Synthesis and antioxidant activity of peptide-based ebselen analogues

Article abstract of DOI:10.1002/chem.201003417

A series of di- and tripeptide-based ebselen analogues has been synthesized. The compounds were characterized by ¹H, ¹³C, and ⁷⁷SeNMR spectroscopy and mass spectral techniques. The glutathione peroxidase (GPx)-like antioxi

Full text of DOI:10.1002/chem.201003417

FULL PAPER
DOI: 10.1002/chem.201003417
Synthesis and Antioxidant Activity of Peptide-Based Ebselen Analogues
Kandhan Satheeshkumar and Govindasamy Mugesh*[a]
Abstract: A series of di- and tripep-
tide-based ebselen analogues has been
synthesized. The compounds were char-
acterized by 1H, 13C, and 77Se NMR
spectroscopy and mass spectral tech-
niques. The glutathione peroxidase
(GPx)-like antioxidant activity has
been studied by using H2O2, tert-butyl
hydroperoxide (tBuOOH), and cumene
hydroperoxide (Cum-OOH) as sub-
strates, and glutathione (GSH) as a co-
substrate. Although all the peptide-
based compounds have a selenazole
ring similar to that of ebselen, the GPx
activity of these compounds highly de-
pends on the nature of the peptide
moiety attached to the nitrogen atom
of the selenazole ring. It was observed
that the introduction of a phenylala-
nine (Phe) amino acid residue in the
N-terminal reduces the activity in all
three peroxide systems. On the other
hand, the introduction of aliphatic
amino acid residues such as valine
(Val) significantly enhances the GPx
activity of the ebselen analogues. The
difference in the catalytic activity of di-
peptide-based ebselen derivatives can
be ascribed mainly to the change in the
reactivity of these compounds toward
GSH and peroxide. Although the pres-
ence of the Val-Ala-CO2Me moiety fa-
cilitates the formation of a catalytically
active selenol species, the reaction of
ebselen analogues that has a Phe-Ile-
CO2Me residue with GSH does not
generate the corresponding selenol. To
understand the antioxidant activity of
the peptide-based ebselen analogues in
the absence of GSH, these compounds
were studied for their ability to inhibit
peroxynitrite (PN)-mediated nitration
of bovine serum albumin (BSA) and
oxidation of dihydrorhodamine 123. In
contrast to the GPx activity, the PN-
scavenging activity of the Phe-based
peptide analogues was found to be
comparable to that of the Val-based
compounds. However, the introduction
of an additional Phe residue to the eb-
selen analogue that had a Val-Ala di-
peptide significantly reduced the po-
tency of the parent compound in PN-
mediated nitration.
Keywords: antioxidants · ebselen ·
glutathione · peptides · selenium
Introduction
Organoselenium compounds that mimic the function of the
antioxidant selenoenzyme glutathione peroxidase (GPx) are
currently attracting considerable interest.[1,2] The initial ob-
servations by Sies et al.[3] and Wendel et al.[4] that the cyclic
selenazole-based compound ebselen (1) mimics the activity
of GPx led to the development of several organoselenium
compounds that can catalytically reduce hydroperoxides in
the presence of thiols. These include compounds 2 and 3
[5]
À
that have cleavable Se N bonds, amine- and amide-based
diselenides (4–7),[6] diaryl diselenides that have Se···O inter-
actions (8 and 9),[7] the cyclodextrin-based diselenide (10),[8]
several monoselenides (11–14),[9] cyclic selenelate ester
(15),[10] and spirodiazaselenurane (16) (Scheme 1).[11] Al-
though the catalytic mechanisms for the GPx-like activity of
many of these compounds are considerably different from
that of the natural enzyme or ebselen, the selenium center
Scheme 1. Some representative examples of small-molecule GPx mimics
1–16.
[a] K. Satheeshkumar, Prof. Dr. G. Mugesh
Department of Inorganic and Physical Chemistry
Indian Institute of Science, Bangalore 560 012 (India)
E-mail: mugesh@ipc.iisc.ernet.in
in these compounds undergoes redox reactions in the pres-
ence of peroxides and thiols.
Although ebselen (1) has been shown to be the first suc-
cessful GPx mimic, the mechanism by which ebselen exerts
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/chem.201003417.
Chem. Eur. J. 2011, 17, 4849 – 4857
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4849

Scheme 2. Proposed mechanism for the GPx activity of ebselen.
its catalytic activity is completely different from that of GPx
(Scheme 2). According to the revised catalytic mecha-
nism,[12] the reaction of ebselen with a thiol produces the
corresponding selenenyl sulfide intermediate (6a), which
undergoes a disproportionation reaction to afford diselenide
6. It has been shown that the formation of diselenide 6 is
the rate-determining step, and this reaction depends on the
nature of thiol used for the GPx assay.[12] The reaction of
diselenide 6 with peroxides such as H2O2 produces the cor-
responding selenenic acid (6b) and seleninic acid (6c) both
of which react with the thiol to regenerate 6a. In the ab-
sence of thiols, the selenenic acid (6b) undergoes a facile
cyclization to produce ebselen. The regeneration of ebselen
from its catalytically active and inactive forms under a varie-
ty of conditions protects the selenium moiety in 6b and 6c
from an overoxidation to other oxidized selenium species
such as selenonic acid.
The available data suggest that ebselen does not follow
the classical GPx catalytic cycle that involves selenol, sele-
nenic acid, and selenenyl sulfide intermediates, although the
formation of selenol has been observed during the reaction
of ebselen with thiols.[13] It is still not clear whether the facil-
itation of selenol formation by modification in the basic
structure can enhance the GPx activity of ebselen deriva-
tives. In this paper, we report the synthesis and GPx activity
of a series of ebselen analogues with di- and tripeptide moi-
eties. We also show that not only the overall catalytic activi-
ty of these compounds but also the formation of selenol
highly depend on the nature of peptide moiety attached to
the nitrogen atom of the selenazole ring. Although some eb-
selen analogues that have amino acid residues have been re-
ported,[14] there is no report on the synthesis and GPx activi-
ty of peptide-based ebselen derivatives.
