Mechanistic investigations on the efficient catalytic decomposition of peroxynitrite by ebselen analogues

Article abstract of DOI:10.1039/c0ob01234c

In this study, ebselen and its analogues are shown to be catalysts for the decomposition of peroxynitrite (PN). This study suggests that the PN-scavenging ability of selenenyl amides can be enhanced by a suitable substitution at the phenyl ring in ebselen

Full text of DOI:10.1039/c0ob01234c

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PAPER
Mechanistic investigations on the efficient catalytic decomposition of
peroxynitrite by ebselen analogues†
Krishna P. Bhabak, Amit A. Vernekar, Surendar R. Jakka, Gouriprasanna Roy and Govindasamy Mugesh*
Received 22nd December 2010, Accepted 20th April 2011
DOI: 10.1039/c0ob01234c
In this study, ebselen and its analogues are shown to be catalysts for the decomposition of peroxynitrite
(PN). This study suggests that the PN-scavenging ability of selenenyl amides can be enhanced by a
suitable substitution at the phenyl ring in ebselen. Detailed mechanistic studies on the reactivity of
ebselen and its analogues towards PN reveal that these compounds react directly with PN to generate
highly unstable selenoxides that undergo a rapid hydrolysis to produce the corresponding seleninic
acids. The selenoxides interact with nitrite more effectively than the corresponding seleninic acids to
produce nitrate with the regeneration of the selenenyl amides. Therefore, the amount of nitrate formed
in the reactions mainly depends on the stability of the selenoxides. Interestingly, substitution of an
oxazoline moiety on the phenyl ring stabilizes the selenoxide, and therefore, enhances the isomerization
of PN to nitrate.
against PN. Furthermore, Sies et al. have shown that a number
of sulfur- and selenium-containing compounds can act as efficient
Introduction
Peroxynitrite (PN, ONOO-), a strong biological oxidizing and
inhibitors of PN-mediated oxidation and nitration reactions.10,14
The GPx-like properties of these compounds have been shown
to be responsible for their PN scavenging effects. It is well
known that the organoselenium compound ebselen (1) acts as
an efficient scavenger of PN and provides defense against PN-
mediated toxicity.15 However, the mechanism by which ebselen
scavenges PN is still not clear. It has been shown that ebselen
reacts with PN to produce the corresponding selenoxide (4) as
the only selenium-containing product.15 In this reaction, PN is
nitrating species, is generated in vivo by the diffusion-controlled
reaction of superoxide anion radical (O2 -) and nitric oxide (NO∑).1
∑
PN is a major component of the reactive nitrogen species (RNS)
and reactive oxygen species (ROS) systems which are involved in
nitrosative and oxidative stress.2 The formation of PN has been
detected in activated macrophages3 and endothelial cells.4 It has
been shown that the overproduction of PN induces DNA damage5
and initiates lipid peroxidation in biomembranes or low-density
lipoproteins.6 In addition, it can oxidize protein/non-protein
thiols7 and is known to inactivate a variety of enzymes by tyrosine
nitration, and thereby, hampers signal transduction process.8,9 As
the PN-mediated nitration of tyrosine residues as well as the
oxidation of bio-components are harmful to biological systems
in general and proteins and peptides having tyrosyl residues in
particular, it is important to develop synthetic compounds that
can effectively inhibit the PN-mediated nitration and oxidation
reactions.
-
-
converted to nitrite (NO2 ) and the amount of NO2 produced in
the reaction depends on the amount of ebselen used in the reaction.
A number of studies reveal that some amount of NO2- is converted
-
to NO3 , indicating that ebselen may catalyze the isomerization of
PN to NO3 .15a A few years ago, Musaev et al. reported, with
-
the help of theoretical calculations, that ebselen and its analogues
-
(1–3) cannot catalyze the isomerization of PN to NO3 (Fig. 1).16
This conclusion was based on the assumption that the reactions of
compounds 1–3 with PN produce the corresponding selenoxides
4–6, which are incapable of transferring oxygen from selenium to
NO2- and the energy barrier for the conversion of these selenoxides
A number of biologically occurring small-molecules such as
CO2, ascorbate, methionine, cysteine etc. have been shown to
react with PN and thus protect the biomolecules from PN-
mediated damage.10 The antioxidant mammalian selenoenzymes
such as glutathione peroxidase (GPx),11 selenoprotein P12 and
thioredoxin reductase (TrxR)13 also provide enzymatic defense
Department of Inorganic and Physical Chemistry, Indian Institute of Science,
Bangalore 560 012, India. E-mail: mugesh@ipc.iisc.ernet.in; Fax: (+)91-
80-2360 1552/2360 0683; Tel: (+)91-80- 2360 2566
† Electronic supplementary information (ESI) available. CCDC reference
number 765980. For ESI and crystallographic data in CIF or other
electronic format see DOI: 10.1039/c0ob01234c
Fig. 1 Chemical structures of ebselen and related compounds.
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to the selenenyl amides is very high. Therefore, the dissociation of
selenoxide-nitrite complex has been shown to be more favored
than the transfer of oxygen from the selenoxide to nitrite.
