Modeling Thioredoxin Reductase-Like Activity with Cyclic Selenenyl Sulfides: Participation of an NH???Se Hydrogen Bond through Stabilization of the Mixed Se?S Intermediate

Article abstract of DOI:10.1002/chem.201902230

At the redox-active center of thioredoxin reductase (TrxR), a selenenyl sulfide (Se?S) bond is formed between Cys497 and Sec498, which is activated into the thiolselenolate state ([SH,Se^?]) by reacting with a nearby dithiol motif ([SH^Cys59,SH^Cys64]) present in the other subunit. This process is achieved through two reversible steps: an attack of a cysteinyl thiol of Cys59 at the Se atom of the Se?S bond and a subsequent attack of a remaining thiol at the S atom of the generated mixed Se?S intermediate. However, it is not clear how the kinetically unfavorable second step progresses smoothly in the catalytic cycle. A model study that used synthetic selenenyl sulfides, which mimic the active site structure of human TrxR comprising Cys497, Sec498, and His472, suggested that His472 can play a key role by forming a hydrogen bond with the Se atom of the mixed Se?S intermediate to facilitate the second step. In addition, the selenenyl sulfides exhibited a defensive ability against H₂O₂-induced oxidative stress in cultured cells, which suggests the possibility for medicinal applications to control the redox balance in cells.

Full text of DOI:10.1002/chem.201902230

A Journal of
Accepted Article
Title: Modeling Thioredoxin Reductase-like Activity Using Cyclic
Selenenyl Sulfides: Participation of an NH•••Se Hydrogen Bond
through Stabilization of the Mixed Se–S Intermediate
Authors: Kenta Arai, Takahiko Matsunaga, Haruhito Ueno, Nozomi
Akahoshi, Yuumi Sato, Gaurango Chakrabarty, Govindasamy
Mugesh, and Michio Iwaoka
This manuscript has been accepted after peer review and appears as an
Accepted Article online prior to editing, proofing, and formal publication
of the final Version of Record (VoR). This work is currently citable by
using the Digital Object Identifier (DOI) given below. The VoR will be
published online in Early View as soon as possible and may be different
to this Accepted Article as a result of editing. Readers should obtain
the VoR from the journal website shown below when it is published
to ensure accuracy of information. The authors are responsible for the
content of this Accepted Article.
To be cited as: Chem. Eur. J. 10.1002/chem.201902230
Link to VoR: http://dx.doi.org/10.1002/chem.201902230
Supported by

Chemistry - A European Journal
10.1002/chem.201902230
FULL PAPER
Modeling Thioredoxin Reductase-like Activity Using Cyclic
Selenenyl Sulfides: Participation of an NH•••Se Hydrogen Bond
through Stabilization of the Mixed Se–S Intermediate
[
a]
[a]
[a]
[a]
[a]
Kenta Arai, Takahiko Matsunaga, Haruhito Ueno, Nozomi Akahoshi, Yuumi Sato, Gaurango
[b]
[b]
[a]
Chakrabarty, Govindasamy Mugesh,* Michio Iwaoka*
Abstract: At the redox active center of thioredoxin reductase (TrxR),
His472 and Glu477 together with Sec498 residues make a
catalytic triad structure, contributing to smooth deprotonation of
a selenenyl sulfide (Se-S) bond is formed between Cys497 and
-
-
Sec498, which is activated into the thiolselenolate state ([SH,Se ]) by
the selenol (SeH) and stabilization of the generated Se in the
Cys59
Cys64
activated state.[6] Existence of His472 side chain close to
Sec498 was indeed observed by Arnér and coworkers in the
reacting with a nearby dithiol motif ([SH
,SH
]) present in the
other subunit. This process completes through two reversible steps,
i.e., an attack of a cysteinyl thiol of Cys59 at the Se atom of the Se-S
bond and a subsequent attack of a remaining thiol at the S atom of
the generated mixed Se-S intermediate. However, it is not clear how
the kinetically unfavorable second step progresses smoothly in the
catalytic cycle. Model study using synthetic selenenyl sulfides, which
mimic the active site structure of human TrxR comprising Cys497,
Sec498, and His472, suggested that His472 can play a key role by
forming a hydrogen bond to the Se atom of the mixed Se-S
intermediate to facilitate the second step. In addition, the selenenyl
[
7]
crystal structure of the resting state of rat TrxR .
Activated state
2 2
sulfides exhibited a defensive ability against H O -induced oxidative
stress in cultured cells, suggesting the possibility for medicinal
applications to control redox balance in cells.
Resting state
Introduction
Thioredoxin reductase (TrxR),
a homodimeric flavoprotein
having a selenocysteine residue (Sec, U) at the active site,
crucially contributes to regulation of the redox state of
thioredoxin (Trx), which is involved in various cellular functions,
such as proliferation, antioxidative defense, and protein
Activated state
[
1]
folding/unfolding. TrxR reduces the disulfide (S-S) bond of
oxidized Trx to the corresponding dithiol state ([SH,SH]) through
a NADPH-dependent electron transfer pathway (Figure 1a).[
2]
-
The highly reactive selenolate (Se ) in the activated state
ox
reduces not only Trx but also a variety of substrates (Sub ),
[
2a,3]
such as hydrogen peroxide (H
(
2
2
O ),
lipid hydroperoxide
Resting state
LOOH),[4] and dehydroascorbic acid (DHAA), to the reduced
[5]
states (i.e., Subred). Thus, TrxR plays roles in maintenance of
redox homeostasis in cells. Wessjohan and coworkers
previously proposed that at the active site of human TrxR,
Figure 1. (a) Enzymatic activity of human TrxR and the proposed catalytic
triad Sec•••His•••Glu. (b) TrxR-like catalytic reaction of a cyclic selenenyl
red
sulfide driven by
a dithiol activator, such as dithiothreitol (DTT ) or
[
a]
Dr. K. Arai, T. Matsunaga, H. Ueno, N. Akahoshi, Y. Sato, Prof. Dr.
M. Iwaoka
dihydrolipoic acid (DHLA).
Department of Chemistry, School of Science, Tokai University
Kitalaname, Hiratsuka-shi, Kanagawa 259-1292, Japan
E-mail: miwaoka@tokai.ac.jp
Dr. G. Chakrabarty, Prof. Dr. G. Mugesh
Department of Inorganic and Physical Chemistry
Indian Institute of Science, Bangalore-560012, India
E-mail: mugesh@ipc.iisc.ernet.in
Unique biological functions of selenoenzymes, such as
[
8]
[
b]
glutathione peroxidase (GPx) and iodothyronine deiodinase
ID),[9] have frequently been focused in biomimetic studies. In
(
this context, design of TrxR mimics would also be of interest in
two points: (1) elucidation of a detailed molecular mechanism for
the enzymatic activity and (2) development of novel drugs for
Supporting information for this article is given via a link at the end of
the document.
This article is protected by copyright. All rights reserved.

