitrite

Article abstract of DOI:10.1021/tx950135w

The kinetics and products of the reaction of peroxynitrite with the phenolic compounds phenol, tyrosine, and salicylate were studied as a function of pH. All reactions are first-order in peroxynitrite and zero- order in the phenolic compound. Relative to

Full text of DOI:10.1021/tx950135w

232
Chem. Res. Toxicol. 1996, 9, 232-240
Nitr a tion a n d Hyd r oxyla tion of P h en olic Com p ou n d s by
P er oxyn itr ite
Merrikh S. Ramezanian,† Sarojini Padmaja,† and Willem H. Koppenol*,†,‡
Department of Chemistry, Louisiana State University, Baton Rouge, Louisiana 70803, and Institut
fu¨r Anorganische Chemie, Eidgeno¨ssische Technische Hochschule, CH-8092 Zu¨rich, Switzerland
Received August 1, 1995X
The kinetics and products of the reaction of peroxynitrite with the phenolic compounds phenol,
tyrosine, and salicylate were studied as a function of pH. All reactions are first-order in
peroxynitrite and zero-order in the phenolic compound. Relative to the hydroxyl group,
electrophilic substitution in the 2- and 4-positions (if available) leads to hydroxylated and
nitrated products. The total yield of the products is proportional to the concentration of
peroxynitrite. The sum of the rates of hydroxylation and nitration of phenol, determined by
the stopped-flow technique, is approximately equal to the rate constant for the isomerization
of peroxynitrite to nitrate. The rate vs pH profiles of the nitration and hydroxylation reactions
parallel the yield vs pH profile with nitration maxima at pH 1.8 and 6.8, while hydroxylation
is dominant between these two pH values. The activation energies for both hydroxylation
and nitration are 18.8 ( 0.3 kcal mol-1, identical to that of the isomerization of peroxynitrite
to nitrate. Ethanol decreases the yield of hydroxylation, but has less effect on the nitration.
The rate of reaction in the presence of metal complexes is first-order in metal complex and
peroxynitrite and zero-order in the phenolic compound. The enhancement of the nitration of
phenol by Fe(III)-edta and -nta is pH-dependent, with a maximum near pH 7, while
Fe(III)-citrate, Cu(II)-edta, and CuSO4 affect the nitration much less. The second-order rate
constants for Fe(III)-edta at pH 4.8 and 7.2 are 1.4 × 103 and 5.5 × 103 M-1 s-1, respectively,
at 25 °C. The activation energies for the nitration reaction in the presence of Fe(III)-edta
are 11.5 and 12.2 kcal mol-1 at pH 4.8 and 7.2, respectively. The nitration of tyrosine and
salicylate by peroxynitrite is maximally enhanced by Fe(III)-edta.
powerful oxidants (8) that damage biological compounds
(8, 11-14). The reaction of hydrogen oxoperoxonitrate
with aromatic compounds produces both nitrated and
hydroxylated products. These reactions proceed as fast
as the isomerization to nitrate and do not depend on the
concentration of the aromatic compound. As will be
shown here, these reactions have the same activation
energy as the isomerization to nitrate, 18 kcal mol-1. In
contrast, oxoperoxonitrate(1-) oxidizes methionine (15),
sulfhydryls (16), and ascorbate (17) in second-order
reactions. Given the respective bimolecular rate con-
stants, these compounds have to be present in the
millimolar range in order to compete with the first-order
isomerization reaction of oxoperoxonitrate(1-). At lower
concentrations, these compounds can be expected to
undergo one-electron oxidations by the same intermedi-
ate that is responsible for the nitration and hydroxylation
of phenolic compounds and that, in the absence of a
substrate, would form nitrate. Indeed, one-electron
oxidations of ascorbate (17) and methionine (15) have
been observed.
In tr od u ction
Nitrogen monoxide (1) has been identified as a biologi-
cally important molecule involved in a number of physi-
ological processes, including relaxation of vascular smooth
muscle, neurotransmission, platelet inhibition, and im-
mune regulation (2-4). One of the important reactions
of nitrogen monoxide is that with superoxide to form the
toxic compound peroxynitrite [oxoperoxonitrate(1-),
OdNOO-] (1) (eq 1). The rate constant for this reaction
is close to diffusion-controlled, 6.7 × 109 (5):
•NO + O2•- f OdNOO-
(1)
Oxoperoxonitrate(1-) is a relatively stable species, but
its protonated form, OdNOOH, readily isomerizes (6, 7)
to NO3- and H+ at a rate of 1.3 s-1 at 25 °C (8). Raman
studies indicate (9) that the oxoperoxonitrate(1-) anion
is present in alkaline solutions in the cis form. The
isomerization to nitrate is thought to require a confor-
mational change to the trans form (8-10). Most higher
level ab initio calculations place the trans forms of the
acid and the anion 1 and 3 kcal above the respective cis
forms; the activation energy for interconversion between
these two forms is considerable, approximately 12-15
kcal mol-1 for the acid and 21-24 kcal mol-1 for the anion
(9). Both hydrogen oxoperoxonitrate and its anion are
The first evidence for the nitration of aromatic com-
pounds by peroxynitrite comes from the work of Tri-
fononow (18). He devised a test for nitrite in which
aniline and hydrogen peroxide were added to the solution
to be analyzed, followed by acidification; the yellow color
of nitroaniline indicated nitrite. Mutatis mutandis, he
developed tests for hydrogen peroxide and aromatic
compounds (18). In 1952, Halfpenny and Robinson (19,
20) reported the nitration and hydroxylation of aromatic
compounds by “pernitrous acid” (hydrogen oxoperoxoni-
trate) at pH 1.4 and proposed a radical mechanism for
these reactions. Beckman and co-workers showed that
* Author to whom correspondence should be addressed: telephone,
41-1-632-2875; fax, 41-1-632-1090; e-mail, koppenol@inorg.chem.ethz.ch.