Recently, we have shown that the reaction of ebselen with H2O2
does not produce the selenoxide (4) as previously reported, but
that it produces the corresponding seleninic acid (7) as the only
selenium-containing product.17 As the reaction of ebselen with
PN may also produce the seleninic acid 7, a re-investigation
of the mechanism is required for a better understanding of
the antioxidant activity of ebselen. In this paper, we show that
the reaction of ebselen with PN produces the seleninic acid 7
via selenoxide (4) and the rapid hydrolysis of the selenoxide
4 to the seleninic acid (7) is a deactivating pathway for the
-
isomerization of PN to NO3 . We also show for the first time that
any substituent capable of stabilizing the selenoxides can enhance
the isomerization reaction by preventing the hydrolysis.
Results and discussion
Inhibition of PN-mediated reactions
Ebselen (1) and its analogues 8–13 used in the present study
were synthesized by treating 2-chloro-selenobenzoyl chloride with
appropriate primary amines (Fig. 2).18 The diselenides 15–17 were
synthesized in almost quantitative yields by treating the corre-
sponding selenenyl amides (1, 11 and 12) with triphenylphosphine.
Compound 14 was synthesized by following a lithiation route
as previously described.19 To understand the inhibition potency
of these selenium compounds towards PN-mediated nitration
reactions, we have chosen some representative compounds and
carried out the inhibition of PN-mediated nitration of bovine
serum albumin (BSA). The nitration of tyrosine residues in BSA
has been used extensively as a biomarker for oxidative and
nitrosative stress.20 The nitration was studied by immunoblotting
method and the nitrated tyrosine residues in the protein were
detected using an antibody against 3-nitro-L-tyrosine.
Fig. 3 Immunoblotting for the inhibition of PN-mediated nitration of
BSA. BSA (133.3 mM) was incubated with PN (1.5 mM) and inhibitors
(80.0 mM) in phosphate buffer (500 mM, pH: 6.9) at 20 ◦C for 7 min and
then subjected to gel electrophoresis. The nitrated proteins were detected
by immunoblotting using the antibody against 3-nitro-L-tyrosine.
ebselen analogues in which substitution at the nitrogen atom has
a considerable effect on the GPx activity.18 On the other hand, the
6-oxazoline substituted selenenyl amide 14 showed significantly
higher inhibition activity than other selenenyl amides indicating
some positive role of the additional oxazolyl group in 14 towards
PN scavenging. The diselenide 15 has been found to be the
strongest PN scavenger among all the compounds studied for the
inhibition of PN-mediated nitration of BSA. As shown in Fig.
3, the diselenide 15 corresponding to ebselen is almost two times
more active than ebselen. The higher activity of the diselenide
can be ascribed to the availability of two selenium moieties in the
diselenide unit for scavenging PN.
To understand the relative activities of all the selenenyl amides
and diselenides towards PN-mediated reactions, we have carried
out the PN-mediated nitration of free L-tyrosine. The nitration of
L-tyrosine was followed by a reverse-phase HPLC method and the
formation of 3-nitro-L-tyrosine was monitored at 275 nm. The IC50
values (concentration of the test compound required to inhibit
50% of the nitration) were calculated from the peak area of 3-
nitro-L-tyrosine at various concentrations of selenium compounds
(Table 1). In addition to the nitration, we have also studied
the PN-mediated oxidation of dihydrorhodamine 123 (DHR) by
fluorescence spectroscopy. In this assay, the IC50 values represent
the concentrations of selenium compounds required to inhibit 50%
of the conversion of DHR to rhodamine 123 (Table 1).
Fig. 2 Chemical structures of selenenyl amides (8–14) and diselenides
(15–17).
As can be seen from Fig. 3, all the selenenyl amides and
diselenides significantly inhibited the nitration reactions. The
inhibitory activity of selenenyl amides 2, 8 and 13 were found
to be almost comparable to that of ebselen. This indicates that
substitution at the nitrogen atom in selenenyl amides does not
have a significant effect on the PN-mediated nitration of BSA.
This is in contrast to the GPx-like antioxidant activities of these
From Table 1, it is clear that ebselen and related compounds
effectively inhibit both the PN-mediated nitration of free L-
tyrosine and the oxidation of DHR. For example, at 1500 mM
concentration of PN, ebselen showed an IC50 value of 63.1
0.3 mM for the nitration reaction. Compound 2, which lacks the
phenyl group of ebselen, was found to be two times more active
than ebselen. This is in contrast to the inhibition of PN-mediated
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Table 1 A comparison of IC50 values for the inhibition of PN-mediated
nitration of L-tyrosine and oxidation of DHR by compounds 1–2 and 8–17
an increase in the concentration of PN till 1500 mM (Figure S1,
ESI†), ebselen at 64 mM concentration is expected to show ~1–2%
inhibition. Similarly, an IC50 value of 4.0 0.2 mM for compound
15 is remarkable as a concentration of ~500 mM is required for
a stoichiometric inhibition. These observations indicate that all
the selenenyl amides and diselenides catalytically scavenge PN. In
other words, all these compounds catalyze the isomerization of
IC50 value (mM)a
Nitration of
Compound
L-tyrosineb
Oxidation of DHRc
1, Ebselen
63.1 0.3
35.2 0.1
42.1 0.1
35.4 0.1
29.3 0.3
35.5 0.3
23.4 0.3
21.5 0.4
5.9 0.1
0.8 0.01
49.2 1.10
1.3 0.01
1.2 0.02
1.6 0.02
1.5 0.01
0.7 0.01
1.8 0.01
15.5 0.03
2.8 0.03
16.3 0.01
1.2 0.01
-
2
PN to NO3 during the nitration of L-tyrosine.