Scheme 3. Ebselen analogues with different di- and tripeptide moieties.
17–24 (Scheme 3) were synthesized in moderate yields by
treating 2-(chloroseleno)benzoyl chloride with appropriate
peptides that have free amino groups. Although 1:1 reac-
tions of 2-(chloroseleno)benzoyl chloride with simple pri-
mary amines generally afford the corresponding cyclic sele-
nazoles as the only products,[15] the nature of peptides in this
study appears to affect the reaction products. Whereas the
reactions of 2-(chloroseleno)benzoyl chloride with H2N-Tyr-
Val-CO2Me, H2N-Trp-Ala-CO2Me, H2N-Val-Ala-CO2Me,
and H2N-Val-Ala-Phe-CO2Me afforded only the cyclic com-
pounds 17, 21, 23, and 24, respectively, similar reactions with
H2N-Phe-Val-CO2Me, H2N-Phe-Ile-CO2Me, H2N-Phe-Ala-
CO2Me, and H2N-Trp-Gly-CO2Me afforded considerable
amounts of the diselenides 25, 26, 27, and 28, respectively
(Scheme 4), in addition to the expected cyclic compounds
(18–20 and 22).
The structure of compound 23 was further studied by
single-crystal X-ray diffraction analysis. This compound crys-
tallized in a non-centrosymmetric space group (P1) with a
Flack parameter[16] of 0.01. This indicates that the crystals of
compound 23 are enantiomerically pure. The ebselen moiety
is almost perpendicular to the side chain of the peptide
backbone as shown in Figure 1. The crystal packing indicates
that there are significant hydrogen-bonding interactions be-
tween two molecules. The carbonyl oxygen atom of the sele-
nazole ring forms hydrogen bonds with the peptidic NH
group of the adjacent molecule. These hydrogen bonds may
play an important role in stabilizing some of the intermedi-
ates in the catalytic cycle.
Results and Discussion
Synthesis of peptide-substituted ebselen analogues:
A
number of di- and tripeptide-containing ebselen analogues
4850
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Peptide-Based Ebselen Analogues
FULL PAPER
mation). These observations indicate that the peptide-based
cyclic compounds and diselenides can be readily intercon-
verted with appropriate reagents.
Glutathione peroxidase (GPx) activity: The GPx-like cata-
lytic activity of compounds 17–24 was studied using gluta-
thione (GSH) as thiol cofactor and hydrogen peroxide
(H2O2), tert-butyl hydroperoxide (tBuOOH), and cumene
hydroperoxide (Cum-OOH) as substrates. Initial rates (v0)
were determined for the reduction of peroxides at 2 mm
concentrations of the selenium catalysts. For comparison,
the initial rates for the reduction of peroxides by ebselen
were also determined. From Table 1, it is evident that the
Scheme 4. Peptide-based diselenides with a Phe or Trp moiety.
Table 1. Initial rates for the reduction of peroxides by GSH in the pres-
ence of compounds 1 and 17–24.
[a]
Compound
Initial rate v0 [mmminÀ1
]
À
À
H2O2
tBu OOH
Cum OOH
1
85.2Æ1.0
54.9Æ1.5
66.3Æ1.5
38.5Æ2.7
183.8Æ1.6
145.7Æ4.5
100.6Æ2.9
149.7Æ2.1
145.7Æ4.5
27.5Æ7.6
19.5Æ5.3
8.4Æ4.6
5.6Æ0.3
5.6Æ0.2
22.4Æ1.9
18.6Æ0.8
55.0Æ5.3
17.4Æ6.3
44.3Æ0.5
83.7Æ8.5
20.0Æ6.2
20.5Æ1.4
38.6Æ1.5
67.3Æ6.9
61.9Æ7.7
154.5Æ1.6
31.4Æ6.7
17
18
19
20
21
22
23
24
[a] The reactions were carried out in phosphate buffer (100 mm, pH 7.5)
at 238C. Catalyst: 80.0 mm; glutathione reduced 2.0 mm; NADPH:
0.4 mm; glutathione disulfide reductase 1.7 unitmLÀ1; peroxide: 1.6 mm.
The initial rates were corrected for the background reaction between per-
oxide and thiol.
À
Figure 1. Crystal structure of compound 23 showing C=O···H N hydro-
gen bonds.
GPx activity of the ebselen analogues highly depends on the
nature of peptides attached to the nitrogen atom. The
nature of peroxide also has a significant effect on the reac-
tion rates. Whereas compound 17, which has a Tyr-Val di-
peptide, was found to be a better catalyst than ebselen in
the Cum-OOH assay, a significant decrease in the activity
was observed when H2O2 or tBuOOH was used as the sub-
strate. The introduction of a Phe residue appears to de-
crease the activity as compounds 18–20 with Phe-Val, Phe-
Ile, or Phe-Ala exhibited poor activity when tBuOOH and
Cum-OOH were used as substrates. In the H2O2 assay, only
compound 20 exhibited higher activity compared to ebselen.
