Reaction between peroxynitrite and triphenylphosphonium-substituted arylboronic acid isomers: Identification of diagnostic marker products and biological implications

Article abstract of DOI:10.1021/tx300499c

Aromatic boronic acids react rapidly with peroxynitrite (ONOO^-) to yield phenols as major products. This reaction was used to monitor ONOO ^- formation in cellular systems. Previously, we proposed that the reaction between ONOO^- and arylboronates (PhB(OH)₂) yields a phenolic product (major pathway) and a radical pair PhB(OH)₂O ^?-...^?NO₂ (minor pathway). [ Sikora, A. et al. (2011) Chem. Res. Toxicol.24, 687-697 ]. In this study, we investigated the influence of a bulky triphenylphosphonium (TPP) group on the reaction between ONOO^- and mitochondria-targeted arylboronate isomers (o-, m-, and p-MitoPhB(OH)₂). Results from the electron paramagnetic resonance (EPR) spin-trapping experiments unequivocally showed the presence of a phenyl radical intermediate from meta and para isomers, and not from the ortho isomer. The yield of o-MitoPhNO₂ formed from the reaction between o-MitoPhB(OH)₂ and ONOO^- was not diminished by phenyl radical scavengers, suggesting a rapid fragmentation of the o-MitoPhB(OH) ₂O^?- radical anion with subsequent reaction of the resulting phenyl radical with ^?NO₂ in the solvent cage. The DFT quantum mechanical calculations showed that the energy barrier for the dissociation of the o-MitoPhB(OH)₂O^?- radical anion is significantly lower than that of m-MitoPhB(OH)₂O ^?- and p-MitoPhB(OH)₂O^?- radical anions. The nitrated product, o-MitoPhNO₂, is not formed by the nitrogen dioxide radical generated by myeloperoxidase in the presence of the nitrite anion and hydrogen peroxide, indicating that this specific nitrated product may be used as a diagnostic marker product for ONOO^-. Incubation of o-MitoPhB(OH)₂ with RAW 264.7 macrophages activated to produce ONOO^- yielded the corresponding phenol o-MitoPhOH as well as the diagnostic nitrated product, o-MitoPhNO₂. We conclude that the ortho isomer probe reported here is most suitable for specific detection of ONOO^- in biological systems.

Full text of DOI:10.1021/tx300499c

Article
pubs.acs.org/crt
Reaction between Peroxynitrite and Triphenylphosphonium-
Substituted Arylboronic Acid Isomers: Identification of Diagnostic
Marker Products and Biological Implications
Adam Sikora,*,†,‡ Jacek Zielonka,† Jan Adamus,‡ Dawid Debski,‡ Agnieszka Dybala-Defratyka,‡
Bartosz Michalowski,‡ Joy Joseph,† Richard C. Hartley,§ Michael P. Murphy,∥
and Balaraman Kalyanaraman*,†
†Department of Biophysics and Free Radical Research Center, Medical College of Wisconsin, Milwaukee, Wisconsin, United States
‡Institute of Applied Radiation Chemistry, Lodz University of Technology, Lodz, Poland
§Centre for the Chemical Research of Ageing, WestCHEM School of Chemistry, University of Glasgow, Glasgow G12 8QQ, United
Kingdom
∥Mitochondrial Biology Unit, Medical Research Council, Cambridge, United Kingdom
S
* Supporting Information
ABSTRACT: Aromatic boronic acids react rapidly with
peroxynitrite (ONOO−) to yield phenols as major products.
This reaction was used to monitor ONOO− formation in
cellular systems. Previously, we proposed that the reaction
between ONOO− and arylboronates (PhB(OH)2) yields a
phenolic product (major pathway) and a radical pair
PhB(OH)2O•−···•NO2 (minor pathway). [Sikora, A. et al.
(2011) Chem. Res. Toxicol. 24, 687−697]. In this study, we
investigated the influence of a bulky triphenylphosphonium
(TPP) group on the reaction between ONOO− and
mitochondria-targeted arylboronate isomers (o-, m-, and p-
MitoPhB(OH)2). Results from the electron paramagnetic
resonance (EPR) spin-trapping experiments unequivocally showed the presence of a phenyl radical intermediate from meta and
para isomers, and not from the ortho isomer. The yield of o-MitoPhNO2 formed from the reaction between o-MitoPhB(OH)2
and ONOO− was not diminished by phenyl radical scavengers, suggesting a rapid fragmentation of the o-MitoPhB(OH)2O•−
•
radical anion with subsequent reaction of the resulting phenyl radical with NO2 in the solvent cage. The DFT quantum
mechanical calculations showed that the energy barrier for the dissociation of the o-MitoPhB(OH)2O•− radical anion is
significantly lower than that of m-MitoPhB(OH)2O•− and p-MitoPhB(OH)2O•− radical anions. The nitrated product, o-
MitoPhNO2, is not formed by the nitrogen dioxide radical generated by myeloperoxidase in the presence of the nitrite anion and
hydrogen peroxide, indicating that this specific nitrated product may be used as a diagnostic marker product for ONOO−.
Incubation of o-MitoPhB(OH)2 with RAW 264.7 macrophages activated to produce ONOO− yielded the corresponding phenol
o-MitoPhOH as well as the diagnostic nitrated product, o-MitoPhNO2. We conclude that the ortho isomer probe reported here is
most suitable for specific detection of ONOO− in biological systems.
detecting cellular hydrogen peroxide.5,6 Mitochondria-targeted
INTRODUCTION
■
TPP chemical probes were designed to detect mitochondrial
Recent research has focused on the development of targeted
ROS (MitoPY, MitoTEMPO-H).7,8 Arylboronic-based TPP
probes for detecting mitochondrial reactive oxygen and
nitrogen species (ROS/RNS).1,2 One of the ways in which a
probe MitoB [m-MitoPhB(OH)2, (2-boronobenzyl)-
triphenylphosphonium salt] (Figure 1) was used to detect
small molecule probe can be transported to mitochondria is
H2O2 in living Drosophila.9,10 In this study, we investigated the
through covalent attachment to a lipophilic, delocalized
reaction between peroxynitrite (ONOO−) and the three
isomers of MitoB.
positively charged triphenylphosphonium (TPP) cation.3
Because of both the cationic and lipophilic character, chemical
probes tagged with TPP cation accumulate preferentially within
mitochondria.