8
9
In contrast to the catalytic nature of the inhibition of nitra-
tion reactions, the selenium compounds act as stoichiometric
scavengers of PN in the oxidation of DHR (Table 1). The
concentrations required for 50% inhibition by some selenium
compounds (2, 14 and 16) were found to be much higher than
the PN concentration (0.97 mM). As the oxidation of DHR to
rhodamine 123 by PN is extremely fast, the selenium compounds
10
11
12
13
14
15
16
17
4.0 0.2
13.4 0.3
22.6 0.1
-
are unable to catalyze isomerization of PN to NO3 . Therefore,
the oxidation of the selenium center in ebselen and related
compounds alone is responsible for the observed IC50 values.
It should be noted that the nitration reactions were relatively
slow and there was sufficient time for the inhibitors to interact
with PN. These observations indicate that the isomerization of
PN to NO3 is important for slow reactions, but it does not
contribute to the overall activity of selenium compounds for fast
reactions.
a The concentration of inhibitor required to inhibit 50% of the nitration
or oxidation. b The test mixture with each concentration of inhibitor was
incubated at 22 ◦C for 7 min for the nitration assay. Assay conditions: L-
tyrosine (1.0 mM), PN (1.5 mM), inhibitors (variable) in phosphate buffer
(100 mM, pH: 7.5). c Assay conditions: DHR (0.5 mM), PN (0.97 mM) and
inhibitors (variable) with 100 mM DTPA in phosphate buffer (100 mM,
pH: 7.5) at 22 ◦C. The fluorescence emission intensity due to rhodamine
123 was monitored immediately after the addition of PN at 526 nm.
-
nitration of BSA in which the activities of ebselen and analogue 2
were comparable. Compounds 8–10 having alkyl substituents on
the nitrogen were found to be more effective than ebselen. The
activity of the bromo-substituted compound 11 was also found to
be higher than that of ebselen. These observations indicate that the
IC50 values for the nitration of L-tyrosine do not correlate well with
the inhibition potencies toward PN-mediated nitration of BSA and
also with the GPx-like antioxidant activities. However, compound
12 having an m-hydroxy substituent on the phenyl ring exhibited
much better activity than ebselen, which is in agreement with the
GPx activity.18 Compound 13 having an aliphatic alcohol side
chain also exhibited good PN-scavenging activity. Interestingly,
compound 14 that contains an oxazoline moiety in the 6-
position of the phenyl ring was found to be almost 10 times
more active than ebselen. This compound was also more active
than compound 13 that lacks the oxazoline group, which is in
agreement with the inhibition of protein nitration. The diselenides
15–17 also exhibited good PN-scavenging activity, although the
activity of compound 17 was almost identical to that of the
corresponding selenenyl amide 12. These observations indicate
that the PN-scavenging activity of ebselen and related compounds
not only depends on the nature of the selenium moiety, but also
depends on the nature of the substrate used for nitration and the
assay conditions. However, the inhibition of nitration by various
ebselen analogues (Fig. 3) at 80 mM concentration of selenium
compounds and 1500 mM concentration of PN indicates that
these compounds effectively catalyze the isomerization of PN to
Reaction of ebselen analogues with PN
To understand the reactivity of ebselen analogues towards PN,
we have analyzed the reactions by 77Se NMR spectroscopy. When
ebselen was treated with an excess amount of PN (10–12 equiv), the
peak at 964 ppm corresponding to ebselen disappeared completely
and a new signal was observed at 1137 ppm. Similarly, the reaction
of 13 with PN produced a new signal at 1136 ppm. In this reaction,
the peak corresponding to the starting material (885 ppm)
disappeared completely. The isolation and characterization of
products from these reactions of ebselen and 13 with PN indicate
the formation of the corresponding seleninic acids 7 and 19,
respectively. These observations suggest that the hydrolysis of
selenoxides 4 and 18 leads to the formation of the corresponding
seleninic acids 7 and 19, respectively (Scheme 1). It should be noted
that the formation of the seleninic acid 7 as the only selenium-
containing compound has been observed previously in the reaction
-
NO3 .
A detailed analysis of the IC50 values (range from 4.0 0.2 to
63.1 0.1 mM) and the concentrations of PN used for the nitration
reactions (1500 mM) indicates that these compounds inhibit
the nitration reaction in a catalytic manner. For stoichiometric
reactions, the IC50 values are expected to be in the mM range.
As the concentration of 3-nitro-L-tyrosine increases steadily with
Scheme 1 Reaction of ebselen (1) and its analogue 13 with PN to produce
the corresponding seleninic acids 7 and 19, respectively. A similar reaction
of compound 14 with PN produces the corresponding selenoxide 20 as the
only stable product.
