Ketone-catalyzed decomposition of peroxynitrite via dioxirane intermediates

Article abstract of DOI:10.1021/ja984358v

Ketones are found to be catalysts for the decomposition of peroxynitrite. Kinetics, product studies, and B3LYP transition-state calculations together provide consistent evidence for a mechanism involving the formation of dioxirane intermediates.

Full text of DOI:10.1021/ja984358v

11976
J. Am. Chem. Soc. 1999, 121, 11976-11983
Ketone-Catalyzed Decomposition of Peroxynitrite via Dioxirane
Intermediates
,
†
†
†
†
‡
Dan Yang,* Yeung-Chiu Tang, Jian Chen, Xue-Chao Wang, Michael D. Bartberger,
,‡
§
K. N. Houk,* and Leif Olson
Contribution from the Department of Chemistry, The UniVersity of Hong Kong, Pokfulam Road,
Hong Kong, Department of Chemistry and Biochemistry, UniVersity of California,
Los Angeles, California 90095, and Eastman Kodak Co., 1999 Lake AVe.,
Rochester, New York 14650-2102
ReceiVed December 21, 1998. ReVised Manuscript ReceiVed October 25, 1999
Abstract: Ketones are found to be catalysts for the decomposition of peroxynitrite. Kinetics, product studies,
and B3LYP transition-state calculations together provide consistent evidence for a mechanism involving the
formation of dioxirane intermediates.
-
Introduction
which can isomerize to form nitrate (NO3 ) spontaneously, or
1
1
more rapidly upon the reaction with CO2 (Scheme 1). These
pathways produce reactive intermediates that can nitrate and
hydroxylate tyrosine residues of proteins, interfering with
1
-
Peroxynitrite (ONOO ) is a potent oxidant generated in cells
from the nearly diffusion-controlled reaction between nitric
•
-
9
-1
oxide (NO) and superoxide (O2 ) [k ) (4.3-6.7) × 10 M
12,13
signaling processes.
Therefore it is important to find reagents
that scavenge peroxynitrite in harmless pathways and thus
reduce its toxicity in vivo.
Some organic selenium-containing compounds such as eb-
selen [2-phenyl-1,2-benzisoselenazol-3(2H)-one] react very fast
with peroxynitrite stoichiometrically and preempt the formation
-1 2
s ]. It has been shown to contribute to tissue injury in a
3
number of human diseases such as rheumatoid arthritis, heart
4
5
6
7
disease, septic shock, atherosclerosis, and stroke.
•
NO + O2•- f ONOO-
14
of oxidizing species from peroxynitrite. Recently, several iron
and manganese porphyrin complexes were reported to decom-
-
Under physiological conditions, ONOO can oxidize biologi-
cal substrates such as sulfhydryls, sulfides, and lipids, and
it decomposes through the formation of peroxynitrous acid
8
9
10
-
6
-1 -1 15a-d
pose ONOO at a rate up to 2.2 × 10 M s .
In
particular, the iron porphyrin complexes are efficient catalysts
†
15d
The University of Hong Kong.
University of California.
Eastman Kodak Co.
for the decomposition of peroxynitrite, and they can protect
‡
culture cells from damage by exogenously added peroxynitrite
and reduce the carrageenan-induced inflammation in rat paws.1
Some short-chain aliphatic aldehydes were found to decompose
ONOO catalytically, although not efficiently, and a radical
mechanism was proposed. Now we have found that ketones
§
5e
(1) For recent reviews on peroxynitrite chemistry, see: (a) Beckman, J.
S.; Koppenol, W. H. Am. J. Physiol. (Cell Physiol.) 1996, 271, C1424-
C1437. (b) Edwards, J. O.; Plumb, R. C. Prog. Inorg. Chem. 1994, 41,
-
5
99-635. (c) Pryor, W. A.; Squadrito, G. L. Am. J. Physiol. (Lung Cell.
16
Mol. Physiol.) 1995, 268, L699-L722.
(
2) (a) Zafiriou, O. G.; Blough, N. V. Inorg. Chem. 1985, 24, 3502-
(11) Lymar, S. V.; Hurst, J. K. J. Am. Chem. Soc. 1995, 117, 8867-
8868.
3
1
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99. (c) Goldstein, S.; Czapski, G. Free Rad. Biol. Med. 1995, 19, 505-
(12) (a) Beckman, J. S.; Ischiropoulos, H.; Zhu, L.; Van der Woerd, M.;
Smith, C.; Chen, J.; Harrison, J.; Martin, J. C.; Tsai, M. Arch. Biochem.
Biophys. 1992, 298, 438-445. (b) Van der Vliet, A.; O’Neill, C. A.;
Halliwell, B.; Cross, C. E.; Kaur, H. FEBS Lett. 1994, 339, 89-92. (c)
Van der Vliet, A.; Eiserich, J. P.; O’Neill, C. A.; Halliwell, B.; Cross, C.
E. Arch. Biochem. Biophys. 1995, 319, 341-349.
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Biophys. 1996, 327, 335-343. (b) Lemercier, J.-N.; Padmaja, S.; Cueto,
R.; Squadrito, G. L.; Uppu R. M.; Pryor, W. A. Arch. Biochem. Biophys.
1997, 345, 160-170. (c) Lymar, S. V.; Jiang, Q.; Hurst, J. K. Biochemistry
1996, 35, 7855-7861. (d) Meli, R.; Nauser, T.; Koppenol, W. H. HelV.
Chim. Acta 1999, 82, 722-725. (e) Bonini, M. G.; Radi, R.; Ferer-Sueta,
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10806.
10.
(
3) Kaur, H.; Halliwell, B. FEBS Lett. 1994, 350, 9-12.
