The reaction of ebselen with peroxynitrite

Article abstract of DOI:10.1021/tx950115u

Ebselen, 2-phenyl-1,2-benzisoselenazol-3(2H)-one, rapidly reacts with peroxynitrite, the rate constant being of the order of 10⁶ M^-1 s^-1; the reaction yields the selenoxide of the parent molecule, 2-phenyl-1,2- benzisosele

Full text of DOI:10.1021/tx950115u

2
62
Chem. Res. Toxicol. 1996, 9, 262-267
Th e Rea ction of Ebselen w ith P er oxyn itr ite
†
Hiroshi Masumoto and Helmut Sies*
Institut f u¨ r Physiologische Chemie I, Heinrich-Heine-Universit a¨ t D u¨ sseldorf, Postfach 101007,
D-40001 D u¨ sseldorf, Germany
Received J une 26, 1995X
Ebselen, 2-phenyl-1,2-benzisoselenazol-3(2H)-one, rapidly reacts with peroxynitrite, the rate
6
-1 -1
constant being of the order of 10 M
s ; the reaction yields the selenoxide of the parent
molecule, 2-phenyl-1,2-benzisoselenazol-3(2H)-one 1-oxide, as the sole selenium-containing
product; a stoichiometry of 1 mol of ebselen reacted and of the selenoxide formed per mole of
peroxynitrite was observed. The reaction was studied in detail at neutral and alkaline pH
(
pH 10-11). It also proceeds at acidic pH where peroxynitrous acid (ONOOH) is predominant,
the yield of the selenoxide being lower because peroxynitrous acid (pK ) 6.8) decays rapidly.
a
Reduction of the selenoxide in cells to regenerate ebselen would allow for a sustained defense
against peroxynitrite. This novel reaction constitutes a potential cellular defense line against
peroxynitrite, one of the important reactive species in inflammatory processes.
In tr od u ction
Peroxynitrite is one of the reactive species generated
control of the two enzymes mentioned above and the
repair activities operative after damage has occurred.
In previous work, we noted that peroxynitrite-induced
luminol chemiluminescence emitted from Kupffer cells
1
in the inflammatory process, e.g., by the reaction of the
•
- 2
•
superoxide anion radical (O
2
) and nitric oxide ( NO)
(16) was inhibited in the presence of low concentrations
•
-
•
(
2
1, 2). Superoxide (O ) and NO are generated upon
of ebselen (2) (17). These observations suggested that
ebselen (2) reacts with peroxynitrite and prompted us to
investigate the reaction further.
activation of the NADPH oxidase and NO synthase
enzymes, respectively, which are present in macrophages
and other cell types. Peroxynitrite can undergo a number
of reactions of pharmacological and toxicological interest
In the present work, we describe the reactivity of the
selenoorganic compound ebselen (2) with peroxynitrite.
Ebselen (2) has been identified as an antiinflammatory
agent (18-22), and it exhibits glutathione peroxidase
(
1, 3, 4). It initiates lipid peroxidation, oxidizes sulfhy-
dryls at a substantially faster rate than its first-order
decay (5), reacts with hydrogen peroxide to form singlet
molecular oxygen (6), and reacts with methionine (7, 8)
and ascorbic acid (9). Also it nitrates and hydroxylates
aromatic compounds (10, 11) and leads to the nitration
of protein tyrosines (12).
(GSH-Px) activity which may explain some of its actions
(23-26).
Ma ter ia ls a n d Meth od s
Rea gen ts. Ebselen, 2-phenyl-1,2-benzisoselenazol-3(2H)-one
-
The peroxynitrite anion (ONOO , 1) is relatively stable
(2), was a kind gift from Rh oˆ ne-Poulenc-Nattermann, Cologne,
in alkaline solution, and its conjugate acid, peroxynitrous
FRG. Chemicals, solvents, and silica gel TLC plate (silica gel
acid (ONOOH, 1a ), with pK
nitrate (the apparent half-life of peroxynitrite, t1/2, is
.1-1 s at pH 7.4 and 37 °C (1, 13, 14)). Peroxynitrous
acid (1a ) has been suggested to decompose by homolytic
a
) 6.8 (13) can isomerize to
6
0 F254, 20 × 20 cm, 0.25 mm thickness) were from E. Merck,
Darmstadt, FRG.