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of ebselen with H2O2.17 In contrast, the reaction of oxazoline-
based compound 14 with PN did not produce the seleninic acid
21, but it produced the selenoxide 20 in nearly quantitative yield
(Scheme 1).
The reaction of 14 with PN produced a new signal at 1102 ppm,
which can be ascribed to the selenoxide 20. The isolation
and characterization of this compound by single crystal X-ray
diffraction studies† indicate that the oxazoline nitrogen strongly
corresponding selenenic acids (Ar-SeOH) with the formation
of NO3 . The selenenic acids thus produced may eliminate a
-
water molecule to regenerate the corresponding selenenyl amides.
However, our experimental results suggest that selenoxides are
more efficient than the seleninic acid for the conversion of NO2- to
-
NO3 . As the hydrolysis of selenoxides disfavors the isomerization
process, it is important to prevent the hydrolysis to enhance the
catalytic isomerization. In agreement with this, the selenoxide
20, which does not undergo any hydrolysis, rapidly regenerates
the selenenyl amide 14 under identical conditions. While ~50%
regeneration of ebselen from the seleninic acid 7 was observed
after ~24 h, a complete regeneration of selenenyl amide 14 from
the corresponding selenoxide was observed (Figures S48 and S51,
ESI†). Therefore, the isomerization of PN to NO3- is favored in the
presence of compound 14. This is also reflected in the IC50 values
obtained for this compound in the nitration reactions (Table 1,
Fig. 3).
˚
interacts with the selenium center (rSe1 ◊ ◊ ◊ N2 = 2.775 A). This
interaction is slightly weaker than that observed for the selenenyl
amide 14.19a However, the non-bonded Se ◊ ◊ ◊ N interaction does
not alter the hydrolysis of Se1–N1 bond in compound 20.
In the crystal structure, the selenoxide (–Se O) moiety lies
perpendicular to the selenazole ring plane (Fig. 4). The steric
hindrance of the oxazoline moiety may also play an important
role in the stabilization of selenoxide 20. Although the Se1 ◊ ◊ ◊ N2
interaction in compound 20 may activate the Se1–N1 bond
towards hydrolysis, similar interaction in the seleninic acid (21)
is expected to destabilize the compound. Therefore, a rapid
elimination of a water molecule would regenerate the selenoxide
(20). In the absence of such interactions in compounds 7 and 19,
the regeneration of selenoxides 4 and 18 appears to be very slow.
-
To further confirm the isomerization of PN to NO3 in the
presence of selenium compounds, we have determined the con-
-
centration of NO3 after keeping the reaction mixture of PN and
selenium compounds for 30 min in the assay buffer. The amount of
NO3- formed in the reaction was then determined by measuring the
concentration of nitro-resorcinol in the presence of concentrated
H2SO4 by following the literature procedure.21 In the presence of
-
concentrated H2SO4, the NO3 produced by the isomerization of
PN nitrates resorcinol and the measurement of the concentration
of nitro-resorcinol at 505 nm by UV–vis spectrophotometry
-
allowed the quantification of NO3 in the solution. A significant
enhancement in the absorbance due to nitro-resorcinol was
observed when resorcinol was treated with PN in the presence of
selenium compounds (Table S25, ESI†). Although there is no clear
correlation between the amount of nitro-resorcinol formed and the
IC50 values for the nitration reactions, these observations suggest
that the catalytic PN-scavenging activity of selenium compounds
-
is mainly due to the isomerization of PN to NO3 .
To confirm that the selenoxide 20 is the main species that
catalyzes the isomerization of PN to NO3- by interacting with the
-
eliminated NO2 ion from PN, we have carried out a series of ex-
Fig. 4 ORTEP diagram of compound 20. Displacement ellipsoids are
drawn at the 50% probability level and hydrogen atoms are shown as small
spheres of arbitrary radii.
periments with the isolated selenoxide 20 and NaNO2 in the assay
buffer. Interestingly, similarly to the reaction of selenenyl amide 14
with PN, the reaction of pure selenoxide 20 with NaNO2 produced
nitro-resorcinol under identical experimental conditions. This
indicates the direct reactivity of the selenoxide 20 towards NO2
leading to the formation of NO3 . To understand whether the
conversion of NO2 to NO3 by selenoxide 20 is catalytic or not,
the formation of nitro-resorcinol was measured with an increasing
concentration of selenoxide 20. An enhancement in the absorbance
due to nitro-resorcinol was observed with increasing concentration
of selenoxide 20 (Table S26 and Figure S26, ESI†), indicating the
Interestingly, when the solution of ebselen with PN was kept
for a few hours, the regeneration of ebselen was observed. The
intensity of the peak for ebselen increased with time, and after
~24 h around 50% of the seleninic acid 7 was converted to ebselen
(Figure S48, ESI†). This indicates the possibility that the hydrolysis
of selenoxide 4 is reversible and a considerable amount of seleninic
acid 7 may undergo cyclization to produce the selenoxide 4
(Scheme 1). Similarly, compound 13 was regenerated almost
quantitatively when the solution of seleninic acid 19 was kept for
-
-
-
-
-
catalytic nature of the isomerization of PN to NO3 .
