Modelling the Inhibition of Selenoproteins by Small Molecules Using Cysteine and Selenocysteine Derivatives

Article abstract of DOI:10.1002/chem.201901363

Small molecule-based electrophilic compounds such as 1-chloro-2,4-dinitrobenzene (CDNB) and 1-chloro-4-nitrobenzene (CNB) are currently being used as inhibitors of cysteine- and selenocysteine-containing proteins. CDNB has been used extensively to determine the activity of glutathione S-transferase and to deplete glutathione (GSH) in mammalian cells. Also, CDNB has been shown to irreversibly inhibit thioredoxin reductase (TrxR), a selenoenzyme that catalyses the reduction of thioredoxin (Trx). Mammalian TrxR has a C-terminal active site motif, Gly-Cys-Sec-Gly, and both the cysteine and selenocysteine residues could be the targets of the electrophilic reagents. In this paper we report on the stability of a series of cysteine and selenocysteine derivatives that can be considered as models for the selenoenzyme–inhibitor complexes. We show that these derivatives react with H₂O₂ to generate the corresponding selenoxides, which undergo spontaneous elimination to produce dehydroalanine. In contrast, the cysteine derivatives are stable towards such elimination reactions. We also demonstrate, for the first time, that the arylselenium species eliminated from the selenocysteine derivatives exhibit significant redox activity by catalysing the reduction of H₂O₂ in the presence of GSH (GPx (glutathione peroxidase)-like activity), which suggests that such redox modulatory activity of selenium compounds may have a significant effect on the cellular redox state during the inhibition of selenoproteins.

Full text of DOI:10.1002/chem.201901363

A Journal of
Accepted Article
Title: Modelling the Inhibition of Selenoproteins by Small Molecules
using Cysteine and Selenocysteine Derivatives
Authors: Kishorkumar M Reddy and Govindasamy Mugesh
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Modelling the Inhibition of Selenoproteins by Small Molecules
using Cysteine and Selenocysteine Derivatives
Kishorkumar M. Reddy and Govindasamy Mugesh*[a]
Abstract: Small molecule-based electrophilic compounds that can be considered as models for the selenoenzyme-
such as 1-chloro-2,4-dinitrobenzene (CDNB), 1-chloro-4- inhibitor complexes. We show that these derivatives react
nitrobenzene (CNB) are being used as inhibitors of cysteine- with H2O2 to generate the corresponding selenoxides, which
and selenocysteine-containing proteins. CDNB has been undergo spontaneous elimination to produce dehydroalanine.
used extensively to determine the activity of glutathione S- In contrast, the cysteine derivatives are stable against such
transferase and to deplete glutathione (GSH) in mammalian elimination reactions. We also demonstrate, for the first time,
cells. Also, CDNB has been shown to irreversibly inhibit the that the arylselenium species eliminated from the
thioredoxin reductase (TrxR), a selenoenzyme that catalyzes selenocysteine derivatives exhibit significant redox activity by
the reduction of thioredoxin (Trx). The mammalian TrxR has catalysing the reduction of H2O2 in the presence of GSH
a C-terminal active site motif Gly-Cys-Sec-Gly and both the (GPx-like activity), suggesting that such redox modulatory
cysteine and selenocysteine residues could be the targets for activity of selenium compounds may have significant effect
the electrophilic reagents. In this paper, we report on the on the cellular redox state during the inhibition of
stability of a series of cysteine and selenocysteine derivatives selenoproteins.
which is an integral part of the antioxidant system in the cells. As
TrxR plays a key role in the regulation of cancer cell apoptosis, it
Introduction
is an attractive target for the design of new anticancer drugs.
Electrophilic compounds such as 1-chloro-2,4-dinitrobenzene
The active site of TrxR comprises of a C- terminal motif Gly-Cys-
(CDNB), 1-chloro-4-nitrobenzene (CNB) (Figure 1A), which are
Sec-Gly. The selenenyl sulphide bond is critical for the redox
arylating agents, are being used as substrates for the assays
activity. The enzyme reduces the disulphide bond in thioredoxin
determining the levels of glutathione S-transferase.[1] CDNB has
(Trx) and the reduced Trx mediates the reduction of many other
been used as GSH-depleting agent in the cell culture
protein disulphides (Figure 1B).[6] Although the main function of
experiments. An early study by Wahlländer and Sies showed
TrxR is to reduce the disulphide bonds in Trx, it also reduces
that CDNB can be used to understand the formation of a
glutathione S-conjugate in perfused rat liver and monitor the
activity of glutathione S-transferases.[1b] It has also been
proposed that CDNB acts as immunostimulatory agent in AIDS
treatments.[2] Holmgren and coworkers reported that CDNB acts
as specific irreversible inhibitor of the thioredoxin reductase
(TrxR) in the presence of NADPH, which can increase the
NADPH oxidase activity in the modified enzyme.[3] In the
absence of NADPH, it was observed that CDNB does not have
any effect on TrxR activity, suggesting that CDNB inhibits the
enzyme only after the reduction of the active site selenenyl
sulfide bond in the enzyme by NADPH. Further, a detailed mass
spectral analysis indicated that the cysteine thiol groups of the
enzyme were modified covalently by CDNB to produce the
corresponding S-aryl derivatives.[4] The covalent bond formation
between the enzyme and inhibitor has been shown to
irreversibly block the enzyme activity.
Arnér and coworkers reported that the small molecule CDNB
can induce cell apoptosis by inhibiting the activity of TrxR,[5]
[a]
K. M. Reddy and Prof. Dr. G. Mugesh
Department of Inorganic and Physical Chemistry
Indian Institute of Science
Bangalore 560012, India
E-mail: mugesh@iisc.ac.in
Figure 1. (A) Chemical structures of some commonly used selenoenzyme
inhibitors. (B) Mechanism of the inhibition of mammalian thioredoxin reductase
(TrxR) by CDNB. The covalent bond formation between TrxR and the inhibitor
inactivates the enzyme.