0
Peroxynitrite was synthesized from sodium nitrite and hy-
drogen peroxide (H
2 2
O ) using a quenched-flow reactor as previ-
•
ously described (1, 6). Residual H O was eliminated by passage
scission into nitrogen dioxide ( NO
2
) and hydroxyl radical
2
2
•
of the peroxynitrite solution over MnO
2
powder. Peroxynitrite
(
OH), and 1a generates a strong oxidant with reactivity
concentration was determined spectrophotometrically at 302 nm
similar to that of the hydroxyl radical (1, 15). Peroxy-
nitrite is thought to play an important role in a potential
pathway of tissue damage, but there is no known endog-
enous defense line against its toxicity, except for the
-1
-1
(
ꢀ ) 1670 M ‚cm ). Peroxynitrite was also synthesized by the
autoxidation of hydroxylamine in 0.5 M aqueous NaOH solution
9, 27). Oxygen gas was bubbled at a flow rate of ca. 0.2 L/min
into a solution (200 mL) of 10 mM NH OH in 0.5 M aqueous
NaOH containing 100 µM diethylenetriaminepentaacetic acid
DTPA) for 3 h at room temperature. The resulting solution
was treated with granular MnO as described above and filtered.
(
2
(
*
Address correspondence to this author at the Institut f u¨ r Physi-
ologische Chemie I, Heinrich-Heine-Universit a¨ t D u¨ sseldorf, Postfach
2
1
+
01007, D-40001 D u¨ sseldorf, Germany. Phone +49-211-811-2707; fax
Peroxynitrite was concentrated by freeze fractionation.
Ebselen Se-oxide, 2-phenyl-1,2-benzisoselenazol-3(2H)-one
49-211-811-3029. e-mail: helmut.sies@uni-duesseldorf.de.
†
On leave from Daiichi Pharmaceutical Co., Ltd., Tokyo, J apan.
Abstract published in Advance ACS Abstracts, December 1, 1995.
Note that the recommended IUPAC nomenclature for peroxynitrite
1
2 2
-oxide (3), was synthesized from ebselen (2) and H O as
X
1
previously described (28). The structure was confirmed by IR,
1
is oxoperoxonitrate; for peroxynitrous acid, hydrogen oxoperoxonitrate;
for nitric oxide, nitrogen monoxide. The term peroxynitrite is used in
the text to refer generically to both peroxynitrite anion (ONOO , 1)
and its conjugate acid, peroxynitrous acid (ONOOH, 1a ). When the
discussion is limited to either the anion or the acid, its specific name
is used.
MS, H NMR (500 MHz) spectra, and elemental analysis. Small
signals which correspond to the hydrolyzed ring-opened form
-
of ebselen Se-oxide (3), 2-(phenylcarbamoyl)phenylseleninic acid
1
(
3a , Se(O)OH), were observed in the H NMR spectrum (DMSO-
d
6
/TMS). These signals were significantly increased after
2
•-
•
Abbreviations: O
2
, superoxide anion radical; NO, nitric oxide
addition of H O (1 drop) to the sample solution, and the mixture
2
-
(nitrogen monoxide); ONOO , peroxynitrite anion (oxoperoxonitrate);
gave two spots on silica gel TLC tracing (solvent for develop-
ment: ethyl acetate/methanol/acetic acid, 10/5/1 v/v). Ebselen
Se-oxide (3) and its hydrolyzed form (3a ) were found to be in
equilibrium.
•
2
ONOOH, peroxynitrous acid (hydrogen oxoperoxonitrate); NO , nitric
•
dioxide; OH, hydroxyl radical; DTPA, diethylenetriaminepentaacetic
acid; DMSO, dimethyl sulfoxide; GSH, glutathione; GSH-Px, gluta-
thione peroxidase, SOD, superoxide dismutase.
0
893-228x/96/2709-0262$12.00/0 © 1996 American Chemical Society
+
+

Reaction of Ebselen with Peroxynitrite
Chem. Res. Toxicol., Vol. 9, No. 1, 1996 263
Gen er a l. Prior to experiments, the concentration of the stock
peroxynitrite solution was determined by measuring the absorb-
ance at 302 nm of an aliquot of the stock peroxynitrite solution
diluted in 0.1 M aqueous NaOH versus an aliquot of the stock
solution that was allowed to decompose in 0.5 M sodium
phosphate buffer (pH 7.4). The first-order rate constant of
peroxynitrite decay (k ) under these conditions was
d
-3 -1
calculated to be ca. 1 × 10
s
by exponential fitting.