The Trp-based compounds 21 and 22 were found to be
much better catalysts than ebselen in both H2O2 or Cum-
OOH systems, but the activity of these compounds in
tBuOOH was slightly lower than that of ebselen. Compound
23, which has a Val-Ala peptide, exhibited the highest activi-
ty in the series in all three peroxide assays. Interestingly,
compound 24 with an additional Phe residue exhibited
much lower catalytic activity than compound 23 when
tBuOOH or Cum-OOH was employed. However, there was
no significant change in the activity upon the introduction of
the Phe residue when H2O2 was used as the substrate. These
observations indicate that the GPx activity of the peptide-
based ebselen analogues depends not only on the nature of
It is known that the treatment of ebselen and its ana-
logues with triphenylphosphine leads to the formation of
the corresponding diselenides.[17] To check the reactivity of
peptide-based ebselen analogues, compound 23 was treated
with different concentrations of triphenylphosphine, and the
reaction was followed by 77Se NMR spectroscopy
(Scheme 5). When four equivalents of triphenylphosphine
Scheme 5. Triphenylphosphine-mediated conversion of 23 to 29.
was added, a complete conversion of 23 to the correspond-
ing diselenide (29) was observed (Figure S41 in the Support-
ing Information). This indicates that the diselenide could be
obtained in stable form even for the Val-Ala moiety, which
appears to favor the formation of a cyclic compound. Simi-
larly, the reaction of diselenide 26 with an excess amount
(5 equiv) of H2O2 afforded the cyclic compound 19 in quan-
titative yield (Figures S42 and S42a in the Supporting Infor-
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G. Mugesh and K. Satheeshkumar
peptide moiety, but also the nature of peroxide used for the
assay.
Mechanistic studies: To understand the reason for the differ-
ence in the activity of various peptide-based ebselen ana-
logues, we have studied the reactivity of compound 19 (the
least active compound) and 23 (the most active compound)
toward GSH and peroxide. We have shown previously that
the reaction of ebselen with thiols such as PhSH produces
the corresponding selenenyl sulfide (6a, R=Ph), which does
not produce any selenol upon further treatment with an
excess amount (5 equiv) of PhSH. Alternatively, compound
6a has been shown to undergo a disproportionation reaction
to produce diselenide 6 and PhSSPh. This is due to the pres-
ence of a strong Se···O interaction in compound 6a, which
enhances a thiol exchange reaction at selenium.[18] There-
fore, the rate of disproportionation depends on the nature
of thiols and strength of the Se···O interactions. When GSH
was employed instead of PhSH, the disproportionation sele-
nenyl sulfide that led to the formation of diselenide 6 was
significantly faster than that of 6a.[12] The facile dispropor-
tionation has been shown to be responsible for the higher
activity of ebselen in the presence of GSH relative to that of
PhSH.[12]
Scheme 7. Reaction of compound 19 with GSH.
selen. This may account for the lower GPx activity of com-
pound 19 compared to that of ebselen in different peroxide
systems. The selenenyl sulfides derived from other ebselen
analogues may undergo different degrees of disproportiona-
tion reactions depending upon the nature of peptide moiety.
These observations indicate that the Val-Ala moiety in com-
pound 31 facilitates the formation of selenol and therefore
increases the GPx activity of compound 23.
The reaction of selenol 32 with two equivalents of H2O2
produced selenoxide 37 as the major product (Scheme 8).
However, at lower concentrations of H2O2 a considerable
Treatment of 23 with one equivalent of GSH afforded the
expected selenenyl sulfide 31, which upon treatment with
another equivalent of GSH produced selenol 32. Although
32 was too unstable to isolate and characterize, this species
could be trapped by treating the reaction mixture with iodo-
acetic acid. When selenenyl sulfide 31 was treated with one
equivalent of GSH in the presence of iodoacetic acid, a
quantitative conversion of 31 to 33 was observed
(Scheme 6). The reactivity of compound 23 towards GSH
appears to be completely different from that of the selenen-
yl sulfide obtained from the reaction of ebselen with GSH.
This may account for the higher GPx activity of compound
23 compared to that of ebselen.
Scheme 8. Formation of selenoxide 37 from the selenol 32.
amount of selenenyl amide 23 was produced possibly
through the formation of selenenic acid 36 as previously
shown for ebselen. The reactions of compounds 19 and 23
with H2O2 led to some interesting observations. When H2O2
(1 equiv) was added to compound 23, the formation of 37
was observed as the major product. An independent HPLC
analysis of the reaction of 23 with H2O2 indicated that this
reaction initially produces selenoxide 37, which finally leads
to the formation of selenenyl amide 23 (Figures S48 and S49
in the Supporting Information). It should be noted that the
selenoxide produced from the reaction of ebselen with H2O2
is highly unstable and it undergoes a rapid hydrolysis to pro-
duce the corresponding seleninic acid (6c).[12] This indicates
that the introduction of Val-Ala moiety leads to the stabili-
zation of selenoxide 37.
In contrast, reaction of the Phe-Ile-based selenenyl amide
19 with H2O2 produced the corresponding selenonic acid 40
as the major product (Scheme 9). Although the formation of
selenoxide 38 was detected by mass spectroscopy, the rapid
hydrolysis of this compound led to the formation of selenin-
ic acid 39, which upon reaction with H2O2 produced sele-
nonic acid 40. Interestingly, the regeneration of 19 was not
observed in these reactions. The HPLC analysis of the reac-
Scheme 6. Reaction of compound 23 with GSH.
The reaction of compound 19 with GSH generated the
corresponding selenenyl sulfide 34, which is similar to the
reaction of 23 with GSH. However, further reaction of 34
with GSH did not produce the expected selenol 35, but it
produced diselenide 26 in nearly quantitive yield
(Scheme 7). As only the diselenide was observed even in the
presence of iodoacetic acid, the disproportionation of 34 to
26 appears to be more favored than the formation of sele-
nol. The disproportionation of 34 to 26 was found be slower
than that observed for the selenenyl sulfide derived from eb-
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Peptide-Based Ebselen Analogues
FULL PAPER
pound 36 undergoes a facile cyclization to regenerate 23. Al-
though the cyclization of compound 36 is very similar to
that of 6b, the reactivity of selenenyl sulfide 31 toward GSH
is strikingly different from that of compound 6a, which does
not produce any selenol upon reaction with PhSH or
GSH.[12] In contrast to compound 23, the ebselen analogues
that have a Phe residue such as compound 19 follow a
mechanism similar to that of ebselen (Scheme 2). In this
case, the disproportionation of selenenyl sulfides to the cor-
responding diselenides is the rate-determining step.