Supporting information for this article is given via a link at the end
of the document.
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hydroperoxides and regenerates some antioxidants such as
lipoate, various selenium compounds, and ubiquinone. All the
biological effects of TrxR would be altered when the TrxR is
inhibited in cells by CDNB.[7]
intermediates for the synthesis of title compounds were
synthesised following the literature procedures with slight
modifications. 5-Membered cyclic sulphamidate (1) and O-tosyl
serine (2) were synthesised starting from serine. The amine
group of serine was protected with Boc group and acid group
was protected by converting it to a methyl ester to obtain Boc-
Ser-OMe. The side chain hydroxyl group was made as a good
leaving group by protecting with a tosyl group to afford the
intermediate 2 (Figure 2). The other intermediate (1) was also
synthesised from Boc-Ser-OMe. Freshly distilled thionyl chloride
and pyridine was added to Boc-Ser-OMe at -40 °C in acetonitrile.
The intermediate produced was isolated and treated with NaIO4
and catalytic amount of RuCl3.3H2O in acetonitrile and water
(1:1) at 0 °C about 2 h to afford the 5-memebered serine cyclic
sulphamidate (1).[16] The fully protected selenocystine 3 was
obtained from O-tosyl serine (2).[14] Briefly, the tosylated
compound 2 was treated with pMob (p-methoxy benzyl) selenol,
which was generated in-situ by treating pMob diselenide with
Owing to their high proliferation and metabolism, cancer
cells generate more amounts of reactive oxygen species (ROS)
as compared to that of normal cells. The increase in the ROS
levels in the cells leads to an imbalance between the ROS
generating and scavenging systems, resulting in a condition
called oxidative stress.[8] To combat the deleterious effects of
ROS and oxidative stress, the levels of Trx system are
elevated.[9] Therefore, it has been proposed that targeting
TrxR/Trx is a promising strategy for cancer treatment.[10] It has
also been reported that the native TrxR provide beneficial effects
to the cancer cells, whereas the irreversibly inhibited TrxR by
arylating agents such as CDNB triggers apoptosis in cancer
cells.[5] Arnér and co-workers proposed that the mechanism by
which these electrophiles inactivate the enzyme involves the
reactions of both selenolate and thiolate in the active site with
the electrophiles to form S-C and Se-C bonds with the inhibitors
as shown in Figure 1B.[4]
NaBH4 in deoxygenated DMF. The
pMob-protected
selenocysteine obtained in this reaction was treated with iodine
in methanol and water to afford the protected selenocystine 3
(Supporting Information, Scheme S1 and S2). The reported
methods for the synthesis of cysteine derivatives involves an
attack of thiol of cysteine at the aryl halides or aryl triflates.[17]
However, these methods require metal complexes as
catalysts.[18] Therefore, we employed a simple method which
involves the serine-based cyclic sulphamidate 1 as a precursor.
The thiol moiety in the aryl thiols attacks at the high electrophilic
β-carbon of 1, leading to the formation of substituted-cysteine
derivatives. Briefly, thiophenol (4), 2-mercaptopyridine (5) or 2-
mercaptopyrimidine (6) was treated with the 5-memebered
serine cyclic sulphamidate 1 in the presence of K2CO3 in DMF.
The reactions were monitored by thin layer chromatography.
After completion of the reactions, the sulphonic group was
cleaved from the amine group by acidic hydrolysis to afford the
aryl cysteine derivatives 7-9, respectively (Figure 2).
After the discovery that small electrophilic reagents can
irreversibly block the enzyme activity by forming covalent bonds
with the active site residues, several research groups utilized the
inhibition of TrxR as a potential approach to kill tumour cells
using several synthetic molecules, natural products and metal
complexes.[11] The reactivity of the cysteine and selenocysteine
residues in TrxR towards inhibitors has been studied and it has
been found that the inhibitors preferentially modify the
selenocysteine residue over cysteine residue. However, the
relative stability of the S-C and Se-C covalent bonds formed
between selenocysteine/cysteine and inhibitor is not clear and it
is not known whether the oxidative stress conditions can affect
the stability of the enzyme-inhibitor complex as both sulfur and
selenium atoms are redox active and can react with reactive
oxygen species such as hydrogen peroxide. Therefore, in this
paper, we present our experimental studies on the reactivity of
thiol and selenol moieties towards electrophilic arylating agents
and the stability of the arylated cysteine and selenocysteine
derivatives in the presence of hydrogen peroxide.
Results and Discussion
Synthesis of aryl Cysteine and Selenocysteine derivatives
The aryl cysteine/selenocysteine derivatives were synthesised
usually by following two methods: The first method involves
reaction of selenocysteine selenolate / cysteine thiolate with the
aryl halides[12] and the second one involves the reaction of
Figure 2. The starting materials 1-3 used for the synthesis of cysteine and
selenocysteine derivatives and the reaction of compound 1 with thiols 4-6 to
produce the aryl-substituted cysteine derivatives 7-9, respectively.
Experimental conditions: a) K2CO3, DMF, 0 °C, 5 min, 27 °C, 6 h. b) 10 %
NaH2PO4, EtOAc, 50 °C, 2 h.
substituted selenolate
intermediates such as β-haloalanine,[13] O-tosyl serine,[14] and
serine-β-lactone[15]
to produce the corresponding
cysteine/selenocysteine derivatives. Starting materials or
/
thiolate with the electrophilic
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Recently, Chandrasekaran et al. reported an open-flask
procedure and a mild chemoselective method for the synthesis
of S-arylation of cysteine under metal-free conditions in
methanol. In this method, arenediazonium salts and Boc-Cys-
OMe were used as the precursors.[19] They showed that the
reaction, which probably takes place under a radical chain
mechanism, is dependent on the use of methanol as solvent.
Interestingly, this method was applied to the synthesis of
biologically important tripeptide glutathione (GSH) and they were
able to isolate the S-aryl glutathione derivatives in good yields.