(
4) Matheis, G.; Sherman, M. P.; Buckberg, G. D.; Haybron D. M.;
Young, H. H.; Ignarro, L. J. Am. J. Physiol. (Heart Circ. Physiol.) 1992,
2
62, H616-H620.
5) Szabo, C.; Salzman, A. L.; Ischiropoulos, H. FEBS Lett. 1995, 363,
35-238.
6) (a) White, R. C.; Brock, T. A.; Chang, L.-Y.; Crapo, J.; Briscoe, P.;
(
2
(
Ku, D.; Bradley, W. A.; Gianturco, S. H.; Gore, J.; Freeman, B. A.; Tarpey,
M. M. Proc. Natl. Acad. Sci. U.S.A. 1994, 91, 1044-1048. (b) Hogg, N.;
Darley-Usmar, V. M.; Graham, A.; Moncada, S. Biochem. Soc. Trans. 1993,
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1, 358-362.
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(
2
(14) (a) Masumoto, H.; Kissner, R.; Koppenol, W. H.; Sies, H. FEBS
Lett. 1996, 398, 179-182. (b) Briviba, K.; Roussyn, I.; Sharov, V. S.; Sies,
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Bredt, D. S.; Snyder, S. H. Proc. Natl. Acad. Sci. U.S.A. 1991, 88, 6368-
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1
1
371.
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(15) (a) Groves, J. T.; Marla, S. S. J. Am. Chem. Soc. 1995, 117, 9578-
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(c) Lee, J.; Hunt, J. A.; Groves, J. T. J. Am. Chem. Soc. 1998, 120, 6053-
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G.; Misko, T. P. Proc. Natl. Acad. Sci. U.S.A. 1998, 95, 2659-2663.
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991, 266, 4244-4250.
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(
2
1
0.1021/ja984358v CCC: $18.00 © 1999 American Chemical Society
Published on Web 12/09/1999

Ketone-Catalyzed Decomposition of Peroxynitrite
J. Am. Chem. Soc., Vol. 121, No. 51, 1999 11977
Scheme 1
Scheme 2. Hypothetical Catalytic Cycle
also react with peroxynitrite: experiments and quantum me-
chanical calculations indicate a novel mechanism for the
decomposition of peroxynitrite, involving dioxirane intermedi-
ates.
Figure 1. Stopped-flow kinetic traces of peroxynitrite decay at different
-
ratios of methyl pyruvate to ONOO in 0.125 M phosphate buffer at
Oxone (2KHSO5‚KHSO4‚K2SO4, a commercial source of
-
pH 7.4 and 22 °C where [ONOO ]
0
) 0.26mM.
1
7
peroxymonosulfate) reacts with ketones to form dioxiranes.
-
-
Given the structural similarity between ONOO and HO3SOO ,
as well as the fact that dioxiranes are excellent reagents for
heteroatom oxidation,18 we hypothesized that ONOO might
-
-
react with ketones to form dioxiranes and NO2 (Scheme 2),
which could then react to give ketones and NO3 , thereby
-
completing a catalytic cycle. Indeed, we have found that ketones
can catalyze the decomposition of peroxynitrite.19
Results and Discussion
Kinetic Studies. At pH 7.4, addition of ketones accelerated
the decomposition of peroxynitrite. As shown in Figures 1 and
Figure 2. A plot of pseudo-first-order rate constants for the decom-
position of peroxynitrite (0.14 mM) in 0.125 M phosphate buffer at
pH 7.4 and 22 ( 1 °C, containing 0-5.4 mM methyl pyruvate. Each
data point represents the mean of five measurements.
2
, the pseudo-first-order rate for the decay of peroxynitrite
increases linearly with the initial concentration of methyl
pyruvate (1).20 This reveals a bimolecular reaction between
peroxynitrite and the ketone. Since the ketones in aqueous
solutions exist in an equilibrium of free (RCOR′) and hydrated
Compared with acetone (8), ketones 1, 2, and 5 are activated
toward nucleophilic addition by the adjacent electron-withdraw-
ing substituents. Ketones 3-4 and 6-7 are all 4-heterocyclo-
hexanones, and their electrophilicity is enhanced by ring strain
and by through-space electrostatic repulsion with the heteroatom
[RC(OH)2R′] forms, the bimolecular reaction may be due to
either form. The apparent second-order rate constants k2,app for
ketones 1-8 were determined at room temperature (Table 1).
(17) For excellent reviews on dioxirane chemistry, see: (a) Murray, R.
21
at the 1-position. The data shown in Table 1 reveal the
W. Chem. ReV. 1989, 89, 1187-1201. (b) Curci, R. In AdVances in
Oxygenated Processes; Baumstark, A. L., Ed.; JAI Press: Greenwich, CT,
importance of electronic factors in accelerating the decomposi-
tion of peroxynitrite. On the other hand, steric hindrance at the
R-position retards the reaction rate (compare ketone 4 with 6).
Both results indicate that the observed reaction is a nucleophilic
addition of peroxynitrite to the electron-deficient carbonyl group
of the free ketone. As the hydrated form is unreactive toward
peroxynitrite, the effective concentration of the ketone is less
than the nominal value. After hydration equilibria of the ketones
were taken into account, the second-order rate constants, k2,
1
990; Vol. 2, Chapter 1. (c) Adam, W.; Curci, R.; Edwards, J. O. Acc.
Chem. Res. 1989, 22, 205-211. (d) Adam, W.; Curci, R.; D’Accolti, L.;
Dinoi, A.; Fusco, C.; Gasparrini, F.; Kluge, R.; Paredes, R.; Schulz, M.;
Smerz, A. K.; Veloza, L. A.; Weinkotz, S.; Winde, R. Chem. Eur. J. 1997,
3
, 105.
18) (a) Wittman, M. D.; Halcomb, R. L.; Danishefsky, S. J. J. Org.