Our previous studies have shown that arylboronates can be
oxidized not only by H2O2 but also by other biologically
Arylboronic acids react with hydrogen peroxide stoichio-
metrically to form the corresponding phenols.4 Recently,
boronate-based fluorogenic probes were synthesized for
Received: December 12, 2012
© XXXX American Chemical Society
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formed from the reaction of o-MitoPhB(OH)2 with ONOO− is
unaffected by the phenyl radical scavengers, suggesting fast
fragmentation of the o-MitoPhB(OH)2O•− radical anion with
•
subsequent reaction of the resulting phenyl radical with NO2
in the solvent cage. Moreover, even in the presence of 2-
propanol, o-MitoPhNO2 is formed in relatively high yield (9%).
This is 9 times higher than that of m-MitoPhNO2 and p-
MitoPhNO2 under the same conditions. We conclude that
determination of both major and minor products from o-
MitoPhB(OH)2 will serve as a unique tool providing a
diagnostic marker for cellular ONOO− formation.
EXPERIMENTAL PROCEDURES
■
Chemicals. MitoPhB(OH)2 isomers, MitoPhNO2, and MitoPh
were synthesized according to a published procedure of triphenyl-
phosphine benzylation.17 All solutions were prepared using deionized
water (Millipore Milli-Q system). ONOO− was prepared according to
the published procedure,18 by reacting nitrite with H2O2. The
concentration of ONOO− was determined by measuring the
absorbance at 302 nm (ε = 1.7 × 103 M−1cm−1) in alkaline aqueous
solutions (pH > 12).18 DPTA-NONOate19,20 was from Cayman
Chemicals. Xanthine oxidase (XO) and myeloperoxidase (MPO) were
obtained from Sigma, and catalase was from Boehringer. All other
reagents (of the highest purity available) were from Sigma-Aldrich
Corp.
Figure 1. Structures of MitoPhB(OH)2 isomers and the major and
minor products of their reactions with peroxynitrite.
relevant oxidants including hypochlorite and ONOO−.11,12
Results showed that boronate-based fluorogenic probes are
suitable for real time monitoring of ONOO− formed in cell-free
systems and in activated macrophages.13,14 Peroxynitrite15,16
reacts with arylboronates rapidly (k ∼106 M−1s−1 at pH 7.4)
and stoichiometrically. The major oxidation products are
phenols (yield ∼85−90%).11 The minor products (10−15%)
are formed via a radical pathway of the reaction.12 The
proposed mechanism for the reaction between ONOO− and
arylboronates is presented in Scheme 1. The initial step of the
reaction involves the formation of an anionic adduct between
ONOO− and the boronate. Further cleavage of the O−O bond
results in the formation of phenol and nitrite (major, heterolytic
O−O cleavage pathway) or caged radical pair PhB-
(OH)2O•−···•NO2 (minor, homolytic O−O cleavage pathway).
The subsequent fragmentation of the PhB(OH)2O•− radical
anion leads to the formation of phenyl radical Ph• that reacts
with H-atom donors, nitrogen dioxide •NO2, or molecular
oxygen.
•−
•
•
•−
Determination of O2 and NO Fluxes. NO and O2 fluxes
were determined in phosphate buffer (100 mM, pH 7.4) containing
dtpa (100 μM) as described previously.14 Briefly, •NO fluxes were
determined by measuring the rate of the decomposition of DPTA-
NONOate by following the decrease in absorbance at 250 nm (ε = 8 ×
19
103 M−1 cm−1). Assuming that two molecules of NO are released
from the nitric oxide donor, the measured rate of NONOate
decomposition was multiplied by a factor of 2 to obtain the rate of
•NO release. The superoxide was generated from XO catalyzed
oxidation of hypoxanthine (HX). The flux of O2•− was determined by
monitoring the superoxide dismutase-inhibitable reduction of
cytochrome c following the increase in absorbance at 550 nm (Δε =
2.1 × 104 M−1cm−1).21
•
HPLC and UPLC/MS Analyses. HPLC analyses were performed
using an Agilent 1100 HPLC system equipped with fluorescence and
UV−vis absorption detectors. In the studies on the reaction profile of
peroxynitrite oxidation of MitoPhB(OH)2 isomers, 100 μL of sample
was injected into the HPLC system equipped with a C18 column
(Phenomenex, Kinetex, 100 × 4.6 mm, 2.6 μm) equilibrated with 10%
CH3CN [containing 0.1% (v/v) trifluoroacetic acid (TFA)] in 0.1%
TFA aqueous solution. The compounds were separated by a linear
Here, we compared the reaction profiles of ONOO− with
three isomeric TPP+-substituted phenylboronic acids (o-
MitoPhB(OH)2, m-MitoPhB(OH)2, and p-MitoPhB(OH)2)
(Figure 1). We determined that the yield of o-MitoPhNO2
Scheme 1
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Figure 2. UPLC analyses of products formed from the reaction between triphenylphosphonium-substituted arylboronic acids and peroxynitrite. (A)
o-MitoPhB(OH)2, (B) m-MitoPhB(OH)2, and (C) p-MitoPhB(OH)2. Incubation mixtures consisting of 200 μM o-, m-, and p-MitoPhB(OH)2 in
phosphate buffer (pH 7.4, 50 mM) containing dtpa (100 μM) and peroxynitrite at the indicated concentration. o-, m-, and p-MitoPhB(OH)2 and
their corresponding oxidation products were detected using UV absorption detection at 268 1.2 nm.
increase in CH3CN phase concentration from 10 to 100% over 7 min,
using a flow rate of 1.5 mL/min. Under these conditions, the following
retention times (in min) were observed: o-MitoPhB(OH)2, 4.31; o-
MitoPhOH, 4.50; o-MitoPhNO2, 4.61; MitoPh, 4.82; m-MitoPhB-
(OH)2, 4.05; m-MitoPhOH, 4.2; m-MitoPhNO2, 4.70; p-MitoPhB-
(OH)2, 3.98; p-MitoPhOH, 4.24; and p-MitoPhNO2, 4.71. The peak
areas detected by monitoring the absorption at 268 nm were used for
the quantitation.
linear increase in CH3CN phase (containing 0.1% (v/v) TFA)
concentration from 0 to 5% over 20 min, then further linear increase in
CH3CN phase concentration from 5 to 100% over the next 30 min,
using a flow rate of 1.0 mL/min. Under those conditions, tyrosine
eluted at 19.7 min, nitrotyrosine at 27.9 min, and dityrosine at 25.8
min. Formation of dityrosine was monitored with the use of a
fluorescence detector (λex.= 284 nm; λem. = 410 nm), and nitrotyrosine
was monitored at 350 nm with the use of absorption detector.