† Louisiana State University.
‡ Eidgeno¨ssische Technische Hochschule.
X Abstract published in Advance ACS Abstracts, December 1, 1995.
0893-228x/96/2709-0232$12.00/0 © 1996 American Chemical Society
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Reactivity of Peroxynitrous Acid
Chem. Res. Toxicol., Vol. 9, No. 1, 1996 233
standards. The retention times, relative to phenol, of 4-hydroxy-
phenol (hydroquinone), 2-hydroxyphenol (catechol), 4-nitrophe-
nol, and 2-nitrophenol are 0.45, 0.61, 1.71, and 2.37, respectively.
Removal of solvent refers to the use of a rotary evaporator
operating at aspirator pressure. Mass spectra were obtained
4-hydroxyphenylacetate, a tyrosine analog, is nitrated
most efficiently at pH 7.5 (21). Van der Vliet et al. (22,
23) studied the reaction of phenylalanine and tyrosine
with peroxynitrite and concluded that hydroxylation and
nitration are caused by hydroxyl and nitrogen dioxide
radicals obtained from homolysis of hydrogen oxoperox-
onitrate. However, Koppenol et al. (8) used thermody-
namic calculations and kinetic considerations to show
that hydrogen oxoperoxonitrate is unlikely to yield free
hydroxyl radical and nitrogen dioxide and proposed that
OdNOOH itself is a strongly oxidizing agent. The
oxidizing species is believed to be an intermediate closely
related to the transition state for the isomerization of
hydrogen oxoperoxonitrate to nitrate (8).
with
a Hewlett-Packard 5971A GC-MS. Products of the
reaction of oxoperoxonitrate(1-) with tyrosine were detected at
280 nm, like those of phenol, and those of salicylate were
detected at 310 nm. The mobile phase consisted of a 30 mM
sodium citrate and 27 mM sodium acetate solution in water,
adjusted to pH 5.1 for tyrosine or to pH 4.7 for salicylate with
1 M H2SO4. 2,5-Dihydroxy- and 2,3-dihydroxybenzoate elute
within 10 min with this mobile phase. After 10 min, the mobile
phase was gradually changed to 40% acetonitrile, which resulted
in the elution of 2-hydroxy-5-nitrobenzoate within 20 min.
Kin etics. We studied the kinetics of the reactions with an
OLIS stopped-flow instrument equipped with an OLIS-RSM
1000 rapid scanning monochromator (OLIS, Bogart, GA). With
the help of this instrument, 1000 spectra are collected per
second. The decay of oxoperoxonitrate(1-) was monitored from
250 to 350 nm. Rates of hydroxylation were monitored at 290
nm and rates of nitration at 380 nm. During all kinetic
experiments the phenolic compound was present in 5-10-fold
excess. The kinetics of metal-enhanced nitrations were followed
by monitoring the buildup of nitrophenol at 380 nm, because
the iron(III) complexes absorb strongly in the region 280-340
nm. The data were analyzed by OLIS RSM 1000 global fit
software. Results from six experiments were averaged to obtain
each rate constant. Temperatures were maintained to (1 °C
with a VWR Model 1160 circulating water bath.
Complexes of transition metals such as iron and copper
catalyze the formation of oxyradicals, and low molecular
weight complexes of iron may be present in vivo (24-
26). Such complexes may enhance the nitration of
phenolic compounds. In fact, Fe(III)-edta enhances the
nitration of 4-hydroxyphenylacetate quite effectively (21).
Furthermore, bovine Cu,Zn superoxide dismutase carries
out a similar reaction, which results in the modification
of its tyrosine 108 to 3-nitrotyrosine (27, 28). Removal
of the copper from the Cu,Zn superoxide dismutase
prevented this reaction. The chemistry of peroxynitrite
has recently been reviewed (29, 30).
With the stopped-flow technique, we have been able
to directly measure the formation of nitrated and hy-
droxylated products from phenol, tyrosine, and salicylate.
Here we report on the kinetics and products of these
reactions in the presence and absence of iron and copper
complexes.
Rea ction P r od u cts of Oxop er oxon itr a te(1-) w ith P h e-
n ol. Oxoperoxonitrate(1-) was added dropwise (final concen-
tration if unreacted: 0.87 mM) to a solution containing excess
phenol (5.0 mM), dtpa (0.2 mM), and a buffer solution (0.25 M)
with stirring for 30 min at room temperature. Experiments in
the presence of ethanol were carried out as follows. Oxo-
peroxonitrate(1-) (final concentration if unreacted: 3.0 mM)
was added dropwise to a mixture of phenol (5.0 mM), phosphate
buffer (125 mM), dtpa (0.2 mM), and ethanol (0.0-0.7 M) with
stirring. The final pH was 6.8. The yield of p-hydroxyphenol
decreased from 3% to 1.5% in the presence of 0.7 M ethanol.
Similarly, the yields of o-hydroxyphenol (0.2% f 0.0%), p-
nitrophenol (2.2% f 1.5%), and o-nitrophenol (3.0% f 2.2%)
decreased as indicated. The total yield of reaction products,
relative to oxoperoxonitrate(1-), decreased from 8% to 5% at
0.7 M ethanol. Preparative HPLC resulted in four different
products. These were extracted with ether, dried over magne-
sium sulfate, and concentrated in a rotary evaporator. Three
of the products (Rf values of 0.45, 0.61, and 1.71) were deriva-
tized with bstfa before GC-MS spectra were recorded.
