Chemiluminescent aldehyde and β-diketone reactions promoted by peroxynitrite

Article abstract of DOI:10.1021/tx990176i

Peroxynitrite is shown here to promote the aerobic oxidation of isobutanal (IBAL) and 3-methyl-2,4-pentanedione (MP) in a pH 7.2 phosphate buffer into acetone plus formate and biacetyl plus acetate, respectively. These products are expected from dioxetane intermediates, whose thermolysis is known to be chemiluminescent (CL). Accordingly, the extent of total oxygen uptake by IBAL at different concentrations parallels the corresponding CL maximum intensities. The pH profile based on oxygen uptake data for the MP reaction matches the titration curve of peroxynitrous acid (pK(a) ~ 7), indicating that peroxynitrite anion is the oxidizing agent. Energy transfer studies with IBAL and the 9,10-dibromoanthracene-2-sulfonate ion, a triplet carbonyl detector, indicates that triplet acetone (τ = 19 μs) is the energy donor. It is postulated that IBAL- or MP-generated triplet carbonyls are produced by the thermolysis of dioxetane intermediates, which are formed by the cyclization of α-hydroperoxide intermediates produced by insertion of dioxygen into the IBAL or MP enolyl radicals, followed by their reduction. Accordingly, EPR spin-trapping studies with 3,5-dibromo-4- nitrosobenzenesulfonic acid (DBNBS) and 2-methyl-2-nitrosopropane (MNP) revealed the intermediacy of carbon-centered radicals, as expected for one- electron abstraction from the enol forms of IBAL or MP by peroxynitrite. The EPR data obtained with IBAL also reveal formation of the isopropyl radical produced by competitive nucleophilic addition of ONOO^- to IBAL, followed by homolytic cleavage of this adduct and β-scission of the resulting Me₂CHCH(O^-)O·. Superstoichiometric formation of fragmentation products from IBAL or MP attests to the prevalence of an autoxidation chain reaction, here proposed to be initiated by one-electron abstraction by ONOO^- from the substrate. This work reveals the potential role of peroxynitrite as a generator of electronically excited species that may contribute to deleterious and pathological processes associated with excessive nitric oxide and aldehyde production.

Full text of DOI:10.1021/tx990176i

MAY 2000
VOLUME 13, NUMBER 5
© Copyright 2000 by the American Chemical Society
Articles
Ch em ilu m in escen t Ald eh yd e a n d â-Dik eton e Rea ction s
P r om oted by P er oxyn itr ite
Fernanda S. Knudsen,† Carlos A. A. Penatti,† Leandro O. Royer,†
Karine A. Bidart,† Marcelo Christoff,† Denise Ouchi,‡ and
Etelvino J . H. Bechara*,†
Departamento de Bioqu´ımica and Departamento de Qu´ımica Fundamental, Instituto de Qu´ımica,
Universidade de Sa˜o Paulo, CP 26077, 05599-970 Sa˜o Paulo, SP, Brazil
Received October 28, 1999
Peroxynitrite is shown here to promote the aerobic oxidation of isobutanal (IBAL) and
3-methyl-2,4-pentanedione (MP) in a pH 7.2 phosphate buffer into acetone plus formate and
biacetyl plus acetate, respectively. These products are expected from dioxetane intermediates,
whose thermolysis is known to be chemiluminescent (CL). Accordingly, the extent of total oxygen
uptake by IBAL at different concentrations parallels the corresponding CL maximum
intensities. The pH profile based on oxygen uptake data for the MP reaction matches the
titration curve of peroxynitrous acid (pKa ∼ 7), indicating that peroxynitrite anion is the
oxidizing agent. Energy transfer studies with IBAL and the 9,10-dibromoanthracene-2-sulfonate
ion, a triplet carbonyl detector, indicates that triplet acetone (τ ) 19 µs) is the energy donor.
It is postulated that IBAL- or MP-generated triplet carbonyls are produced by the thermolysis
of dioxetane intermediates, which are formed by the cyclization of R-hydroperoxide intermedi-
ates produced by insertion of dioxygen into the IBAL or MP enolyl radicals, followed by their
reduction. Accordingly, EPR spin-trapping studies with 3,5-dibromo-4-nitrosobenzenesulfonic
acid (DBNBS) and 2-methyl-2-nitrosopropane (MNP) revealed the intermediacy of carbon-
centered radicals, as expected for one-electron abstraction from the enol forms of IBAL or MP
by peroxynitrite. The EPR data obtained with IBAL also reveal formation of the isopropyl
radical produced by competitive nucleophilic addition of ONOO- to IBAL, followed by homolytic
cleavage of this adduct and â-scission of the resulting Me2CHCH(O-)O•. Superstoichiometric
formation of fragmentation products from IBAL or MP attests to the prevalence of an
autoxidation chain reaction, here proposed to be initiated by one-electron abstraction by ONOO-
from the substrate. This work reveals the potential role of peroxynitrite as a generator of
electronically excited species that may contribute to deleterious and pathological processes
associated with excessive nitric oxide and aldehyde production.
× 1010 M-1 s-1; pKa(ONOOH) ) 6.8] (1), has been shown
In tr od u ction
to perform one- and two-electron oxidations, nitrations,
Peroxynitrite, formed by a diffusion-controlled reaction
of nitric oxide with the superoxide anion radical [k ) 2
and nucleophilic addition to carbonyls (2 and references
therein). A significant fate of peroxynitrite in biological
systems appears to be its reaction with dissolved CO2
* To whom correspondence should be addressed: Instituto de
Qu´ımica, Universidade de Sa˜o Paulo, Caixa Postal 26077, 05599-970
Sa˜o Paulo, SP, Brazil. Telephone: 55-11-818-3869. E-mail: ebechara@
quim.iq.usp.br.