(
Chem. 1990, 55, 1981-1983. (b) Baboul, A. G.; Schlegel, H. B.;
Glukhovtsev, M. N.; Bach, R. D.; J. Comput. Chem. 1998, 19, 1353 and
references therein.
(19) Pyruvate anion was reported to react with peroxynitrite to give
acetate, carbon dioxide, and carbon dioxide radical anion: V a´ squez-Vivar,
-
were found to be up to half of that between ONOO and CO2
J.; Denicola, A.; Radi, R.; Augusto, O. Chem. Res. Toxicol. 1997, 10, 786-
4
-1 -1
11
(
k2 ) 2.9 × 10 M
s
at 24 °C).
7
94.
(20) Atmospheric carbon dioxide CO2 (from air-equilibrated solutions)
Figure 3 shows the pH-rate profiles for ketones 2 and 4, which
was not excluded from the reaction mixtures, but the presence of CO2 does
not affect the kinetic results for the reactions of peroxynitrite with ketones,
nor the conclusion that dioxirane intermediates are involved in these
reactions.
give an unsymmetrical bell-shaped curve with a maximum
(21) Conroy, J. L.; Sanders, T. C.; Seto, C. T. J. Am. Chem. Soc. 1997,
119, 4285-4291.

11978 J. Am. Chem. Soc., Vol. 121, No. 51, 1999
Yang et al.
Table 1. Rate Constants for Peroxynitrite Decomposition Catalyzed by Ketonesa
a
The experiments were carried out by mixing peroxynitrite solution with phosphate buffer in equal volumes. the conditions after mixing were:
-
b
0
.12-0.3 mM ONOO and 0.6-6 mM 1-7 or 0.08-1.4 M acetone in a mixture of CH
hydrate/ketone ratio was measured by H NMR under the conditions used for the kinetic experiments before mixing (0.25 M phosphate buffer in
3
CN and 0.125 M phosphate buffer (v/v 1:19) at pH 7.4. The
1
c
d
CD
3
CN/D
2
O v/v 1:9).
k
2
) k2,app × (1 + hydrate/ketone). Ketone 3 was mostly protonated in the above conditions.
0
Figure 3. pH dependence of the difference between the pseudo-first-order rate constants in the presence (kobs) and absence (k ) of (A) ketone 2 (6
mM) or (B) ketone 4 (6 mM). Peroxynitrite (A: 0.45 mM; B: 0.37 mM) was allowed to react at (A) 24 °C or (B) 22 °C, and pH 5.5-10.5 in 0.125
M phosphate buffer.
-
between pH 6.8 and 7.5. Two possible rate laws (eqs 1 and 2),
together with the corresponding mechanisms (Schemes 3 and
and H2PO4 are 6.8 and 7.2, respectively. However, the observed
curve is unsymmetrical, and the rate at high pH is much higher
than that at low pH, which indicates a pathway that occurs under
4), can be proposed to explain the bell shape. Both rate laws
-
will give the same symmetrical bell-shaped pH-rate profile with
basic conditions; this must be the direct addition of ONOO to
a maximum at about pH 7 because the pKa values of HOONO
the carbonyl group. As a result, the general-acid-catalyzed

Ketone-Catalyzed Decomposition of Peroxynitrite
J. Am. Chem. Soc., Vol. 121, No. 51, 1999 11979
Scheme 3
effects.23 The evidence for the formation of dioxiranes from
peroxynitrite and ketones is summarized in Table 2. In the
absence of ketone, 1,3-dimethylcyclohexene and ONOO did
-
not yield a detectable amount of epoxide. Upon addition of 1
or 2, a significant amount of epoxide products was formed.
2
4
-
Either Oxone or ONOO in combination with 1 or 2 gave
the identical trans/cis ratios of epoxides, that is, the same
diastereoselectivity was obtained when the same ketone was
employed, irrespective of the terminal oxidant. This implicates
a common dioxirane intermediate.
Regeneration of Ketones. As shown in Scheme 2, the
addition of peroxynitrite to ketone generates dioxirane inter-
mediate, which reacts further with nitrite to give back ketone
as the only carbon-containing product; that is, ketone should
be regenerated in the reaction process. Indeed, ketones 2 and 3
were recovered almost quantitatively in their reactions with
peroxynitrite, where a 4.5-fold amount of ketone was employed
to ensure that more than 90% of peroxynitrite was consumed
by the ketone (see Table 3).
Scheme 4
Analysis of Nitrite/Nitrate Ratio. At neutral pH, nitrite and
nitrate are the nitrogen-containing products from self-decom-
25
position of peroxynitrite. The data in Table 4 reveal that nitrate
and nitrite are the only nitrogen-containing products in the
reactions of peroxynitrite with ketones 1 and 2, and the nitrite/
nitrate ratio increases with the amount of ketone added.
According to Scheme 2, the adduct of peroxynitrite and ketone
collapses to nitrite and dioxirane, and the oxidation of nitrite
by dioxirane leads to nitrate. Meanwhile, the formation of nitrite
can be explained by proposing another pathway shown in
Scheme 6, where dioxirane reacts with peroxynitrite to give
ketone, nitrite, and oxygen. Kinetic studies on the reaction of
dioxirane with peroxynitrite or nitrite may provide some
evidence for the above proposed pathways.
Scheme 5
Reaction of Dimethyldioxirane with Nitrite or Peroxyni-
trite. Due to the difficulty in obtaining the corresponding
isolated dioxiranes derived from ketones 1-7, dimethyldioxirane
(DMD) was used as a model to examine the reactions of
dioxiranes with nitrite or peroxynitrite. Nitrite was found to be
oxidized quantitatively (see Table 5) to nitrate by DMD with a
addition of ONOO- shown in Scheme 4 is more likely than
the pathway in Scheme 3.