Exp er im en ta l P r oced u r es
Gen er a l. Oxoperoxonitrate(1-) was prepared by ozonizing
a solution of sodium azide in sodium hydroxide (31, 32). The
concentration of oxoperoxonitrate(1-) was determined with a
Beckman (Irvine, CA) DU-7HS spectrophotometer at 302 nm
(ꢀ ) 1670 ( 50 M-1 cm-1) (33). Phenol was obtained from Sigma
Chemical Co. (St. Louis, MO), and sodium azide was from EM
Science (Gibbstown, NJ ). The pH of the solutions was adjusted
with hydrochloric acid (pH 1.0-2.5), acetate buffer (pH 3.6-
5.6), phosphate buffer (pH 6.0-8.0), and glycine/sodium hy-
droxide buffer (pH 8.0-10.0). Deionized water was used for
preparing solutions. The metal complexes Fe(III)-edta1 and
Cu(II)-edta were prepared by mixing solutions of the metal ions
and edta in a 1:1.1 ratio, Fe(III)-citrate was prepared by adding
citric acid in 4-fold excess, and Fe(III)-nta was prepared in a
1:2 ratio. In experiments with Fe(III)-atp and Fe(III)-adp
involving tyrosine, the ligand was present in 4-fold excess.
Where indicated, the excess ligand was necessary to ensure
complete complexation of iron(III).
An a lysis of Rea ction P r od u cts. Reaction products were
identified and quantified by HPLC and GC-MS. We used a
Milton Roy (presently Thermo Separation Products, Riviera
Beach, FL) CM 4000 liquid chromatograph with an Alltech
(Deerfield, IL) Microsphere 300 C18 250 × 4.6 mm reverse-
phase column. The flow rate was 1 mL per minute. Hydroxyl-
ated products derived from phenol and tyrosine were detected
at 280 nm. In the case of phenol, the mobile phase consisted of
a 30 mM sodium citrate and 27 mM sodium acetate solution in
water with acetonitrile (80:20), adjusted to pH 3.2 with 1 M
H2SO4. Peaks were identified on the basis of coelution and mass
spectrometry and quantified by peak area using external
Rea ction of Oxop er oxon itr a te(1-) w ith P h en ol in th e
P r esen ce of Meta l Com p lexes. Oxoperoxonitrate(1-) was
added dropwise (final concentration if unreacted: 0.9 mM) to a
solution of phenol (5.0 mM), metal complex (0.0-2.5 mM), and
a buffer solution (0.08 M) with stirring for 30 min at room
temperature.
Rea ction of Oxop er oxon itr a te(1-) w ith Tyr osin e. Oxo-
peroxonitrate(1-) was added dropwise (final concentration: 0.74
mM) to a solution containing tyrosine (1.2 mM), dtpa (0.2 mM),
and a buffer solution (0.13 M) with stirring for 30 min at room
temperature.
Rea ction of Oxop er oxon itr a te(1-) w ith Tyr osin e in th e
P r esen ce of Meta l Com p lexes. Oxoperoxonitrate(1-) was
added dropwise (final concentration if unreacted: 0.8 mM) to a
solution of tyrosine (1.2 mM) and metal complex (0.0-0.55 mM)
at pH 7.3 ( 0.3 with stirring for 30 min at room temperature.
The concentration of the phosphate buffer was 0.16 M.
Rea ction of Oxop er oxon itr a te(1-) w ith Sa licyla te. Oxo-
peroxonitrate(1-) was added dropwise (final concentration if
unreacted: 8.0 mM) to a solution containing salicylate (10.0
mM), dtpa (0.2 mM), and a buffer solution (0.1 M) with stirring
for 30 min at room temperature.
1
Abbreviations: adp, adenosine 5′-diphosphate anion; atp, adenos-
ine 5′-triphosphate anion; bstfa, N,O-bis(trimethylsilyl)trifluoroac-
etamide; dtpa, [[(carboxymethyl)imino]bis(1,2-ethanediylnitrilo)]tet-
raacetate anion; edta, 1,2-ethylenedinitrilotetraacetate anion; nta,
nitrilotriacetate.
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234 Chem. Res. Toxicol., Vol. 9, No. 1, 1996
Ramezanian et al.
F igu r e 1. Arrhenius plot for the reaction of oxoperoxonitrate-
(1-) with phenol at pH 7.2: [ONOO-] ) 0.5 mM, [phenol] )
5.0 mM, and a buffer concentration of 50 mM.
F igu r e 2. Rate of nitration (b) and hydroxylation (3) vs pH
for the reaction of oxoperoxonitrate(1-) with phenol at 298 K:
[ONOO-] ) 0.5 mM, [phenol] ) 5.0 mM, and a buffer concentra-
tion of 50 mM.
R ea ct ion of Oxop er oxon it r a t e(1-) w it h Sa licyla t e in
th e P r esen ce of Meta l Com p lexes. Oxoperoxonitrate(1-)
was added dropwise (final concentration if unreacted: 6.2 mM)
to a solution of salicylate (8.0 mM), metal complex (0.0-8.0 mM),
and phosphate buffer (0.1 M) at pH 6.5 ( 0.3 with stirring for
30 min at room temperature.
Resu lts
Kin etics of P h en ol. The reaction of oxoperoxoni-
trate(1-) with phenol was studied at pH 4.0 and pH 7.2
by monitoring the buildup of nitrophenol at 380 nm and
that of hydroxyphenol at 290 nm. The rate of the
reaction is first-order in oxoperoxonitrate(1-) and inde-
pendent of phenol concentration over the entire concen-
tration range of 5.0-25.0 mM. The effect of temperature
was studied by monitoring the buildup of nitrophenol at
pH 7.2 over the temperature range 283-323 K. The
activation energy calculated from the slope of the Arrhe-
nius plot (Figure 1) is 18.8 ( 0.3 kcal mol-1, which is
identical to that of the isomerization to nitrate. The
activation energy of the hydroxylation reaction, deter-
mined in a similar fashion, is the same within the error.