UV−Vis Absorption Measurements. The UV−vis absorption
spectra were collected using an Agilent 8453 spectrophotometer
equipped with a photodiode array detector and thermostatted cell
holder.
To characterize the synthesized triphenylphosphonium compounds
and to identify the products of the reaction of peroxynitrite with
triphenylphosphonium-substituted arylboronic acids, the UPLC/MS
analyses were performed using an Acquity UPLC Waters Ltd. system
coupled online to an LCT Premier XE (Waters) mass spectrometer
with a ToF mass detector. One microliter of sample was injected into
the UPLC system equipped with a C18 column (Waters Acquity BEH
C18, 100 × 2.1 mm, 1.7 μm) maintained at 40 °C and equilibrated
with 30% CH3CN [containing 0.1% (v/v) trifluoroacetic acid (TFA)]
in 0.1% TFA aqueous solution. The compounds were separated by a
linear increase in CH3CN phase concentration from 30 to 60% from
0.8 to 4 min, using a flow rate of 0.35 mL/min. Under these
conditions, the following retention times (in min) were observed: o-
MitoPhB(OH)2, 3.30; o-MitoPhOH, 3.52; o-MitoPhNO2, 3.59;
MitoPh, 3.91; m-MitoPhB(OH)2, 2.86; m-MitoPhOH, 3.16; m-
MitoPhNO2, 3.69; p-MitoPhB(OH)2, 2.76; p-MitoPhOH, 3.11; and
p-MitoPhNO2, 3.72. The compounds were detected by monitoring the
absorption at 268 1.2 nm. The mass spectrometer was operated in
W mode with lock mass correction. Lock mass solution (leucine-
enkephalin, reference mass: [M + H]+ 556.2771; [M + H]+ 557.2801)
was prepared at a concentration of 0.5 ng/μL in 50:50 (v/v), water/
acetonitrile solution and stored in 4 °C until further use. Data
acquisition and analyses were performed using the MassLynx 4.1 data
system software (Waters) and OriginPro 8.6 (OriginLab).
EPR Experiments. All electron paramagnetic resonance (EPR)
spectra were collected using a Bruker EMX spectrometer.
ABTS Oxidation. Typically, incubation mixtures used in EPR
experiments on ABTS oxidation consisted of 500 μM ABTS and 250
μM MitoPhB(OH)2 isomer in phosphate buffer (100 mM, pH 7.4)
containing dtpa (100 μM) and were rapidly mixed with bolus ONOO−
(250 μM). Samples were subsequently transferred to a 100 μL
capillary tube, and EPR measurements were started within 30 s after
the bolus addition of peroxynitrite. Spectrometer parameters were as
follows: scan range, 60 G; field set, 3505 G; time constant, 1.28 ms;
scan time, 42 s; modulation amplitude, 0.2 G; modulation frequency,
100 kHz; receiver gain, 2 × 105; and microwave power, 20 mW. The
spectra shown are the averages of 10 scans.
EPR Spin-Trapping Experiments. Typically, incubation mixtures
used in spin-trapping experiments consisted of 250 μM MitoPhB-
(OH)2 isomer and MNP spin trap (40 mM) in phosphate buffer (100
mM, pH 7.4) containing dtpa (100 μM) and 5% CH3CN. Solutions
were rapidly mixed with bolus ONOO− (200 μM). Samples were
subsequently transferred to an EPR capillary tube, and spectra were
recorded. Spectrometer parameters were as follows: scan range, 75 G;
modulation amplitude, 1.0 G; and receiver gain, 1 × 105. The spectra
shown are the averages of 10 scans.
To investigate the effect of MitoPhB(OH)2 isomers on the
oxidation and nitration of tyrosine, 100 μL of sample was injected
into the HPLC system equipped with a Phenomenex Synergi 4u
Hydro-RP 80 Å column (250 × 4.6 mm, 4 μm) equilibrated with water
containing 0.1% (v/v) TFA. The compounds were separated by a
Theoretical Studies. All calculations were performed with the use
of the Gaussian 09, rev.A.02 (G09), package.22 The electronic
structure calculations were carried out using the M06−2X function
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Figure 3. Relationship between substrate depletion and major/minor product formation during the peroxynitrite reaction with MitoPhB(OH)2. (A)
Incubation mixtures consisted of 200 μM MitoPhB(OH)2 in phosphate buffer (pH 7.4, 100 mM) containing dtpa (100 μM), 10% 2-PrOH, and
catalase (100 U/mL). After bolus addition of peroxynitrite, the reaction mixtures were analyzed using the HPLC/UV method. MitoPhB(OH)2 and
the products were detected at 268 nm. Each point represents the average value of three samples. The concentrations were determined based on the
calibration curves obtained for authentic standards. MitoPhOH standards were prepared by oxidation of MitoPhB(OH)2 in the presence of excess of
H2O2 (incubation mixture contained MitoPhB(OH)2 in phosphate buffer (pH 7.4, 100 mM) and dtpa (100 μM), 10% 2-PrOH, and 10 mM H2O2).
The standard deviations are smaller than the point’s size. (B) Analysis of the ratio of minor products (sum of MitoPh + MitoPhNO2) and the major
product (MitoPhOH). The ratio of the rate constants of homolytic (k2) and heterolytic (k1) cleavage pathways was estimated.
of Truhlar and co-workers23,24 with the 6-31+G(d,p) basis set.25,26
RESULTS
■
Initial geometry of the three isomeric MitoPhB(OH)2O•− radical
Reaction of MitoPhB(OH)2 with ONOO−. The second-
anions was fully optimized and used for the relaxed potential energy
order rate constants for the reaction between peroxynitrite and
scan performed along the reaction coordinate defined as an elongating
mitochondria-targeted arylboronates for ortho, meta, and para
isomers were determined to be (3.5 0.5) × 105, (1 0.1) ×
boron−carbon bond length. All structures on the potential energy
106, and (1
0.1) × 106 M−1s−1, respectively. The rate
surfaces were fully optimized, and the stationary points were
confirmed by performing harmonic vibrational analysis. Local minima
were characterized by 3N-6 real normal modes of vibrations, whereas
the transition states had exactly one imaginary frequency. The
influence of the environment was modeled using the polarizable
continuum solvent model (PCM)27 with parameters for water as
implemented in G09. Open shell species were treated using the
unrestricted Hartree−Fock (UHF) method.28 We have used the
default convergence and optimization criteria in all calculations
performed using the Gaussian package.