Rea ctivity of Ebselen w ith P er oxyn itr ite. The
reaction of ebselen (2) with peroxynitrite in 0.05 M
sodium phosphate buffer (pH 10.9) was followed spec-
trophotometrically. The spectrum of 2 (50 µM) with an
absorbance maximum at 325 nm was altered substan-
tially after addition of peroxynitrite (30 µM), as shown
in Figure 1A. The spectrum of the reaction mixture after
addition of a slight excess of peroxynitrite (60 µM) was
similar to that of authentic ebselen Se-oxide (3) (Figure
d
spontaneous peroxynitrite decay (k ) was determined by least-
squares fitting of absorbance at 302 nm versus time. Phosphate
buffer is the least reactive solvent in terms of accelerating
peroxynitrite decomposition (29). The buffer was 0.05 or 0.5 M
sodium phosphate (pH 10-11) as indicated.
Rea ction System . Typically, peroxynitrite diluted in 0.5 M
aqueous NaOH was added to a solution of ebselen in sodium
phosphate buffer (1.0 mL) while vigorously vortexing to start
the reaction at room temperature (22 °C). For HPLC analysis,
methanol (2.0 mL) was added to the reaction mixture, and the
resulting mixture was cooled to -20 °C to precipitate buffer
salts. After centrifugation, the supernatant fraction was sepa-
rated and stored at -20 °C until analysis. Ebselen (2) was used
as a solution in methanol or DMSO, and the concentration of
organic solvent did not exceed 1% v/v in the reaction mixture.
Controls received solvent only. The pH was measured after
peroxynitrite addition to account for the slight alkaline shift
caused by the NaOH used to stabilize peroxynitrite. Recovery
of selenium compounds after the workup procedure was quan-
titative (>97%).
1
A). Further addition of peroxynitrite had no effect on
the spectrum. The spectral changes were very rapid and
were complete within <1 s; the rate constant is of the
6
-1
-1
order of 10
M
s
as determined by stopped-flow
measurement.3 The spectrum of authentic ebselen Se-
oxide (3, 50 µM) under the same conditions was not
changed upon addition of peroxynitrite (60 µM) (data not
shown).
Difference spectra of the reaction mixtures were almost
identical to that of authentic ebselen Se-oxide (3) (Figure
1
B). The relative absorbance change at 279 nm against
that at 327 nm, ∆A (279 - 327 nm), resulting from the
reaction with peroxynitrite (0-25 µM) was in proportion
to the peroxynitrite concentration (Figure 2) and was
completed at excess peroxynitrite over the 25 µM ebselen
Alternatively, solutions of peroxynitrite (initial 20 µM) in 0.5
M sodium phosphate buffer (pH 10.2) were prepared. A slight
excess amount of 2 (25 µM) was added separately at various
times after initial peroxynitrite addition to start the reaction.
Reactions were incubated for 10 min at room temperature (22
(2) present. This indicates a stoichiometry of 1:1.
Id en tifica tion of Rea ction P r od u cts. Peroxynitrite
°
C) and then worked up for HPLC as described above.
Sp ectr op h otom etr ic An a lysis. Absorbance measurements
(1.1 mM) was added dropwise to a solution of ebselen (2,
0.5 mM) in 0.1 M sodium phosphate buffer (pH 10.0)/
ethanol ) 7:3 (200 µL) and left standing at room
temperature. For this preparative reaction, ethanol was
included to obtain a higher concentration of 2. TLC
analysis and the comparison with authentic samples
showed nearly complete consumption of 2 and production
of its Se-oxide (3) as the sole product. HPLC analysis
and the coinjection with authentic samples showed nearly
complete consumption of 2 and production of 3 as well.
The HPLC analysis was done with an eluent of 0.1 M
aqueous ammonium acetate/acetonitrile, for which the
gradient was from 80/20 to 5/95 over 15 min at a flow
rate of 1.0 mL/min. Ebselen Se-oxide (3) appeared as
twin peaks which correspond to 3 and its hydrolyzed
form, 3a .
Yield of Ebselen Se-Oxid e a n d Stoich iom etr y. For
further analysis of the peroxynitrite-mediated oxidation
of ebselen (2), 10 mM sodium phosphate buffer (pH 7.4)/
acetonitrile was chosen as an eluent (details, see Materi-
als and Methods), and good separation was obtained
(Figure 3). The yield of ebselen Se-oxide (3) by peroxy-
nitrite-mediated oxidation of 2 was observed under
various peroxynitrite concentrations. As shown in Figure
were performed with a Perkin Elmer Lambda 2 spectrophotom-
eter. Absorbance spectra of the reaction mixture in a quartz
cuvette (light path 1 cm) were recorded immediately after
addition of peroxynitrite against buffer solution as reference.