-
a long time. In solution, the selenoxides 4 and 18 react with NO2
To analyze the catalytic intermediates involved in the isomeriza-
tion process, we have studied the reactivity of pure selenoxide 20
with NaNO2 and/or PN. The intermediates and products of the
reactions were analyzed by 77Se NMR spectroscopic and ESI-MS
spectrometric techniques. Treatment of 20 with NaNO2 produced
a new signal at 1115 ppm in the 77Se NMR experiment along
with the peak for the selenoxide at 1100 ppm. The new signal
can be ascribed to the corresponding selenoxide–nitrite complex
24. Formation of complex 24 was further confirmed by ESI-MS
to regenerate the selenenyl amides 1 and 13 with an elimination of
-
NO3- anion and thereby catalyze the conversion of NO2- to NO3
(Scheme 1). Therefore, the stability of the selenoxides determines
-
the conversion of NO2- to NO3 . In other words, the isomerization
of PN to NO3- depends on the stability of the selenoxides produced
in the reactions. The seleninic acids such as 7 and 19 may also
-
-
catalyze the conversion of NO2 to NO3 by interacting directly
-
with NO2 . In this case, the seleninic acids may generate the
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spectrometric analyses (Figures S55 and S56, ESI†). The reaction
mixture slowly regenerated the selenenyl amide 14 upon keeping
the solution for a longer time. Interestingly, the peak at 1115 ppm
due to complex 24 disappeared completely upon addition of PN.
This observation indicates that the selenoxide–nitrite complex 24
reacts with PN to regenerate the selenoxide 20, which can further
Catalytic isomerization of PN
Based on our experimental evidences, a catalytic mechanism for
the isomerization of PN to NO3- by compound 14 can be proposed
(Scheme 3). According to this mechanism, the selenenyl amide 14
reacts with PN to produce the corresponding selenoxide 20 via
the selenenyl amide-(O-ONO) complex 23. The oxidation of the
selenium center in compound 14 is less favored as compared to
that of ebselen and 13. This is due to the presence of a Se ◊ ◊ ◊ N
interaction that considerably reduces the reactivity of selenium
-
react with NO2 in a catalytic fashion. As the regeneration of
selenenyl amide 14 is observed only upon keeping the reaction
mixture for at least 24 h (Figure S59, ESI†), the selenenyl amide
14 may lie off the main catalytic cycle.
-
towards PN. The elimination of NO2 from complex 23 produces
Although a number of ebselen analogues and related monose-
lenides have been studied as PN-scavengers,10,14 the reactivity of
the corresponding diselenides towards PN has not been studied.
Therefore, we treated the diselenide 15 with PN and the reaction
was followed by 77Se NMR spectroscopy. In this reaction, a peak
at 1133 ppm corresponding to the seleninic acid 7 was obtained
along with a peak for ebselen. When a large excess of PN (~10–
15 equiv) was used, a quantitative conversion of diselenide 15 to
the seleninic acid 7 was observed (Figures S51 and S52, ESI†).
This indicates that the reaction of diselenides with PN produces
a mixture of selenenic and seleninic acids (22 and 7) as shown in
Scheme 2. The formation of ebselen in the reaction of diselenide
with PN is due to a rapid cyclization of 22 (Scheme 2). In the
presence of an excess amount of PN, ebselen undergoes further
reaction with PN to give the seleninic acid 7 via the formation of
selenoxide 4. As these processes utilize a greater amount of PN,
the diselenide 15 was found to be more active than ebselen (Table
1). The generation of ebselen in the reaction of 15 with H2O2 has
been reported earlier.17
the selenoxide 20. The Se ◊ ◊ ◊ N interaction is retained in the se-
lenoxide structure and is, therefore, responsible for the stability of
selenoxide 20. The next step is the interaction between selenoxide
-
-
20 and NO2 to produce the selenoxide–NO2 complex 24. In the
presence of an excess amount of PN, complex 24 reacts further with
-
PN to regenerate the selenoxide 20 with the elimination of NO2
-
and NO3 . When PN is depleted in the reaction mixture, a slower
transfer of oxygen from selenium to NO2 in complex 24 leads to
the elimination of NO3 with the regeneration of compound 14
-
-
(Scheme 3). It should be noted that the first half of the reaction is
probably responsible for the inhibition of PN-mediated oxidation
of DHR and the second half of the reaction i.e., NO3- formation, is
important for the inhibition of PN-mediated nitration reactions.
The presence of the oxazoline moiety at the 6-position of the
-
phenyl ring favors the formation of NO3 .
Scheme 3 Proposed mechanism for the catalytic isomerization of PN to
-
NO3 by the 6-oxazoline-substituted selenenyl amide 14.
Scheme 2 The proposed reaction pathways of ebselen diselenide 15 with
PN to produce the corresponding seleninic acid 7 as the main oxidation
product.
The catalytic mechanism shown in Scheme 3 is similar to the
one proposed for an iron–porphyrin complex.21 In this case, the
FeIII–L cationic complex reacts with PN to produce an L–FeIII–O–
ONO species, which undergoes homolysis to produce an O FeIV–
L intermediate. The recombination of this intermediate with nitrite
generates a L–FeIII–O–NO2 complex, which upon elimination of
nitrate regenerates the FeIII–L cationic complex. Therefore, the two
electron redox processes at the selenium centers in the selenenyl
amides appear to be quite similar to those of the FeIII–L cationic
complex.