constants were determined by competition kinetics with
resorufin boronate as a standard (k = (1
0.1) × 106
M−1s−1) using pulse radiolysis; the identity of the oxidizing
species was confirmed by the inhibitory effect of superoxide
dismutase on the yield of resorufin (unpublished data). Both
para and meta isomers react with ONOO− with the rate
constant close to the value of 1 × 106 M−1s−1, previously
determined for phenylboronic acids.11,13 However, the rate
constant of the reaction of peroxynitrite with the ortho isomer is
3-fold lower, possibly due to the steric hindrance of the bulky
triphenylphosphonium moiety. Nonetheless, the rate constants
of the reaction of peroxynitrite with all three isomers of
MitoPhB(OH)2 are several orders of magnitude higher than
that reported for the reaction of m-MitoPhB(OH)2 with
hydrogen peroxide (k = 3.8 M−1s−1 at pH 8; T = 25 °C).9
Identification and Quantitation of Products Formed
from Oxidation of MitoPhB(OH)2 Isomers. The structures
of the oxidation products of MitoPhB(OH)2 were determined
by mass spectral analysis and by comparison with synthesized
Cell Culture. RAW 264.7 macrophages were cultured in DMEM
medium supplemented with 10% FBS. Details on the culturing
conditions and protocol for stimulation of macrophages to produce
ONOO− have been published elsewhere.14 For stimulation of NO
•
production, the cells were incubated overnight (12−16 h) with LPS (1
•−
μg/mL) and IFNγ (50 U/mL). For stimulation of O2 production,
•
•−
cells were treated with 1 μM PMA. Co-stimulation of NO and O2
production leads to the generation of ONOO−, as shown
previously.13,14
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authentic standards (Figure 2). Table S1 (see Supporting
Information) lists the mass spectral parameters for the isomers
of MitoPhB(OH)2 and their major and minor oxidation
products. As shown in Figure 2A, oxidation of o-MitoPhB-
(OH)2 by peroxynitrite in phosphate buffer (pH 7.4) leads to
the formation of two oxidation products: o-MitoPhOH (the
major product) and o-MitoPhNO2, whereas the oxidation of
two other MitoPhB(OH)2 isomers results mainly in the
formation of corresponding phenols, with much lower yields
of the nitrated products (Figures 2B and C). The reaction
between aryl boronic acids and ONOO− is stoichiometric, as
the yield of boronate consumption is close to 100% with
respect to the amount of peroxynitrite (Figure 3A).
To characterize the reactions in more detail, the profiles of
formation of the major and minor products (MitoPhOH,
MitoPhNO2, and MitoPh, Figure 1) formed during the reaction
between ONOO− and MitoPhB(OH)2 isomers were inves-
tigated. We used the HPLC technique to determine the amount
of major and minor products formed during the reaction with
peroxynitrite in the presence of 2-PrOH, an efficient phenyl
radical scavenger. The consumption of substrates and the
amount of products formed in the reaction with peroxynitrite
were determined based on the calibration curves obtained for
the boronate isomers MitoPhB(OH)2 and the oxidation
products MitoPhOH, MitoPhNO2 isomers, and MitoPh.
Figure 3A shows substrate depletion and product formation
during the reaction between MitoPhB(OH)2 isomers and
ONOO−. Boronates (250 μM) in phosphate buffer (100 mM,
pH 7.4) containing dtpa (100 μM), 10% 2-PrOH, and catalase
(100 U/mL) were rapidly mixed with ONOO− (10−300 μM).
Previously, we have shown that the addition of phenyl radical
scavengers (e.g., 2-PrOH) to the reaction mixtures is a
convenient way to estimate the yield of minor products formed
during the reaction between arylboronates and peroxynitrite.12
In the presence of 2-propanol, the phenyl radical formed in the
radical pathway is almost quantitatively converted into the
product in which the boronate moiety is replaced by a
hydrogen atom, with a smaller fraction undergoing a
Figure 4. UPLC analyses of products formed from o-MitoPhB(OH)2
in the MPO/H2O2/nitrite system. Incubation mixtures contained o-
MitoPhB(OH)2 (100 μM), H2O2 (500 μM), MPO (5 U/mL), and
NaNO2 (500 μM) in phosphate buffer (pH 7.4, 50 mM) with dtpa
(100 μM). The reaction was initiated by the addition of H2O2.
Products were determined 40 min after adding H2O2 at 268 1.2 nm.
A and B indicate two isomeric nitration products of o-MitoPhOH
(general formula, C25H21NO3P) as determined with the use of an MS
detector (see Table S2 in Supporting Information).
the nonradical pathway and is not consumed under the
experimental conditions, and that all the minor products
(MitoPh and MitoPhNO2) are formed through the radical
pathway (from the fragmentation of MitoPhB(OH)2O•−), the
ratios of the rate constants of the radical (homolytic cleavage of
the O−O bond) and nonradical (heterolytic cleavage of the
O−O bond) pathways were determined from the plot of the
sum of the minor products versus the amount of MitoPhOH
formed (Figure 3B).
The selective formation of o-MitoPhNO2 as the predominant
minor product from the ONOO−/o-MitoPhB(OH)2 reaction is
unique and significant from a diagnostic perspective involving
the detection of ONOO−. Therefore, we also investigated the
influence of some biologically relevant reductants, glutathione,
NADH, and ascorbate, on minor oxidation products’ profiles
and found an effect similar to that seen with 2-PrOH.
Glutathione and ascorbate are known to react with
peroxynitrite (the reported rate constants are k = 1.3 × 103
M−1s−1 for glutathione, at 37 °C and pH 7.5,29 and k = 1 × 106
M−1s−1 for the formation of an adduct of ONOOH to
monohydrogen ascorbate at pH 5.830). Therefore, although
GSH decreased the consumption of o-MitoPhB(OH)2 by ca.
15% (Figure 5A), this cannot be explained by simple
competition with boronate for ONOO−. Of note, the reported
rate constant of the reaction of ONOOH with monohy-
droascorbate30 seems to be overestimated, as one would expect
•
recombination reaction with NO2. We have shown that the
sum of MitoPh and MitoPhNO2 formed reflects the total
amount of phenyl radicals produced. The enzyme, catalase, was
used to inhibit the oxidation of boronates by H2O2 that can be
formed after the hydrogen atom abstraction from 2-PrOH by
phenyl radicals in the presence of oxygen.