† Departamento de Bioqu´ımica.
‡ Departamento de Qu´ımica Fundamental.
10.1021/tx990176i CCC: $19.00 © 2000 American Chemical Society
Published on Web 04/06/2000

318 Chem. Res. Toxicol., Vol. 13, No. 5, 2000
Knudsen et al.
Sch em e 1. Gen er a tion of Tr ip let Aceton e fr om Isobu ta n a l (IBAL) In itia ted by HRP /H2O2 or P er oxyn itr itea
a
Route A, from Uppu et al. (21) and Va´squez-Vivar et al. (23). Route B, from Bechara et al. (24). Route C, from this work.
(k ) 1.0 × 104 M-1 s-1) (3), whose concentration may be
as high as 1-2 mM (4). However, peroxynitrite has been
observed to react in vivo directly with lipids, DNA,
proteins, and several other biomolecules such as deoxy-
ribose, glutathione, R-tocopherol, phenols, and amino
acids (5-16). Such reactions can trigger important
deleterious, protective, or regulatory biological responses
(4, 17). In addition, peroxynitrite-mediated reactions may
involve reactive intermediates resulting from the decom-
position of the peroxynitrite-CO2 adduct (14, 18-20).
tively, the fragmentation products expected from the
thermolysis of dioxetane intermediates. The R-methyl
substitution in MP precludes its subsequent nitration,
as reported to occur with ethyl acetoacetate treated with
peroxynitrite (29). Superstoichiometric formation of sub-
strate fragmentation products (based on added peroxy-
nitrite), as well as formation of tertiary carbon-centered
radical intermediates that can be detected by EPR spin
trapping, can be predicted by this route.
Recent studies have shown that the nucleophilic ad-
dition of ONOO- to linear aliphatic aldehydes produces
mainly reformed aldehyde and nitrate, plus minor prod-
ucts such as H2O2 and organic fragmentation products
(organic acids) (21, 22). Nucleophilic addition of ONOO-
to the keto group of pyruvate has also been observed to
result in decarboxylation of pyruvate into acetate, with
Ma ter ia ls a n d Meth od s
Rea gen ts a n d P r ep a r a tion s. All the reagents, purchased
from Sigma-Aldrich, were of purest analytical grade. Peroxyni-
trite was prepared in 0.6 M HCl from NaNO2 and H2O2 in a 1.5
M NaOH quenched-flow reaction according to Beckman et al.
(30) and quantified spectrophotometrically at 302 nm (ꢀ ) 1670
M-1 cm-1). Sodium 9,10-dibromoanthracene-2-sulfonate (DBAS)
and sodium 9,10-diphenylanthracene-2-sulfonate (DPAS) (ꢀ405
) 7750 M-1 cm-1 and ꢀ397 ) 8050 M-1 cm-1, respectively) were
prepared according to the methods of Catalani et al. (31).
Solutions were prepared in Milli-Q-purified water and the
buffers pretreated with Chelex.
•-
the production of the CO2 radical (23). These studies
are relevant, considering the ubiquity of aldehydes and
ketones in biological systems, where they may act as
reactive intermediate sources.
We examine here whether peroxynitrite, in competition
with nucleophilic addition to easily enolizable carbonyl
compounds, can act as a one-electron oxidant of the
corresponding enols, initiating an oxygen-dependent
chemiluminescent reaction similar to those previously
reported for the horseradish peroxidase-catalyzed (HRP/
H2O2) oxidation of isobutanal (IBAL) and 3-methyl-2,4-
pentanedione (MP) (24, 25). Scheme 1 illustrates the two
oxidation pathways initiated by peroxynitrite, nucleo-
philic addition (route A) versus electron abstraction
(routes B and C), in the case of IBAL. The HRP-catalyzed
reactions of IBAL and MP have been shown to produce
electronically excited triplet acetone and triplet biacetyl,
respectively. Triplet species have been reputed to be of
significant biological relevance because triplet carbonyls
can transfer energy to several biomolecules and, being
endowed with the chemical behavior of radicals, can
trigger typically photochemical reactions in the dark (26).
Our hypothesis is that carbonyl compounds that (i) are
capable of fast enolization either directly, as is the case
of MP [2.8% enol form exists in aqueous medium (27)],
or indirectly, by phosphate catalysis as with IBAL (28),
and (ii) bear an additional R-alkyl substituent, which is
expected to lower the reduction potential (in the case of
both MP and IBAL), may prefer to react with peroxyni-
trite via route C in Scheme 1. For comparison, parallel
studies were carried out with 3-methyl-2-butanone and
nonsubstituted substrates such as 2-pentanone and 2,4-
pentanedione. If route C (Scheme 1) were operative, the
reactions with IBAL and MP would lead to triplet acetone
plus formate and triplet biacetyl plus acetate, respec-
Tech n iqu es. Oxygen uptake was followed in a Yellow Spring
Instruments model 53 oxygen monitor and chemiluminescence
measured with a Hamamatsu TVC 767 photon counter equipped
with a red-sensitive Thorn EMI (9658AM) photomultiplier
cooled to -12 °C by a Thorn EMI (model Fact MKIII) thermo-
electric cooler. Acetone was analyzed by GC with a Shimadzu
CH-14A chromatograph with flame ionization detection. A
SPB-1 polymethylsilicone capillary column (25 m × 0.25 mm
o.d. × 0.25 µm i.d.) was used. Temperatures were as follows:
injection, 200 °C; detector, 250 °C; and column oven, 35 °C.