Inhibition of peroxynitrite-mediated nitration and oxidation:
Peroxynitrite (PN) is a highly reactive oxygen and nitrogen
species that plays a key role in a variety of inflammatory, al-
lergic, and other diseases.[19] In biological medium, PN is
Scheme 9. Reaction of 19 with H2O2 to produce the corresponding selen-
oxide, selenenic, and seleninic acids.
generated by the diffusion-controlled reaction of superoxide
[20]
CÀ
C
anion radical (O2 ) and nitric oxide (NO ). It is known
that PN can induce DNA damage[21] and initiate lipid perox-
idation in biomembranes or low-density lipoproteins.[22] In
addition, PN can deactivate a variety of enzymes by tyrosine
nitration.[23] To understand the protective effects of peptide-
based ebselen analogues against PN, we have used the PN-
mediated nitration of bovine serum albumin (BSA).[24] The
inhibition of tyrosine nitration was followed by immunoblot-
ting methods using antibody against 3-nitro-l-tyrosine. In a
typical experiment, BSA (100 mm) was incubated with PN
(1.2 mm) and inhibitor (133 mm), and the reaction was incu-
bated at 208C for 20 min. At 133 mm concentration, the
effect of various ebselen analogues on protein tyrosine nitra-
tion is given in Figure 2. From the percentage of nitration, it
is clear that all the ebselen analogues significantly inhibit
the PN-mediated nitration reaction. Compound 19, which
exhibited poor GPx activity, was found to be the most effec-
tive compound in inhibiting the PN-mediated nitration. The
Val-Ala-based compound 23 also strongly inhibited the ni-
tion mixture indicated the formation of diselenide 26 as the
major product (Figure S52 in the Supporting Information).
As previously mentioned, the formation of diselenide 26
was also observed during the synthesis of compound 19.
However, no such diselenide formation was observed during
the synthesis of ebselen or compound 23. These observa-
tions indicate that the Phe-Ile moiety may disfavor the cycli-
À
zation process by preventing the interactions between the
À
NH and selenium moieties. Therefore, the selenium center
in compound 39 undergoes a further oxidation to produce
selenonic acid 40, which may also account for the lower
GPx activity of compound 19 compared to that of 23.
On the basis of above experimental observations, a mech-
anism for the GPx activity of compound 23 can be proposed
(Scheme 10). According to this mechanism, the cleavage of
Scheme 10. Proposed mechanism for the GPx activity of compound 23.
À
the Se N bond in selenenyl amide 23 by GSH produces se-
lenenyl sulfide 31, which upon reaction with a second equiv-
alent of GSH generates the corresponding selenol 32. Simi-
lar to the selenol moiety in GPx, selenol 32 reacts with
H2O2 to produce the selenenic acid 36, which further reacts
with GSH to regenerate selenenyl sulfide 31. When the con-
centration of GSH is much lower than that of H2O2, com-
Figure 2. Immunoblots for the inhibition of PN-mediated nitration of
BSA. BSA (100 mm) was incubated with inhibitor (133 mm) and PN
(1.2 mm) for 30 min at 208C and then subjected to gel electrophoresis.
Chem. Eur. J. 2011, 17, 4849 – 4857
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4853

G. Mugesh and K. Satheeshkumar
tration, thereby indicating that this compound is a good can-
didate for both GPx and PN-scavenging activities. In agree-
ment with the effects of Phe residue on GPx activity, the in-
troduction of an additional Phe residue to compound 23 sig-
nificantly reduced its PN-scavenging activity.
In addition to the nitration of tyrosine residues in pep-
tides and proteins, PN is also known to oxidize biomole-
cules. PN mediates the oxidation of dihydrorhodamine 123
(DHR) to rhodamine 123 (Scheme 11) in a linear fashion
Conclusion
In this study, we have shown for the first time that the GPx
activity of peptide-based ebselen analogues depends on the
nature of peptide moiety attached to the nitrogen atom of
the selenazole ring. Whereas the introduction of a Phe
amino acid residue reduces the GPx activity, the introduc-
tion of aliphatic amino acid residues such as Val significantly
enhances the activity. The difference in the antioxidant ac-
tivity of peptide-substituted selenenyl amides is due to the
difference in the reactivity of these compounds toward GSH
and peroxide. Although dipeptides such as Val-Ala that
have aliphatic amino acid residues facilitates the formation
of a catalytically active selenol, the introduction of aromatic
amino acid such as Phe appears to prevent the generation of
selenol. Furthermore, the Phe-based peptides prevent the
cyclization of selenenic acids to produce the corresponding
selenenyl amides, which leads to the overoxidation of sele-
nenic and seleninic acids to selenonic acids. In addition to
the GPx activity, the peptide-based ebselen analogues effec-
tively inhibit the peroxynitrite-mediated nitration of protein
tyrosine residues and oxidation of dihydrorhodamine 123. In
contrast to the GPx activity, the PN scavenging activity of
the Phe-based peptide analogues was found to be compara-
ble to that of the Val-based compounds.
Scheme 11. Oxidation of dihydrorhodamine 123 to rhodamine 123 by per-
oxynitrite.
over the range of 0–1000 nm, which is generally monitored
by fluorescence spectrophotometric methods. To understand
the effect of ebselen analogues on PN-mediated oxidation
reactions, we have studied the conversion of DHR to rhoda-
mine 123 in the presence of various concentrations of seleni-
um compounds. The IC50 values (the concentration of test
compounds required to inhibit 50% of the control activity)
obtained for ebselen and compounds 17–24 are summarized
in Table 2. Although there is no correlation between the
Experimental Section
General procedure: All the chemical reactions were carried out under ni-
trogen atmosphere using standard vacuum-line techniques. Due to the
unpleasant odors of several of the reaction mixtures involved, most ma-
nipulations were carried out in a well-ventilated fume hood. Thin-layer
chromatography (TLC) analyses were carried out on precoated silica gel
plates (Merck), and spots were visualized by UV irradiation. Column
chromatography was performed on glass columns loaded with silica gel
or using an automated flash chromatography system (Biotage) by using
preloaded silica cartridges. 1H (400 MHz), 13C (100.56 MHz), and 77Se
(76.29 MHz) NMR spectra were obtained using a Bruker 400 MHz
NMR spectrometer. Chemical shifts are cited with respect to SiMe4 as in-
ternal (1H and 13C) and Me2Se as external (77Se) standard. Mass spectral
studies were carried out using a Q-TOF micro mass spectrometer or
Bruker Daltonics Esquire 6000plus mass spectrometer with ESI-MS
mode analysis. The melting point was determined in open capillary using
an ANALAB melting-point apparatus.