3
-1 -1
rate constant of 3.2 × 10 M
s
(at 25.6 ( 0.1 °C and pH
7
.4), and DMD decomposed peroxynitrite efficiently at a rate
The two parallel pathways are summarized in Scheme 5, and
the pH-rate profile can be approximated by eq 4.
4
-1 -1
of 2.5 × 10 M
s
(at 25.6 ( 0.1 °C and pH 7.4), about
1
0-fold faster than that for the former reaction. These kinetic
-
Below pH 6.8, the concentration of ONOO decreases, giving
studies suggest that dioxirane generated in situ from ketone and
peroxynitrite may react further with peroxynitrite and nitrite,
giving nitrite and nitrate respectively, and nitrite formation may
compete with nitrate formation in these processes.
a low reaction rate. Above pH 7.5, the general-acid-catalyzed
-
addition becomes less important as the concentration of H2PO4
decreases, and thus the rate declines, finally leveling off above
-
pH 9, where only uncatalyzed addition of ONOO occurs.
Nitration of Phenolic Compounds by Peroxynitrite. One
potentially damaging biochemical reaction of peroxynitrite is
Eq 4 shows a linear relationship between kobs - k0 and
1
2
-
-
the nitration of phenolic compounds, which has been shown
[
H2PO4 ]. At a fixed pH, [H2PO4 ] is proportional to the total
1
3
to be enhanced by the presence of CO2. However, in the
presence of excess amounts of ketones 1-4, the nitration yield
of 4-methylphenol was found to decrease significantly to 4-6%
while the yield was 10% in the absence of ketone (Table 6).
Figure 5 shows the yields of nitration of 4-hydroxypheny-
lacetate (4-HPA) by peroxynitrite with 0-15 mM ketones 3
and 4 at pH 7.4 and room temperature. In the absence of ketone,
buffer concentration. Therefore, a plot of kobs - k0 against buffer
concentration at a constant ketone concentration should give a
straight line. Figure 4 shows this relationship for ketones 2 and
4
, which further confirms the general-acid catalysis.
Dioxirane Formation. Dioxiranes are mild and versatile
17
oxidants under neutral conditions. A wide variety of olefins
can be epoxidized by dioxiranes generated in situ from Oxone
and ketones.22 Dioxiranes usually give diastereoselectivities
different from those given by peracids, and different dioxiranes
give distinct stereoselectivities due to steric and electronic
(23) (a) Murray, R. W.; Singh, M.; Williams, B. L.; Moncrieff, H. M. J.
Org. Chem. 1996, 61, 1830. (b) Yang, D.; Jiao, G.-S.; Yip, Y.-C.; Wong,
M.-K. J. Org. Chem. 1999, 64, 1635-1639.
(24) As shown in Table 2, Oxone gave a lower epoxidation yield in the
presence of ketone 1 or 2, which is because the dioxiranes formed from
these ketones also consumed Oxone and the amount of Oxone was limited.
See: Montgomery, R. E. J. Am. Chem. Soc. 1974, 96, 7820-7821.
(25) Pfeiffer, S.; Gorren, A. C. F.; Schmidt, K.; Werner, E. R.; Hansert,
B.; Bohle, D. S.; Mayer. B. J. Biol. Chem. 1997, 272, 3465-3470.
(
22) (a) Yang, D.; Wong, M.-K.; Yip, Y.-C. J. Org. Chem. 1995, 60,
3
887-3889. (b) Yang, D.; Yip, Y.-C.; Tang, M.-W.; Wong, M.-K.; Zheng,
J.-H.; Cheung K.-K. J. Am. Chem. Soc. 1996, 118, 491-492. (c) Denmark,
S. E.; Wu. Z. Synlett 1999, 847-859.

11980 J. Am. Chem. Soc., Vol. 121, No. 51, 1999
Yang et al.
Figure 4. Effect of phosphate buffer concentration on kobs - k
0
for the reaction between peroxynitrite (A: 0.35 mM; B: 0.22 mM) and (A) ketone
(4 mM) or (B) ketone 4 (6 mM) in 0.125-0.5 M phosphate buffer at 24 °C and pH 7.4.
2
Table 2. Epoxidation of 1,3-Dimethylcyclohexenea
Table 4. Shifts in [NO
-]/[No
-] Ratios by Ketones 1 and 2 at 25
2
3
a
°
C
mM
-]/[NO
3
ratio
-]
[
NO
2
ketone [ketone]:[ONOO-
] b
[NO
-]c
[NO
-]d
3
0
2
0
1
:1
:1
0.080
0.108
0.125
0.144
0.153
0.254
0.237
0.211
0.198
0.193
0.32
0.46
0.59
0.72
0.79
1
2
2.5:1
:1
0:1
5
%
conv
%
conv
1
oxidant
trans/cis
oxidant
trans/cis
0
1
:1
:1
0.070
0.151
0.182
0.201
0.240
0.325
0.235
0.211
0.195
0.146
0.22
0.64
0.85
1.03
1.64
-
oxone only
Oxone + 1
Oxone + 2
77
42
15
2.1:1
4.6:1
ONOO only
0
16
23
-
-
ONOO + 1
4.8:1
2.5:1
:1
10:1
b
-
only transb
only trans ONOO + 2
5
a
Conditions: 1.24 mM of olefin, 1.24 mM of NaOONO or Oxone
•
(
2KHSO
5
KHSO
‚K SO
4 2 4
) and 6.19 mM of ketone 1 or 2 in a total
CN:H
a
Reaction was conducted in 0.160 M phosphate buffer at pH 7.4 in
a total volume of 11 mL. All data are the mean values of three
volume of 33 mL phosphate buffer (CH
Phosphate buffer concentration is 0.16-0.17 M at pH 7.4. The %
conversion and trans/cis ratios were determined by GC after extraction
3
2
O ) 1:10 v/v).
b
-
) 0.322 mM. c After subtracting [NO
-
2 0
experiments. [ONOO ]
0
] .
d
-
0
[
NO
3
]
was found negligible.
b
with CH
2
Cl
2
.