Helium was used as a carrier gas; the sample size (headspace)
was 1.0 mL. The integrator was a Shimadzu chromatopac
C-R4A. Biacetyl was detected by two methods: (a) derivation
with 6-hydroxy-2,4,5-triaminopyridine, followed by HPLC/
fluorescence detection analysis, according to the method of
Mansilla-Espinosa et al. (32); and (b) GC on a Shimadzu CH
14A chromatograph with flame ionization detection. The ex-
perimental conditions employed were the same as those used
for the analysis of acetone. EPR spectra were recorded at room
temperature using a Bruker EMX spectrometer, 4 min after the
addition of peroxynitrite.
Capillary electrophoresis was performed on a Perkin-Elmer
model 270A-HT apparatus equipped with a UV/vis detector set
at 254 nm, a temperature control device maintained at 25 °C,
and a data acquisition and analysis system (Turbochrom). A
fused-silica capillary (Polymicro Technologies) was employed (75
µm i.d., 375 µm o.d., and 72 cm length). A detection window of
approximately 0.2 cm was created at 50 cm from the capillary
inlet by removing the polyamide coating. Samples were injected
by the hydrodynamic method, at 5 mmHg for 1-5 s. The
electrolyte was 0.2 mM CTAB (hexadecyltrimethylammonium
bromide) and 10 mM Na2CrO4. The electrophoresis system was
operated under reversed polarity and a constant voltage of -30

Carbonyl Oxidation into Triplets by Peroxynitrite
Chem. Res. Toxicol., Vol. 13, No. 5, 2000 319
relatively high concentrations of the enol form in aqueous
medium (2.8 and 15.9%, respectively, from Table 1), and
the aldehyde IBAL (Ke ) 1.8 × 10-4 in water) (27), which
undergoes fast enolization under bifunctional phosphate
catalysis (28), are readily oxidized by the dissolved
oxygen. The “bifunctional” phosphate catalysis of IBAL
enolization mentioned here results from the concomitant
proton donor (to the carbonyl oxygen of IBAL) and proton
acceptor (from the methyne hydrogen of IBAL) capacity
of the H2PO4- anion. The poorly enolizable monoketones
(Ke ∼ 10-8 in water) (36-39), although prone to nucleo-
philic addition of peroxynitrite, do not appreciably con-
sume oxygen. This is also the case of 2,2-dimethylpro-
panal, a nonenolizable IBAL homologue, treated with
peroxynitrite under identical experimental conditions
(not shown). Similarly, acetaldehyde (22), whose Ke is
10-5 in water (36), and pyruvate (23) consume little
oxygen when treated with peroxynitrite. The reactivity
order toward oxygen (â-diketones > IBAL > acetalde-
hyde, 2,2-dimethylpropanal, 2-pentanone, 3-methylbu-
tanone, and pyruvate) appears to be dictated by the easy
enolization of substrate and of one-electron oxidation (see
anodic oxidation peaks in Table 1). Moreover, the lack of
oxygen consumption by acetaldehyde and monoketones,
which might be expected to undergo peroxynitrite nu-
cleophilic addition followed by adduct homolytic cleavage,
â-scission, oxygen addition to resulting carbon-centered
radicals, and Russell reactions (21, 22), suggests that the
oxygen uptake observed with â-diketones and IBAL
cannot be associated with the operation of route A in
Scheme 1. Partial oxygen depletion by IBAL may be
explained by the competition between nucleophilic attack
of peroxynitrite on IBAL and electron abstraction (route
A vs routes B and C in Scheme 1).
The extent of total oxygen consumption by IBAL as a
function of either peroxynitrite or IBAL concentration
roughly parallels the accompanying chemiluminescence
(maximum intensity) elicited upon addition of a triplet
energy detector (31), the 9,10-dibromoanthracene-2-sul-
fonate ion (DBAS) (panel A or B of Figure 2, respectively).
A double-reciprocal plot of the DBAS-sensitized chemi-
luminescence intensity versus DBAS concentration
(1-10 µM) gave a straight line, the slope of which reflects
the product of the excited donor lifetime, τ, and the rate
constant kET of triplet-singlet energy transfer from
acetone to DBAS (31). The slope kETτ was determined to
be 7.5 × 104 M-1, from which the lifetime of triplet
acetone could be estimated to be roughly 19 µs assuming
kET ) 4 × 109 M-1 s-1 (31). This agrees well with triplet
acetone lifetime values reported in the literature (31),
which range from 2 to 20 µs, depending on the concentra-
tion of dissolved oxygen, a triplet quencher. Addition of
9,10-diphenyl-2-anthracenesulfonate ions, capable of de-
tecting excited singlets but not triplets (31), did not
enhance chemiluminescence, attesting to the triplet
nature of the excited donor. The reaction of MP with
peroxynitrite in a phosphate buffer also emits light, but
the rapid kinetics (>90% CL is released within 4 s) did
not allow for accurate measurements by our photon-
counting equipment (not shown).
F igu r e 1. Total oxygen uptake by aldehydes and ketones (all
at 10 mM) treated with peroxynitrite (500 µM) in 100 mM
phosphate buffer (pH 7.2) at 25 °C. 2-P, pentanone; MB,
3-methyl-2-butanone; IBAL, isobutanal; MP, 3-methyl-2,4-pen-
tanedione; PD, 2,4-pentanedione.