We employed this method with slight modification for the
synthesis of compound 11, as the 1-chloro-2,4-dinitrobenzene
(CDNB) is highly electrophilic and a direct attack of cysteine thiol
is possible. This reaction afforded 2,4-dinitrobenzyl-substituted
cysteine 11 in moderate yield (Figure 3A).
We reported earlier that N-Boc-L-serine methyl ester may be
used as starting material for the synthesis of substituted
selenocysteine derivatives. In this method, the phenyl-
substituted selenocysteine was synthesized from Boc–Ser–OMe
by converting it to the corresponding bromide, and subsequent
reaction with lithium benzene selenolate (PhSe−Li+), which in
turn was generated in situ by reducing diphenyl diselenide
(PhSeSePh) by superhydride.[20] In the present study, the phenyl
derivative of selenocysteine (22) was synthesized following
method 2, where the commercially available diphenyl diselenide
was reduced with NaBH4 in DMF to generate the free
selenol/selenolate, which was added to O-tosyl L-serine (2) in
DMF to afford the protected phenyl selenocysteine 22 (Figure
3B, inset).
All the selenocysteine derivatives were well characterized by
NMR spectroscopy. From Table 1, it is evident that the 77Se
NMR chemical shifts values are shifted downfield upon
increasing the number of nitrogen atom or nitro groups in the
phenyl ring. This indicates that the selenium center in these
compounds becomes more electrophilic upon substitution of the
phenyl ring with pyridine, pyrimidine or nitrophenyl substituents.
Given the electron-deficient nature of the selenium atoms in
these compounds, particularly in the dinitrophenyl derivative 21,
potential reactions can take place between the selenium centers
and nucleophiles.
Table 1. Experimental 77Se NMR chemical shifts values for the selenocysteine
derivatives 17-22.
Compd.
17
18
77Se NMR δ (ppm)a
Compd.
20
21
77Se NMR δ (ppm)a
330
354
224
279
374
265
19
22
aThe 77Se NMR chemical shifts are cited with respect to Me2Se in CDCl3.
Figure 3. (A) Synthesis of the cysteine derivative 11 from compound 10 using
1-chloro-2,4-dinitrobenzene (CDNB). a) K2CO3, DMF, 0 °C, 5 min, 27 °C, 6 h.
(B) Synthesis of the selenocysteine derivatives 17-21 from the protected
selenocysteine 3, by reduction followed by treatment with appropriate aryl
Reactivity of Cysteine/Selenocysteine derivatives towards
H2O2
halides. a) NaBH4, MeOH,
5 min 0 °C. b) 2-Bromopyridine (13), 2-
bromopyrimidine (14), 1-chloro-2-nitrobenzene (15) or 1-chloro-4-nitrobenzene
(16) 13-16 or CDNB in THF, 27 0 °C, 6 h. Inset: Compound 22 synthesized
from PhSeH and O-tosyl-L-serine (2).
It has been shown that the reactions of some of the aryl halides
with TrxR generate adducts with the selenocysteine moiety. The
formation of such adducts has been confirmed by ESI-MS
analysis. In fact, ebselen, a known GPx mimic, forms a stable
selenenyl sulphide species upon reactions with GSH or any of
the free thiols on proteins.[21] The irreversible formation of
selenenyl sulfide with protein thiols inhibit the enzyme activity.[21]
To understand the reactivity of the aryl sulfide and selenide
towards hydrogen peroxide, we carried a simple experiment by
incubating the cysteine / selenocysteine derivatives with H2O2
and monitored the reaction by spectroscopy. In the initial
experiments, we treated all the key compounds with four
equivalents of H2O2 and found that they indeed undergo reaction
with H2O2 to produce dehydroalanine (Dha) with elimination of
The aryl selenocysteine derivatives were synthesised by
following method 1, which involves the attack of highly
nucleophilic selenol/ selenolate of selenocysteine at aromatic
halides. However, the yield of the reaction depends on the
electrophilicity of the aryl halides. When the highly electrophilic
1-chloro-2,4-dinitrobenzene (CNDB, 11) was used as aryl halide,
the reaction afforded compound 21 in 80% yield. In other cases,
particularly with the aryl halides that are less electrophilic, we
observed lower yields. Typically, the procedure involves the
initial reduction of the protected selenocystine derivative 3 to
generate the free selenol by NaBH4 in situ in methanol. Addition
of the selenol or selenolate to the aryl halides 2-bromopyridine
(13), 2-bromopyrimidine (14), 1-chloro-2-nitrobenzene (15) or 1-
chloro-4-nitrobenzene (16) in THF afforded the aryl-substituted
selenocysteine derivatives 17-20, respectively (Figure 3B).
1
the selenium moiety. The formation of Dha was confirmed by H
NMR and HPLC analysis. All the cysteine derivatives 7-9 and 12
were found to be very stable and did not react with H2O2 even at
higher concentrations of the peroxide. On the other hand, the
selenocysteine derivatives 17-22 reacted readily with H2O2 in a
time as well as concentration-dependent manner to generate the
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corresponding dehydroalanine derivatives. For example, the 4-
nitrophenyl-substituted selenocysteine derivative 20 reacted
rapidly with H2O2 to produce 23 quantitatively (Figure 4A). The
reactivity towards H2O2. We found that compound 21 reacts with
4 equiv. of H2O2 very slowly to generate Dha and the reaction
takes about 14h for completion. When compound 21 was treated
with 10 equiv. of H2O2, the reaction produced Dha within 3h,
which was confirmed by 1H NMR and HPLC experiments
(Supporting Information, Figure S1). These observations
indicated that the reaction of compound 21 with hydrogen
peroxide is found to be much slower than that observed for the
phenyl, pyridyl, piperidyl or 4-nitrophenyl-substituted compounds
(Supporting Information, Figure S2). However, the elimination of
the selenium moiety from the selenocysteine derivatives is
consistent with the proposal that the selenium moiety may be
eliminated from the TrxR-inhibitor complex. Although the
inhibition of TrxR by dinitrohalobenzenes such as CDNB is
characterised by an arylation of both the -SeH of the active site
selenocysteine and the -SH of the adjacent cysteine, only the
loss of Se-dinitrophenol group from the peptide may occur,
leading to the formation of a dehydroalanine. This is in
agreement with the report of Anestål and Arnér that the
enzymatically fully active selenocysteine-containing TrxR1 does
not have any cell death-promoting effects, but the truncated or
selenium-compromised forms of TrxR1 may directly promote
apoptosis.[4-5] The Dha derivative of TrxR1 may be considered
as the selenium-compromised form and such derivative cannot
maintain the Trx-mediated redox balance in the cells.