F igu r e 3. Plot of k vs concentration of Fe(III)-edta for the
reaction of ONOO- with phenol at various temperatures.
Conditions: [ONOO-] ) 0.5 mM; [phenol] ) 5.0 mM; buffer
concentration 50 mM (pH ) 4.8). Symbols: b, 288 K; 3, 298 K;
1, 308 K; 0, 318 K.
+d[hydroxyphenol]/dt ) kh[OdNOOH]
+d[nitrophenol]/dt ) kn[OdNOOH]
(3)
(4)
At all pH values, the sum of the rate constants of
hydroxylation and nitration is close to the rate constant
for the isomerization of hydrogen oxoperoxonitrate to
nitrate. A rate law to express the observed rate data is
given in eq 2:
Thus, the overall rate constant k is equal to the sum of
kh and kn and is close to the rate of isomerization of
hydrogen oxoperoxonitrate at that pH. Above pH 8.0,
the rate of nitration is essentially zero.
-d[OdNOOH]/dt ) k[OdNOOH]1[phenol]0 (2)
Kin etics of P h en ol in th e P r esen ce of Meta l
Com p lexes. The rate of nitration is first-order in metal
complex and in oxoperoxonitrate(1-), but zero-order in
phenol. Figure 3 shows the dependence of the pseudo-
first-order rate constant (kobs) on the metal complex
concentration at pH 4.8 at different temperatures. The
rate constants are calculated from the slopes of the lines
(Figure 3) at pH 4.8 and 7.2 (Table 1). The activation
energies are 11.5 and 12.2 kcal mol-1 at pH 4.8 and 7.2,
respectively.
The pH dependence of the rate constant in the presence
of Fe(III)-edta is shown in Figure 4. The rate of
nitration is maximal at pH 7.0 ( 0.2. The rate constants
at room temperature in the presence of other Fe(III) and
Cu(II) complexes are shown in Table 2. Clearly, all of
these complexes affect the rate of nitration, even though
Hydrogen oxoperoxonitrate has a pKa of 6.8 at 25 °C (8).
Below pH 3.0, the rate of isomerization of hydrogen
oxoperoxonitrate increases (34). The variation in the
rates of hydroxylation and nitration with pH at 25 °C is
shown in Figure 2. Over the pH range 1.0-10.0, the rate
of nitration is maximal at pH 1.5 and 6.8, with a
minimum in between. In the pH range 1.5-3.5, no
hydroxylated products are observed by UV detection in
the stopped flow (Figure 2), although product analysis
showed that some are formed. Monitoring of these
products was difficult due to the absorption of hydrogen
oxoperoxonitrate and the weak absorption of the hydrox-
yphenols. The rate of hydroxylation is maximal at pH
4.8. Beyond pH 4.8 the rate decreased, and no product
buildup was observed above pH 8.0. The rate equations
to explain the observed data are given in
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Reactivity of Peroxynitrous Acid
Chem. Res. Toxicol., Vol. 9, No. 1, 1996 235
F igu r e 4. (b) Rate constant vs pH for the reaction of ONOO-
with phenol in the presence of Fe(III)-edta. Conditions: [ONOO-]
) 0.5 mM; [phenol] ) 5.0 mM; [Fe(III)-edta] ) 0.1-0.5 mM;
buffer concentration 50 mM. (1) Yield vs pH phenol in the
presence of Fe(III)-edta. Conditions: [phenol] ) 5.0 mM;
[ONOO-] ) 0.8 mM; [Fe(III)-edta] ) 4.0 mM; buffer concentra-
tion 125 mM.
F igu r e 5. Yield of hydroxylated and nitrated products vs pH
for the reaction of oxoperoxonitrate(1-) with phenol: 1, o-
hydroxyphenol; 3, p-hydroxyphenol; b, o-nitrophenol; O, p-
nitrophenol. Conditions: [ONOO-] ) 0.8 mM; [phenol] ) 5.0
mM; buffer concentration 125 mM.
Ta ble 1. Secon d -Or d er Ra te Con sta n ts for th e Rea ction
of Oxop er oxon itr a te(1-) w ith P h en ol in th e P r esen ce of
F e(III)-ed ta a t p H 4.8 a n d 7.2
k (M-1 s-1
)
T (K)
pH 4.8
pH 7.2
288
298
308
318
(5.5 ( 0.1) × 102
(1.4 ( 0.1) × 103
(2.5 ( 0.2) × 103
(4.8 ( 0.2) × 103
(2.9 ( 0.2) × 103
(5.5 ( 0.1) × 103
(1.2 ( 0.3) × 104
(1.7 ( 0.5) × 104
Ta ble 2. Ra te Con sta n ts for th e Rea ction of
Oxop er oxon itr a te(1-) P h en ol in th e P r esen ce of Va r iou s
Meta l Com p lexes a t p H 7.2
metal complexes
k298 (M-1 s-1
)
Fe(III)-edta
Fe(III)-nta
Cu(II)-edta
CuSO4
(5.5 ( 0.1) × 103
(1.5 ( 0.1) × 103
(9.3 ( 0.1) × 102
(6.5 ( 0.1) × 102
F igu r e 6. Yield of hydroxylated and nitrated products vs
concentration of ONOO- for the reaction of oxoperoxonitrate-
(1-) with phenol at pH 6.8 ( 0.2. Conditions: [phenol] ) 5.0
mM; buffer concentration 125 mM. Symbols: 0, total yield; 1,
o-hydroxyphenol; 3, p-hydroxyphenol; b, o-nitrophenol; O, p-
nitrophenol.
they behave differently. Fe(III)-edta and Fe(III)-nta
enhance the rate without showing any saturation at pH
7.0 ( 0.2. The influence of Fe(III)-citrate on the rate of
the reaction at pH 7.0 ( 0.2 is very small compared to
other complexes. A control experiment was carried out
at pH 7.0. A known amount of Fe(III)-edta and phenol
in one syringe of the stopped flow was mixed with
oxoperoxonitrate(1-) in the other syringe. The rate of
the reaction was determined. The reactants were also
mixed outside the stopped flow, and the stopped-flow
experiment was repeated by reacting oxoperoxonitrate-
(1-) with this solution. The pseudo-first-order rate
constant was smaller. By repeating this experiment we
observed that the pseudo-first-order rate constant de-
creased continuously and reached the same value as that
of the decay of oxoperoxonitrate(1-) in the absence of
metal complexes.