Ta ble 1. En oliza tion of Ca r bon yls in a n Aqu eou s Med iu m
3-methyl-
2,4-pentanedione 2,4-pentanedione
propertya
isobutanal
Ke
Epa (V)
1.8 × 10-4 b
0.19c
0.029c
1.99
d
>2.75
2.53
pKa
11.6e
8.87f
10.65f
17e
k (M-1 s-1
)
8.5 × 10-5 g
2.3 × 103
e
a
Ke, enolization equilibrium constant; pKa, enol dissociation
constant; Epa, anodic potential; k, second-order rate constant (in
the presence of phosphate). b Ref 33. c Ref 27. Epa was determined
d
by cyclic voltammetry in acetonitrile containing 0.1 M NaClO4 with
Pt bead (working), Ag/AgI (reference), and Pt plate (auxiliary)
electrodes and expressed vs NHE (this work). e Ref 34. f Ref 28.
g
Ref 35.
kV. External calibration was carried out with stock solutions
of organic solutes: formate (retention time of 2.3 min), acetate
(2.8 min), nitrite (1.9 min), nitrate (2.0 min), and carbonate (2.5
min). Aliquots of peroxynitrite and HCl (sufficient to obtain a
given pH in the reaction mixture) were deposited separately onto
the inner walls of an Eppendorf tube containing 1.5 mL of
aqueous substrate, followed by homogenization via vigorous
vortexing. The desired final pH was checked in each case.
An Applied Photophysics (model SX 18 MV) stopped-flow
spectrophotometer was used for kinetic measurements. Peroxy-
nitrite decay was monitored at 340 nm, where interference due
to the IBAL and MP substrates is minimal. Stock solutions of
substrates in deionized water were mixed with the phosphate
buffer less than 1 h before being used.
Resu lts a n d Discu ssion
Oxygen Up ta k e a n d Ch em ilu m in escen ce. Total
oxygen depletion occurs within seconds when IBAL, MP,
or 2,4-pentanedione (but not 2-pentanone or 3-methyl-
2-butanone) (Figure 1) is treated with peroxynitrite under
standard experimental conditions, i.e., 10 mM carbonyl
compound and 500 µM peroxynitrite in normally aerated
0.10 M phosphate buffer (pH 7.2). Relevant physicochem-
ical constants of these compounds such as enolization
equilibrium constants (Ke), enolization rate constants (k1),
enol pKa values, and anodic potentials (Epa) are sum-
marized in Table 1 (27, 28, 33-35). The Ke values of
â-diketones (such as MP and 2,4-pentanedione) are much
higher than those of monocarbonyls because their enol
forms are stabilized by intramolecular hydrogen bonds
and by conjugation of the carbon-carbon double bond
with the carbonyl function. Figure 1 shows that the two
â-diketones, MP and 2,4-pentanedione, which exhibit
The extent of total oxygen consumption by the IBAL/
peroxynitrite system is 12-fold higher in 100 mM arsen-
ate buffer (pH 7.2) than in phosphate buffer at the same
concentration (not shown). Arsenate is known to act more
efficiently than phosphate as a bifunctional catalyst of
carbonyl enolization (24, 28).

320 Chem. Res. Toxicol., Vol. 13, No. 5, 2000
Knudsen et al.
F igu r e 4. pH dependence of the observed pseudo-first-order
rate constant (kobs) for isobutanal reaction in 500 (1), 250 (2),
and 100 mM (3) phosphate buffer (pH 7.2). Peroxynitrite (500
µM) was allowed to react at 25 °C with 10 mM IBAL. The points
were adjusted using the following Lorentzian function: y ) yo
+ (2A/π)[W/[4(X - Xc)2 + W2]], where yo ) offset, Xc ) center,
W ) width, and A ) area.
F igu r e 2. (A) Total oxygen consumption and maximum chemi-
luminescence intensity by 20 mM isobutanal (IBAL) at different
peroxynitrite concentrations in 100 mM phosphate buffer (pH
7.2) at 25 °C. (B) Oxygen consumption and maximum chemi-
luminescence by 500 µM peroxynitrite at different IBAL con-
centrations, in 100 mM phosphate buffer (pH 7.2).
F igu r e 5. pH dependence of the observed pseudo-first-order
rate constant (kobs) for 3-methyl-2,4-pentanedione (MP) reaction
in 500 mM phosphate buffer (pH 7.2). Peroxynitrite (500 µM)
was allowed to react with 10 mM MP at 25 °C.
keto form. The IBAL pH profile shown in Figure 3 also
suggests that both the peroxynitrite anion and the
peroxynitrous acid act as oxidants in this case.