-
Although ebselen analogues catalyze the formation of NO3
-
from NO2 , the isomerization reactions do not play an important
role in the inhibition of PN-mediated oxidation of DHR. As the
oxidation of DHR by PN is extremely fast, the rapid oxidation of
the selenium centers in the selenenyl amides and diselenides alone
is responsible for the inhibition. The lower activity of compound
14 as compared to that of compound 13 in the inhibition of PN-
mediated oxidation of DHR is due to the presence of a strong
Se ◊ ◊ ◊ N interaction in compound 14, which significantly decreases
the reactivity of selenium towards PN. Therefore, the oxazoline
substituent plays important roles in the nitration reactions than
the oxidation of DHR.
Conclusions
In this study, we show that the reactions of selenenyl amides with
peroxynitrite (PN) produce the corresponding seleninic acids via
selenoxides. Hydrolysis of selenoxides to the seleninic acids is a
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1
deactivating pathway, which is probably due to the lower reactivity
of seleninic acids towards NO2 as compared to selenoxides. This
Compound 16: H NMR (DMSO-d6) d (ppm): 7.12–7.20 (m,
2H), 7.31 (d, 2H, J = 8.0 Hz), 7.45 (d, 2H, J = 8.0 Hz), 7.50
(d, 1H, J = 8.0 Hz), 7.65 (d, 1H, J = 8.0 Hz), 10.44 (s, 1H).
13C NMR (DMSO-d6) d (ppm): 117.0, 123.5, 127.6, 129.5, 131.2,
132.5, 132.8, 133.1, 134.4, 138.7, 167.4. 77Se NMR (DMSO-d6)
d (ppm): 441. ESI-MS m/z calcd. for C26H19Br2N2O2Se2 [M+H]+
708.81; found: 708.78.
-
study also reveals that the introduction of an oxazoline substituent
at the 6-position of the phenyl ring in ebselen derivative can prevent
the hydrolysis by stabilizing the selenoxide. The stabilization of
selenoxide leads to an efficient catalytic isomerization of PN
to nitrate. The overall activity of ebselen and its analogues on
PN-mediated reactions depends on the nature of substrate used
for the oxidation or nitration reaction. While the isomerization
plays an important role in the inhibition of PN-mediated tyrosine
nitration, such isomerization does not have any effect on PN-
mediated oxidation of dihydrorhodamine 123. These observations
indicate that the selenium compounds that can enhance the PN
↔ nitrate isomerization would provide a better protection against
PN-mediated reactions.
1
Compound 17: H NMR (DMSO-d6) d (ppm): 6.52 (d, 3H,
J = 8.0 Hz), 7.07–7.14 (m, 2H), 7.27 (s, 1H), 7.33–7.41 (m, 2H),
7.72 (d, 1H, J = 8.0 Hz), 7.84 (d, 1H, J = 8.0 Hz), 10.42 (s, 1H).
13C NMR (DMSO-d6) d (ppm): 108.6, 112.4, 127.6, 129.4, 130.4,
131.1, 132.7, 132.9, 134.9, 140.4, 158.3, 167.3. 77Se NMR (DMSO-
d6) d (ppm): 443. ESI-MS m/z calcd. for C26H21N2O4Se2 [M+H]+
584.98; found: 584.81.
Synthesis of compound 19
To a stirred solution of 13 (0.025 g, 0.092 mmol) in methanol was
added an alkaline solution of peroxynitrite in excess amount (10–
12 equiv) and the reaction mixture was stirred at room temperature
for 2 h. The solvent was evaporated to obtain crude solid which was
purified by a reverse phase C18 column using water and methanol
as eluent. The solvent was evaporated in vacuo to obtain the
pure compound 19 as white solid. Yield: 9.7 mg (37%). 1H NMR
(MeOH-d4) d (ppm): 1.29 (s, 6H), 3.66 (s, 2H), 7.40 (t, 1H, J =
8.0 Hz), 7.51–7.61 (m, 2H), 7.81 (s, 1H), 8.10 (d, 1H, J = 8.0 Hz) 13C
NMR (MeOH-d4) d (ppm): 23.4, 56.3, 67.3, 125.2, 127.7, 130.3,
131.1, 135.9, 152.3, 169.3. 77Se NMR (MeOH-d4) d (ppm): 1145.
ESI-MS m/z calcd. for C11H15NO4Se [M+2H]2+ 307.01; found:
307.60.