P r od u cts of P h en ol. Products of the reaction of
phenol with oxoperoxonitrate(1-) in the pH range 1.0-
10.0 were determined by HPLC and mass spectrometry.
The mass spectrum of the derivatized sample with Rf )
0.45 yields peaks at m/ z 254 (M•+), 239 (M - CH3•+), and
73 [(Si(CH3)3•+]. Rf ) 0.61: m/ z 254 (M•+), 239 (M -
CH3•+), 91 (C7H7•+), 77 (C6H5•+), and 73 [Si(CH3)3•+]. Rf
) 1.71: m/ z 211 (M•+), 196 (M - CH3•+), 91 (C7H7•+), 77
(C6H5•+), and 73 [Si(CH3)3•+]. The mass spectrum of the
underivatized sample with Rf ) 2.37 yields peaks at m/ z
139 (M•+), 109(M - NO•+), 93 (M - NO2•+), 81 [M - (NO,-
CO)•+], 78 (C6H6•+), and 65 (C5H5•+). The following
compounds were identified: p-hydroxyphenol (Rf ) 0.45),
o-hydroxyphenol (Rf ) 0.61), p-nitrophenol (Rf, ) 1.71),
and o-nitrophenol (Rf ) 2.37).
The yield of o-hydroxyphenol increases rapidly with
increasing pH to a maximum near pH 4.0, followed by a
decrease and another rise above pH 8.0 (Figure 5). o-
and p-nitrophenol show maxima near pH 2.0 and 6.6,
while little is formed near pH 4.7. Formation of p-
hydroxyphenol is maximal around pH 5.0, while above
pH 9.0 only trace amounts are observed. o-Hydroxyphe-
nol appears to be the only product formed at alkaline pH.
The maximal yield of reaction products is 24% near pH
2.0, relative to oxoperoxonitrate(1-) (Figure 6). Around
this pH the yields of nitrated and hydroxylated products
are similar. Overall, the yields of o-hydroxyphenol and
o-nitrophenol are greater than those of para-substituted
compounds (Figure 5). Beckman et al. (21) reported
maximal nitration of 4-hydroxyphenylacetate at pH 7.4.
Because the phosphate concentration in their experi-
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236 Chem. Res. Toxicol., Vol. 9, No. 1, 1996
Ramezanian et al.
F igu r e 7. Total yield of hydroxylated and nitrated products
vs concentration of ethanol for the reaction of oxoperoxonitrate-
(1-) with phenol at pH 6.8 ( 0.2. Conditions: [ONOO-] ) 3.0
mM; [phenol] ) 5.0 mM; buffer concentration 125 mM. Sym-
bols: b, o- and p-nitrophenol; O, o- and p-hydroxyphenol.
F igu r e 8. Yield of nitrophenol as a function of the concentra-
tion of metal complex for the reaction of ONOO- with phenol.
Symbols: O, Fe(III)-edta; 0, Fe(III)-nta; b, CuIISO4; 3, Cu-
(II)-edta. Conditions: pH 7.0 ( 0.2; [phenol] ) 5.0 mM;
[ONOO-] ) 0.8 mM. The buffer concentration was 125 mM.
ments was lower than that in ours, we varied the
phosphate concentration from 50 to 250 mM. However,
no change in the pH maximum was observed (not shown).
The effect of the concentration of oxoperoxonitrate(1-)
on the total yield of products is shown in Figure 6.
Generally, the yield increases linearly with the oxoper-
oxonitrate(1-) concentration. However, the yield of o-
hydroxyphenol decreases with increasing concentrations
of oxoperoxonitrate(1-). It is possible that o-hydrox-
yphenol is further oxidized by oxoperoxonitrate(1-). The
ratio of o-nitrophenol to p-nitrophenol (1.4:1) remained
the same throughout all experiments.
Figure 7 presents the effect of ethanol on the yield of
products at pH 6.8 ( 0.2. The yield of o- and p-
hydroxyphenol decreases with increasing ethanol con-
centration. At concentrations of ethanol greater than 0.4
M, only trace amounts of o-hydroxyphenol are formed.
Formation of nitrophenol products also decreases, but the
effect of ethanol on hydroxylation is much greater than
that on nitration.
Since nitrate is the product of isomerization of oxop-
eroxonitrate(1-), a control experiment was carried out.
Phenol was mixed with nitrate at various pH values
between 1.0 and 7.0, but no nitration was observed.
Similar experiments with nitrite near neutral pH also
yielded negative results. At low oxoperoxonitrate(1-)
concentrations (50-100 µM) and neutral pH, we observed
the formation of small amounts of 4-nitrosophenol. These
findings are currently under investigation.
P r od u ct s of P h en ol in t h e P r esen ce of Met a l
Com p lexes. By coelution the following compounds were
identified: p-hydroxyphenol, o-hydroxyphenol, p-nitro-
phenol, and o-nitrophenol. Nitration as a function of pH
in the presence of Fe(III)-edta is shown in Figure 4. The
yield remains the same from pH 1.0 to 4.0 and then
increases, with a maximum at pH 7.0 ( 0.2. At pH 7.0
( 0.2, when the ratio of oxoperoxonitrate(1-), phenol,
and Fe(III)-edta is approximately 1:1:3.5, 45% of all
phenol is nitrated. The increase in yield of nitrated
products is at the expense of hydroxylated products.