The rate of disappearance of peroxynitrite upon addi-
tion of excess MP or IBAL (both of which absorb strongly
in the 200-320 nm range), monitored in a stopped-flow
instrument at 340 nm, follows first-order kinetics. The
slope gave an apparent second-order constant of 166 M-1
s-1 for MP and 344 M-1 s-1 for IBAL, in 100 mM
phosphate buffer (pH 7.2) at 25 °C. These values are
lower than those obtained for simple aliphatic alde-
hydes: propionaldehyde, 530 M-1 s-1 (21); and acetalde-
hyde, 680-830 M-1 s-1 (21, 22). The plot of the first-order
constant (kobs) for peroxynitrite disappearance, obtained
by subtracting the rate constant values for spontaneous
peroxynitrite decomposition from the values measured
with the complete system, versus the pH roughly delin-
eates a bell-shaped curve for both IBAL (Figure 4) and
MP (Figure 5). As previously reported in the literature,
the pH profile for spontaneous peroxynitrite decomposi-
tion follows the titration curve of peroxynitrous acid, with
observed rate constants of 0.55 s-1 at pH 7.0, 1.0 s-1 at
F igu r e 3. pH profile of isobutanal (IBAL) and 3-methyl-2,4-
pentanedione (MP) treated with 500 µM peroxynitrite in 500
mM phosphate buffer (pH 7.2) at 25 °C with 10 mM IBAL and
1 mM MP.
p H P r ofile. The pH profiles of the peroxynitrite-
promoted oxidations of IBAL and MP were obtained by
two methods: total oxygen consumption and stopped-flow
spectrophotometry. The pH profile of the MP oxygen-
consuming reaction (Figure 3) roughly describes the
peroxynitrous acid titration curve (pKa ) 6.8 in 100 mM
phosphate) (11), pointing to peroxynitrite anion as the
initiator of MP aerobic oxidation (route B in Scheme 1).
With IBAL, a subtle increase in the extent of oxygen
uptake in the pH region close to the peroxynitrous acid
pKa value may be related to the loss of enol substrate by
nucleophilic addition of peroxynitrite to the substrate
pH 6.5, and 1.1 s-1 at pH 6.0 (0.43, 0.9, and 1.3 s-1
,
respectively, from ref 14). The catalytic effect of increas-
ing phosphate concentration (100-500 mM) on the rate
constants of the IBAL reaction with peroxynitrite is
ratified by this experiment (differences in the ionic
strength were compensated by addition of NaCl).
Unlike the profiles reported for acetaldehyde and
pyruvate (21, 22), which match the peroxynitrite titration
curve, the bell-shaped pH profiles of the reaction of IBAL
and MP with peroxynitrite may result from the contribu-

Carbonyl Oxidation into Triplets by Peroxynitrite
Chem. Res. Toxicol., Vol. 13, No. 5, 2000 321
Ta ble 2. P r od u ct An a lysis for th e Ca r bon yl (10 m M)/
P er oxyn itr ite (500 µM) Rea ction s
no. of
experiments
[RCO2-] (mM)
formate
[R2CdO] (mM)
substrate
acetone
IBAL control
IBAL (pH 7.5)
IBAL (pH 11)
3
3
6
0.06 ( 0.01
0.32 ( 0.03
0.66 ( 0.11a
0.80
no. of
[RCO2-] (mM) [R2CdO] (mM)
substrate
experiments
acetate
biacetyl
MP control
MP (pH 7.5)
MP (pH 11)
3
3
6
0.78 ( 0.04
1.40 ( 0.30
6.36 ( 0.87b
9
no. of
experiments
[RCO2-] (mM)
acetate
substrate
2,4-pentanedione (pH 7.5)
2,4-pentanedione (pH 11)
3
3
0.50 ( 0.02
1.20 ( 0.10
no. of
experiments
[RCO2-] (mM)
acetate
substrate
2-pentanone (pH 11)
2-pentanone (pH 7.5)
3
3
ND
0.14 ( 0.04
a
Acetone detected by gas chromatography within 20% error.
b Biacetyl detected by gas chromatography and HPLC/fluorescence
within 20% error.
F igu r e 6. EPR spectra of DBNBS radical adducts obtained
after incubation for 3 min at room temperature of 20 mM
DBNBS and 500 µM peroxynitrite in phosphate buffer (pH 7.2)
containing 0.1 mM diethylenetriaminepentaacetic acid (DTPA)
in the presence of 10 mM IBAL (A, C, and D), 10 mM 3-methyl-
2-butanone (MB), and 20 mM HCO3-. Instrumental conditions
were as follows: microwave power, 2 mW; modulation ampli-
tude, 1 G; time constant, 0.32 s; and gain, 1 × 106.
tion of opposite effects: (i) The ascending part of the
curve can be related to the increase in the ONOO-
concentration, here assumed to be a one-electron oxidant
of the substrate in route B in Scheme 1 or a nucleophilic
agent in route A, while (ii) the descending part of the
-
curve may be determined by either a decrease in H2PO4
concentration above pH 7 (pKa2 for phosphoric acid ) 7.2,
-
H2PO4 presumably being a more effective bifunctional
Due to the presence of CO2 in the reaction mixtures,
resulting from bicarbonate contamination in both the
buffer and peroxynitrite stock solution, one might at-
tribute the initiation of the oxygen consumption to
CO2ONOO- or to one of its oxidizing decomposition
products (3, 18). The possibility that this is not the case,
however, is suggested by the reduction (30 and 50%) of
the extent of total oxygen uptake caused by the addition
of bicarbonate (1 and 5 mM) to the MP (0.5 mM)- and
IBAL (10 mM)-containing standard reaction mixtures.