Difference spectra were recorded against an ebselen reference
solution. Ebselen oxidation was measured by the absorbance
difference, ∆A (279 - 327 nm).
HP LC An a lysis. Aliquots of samples (typically, 20 µL) were
injected onto a reversed-phase column (RP C18, 150 mm × 4.6
mm i.d., Capcell Pak C18 (SG120) 5 µm; Shiseido Co., Tokyo).
Separation was performed with a 10 mM sodium phosphate
buffer, (pH 7.4)/acetonitrile gradient on a Merck-Hitachi L-6200A
HPLC unit, at a flow rate of 1.0 mL/min. The linear gradient
was typically from 80/20 to 24/76 over 20 min. The seleno-
organic compounds were monitored with a Merck-Hitachi 655A
UV detector equipped with D-2500 Chromato-Integrator at 254
nm. Ebselen Se-oxide (3) was fully separated from its parent
substrate, ebselen (2), and appeared as single peak (see Figure
3
). Calibration curves were calculated from peak areas versus
concentration values of authentic samples carried through the
extraction procedure and HPLC analysis. The calibration
curves were used to calculate the concentrations of the com-
pounds in the samples from their peak areas.
Nitr ite Qu a n tita tion . Nitrite (NO
2
-) concentration was
measured by the Griess reaction (30) with minor modifications.
Sample (1.0 mL) was added to 1% w/v sulfanilamide solution
4
, 2 was almost quantitatively converted to its Se-oxide
(3), and the yield was in proportion to the peroxynitrite
(1.0 mL in 2 M HCl). Thereafter, 1% w/v N-(1-naphthyl)-
concentration as long as 2 was available. Spontaneous
oxidation of ebselen (measured as 1.4 ( 0.4 µM) appears
as a small nonzero intercept. A stoichiometry of 1:1 was
determined from the slope of the plot of product yield
ethylenediamine solution (1.0 mL in 2 M HCl) was added to
the mixture. After 10 min at room temperature, the absorbance
of the resulting mixture at 540 nm was measured. Calibration
curves were calculated from the absorbance of standard NaNO
solutions.
2
(Figure 4). Complete oxidation of 2 was observed with
excess peroxynitrite addition.
The relationship between the product yield and the
stability of peroxynitrite in the reaction mixture was
investigated. For this purpose, 2 was added to buffer
solutions of peroxynitrite at various times after initial
Resu lts
Sta bility of P er oxyn itr ite. Spontaneous decomposi-
tion of peroxynitrite in 0.5 M sodium phosphate buffer
pH 10.2) at room temperature (22 °C) was measured by
(
monitoring the absorbance at 302 nm of the peroxynitrite
solution. The first-order rate constant of spontaneous
3
H. Masumoto, R. Kissner, W. H. Koppenol, and H. Sies, unpub-
lished work.
+
+

2
64 Chem. Res. Toxicol., Vol. 9, No. 1, 1996
Masumoto and Sies
F igu r e 2. Relationship between ∆A (279 - 327 nm) and
peroxynitrite concentration added. Ebselen (2, 25 µM) was
reacted with peroxynitrite as in Figure 1. Difference spectra
were monitored immediately after peroxynitrite addition. Linear
least-squares regression line is shown.
F igu r e 3. HPLC chromatograms of ebselen and ebselen Se-
oxide (A) and reaction mixture samples (B). (A) Authentic
ebselen (2) and ebselen Se-oxide (3) (0.15 and 0.13 nmol,
respectively). (B) Ebselen (2, 25 µM) reacted with peroxynitrite
F igu r e 1. Absorption spectra from the reaction of ebselen with
peroxynitrite. (A) Ebselen (2, 50 µM) was reacted with peroxy-
nitrite in 0.05 M sodium phosphate buffer (pH 10.9) at room
temperature (22 °C). Spectra were recorded immediately before
(20 µM). HPLC condition is as in Materials and Methods.
(a) and after peroxynitrite addition (30 µM, b; 60 µM, c).
Spectrum of authentic ebselen Se-oxide (3, 50 µM) is shown as
d. (B) Difference spectra were recorded immediately before (e)
and after peroxynitrite addition (10 µM, f; 25 µM, g) to a solution
of ebselen (2, 25 µM).