The yield of nitration for the reaction of oxoperoxoni-
trate(1-) with phenol in the presence of other metal
complexes at pH 7.0 ( 0.2 is shown in Figure 8. At this
pH the yields increase with the concentrations of Fe(III)-
nta and Fe(III)-edta (also shown in Figure 8). However,
Fe(III)-citrate, copper sulfate, and Cu(II)-edta have
little effect.
F igu r e 9. First-order rate constant vs pH for the reaction of
oxoperoxonitrate(1-) with salicylate to form 2-hydroxy-5-ni-
trobenzoate. Conditions: [ONOO-] ) 1.0 mM; [salicylate] ) 20
mM; buffer concentration 50 mM.
Kin etics of Tyr osin e a n d Sa licyla te. The kinetics
of the reaction of oxoperoxonitrate(1-) with tyrosine
could not be studied due to the limited solubility of
tyrosine. The kinetics of the reaction of oxoperoxonitrate-
(1-) with salicylate was studied at pH 7.1, with the latter
present in excess. The reaction is followed by monitoring
the buildup of the product, 2-hydroxy-5-nitrobenzoate, at
400 nm. The rate of the reaction is independent of the
concentration of salicylate over the entire concentration
range, and the observed first-order rate constant is
approximately the same as that of the decomposition of
oxoperoxonitrate(1-) at that pH.
The pH dependence of the rate of the reaction at room
temperature was studied over the pH range 2.0-9.0. The
rate of nitration is highly pH-dependent. The rate vs pH
curve (Figure 9) shows a maximum around pH 6.0, while
above pH 8.0 the rate of nitration is almost zero. No
hydroxylated product could be detected by UV absorption
during the stopped-flow experiment, and therefore the
rate of hydroxylation could not be measured. This may
be due to the very low yield of hydroxylated products.
P r od u cts of Tyr osin e a n d Sa licyla te. The reaction
of peroxynitrite with salicylate and tyrosine in the pH
range 4.0-9.0 leads to three major products from sali-
cylate, 2,3-dihydroxybenzoate, 2,5-dihydroxybenzoate,
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Reactivity of Peroxynitrous Acid
Chem. Res. Toxicol., Vol. 9, No. 1, 1996 237
F igu r e 12. Yield vs pH for the reaction of oxoperoxonitrate-
(1-) with salicylate. Symbols: 0, total yield; 1, 2-hydroxy-5-
nitrobenzoate; 3, 2,3-dihydroxybenzoate; b, 2,5-dihydroxyben-
zoate. Conditions: [ONOO-] ) 8.0 mM; [salicylate] ) 10.0 mM;
[dtpa] ) 0.2 mM2; buffer concentration 0.10 M.
F igu r e 10. HPLC chromatograms of reaction products of
oxoperoxonitrate(1-) (0.74 mM) with tyrosine (1.2 mM): A,
3-hydroxytyrosine; B, tyrosine; C, 3-nitrotyrosine; D and E,
unidentified compounds; I, pH 4.0-5.0; II, pH 7.0.
F igu r e 11. Yield vs pH for the reaction of oxoperoxonitrate-
(1-) with tyrosine. Conditions: [ONOO-] ) 0.74 mM; [tyrosine]
) 1.2 mM; [dtpa] ) 0.2 mM; buffer concentration 0.13 M.
Symbols: b, 3-hydroxytyrosine; 3, 3-nitrotyrosine.
F igu r e 13. HPLC chromatograms of the reaction products of
oxoperoxonitrate(1-) with tyrosine in the presence of Fe(III)-
edta at pH 7.3 ( 0.3: A, 3-hydroxynityrosine; B, tyrosine; C,
3-nitrotyrosine; D and E, unidentified compounds; I, 0.11 mM
Fe(III)-edta; II, 0.55 mM Fe(III)-edta.
and 2-hydroxy-5-nitrobenzoate, and to two major prod-
ucts from tyrosine, 3-hydroxytyrosine and 3-nitrotyrosine.
all pH’s more nitrated than hydroxylated products were
formed. 2,3-Dihydroxybenzoate is formed between pH
4.0 and 5.0, but not above pH 6.0. The yields of
2-hydroxy-5-nitrobenzoate and 2,5-dihydroxybenzoate
were maximal at pH 6.6 and decreased at higher pH.
P r od u cts of Tyr osin e a n d Sa licyla te in th e P r es-
en ce of Meta l Com p lexes. Transition metal com-
plexes, especially Fe(III)-edta, enhance the nitration of
tyrosine and salicylate by oxoperoxonitrate(1-). Fig-
ure 13 shows the effect of Fe(III)-edta. At 0.11 (Figure
13, panel I) and 0.55 mM (Figure 13, panel II) Fe(III)-
The HPLC chromatogram (Figure 10, panel I) shows
that, between pH 4.0 and 5.0, only 3-hydroxy- and
3-nitrotyrosine are formed. The distribution of products
is pH-dependent: formation of 3-hydroxytyrosine is
maximal at pH 4.2, while 3-nitrotyrosine is formed mostly
at pH 7.4 (Figure 11). At pH greater than 4.2, two
unidentified products (Figure 10, panel II) were also
observed. These unidentified compounds may be isomers
of hydroxy- and nitrotyrosine. No products were detected
above pH 9.0. The effect of pH on the reaction of
salicylate with peroxynitrite is shown in Figure 12. At
+
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238 Chem. Res. Toxicol., Vol. 9, No. 1, 1996
Ramezanian et al.
is, at best, 24% relative to oxoperoxonitrate(1-), the bulk
of this anion isomerizes to nitrate, since the rate of its
disappearance does not depend on the phenol concentra-
tion. Thus, the mechanism of the reaction involves a
rate-limiting conformational change to an intermediate,
which reacts with phenol or isomerizes further to nitrate.