Spin-trapping studies described below confirm that the
addition of bicarbonate (20 mM) to the IBAL (10 mM)-
containing system does lead to higher amounts of
CO2ONOO-, which competes with nucleophilic addition
of peroxynitrite to IBAL.
catalyst of IBAL enolization than its conjugate base,
which favors of route B) or a decrease in carbonyl
concentration due to base-catalyzed enolization at in-
creased pH values (which would favor route A). The latter
hypothesis, i.e., an increased level of enolization upon the
pH being raised, is more attractive given that, when enol/
enolate formation is followed spectrophotometrically at
280 nm, a batochromic shift and a strong increase in
absorbance with increasing pH can be observed above pH
7. Ascribing the descending part of the pH profile to
deprotonation of the substrate (enol IBAL), giving a less
reactive form (enolate IBAL), as suggested previously in
the case of the reaction of peroxynitrite with ascorbate
(pKa ) 4.25) (40), collides with the fact that enol IBAL is
a very weak acid (pKa ) 11.6; Table 1). We are, thus,
inclined to conclude that the pH profiles for the reaction
of peroxynitrite with IBAL and MP, accompanied by
following the disappearance of peroxynitrite in the
stopped-flow apparatus, reflect the influence of pH on the
nucleophilic addition of peroxynitrite to carbonyl.
For comparative purposes, we reproduced the pH
profile for the reaction of peroxynitrite with acetaldehyde
in 500 mM phosphate buffer, previously shown by Nakao
et al. (22) to mirror the peroxynitrous titration curve, in
100 mM phosphate buffer under comparable experimen-
tal conditions (not shown). One can see a descending
trend at pHs above 7, as in the case of IBAL and MP,
which might result from the loss of the aldehyde form
by phosphate-catalyzed enolization at a high phosphate
concentration. On the other hand, the higher kobs values
for peroxynitrite disappearance obtained at higher phos-
phate concentrations may be attributed to phosphate-
assisted addition (base catalysis) of peroxynitrite to
acetaldehyde.
P r od u ct An a lysis. Formate and acetate concentra-
tions were determined by capillary electrophoresis in the
spent reaction mixtures of air-equilibrated aqueous IBAL
and MP (both at 10 mM) treated with 500 µM peroxyni-
trite (containing 0.5 M NaOH) under vigorous stirring
at final pHs of 7-8 (adjusted with HCl) and 11 (Table
2). Capillary electrophoresis also showed the presence of
nitrate (0.4-0.7 mM), nitrite (0.5-2.5 mM), and carbon-
ate (1-2 mM) in the spent reaction mixture. The latter
values are not reliable in a discussion of the data because
peroxynitrite preparations are normally contaminated
with these ions at concentrations in the millimolar range
and carbonate varies considerably in buffers and peroxy-
nitrite preparations.
Acetate was analyzed in the reaction mixtures of 2,4-
pentanedione and 2-pentanone, for comparison. Acetone
was identified in the phosphate (100 mM, pH 7.2)-
buffered IBAL-containing reaction mixture by mass
spectroscopy by comparison with an authentic sample of

322 Chem. Res. Toxicol., Vol. 13, No. 5, 2000
Knudsen et al.
Ta ble 3. Hyp er fin e Sp littin g Con sta n ts (G) for Ad d u ct Ra d ica ls w ith eith er DBNBS or MNP Obta in ed by Tr ea tm en t of
Ca r bon yls w ith P er oxyn itr itea
b
a
b
The spectra were obtained at room temperature in 100 mM phosphate buffer (pH 7.2). MB, 3-methyl-2-butanone.
acetone, and quantified by gas chromatography; 0.8 mM
acetone was found in the spent reaction mixture obtained
from 10 mM IBAL and 500 µM peroxynitrite. Acetone
was not found in unbuffered reaction mixtures, probably
because of the importance of phosphate catalysis in the
oxygen-consuming reaction. In addition, biacetyl was
analyzed in the MP (10 mM)-containing reaction mixture
by both HPLC/fluorescence detection (32) and gas chro-
matography/flame ionization detection, calibrated against
authentic samples of biacetyl; values of 9.0 and 8.8 mM
were found with the two methods, respectively.
Examination of product yields (listed in Table 2) and
the enolization properties of IBAL, MP, and 2,4-pen-
tanedione (given in Table 1) allows for the following
conclusions. (1) In every case, the observed yield of
carboxylic products (Table 2) is greater than the upper
limit predicted from the quantitative stoichiometric reac-
tion of peroxynitrite (500 µM added peroxynitrite) with
the substrate, suggesting that a chain reaction occurs in
the presence of oxygen. (2) The more readily enolizable
and oxidizable the carbonyl substrate (Table 1), the
higher the product yield; thus, reactivity decreases in the
following order: MP > 2,4-pentanedione > IBAL >
2-pentanone (Figure 1 and Table 2). (3) The observed
stoichiometries for acetone/formate (roughly 1:1) and
biacetyl/acetate (roughly 1.5:1) mixtures formed in the
IBAL and MP reactions, respectively, do not support
product formation exclusively in reactions initiated by
ONOO- nucleophilic addition (Table 2).
nates from two spin adducts: a six-line spectrum (aN )
•
14.5 G; aH ) 9.7 G) that can be assigned to a CHR2
[•CH(CH3)2]-DBNBS adduct (Table 3) and a triplet (aN
) 14.5 G) that can be attributed to an adduct with a
tertiary radical (IBAL•). Formation of an isopropyl radical
is predicted by route I in Scheme 2 (reaction initiated by
addition of ONOO- to IBAL) and the tertiary radical by
electron abstraction from IBAL enolate (route II in
Scheme 2). Because the aH constant of the six-line
spectrum does not agree with the only value reported for
an isopropyl adduct (aH ) 5.85 G) (41), we carried out
the same experiment with 3-methyl-2-butanone (MB), an
IBAL analogue, which is expected to yield the isopropyl
radical by route I in Scheme 2, initiated by the addition
of ONOO- to the carbonyl group. Indeed, an equivalent
six-line spectrum appears to favor our assignment. The
triplet signal seen after 4 min does not appear at 4 min
in the control experiment without peroxynitrite and tends
to increase with time (30 min) in the complete reaction
mixture, probably due to the accumulation of the oxidized
“ene” adduct of enol IBAL with DBNBS, which is
expected to exhibit the same EPR adduct signal. The fact
that the six-line spectrum may be due to an adduct with
an isopropyl radical formed from route I (Scheme 2) is
indicated by quenching of its signal upon addition of 20
mM bicarbonate, a source of CO2, that should compete
with IBAL in ONOO- nucleophilic carbonyl addition (21).