Interestingly, the inhibitor bound to the cysteine residue may
remain intact under oxidative stress conditions (Figure 5). This is
in agreement with our observations that the cysteine derivatives
7-9 and 12 are very stable and they do not react with H2O2 even
at higher concentrations of the peroxide.
1
formation of Dha can be followed conveniently by HPLC and H
NMR spectroscopy. The HPLC analysis showed that the peak
corresponding to the selenocysteine derivative 20 disappeared
upon addition of hydrogen peroxide, with a new peak appearing
1
at 7.3 min for the Dha derivative 23 (Figure 4B). The H NMR
spectrum of authentic Dha in CD3OD shows characteristic peaks
at δ 5.69-5.71 (Ha) as doublet and at δ 6.0 (Hb) as singlet. As all
the selenocysteine derivatives have a hydrogen atom attached
to the chiral carbon (α-C) exhibiting a peak around δ 4.5 as
multiplet, the disappearance of this peak and appearance of the
1
alkene (Ha and Hb) peaks can be monitored conveniently by H
NMR spectroscopy (Figure 4C).
Figure 5. The formation of a relatively stable C-Se bond with the inhibitor and
the facile oxidation of selenium leads to the elimination of the selenium moiety
from TrxR, possibly under oxidative stress conditions. The arylated cysteine
residue appears to be stable under these conditions.
Figure 4. (A) Reaction of compound 20 with H2O2 to produce the
dehydroalanine derivative 23. (B) HPLC chromatograms obtained after
treating compound 20 with 4 equiv. of H2O2. (C) Partial 1H spectra of
compound 20 after incubating with H2O2. In both the experiments, the
formation of Dha was clearly observed.
Although the above studies clearly indicated the formation of
Dha derivatives upon reactions of the selenocysteine derivatives
with hydrogen peroxide, it is important to understand the nature
of selenium species eliminated from these reactions. It is known
that alkyl/aryl selenides can react with hydrogen peroxide and
organic peroxides to produce the corresponding selenoxides.[20]
The reactions of compounds 17-22 with hydrogen peroxide are
expected to produce the corresponding selenoxides 24-29,
which may undergo spontaneous elimination of Dha (23) to
produce the corresponding selenenic acids 30-35 (Figure 6).
Alternatively, the further reactions of selenoxides with H2O2 to
produce the corresponding selones 36-41, followed by the
The dinitro-based compound 21, which has two highly
electron withdrawing NO2 groups at ortho and para positions on
the aryl ring, is used as the model compound for the irreversible
TrxR inhibition. It is expected that the formation of Dha from 21
is more facile as compared to that of other selenocysteine
derivatives because of the presence of highly electron
withdrawing groups. However, the reactivity of 21 with H2O2 was
found to be very slow. This may be ascribed to the fact that the
electron withdrawing effect of the nitro groups makes the
selenium center electron deficient, which may reduce its
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elimination of Dha can produce the seleninic acids 42-47. In
addition, the selenenic acids 30-35 can also generate the
seleninic acids 42-47 upon reaction with H2O2 and further
oxidation by H2O2 can produce the corresponding selenonic
acids 48-53. The formation of the overoxided selenonic acid
derivatives requires a very strong oxidizing condition. To
understand the nature of oxidized selenium species formed in
the reactions of the selenocysteine derivatives with H2O2, we
carried out 77Se NMR spectroscopic studies. These reactions
were carried out in an NMR tube by treating the selenocysteine-
derivatives with various amounts of H2O2 in CD3OD. When 18
was treated with one or two equivalents of H2O2, only a peak at
δ 352 ppm corresponding to the starting material was observed.
However, when compound 18 was treated with three equivalents
of H2O2, the peak observed at δ 352 was disappeared
completely, with two new peaks appearing at δ 1156 and δ 1201
ppm. These two signals can be ascribed to the corresponding
selenenic acid 31 and seleninic acid 43, respectively (Supporting
Information, Figure S3). The ortho-nitro derivative 19 underwent
a rapid oxidation by two equivalents of H2O2 to produce three
selenium-based species. The three signals observed in the 77Se
NMR spectrum at δ 1158, 1199 and 1213 ppm can be ascribed
Interestingly, the formation of the selenonic acid 52 was not
observed even at higher concentrations of H2O2, indicating that
the formation of overoxidized selenium species is highly
dependent on the nature of substituents present in the phenyl
ring. When nitro group is presents at ortho or para positions, the
selenocysteine derivatives produce Dha at a relatively low
concentration of H2O2, whereas higher amounts of H2O2 are
required to obtain the Dha derivative from 21 having two nitro
groups in the phenyl ring. These observations suggest that 1-
chloro-2,4-dinitrobenzene (CDNB) may react rapidly with the
selenocysteine moiety in TrxR or other selenoenzymes due to
the highly nucleophilic nature of the selenolate, but the high
oxidative stress conditions in cancer cells is probably
responsible for the elimination of the Se-dinitrophenyl moiety
from the enzyme-inhibitor complex.