No detectable amount of the cis-epoxide was found in
the gas chromatogram.
Scheme 6
Table 3. Recovery of Ketones 2 and 3a
a
-
-
3
Table 5. HPLC Analysis of NO
2
and NO
-] (mM)
reactionb
[NO
[NO
0
5.87 ( 0.02
-] (mM)
3
2
-
NO
NO
2
+ acetone
+ DMD
5.96 ( 0.03
0
-
2
a
HPLC conditions: Hamilton PRP-X100 anion-exchange column,
aqueous potassium hydrogen phthalate (2.0 mM) at pH 5.0 as the mobile
phase. b Reaction conditions: [NO
-
] ) 6.00 mM, 5 equiv of DMD,
2 0
a
Decomposition of ONOO- in the absence or presence of ketone
room temperature, and 0.5 h. All data are the mean values of two
experiments.
was conducted at room temperature in 0.160 M phosphate buffer at
b
pH 7.4 in a total volume of 11 mL. Determined by GC analysis after
c
the extraction of ketone with CH
2
Cl
2
.
The control experiment was
nitration in the case of ketones may be due to the oxidation of
the phenols by RR′C(OH)O .
Theoretical Studies. Theoretical evidence for this mechanism
•
carried out under similar conditions except adding ketone 10 min after
mixing peroxynitrite with buffer alone.
was obtained from density functional theory (DFT) calculations
the yield of 3-nitro-4-hydroxyphenylacetate (3-NO2-4-HPA) was
26
(B3LYP/6-31G*). Transition structures were found for con-
8
-10 mol % based on the concentration of peroxynitrite. In
certed oxygen atom transfer to acetone from peroxynitrite
the presence of ketone, the yield progressively decreased to a
steady value, at which the ketone reached saturating conditions,
that is, all peroxynitrite present reacted with the ketone. The
maximum possible inhibitions of 4-HPA nitration by ketones 3
and 4, calculated using Scatchard-like plots, are 69 and 42%,
respectively.
(
Figure 6) and peroxynitrous acid. The B3LYP/6-31G* transition
-
structures for epoxidations of ethylene by ONOO and ONOOH
were reported earlier and are quite similar.
structure for ONOO oxidation of acetone has considerable
27-29
The transition
-
(26) Frisch, M. J.; Trucks, G. W.; Schlegel, H. B.; Gill, P. M. W.;
Johnson, B. G.; Robb, M. A.; Cheeseman, J. R.; Keith, T.; Petersson, G.
A.; Montgomery, J. A.; Raghavachari, K.; Al-Laham, M. A.; Zakrzewski,
V. G.; Ortiz, J. V.; Foresman, J. B.; Cioslowski, J.; Stefanov, B. B.;
Nanayakkara, A.; Challacombe, M.; Peng, C. Y.; Ayala, P. Y.; Chen, W.;
Wong, M. W.; Andres, J. L.; Replogle, E. S.; Gomperts, R.; Martin, R. L.;
Fox, D. J.; Binkley, J. S.; Defrees, D. J.; Baker, J.; Stewart, J. P.; Head-
Gordon, M.; Gonzalez, C.; Pople, J. A. Gaussian 94 (Revision A.1);
Gaussian, Inc., Pittsburgh, PA, 1995.
These results show that the nitration of phenolic compounds
by peroxynitrite can be partially suppressed by ketones. On the
other hand, propionaldehyde was reported to inhibit 90% of
1
6
4
-HPA nitration. Similar to the case of CO2 and aldehydes,
the protonated tetrahedral adduct of ketones and peroxynitrite
RR′C(OH)OONO] may undergo homolytic cleavage, yielding
[
(27) Houk, K. N.; Condroski, K. R.; Pryor, W. A. J. Am. Chem. Soc.
•
•
a radical pair RR′C(OH)O and NO2. The residual yield of
1996, 118, 13002-13006.

Ketone-Catalyzed Decomposition of Peroxynitrite
J. Am. Chem. Soc., Vol. 121, No. 51, 1999 11981
Table 6. Nitration of 4-Methylphenol by Peroxynitrite and
a
Ketones
a
Nitration of 4-methylphenol (0.9-1.5 mM) by peroxynitrite (1
equiv) was performed in 0.45 M phosphate buffer at pH 7.4 and room
temperature. The data shown are the mean of the results from two
experiments. Ketone 1 was in 5-fold excess; the others were in 10-
b
fold excess.
nucleophilic character, but gives NO2- and dimethyldioxirane
directly. While acetone (used for convenience in the calcula-
tions) is a relatively electron-rich ketone, a nucleophilic attack
transition state is consistent with the observation that electron-
-
Figure 5. Nitration of 4-HPA (4 mM) by peroxynitrite (1 mM) in the
presence of 0-15 mM (A) ketone 3 or (B) ketone 4 in 4 mL of 0.17
M phosphate buffer at pH 7.4 and room temperature. Each data point
represents the mean ((SD) of the results from two experiments. Inset
is a double-reciprocal plot of fractional inhibition of 4-HPA nitration
against the concentration of (A) ketone 3 or (B) ketone 4.
deficient ketones react most readily with ONOO . This reaction
is predicted to be especially facile; peroxynitrite and acetone
form an ion-molecule complex followed by a transition
structure which is 1.1 kcal/mol lower in energy than the
separated reactants in the gas phase, an energetic profile similar
to the reactions of other anionic nucleophiles with aldehydes
and ketones.30 In solution, activation barriers are increased due
to solvation of the nucleophile.31
to release singlet oxygen in the reaction of amine N-oxides.34
Experiments to detect singlet O2 in the ketone-catalyzed
The second step in the catalytic process occurs with no barrier
-
decomposition of ONOO are currently underway.