As already mentioned, the inhibitory effect of bicarbonate
as a sink for removing ONOO- can also be evaluated by
the observed decline in the extent of oxygen consumption
by IBAL plus peroxynitrite (∼50% with 5 mM bicarbon-
ate).
EP R Sp in -Tr a p p in g Stu d ies. EPR spin-trapping
studies with 3,5-dibromo-4-nitrosobenzenesulfonic acid
(DBNBS) and 2-methyl-2-nitrosopropane (MNP) were
conducted to detect and possibly identify carbon-centered
radical intermediates. Figure 6 summarizes the spin-
trapping studies conducted with IBAL/peroxynitrite in
the presence of DBNBS. The observed spectrum origi-
Spin-trapping experiments with the MP/peroxynitrite/
DBNBS system revealed only a triplet signal (Table 3),
which can be assigned to a tertiary radical adduct with
MP• (route II in Scheme 3). Control experiments in the

Carbonyl Oxidation into Triplets by Peroxynitrite
Chem. Res. Toxicol., Vol. 13, No. 5, 2000 323
Sch em e 2. Gen er a tion of Tr ip let Aceton e by Rea ction of Isobu ta n a l (IBAL) w ith P er oxyn itr ite
absence of peroxynitrite show no triplet signal at 4 min,
although this signal does develop with time due to
accumulation of the oxidized “ene” adduct of enol MP
with DBNBS and is enhanced upon addition of 20 µM
ferricyanide (not shown). Similar experiments carried out
with MNP (Figure 7) revealed further significant details
that are important to confirm the contribution of route
II (Scheme 3) to the chemiluminescent reaction. Besides
a triplet signal (aN ) 15.2 G), which can be attributed to
an adduct with the tertiary MP• radical, these experi-
ments revealed the presence of a triplet that can be
assigned to an adduct with acetyl radical (aN ) 8.3 G),
previously characterized (aN ) 8.3 G) during treatment
of 2,4-pentenedione with HRP/H2O2 (42). The “ene”
adduct is almost absent. Without peroxynitrite, a triplet
signal (aN ) 17.0 G) that can be assigned to the MNP
oxidation product can be detected; extensive MNP oxida-
tion may be coupled to substrate autoxidation.
shown in Figure 7 and illustrated in Scheme 3. Biacetyl
in the triplet state has previously been shown to be a
major product of the MP/HRP/H2O2 system (25). We
propose here that EPR-detected acetyl radicals from
MP/HRP/H2O2 or MP/peroxynitrite may arise from car-
bonyl-carbonyl cleavage of electronically excited triplet
biacetyl (43), thus reinforcing the occurrence of route II
in Scheme 3.
Con clu sion s
Our hypothesis is that, as in the reaction of IBAL and
MP with HRP/H2O2, a reaction with ONOO- can be
initiated by phosphate-catalyzed enolization of the alde-
hyde or ketone, followed by one-electron oxidation of the
enol by peroxynitrite to give the resonance-stabilized
enolyl radical (route II in Schemes 2 and 3). With MP,
the phosphate-catalyzed enolization is not as strong a
determinant as with IBAL because the MP enol form is
already present to the extent of about 2.8% in water
(Table 1) (hence, the ease with which its products can be
analyzed by capillary electrophoresis and the product
yield data correlated using GC and HPLC techniques).
For comparison, MP oxidation was also conducted
with HRP/H2O2 as an oxidant in the presence of MNP,
and the same acetyl radical adduct was detected
(triplet, aN ) 8.3 G). This triplet can also be obtained in
high yields by reaction of biacetyl with peroxynitrite, as

324 Chem. Res. Toxicol., Vol. 13, No. 5, 2000
Knudsen et al.
Sch em e 3. Gen er a tion of Tr ip let Bia cetyl by Rea ction of 3-Meth yl-2,4-p en ta n ed ion e w ith P er oxyn itr ite
One-electron oxidation of either IBAL or MP appears
to compete with nucleophilic addition of ONOO- to the
carbonyl substrate, followed by fragmentation to radicals
(route I in Schemes 2 and 3). The greater the facility of
substrate enolization (in the case of â-diketones and
phosphate catalysis) and/or oxidation (in the case of
R-methyl substitution), the more important the contribu-
tion of one-electron abstraction. For both MP and IBAL,
bell-shaped pH profile curves suggest two rate-limiting
reactions. The ascending curve matches the onset of the
titration curve of peroxynitrous acid and, hence, points
to the ONOO- concentration as rate-limiting. This de-
pendence is also shown when the reaction is monitored
by oxygen uptake, which indicates that ONOO- is the
one-electron oxidant of the substrates. The curve obtained
spectrophotometrically following the rate of peroxynitrite
disappearance reaches a maximum at pH 7.2 (IBAL) or
7.0 (MP) and then decreases, accompanying both the
second half of the H2PO4- titration curve and the loss of
carbonyl substrate by base-catalyzed enolization. In
contrast, linear aldehydes exhibit a pH profile (20) that
is interpreted from the reaction kinetics as being domi-
nated by nucleophilic addition to the carbonyl compound.