Table 2. Inhibition of PN-mediated oxidation of dihydrorhodamine 123
by ebselen and the peptide-based analogues.[a]
Compound
IC50 [mm]
Compound
IC50 [mm]
1
0.9Æ0.01
2.6Æ0.02
0.9Æ0.08
1.7Æ0.07
1.0Æ0.03
21
22
23
24
3.5Æ0.18
1.9Æ0.03
2.0Æ0.02
1.1Æ0.04
17
18
19
20
[a] Assay conditions: dihydrorhodamine 123 (DHR; 0.1 mm), peroxyni-
trate (0.69 mm), and inhibitors (variable) in sodium phosphate buffer
(100 mm, pH 7.5) with DTPA (100 mm) at 238C.
Synthesis of 17: A solution of NH2-Tyr-Val-OMe (0.12 g, 0.39 mmol) in
dry acetonitrile (5 mL) was added dropwise with stirring over a period of
effect of these compounds on protein tyrosine nitration and
the IC50 values for the oxidation of DHR, all the compounds
inhibited the oxidation of DHR in the low micromolar
range. Interestingly, compounds 18, 20, and 24, which exhib-
ited poor GPx activity in the tBuOOH and Cum-OOH
assays, were found to be very effective as inhibitors of PN-
mediated oxidation. On the other hand, compound 23,
which exhibited much higher GPx activity, was found to be
almost two times less active than ebselen in the DHR assay.
Compound 19 was also found to be a good scavenger of PN
as this compound effectively inhibited both the nitration
and oxidation reactions.
10 min to
a solution of 2-(chloroseleno)benzoyl chloride (0.10 g,
0.39 mmol) in dry acetonitrile (5 mL). Triethylamine (0.10 mL,
0.78 mmol) was added, and the reaction mixture was stirred at 258C for
about 3 h. Solvent was evaporated in vacuo to give a yellow oil, which
was purified on a silica gel column using ethyl acetate and petroleum
ether as eluent to give the desired compound as a yellow oil. Yield:
0.091 g (49%); [a]2D5 =À15 (c=0.2 in MeOH); 1H NMR (CDCl3): d=
0.76–0.77 (d, J=5.6 Hz, 6H), 1.38–1.52 (m, 1H), 2.97–3.21 (m, 2H), 3.67
(s, 3H), 4.50–4.53 (t, J=13.6 Hz 1H), 5.49–5.53 (t, J=14.4 Hz, 1H), 6.66–
6.68 (d, J=7.6 Hz, 2H), 6.99–7.01 (d, J=6.4 Hz, 3H), 7.32–7.36 (t, J=
15.4 Hz, 1H), 7.53–7.56 (t, J=14.8 Hz, 1H), 7.62–7.64 (d, J=8 Hz, 1H),
7.96–7.98 ppm (d, J=7.6 Hz, 1H); 13C NMR (CDCl3) d=22.2, 22.9, 25.2,
39.2, 41.5, 51.5, 52.9, 59.0, 116.1, 124.2, 126.6, 127.1, 127.6, 128.9, 130.8,
132.7, 141.1, 155.9, 168.4, 170.2, 173.1 ppm; 77Se NMR (CDCl3): d=
4854
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Peptide-Based Ebselen Analogues
FULL PAPER
896 ppm; HRMS: m/z: calcd for C22H24N2O5Se: 498.3862 [M+Na]+;
found: 497.0955.
77Se NMR (CDCl3): d=884 ppm; HRMS: m/z: calcd for C22H21N3O4Se:
493.3696 [M+Na]+; found: 494.0595.
Synthesis of 22: A solution of NH2-Trp-Gly-COOMe (0.11 g, 0.39 mmol)
Synthesis of 18: A solution of NH2-Phe-Val-OMe (0.11 g, 0.39 mmol) in
in dry acetonitrile (5 mL) was added dropwise with stirring over a period
dry acetonitrile (5 mL) was added dropwise with stirring over a period of
of 10 min to
a solution of 2-(chloroseleno)benzoyl chloride (0.10 g,
10 min to
a solution of 2-(chloroseleno)benzoyl chloride (0.10 g,
0.39 mmol) in dry acetonitrile (5 mL). After addition of triethylamine
(0.10 mL 0.78 mmol), the reaction mixture was stirred at room tempera-
ture for about 3 h, and then the solvent was evaporated reduced pressure.
Purification of the crude compound on a silica gel column using ethyl
acetate and petroleum ether (1:3) as eluent afforded the desired com-
pound as white solid. Yield: 0.086 g (48%); m.p. 183–1858C; [a]2D5 =À25
(c=0.2 in MeOH); 1H NMR (CDCl3): d=3.27–3.44 (m, 2H), 3.58 (s,
3H), 3.82–3.90 (m, 2H), 5.62–5.59 (t, J=12 Hz, 1H), 7.03–7.13 (m, 3H),
7.26–7.39 (m, 2H), 7.55–7.69 (m, 3H), 7.99–8.02 (d, J=4 Hz, 1H),
8.36 ppm (s, 1H); 13C NMR (CDCl3): d=29.7, 41.7, 52.8, 57.9, 110.4,
111.7, 119.1, 120.0, 122.6, 123.8, 124.3, 126.5, 127.3, 127.8, 128.9, 132.5,
136.6, 141.0, 168.2, 170.1, 170.9 ppm; 77Se NMR (CDCl3): d=900.03 ppm;
HRMS: m/z: calcd for C21H19N3O4Se: 479.3431 [M+Na]+; found:
480.0438.