Chemistry - A European Journal
10.1002/chem.201902230
FULL PAPER
controlling the cellular redox balance. However, only a few TrxR
mimics have been reported. Dawson et al.[10] previously
demonstrated that seleno-glutaredoxin 3, which has an amino
acid sequence of C-X-X-U (or U-X-X-C) in place of C-X-X-C at
the redox active site of glutaredoxin 3, behaves like TrxR in
reduction of oxidized Trx. Sarma and Mugesh[11] proposed,
based on the model study using aromatic selenenyl sulfides
having a tertiary amino group, that the direct interaction between
the basic amino group and the sulfur atom of the active-site
selenenyl sulfide (Se-S) bond facilitates a nucleophilic attack of
a thiol at the intrinsically less electrophilic S atom, rather than at
was regioselectively substituted by an acetylthio group.
Produced 6 was subsequently converted into 7 by reacting with
p-methoxybenzyl diselenide ((PMBSe)
2
) in the presence of
NaBH and saponification of the acetylthio group. Applying a
4
similar protocol, 9 having the SH and SePMB functional groups
in the inverted positions was prepared. Compounds 7 and 9
were subsequently treated with I to obtain 10 and 11, which
2
were further converted to 2a and 3a, respectively, by treatment
with HCl. Identities and purities of the obtained compounds were
confirmed by NMR and reverse-phase (RP) HPLC analyses. In
addition, the relative positions of the S and Se atoms were
unambiguously determined by X-ray analysis for the synthetic
intermediate 11 as shown in Figure 3. Acetamides 2b and 3b
were obtained by reacting 2a and 3a with acetyl chloride.
Acetylation was also performed for diselenide 1a and disulfide
4a to give 1b and 4b, respectively.
-
the Se atom, generating the desired active selenolate (Se ).
Furthermore, it is well established that in the resting state of
TrxR a thiol of Cys59 of the dithiol motif ([SHCys59,SHCys64]) in
one subunit predominantly attacks at the more electrophilic Se
atom of the Se-S bond located in the other subunit, transiently
forming a mixed Se-S intermediate[7,12]. A subsequent attack of a
remaining thiol of Cys64 at the S atom of the mixed Se-S bond
-
would produce an active thiolselenolate state ([SH,Se ]).
However, it is not clear how the kinetically unfavorable second
step, i.e., a nucleophilic attack at the S atom rather than at the
Se atom, progresses smoothly in the catalytic cycle.
To imitate the enzymatic activity of TrxR and further provide
mechanistic information about the TrxR catalytic cycle, we
herein utilize redox reactions of small cyclic selenenyl sulfides
having an amino substituent, 2a and 3a (Figure 2), which
models the topology of the TrxR-active center including Cys497,
Sec498, and His472, in the presence of a dithiol activator as
illustrated in Figure 1b. In the catalytic cycle, [Se–S] (a resting
state) is activated by a dithiol, such as dithiothreitol (DTTred) or
dihydrolipoic acid (DHLA), to the corresponding open-chain
Scheme 1. Synthetic routes for cyclic selenenyl sulfides 2ab and 3ab.
-
thiolselenolate state ([SH,Se ], I), which should subsequently
ox
reduce various substrates (Sub ), such as a disulfide (S-S)-
containing protein and a hydroperoxide. Diselenide 1a, which
behaves like ER-resident oxidoreductases,[13] and disulfide 4a as
well as acetylated 1b–4b are employed as reference
compounds in order to elucidate importance of the interaction
between the Se-S unit and a nearby basic amino group in the
TrxR-like activity. Kinetics of the redox reactions of 2ab and 3ab
and their defensive capacities against an oxidative stress in
cultured cells are also investigated to gain an insight of the
catalytic mechanism and the possibility for pharmaceutic
applications.
C22
C7
O16
Se1
S3
C21
C24
C15
N12
C6
C5
C4
O29
C23
Figure 3. X-ray crystal structure of 11. Thermal ellipsoids of 50% probability
are presented.
The ability of 2a and 3a as a TrxR-like catalyst was evaluated by
the S-S reduction assay using bovine pancreatic insulin (BPIns)
ox
as a substrate (Sub ). BPIns, which has two S-S bonds
Figure 2. Cyclic diselenide (1), selenenyl sulfide (2 and 3), and disulfide (4)
compounds employed in this study.
A7
B7
A20
B19
between the A- and B-chains (Cys ‒Cys , Cys ‒Cys ) in
addition to one S-S bond in the A-chain (CysA6‒CysA11), is
frequently employed as a substrate to investigate enzymatic
activities of disulfide-reductases.[14] The reaction was carried out
Results and Discussion
red
in the presence of a catalytic amount of 1–4 by using DTT as a
Selenenyl sulfides 2a and 3a were prepared according to
Scheme 1. First, one of the two mesyloxy groups of 5 (i.e., R )
dithiol activator. Reductive cleavage of the disulfide bonds of
native insulin (N) was monitored by RP HPLC analysis (Figure
2
This article is protected by copyright. All rights reserved.

Chemistry - A European Journal
10.1002/chem.201902230
FULL PAPER
S1). The times required for 50% conversion of N (τ50) (see
Figure 4a and Table S1) clearly showed that the reduction rate
for N was significantly increased in the presence of 2 and 3. In
addition, acetylation of the amino group did not affect the
capacity as a disulfide reductase-like catalyst although the
catalytic ability of 1a was slightly enhanced by acetylation. A
similar trend was observed when DHLA, a small molecule dithiol
existing in living cells, was used as a dithiol activator instead of
DTTred (Figure S2).
catalytic reduction of H
to the results from the experiment using insulin as a substrate
(Figure 4a).
When GSH was used as a monothiol activator, 2a and 3a
exhibited a lower catalytic ability than diselenide 1a (Figure 5b
and Table S1), indicating that monothiol GSH is not suitable as
an activator for cyclic selenenyl sulfides. It is interesting to note
that N-acetylation again decreased the capacity of 1a, 2a, and
3a as an antioxidant catalyst.
2
O
2
. This observation is in sharp contrast
(
a)
(a)
(b)
(
b)
No catalyst
4a
0
.14
0
.00
0
.0
1
00
No catalyst
4a, 4b
0.12
0.10
0.08
0.06
0.04
0.02
No cat.