Catalytic reduction of H2O2 by aryl selenocysteine
derivatives
As all the selenocysteine derivatives 17-22 undergo oxidation by
hydrogen peroxide to produce the Dha derivative (23) and the
corresponding selenenic and seleninic acids, it was thought
to the selenenic acid 32, seleninic acid 44 and selenonic acid 50, worthwhile to study the redox behaviour of the selenium
respectively (Supporting Information, Figure S4). These
observations indicate that the presence of ortho-nitro group
facilitates the formation of overoxidized selenium species
selenonic acid.
compounds in the presence of hydrogen peroxide and
glutathione (GSH). It has been reported that aryl diselenides act
as glutathione peroxidase (GPx) mimetics by catalytically
reducing hydrogen peroxide in the presence of GSH. The
catalytic cycle of these compounds involves the formation of
selenenic and seleninic acids.[22] It has been reported from our
laboratory that the aryl-substituted selenocysteine derivatives
having basic amino groups act as pro-catalysts that can
generate catalytically active species upon reaction with
hydrogen
peroxide.[20]
To
understand
whether
the
selenenic/seleninic acids produced in the reaction of aryl
selenocysteine derivatives 17-22 with hydrogen peroxide can act
as GPx mimetics, we studied the GPx mimetic activity by using
an enzymatic method. The catalytic reduction of H2O2 was
evaluated by using GSH as cofactor. The initial rate (ν0) of the
reaction was determined by following the oxidation of NADPH at
340 nm in a GSH-GSSG coupled assay in the presence of
glutathione reductase (GR) in phosphate buffer (Figure 7A).[23]
GR catalytically reduces GSSG in the presence of NADPH.
From the initial rates (ν0) for the reduction of hydrogen
peroxide by GSH in the presence 17-22 (Figure 7B), it can be
observed that the GPx-like activity strongly depends on the
nature of substituents attached to the selenium. Compound 21
having a dinitrophenyl substituent was found to be the least
active compound. Similarly, the selenocysteine derivatives 17,
18 and 23 having pyridyl, piperidyl and phenyl substituents,
respectively, were also found to be poor catalysts. The activity of
these compounds appears to correlate well with their ability to
release Dha and selenenic acids in the presence of H2O2.
Interestingly, compounds 19 and 20 having a nitrophenyl group
(ortho or para position) exhibited very good GPx-like activity and
the initial rates observed for these compounds (19: 78 µM min-1
and 20: 84 µM min-1) were almost comparable to that of
Figure 6. (A) Reaction of 17-22 with H2O2 to produce the corresponding
selenenic acids 30-35, seleninic acids 42-47 and selenonic acids 48-53 in the
presence of excess H2O2. (B) The selenoxides 24-29 and selones 36-41 serve
as the precursors for the selenenic and seleninic acids, respectively. The
selenonic acids 48-53 are produced from the seleninic acids by overoxidation.
The dinitro derivative 21 also reacts readily with four
equivalents of H2O2 to produce two selenium-based species that
exhibited 77Se NMR signals at δ 1156 and 1193 ppm for the
selenenic acid 34 and seleninic acid 46, respectively.
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acid were disappeared completely indicating that these species
react with GSH during the GPx cycle.
Based on the above observations, a catalytic cycle for the
GPx-like activity of the aryl-substituted selenocysteine such as
20 can be proposed. According to this mechanism, the reaction
of 20 with hydrogen peroxide produces the selenenic acid 33
through the formation of a selenoxide intermediate, followed by
a -hydride elimination as described in Figure 6. When enough
glutathione (GSH) is present in the reaction mixture, the
selenenic acid thus produced reacts with GSH to generate the
corresponding selenenyl sulfide 55, which upon reaction with
another equivalent of GSH produces the catalytically active
selenol 56. The oxidation of the selenol by hydrogen peroxide
regenerates the selenenic acid with the elimination of water. At
higher peroxide concentrations, the selenenic acid 33 is oxidized
further to the corresponding seleninic acid 45, which can again
participate in the catalytic cycle in the presence of an excess
amount of GSH as shown in Figure 8. The reduction of GSSG
by GR and NADPH maintains the redox balance and the
catalytic cycles continues till all the H2O2 is consumed.
Figure 7. (A) The reduction of H2O2 by GSH in the presence of ebselen and
17-22. The decrease in the absorbance of NADPH was followed at 340 nm in
a GSSG/GR coupled assay. (B) A comparison of the GPx-like activity of 17-22
with that of ebselen. Assay conditions: Phosphate buffer pH 7.4, 0.4 mM GSH,
0.4mM NADPH, 1.74 U glutathione reductase (GR) and 1.5 mM of H2O2 (C)
The reaction of 18 with H2O2 generates Dha and selenenic acid, which reacts
with GSH to produce the selenenyl sulfide 54.
ebselen (103 µM min-1), a well-known GPx mimetic (Figure 7B).
Ebselen has been reported to have anti-inflammatory, anti-
oxidant and cytoprotective activities and it can very efficiently
scavenge peroxynitrite.[21,24] It is being studied as a possible
candidate in the treatment of reperfusion injury and stroke,
hearing loss and tinnitus, and bipolar disorder.[25] Several other
organoselenium compounds have been reported to exhibit GPx-
like activity.[26]
The large variation in the initial rates also indicate that the
GPx-like activity depends not only on the H2O2 concentration,
but also on the GSH concentration. For example, for compound
20, a concentration-dependent activity was observed for both
peroxide and thiol (Supporting Information, Figure S5). As the
conversion of the oxidized products to the corresponding
selenenyl sulfides and the subsequent formation of selenol
species are important for the GPx-like activity, the reaction
mixture containing selenenic acids and other overoxidized
products was treated with GSH to understand their reactivity
towards thiols. The selenenic and seleninic acids generated
from compound 18 reacted rapidly with GSH to produce the
corresponding selenenyl sulfide, which exhibited a 77Se NMR
signal at δ 525 ppm (Supporting Information, Figure S6).
Similarly, the 77Se NMR spectrum obtained after treatment of 19
with 4 equivalents of H2O2, following by addition of 3 equivalents
of GSH showed a peak at 540 ppm for the corresponding
selenenyl sulfide. Although the formation of selenenyl sulfide
intermediates was not observed by 77Se NMR spectroscopy for
compounds 17, 20, 21 and 22 even at higher concentrations of
GSH, the peaks corresponding to the selenenic and seleninic
Figure 8. Proposed catalytic mechanism for the GPx-like activity of
compound 20
.