0.39 mmol) in dry acetonitrile (5 mL). Triethylamine (0.10 mL
0.78 mmol) was added to this, and the reaction mixture was stirred at
258C for about 3 h. Removal of the solvent under reduced pressure af-
forded the crude product, which was purified on a silica gel column using
ethyl acetate and petroleum ether as eluent to give the desired com-
pound as a yellow solid. Yield: 0.082 g (45%); m.p. 135–1378C; [a]D25
=
À35 (c=0.2 in MeOH); 1H NMR (CDCl3): d=0.84–0.85 (d, J=2.4 Hz,
7H), 2.06 (m, 1H), 3.11–3.31 (m, 2H), 3.67 (s, 3H), 4.43–4.45 (t, J=
8.2 Hz, 1H), 5.44–5.48 (t, J=14.8 Hz, 1H), 6.42–6.44 (d, J=8.4 Hz, 1H),
7.20–7.31 (m, 5H), 7.41–7.43 (t, J=7.6 H, 1Hz), 7.60–7.64 (m, 1H), 8.02–
8.04 ppm (d, J=7.6 Hz, 1H); 13C NMR (CDCl3): d=18.2, 19.3, 25.4, 31.8,
39.4, 52.7, 57.8, 58.9, 124.3, 126.5, 127.3, 127.5, 129.1, 129.7, 132.6, 136.4,
140.5, 168.2, 169.9, 171.7 ppm; 77Se NMR (CDCl3): d=899 ppm; HRMS:
m/z: calcd for C22H24N2O4Se: 482.3868 [M+Na]+; found: 483.0799.
Synthesis of 23: A solution of NH2-Val-Ala-COOMe (0.081 g, 0.04 mmol)
in dry acetonitrile (5 mL) was added dropwise with stirring over a period
Synthesis of 19: A solution of NH2-Phe-Ile-OMe (0.12 g, 0.39 mmol) in
dry acetonitrile (5 mL) was added dropwise with stirring over a period of
of 10 min to
a solution of 2-(chloroseleno)benzoyl chloride (0.10 g,
10 min to
a solution of 2-(chloroseleno)benzoyl chloride (0.10 g,
0.39 mmol) in dry acetonitrile (5 mL). Triethylamine (0.10 mL
0.78 mmol) was added and the reaction mixture was stirred at 258C for
about 3 h. Removal of solvent under reduced pressure afforded the de-
sired compound, which was purified by flash chromatography on a silica
gel column by using ethyl acetate and petroleum ether as eluent to give a
yellow solid. Yield : 0.08 g (51%); m.p. 188–1918C; [a]2D5 =À70 (c=0.2 in
MeOH); 1H NMR (CDCl3): d=0.89–0.90 (d, J=4 Hz, 3H), 1.13–1.15 (d,
J=8.0 Hz, 4H), 1.42–1.44 (d, J=8 Hz, 3H), 2.30–2.34, (m, 1H), 3.78 (s,
3H) 4.61–4,65 (q, 1H), 5.31–5.33 (d, J=10 Hz, 1H),7.39–7.43 (t, J=
7.2 Hz, 1H), 7.56–7.60 (d, J=8 Hz, 1H), 8.07–8.09 (d, J=7.6 Hz, 1H),
8.23–8.24 ppm (d, J=6.8 Hz, 1H); 13C NMR (CDCl3): d=18.1, 19.3, 19.6,
33.25, 48.5, 52.8, 62.9, 124.2, 126.3, 127.2, 129.0, 132.4, 141.2, 168.2, 170.6,
173.5 ppm; 77Se NMR (CDCl3): d=900 ppm; HRMS: m/z: calcd for
C17H22N2O4Se: 406.2898 [M+Na]+; found: 407.0486.
0.39 mmol) in dry acetonitrile (5 mL). Triethylamine (0.10 mL
0.78 mmol) was added to the reaction mixture, and it was stirred at 258C
for about 3 h. Removal of solvent under reduced pressure afforded
yellow residue, which was purified on a silica gel column using ethyl ace-
tate and petroleum ether as eluent to yield compound 19 as a yellow
powder. Yield: 0.074 g (40%); m.p. 146–1488C; [a]2D5 =À35 (c=0.2 in
MeOH); 1H NMR (CDCl3): d=0.77–0.78 (d, J=5.2 Hz, 6H), 1.43–1.54
(m, 3H), 3.15–3.20 (m,2H), 3.64 (s, 3H), 4.47–4.47 (d, J=4.8 Hz, 1H),
4.89–4.91 (q, J=7.2 Hz, 1H), 6.41–6.43 (d, J=8.0 Hz, 1H), 7.06–7.25 (m,
7H), 7.37–7.37 (d, J=1.2 Hz, 1H), 7.70–7.72 ppm (d, J=7.6 Hz, 1H);
13C NMR (CDCl3): d=22.4, 23.2, 25.2, 38.8, 41.9, 51.6, 52.8, 55.3, 126.7,
127.5, 127.7, 129.2, 129.9, 131.7, 132.6, 133.7, 136.8, 168.4, 170.9,
173.1 ppm; 77Se NMR (CDCl3): d=904.346 ppm; HRMS: m/z: calcd for
C23H26N2O4Se: 496.4133 [M+Na]+; found: 497.0955.