As shown in Figure 3A, the extent of boronate consumption
in the presence of ONOO− and the formation of major
phenolic product, MitoPhOH, are very similar for all isomers.
However, the relative yields of the minor product of the
reaction between ONOO− and MitoPhB(OH)2 are different.
With para and meta isomers, the dominant minor product
formed from the radical pathway in the presence of 10% 2-
PrOH (an effecient phenyl radical scavenger) is MitoPh,
whereas with the ortho isomer, the only minor product detected
was o-MitoPhNO2. More importantly, o-MitoPhNO2 formation
−
was not observed in the MPO/H2O2/NO2 system generating
•NO2 (Figure 4). In this system, o-MitoPhOH formed in the
reaction between o-MitoPhB(OH)2 and H2O2 is efficiently
•
−
nitrated by NO2 produced by the MPO/H2O2/NO2 system,
as evidenced by the formation of two isomeric nitration
products of o-MitoPhOH (e.g., C25H21NO3P) (see Figure 4
and Table S2 in Supporting Information).
Assuming that MitoPhOH, the major product of MitoPhB-
(OH)2 oxidation by peroxynitrite, is produced exclusively via
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Figure 5. Effects of ascorbate, glutathione, and NADH on substrate
depletion and major/minor product formation. Incubation mixtures
consisted of 250 μM o-MitoPhB(OH)2 in phosphate buffer (pH 7.4,
100 mM) containing dtpa (100 μM), catalase (100 U/mL), and the
reductant (as indicated). After a bolus addition of peroxynitrite
(resulting in the 150 μM peroxynitrite concentration in the sample),
the reaction mixtures were analyzed by HPLC/UV. Each bar
represents the average value of three samples. The error bars represent
standard deviations.
much stronger inhibitory effects of ascorbate on the yield of o-
MitoPhOH and o-MitoPhNO2 than those observed. One can
assume that all of these reductants are also able to react with
the nitrogen dioxide radical (•NO2) and/or phenyl radicals. As
shown in Figure 5, all of the reductants had little or no effect on
the product yield ratios of o-MitoPhOH and o-MitoPhNO2
formed from the o-MitoPhB(OH)2 reaction with peroxynitrite.
To fully understand the mechanistic basis for the differences
in product formation from the reaction between ONOO− and
MitoPhB(OH)2 isomers, we quantitated the amount of
oxidizing radicals using ABTS, a potent radical scavenger.
ABTS can be oxidized to its stable radical cation, ABTS•+, by
radical species generated in the boronic acid/peroxynitrite
system: phenylperoxyl, phenoxyl, and •NO2 radicals.31−36
Formation of the ABTS radical cation can be monitored by
EPR or spectrophotometry (at 420 nm, ε = 3.6 × 104 M−1cm−1,
and at 735 nm, ε = 1.6 × 104 M−1cm−1). As shown in Figure 6A
and B, the oxidation of ABTS by ONOO−-derived radicals was
decreased in the presence of para and meta isomers of
MitoPhB(OH)2 and was almost completely suppressed by o-
MitoPhB(OH)2. Of note, in the absence of boronates, the yield
of ABTS radical cation decreased with increasing concentration
of peroxynitrite (Figure 6B). This can be explained by the
Figure 6. Oxidation of ABTS by oxidants formed from the
MitoPhB(OH)2 reaction with peroxynitrite. (A) Incubation mixtures
consisted of 500 μM ABTS and 250 μM MitoPhB(OH)2 (if indicated)
in phosphate buffer (pH 7.4, 100 mM) containing dtpa (100 μM).
The reaction mixture was transferred to an EPR capillary immediately
after bolus addition of ONOO− (resulting in the 200 μM peroxynitrite
concentration in the sample), and the spectra were recorded at room
temperature. (B) Incubation mixtures consisted of 500 μM ABTS and
250 μM MitoPhB(OH)2 (as indicated) in phosphate buffer (pH 7.4,
100 mM) containing dtpa (100 μM). The absorption spectra were
recorded immediately after bolus addition of ONOO− (resulting in the
0−200 μM peroxynitrite concentration in the sample). The
concentration of the ABTS radical cation was calculated based on
the measured absorbance at 420 nm (ABTS radical cation molar
absorption coefficient ε420 nm = 3.6 × 104 M−1cm−1). Each point
represents the average value of three samples. The error bars represent
standard deviations.
reaction between ONOO− and ONOOH (k ∼3 × 104 M−1s−1).
37
Elucidation of Radical Pathway: Spin-Trapping of
Phenyl Radical Intermediates. The EPR study using MNP
spin-trap was performed primarily to detect and characterize
radical intermediates formed during the reaction between
peroxynitrite and isomers of MitoPhB(OH)2. Previously, we
have shown that the phenyl radicals generated during the
ONOO−/arylboronate reaction can be trapped with MNP or
DEPMPO spin-traps. The EPR spectrum of the MNP-phenyl
adduct is more informative (i.e., reveals hyperfine couplings
from the aromatic ring protons) than the EPR spectrum of the
DEPMPO-phenyl adduct. The addition of a bolus amount of
peroxynitrite to incubations containing p-MitoPhB(OH)2 or m-
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MitoPhB(OH)2, and MNP (pH 7.4) resulted in a multiline
spectrum that can be assigned to the corresponding MNP-
phenyl radical adduct based on the hyperfine coupling values
(Figure 7; Table 1).12 The multiline EPR spectrum of the
contained 2.5 mM tyrosine and 500 μM MitoPhB(OH)2 in
phosphate buffer (pH 7.4, 100 mM) containing dtpa (100 μM).
The reaction was initiated with the rapid addition of ONOO−.
The absorption spectra were recorded after a bolus addition of
ONOO− (final concentration in the 0−400 μM range), and the
absorbance at 440 nm was plotted as a function of ONOO−
concentration (Figure 8A). The HPLC analyses were
performed using the same conditions. Both nitrotyrosine and
dityrosine were quantified by HPLC analyses of the reaction
mixtures (Figure 8B). Figure 8A and B shows the inhibitory
effect of isomers of MitoPhB(OH)2 on tyrosine nitration and
oxidation. Nitrotyrosine formation was attenuated by 87 6%,
59 5%, and 56 5% in the presence of o-MitoPhB(OH)2, m-
Figure 7. Spin-trapping of phenyl radicals formed from the reaction
between ONOO− and MitoPhB(OH)2. Incubation mixtures contained
the following compounds: MitoPhB(OH)2 (250 μM), MNP (40 mM)
in phosphate buffer (100 mM, pH 7.4) containing dtpa (100 μM), and
5% CH3CN. The reaction mixture was transferred to an EPR capillary
immediately after bolus addition of ONOO− (resulting in the 200 μM
peroxynitrite concentration in the sample), and the spectra were
recorded at room temperature.