1a
2b
8
0
0
‒0.01
‒0.02
3b
3a
–
0.2
4b
2a
2
1
3
b
b
a
6
3b
1b
2
a
b
1
a, 4a, 4b
2
–0.4
–0.6
–0.8
4
0
0
0
3a
3b
1
b
4a
‒
0.03
4b
1a
2a
2
2
a, 3a, 2b, 3b
1a
1b
No catalyst
‒
0.04
0.00
0
30
60
90
0
200
400
600
0
100
200
300
400
0
600
2
00
400
Reaction time (min)
Reaction time (sec)
Reaction time (sec)
Reaction time (sec)
Figure 4. Catalytic S-S reduction of BPIns in the presence of DTTred (a) or
GSH (b) as a thiol activator. (a) Population changes of native BPIns (N). The
2 2
Figure 5. Catalytic reduction of H O
using DTTred (a) or GSH (b) as a thiol
activator in the presence or absence of 1–4 as a catalyst. (a) UV absorbance
reaction conditions were [BPIns]
0 μM, at 25 °C and pH 7.5. (b) UV absorbance changes at 340 nm due to the
consumption of NADPH. Reaction conditions were [BPIns] = 60 μM, [GSH]
= 0.12 mM, [GR] = 4 unit mL , and [catalyst] = 45 μM at
0 0
= 100 μM, [DTT] = 300 μM, and [catalyst] =
ox
changes at 310 nm due to the generation of DTT . Reaction conditions were
3
red
[
DTT ] = 2.0 mM, [H O ] = 2.0 mM, and [Catalyst] = 0.2 mM at pH 7.5 and
0 2 2 0
0
0
=
-
1
25 °C. (b) UV absorbance changes at 340 nm due to the consumption of
NADPH. Reaction conditions were [GSH] = 4.0 mM, [NADPH] = 0.3 mM,
= 0.25 mM, [GR] = 4 unit mL , and [catalyst] = 0.1 mM at pH 7.0 and
3.7 mM, [NADPH]
0
0
0
pH 7.5 and 25 °C
-
1
2 2 0
[H O ]
[
15]
25 °C. The protocol was followed by literature method.
Catalytic activities of selenenyl sulfides 2ab and 3ab
investigated by using glutathione (GSH) instead of DTTred
showed a different behavior. The reaction was monitored by the
decrease of UV absorption at 340 nm due to consumption of
NADPH (Figure 4b), which was added with glutathione
reductase (GR) to reduce oxidized glutathione (GSSG).[13] The
initial velocities (v Ins
Figure 4b and Table S1) showed that selenenyl sulfides 2ab
) for the reduction of insulin S-S bonds
0
(
and 3ab less enhanced the velocity than diselenides 1ab, and
also that disulfide analogues 4ab are essentially inactive for this
reaction. It is of note that the catalytic activities of diselenides
Working Activation
1ab and selenenyl sulfides 2ab and 3ab were reverse in order
from those observed when dithiol activators were used (Figure
4a).
TrxR intrinsically possesses hydroperoxidase activity too.[2a,3,4]
Hence, antioxidative activity of 1–4 against
representative source of oxidative stress, was next investigated
by using DTTred as an activator. The velocities of H
reduction
H
2
O
2
,
a
2
O
2
Figure 6. A catalytic cycle proposed for the TrxR-like activity of a cyclic
selenenyl sulfide using a dithiol activator through a mixed Se-S intermediate I′
and an open-chain selenolate intermediate I.
were evaluated by monitoring UV absorbance change at 310 nm
ox
due to formation of oxidized DTT (DTT ) (Figure 5a). The initial
dithiol
reaction velocities (v
0
) showed that 2a had a greater catalytic
activity than 1a and also that 4ab did not promote the reaction.
Similarly, when DHLA was used as a dithiol activator instead of
DTTred, the initial velocity of the reaction in the presence of 2a
was higher than that in the presence of 1a (Table S1).
Furthermore, acetylation of the amino group in 1a, 2a, and 3a
significantly decreased their hydroperoxidase-like ability,
suggesting that the amino group should play a role in the
In the catalytic reactions demonstrated above, cyclic selenenyl
sulfides (2 and 3) should be reduced by a dithiol activator to
active selenolate species I (Figure 1b). This process would be
achieved by two reversible steps (Figure 6). In the first step, a
dithiol preferentially attacks at the intrinsically more electrophilic
Se atom of the Se-S bond of 2 or 3, providing mixed Se-S
This article is protected by copyright. All rights reserved.

Chemistry - A European Journal
10.1002/chem.201902230
FULL PAPER
ox
intermediate I′, which is essentially inactive against Sub . In this
step, the attack on the S atom should be unfavorable unless the
S atom coordinates with a heteroatom, such as N or O,
intramolecularly.[16] In the second step, the other thiol group of
the reacted dithiol attacks to the S atom of the Se-S bond of I′ to
produce I liberating a disulfide. Since I′ was not observed in the
Similarly, the apparent reduction velocity for acetylated 2b was
slightly higher than 3b. Interestingly, the order of the apparent
velocities, i.e., 2a > 3a and 2b > 3b, matched with that of the
catalytic activities observed in Figure 5a. This suggested that the
activation process of Se-Sox becomes a rate-determining step in
2 2
the catalytic cycle when H O is employed as a substrate. On
7
7
Se NMR and ESI(+)-TOF-MS spectra for the reaction mixture
the other hand, the trends of the apparent velocities did not
influence the catalytic activities observed for insulin substrate
(Figure 4a), suggesting the rate-determining step of the catalytic
cycle to be assigned to the working process in this case (i.e.
of 2a and DTTred (Figures S3 and S4), this intermediate should
be transiently formed and smoothly converted to I through an
intramolecular nucleophilic attack of a thiol at the S atom of the
Se-S bond. When a monothiol is used as an activator, on the
other hand, the reverse reaction of the first step would be more
dominant than the second bimolecular process, thereby resulting
in the lower catalytic activities. In the case of diselenides 1ab,
ox
red
Sub → Sub ).
Two parameters controlling the reaction rate of eq 1 are
ox
considerable for Se-S . One is the electrophilicity of the Se
atom, which relates to the ability to accept electron donation
from a thiol. However, ab initio calculations for 2a and 3a in
water showed that in the most stable conformation there is no
interaction between the Se-S bond and amino group and the
LUMO energy levels are almost comparable with each other
(see supporting information). The other is the presence of an
intramolecular NH•••Se hydrogen bond for transient intermediate
I′ as shown in Figure 8a, which would suppress the reverse
reaction to Se-Sox and promote the nucleophilic attack of a
second thiol at the S atom to produce I. Iwaoka and coworkers
previously proposed that a similar NH•••Se hydrogen bond in a
selenenyl sulfide species can accelerate a cleavage of the Se-S
bond during the GPx-like catalytic cycle of diselenide
-
however, the Se species is formed by the first attack of the thiol
to the diselenide (Se-Se) bond.[13] Thus, the diselenide
compounds would have exhibited a greater catalytic activity than
2
and 3.
As mentioned above, the effects of the N-acetylation on the
catalytic activities were significantly different between the two
substrates, i.e., insulin S-S bonds and H . This would arise
from a shift of the rate-determining step in the catalytic cycle
from the working process to the activation process (Figure 6). To
confirm this hypothesis, thermodynamic and kinetic analyses for
the redox reaction (eq 1) were carried out.
2
O
2
18]
[19]
…
(1)
selenoglutathione (GSeSeG)[
and dipeptide Sec-Cys.