Synthesis of 24:
A solution of NH2-Val-Ala-Phe-COOMe (0.14 g,
Synthesis of 20: A solution of 2-(chloroseleno)benzoyl chloride (0.10 g,
0.39 mmol) in dry acetonitrile (5 mL) was added dropwise with stirring a
solution of NH2-Phe-Ala-OMe (0.09 g, 0.39 mmol) in dry acetonitrile
(5 mL) over a period of 10 min. After addition of triethylamine (0.10 mL
0.78 mmol), the reaction mixture was stirred at 258C for about 3 h. The
solvent was removed under reduced pressure. Purification of the crude
compound on a silica gel column using ethyl acetate and petroleum ether
as eluent gave the expected compound as yellow oil. Yield: 0.090 g
(53%); [a]2D5 =À55 (c=0.2 in MeOH); 1H NMR (CDCl3): d=1.31–1.33
(d, 3H, J=7.2 Hz), 3.09–3.30 (m, 2H), 3.68 (s, 3H), 4.46–4.50 (t, J=
14.4 Hz, 1H), 5.52–5.56 (t, J=14.8 Hz, 1H), 6.90–6.91 (d, J=6.8 Hz, 1H),
7.16–7.20 (m, 5H), 7.26–7.30 (t, J=13.6 Hz, 1H), and 7.37–7.40 ppm (t,
J=14.4 Hz, 1H); 3C NMR (CDCl3): d=18.7, 30.2, 39.0, 48.9, 53.1, 55.3,
126.7, 127.5, 127.7, 129.3, 129.9, 131.7, 132.7, 136.7, 168.3, 170.6,
173.1 ppm; 77Se NMR (CDCl3): d=901 ppm; HRMS: m/z: calcd for
C20H20N2O4Se: 454.3336 [M+Na]+; found: 455.0489.
0.39 mmol) in dry acetonitrile (5 mL) was added dropwise with stirring to
a solution of 2-(chloroseleno)benzoyl chloride (0.10 g, 0.39 mmol) in dry
acetonitrile (5 mL). After addition of triethylamine (0.10 mL 0.78 mmol),
the reaction mixture was stirred at 258C for about 3 h, and then the sol-
vent was evaporated under reduced pressure. Purification of the crude
compound on a silica gel column using ethyl acetate and petroleum ether
as eluent gave a yellow solid. Yield: 0.120 g (56%); m.p. 126–1288C;
[a]2D5 =À20 (c=0.2 in MeOH); 1H NMR (CDCl3): d=0.85–0.86 (d, J=
4 Hz, 3H), 0.96–0.98, (d, J=8 Hz, 4H), 1.66 (s, 1H), 2.10–2.30 (m, 1H),
3.12–3.13 (t, J=4 Hz, 2H), 3.72 (s, 3H), 4.48–4.50, (t, J=8 Hz, 1H),
4.86–4.89 (t, J=12 Hz, 2H), 6.55–6.56 (d, J=4 Hz, 1H), 7.04–7.06 (q, J=
8 Hz, 3H), 7.24–7.31 (m, 3H), 7.41–7.43 (t, J=8 Hz, 1H), 7.43–7.65 (m,
1H), 8.05 ppm (d, J=4 Hz, 1H); 13C NMR (CDCl3): d=18.3, 32.9, 38.4,
49.4, 52.9, 54.1, 63.1, 124.2, 126.5, 127.6, 129.1, 129.8, 132.5, 136.2, 140.9,
168.3, 170.5, 171.9, 172.2 ppm; 77Se NMR (CDCl3): d=894 ppm; HRMS:
m/z: calcd for C25H29N3O5Se: 553.4647 [M+Na]+; found: 554.1170.
Synthesis of 21: a solution of NH2-Trp-Ala-OMe (0.11 g, 0.39 mmol) in
dry acetonitrile (5 mL) was added dropwise with stirring over a period of
Measurement of GPx activity: The GPx activity of ebselen and all the
peptide-based compounds was studied spectrophotometrically. The test
mixture contained glutathione (2.0 mm), EDTA (1 mm), glutathione disul-
fide reductase (1.7 unitsmLÀ1), and nicotinamide adenine dinucleotide
phosphate-oxidase (NADPH; 0.4 mm) in 0.1m potassium phosphate
buffer of pH 7.5. The samples (80 mm) were added to the test mixture at
room temperature (258C), and the reaction was started by addition of
H2O2, tBuOOH, or Cum-OOH (1.6 mm). The initial reduction rates (v0)
were calculated from the rate of NADPH oxidation at 340 nm. Each ini-
tial rate was measured at least 3 times and calculated from the first 5–
10% of the reaction by using the molar extinction coefficient
(6.22 mmÀ1 cmÀ1) for NADPH. For the peroxidase activity, the rates were
corrected for a background reaction between peroxide and glutathione.
10 min to
a solution of 2-(chloroseleno)benzoyl chloride (0.10 g,
0.39 mmol) in dry acetonitrile (5 mL). After the addition of triethylamine
(0.10 mL 0.78 mmol), the reaction mixture was stirred at 258C for about
3 h. Solvent was evaporated under reduced pressure and the crude com-
pounds was purified on a silica gel column using ethyl acetate and petro-
leum ether as eluent to give the desired compound as a yellow solid.