Table 1. Hyperfine Coupling Constants of MNP Spin
Adducts
hyperfine splitting constants [G]
m-
MitoPhB(OH)2
a(N) = 14.58; a(1H) = 1.82; a(1H) = 1.48; a(1H) = 1.38
p-
MitoPhB(OH)2
a(N) = 14.43; aortho(2H) = 1.86; ameta(2H) = 0.90; a(P) =
6.46
adduct of p-MitoPh radical to MNP trap shows an additional
hyperfine coupling (aP = 6.46 G) due to the presence of a
phosphorus atom (I = 1/2) in the TPP cation and a significant
spin density at the para position (Figure 7). Incubation of
peroxynitrite with o-MitoPhB(OH)2 did not result in the
formation of a similar multiline EPR spectrum. This further
suggests a rapid radical−radical recombination mechanism
Figure 8. Inhibition of peroxynitrite-induced tyrosine nitration and
oxidation by MitoPhB(OH)2. (A) Formation of nitrotyrosine was
monitored spectroscopically at 440 nm. Incubation mixtures consisted
of 2.5 mM tyrosine and 500 μM MitoPhB(OH)2 (as indicated) in
phosphate buffer (pH 7.4, 100 mM) containing dtpa (100 μM). The
absorption spectra were recorded after bolus addition of ONOO−
(resulting in the 0−400 μM peroxynitrite concentration in the
sample). Each point represents the average value of three samples. The
error bars represent standard deviations. (B) Formation of nitro-
tyrosine and dityrosine was monitored by HPLC. Incubation mixtures
consisted of 2.5 mM tyrosine and 500 μM MitoPhB(OH)2 (as
indicated) in phosphate buffer (pH 7.4, 100 mM) containing dtpa
(100 μM). The HPLC analyses were performed after bolus addition of
ONOO− (resulting in the 400 μM peroxynitrite concentration in the
sample). Each bar represents the average value of three samples. The
error bars represent standard deviations. Student’s unpaired t test was
used to determine the statistical significance of differences between the
formation of nitrotyrosine (or dityrosine) in the absence and presence
of the appropiate MitoPhB(OH)2 isomer. P values of <0.01 and
<0.001 were considered as significant and highly significant,
respectively (marked with ** and ***, respectively).
•
between o-MitoPh• radicals and NO2 inside the solvent cage.
Arylboronate Isomers as Potential Antinitration
Antioxidants. Previously, we showed that boronates are
efficient scavengers of ONOO− and other oxidants.11,13,38
Nitration of tyrosyl residues in proteins perturb their functional
activity.39−43 Thus, the ability to prevent ONOO−-mediated
tyrosyl nitration is very significant. The finding that o-
MitoPhB(OH)2 almost completely blocked ONOO−-depend-
ent oxidation of ABTS implies that this class of compounds
could be used as potent antioxidants/antinitration agents. To
this end, we investigated the effect of isomers of MitoPhB-
(OH)2 on ONOO−-induced nitration and oxidation of
tyrosine. Tyrosine nitration was monitored spectroscopically
at 440 nm, while HPLC was used to follow both nitration and
oxidation of tyrosine induced by ONOO−. Incubation mixtures
G
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MitoPhB(OH)2, and p-MitoPhB(OH)2, respectively. The
dityrosine formation was also inhibited by 89%, 85%, and
76% in the presence of o-MitoPhB(OH)2, m-MitoPhB(OH)2,
and p-MitoPhB(OH)2, respectively.
Theoretical Studies. DFT quantum mechanical calcu-
lations were performed to characterize the postulated key
intermediates formed on the radical pathway of MitoPhB-
(OH)2 reaction with peroxynitrite, namely, the MitoPhB-
(OH)2O•− radical anions. We assumed that the radical anions
MitoPhB(OH)2O•− formed upon the homolytic O−O bond
cleavage undergo further fragmentation resulting in MitoPh•
phenyl radical formation. Starting from the optimized structures
of the appropriate MitoPhB(OH)2O•− radical anion, we
computed a potential energy curve based on which the
respective transition state of the fragmentation process was
found. According to the PCM/M06-2X/6-31+G(d,p) calcu-
lations, the Gibbs energies of activation for the boron−carbon
(B−C) bond cleavage in the MitoPhB(OH)2O•− radical anions
are 16.3, 26.4, and 27.2 kcal/mol for the ortho-, meta-, and para-
isomers, respectively. Table 2 contains the most important
Table 2. Structural Parameters of MitoPhB(OH)2O•−
Radical Anions and the Transition States (TS) of the
a
Fragmentation Reactions
MitoPhB(OH)2O•−
TS
ΔE#
[kJ/
mol]
B−C O spin C spin B−C O spin C spin
(Å)
density density
(Å)
density density
para-
meta-
ortho-
1.65
1.65
1.67
0.87
0.87
0.93
0.12
0.11
0.05
2.21
2.19
1.99
0.25
0.27
0.48
0.80
0.79
0.49
27.2
26.4
16.3
a
ΔE# represents the Gibbs energy of activation of C−B bond cleavage,
according to the DFT calculations performed.
structure parameters of MitoPhB(OH)2O•− radical anions and
the transition states of their fragmentation reactions. Analysis of
spin densities clearly indicates that the majority of radical
character in MitoPhB(OH)2O•− radical anions is located on the
O•− oxygen atom, whereas in the transition state, it is partially
carried by the carbon atom of the phenyl ring being attached to
boron. Complete geometries and calculated energies for
MitoPhB(OH)2O•− isomers and corresponding transition
states are included in Supporting Information.
Figure 9. HPLC analyses of products formed from oxidation of o-
MitoPhB(OH)2 by ONOO− generated in situ in cell-free and cellular
systems. (A) Incubation mixtures contained 100 μM o-MitoPhB-
(OH)2, HX (40 μM), and XO (1 mU/mL) in phosphate buffer (pH
7.4, 50 mM) containing 100 μM dtpa, in the presence or absence of
DPTA-NONOate (250 μM). After a 1 h incubation at room
temperature, the products were analyzed as described in the
Experimental Procedures section. (B) RAW 264.7 macrophages were
activated using LPS (1 μg/mL), IFNγ (50 U/mL), and PMA (1 μM),
and incubated for 1 h with o-MitoPhB(OH)2 (50 μM) in DPBS
supplemented with glucose and pyruvate as described in the
Experimental Procedures section. After incubation, the cells were
lysed and the lysate deproteinized before HPLC analysis. (C) The
same as in panel B, but cell media were analyzed.