Ab
initio calculation at HF/6-31++G(d,p) in water revealed that the
most stable conformation has a NH•••Se hydrogen bond for both
selenenyl sulfides 12 and 13 as shown in Figure 8a. The relative
stabilities of 12 and 13 were comparable with each other (∆E = +
0.70 kcal mol-1 for 12).
Here, Se-Sox and Se-Sred are 2a, 3a, 2b, or 3b and the
corresponding reduced form (i.e., I in Figure 6), respectively.
The concentration of DTTox in the reaction mixture, which was
estimated from the observed peak area on the HPLC chart
(
Figure S5), is plotted against the reaction time in Figure 7.
(a)
(b)
1
1
1
4
2
0
8
6
4
2
0
2
a
3
a
1
0
9
8
2a
2b 7
3
a
6
5
4
2b
Figure 8. (a) Intramolecular NH•••Se hydrogen bond of mixed Se-S
intermediates 12 and 13 derived from 2a and 3a, respectively. (b) Putative
NH•••Se hydrogen bond formed in a mixed Se-S intermediate of TrxR.
3b 3
3
b
2
1
0
0
15 30 45 60
Figure 7 also showed that the redox reaction (eq. 1) reaches an
equilibrium within 6 h. From the obtained equilibrium constants
(K), the redox potentials (E°′) were determined for 2a (‒342 mV),
0
60 120 180 240 300 360
Reaction time (min)
2b (‒362 mV), 3a (‒344 mV), and 3b (‒366 mV). The results
Figure 7. The concentration of DTTox during the reduction of selenenyl
revealed that acetylation of the amino group decreases the
redox potentials by ca. 20 mV, indicating that 2a and 3a having
a free amino group are more readily reduced not only by a
kinetic factor but also by a thermodynamic factor, which would
be assessed to stabilization of intermediate I via intramolecular
NH•••Se hydrogen bond. Indeed, ab initio calculation in water
supported the presence of such a hydrogen bond for I (see
red
ox
= 30 μM and [DTTred
sulfides by DTT . Reaction conditions were [Se-S
0 μM at pH 7.0 and 25 °C under argon atmosphere.
] ] =
0 0
3
Comparison of the apparent velocity of DTTox (or Se-Sred
)
generation (Table S2) clearly showed that selenenyl sulfide 2a is
more promptly reduced by DTTred than the regioisomer (3a).
This article is protected by copyright. All rights reserved.

Chemistry - A European Journal
10.1002/chem.201902230
FULL PAPER
supporting information). However, it should be noted that this
thermodynamic effect cannot fully explain the catalytic activities
observed in Figure 5 as the E°′ values are almost comparable
with each other for 2a and 3a while the activity for 2a was
obviously higher than that for the corresponding regioisomer 3a.
A recent literature indeed suggested that the reactivities of the
Se-S motifs in selenoenzymes must be considered not only from
the redox potentials but also from structures and flexibilities of
the motifs.[20]
4
3
3
0
5
0
**
*
*
25
≈
0
Previous studies assumed that roles of His472 at the active
2a
14
15
16
2a
+ His
site of TrxR include deprotonation of the SeH group in the
activated [SH,SeH] state to stabilize the reactive Se- via
Compound
formation of a hydrogen bond (Figure 1a)[6] and/or proton
acceptance from the dithiol motif of [SHCys59,SHCys64] to activate
the thiol.[7] On the other hand, our model study here suggested
2 2
Figure 9. Comparison of the initial velocities for catalytic reduction of H O
red
using DTT between the catalysts 2a and its amino acid conjugates (14–16).
another possible role that His472 interacts with the Se atom in
the mixed Se-S intermediate via formation of an NH•••Se
hydrogen bond as shown in Figure 8b, thereby accelerating the
catalytic cycle of TrxR.
red
Reaction conditions were [DTT
0.2 mM, and [His] = 0 or 0.2 mM at pH 7.5 and 25 °C. ** represents P<0.01, P
value was obtained by Tukey's post-hoc test.
0 2 2 0
] = 2.0 mM, [H O ] = 2.0 mM, [Catalyst] =
A small molecule TrxR-model would have another noteworthy
advantage as a potential drug in addition to their applicability as
a mechanistic probe for the enzymatic reaction. To demonstrate
a possibility of selenenyl sulfides for pharmaceutical applications,
2 2
the capacity of 2ab and 3ab to tackle against H O -induced
oxidative stress was investigated using HeLa cell as a model
human cell. When the cells were preincubated with 2ab or 3ab
before inducing the oxidative stress, the cell viabilities estimated
by 3-(4,5-di-methylthiazol-2-yl)-2,5-diphenyltetrazolium bromide
(
MTT) assay were obviously higher than those preincubated with
vehicle, diselenides 1ab, disulfides 4ab, or ebselen,[23]
a
Scheme 2. Preparation of the amino acid-conjugates (14–16) of 2a.
representative organoselenium antioxidant (Figure 10a). The
result suggested that cyclic selenenyl sulfides have antioxidative
properties at the applied concentrations (~20 μM), at which the
cytotoxicity of the selenenyl sulfides is negligible (Figure 10b).
The mechanisms of the antioxidative activities would be
explained by two scenarios. One is the direct contribution to
antioxidation. Namely, the selenenyl sulfides would regulate a
redox balance through the TrxR-like catalytic cycle as shown in
Figure 6 in the cultured cells, where dithiol activators, such as
DHLA, exist. The other is the indirect contribution to the increase
in levels of antioxidant enzymes. Indeed, it is known that several
organoselenium compounds work as a “thiol modifier”, which
can oxidize the cysteinyl thiols of Keap1 to stimulate a signaling
pathway for the cellular adaptive responses to the oxidative
stress.[24] At this moment, details of observed antioxidative
functions of 2ab and 3ab are largely left for future elucidation.
To support the importance of hydrogen bond formation
between Se and a His side chain in intermediate I’ (Figure 8b),
amino acid conjugates of 2a (14–16) were prepared following
Scheme 2, and their TrxR-like activities were investigated by
means of DTT-assay under the same conditions as Figure 5a.
Comparison of the v dithiol
for the generation of DTTox showed
0
that by conjugation of His (i.e., for 14) the activity is slightly
enhanced whereas Ser or Ala conjugation (i.e., 15 and 16,
respectively) decreases the activity (Figure 9). The result was
consistent with our model that His472 would participate in
stabilization of the mixed Se–S intermediate via NH•••Se
hydrogen bond formation. Indeed, ab initio calculation in water
supported possible formation of NH•••Se hydrogen bond in the
mixed Se–S intermediate derived from 14 (see supporting
information). Interestingly, Ser did not positively affect the
activity despite a potential of the side-chain OH group to form
hydrogen bond with Se.[21] This behavior is consistent with the
previous observation that mutation of the flanking Gly residues
to Ser at the active site of mammalian TrxR (i.e., Gly-Cys-Sec-
Gly) did not affect the enzymatic activity.[22]
This article is protected by copyright. All rights reserved.