(Figure 5). The slope of the line in Figure 5 corresponds
-
3
-1
to 1.2 × 10
s
. The value compares well with the rate
) in the
). The yield was
constant of spontaneous peroxynitrite decay (k
d
-3
-1
peroxynitrite addition to start the reaction (for details,
see Materials and Methods). The yield of 3 followed an
exponential decrease after initial peroxynitrite addition
solution obtained above (ca. 1 × 10
s
dependent on the peroxynitrite concentration. The in-
tercept of the extrapolated line with the ordinate, 1.3,
+
+

Reaction of Ebselen with Peroxynitrite
Chem. Res. Toxicol., Vol. 9, No. 1, 1996 265
F igu r e 4. Oxidation of ebselen and formation of ebselen Se-
oxide as a function of peroxynitrite concentration. Ebselen (2,
F igu r e 6. Effect of pH on peroxynitrite-mediated oxidation of
ebselen. Ebselen (2, 25 µM) was reacted with peroxynitrite (30
µM) in 0.5 M sodium phosphate buffer. Reactions were incu-
bated for 30 min and assayed for the substrate (2, O), the
product (ebselen Se-oxide, 3, b), and 2 plus 3 (0) by HPLC. The
pH values were determined after completion of the reaction.
Values are means ( SD (n ) 3).
2
5 µM) was oxidized by peroxynitrite in 0.5 M sodium phosphate
buffer (pH 10.2) at room temperature (22 °C). Reactions were
incubated for 30 min and assayed for the substrate (2, O) and
the product (ebselen Se-oxide, 3, b) by HPLC. Lines are linear
least-squares regression lines. Values are means ( SD (n ) 3).
(9.0 and 18.5 µM at pH 5.3 and 10.2, respectively) was
dissolved in sodium phosphate buffer without organic
solvent as a vehicle and reacted with peroxynitrite (30
µM) at room temperature for 15 min. Methanol (123
mM), DMSO (70 mM), and ethanol (103 mM) did not
affect the yield of 3. Likewise, the radical scavenger
mannitol (100 mM) had no effect on the yield: relative
yield, 105 ( 6% and 93 ( 5% of control at pH 5.3 and
1
0.2, respectively. The metal chelator DTPA (100 µM)
also had no effect on the yield: relative yield, 102 ( 4%
and 95 ( 9% of control at pH 5.3 and 10.2, respectively.
Nitr ite P r od u ction . A peroxynitrite solution syn-
thesized from hydroxylamine was used for this experi-
ment since this preparation contains low nitrite contami-
nation as byproduct (27) as compared to the preparation
with the quenched-flow reactor. In order to distinguish
nitrite derived from spontaneous decomposition of per-
oxynitrite and/or nitrite initially present in the reaction
mixture from nitrite produced during the reaction with
ebselen (2), various concentrations of ebselen (0-40 µM)
were reacted with a fixed concentration of peroxynitrite
(58 µM) in 0.5 M sodium phosphate buffer (pH 10.2) and
were quantitatively oxidized. At this peroxynitrite con-
centration, nitrite contamination in the reaction mixture
F igu r e 5. Estimation of theoretical yield of ebselen Se-oxide.
Ebselen (2, 25 µM) was added to a solution of peroxynitrite
(
initial 20 µM) as in Figure 4 at the indicated time after initial
peroxynitrite addition. Reactions were incubated for 10 min and
assayed for the product (ebselen Se-oxide, 3) by HPLC. Non-
linear least-squares regression line is shown. Values are means
(background level) was 60 µM. Nitrite production in the
(
SD (n ) 3).
reaction with 2 was estimated as the increase of nitrite
concentration over the background level. The nitrite
production was in proportion to the substrate concentra-
tion. A yield of 0.41 mol of nitrite/mol of 2 added was
determined from the slope in the plot of nitrite production
versus ebselen concentration (data not shown).
corresponds to the theoretical yield of 3, 19.9 µM, in the
reaction mixture and is quantitative with respect to the
initial concentration of peroxynitrite (20 µM).
Yield of Ebselen Se-Oxid e a n d p H. The reaction
occurred also at acidic pH, but was greater at physiologi-
cal and alkaline pH, where the yield of 3 was almost
quantitative (Figure 6). At pH 6.5-10.2, the yield of 3
based on converted 2 was quantitative; i.e., ebselen (2)
which reacted with peroxynitrite was fully oxidized to 3.