Oxidation of o-MitoPhB(OH)2 by in Situ Generated
ONOO−. The oxidation of o-MitoPhB(OH)2 was investigated
•−
in the presence of O2 and •NO generating systems. o-
MitoPhB(OH)2 (100 μM) was incubated with HX (40 μM)
and XO (1 mU/mL) in phosphate buffer (50 mM, pH 7.4)
containing dtpa (100 μM) for 1 h at room temperature. Similar
incubations were performed in the presence of nitric oxide
donor, DPTA-NONOate (250 μM). The products were
analyzed by HPLC (Figure 9A). In the absence of DPTA-
NONOate, there was a modest conversion of o-MitoPhB(OH)2
to o-MitoPhOH that was attributed to a slow oxidation of
boronate by H2O2. In incubation mixtures containing DPTA-
NONOate and HX/XO, the rate of o-MitoPhB(OH)2
oxidation was significantly enhanced along with the formation
of o-MitoPhNO2. These results further confirm our notion that
ONOO−, whether added as a bolus or generated in situ, forms
the specific nitrated product o-MitoPhNO2 in the presence of
the ortho isomer.
Cell Culture Study. As a proof of concept, we used the
macrophage-like RAW 264.7 cells as a model system. Activated
RAW 264.7 cells produce ONOO−. We tested whether
incubation of o-MitoPhB(OH)2 in activated RAW cells would
form this nitrated product, o-MitoPhNO2 (Figures 9B and C).
We measured by HPLC both cell extracts and extracellular
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Scheme 2
medium. Using boronate-based fluorogenic probes, we have
shown previously that macrophages produce ONOO− after
stimulation with lypopolysaccharide (LPS), interferon γ
(IFNγ), and phorbol 12-myristate-13-acetate (PMA). As
shown in Figure 9B, no oxidation of o-MitoPhB(OH)2 is
observed in nonstimulated cells. However, when the macro-
phages are activated to produce peroxynitrite, both o-
MitoPhOH and o-MitoPhNO2 can be detected. As o-
MitoPhNO2 is not formed during the oxidation of o-
case of all isomers are the corresponding phenols (∼90% yield),
but the relative yields of the minor product of the reaction
between ONOO− and MitoPhB(OH)2 are different. With para
and meta isomers, the dominant minor product formed from
the radical pathway in the presence of 10% 2-PrOH (an
effective phenyl radical scavenger) is MitoPh, which is formed
by a hydrogen atom abstraction from 2-PrOH by MitoPh•
radicals. With the ortho isomer, the only minor product
detected was o-MitoPhNO2, suggesting a rapid fragmentation
of MitoPhB(OH)2O•− and subsequent recombination of the
phenyl radical with •NO2 in the solvent cage. The ortho isomer
forms a diagnostic marker product in the presence of ONOO−,
which strongly suggests that there are significant differences in
the decomposition pathway of the boronate−peroxynitrite
intermediate in the radical cage. Support for the phenyl radical
intermediate came from EPR spin-trapping experiments.
Previously, it was reported that m-MitoPhB(OH)2 (MitoB)
can be used for the detection and quantitation of mitochondria-
•
MitoPhB(OH)2 by other oxidants (or by NO2 formed by
MPO), this means that ONOO− is actually produced in this
system.
DISCUSSION
■
Here, we report the study on the product formation profile for
the reactions of isomeric mitochondria-targeted arylboronates
with peroxynitrite. HPLC analyses showed that the major
products of MitoPhB(OH)2 oxidation by peroxynitrite in the
I
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derived hydrogen peroxide.9,10 In this study, we show that m-
MitoPhB(OH)2 and other isomers (o- and p-MitoPhB-
(OH)2)11,13,38 also react with ONOO−. As with simple
arylboronates, the peroxynitrite adduct to the boronate moiety
decays via two pathways: the heterolysis of the O−O bond (the
major pathway) leading to the formation of the corresponding
phenols and nitrite and the homolysis of the O−O bond (the
minor reaction) that results in the formation of a radical anion,
MitoPhB(OH)2O•−, and nitrogen dioxide. According to the
DFT quantum mechanical calculations, the energy barrier for
the decomposition of the o-MitoPhB(OH)2O•− radical anion,
leading to the formation of o-MitoPh• radicals (∼16 kJ/mol), is
lower than the energy barrier for the fragmentation of meta and
para isomeric MitoPhB(OH)2O•− radical anions (∼27 kJ/
mol). This strongly suggests the possibility of rapid and
spontaneous o-MitoPhB(OH)2O•− fragmentation, resulting in
the formation of o-MitoPh• radical and its subsequent reaction
antinitration agent for inhibiting tyrosyl nitration of cellular/
mitochondrial proteins.
In this study, we report that substitution with a charged,
bulky group at the ortho position (not meta or para position) of
phenylboronates dramatically alters product formation (nitro-
benzene derivative) in the presence of ONOO− via the minor
radical pathway. All of the isomers yielded nearly the same
amount of the major product, the corresponding phenol. These
differences in reaction pathways are attributed to the steric
hindrance of the bulky group on radical reactions in the solvent
cage of the boronate−peroxynitrite intermediate. Results from
the EPR spin-trapping experiments showed that MitoPh•
radicals formed from the reaction between ONOO− and meta
and para isomers (m-MitoPhB(OH)2 and p-MitoPhB(OH)2)
and not from the ortho isomer (o-MitoPhB(OH)2) could be
detected with 2-methyl-2-nitrosopropane (MNP) spin trap.
Regardless of the presence of any biological reductants, the
ONOO−/o-MitoPhB(OH)2 reaction yields the same amount of
o-MitoPhNO2. DFT quantum mechanical calculations suggest
that the energy barrier for the dissociation of the o-
MitoPhB(OH)2O•− radical anion is lower than that for m-
MitoPhB(OH)2O•− and p-MitoPhB(OH)2O•− radical anions,
which explains the fast recombination of radicals formed within
the solvent cage. Future studies will focus on the applicability of
the ortho isomer probe described here or its long-chain
analogue for the detection of mitochondrial ONOO−
generation.