Chemistry - A European Journal
10.1002/chem.201902230
FULL PAPER
T100LP mass spectrometer under atmospheric pressure chemical
ionization (APCI+) or electrospray ionization (ESI+) conditions. All
reactions for the synthesis were monitored by thin-layer chromatography
(TLC), which was performed on precoated sheets of silica gel 60
purchased from Merck Millipore. Gel permeation chromatography (GPC)
(a)
1
00
*
8
0
0
**
*
*
*
*
*
*
*
*
6
3
was performed with a JAI LC-918 HPLC system using CHCl as an
4
0
eluent. RP HPLC analyses for reductive unfolding of insulin and
measurement of the redox potentials were performed with a SHIMADZU
HPLC Prominence Series equipped with a 1 mL sample solution loop
and a Tosoh TSKgel ODS-100V reverse-phase column (φ4.6 × 150 mm).
Mesylate 5 was prepared by following the literature.[25] Compounds 1a,[13]
2
0
0
1
0
20 10 20 10 20 10 20 10 20 10 20 10 20 10 20 10 20
a
2a 3a
4a
1b
2b 3b
4b ebselen
1
4
a,[13] and 2-aminoethyl methanethiosulfonate (AEMTS)[26] was
Compound (μM)
(b)
synthesized according to previous procedures. Bovine pancreatic insulin
(BPIns) was purchased from Sigma-Aldrich Japan. The concentration of
BPIns was determined by using a molar extinction coefficient at 278 nm
of 6,090 cm-1 M . All buffer solutions were fully purged with argon gas by
bubbling over 30 min. All other chemicals were used as purchased
without further purification.
1
20
1
00
-1
8
0
0
6
4
0
2
0
(S)-S-(3-((tert-Butoxycarbonyl)amino)-4-((methylsulfonyl)oxy)butyl)
ethanethioate (6)
0
10 20 50 10 20 50 10 20 50 10 20 50 10 20 50 10 20 50 10 20 50 10 20 50 10 20 50
1
a
2a
3a
4a
1b
2b
3b
4b
ebselen
Compound (μM)
A DMF solution (21 mL) of mesylate 5 (0.300 g, 0.830 mmol) was added
with potassium thioacetate (0.114 g, 0.998 mmol) and subsequently
stirred for 3 h at 60 °C. The resulting solution was evaporated to remove
DMF in vacuo. To the residual yellow material was added water (30 mL)
and extracted with EtOAc (30 mL × 4). The combined organic layers
Figure 10. (a) Cell viability of HeLa cell (1×104 cell/well) cultured with H
2 2
O
0.4 mM) after the treatment in the presence of compounds 1−4 or ebselen (10
or 20 μM) at 37 ˚C and 5% CO for 15 h. Bars are shown as means ±SEM
2
(
(n=3). * represents P<0.05. ** represents P<0.01. P value was obtained by t-
4
were washed with brine (20 mL× 2), dried over MgSO , and concentrated
test. (b) Cytotoxicity of compounds 1–4 and ebselen at different
concentrations determined by MTT assay with HeLa cells (1×10 cell/well)
cultured in the presence of compounds 1−4 or ebselen (10−50 μM) at 37 ˚C
2
and 5% CO for 4 h. Bars are shown as means ±SEM (n=3).
in vacuo. The residual yellow oil was purified by silica gel column
4
chromatography (EtOAc:n-hexane = 1:1) to give a white solid of 6. Yield:
: 0.48 (EtOAc/n-hexane 1:1); 1H NMR
0
.145 g (51%); mp: 80−82 °C; R
f
(
500 MHz, CDCl ): δ 4.76 (br d, J = 8.4 Hz 1H), 4.26−4.10 (m, 2H),
3
3
2
.86−3.84 (m, 1H), 2.98 (s, 3H), 2.97−2.90 (m, 1H), 2.80−2.72 (m, 1H),
.27 (s, 3H), 1.83−1.69 (m, 2H), 1.38 (s, 9H) ppm; 13C NMR (125.8 MHz,
CDCl
ppm; HRMS (APCI-TOF) m/z: [M
42.1040; found, 342.1074.
3
): δ 195.8, 155.3, 80.1, 70.9, 49.0, 37.3, 31.4, 30.6, 28.3, 25.4
Conclusions
+
H]+ calcd for +,
12 6 2
C H24NO S
3
Our model study proposes for the first time that His472, which is
present close to the redox center of TrxR, i.e., [Se–S], would
play an important role by forming a hydrogen bond with the Se
tert-Butyl (S)-(4-mercapto-1-((4-methoxybenzyl)selanyl)butan-2-
yl)carbamate (7)
atom in
a
transient mixed Se-S intermediate during the
-
activation process of [Se–S] to [SH,Se ]. Thus, His472 would be
a player not only as a stabilizer of the selenolate (Se ) form
Sodium borohydride (90%, 17.9 mg, 0.426 mmol) and p-methoxybenzyl
diselenide ((PMBSe) ) (85.2 mg, 0.213 mmol) were placed in a two-
-
[6]
2
and/or a proton acceptor from the proximate dithiol motif of
necked round-bottom flask. After replacement of air to argon gas in the
flask, anhydrous THF/EtOH (2:1, 4.5 mL) was rapidly added via a glass
syringe, and the solution was stirred for 10 min at room temperature. To
the obtained colorless solution, a solution of 6 (0.132 g, 0.387 mmol) in
anhydrous THF (2.5 mL) was then added. The mixture solution was
stirred at 60 °C for 1 h and then concentrated under in vacuo. The
Cys64 [7]
[
SHCys59,SH
]
but also as an accelerator for the activation of
the Se-S bond. Nonetheless, more studies are necessary to
confirm this new role. Furthermore, the synthesized cyclic
selenenyl sulfides (2ab and 3ab) exhibited cytoprotection
against
2 2
H O -induced oxidative stress in cultured cells,
obtained yellow-red solid was dissolved in MeOH/H
the solution was then added with Na CO (0.247 g, 2.33 mmol) and
NaOH (31 mg, 0.776 mmol) and stirred at room temperature for 30 min.
The solution was added with saturated aqueous NH Cl (10 mL), acidified
with 2N H SO to ca. pH 3, and evaporated to remove MeOH. The
resulting liquid was added with water (30 mL) and extracted with EtOAc
15 mL × 3). The combined organic layers were washed with brine (30
mL), dried over MgSO , and concentrated in vacuo. The residual yellow
solid was purified by silica gel column chromatography (EtOAc:n-hexane
1:2) and the recrystallization from cyclohexane to give an white crystal
2
O (6:1, 12 mL), and
suggesting the possibility for pharmaceutical applications to
control redox balance in cells.