Recovery of 2 plus 3 was less than quantitative at pH
Discu ssion
The results obtained here demonstrate that ebselen (2)
is oxidized by peroxynitrite rapidly and quantitatively,
forming its Se-oxide (3) as the sole selenium-containing
product at alkaline pH (Figures 3 and 4; Scheme 1). The
yield of 3 is governed by the amount of peroxynitrite
present in the reaction medium (Figure 5). At physi-
3
.6-5.2 (Figure 6).
Effect of An tioxid a n ts. Peroxynitrite-mediated oxi-
dation of ebselen (2) was evaluated in the presence of
antioxidants at pH 5.3 and 10.2. In this experiment, 2
+
+

2
66 Chem. Res. Toxicol., Vol. 9, No. 1, 1996
Masumoto and Sies
Sch em e 1. P oten tia l Detoxica tion Mech a n ism of
Ebselen a ga in st P er oxyn itr itea
mechanism of oxidation at the sulfur atom of methionine
(7, 8). The mechanism is to be studied by kinetic
analysis.
A novel principle of the antiinflammatory action of 2
resides in its high reactivity with peroxynitrite which,
importantly, leads to oxidation to 3 that can revert to 2
by reducing equivalents such as GSH. This could po-
tentially establish a sustained defense line against per-
oxynitrite.
Ack n ow led gm en t. We thank Drs. Karlis Briviba
and Wilhelm Stahl for valuable discussion. We also
thank Fonds der Chemischen Industrie, Frankfurt, for
support of this study.
Refer en ces
(
1) Beckman, J . S., Beckman, T. W., Chen, J ., Marshall, P., and
Freeman, B. A. (1990) Apparent hydroxyl radical production by
peroxynitrite: Implications for endothelial injury from nitric oxide
and superoxide. Proc. Natl. Acad. Sci. U.S.A. 87, 1620-1624.
2) Huie, R. E., and Padmaja, S. (1993) The reaction of NO with
superoxide. Free Radical Res. Commun. 18, 195-199.
3) Ischiropoulos, H., Zhu, L., and Beckman, J . S. (1992) Peroxynitrite
formation from macrophage-derived nitric oxide. Arch. Biochem.
Biophys. 298, 446-451.
a
The peroxynitrite-dependent oxidation of ebselen (2) to eb-
selen Se-oxide (3) is shown at left. The reduction of ebselen Se-
oxide (3) to 2 as mediated by GSH, for example, is shown at right.
(
(
ological pH, peroxynitrite (1) becomes protonated to
peroxynitrous acid (ONOOH, 1a ), and 1a decays rapidly
-
1
with the first-order rate constant, 1.3 s , at 25 °C (13).
Ebselen (2) reacts with peroxynitrite (1/1a ) almost quan-
titatively at pH 7.5 (Figure 6). The rate constant of the
(
4) Radi, R., Beckman, J . S., Bush, K. M., and Freeman, B. A. (1991)
Peroxynitrite-induced membrane lipid peroxidation: The cytotoxic
potential of superoxide and nitric oxide. Arch. Biochem. Biophys.
288, 481-487.
order 106
M
-1
s
-1
is one of the fastest known for
3
(5) Radi, R., Beckman, J . S., Bush, K. M., and Freeman, B. A. (1991)
peroxynitrite. Even at acidic pH, where 1a is predomi-
nant, 2 reacts significantly with peroxynitrite (1/1a ),
giving 3 as a main product. For example, 3 was obtained
as 56% yield at pH 5.2 (Figure 6). In view of the pK
.8 for 1a , the lower yield of 3 seems to be due to the
reaction rate that is competitive both with the rate of
the spontaneous decay process of 1a at acidic pH and
with the rate of pH-dependent formation of byproduct(s)
from 2. The evidence suggests that the reaction between
Peroxynitrite oxidation of sulfhydryls. J . Biol. Chem. 266, 4244-
4
250.
(6) Di Mascio, P., Bechara, E. J . H., Medeiros, M. H. G., Briviba, K.,
and Sies, H. (1994) Singlet molecular oxygen production in the
reaction of peroxynitrite with hydrogen peroxide. FEBS Lett. 355,
a
of
6
2
87-289.
(
7) Moreno, J . J ., and Pryor, W. A. (1992) Inactivation of R-1-
proteinase inhibitor by peroxynitrite. Chem. Res. Toxicol. 5, 425-
4
31.
(
8) Pryor, W. A., J in, X., and Squadrito, G. L. (1994) One- and two-
electron oxidations of methionine by peroxynitrite. Proc. Natl.
Acad. Sci. U.S.A. 91, 11173-11177.