The above observations reveal an important aspect related
to the inhibition of TrxR or other selenoproteins by 1-chloro-2,4-
dinitrobenzene (CDNB) and 1-chloro-4-nitrobenzene (CNB) and
their analogues. Although these compounds irreversibly inhibit
the TrxR and related enzymes, it is important to study the
possible elimination of arylselenium moiety from the enzyme-
inhibitor complexes. The arylselenium species produced in these
reactions can possess significant antioxidant (GPx-like) activity
in the presence of GSH, which can significantly reduce the level
of hydrogen peroxide and subsequently alters the redox
signalling in mammalian cells.
Conclusions
In this paper, several aryl derivatives of cysteine
/
selenocysteine have been synthesized as chemical models to
understand the inhibition of TrxR and related selenoenzymes.
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52.33, 53.28, 80.05, 127.03, 129.05, 131.02, 134.80 , 155.03, 171.05;
ESI-MS: m/z calcd for C15H21NO4SNa [M+Na]+: 334.1089; found:
334.1154.
We showed that the products obtained from the reactions of
selenocysteine with TrxR inhibitors react with hydrogen peroxide
to generate the corresponding selenoxides, which undergo
spontaneous elimination to produce dehydroalanine. In contrast,
the cysteine derivatives are stable against such elimination
reactions. Although the elimination of selenium moieties from the
selenocysteine derivatives is consistent with the observations
made for TrxR-inhibitor complex, we demonstrated for the first
time that the arylselenium moiety eliminated from the
selenocysteine derivatives exhibit significant redox activity.
These observations indicate that the selenium species
eliminated upon reaction with hydrogen peroxide can control the
redox state during the enzyme inhibition, and therefore, will have
implications in cancer therapy. Overall, this study suggest that
the GPx-like antioxidant activity of the selenium oxidation
products should be considered while designing new inhibitors for
selenoenzymes.
Compound 8: Thick viscous oil, Yield: 93.6 % (0.83 mmol, 260 mg). 1H
NMR (CDCl3), δ (ppm): 1.35 (s, 9H), 3.52-3.62 (m, 2H), 3.64 (s, 3H), 4.53
(q, J = 6.5 Hz, 1H), 6.34 (d, J = 7.0 Hz, 1H), 6.95-6.97 (m, 1H), 7.15 (d, J
= 8.0 Hz, 1H), 7.43 (td, J1 = 15.4 Hz, J2 = 1.8 Hz, 1H), 8.34 (d, J = 4.5 Hz,
1H); 13C NMR (CDCl3), δ (ppm): 28.3, 32.0, 52.4, 54.5, 79.6, 120.1,
122.6, 136.3, 149.2, 155.5, 157.7, 171.5; ESI-MS: m/z calcd for
C14H20N2O4SNa [M+Na]+: 335.1041; found: 335.1237.
Compound 9: Thick viscous oil, Yield: 91.6 % (0.81 mmol, 255 mg). 1H
NMR (CDCl3), δ (ppm): 1.33 (s, 9H), 3.48-3.51 (m, 1H), 3.60-3.65 (m,
1H), 3.67 (s, 3H), 4.57-4.62 (m, 1H), 5.73 (d, J = 7.5 Hz, 1H), 6.95 (t, J =
4.8 Hz, 1H), 8.45 (d, J = 4.8 Hz, 1H); 13C NMR (CDCl3), δ (ppm): 28.30,
32.77, 52.55, 53.74, 79.93, 117.02, 155.32 , 157.36, 171.43; ESI-MS:
m/z calcd for C13H19N3O4SNa [M+Na]+: 336.0994; found: 336.1188.
Synthesis of compound 12: Methyl and Boc protected cysteine (10, 1
equiv., 1.06 mmol) was accurately weighed and dissolved in DMF (5 mL).
K2CO3 (2 equiv., 2.13 mmol) was added to the reaction mixture. Allowed
the reaction mixture to stir for 10 minutes. 1-Chloro 2,4-dinitrobenzene 11
(2 equiv., 2.13 mmol) dissolved in THF (5 mL) was added to the reaction
mixture. Allowed the reaction mixture to stir until the reaction was
completed (8 h). Extracted the compound using ethyl acetate (3 × 20 mL).
Washed the combined organic layer using brine (5 × 25 mL). The organic
layer was dried over Na2SO4, filtered and evaporated. The crude product
was purified over silica gel of mesh size 100-200 using 20% EtOAc / pet.
ether as eluent. Column chromatography yielded the desired compounds
12 as a yellow solid. Yield: 51.9 %(0.55 mmol, 220 mg), 1H NMR (CDCl3),
δ (ppm): 1.41 (s, 9H), 3.41-3.46 (m, 1H), 3.59-3.64 (m, 1H), 3.79 (s, 3H),
4.66 (d, J = 6.4Hz1H), 5.42 (d, J = 6.8 Hz 1H), 7.77 (d, J = 8.9 Hz,1H),
8.37 (dd, J1 = 8.9 Hz, J2 = 2.3 Hz, 1H), 9.02 (d, J = 1.6 Hz 1H); 13C NMR
(CDCl3), δ (ppm): 28.6, 35.3, 52.7, 53.5, 81.1, 122.0, 127.7, 127.9,
132.06, 144.6, 145.8, 155.6, 170.8; ESI-MS: m/z calcd for
C15H19N3O8SNa [M+Na]+: 424.0791; found: 424.1049.