Experimental section
General procedure
Compounds 2, 8–12,18 13,17 1419a and 1517 were synthesized by
following literature methods. 1H (400 MHz), 13C (100 MHz)
and 77Se (76 MHz) NMR spectra were obtained on a Bruker
400 MHz NMR spectrometer. Chemical shifts are cited with
respect to Me4Si (1H and 13C) as internal, and Me2Se (77Se) as
external standards. Mass spectral studies were carried out on a
Bruker Daltonics Esquire 6000plus mass spectrometer with ESI-
MS mode analysis. UV–vis spectroscopic experiments were carried
out on a Varian CARY 300 Bio spectrophotometer. Peroxynitrite
(PN) was synthesized in the laboratory following the literature
procedure.20f,22
Synthesis of compound 20
To a solution of selenazole 14 (0.10 g, 0.272 mmol) in MeOH
was added an alkaline solution of an excess amount of PN (10–
12 equiv). The reaction mixture was stirred for 12 h at room
temperature and then extracted with dichloromethane (3 ¥ 50 mL)
to remove the unreacted starting materials and other non-polar
impurities. The aqueous solution was then neutralized with 1 N
HCl and the solvent was evaporated by lyophilization. The white
solid obtained was dissolved in a minimum amount of methanol
to exclude the excess amount of NaCl. The solution was filtered
and the solvent was evaporated to obtain the product 20 as white
Synthesis of compound 7
To a stirred solution of ebselen (0.02 g, 0.073 mmol) in methanol
was added an alkaline solution of peroxynitrite in excess amount
(10–12 equiv) and the reaction mixture was stirred at room
temperature for 2 h. The solvent was evaporated to obtain crude
solid which was purified by a reverse phase C18 column using water
and methanol as eluent. The solvent was evaporated in vacuo to
obtain the pure compound 7 as white solid. Yield: 9.4 mg (42%).
1H NMR (MeOH-d4) d (ppm): 7.12 (t, 1H, J = 8.0 Hz), 7.33 (t, 2H,
J = 8.0 Hz), 7.57 (t, 1H, J = 8.0 Hz), 7.71 (d, 3H, J = 8.0 Hz), 7.87
(d, 1H, J = 8.0 Hz), 8.33 (d, 1H, J = 8.0 Hz). 13C NMR (MeOH-
d4) d (ppm): 123.7, 127.1, 127.8, 130.1, 131.2, 132.7, 134.3, 137.1,
141.2, 156.2, 169.5. 77Se NMR (MeOH-d4) d (ppm): 1144. ESI-MS
m/z calcd. for C13H11NO3Se [M - H]- 307.98; found: 307.70.
1
powder. Yield: 44 mg (43%). H NMR (CDCl3) d (ppm): 1.41
(s, 3H), 1.47 (s, 3H), 1.69 (s, 3H), 1.73 (s, 3H), 3.58 (d, 1H, J =
11.6 Hz), 4.00 (d, 1H, J = 11.6 Hz), 4.28– 4.44 (m, 2H), 7.75
(t, 1H, J = 7.2 Hz), 7.97 (d, 1H, J = 7.6 Hz), 8.02 (d, 1H, J =
7.6 Hz). 13C NMR (CDCl3) d (ppm): 25.2, 26.3, 28.0, 28.5, 63.3,
67.8, 68.2, 81.0, 125.3, 130.2, 131.2, 133.4, 134.0, 143.8, 160.2,
168.3. 77Se NMR (CDCl3) d (ppm): 1102. ESI-MS m/z calcd. for
C16H20N2O4Se [M+Na]+: 407.0486; found: 407.0506.
Synthesis of compounds 16–17
To a CH2Cl2 solution of the ebselen derivatives (11 and 12)
(0.10 mmol), triphenylphosphine (0.20 mmol) was added and
the reaction mixture was stirred at room temperature for 3 h
to obtain a white precipitate of the corresponding diselenide
compounds. The solvent was evaporated and the solid was washed
3–4 times with petroleum ether and CH2Cl2 mixture to remove the
unreacted triphenylphosphine and its by-products. The residue
was dried under vacuum to obtain the products as white powder
in quantitative yields.
PN-mediated nitration of L-tyrosine
High performance liquid chromatography (HPLC) experiments
were carried out on a Waters Alliance System (Milford, MA)
consisting of a 2695 separation module, a 2996 photodiode-array
detector and a fraction collector. The assays were performed in
1.8 mL sample vials and a built-in autosampler was used for
sample injection. The Alliance HPLC System was controlled with
5198 | Org. Biomol. Chem., 2011, 9, 5193–5200
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EMPOWER software (Waters Corporation, Milford, MA). The
nitration assay of L-tyrosine were analyzed by reverse-phase HPLC
(Lichrosphere-100 RP-C18 60A 5 mm 250 ¥ 4.6 mm) using isocratic
elution with 100% water containing 0.1% TFA and methanol
(8 : 2). In the PN-mediated nitration of L-tyrosine, we employed
a mixture containing L-tyrosine (1 mM) and PN (1.5 mM) in
sodium phosphate buffer (100 mM) of pH 7.5 without and with
increasing concentration of the inhibitor. It was then incubated
for 7 min before injection. The formation of 3-nitro-L-tyrosine
was monitored at the wavelength of 275 nm. The inhibition plots
were obtained using Origin 6.1 software utilizing sigmoidal curve
fitting and these plots were used for the determination of IC50
values.
The final fluorescence intensities were corrected for background
reactions. The inhibition plots were obtained using Origin 6.1
software utilizing sigmoidal curve fitting and these plots were used
for the determination of IC50 values.