2
3
4
2
4
Experimental Section
(
4
General
=
Melting points were measured with a Yanako MP-S3 micro melting-point
system and are uncorrected. 1H (500 MHz), 13C (125.8 MHz), and 77Se
of 7. Yield: 68.0 mg (43%); mp: 98–102 °C; R
f
: 0.51 (EtOAc/n-hexane
1
1
4
2
1
:2); H NMR (500 MHz, CDCl
3
): δ 7.25–7.24 (m, 2H), 6.86–6.83 (m, 2H),
(95.4 MHz) NMR spectra were recorded with a Bruker AV-500
.567 (br d, J = 8.3 Hz 1H), 3.88‒3.87 (m, 1H), 3.81 (s, 3H), 3.79 (s, 2H),
.74–2.64 (m, 4H), 2.00–1.93 (m, 1H), 1.80–1.77 (m, 2H), 1.68 (s, 1H),
.47 (s, 9H) ppm; 13C NMR (125.8 MHz, CDCl
spectrometer at 263 or 298 K. Coupling constants (J) are reported in Hz.
High-resolution mass spectra (HRMS) were recorded with a JEOL JMS-
3
): 158.5, 155.4, 130.9,
This article is protected by copyright. All rights reserved.

Chemistry - A European Journal
10.1002/chem.201902230
FULL PAPER
1
30.0, 114.0, 79.5, 55.3, 49.5, 35.3, 34.7, 29.7, 28.4, 27.4 ppm; 77Se
stirred for 30 min and then concentrated in vacuo. The obtained yellow
material was added with water (10 mL) and extracted with EtOAc (10 mL
× 3). The combined organic layers were washed with brine (10 mL × 2),
NMR (95.4 MHz, CDCl
calcd for C17
3
): δ 205.2 ppm; HRMS (APCI-TOF) m/z: [M + H]+
+
H28NO
3
S80Se , 406.0950; found, 406.0925.
dried over MgSO
was purified by silica gel column chromatography (EtOAc:n-hexane =
:2) and then by GPC to give an off-white solid of 10. Yield: 42.0 mg
98%); mp: 145−148 °C; R
: 0.68 (EtOAc/n-hexane 1:2); 1H NMR (500
): δ 4.91 (br s, 1H), 3.86 (br s, 1H), 3.19−3.10 (m, 2H), 2.93
4
, and concentrated in vacuo. The residual yellow solid
(
S)-2-((tert-Butoxycarbonyl)amino)-4-((4-
1
(
methoxybenzyl)selanyl)butyl methanesulfonate (8)
f
MHz, CDCl
3
2
Sodium borohydride (90%, 32.0 mg, 0.762 mmol) and (PMBSe) (0.102
(
9
2
s, 1H), 2.81−2.71 (m, 1H), 2.20−1.89 (m, 1H), 1.70 (br s, 1H), 1.38 (s,
mg, 0.254 mmol) were placed in a two-necked round-bottom flask. After
replacement of air to argon gas in the flask, anhydrous THF/EtOH (2:1, 3
mL) was rapidly added via a glass syringe, and the solution was stirred
for 10 min at 0 °C. To the obtained colorless solution, a solution of 5
H) ppm; 13C NMR (125.8 MHz, CDCl
): δ 154.6, 79.8, 47.4, 34.9, 32.3,
9.9, 28.4 ppm; HRMS (APCI-TOF) m/z: [M
3
+
H]+ calcd for
C H
9
18NO
2
S80Se , 284.0218; found, 284.0200.
+
(
0.153 g, 0.423 mmol) in anhydrous THF (3 mL) was then added on ice.
tert-Butyl (S)-(1,2-thiaselenan-5-yl)carbamate (11)
The mixture solution was stirred at 50 °C for 20 min and then at room
temperature for 2 h. The resulting solution was concentrated under in
vacuo. The obtained white solid was added with water (20 mL) and
extracted with Et
washed with brine (30 mL), dried over MgSO
A similar protocol to the synthesis of 10. Starting with 9 (0.152 g, 0.376
mmol), 11 was obtained as an off-white solid. Yield: 93.6 mg (88%); mp:
: 0.43 (EtOAc/n-hexane 1:4); 1H NMR (500 MHz, CDCl
113−119 °C; R ):
f 3
2
O (20 mL × 3). The combined organic layers were
4
, and concentrated in vacuo.
The residual white solid was purified by silica gel column
δ 5.09 (br s, 1H), 3.91 (br s, 1H), 3.28–3.20 (m, 2H), 2.99 (m, 1H), 2.87–
2.75 (m, 1H), 2.28–2.24 (m, 1H), 2.05 (br s, 1H), 1.46 (s, 9H) ppm; 13C
NMR (125.8 MHz, CDCl ): δ 154.6, 79.7, 47.3, 37.5, 33.7, 28.4, 22.9
3
chromatography (EtOAc/n-hexane 1:1) to give a white solid of 8. Yield:
0
.104 g (53%); mp: 86−89 °C; R
f
: 0.46 (EtOAc/n-hexane 1:2); 1H NMR
(
500 MHz, CDCl ): δ 7.19–7.09 (m, 2H), 6.80–6.72 (m, 2H), 4.56 (br d, J
3
ppm; 77Se NMR (95.4 MHz, CDCl
3
): δ 379.9 ppm; HRMS (APCI-TOF)
+
S80Se , 284.0218; found, 284.0216.
+
=
3
2
1
7.3 Hz, 1H), 4.20–4.11 (m, 1H), 4.11–4.01 (m, 1H), 3.89–3.78 (m, 1H),
m/z: [M + H] calcd for C
9
H
18NO
2
.72 (s, 3H), 3.69 (s, 2H), 2.94 (s, 3H), 2.47–2.37 (m, 2H), 1.74–1.66 (m,
H), 1.37 (m, 3H) ppm; 13C NMR (125.8 MHz, CDCl
3
): 158.5, 155.3,
(
S)-1,2-Thiaselenan-4-amine hydrochloride (2a)
31.0, 130.0, 114.0, 80.0, 70.8, 55.3, 49.8, 37.3, 32.0, 28.3, 26.8, 19.3
7
7
ppm; Se NMR (95.4 MHz, CDCl
+
3
): δ 255.8. HRMS (APCI-TOF) m/z: [M
Hydrochloric acid (2 mL, 5 M) was added to a solution of 10 (42.0 mg,
.149 mmol) in Et O (2 mL). The mixture was vigorously stirred for 24 h
+
S80Se , 468.0954; found, 468.0929.