Experimental Section
Materials and Methods
Methanol was obtained from Merck. All other chemicals were of the
highest purity available. Most reactions were carried out in a well-
ventilated fume hood to avoid the unpleasant odours and toxic nature of
the reaction mixtures involved. Thin-layer chromatography analyses were
carried out on pre-coated silica gel plates (Merck), and spots were
visualized under UV radiation. Column chromatography was performed
on glass columns loaded with silica gel. 1H (400 MHz), 13C (100.56 MHz),
and 77Se (76.29 MHz) NMR spectra were obtained on a Bruker 400 MHz
NMR spectrometer. Chemical shifts for 1H and 13C NMR spectra are cited
with respect to SiMe4 as internal standard. Chemical shifts are reported
relative to dimethyl selenide (0 ppm) by assuming that the resonance of
the standard is at 461.0 ppm. A Perkin–Elmer Lambda 5 UV/Vis
spectrophotometer and high performance liquid chromatography (HPLC)
with use of a 2695 separation module was used to measure the catalytic
activities and dehydroalanine formation respectively. Mass spectral data
were acquired with an Agilent 6538 Ultra High Definition Accurate Mass
(LC‐HRMS) instrument.
Synthesis of compound 17: Methyl and Boc-protected selenocystine 3
(1 equiv., 0.35 mmol) was accurately weighed and dissolved in methanol
(2 mL) Added NaBH4 (2 equiv., 0.71 mmol) at 0 °C under N2 atmosphere.
Allowed the reaction mixture to stir for 5 minutes. 2-Bromopyridine 13 (2
equiv., 0.71 mmol) in THF (2 mL) was added to the reaction mixture at
the same temperature. Allowed the reaction mixture to attain room
temperature (27 °C) and continued stirring until the completion of the
reaction. Added dil. HCl (10 mL) to the reaction mixture and extracted the
compound with ethyl acetate (3 × 15 mL) and washed the combined
organic layer with brine (3× 15 mL). The organic layer was dried over
Na2SO4, filtered and concentrated. The crude product was purified over
silica gel of mesh size 230-400 using 20% EtOAc / pet. ether as eluent.
Column chromatography yielded the desired compound as a thick
viscous oil. Yield: (60%, 145 mg, 0.42 mmol), 1H NMR (CDCl3), δ (ppm):
1.41 (s, 9H), 3.51-3.61 (m, 2H), 3.70 (s, 3H), 4.66 (q, J = 6.9 Hz 1H),
6.66 (d, J = 6.3 Hz 1H), 7.06-7.09 (m, 1H), 7.36 (d, J = 7.8 Hz, 1H), 7.44-
7.48 (m, 1H), 8.43 (d, J = 4.0 Hz, 1H); 13C NMR (CDCl3), δ (ppm): 27.5,
28.7, 52.8, 55.1, 80.0, 121.3, 126.1, 136.7, 150.3, 154.6, 156.1, 172.2;
77Se NMR (CDCl3), δ (ppm): 331; ESI-MS: m/z calcd for C14H22N2O3Se
[M+1]+: 361.0588; found: 361.1619.
General procedure for the synthesis of cysteine derivatives (7-9)
Thiol compound (4-6) (1 equiv., 0.89 mmol) and K2CO3 (2 equiv., 1.78
mmol) were accurately weighed and transferred to a 100ml RB. Stirred
the contents in DMF for10 minutes, serine cyclic sulphamidate 1 (1 equiv.,
0.89 mmol) dissolved in DMF (5 mL) was added to the reaction mixture.
Allowed the reaction mixture to stir until the reaction was completed (6 h).
Added 10% NaH2PO4 into the reaction mixture until it shows pink colour
in the pH paper. Add 25ml of ethyl acetate and heated at 50 °C for 2 h.
extracted the compound using ethyl acetate (3 × 20 mL). Washed the
combined organic layer using brine (5 × 25 mL). The organic layer was
dried over Na2SO4, filtered and evaporated. The crude product was
purified over silica gel of mesh size 100-200 using 20% EtOAc / pet.
ether as eluent. Column chromatography yielded the titled compounds 7-
9 in quantitative yields.
Compound 18: The same procedure was employed as for the
preparation of compound 17 with 0.26 mmol of compound 3, 0.53 mmol
of NaBH4 and 0.53 mmol of 2-bromopyrimidine (14) to afford compound
18 as thick viscous oil, yield: (65%, 117 mg, 0.346 mmol), 1H NMR
(CDCl3), δ (ppm): 1.37 (s, 9H), 3.48-3.53 (m, 1H), 3.53-3.62 (m, 1H), 3.71
Compound 7:[27] Thick viscous oil, Yield: 90.3% (0.80 mmol, 250 mg). 1H
NMR (CDCl3), δ (ppm): 1.42 (s, 9H), 3.38 (d, J = 4.7 Hz, 2H), 3.53 (s, 3H),
4.55-4.59 (m, 1H), 5.41 (d, J = 7.4 Hz 1H), 7.19-7.23 (m, 1H), 7.27-7.30
(m, 2H), 7.40-7.42 (m, 2H); 13C NMR (CDCl3), δ (ppm): 28.30, 37.16,
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10.1002/chem.201901363
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FULL PAPER
(s, 3H), 4.65-4.70 (m, 1H), 5.93 (d, J = 7.2 Hz 1H), 7.01 (t, J = 4.9 Hz,
1H), 8.46 (d, J = 4.8 Hz, 2H); 13C NMR (CDCl3), δ (ppm): 28.54, 28.71,
52.92, 54.64, 80.24, 118.11, 155.79, 157.76, 170.0, 172.17; 77Se NMR
(CDCl3), δ (ppm): 354.3; ESI-MS: m/z calcd for C13H21N3O3Se [M+1]+:
362.0541; found: 362.1577.
reductase (1.7 units/mL), and NADPH (0.4 mM) in 0.1 M potassium
phosphate buffer of pH 7.4. Compounds (100 μM) were added to the test
mixture at 25 oC temperature and the reaction was started by the addition
of peroxide (1.6 mM). The initial reduction rates were calculated from the
rate of NADPH oxidation at 340 nm in GSH assay. Each initial rate was
measured at least 3 times and calculated from the first 5-10% of the
reaction by using 6.22 mM-1cm-1 as the molar extinction coefficient for
NADPH. For the peroxidase activity, the rates were corrected for
background reaction between peroxide and thiol.