-
Determination of nitrate (NO3 ) concentration
-
The isomerization of PN to NO3 was determined qualitatively
by measuring the formation of nitro-resorcinol by following the
literature procedure with minor modifications.23 The mixture of
PN (0.26 mM) and selenium compounds (73 mM) was incubated
for 30 min in the assay buffer to ensure the isomerization of PN
-
to NO3 by the selenium compounds. The nitration of resorcinol
-
(11.4 mM) by the NO3 produced was initiated by the addition of
concentrated H2SO4 (1 mL) in water. The resultant mixture was
further incubated in the dark for another 30 min. The formation
of nitro-resorcinol was measured by UV–vis spectrophotometric
method at the wavelength of 505 nm with the molar extinction
coefficient (e) of 1.7 ¥ 104 M-1 cm-1 due to nitro-resorcinol. The
control reaction was carried out under identical experimental
conditions but in the absence of selenium compounds.
Inhibition of protein nitration
For bovine serum albumin (BSA), the nitration was performed
by the addition of PN (1.5 mM) to BSA (133.3 mM) in 0.5 M
phosphate buffer of pH 6.9 at 20 ◦C. After the addition of PN,
the final pH was maintained below 7.5. The reaction mixture was
incubated for 7 min at 20 ◦C. Similarly, the reactions of BSA
with PN were performed in the presence of different selenium
compounds (80 mM) as inhibitors. After the reactions, the mixture
was denatured by boiling at 100 ◦C for 5 min in the presence
of sample loading dye and subjected to polyacrylamide gel
electrophoresis and immunoblot analyses.
X-Ray crystallography
Single crystal X-ray diffraction data was collected on a Bruker
AXS SMART APEX CCD diffractometer. The X-ray generator
was operated at 50 KV and 35 mA using Mo-Ka radiation
˚
(l = 0.71073 A). The data was collected using SMART software
Electrophoretic analysis
package.24 The data were reduced by SAINTPLUS,24 an empirical
absorption correction was applied using the package SADABS25
and XPREP24 was used to determine the space group. The crystal
structure was solved by direct methods using SIR9226 and refined
by full-matrix least-squares method using SHELXL97.27 All non-
hydrogen atoms were refined anisotropically and hydrogen atoms
were assigned at idealized locations.
Gel was prepared with 10% polyacrylamide with 6% stacking
gel for BSA. The gel was run in the running buffer of pH 8.3
with glycine and SDS. After separating the proteins, the gel
was analyzed by immunoblotting experiments. The proteins were
transferred to a PVDF membrane and the non-specific binding
sites were blocked by 5% non-fat skimmed milk in PBST (blocking
solution) for 1 h. Then the membrane was probed with rabbit
polyclonal primary antibody against 3-nitro-tyrosine (1 : 20000
dilutions) in blocking solution for 2 h followed by incubation with
horseradish peroxidase-conjugated donkey polyclonal anti-rabbit
IgG (1 : 20000 dilutions) for another 1 h. The probed membrane
was washed three times with blocking solution with 0.1% Tween
20 after each of the above steps and the immunoreactive protein
was then detected by luminol-enhanced chemiluminescence (ECL,
Amersham).
Crystal data for compound 2028
¯
C16H20N2O4Se; Mr = 383.30; triclinic; space group: P1;◦ a =
˚
˚
˚
6.9670(14) A; b = 10.517(2) A; c = 11.826(2) A; a = 87.388(3) ; b =
◦
◦
3
-3
˚
73.485(3) ; g = 85.092(3) ; V = 827.5(3) A ; rc = 1.538 g m ; Z =
2; m Mo-Ka radiation (l = 0.71370 A); T = 291(2)K; Rint: 0.024; R
˚
(observed data): R1 = 0.040; wR2 = 0.108; R (all data): R1 = 0.045;
-3
˚
wR2 = 0.112; GOF = 1.078; Drmin and Drmax (eA ): -0.407 and
1.361.
PN-mediated oxidation assay
Acknowledgements
PN-mediated oxidation of dihydrorhodamine 123 (DHR) was
studied using fluorescence spectroscopy. Fluorescence intensity
was measured using Horiba Jobin Yvon, USA Fluoromax-4 Lu-
minescencespectrometer withexcitation and emissionwavelengths
of 500 nm and 526 nm, respectively. The stock solution of DHR
in dimethylformamide was purged with nitrogen and stored at
-20 ◦C. The working solution of DHR and PN were kept on ice
bath. The assay mixture contained DHR (0.50 mM), PN (0.97 mM)
in 100 mM phosphate buffer of pH 7.4 with 100 mM DTPA and
variable inhibitor concentrations. The fluorescence intensity from
the reaction of DHR with PN was set as 100% and the intensity
after the addition of various inhibitors was expressed as the
percentage of the intensity observed in the absence of inhibitors.
This study was supported by the Department of Science and
Technology (DST), New Delhi, India. GM acknowledges the
DST for the Ramanna and Swarnajayanti Fellowships. KPB and
GR thank Indian Institute of Science, Bangalore for a Research
Associate fellowship. AAV and SRJ acknowledge the Council of
Scientific and Industrial Research (CSIR), New Delhi, India for a
research fellowship.
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