2
and 1/1a is substantially faster than the spontaneous
decay process of 1/1a at physiological pH, even when the
concentration of ebselen is in the micromolar range. In
addition, radical scavengers have no effect on the yield
at acidic as well as alkaline pH. Ebselen (2) seems to
react with peroxynitrite (1/1a ) directly to give 3. Both
the anion (1) and the free acid (1a , ground state and
energized forms) are likely to be reactive species toward
ebselen. Further kinetic study will be needed to specify
which is the reactive species.
The main nitrogen-containing product derived from
peroxynitrite in the reaction is expected to be nitrite.
Nitrite is produced in proportion to the amount of
peroxynitrite reacted with 2, and 40% of peroxynitrite
reacted with 2 was recovered as nitrite. The recovery
was lower than expected. Nitrate is a possible further
product.
(9) Bartlett, D., Church, D. F., Bounds, P. L., and Koppenol, W. H.
1995) The kinetics of the oxidation of L-ascorbic acid by peroxy-
(
nitrite. Free Radical Biol. Med. 18, 85-92.
(
10) Halfpenny, E., and Robinson, P. L. (1952) The nitration and
hydroxylation of aromatic compounds by pernitrous acid. J . Chem.
Soc., 939-946.
(
11) van der Vliet, A., O’Neill, C. A., Halliwell, B., Cross, C. E., and
Kaur, H. (1994) Aromatic hydroxylation and nitration of phenyl-
alanine and tyrosine by peroxynitrite. Evidence for hydroxyl
radical production from peroxynitrite. FEBS Lett. 339, 89-92.
(12) Beckman, J . S., Ye, Y. Z., Anderson, P. G., Chen, J ., Accavitti, M.
A., Tarpey, M. M., and White, C. R. (1994) Extensive nitration of
protein tyrosines in human atherosclerosis detected by immuno-
histochemistry. Biol. Chem. Hoppe-Seyler 375, 81-88.
(13) Koppenol, W. H., Moreno, J . J ., Pryor, W. A., Ischiropoulos, H.,
and Beckman, J . S. (1992) Peroxynitrite, a cloaked oxidant formed
by nitric oxide and superoxide. Chem. Res. Toxicol. 5, 834-842.
14) Pryor, W. A., Cueto, R., J in, X., Koppenol, W. H., Ngu-Schwemlein,
M., Squadrito, G. L., Uppu, P. L., and Uppu, R. M. (1995) A
practical method for preparing peroxynitrite solutions of low ionic
strength and free of hydrogen peroxide. Free Radical Biol. Med.
(
Ebselen Se-oxide (3) is readily reduced by reducing
equivalents such as GSH to revert to 2 (28, 31), consti-
tuting an interesting potential defense line against
peroxynitrite (Scheme 1). Other reduced species derived
from 2 such as selenosulfides (S-[2-(phenylcarbamoyl)-
phenyl]- derivatives) and selenol (2-(phenylcarbamoyl)-
phenylselenol) can be generated in the presence of excess
thiols (32-34). In particular, the selenol is highly
reactive with oxidants (34, 35).
Oxidation reactions of peroxynitrite can involve a two-
electron process (nucleophilic attack of substrate) or an
one-electron transfer reaction (one-electron oxidation by
peroxynitrite). A two-electron process with bimolecular
displacement by the selenium could be feasible as in the
1
8, 75-83.
(
15) Crow, J . P., Spruell, C., Chen, J ., Gunn, C., Ischiropoulos, H.,
Tsai, M., Smith, C. D., Radi, R., Koppenol, W. H., and Beckman,
J . S. (1994) On the pH-dependent yield of hydroxyl radical
products from peroxynitrite. Free Radical Biol. Med. 16, 331-
3
38.
(
(
(
16) Wang, J .-F., Komarov, P., Sies, H., and de Groot, H. (1991)
Contribution of nitric oxide synthase to luminol-dependent chemi-
luminescence generated by phorbol-ester-activated Kupffer cells.
Biochem. J . 279, 311-314.
17) Wang, J .-F., Komarov, P., Sies, H., and de Groot, H. (1992)
Inhibition of superoxide and nitric oxide release and protection
from reoxygenetion injury by ebselen in rat Kupffer cells. Hepa-
tology 15, 1112-1116.
18) Cadenas, E., Wefers, H., M u¨ ller, A., Brigelius, R., and Sies, H.