+
H] calcd for C18H30NO
6
0
2
at 35 °C. To remove the residual organic impurities, the mixture solution
was washed the dichloromethane (10 mL). The acidic aqueous phase
was made alkaline (≈ pH 11) with 5 M NaOH solution, and the solution
tert-Butyl (S)-(1-mercapto-4-((4-methoxybenzyl)selanyl)butan-2-
yl)carbamate (9)
was extracted with Et
2
O (20 mL × 3). The combined organic layers were
A DMF solution (21 mL) of 8 (0.300 g, 0.643 mmol) was added with
washed with brine (20 mL), dried over MgSO
4
, and concentrated in vacuo.
potassium thioacetate (0.113 g, 0.987 mmol) and subsequently stirred for
The resulting material was dissolved in 10 mM HCl (20 mL), and the
3
h at 60 °C. The resulting solution was evaporated to remove DMF in
vacuo. To the residual material was added water (30 mL) and extracted
with Et O (20 mL × 4). The combined organic layers were washed with
brine (20 mL× 2), dried over MgSO , and concentrated in vacuo. The
obtained yellow oil was dissolved in MeOH/H O (6:1, 14 mL), and the
solution was then added with Na CO (0.41 g, 4.88 mmol) and NaOH
62.5 mg, 1.56 mmol) and stirred at room temperature for 30 min. The
solution was added with saturated aqueous NH Cl (20 mL), acidified with
N H SO to ca. pH 3, and evaporated to remove MeOH. The resulting
liquid was added with water (30 mL) and extracted with EtOAc (15 mL ×
). The combined organic layers were washed with brine (30 mL), dried
over MgSO , and concentrated in vacuo. The residual yellow oil was
purified by silica gel column chromatography (EtOAc:n-hexane = 1:2) to
give a white solid of 9. Yield: 0.208 g (80%); mp: 73−75 °C; R : 0.54
EtOAc/n-hexane 1:2); 1H NMR (500 MHz, CDCl
): δ 7.17–7.10 (m, 2H),
.80–6.71 (m, 2H), 4.54 (br d, J = 7.2 Hz, 1H), 3.72 (s, 3H), 3.69 (s, 2H),
.64–2.51 (m, 2H), 2.43–2.34 (m, 2H), 1.78–1.61 (m, 2H), 1.37 (s, 9H),
.18 (s, 1H) ppm; 13C NMR (125.8 MHz, CDCl
): δ 158.4, 155.4, 131.1,
aqueous solution was lyophilized to give an off-white powder of 2a. Yield:
31.1 mg (96%); mp: 175.0 °C (decomp); 1H NMR (500 MHz, D
O): δ
3.64–3.58 (m, 1H), 3.25–3.20 (m, 1H), 3.09–3.00 (m, 3H), 2.29–2.25 (m,
1H), 1.80–1.73 (m, 1H) ppm; 13C NMR (125.8 MHz, D
O): δ 49.7, 32.7,
32.5, 25.5 ppm; 77Se NMR (95.4 MHz, CD
OD/D O (10:1 v/v), 263 K): δ
3 2
2
2
4
2
2
388.0 ppm; HRMS (ESI-TOF) m/z: [M – Cl]+ calcd for C 10NS80Se+,
2
3
4
H
(
183.9694; found, 183.9677.
4
2
2
4
(
S)-1,2-Thiaselenan-5-amine hydrochloride (3a)
3
A similar protocol to the synthesis of 2a. Starting with 11 (31.4 mg, 0.111
4
mmol), 3a was obtained as an off-white powder solid. 23 mg (95%); mp:
1
1
65.5 °C (decomp); H NMR (500 MHz, D
2
O): δ 3.52–3.48 (m, 1H), 3.16–
f
3
.14 (m, 3H), 3.03–2.91 (m, 1H), 2.47–2.34 (m, 1H), 2.07–2.00 (m, 1H)
(
3
ppm; 13C NMR (125.8 MHz, D
O): δ 49.9, 34.0, 32.0, 23.7 ppm; 77Se
2
6
2
1
1
NMR (95.4 MHz, CD
3
OD/D
2
O (10:1 v/v), 263 K): δ 380.4 ppm; HRMS
+
(
APCI-TOF) m/z: [M – Cl]+ calcd for C
83.9724.
4
H
10NS80Se , 183.9694; found,
3
1
29.9, 114.0, 79.6, 55.3, 51.8, 33.9, 29.4, 28.4, 26.7, 19.6 ppm; 77Se
NMR (95.4 MHz, CDCl
calcd for C17
3
): δ 259.2 ppm; HRMS (APCI-TOF) m/z: [M + H]+
S80Se , 406.0950; found, 406.0945.
General procedure for preparation of acetylated compounds 1b–4b
+
H
28NO
3
1
a, 2a, 3a, or 4a was suspended in CHCl (2 mL) in a round-bottom flask.
3
tert-Butyl (S)-(1,2-thiaselenan-4-yl)carbamate (10)
Triethylamine (2 equiv.) was added to the solution cooled on ice. To the
mixture solution was slowly added a solution of acetyl chloride (1.2
Starting material 7 (61.0 mg, 0.151 mmol) was placed in a round-bottom
equiv.) in CHCl
was raised to room temperature, and the reaction solution was further
stirred for 5 h. After addition of CHCl (15 mL), the organic layer was
washed with saturated aqueous NH Cl (20 mL×1), saturated aqueous
4
NaHCO (20 mL×1), and brine (20 mL×1), then dried over MgSO ,
3
(2 mL). After stirring for 10 min at 0 °C, the temperature
flask and dissolved in MeOH/H
with finely crushed I (57.5 mg, 0.227 mmol) and stirred at room
temperature for 45 min. The reaction was quenched by addition of
Na (26 mg, 0.151 mmol) as a reductant. The mixture was further
2
O (95:5, 20 mL). The solution was added
2
3
4
2 2 4
S O
3
This article is protected by copyright. All rights reserved.

Chemistry - A European Journal
10.1002/chem.201902230
FULL PAPER
filtrated, and concentrated under vacuum. The residual material of crude
products was purified by silica gel column chromatography
(S)-2-Amino-3-(1H-imidazol-5-yl)-N-((S)-1,2-thiaselenan-4-
yl)propanamide di-TFA salt (14)
(
EtOAc:MeOH = 8:1). The corrected fractions that contained the target
compound were combined and concentrated to give a solid of 1b or 2b or
b or 4b.
Using Boc-L-His(Boc) (73.7 mg), 14 was obtained as a white powder.
3
Yield: 33.7 mg (90%); 1H NMR (500 MHz, D
1
2
O): δ 8.60 (s, 1H), 7.30 (m,
H), 4.07 (t, J = 7.7 Hz, 1H), 3.96–3.92 (m, 1H), 3.28–3.19 (m, 2H),
3.13–3.08 (m, 1H), 2.94 (t, J = 11.5 Hz, 1H), 2.79–2.63 (m, 2H), 2.04–
.00 (m, 1H), 2.04–2.00 (m, 1H), 1.58 (q, J = 10.8 Hz, 1H) ppm; 13C NMR
(
S)-N-(1,2-Diselenan-4-yl)acetamide (1b)
2
(
2
125.8 MHz, D O): δ 166.4, 134.2, 126.0, 118.3, 52.1, 48.0, 33.5, 32.6,
Starting with 1a (60.0 mg, 0.226 mmol), 1b was obtained as a yellow
27.3, 26.0 ppm; HRMS (ESI-TOF) m/z: [M+H-2TFA]+ calcd for
1
solid. Yield: 58.3 mg (95%); mp: 151–157 °C; H NMR (500 MHz, CDCl
3
):
+
C
10
H
17
N
4
OSSe , 321.0283; found, 321.0251.