In the pH range 1.0-2.5, where we find an increase in
nitrated products, the isomerization of hydrogen oxop-
eroxonitrate is catalyzed by acid (34) and may involve
the following equilibrium:
OdNOOH + H+ S OdNOOH2+ S NO2+ + H2O (5)
The species OdNOOH2+, or even NO2+, could be
responsible for nitration. Thermodynamic calculations
suggest that heterolytic cleavage cannot occur spontane-
ously, because the energy required to produce an initial
charge separation into NO2+ and HO- in water requires
F igu r e 14. Yield as a function of the metalcomplex concentra-
tion for the reaction of oxoperoxonitrate(1-) with tyrosine.
Symbols: O, Fe(III)-edta; 9, Cu(II)-acetate; 3, Fe(III)-nta;
b, Fe(III)-citrate; 0, Fe(III)-adp; 1, Fe(III)-atp. Conditions:
[ONOO-] ) 0.8 mM; [tyrosine] ) 1.2 mM; pH 7.3 ( 0.3; buffer
concentration 0.16 M.
approximately 45 kcal mol-1
. However, protonation
would reduce this barrier considerably (8). Since at low
pH the reaction is still first-order in oxoperoxonitrate-
(1-) and zero-order in phenol, one must assume that
formation of the nitrating species is rate-limiting. Al-
ternatively, since our oxoperoxonitrate(1-) preparations
contain significant amounts of nitrite (32), it is conceiv-
able that at low pH the phenol is first nitrosated by
nitrous acid and that nitrosophenol is subsequently
oxidized by hydrogen oxoperoxonitrate. We have found
that 4-nitrosophenol is oxidized to 4-nitrophenol by
oxoperoxonitrate(1-).2 However, one would expect to
observe small amounts of nitrosophenol, which we did
not. For this reason, we prefer the first explanation.
In the pH range 4.0-6.0, the rate and yield of hy-
droxylation are higher than the rate of nitration (Figures
4 and 5). Since homolysis of the OsO bond of hydrogen
oxoperoxonitrate is unlikely, we propose that an inter-
mediate structurally similar to the transition state
postulated for the isomerization to nitrate (8) reacts with
phenol. Again, the formation of this intermediate is rate-
limiting. The ratio of o- to p-nitrophenol, 1.4:1, is very
similar to that found during the nitration of phenol in
sulfuric acid, which presumably involves NO2+, the nitryl
cation, and to that observed when phenol is nitrated by
nitrogen dioxide in carbon tetrachloride and cyclohexane
(35). In those cases it is assumed that the nitryl cation
and nitrogen dioxide oxidize phenol to the phenoxyl
radical. The standard one-electron reduction potentials
F igu r e 15. Yield as a function of the metal complex concentra-
tion for the reaction of oxoperoxonitrate(1-) with salicylate.
Symbols: b, Fe(III)-edta; 0, copper sulfate. Conditions:
[ONOO-] ) 6.2 mM; [salicylic acid] ) 8.0 mM; pH 6.5 ( 0.3;
buffer concentration 0.1 M.
edta, the yield of 3-nitrotyrosine increases (compare
Figure 10), while there is almost no effect on the yield of
hydroxylated and unidentified products. 3-Nitrotyrosine
was obtained in 15% yield relative to oxoperoxonitrate-
(1-) when the ratio of oxoperoxonitrate(1-) to tyrosine
to Fe(III)-edta was 1:1.2:5.5. Figure 14 shows the effect
of iron and copper complexes on the reaction of oxoper-
oxonitrate(1-) with tyrosine at pH 7.3 ( 0.3. The yield
of 3-nitrotyrosine increases sharply with increasing
concentrations of Fe(III)-edta. Fe(III)-nta, Cu(II)-
acetate, and Fe(III)-citrate also enhance the yield of
nitrotyrosine, but Fe(III)-atp and Fe(III)-adp have little
effect. Fe(III)-edta and copper sulfate, up to 0.2 mM,
increase the yield of salicylate nitration at pH 6.5 ( 0.3,
as shown in Figure 15. However, at higher concentra-
tions of metal complexes the yield decreases. We have
no explanation for this observation.
•
of the NO2+/NO2• and the NO2 /NO2- couples are 1.6 and
•
0.99 V, respectively, while that of the OdNOOH/NO2
(H2O) couple is estimated to be 1.4 V at pH 7 (8). These
values are valid in water, but they do indicate that
oxoperoxonitrate(1-) is oxidizing enough: the reduction
potential of the phenoxyl/phenol couple is 0.9 V at pH 7
(36). The one-electron reduction of hydrogen oxoperox-
onitrate results in water and nitrogen dioxide; the latter
is likely to react with the phenoxyl radical to form
4-nitrocyclohexa-2,5-dien-1-one and 6-nitrocyclohexa-2,4-
dien-1-one. This reaction is likely to be very fast, in
analogy to the reaction of phenoxyl radicals with other
radicals (37). The conversion to p- and o-nitrophenol is
relatively slow (35), and it is possible that the nitrohexa-
dienone intermediates hydrolyze to form catechol and
hydroquinone. In order to preserve the ratio of o- to
p-nitrophenol, one must assume that the rates of hy-
Discu ssion
P h en ol. Hydrogen oxoperoxonitrate is a nitrating and
hydroxylating agent capable of reacting by multiple
mechanisms, which depend on the pH. Given the total
yield of products, which in the absence of metal complexes
2
M. S. Ramezanian, S. Padmaja, and W. H. Koppenol, Unpublished
results.