•
with the NO2 radical within the solvent cage. It has to be
noted that in case of meta and para isomers, the radical anion
MitoPhB(OH)2O•− also undergoes spontaneous fragmenta-
tion, which is consistent with the low energy barrier. However,
in the presence of the phenyl radical scavenger (2-PrOH), the
yield of MitoPhNO2 products is almost 10-fold lower than that
for the ortho isomer, most probably due to the less efficient
radical recombination within the solvent cage. The overall
mechanism can be summarized as shown in Scheme 2. The
hydrogen atom donors (ABTS, GSH, NADH, and ascorbate)
were unable to prevent the formation of o-MitoPhNO2. The
EPR spin-trapping experiments with o-MitoPhB(OH)2 and
ONOO− did not provide major evidence for trappable radical
intermediates, consistent with a rapid recombination of o-
MitoPh• and •NO2 radicals in the solvent cage. In addition, the
formation of other oxidizing and nitrating radicals formed from
the reaction between ONOO− and o-MitoPhB(OH)2 was
minimal, leading to an effective attenuation of ONOO−-
mediated tyrosyl nitration. o-MitoPhNO2 formed in relatively
high yields (η ≈ 9%) can be used as a diagnostic biomarker of
ONOO− in cellular and biological systems. In contrast,
ONOO−-dependent oxidation of meta and para isomers of
MitoPhB(OH)2 results in the formation of free MitoPh•
radicals that can be scavenged by oxygen and hydrogen atom
donors or trapped with MNP and DEPMPO spin traps to give
characteristic EPR spectra of the corresponding phenyl radical
adducts.
ASSOCIATED CONTENT
* Supporting Information
■
S
Optimized geometries of all stationary points; mass spectrom-
etry data identifying the products of peroxynitrite oxidation of
o-, m-, and p-MitoPhB(OH)2, as well as the products formed
from o-MitoPhB(OH)2 in MPO/H2O2/nitrite system. This
material is available free of charge via the Internet at http://
pubs.acs.org.
AUTHOR INFORMATION
Corresponding Authors
■
*Institute of Applied Radiation Chemistry, Lodz University of
Technology, Zeromskiego 116, 90-924 Lodz, Poland. Tel: +48
42 631 31 70. E-mail: adam.sikora@p.lodz.pl.
*Department of Biophysics, Medical College of Wisconsin,
8701 Watertown Plank Road, Milwaukee, WI 53226. Tel: 414-
955-4000. Fax: 414-955-6512. E-mail: balarama@mcw.edu.
The mechanism of the reaction between boronates and
peroxynitrite involving both nonradical and radical pathways
appears to be quite general. However, it is not clear how other
factors, including steric hindrance and electronic effects of
substituents, influence the stability of the key reaction
intermediates (e.g., boronate-ONOO− anionic adduct and R-
B(OH)2O•− radical anion) leading to specific product
formation. On the basis of the present results obtained with
o-MitoPhB(OH)2, we conclude that the o-MitoPhNO2 formed
can be used to unequivocally detect ONOO− formation in cell-
free and cellular systems. Boronate probes containing bulky
substituents at the ortho position may be used in cells to
unequivocally confirm ONOO− formation, via the specific
nitration of the boronate probe. Other oxidants (H2O2 and
HOCl) that can oxidize boronates do not form nitrated
products. The nitration of ortho-substituted boronates can also
Funding
This work was performed with the help of a grant (R01
HL063119) funded by the National Institutes of Health
awarded to B.K. A.S. was supported the grant from the
Foundation for Polish Science (FNP) within the “Homing
Plus” program, and the grant coordinated by JCET, No.
POIG.01.01.02-00-069/09 (both grants are supported by the
European Union from the resources of the European Regional
Development Fund under the Innovative Economy Pro-
gramme).
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
•
be used to distinguish the formation of ONOO− and NO2
Access to supercomputing facilities at Cyfronet (Poland) is
gratefully acknowledged. We thank Mrs. Monika Zielonka for
her assistance with cell culture experiments.
formed from MPO/H2O2/NO2− reaction. o-MitoPhB(OH)2 or
similar sterically hindered boronates may be used as a potent
J
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ABBREVIATIONS
■
2-PrOH, 2-propanol; ABTS, 2,2′-azino-bis(3-ethylbenzothiazo-
line-6-sulfonate); dtpa, diethylenetriaminepentaacetic acid;
DFT, Density Functional Theory; diTyr, dityrosine; DMEM,
Dulbecco’s modified Eagle’s medium; DPBS, Dulbecco’s
phosphate-buffered saline; FBS, fetal bovine serum; HX,
hypoxanthine; IFNγ, interferon γ; LPS, lypopolysaccharide;
m-MitoPhB(OH)2, (3-boronobenzyl)triphenylphosphonium
cation; MitoPh, benzyltriphenylphosphonium cation; m-Mito-
PhOH, (3-hydroxybenzyl)triphenylphosphonium cation; m-
MitoPhNO2, (3-nitrobenzyl)triphenylphosphonium cation;
MNP, 2-methyl-2-nitrosopropane; MPO, myeloperoxidase;
•NO, nitric oxide (IUPAC-recommended names for nitric
oxide are nitrogen monoxide or oxidonitrogen(•)); •NO2,
nitrogen dioxide; o-MitoPhB(OH)2, (2-boronobenzyl)-
triphenylphosphonium cation; o-MitoPhOH, (2-
hydroxybenzyl)triphenylphosphonium cation; o-MitoPhNO2,
(2-nitrobenzyl)triphenylphosphonium cation; O2•−, superoxide
radical anion; ONOO−/ONOOH, peroxynitrite (IUPAC-
recommended names for peroxynitrite anion and peroxynitrous
acid are oxidoperoxidonitrate(1−) and (dioxidanido)-
oxidonitrogen, respectively); PMA, phorbol 12-myristate-13-
acetate; p-MitoPhB(OH)2 , (4-boronobenzyl)-
triphenylphosphonium cation; p-MitoPhOH, (4-
hydroxybenzyl)triphenylphosphonium cation; p-MitoPhNO2,
(4-nitrobenzyl)triphenylphosphonium cation; DPTA-NON-
Oate, ((Z)-1-[N-(3-aminopropyl)-N-(3-ammoniopropyl)-
amino]diazen-1-ium-1,2-diolate); Ph•, phenyl radical; SOD,
superoxide dismutase; TFA, trifluoroacetic acid; TyrNO2,
nitrotyrosine; X, xanthine; XO, xanthine oxidase
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