Yield: 0.110 g (60%); m.p. 193–1968C; [a]2D5 =À10 (c=0.2 in MeOH);
1H NMR (CDCl2): d=1.25–1.26 (d, J=4 Hz, 3H), 3.25–3.39 (m, 2H),
3.58 (s, 3H), 4.38–4.44 (q, 1H), 5.62–5.63 (t, J=4 Hz, 1H), 6.88–6.89 (d,
J=4 Hz, 1H), 7.15–7.20 (m, 3H), 7.30–7.37 (m, 2H), 7.56–7.99 (m, 3H),
7.99–8.01 (d, J=4 Hz, 1H), 8.42 ppm (s, 1H); 3C NMR (CDCl3): d=
18.43, 29.9, 48.8, 52.9, 58.0, 110.4, 111.6, 119.1, 119.9, 122.4, 123.9, 124.3,
126.4, 127.4, 127.9, 128.8, 132.5, 136.6, 141.3, 168.2, 170.4, 172.9 ppm;
Single-crystal X-ray structure determination: X-ray crystallographic stud-
ies were carried out using a Bruker CCD diffractometer with graphite-
Chem. Eur. J. 2011, 17, 4849 – 4857
ꢀ 2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemeurj.org
4855

G. Mugesh and K. Satheeshkumar
monochromatized MoKa radiation (l=0.71073 ꢁ) controlled by a Penti-
um-based PC running the SMART software package.[25] Single crystals
were mounted at room temperature on the ends of glass fibers, and data
were collected at room temperature. The structures were solved by direct
methods and refined using the SHELXTL software package.[26] All non-
hydrogen atoms were refined anisotropically and hydrogen atoms were
assigned idealized locations. Empirical absorption corrections were ap-
plied to all structures using SADABS.[27] The structures were solved by
direct method (SIR-92) and refined by full-matrix least-squares proce-
dure on F2 for all reflections (SHELXL-97).[28]
trogen and stored at À208C. The working solution of DHR and peroxini-
trite were kept in an ice bath. The assay mixture contained DHR
(0.1 mm), peroxynitrite (0.69 mm) in 100 mm phosphate buffer of pH 7.5
with 100 mm DTPA and variable inhibitor concentrations. The fluores-
cence intensity from the reaction of DHR with peroxynitrite was set as
100%, and the intensity after the addition of various inhibitors was ex-
pressed as the percentage of the observed in the absences of inhibitors.
The final fluorescence intensity was corrected for background reactions.
The inhibition plots were obtained by using Origin 6.1 software that uti-
lized sigmoidal curve fitting, and these plots were used for the calculation
of IC50 values.
Crystal data for 23: C16H20N2O4Se2; Mr =921.7; triclinic; space group P1;
a=8.3945(18), b=9.470(2), c=12.166(3) ꢁ; a=85.632(4), b=75.894(3),
g=79.468(4)8; V=921.7(3) ꢁ3; Z=2; 1calcd =1.38 gcmÀ3; MoKa radiation
(l=0.71073 ꢁ); T=293(2) K; R1 =0.069, wR2 =0.118 (I>2s(I)); R1 =
0.045, wR2 =0.106 (all data). CCDC-791202 contains the supplementary
crystallographic data for this paper. These data can be obtained free of
charge from The Cambridge Crystallographic Data Centre via
www.ccdc.cam.ac.uk/data_request/cif.
Acknowledgements
This study was supported by the Department of Science and Technology
(DST), New Delhi, India. We are grateful to the Alexander von Hum-
boldt Foundation, Bonn, Germany for the donation of an automated
flash chromatography system. G.M. acknowledges the DST for the award
of Ramanna and Swarnajayanti fellowships and K.S. thanks the DST for
a research fellowship.
Synthesis of peroxynitrite (PN): Peroxynitrite was synthesized by follow-
ing the literature method with minor modifications.[29] A solution of 30%
(ꢀ8.8m) H2O2 (5.7 mL) was diluted to 50 mL with water, cooled to
about 48C in an ice/water mixture, added to NaOH (5n, 30 mL), and di-
ethylene triamine pentaacetic acid (DTPA; 0.04m, 5 mL) in NaOH
(0.05n) with gentle mixing, and then diluted to a total volume of 100 mL.
The concentration of H2O2 in the final solution was 0.5m; the pH ranged
from 12.5 to 13.0. The buffered H2O2 was stirred vigorously with an equi-
molar amount of isoamyl nitrite (0.05m or 6.7 mL) for 3–4 h at room tem-
perature. The reaction was monitored by withdrawing aliquots at an in-
terval of 15 or 30 min and assaying for peroxynitrite at 302 nm using a
UV/Vis spectrophotometer. When the yield of peroxynitrite reached a
maximum, the aqueous phase was washed with dichloromethane, chloro-
form, and hexane (3ꢂ100 mL) in a separating funnel to remove the con-
taminating isoamyl alcohol and isoamyl nitrite. The unreacted H2O2 was
removed by passing the aqueous phase through a column filled with
granular MnO2 (25 g). The concentration of the stock solution of peroxy-
nitrite was measured after 500 times dilution with 0.1n NaOH solution
and then assaying for peroxynitrite at 302 nm (e=1670mÀ1 cmÀ1) by the
UV/Vis spectrophotometric method.
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Inhibition of protein nitration of BSA: For bovine serum albumin
(BSA), the nitration was performed by the addition of PN (1.2 mm) to
BSA (100 mm) in 0.5m phosphate buffer of pH 7.0 with DTPA (0.1 mm)
at 208C. After the addition of PN, the final pH was maintained below
7.5. The reaction mixture was incubated for 20 min at 208C. Similarly,
the reactions of BSA with PN were performed in the presence of differ-
ent peptide-based ebselen analogues (133 mm) as inhibitors. Upon per-
forming the reactions, the mixture was denatured by boiling at 1008C for
5 min in the presence of sample loading dye and subjected to polyacryl-
amide gel electrophoresis and Western blot analyses.
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buffer of pH 8.3 with glycine and sodium dodecyl sulfate (SDS). After
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a polyvinylidene fluoride
(PVDF) membrane and the nonspecific binding sites were blocked by
5% nonfat skimmed milk in PBST (blocking solution) for 2 h. Then the
membrane was probed with rabbit polyclonal primary antibody against
nitrotyrosine (1:20000 dilutions) in blocking solution for 2 h followed by
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Inhibition of PN-mediated oxidation: PN-mediated oxidation of dihydro-
rhodamine 123 (DHR) was measured by fluorometric techniques by fol-
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with excitation and emission wavelengths of 500 and 526 nm, respectively.
The stock solution for DHR in dimethylformamide was purged with ni-
4856
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Chem. Eur. J. 2011, 17, 4849 – 4857

Peptide-Based Ebselen Analogues
FULL PAPER
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Received: November 26, 2011
Published online: March 11, 2011
Chem. Eur. J. 2011, 17, 4849 – 4857
ꢀ 2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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4857