(1982) Active oxygen metabolites and their action in the hepato-
cyte. Studies on chemiluminescence responses and alkane produc-
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Reaction of Ebselen with Peroxynitrite
Chem. Res. Toxicol., Vol. 9, No. 1, 1996 267
tion. In Agents and Actions Supplements (Parnham, M. J ., and
Winkelmann, J ., Eds.) Vol. 11, pp 203-216, Birkh a¨ user Verlag,
Basel.
(28) Fischer, H., and Dereu, N. (1987) Mechanism of the catalytic
reduction of hydroperoxides by ebselen: A selenium-77 NMR
study. Bull. Soc. Chim. Belg. 96, 757-768.
(
19) Parnham, M. J ., Leyck, S., Dereu, N., Winkelmann, J ., and Graf,
E. (1985) GSH-peroxidase-like organo-selenium compound with
antiinflammatory activity. Adv. Inflammation Res. 10, 397-401.
20) Wendel, A., and Tiegs, G. (1986) A novel biologically active seleno-
organic compound. VI. Protection by ebselen (PZ 51) against
galactosamine/endotoxin-induced hepatitis in mice. Biochem.
Pharmacol. 35, 2115-2118.
(29) Beckman, J . S., Chen, J ., Ischiropoulos, H., and Crow, J . P. (1994)
Oxidative chemistry of peroxynitrite. Methods Enzymol. 233,
2
29-240.
(
(
30) Kosaka, H., Imaizumi, K., Imai, K., and Tyuma, I. (1979)
Stoichiometry of the reaction of oxyhemoglobin with nitrite.
Biochim. Biophys. Acta 581, 184-188.
(
21) Kuhl, P., Borbe, H. O., Fischer, H., R o¨ mer, A., and Safayhi, H.
(31) Glass, R. S., Farooqui, F., Sabahi, M., and Ehler, K. W. (1989)
Formation of thiocarbonyl compounds in the reaction of ebselen
oxide with thiols. J . Org. Chem. 54, 1092-1097.
(1986) Ebselen reduces the formation of LTB
4
in human and
porcine leukocytes by isomerization to its 5S,12R-6-trans-isomer.
Prostaglandins 31, 1029-1048.
(
32) Maiorino, M., Roveri, A., Coassin, M., and Ursini, F. (1988) Kinetic
mechanism and substrate specificity of the glutathione peroxidase
activity of ebselen (PZ 51). Biochem. Pharmacol. 37, 2267-2271.
(
22) Parnham, M. J ., and Graf, E. (1987) Seleno-organic compounds
and the therapy of hydroperoxide-linked pathological conditions.
Biochem. Pharmacol. 36, 3095-3102.
23) M u¨ ller, A., Cadenas, E., Graf., P., and Sies, H. (1984) A novel
biologically active seleno-organic compound. I. Glutathione per-
oxidase-like activity in vitro and antioxidant capacity of PZ 51
(
33) Akerboom, T. P. M., Sies, H., and Ziegler, D. M. (1995) The
oxidation of ebselen metabolites to thiol oxidants catalyzed by
liver microsomes and perfused rat liver. Arch. Biochem. Biophys.
(
3
16, 220-226.
(ebselen). Biochem. Pharmacol. 33, 3235-3239.
(
24) Wendel, A., Fausel, M., Safayhi, H., Tiegs, G., and Otter, R. (1984)
A novel biologically active seleno-organic compound. II. Activity
of PZ 51 in relation to glutathione peroxidase. Biochem. Phar-
macol. 33, 3241-3245.
(34) Cotgreave, I. A., Morgenstern, R., Engman, L., and Ahokas, J .
(1992) Characterization and quantitation of a selenol intermediate
in the reaction of ebselen with thiols. Chem.-Biol. Interact. 84,
69-76.
(
(
(
25) Sies, H. (1993) Ebselen, a selenoorganic compound as glutathione
peroxidase mimic. Free Radical Biol. Med. 14, 313-323.
26) Schewe, T. (1995) Molecular actions of ebselensan antiinflam-
matory antioxidant. Gen. Pharmacol. 26, 1153-1169.
27) Hughes, M. N., and Nicklin, H. G. (1971) Autoxidation of
hydroxylamine in alkaline solutions. J . Chem. Soc. (A), 164-168.
(
35) Morgenstern, R., Cotgreave, I. A., and Engman, L. (1992) Deter-
mination of the relative contributions of the diselenide and selenol
forms of ebselen in the mechanism of its glutathione peroxidase-
like activity. Chem.-Biol. Interact. 84, 77-84.
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