-
at all: if NO2 and dimethyldioxirane are juxtaposed in a
-
The alternative N-atom oxidation of ONOO by dimethyl-
reasonable reacting conformation, oxygen atom transfer is
-
dioxirane to give O2NOO (peroxynitrate) should also be facile,
-
spontaneous, yielding NO3 and acetone. Our calculations
and the oxygen transfer transition state is 2.8 kcal/mol more
stable than the separated reactants in the gas phase (similar to
indicate that dioxiranes might also react with unconsumed
-
ONOO . Oxygen atom transfer from dimethyldioxirane to the
-
the situation with ONOO + acetone). Thus, our DFT calcula-
-
-
terminal peroxide oxygen of ONOO to give acetone, NO2
-
-
tions indicate that O2NOO should, like ONOO , react readily
with ketones and dioxiranes. The B3LYP/6-31G* structures and
and singlet O2 (predicted on the basis of spin conservation)
occurs with no barrier by DFT. Brauer et al. have demonstrated
via direct spectrophotometric detection the quantitative liberation
of singlet oxygen in the ketone-catalyzed decomposition of
peroxymonosulfuric acid;3 dioxiranes have also been shown
-
-
energies of O2NOO reactions with acetone (to give NO3 and
dimethyldioxirane) and with dimethyldioxirane (to give NO3 ,
-
acetone, and singlet O2) are nearly perfect analogues of the
2,33
-
ONOO reactions, suggesting that ketones should catalyze the
decomposition of peroxynitrate. The experimentally observed
(
28) Bartberger, M. D.; Olson, L. P.; Houk, K. N. Chem. Res. Toxicol.
998, 11, 710-711.
29) The validity of B3LYP results has been confirmed by comparisons
-
1
NO3 may arise partly from these peroxynitrate decomposition
(
-
pathways and partly from oxidation of NO2 by dioxirane.
with higher level calculations of epoxidations: see ref 27 and Glukhovtsev,
The general-acid-catalyzed reaction of peroxynitrite with
ketones at neutral pH leads to formation of the peroxynitrous
acid adduct ONOOCR2OH. The tetrahedral intermediate shown
in Figure 7 could form a dioxirane. However, we find no
energetically accessible pathway for an intramolecular nucleo-
philic displacement that would lead to dioxirane formation from
M. N.; Canepa, C.; Bach, R. D. J. Am. Chem. Soc. 1998, 120, 10528-
1
0533.
(
30) Pranata, J. J. Phys. Chem. 1994, 98, 1180-1184 and references
therein.
(
31) Yu, H. A.; Karplus M. J. Am. Chem. Soc. 1990, 112, 5706-5716
and references therein.
(
32) Lange, A.; Hild, M.; Brauer, H.-D. J. Chem. Soc., Perkin Trans. 2
999, 1343-1350.
33) Lange, A.; Brauer, H.-D. J. Chem. Soc., Perkin Trans. 2 1996, 805-
11.
1
(
(34) Ferrer, M.; Sanchez-Baeza, F.; Messenguer, A.; Adam, W.; Golsch,
D.; Gorth, F.; Kiefer, W.; Nagel, V. Eur. J. Org. Chem. 1998, 2527-2532.
8

11982 J. Am. Chem. Soc., Vol. 121, No. 51, 1999
Yang et al.
damaging hydroxyl radical which arises from self-decomposition
of peroxynitrite, or the highly reactive carbonate radical anion
arising from the CO2-mediated decomposition of peroxynitrite,1
and might promote a quite different biochemistry in vivo.
3d,e
Experimental Section
General. All kinetic studies were performed on the Applied
Photophysics SX.18MV stopped-flow spectrophotometer with
a dead time less than 2 ms. NMR spectra were recorded at
ambient temperature on a Bruker AVANCE DPX 300 Fourier
transform spectrometer. Gas chromatograms were recorded on
a Hewlett-Packard 5890A gas chromatograph using a flame
ionization detector (FID), and a capillary column [either a 30
m × 0.32 mm × 0.50 µm HP-INNOWAX (cross-linked poly-
(
ethylene glycol)) polar one, or a 25 m × 0.32 mm × 0.52 µm
HP-Ultra 1 (cross-linked methyl silicone gum) nonpolar one].
All chemicals were used as received; 2-chlorocyclohexanone
was redistilled before use. Acetonitrile was HPLC or AR grade.
All aqueous solutions were prepared in doubly distilled water.
Dry dichloromethane was distilled from calcium hydride.
Figure 6. B3LYP/6-31G* structures of peroxynitrite plus acetone, the
Ketones 4 and 7 were prepared according to the literature
-
35,36
transition state for O transfer, and the structures of NO
dimethyldioxirane, and nitrate plus acetone.
2
plus
procedures.
1,3-Dimethylcyclohexene was prepared by the
dehydration of 2,6-dimethylcyclohexanol in HMPA at 230-
3
7
2
40 °C.
Synthesis of Peroxynitrite. Peroxynitrite was synthesized
either by the reaction between acidified hydrogen peroxide and
sodium nitrite followed by rapid mixing with sodium hydroxide
3
8
in a quenched flow reactor or by the nitrosation of hydrop-
eroxide anion with 2-ethoxyethyl nitrite in basic medium.39 The
yellow peroxynitrite solution was filtered through a layer of
manganese dioxide to remove excess hydrogen peroxide.