Compound 19: The same procedure was employed as for the
preparation of compound 17 with 0.35 mmol of compound 3, 0.71 mmol
of NaBH4 and 0.71 mmol of 2-nitrochlorobenzene 15 to afford compound
1
19 as yellow solid, yield: (25%, 72 mg, 0.178 mmol) H NMR (CDCl3), δ
(ppm): 1.41 (s, 9H), 3.33-3.54 (m, 2H), 3.73 (s, 3H), 4.62 (d, J = 5.8
Hz, ,1H), 5.31 (d, J = 6.7 Hz, 1H), 7.41-7.45 (m, 1H), 7.63-7.67 (m, 1H),
8.30-8.32 (m, 2H); 13C NMR (CDCl3), δ (ppm): 28.7, 32.9, 53.1, 54.4,
80.8, 126.6, 127.7, 130.7, 132.2, 134.9, 146.3, 155.3, 171.3; 77Se NMR
(CDCl3), δ (ppm): 224; ESI-MS: m/z calcd for C15H20N2O6SeNa [M+Na]+:
427.0384; found: 427.0582.
HPLC Method
High performance liquid chromatography experiments were carried out
on Waters Alliance System (Milford, MA) consisting of a 2695 separation
module, 2996 Photodiode-array detector and a fraction collector. A built-
in auto sampler was used for sample injection. The Alliance HPLC
system was controlled with EMPOWER software (Waters corporation,
Milford, MA). Samples were analysed by reverse phase HPLC method
(Princeton C18 column, 4.6 × 150 mm, 5 µm) with 1 mL.min-1 gradient
elution method. The optimized elution parameters were mobile phase A
(ACN) 60 % and mobile phase B (H2O) 40 % for 20 min.
Compound 20: The same procedure was employed as for the
preparation of compound 17 with 0.35 mmol of compound 3, 0.71 mmol
of NaBH4 and 0.71 mmol of 4-nitrochlorobenzene 16 to afford compound
20 as yellow solid, yield: (24%, 69 mg, 0.17 mmol) 1H NMR (CDCl3), δ
(ppm): 1.39 (s, 9H), 3.38-3.53 (m, 2H), 3.64 (s, 3H), 4.71 (d, J = 6.6 Hz,
1H), 5.35 (d, J = 6.5 Hz, 1H), 7.59 (d, J = 8.6 Hz, 2H), 8.06-8.08 (d, J =
8.5 Hz, 2H); 13C NMR (CDCl3), δ (ppm): 28.7, 30.4, 53.1, 53.9, 80.9,
124.3, 132.0, 140.5, 147.1, 155.3, 171.2; 77Se NMR (CDCl3), δ (ppm):
279; ESI-MS: m/z calcd for C15H20N2O6SeNa [M+Na]+: 427.0384; found:
427.1469.
Acknowledgements
This study was supported by the Science and Engineering
Research Board (SERB, EMR/IISc-01/2016), Department of
Science and Technology (DST), New Delhi. KMR acknowledges
the CSIR, New Delhi, for a research fellowship. We thank Ms
Gayatri, K. for her help with the synthesis of some of the
compounds and Aswathi, V. V. for her help in the determination
of GPx-like activity. G. M. thanks the SERB for the award of J. C.
Bose National fellowship (SB/S2/JCB-067/2015).
Compound 21: The same procedure was employed as for the
preparation of compound 17 with 0.35 mmol of compound 3, 0.71 mmol
of NaBH4 and 0.71 mmol of 1-chloro 2,4-dinitrobenzene 11 to afford
1
compound 21 as yellow solid, yield: (75%, 238 mg, 0.53 mmol) H NMR
(CDCl3), δ (ppm): 1.44 (s, 9H), 3.35-3.39 (m, 1H), 3.52-3.56 (m, 1H),
3.80(s, 3H), 4.72 (d, J = 5.9 Hz, 1H), 5.38 (d, J = 5.9 Hz, 1H), 7.90 (d, J =
8.7 Hz, 1H), 8.32-8.35 (dd, J1 = 9.0 Hz, J2 = 2.4 Hz, 1H), 9.10 (d, J = 2.32
Hz, 1H); 13C NMR (CDCl3), δ (ppm): 28.7, 29.6, 53.2, 53.6, 81.3, 122.1,
127.4, 130.6, 142.6, 145.7, 146.8, 155.6, 171.1; 77Se NMR (CDCl3), δ
(ppm): 370.8; ESI-MS: m/z calcd for C15H19N3O8SeNa [M+Na]+:
472.0235; found: 472.0582.
Keywords: cysteine • glutathione • inhibitors • selenocysteine •
thioredoxin reductase
Synthesis of compound 23:[28] Diphenyl diselenide 22 (2.01 mmol,
631mg) was reacted with NaBH4 (4.02 mmol, 153 mg) in DMF (5 mL) for
5 mins at 0 °C. To this was added slowly at the same temperature O-
tosyl serine 2 (1.34 mmol, 500 mg) in DMF (5 mL). Allowed the reaction
mixture to attain room temperature (27 °C) and continued stirring until the
reaction was completed. Excess of dil. HCl is added to the reaction
mixture and extract the compound using ethyl acetate (3 × 15 mL).
Combined organic layer was washed with dil. HCl (3 × 15 mL) followed
by brine solution (3 × 15 mL). The organic layer was dried over Na2SO4
and evaporated. The crude product was purified over silica gel of mesh
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10.1002/chem.201901363
Chemistry - A European Journal
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The reactivity of cysteine and
Kishorkumar M. Reddy
and Govindasamy
Mugesh*
selenocysteine
towards
derivatives
used
commonly
selenoenyzme inhibitors is
reported. The Se-species
eliminated in the reactions
exhibit GPx-like redox activity,
suggesting that such redox
Page No. – Page No.
Modelling the
Inhibition of
Selenoproteins by
Small Molecules using
Cysteine and
Selenocysteine
Derivatives
modulatory
activity
of
selenium compounds may
have significant effect on the
cellular redox state during the
inhibition of selenoproteins.
This article is protected by copyright. All rights reserved.