We propose that the pH profiles of the compounds studied
here, obtained by the stopped-flow technique, also mirror
nucleophilic addition to the substrate (route I in Scheme
2), whereas those determined by oxygen consumption
refer to the competing reaction initiated by electron
abstraction from the enol substrates, leading to triplet
product formation and hence chemiluminescence (route
II in Scheme 2).
Insertion of dissolved molecular oxygen into the enolyl
radical is expected to produce an R-hydroperoxyl carbonyl
radical, which may, in turn, abstract a hydrogen atom
from another IBAL or MP molecule, thereby propagating
a chain reaction and producing a dioxetane intermediate
by cyclization (route II in Scheme 2). Thermolysis of the
dioxetane intermediates then yields triplet acetone plus
formic acid from IBAL and biacetyl plus acetate from MP.
Nucleophilic addition of ONOO- to the substrate should

Carbonyl Oxidation into Triplets by Peroxynitrite
Chem. Res. Toxicol., Vol. 13, No. 5, 2000 325
cytochrome c, myeloperoxidase, lipoxygenase, and pros-
taglandin-H synthase, have been shown to catalyze such
processes.
(ii) The second is dismutation of alkoxyl radicals
formed by one-electron reduction of alkyl hydroperoxides
(Φ3 ) 10%) (45).
(iii) The third is disproportionation of alkylperoxyl
radicals formed in lipid peroxidation chains (Φ3 < 0.01%)
(46, 47).
(iv) The final reaction is one-electron oxidation of “ene”
hydroperoxides by horseradish peroxidase (HRP) or per-
oxynitrite, also via dioxetane intermediates (48).
F in a l Rem a r k s. Considering that (i) aldehydes are
ubiquitous compounds in biological systems under physi-
ological and pathological conditions, (ii) peroxynitrite has
increasingly been implicated in cell toxicity and signaling,
and (iii) n,π* triplet carbonyls may be viewed as alkoxyl
radicals and, therefore, play a deleterious role in biomol-
ecules, it is tentatively proposed that carbonyl compound
reactions with peroxynitrite may constitute another
chemical source of long-living reactive triplets operative
in “photobiochemical processes without light”.
Ack n ow led gm en t. This work was supported by
grants from the Fundac¸a˜o de Amparo a` Pesquisa do
Estado de Sa˜o Paulo (FAPESP), the Programa de Apoio
a Nu´cleos de Exceleˆncia (PRONEX), Conselho Nacional
de Desenvolvimento Cientifico e Tecnolo´gico (CNPq), and
the von Humboldt Foundation. We are grateful to Dr.
Omar El Seoud, Dr. Wilson F. J ardim, and Dr. Lilian R.
F. de Carvalho for providing access to the stopped-flow
spectrophotometer, the gas chromatograph, and capillary
electrophoresis system, respectively. We also thank Dr.
Frank Quina, Dr. Brian Bandy, and Ms. Beatrice Allain
for reading the manuscript and Dr. The´re`se Wilson and
Ohara Augusto for helpful discussions. Samples of MB
were kindly provided by Dr. Bruce Freeman and Dr.
Enrique Cadenas.
F igu r e 7. EPR spectra of MNP radical adducts obtained after
incubation for 4 min at room temperature of 10 mM MNP, 10
µM HRP (C), and 500 µM peroxynitrite in 100 mM phosphate
buffer (pH 7.2) containing 0.1 mM DTPA in the presence of 10
mM MP (A-C and E) and 10 mM biacetyl. Instrumental
conditions were as follows: microwave power, 20 mW; modula-
tion amplitude, 1 G; time constant, 0.32 s (A-C and E) and 0.16
s (D); gain, 5.0 × 104 (A and D) and 5.0 × 105 (B, C, and E).
also generate the same products. However, chemical
yields of biacetyl and acetone greatly exceed the added
peroxynitrite concentration, suggesting that the oxygen-
consuming chain reaction initiated by electron abstrac-
tion from the substrate enols predominates over that
initiated by nucleophilic addition of ONOO-.
Refer en ces
With regard to the weak chemiluminescence associated
with generation of triplet species in the reaction of
peroxynitrite with enolizable carbonyl compounds, it
should be kept in mind that, due to their intrinsically
long lifetimes (microsecond), triplet species have been
considered suitable candidates for participation in bimo-
lecular processes, such as energy transfer and chemical
reactions (e.g., isomerization, cycloaddition, Norrish cleav-
age, and hydrogen abstraction) (26). In addition to the
peroxynitrite-driven reaction described in this work,
various chemical reactions leading to triplet carbonyl
formation may also be of biological relevance, including
the following.
(i) One is heme protein-catalyzed aerobic oxidation of
carbonyl compounds bearing an R-hydrogen, such as
IBAL (24) and MP (25), via dioxetane intermediates.
Dioxetanes have long been known as clean and efficient
sources of triplet carbonyls (Φ3 ∼ 10-60%) (26, 44). Using
the IBAL/HRP system as a source of triplet acetone,
Cilento (26) demonstrated over the past two decades the
feasibility of “photochemistry without light” with, for
example, colchicine, riboflavin, chlorophyll, phytochromes,
lysozyme, isolated or intracellular DNA, and several
other synthetic electronic energy acceptors (e.g., dibro-
moanthracene and xanthene dyes). Not only HRP but
also other proteins and enzymes, including myoglobin,
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