+
+

Reactivity of Peroxynitrous Acid
Chem. Res. Toxicol., Vol. 9, No. 1, 1996 239
drolysis of the two nitrohexadienone intermediates are
the same. This mechanism does not suggest a pH
dependence of the rates of hydroxylation and nitration,
as observed. However, it is possible that either the rate
of hydrolysis or the rate of isomerization of the nitro-
hexadienones to nitrophenols is pH-dependent. This
mechanism accounts for the observation that the activa-
tion energies of the hydroxylation and nitration reactions
are identical. While the yields of o- and p-nitrophenol
as a function of pH parallel one another, as discussed
earlier, this is not the case for the hydroxylated prod-
ucts: the yield of p-dihydroxybenzene is maximal at pH
5.5 and that of o-dihydroxybenzene at pH 3.5. One must
assume that both are formed in the same ratio as the
nitrophenols and that, below pH 5.5, p-dihydroxybenzene
disappears through unknown oxidation processes.
As mentioned earlier, we observed a maximum in
nitration at pH 6.6. An explanation for the nitration
maximum has been offered by Beckman et al. (10), based
on the different pKa values of cis- and trans-hydrogen
oxoperoxonitrate. These investigators observed a maxi-
mum for the yield of nitration, not at pH 6.6 but at pH
7.5, in between the pKa values of 6.8 for the cis form and
8.0 for the trans form. Since the maximum we observed
lies below the pKa of the cis form, the hypothesis of
Beckman et al. (10) does not explain our results.
At pH 6.8 ( 0.2 the hydroxyl radical scavenger ethanol
reduced the yield of hydroxyphenol products by 50%, but
that of nitrophenol only by 30%. In the case of the
reaction of oxoperoxonitrate(1-) with phenylalanine, 100
mM mannitol decreased the yield of hydroxylated prod-
ucts by 56% (22). If OdNOOH were to undergo homoly-
sis and form hydroxyl and nitrogen dioxide radicals, and
given the rate constant for the reaction of the hydroxyl
radical with mannitol (k ) 1.7 × 109 M-1 s-1) (38), the
100 mM mannitol should have scavenged all hydroxyl
radicals and the 40% yield of hydroxylated products (22)
should not have been observed. Furthermore, after the
reaction of oxoperoxonitrate(1-) with phenol, neither
biphenol nor any other byproduct resulting from a radical
mechanism was observed. Beckman and co-workers also
oberved that hydroxyl radical scavengers minimally
affected the yield of nitrated products (21).
Beckman et al. (21) in the reaction of oxoperoxonitrate-
(1-) with phenylacetic acid in the presence of the same
metal complex. The metal withdraws electron density
and may promote the formation of a species that reacts
like NO2+. This would be analogous to the protonation
of OdNOOH proposed earlier.
One could argue that these metal complexes are
catalysts, because they lower the activation energy from
18.8 (see above) to 11.5 kcal mol-1. However, we have
also shown that the activity of these metal complexes
decreases upon repeated exposure to oxoperoxonitrate-
(1-). As the heterolysis would leave the iron(III) with a
coordinated hydroxide anion, it is possible that the
formation of (HO-)Fe(III)-L inhibited the activity of the
Fe(III)-edta complex. Given the pKa of the water
molecule in the seven-coordinate H2O-Fe(III)-edta com-
plex (39), this is quite feasible. It is possible that a
coordination site occupied by water is a requirement for
the enhancement of nitration: Fe(III)-edta is seven-
coordinate and active, while Cu(II)-edta is six-coordinate
and shows no activity. The increases in rate and yield
are small for the Cu(II) complexes compared to those for
the Fe(III) complexes.
Tyr osin e a n d Sa licyla te. As observed with phenol,
the reaction of hydrogen oxoperoxonitrate with tyrosine
and salicylate involves at least two intermediates: one
that leads to nitration and a second that results in
hydroxylation. Again, the formation of these intermedi-
ates is rate-limiting. The yield of the reaction of oxop-
eroxonitrate(1-) with salicylate is much smaller than
with phenol and with tyrosine. While salicylate is the
best aromatic scavenger to detect hydroxyl radicals in
vivo (40, 41), the high concentrations required to obtain
detectable yields make this scavenger less valuable for
obtaining evidence for in vivo oxoperoxonitrate(1-) for-
mation. It is also troublesome that the reaction of the
hydroxyl radical and hydrogen oxoperoxonitrate with this
scavenger yield both 2,3- and 2,5-dihydroxybenzoate.
Were it not for the low yield, one could argue that
previous reports (42, 43) on hydroxyl radical formation
in vivo have more to do with oxoperoxonitrate(1-)
detection.
Ack n ow led gm en t. This work was supported by the
National Institutes of Health (GM48829) and the Council
for Tobacco Research-U.S.A., Inc. We thank Dr. J . S.
Beckman and Dr. H. Ischiropoulos for helpful discussions.
P h en ol a n d Meta l Com p lexes. Transition metal
complexes can increase the rate yield of nitration of
phenol by oxoperoxonitrate(1-). When there is such an
increase, the yield of hydroxylated products is concomi-
tantly decreased. The effect of Fe(III) complexes on the
nitration of phenol by oxoperoxonitrate(1-) is larger than
that of Cu(II) complexes. Our studies on the reaction of
oxoperoxonitrate(1-) with phenol show that the rate
constants vary from 5.5 × 103 M-1 s-1 for Fe(III)-edta
to 6.5 × 102 M-1 s-1 for CuSO4 at pH 7.0 ( 0.2 and at
room temperature. The activities of complexes vary with
the pH of the solution. The rate vs pH curve of the
nitration reaction is very similar to that of the yield vs
pH curve from pH 1.0 to 9.0 in the presence of Fe(III)-
edta (Figure 4). Over the pH range 4.0-9.0, the effect
of pH on nitration shows the same trend as that observed
in the absence of metal complexes. This indicates that
the same intermediate is responsible for the nitration of
phenol.
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TX950135W
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