(EDTA)Na2 was added to chelate any adventitious metal
contaminants. The concentrations obtained by these two methods
were 24-70 mM and 12-40 mM, respectively, which were
-
1
determined spectrophotometrically at 302 nm (ꢀ ) 1670 M
-
1 40
cm ).
Synthesis of Ketone 6 (1,1,3,5-Tetramethyl-4-oxopiperi-
dinium Trifluoromethanesulfonate). 1,3,5-Trimethyl-4-pip-
eridone (1.37 g, 9.7 mmol) was dissolved in dry CH2Cl2 (40
mL) under N2 atmosphere and cooled to 0 °C. MeOTf (1.6 mL,
13.7 mmol, 1.4 equiv) was added via a syringe. The reaction
mixture was allowed to warm to room temperature slowly and
stirred for 21 h. The white precipitate was filtered and
3
Figure 7. B3LYP/6-31G structures of the peroxynitrous acid adduct
with acetone, the radical pair, and the nitric acid adduct with acetone.
such an intermediate. Simple O-O bond homolysis to form
recrystallized (EtOAc/CH CN) to provide 2.14 g (72%) of 6 as
•
•
1
ONO and OC(CH3)2OH (Figure 7), requires only 10.1 kcal/
mol. O-O recombination of the radicals to regenerate starting
material would occur, or the radicals could recombine at N.
N-O bond formation gives the adduct of nitric acid and acetone,
a process calculated to be exothermic by 37.3 kcal/mol. This
latter species will be unstable at moderate pH, since its conjugate
white plates: mp 183-187 °C (EtOAc/CH CN); H NMR (300
3
MHz, acetone-d ) δ 4.04 (dd, J ) 11.2, 6.0 Hz, 2H), 3.78 (t, J
6
) 13.3 Hz, 2H), 3.73 (s, 3H), 3.49 (s, 3H), 3.35 (dqn, J )
1
3
12.9, 6.4 Hz, 2H), 1.06 (d, J ) 6.5 Hz, 6H); C NMR (75.47
MHz, acetone-d ) δ 204.8, 122.2 (q, J ) 321 Hz), 68.2, 56.9,
6
+
49.3, 39.2, 10.9; FAB-MS (+ve) m/z 156 (M , 100); FAB-MS
-
-
base readily reverts to NO3 and acetone. DFT found no barrier
(-ve) m/z149 ( OTf, 100); HRMS for C H NO (FAB, +ve)
9
18
for this reversion. Therefore, the general-acid-catalyzed portion
of the pathway may share similar features with uncatalyzed
m/z calcd 156.1383, found 156.1381; IR (KBr disk) 1733, 1263,
-
1
1166, 1031 cm . Anal. Calcd for C H F NO S: C, 39.34%;
1
0
18
3
4
-
decomposition of ONOOH, or ONOO decomposition mediated
H, 5.94%; N, 4.59%. Found: C, 39.22%; H, 5.99%; N, 4.57%.
Kinetic Studies on Peroxynitrite Decomposition. The rate
of peroxynitrite decomposition was followed at 302 nm or 265
2
8
by CO2. That is, covalent bonding of the terminal peroxide
-
+
oxygen of ONOO to an electrophile such as H or CO2 (or,
in this case, a protonated ketone) facilitates O-O bond
-
28
(35) Denmark, S. E.; Wu, Z. J. Org. Chem. 1997, 62, 8964-8965.
homolysis and, ultimately, formation of NO3 .
(
36) Yang, D.; Yip, Y.-C.; Jiao, G.-S.; Wong, M.-K. J. Org. Chem. 1998,
In summary, we have identified a new mechanism for
catalytic decomposition of ONOO- by ketones, involving
dioxirane intermediates under neutral conditions. In addition,
the general-acid-catalyzed component is likely to occur by the
O-O bond homolysis pathway, similar to the one suggested
earlier by Pryor and co-workers.16 These pathways avoid the
6
3, 8952-8956.
37) Monson, R. S. Tetrahedron Lett. 1971, 12, 567-570.
(38) Beckman, J. S.; Chen, J.; Ischiropoulos, H.; Crow, J. P. Methods
Enzymol. 1994, 233, 229-240.
39) Leis, J. R.; Pe n˜ a, M. E.; R ´ı os, A. J. Chem. Soc., Chem. Commun.
(
(
1
993, 1298-1299.
(40) Hughes, M. N.; Nicklin, H. G. J. Chem. Soc. (A) 1968, 450-452.

Ketone-Catalyzed Decomposition of Peroxynitrite
J. Am. Chem. Soc., Vol. 121, No. 51, 1999 11983
nm at pH below 6. Peroxynitrite (0.25-0.6 mM) in 15 mM
NaOH solution was mixed in equal volume with a mixture of
CH3CN and potassium phosphate buffer (v/v 1:9) (final pH )
nitrite (6.00 mM) to nitrate by DMD (30.00 mM) in acetone
was conducted at room temperature. The control experiment
was carried out under the same conditions except using acetone
only. Nitrite and nitrate were analyzed quantitatively by anion
HPLC (Hamilton PRP-X100 anion-exchange column, serial
number PRP-X100 2857, and aqueous potassium hydrogen
phthalate (2.0 mM) at pH 5.0 as the mobile phase).
5
.5-10.5) at 20-24 °C. Stock solutions of ketones in CH3CN
were mixed with the phosphate buffer at least 5 min before the
addition of peroxynitrite to allow the ketones to reach the
hydration equilibria.
-
1
Pseudo-first-order rates, kobs(s ), were determined by non-
linear least-squares fitting of stopped-flow data to a single-
exponential function with a nonzero offset. Results from five
measurements were averaged to obtain each rate constant.
Apparent second-order rate constants (k2,app) for the reactions
of peroxynitrite with ketones were calculated from the slopes
of the plots of kobs versus the concentration of the ketone. The
self-decomposition rate of peroxynitrite, k0, was subtracted from
kobs before plotting against pH or buffer concentration in the
relevant experiments. Data fitting was performed using Origin
Kinetic Studies on the reaction of DMD with Peroxyni-
trite. Peroxynitrite (0.544 mM) in 0.15 mM NaOH solution was
mixed in equal volume with a solution of DMD (0-11 mM) in
a mixture of acetone and potassium phosphate at pH 7.4 and
25.6 °C. The pseudo-first-order rates were corrected by subtract-
ing the rates of peroxynitrite/acetone reaction. The reported
value of k2 is the mean of the results from three experiments.
Kinetic Studies on the reaction of DMD with Nitrite. The
rate of nitrite oxidation was followed at 356 nm. A solution of
DMD (5-80 mM) in a mixture of acetone and potassium
phosphate was mixed in equal volume with a nitrite solution (5
mM) at pH 7.4 and 25.6 °C. The reported value of k2 is the
mean of the results from two experiments.
3.5 (Microcal Software, Inc.).
Epoxidation of 1,3-Dimethylcyclohexene by Oxone or
Peroxynitrite. Oxone (25.1 mg, 41 µmol) or peroxynitrite (41
µmol) was added to a solution of 1,3-dimethylcyclohexene (41
µmol) in 0.18 M phosphate buffer at pH 7.4, containing 3 mL
of CH3CN and 0.204 mmol of ketone 1or 2 in a total volume
of 33 mL. The solution was stirred for 30 min at room
temperature, and then it was extracted with CH2Cl2 (4 × 5 mL).
After drying over anhydrous magnesium sulfate and filtration,
the solution was subjected to GC analysis (HP-Ultra 1 column;
carrier gas, helium).
GC Analysis of Ketone Recovery. Peroxynitrite was de-
composed by incubation with 0.16 M phosphate buffer, contain-
ing 1 mL of CH3CN and ketone in a total volume of 11 mL,
for an hour at pH 7.4 and room temperature. Extraction of the
reaction mixture with dichloromethane was followed by GC
analysis using toluene and 2-methylcyclohexanone as internal
standard references for ketone 2 and 3 respectively (HP-
INNOWAX column; carrier gas, helium).
Nitration of 4-Methylphenol. Peroxynitrite (0.9-1.5 mM)
was allowed to react with 1 equiv of 4-methylphenol in 0.45
M phosphate buffer at pH 7.4, containing 0-10 equivs of
ketone. The reaction mixture was stirred for an hour at room
temperature. Then it was extracted with diethyl ether (five
times). The extract was dried over anhydrous magnesium sulfate
and evaporated to dryness to give a yellow residue. The residue
1
was analyzed by H NMR in CDCl3. The percentage nitration
was estimated by the integration ratio of the signals of 2-nitro-
4-methylphenol to that of 4-methylphenol in the aromatic region.
Nitration of 4-Hydroxyphenylacetate (4-HPA). An aliquot
of peroxynitrite solution (4 µmol) was mixed rapidly with 0.17
M phosphate buffer at pH 7.4, containing 0.4 mL of CH3CN,
1
4
6 µmol of 4-HPA, and 0-15 mM ketone in a total volume of
mL. Throughout the course of addition of peroxynitrite, the
contents of the reaction mixture were vigorously shaken by using
a vortex mixer, and shaking was continued for an additional 10
s at room temperature. The concentration of 3-nitro-4-hydrox-
yphenylacetate (3-NO2-4-HPA) was estimated spectrophoto-
Analysis of Nitrite/Nitrate Ratio. Peroxynitrite (0.32 mM)
was decomposed by incubation with 0.16 M phosphate buffer,
containing 1 mL of CH3CN and ketone (0-3.22 mM) in a total
volume of 11 mL, for an hour at pH 7.4 and room temperature.
Measurement of nitrite and nitrate was performed following the
ASTM methods.41 Nitrate was reduced to nitrite by passing
nitrate through a copper-coated cadmium column, and nitrite
was determined through UV measurement at 543 nm of an azo
complex produced from the diazotization and subsequent
coupling of sulfanilamide to N-(1-naphthyl)ethylenediamine.
-
1
-1 12a
metrically at 430 nm (ꢀ ) 4400 M cm ) after the addition
of 1 mL of 1.2 M NaOH solution.
Acknowledgment. We are grateful to the U.S. National
Science Foundation and the National Institutes of Health, and
the Hong Kong Research Grants Council for financial support
of this research. X.-C.W. is a recipient of the University
Postdoctoral Fellowship, and J.C. is a recipient of the Hui Pun
Hing Scholarship for Postgraduate Research from The Univer-
sity of Hong Kong. We are grateful to Professor William A.
Pryor for extensive discussions and to Professor C.-M. Che for
the use of the stopped-flow UV spectrometer.
-
-
[
NO2 ]0 and [NO3 ]0 were determined by decomposing per-
oxynitrite under acidic or basic/copper(II) conditions following
4
2
literature methods.
Oxidation of Nitrite to Nitrate by Dimethyldioxirane
(
DMD). DMD was prepared from the reaction of acetone with
4
3
Oxone following the reported procedure. The oxidation of
Supporting Information Available: Typical procedure for
determination of hydrate/ketone ratios for ketones 1-7 (PDF).
This material is available free of charge via the Internet at
http://pubs.acs.org.
(
41) Annual Book of ASTM Standards; International Library Service:
Provo, Utah, 1989; Vol. 11.01, pp 438-446.
42) Plumb R. C.; Edwards, J. O.; Herman, M. A. Analyst 1992, 117,
639-1641.
43) Murray, R. W.; Jeyaraman, R. J. Org. Chem. 1985, 50, 2847-2853.
(
1
(
JA984358V