δ 6.06 (br s, 1H), 4.24–4.14 (m, 1H), 3.30–3.20 (m, 2H), 2.97–2.77 (m,
H), 2.21–2.01 (m, 1H), 2.04–1.97 (m, 1H), 1.96 (s, 3H) ppm; 13C NMR
125.8 MHz, CDCl
): δ 169.0, 45.6, 34.2, 28.0, 23.5, 21.3 ppm; 77Se
NMR (95.4 MHz, CDCl
): δ 291.4 ppm; HRMS (APCI-TOF) m/z: [M + H]+
calcd for C
12NO80Se +, 273.9244; found, 273.9275.
2
(
3
(2S)-2-amino-3-hydroxy-N-(1,2-thiaselenan-4-yl)propanamide
salt (15)
TFA
3
6
H
2
t
Using Boc-L-Ser(O Bu) (35.9 mg), 15 was obtained as a white powder.
(
S)-N-(1,2-Thiaselenan-4-yl)acetamide (2b)
Yield: 26.3 mg (92%); 1H NMR (500 MHz, D
2
O): δ 4.01–3.96 (m, 1H),
.93 (t, J = 5.1 Hz, 1H), 3.86 –3.76 (m, 2H), 3.16 –3.11 (m, 1H), 2.97 (t, J
11.6 Hz, 1H), 2.91–2.82 (m, 2H), 2.07–2.04 (m, 1H), 1.63 (t, J = 11.2,
O): δ 166.2, 60.2, 54.4, 48.0, 33.7,
2.9, 27.7 ppm; HRMS (ESI-TOF) m/z: [M+H-TFA]+ calcd for
3
=
Starting with 2a (40.0 mg, 0.183 mmol), 2b was obtained as a white solid.
Yield: 39.8 mg (97%); mp: 171–173 °C; 1H NMR (500 MHz, CDCl
): δ
.15 (br s, 1H), 4.30–4.24 (m, 1H), 3.29–3.17 (m, 2H), 3.00–2.79 (m, 2H),
1H) ppm; 13C NMR (125.8 MHz, D
2
3
3
C
6
2
+
.19–2.12 (m, 1H), 2.02 (s, 3H), 1.86 (br s, 3H) ppm; 13C NMR (125.8
): δ 169.1, 45.6, 34.2, 31.7, 29.4, 23.4 ppm; HRMS (ESI-
7 15 2 2
H N O SSe , 271.0014; found, 271.0037.
MHz, CDCl
TOF) m/z: [M + H] calcd for C
3
+
12NOS80Se , 225.9799; found, 225.9779.
+
6
H
(S)-2-amino-N-((S)-1,2-thiaselenan-4-yl)propanamide TFA salt (16)
(
S)-N-(1,2-Thiaselenan-5-yl)acetamide (3b)
Using Boc-L-Ala (26.0 mg), 16 was obtained as a white powder. Yield:
1
2
21.9 mg (87%); H NMR (500 MHz, D O): δ 3.98–3.92 (m, 1H), 3.87 (q, J
=
7.1 Hz, 1H), 3.15–3.11 (m, 1H), 2.98 (t, J = 11.9 Hz, 1H), 2.90–2.82 (m,
Starting with 3a (56.1 mg, 0.257 mmol), 3b was obtained as a white solid.
Yield: 49.2 mg (85%); mp: 165–166 °C; 1H NMR (500 MHz, CDCl
): δ
1H), 2.07–2.03 (m, 1H), 1.61 (q, J = 11.0 Hz, 1H), 1.36 (d, J = 7.1 Hz,
3
3
2
H) ppm; 13C NMR (125.8 MHz, D
O): δ 169.3, 48.9, 47.9, 33.7, 33.1,
2
7.6, 16.5 ppm; HRMS (ESI-TOF) m/z: [M+H-TFA]+ calcd for
6
.15 (br s, 1H), 4.20–4.15 (m, 1H), 3.18–3.14 (m, 2H), 2.87 (br s, 1H),
.76–2.72 (m, 1H), 2.19–2.14 (m, 1H), 2.08–2.05 (m, 1H), 1.96 (br s, 3H)
2
+
ppm; 3C NMR (125.8 MHz, CDCl
1
C
H N
7 15 2
OSSe , 255.0065; found, 255.00385.
3
): δ 169.1, 45.3, 37.1, 33.0, 23.5, 22.4
3
): δ 382.8 ppm; HRMS (ESI-TOF) m/z:
7
7
ppm; Se NMR (95.4 MHz, CDCl
+
12NOS80Se , 225.9799; found, 225.9779.
+
Catalytic disulfide reduction of BPIns using DTTred or DHLA as an
activator
6
[M + H] calcd for C H
(
S)-N-(1,2-Dithian-4-yl)acetamide (4b)
Catalytic disulfide-reduction of BPIns was initiated by mixing 125 μM
bovine pancreatic insulin solutions (160 μL), 3 mM DTTred or DHLA
solution (20 μL), and 300 μM catalyst solution (20 μL) in 100 mM Tris-
HCl buffer at pH 7.5 and 25 °C. After a certain period of time, an aliquot
Starting with 4a (20.0 mg, 0.116 mmol), 4b was obtained as a white solid.
Yield: 15.1 mg (73%); mp: 169–171 °C; 1H NMR (500 MHz, CDCl
): δ
3
6
2
.04 (br s, 1H), 4.17 (br s, 1H), 3.06–2.97 (m, 2H), 2.81–2.56 (m, 2H),
-
1
.14–2.04 (m, 1H), 1.96 (m, 3H), 1.89–1.78 (m, 1H) ppm; 13C NMR
(15 μL) was transferred into an aqueous AEMTS solution (8 mg mL ,
00 μL) at 25 °C in a micro-centrifuge tube. After 15 min, the mixture was
diluted with an aqueous TFA solution (0.1%, 835 μL) and stored at
2
(
(
1
125.8 MHz, CDCl
APCI-TOF) m/z: [M + H] calcd for C
78.0377.
3
): δ 169.2, 45.7, 37.9, 33.1, 29.7, 23.4 ppm; HRMS
+
+, 178.0355; found,
H12NOS
6 2
−
30 °C. The collected sample solutions were analyzed by RP HPLC. The
HPLC conditions were followed as described previously.[
27]
General procedure for preparation of amino acid conjugates 14–16
Catalytic disulfide reduction of BPIns using GSH as an activator
The assay was performed by following the literature with slight
2
(
a (15 mg, 68.6 μmol) was addied to a solution of Boc-L-amino acid
0.137 mmol, eq) dissolved in anhydrous DMF (2.2 mL). (7-
Azabenzotriazol-1-yloxy)tripyrrolidinophosphonium hexafluorophosphate
2
[
13]
modifications. A test solution (2.8 mL) was prepared by mixing a 200
mM phosphate/5 mM EDTA buffer solution (2655 μL) at pH 7.5
containing NADPH (1.12 μmol) and GSH (34.4 μmol) with a GR solution
(
(
PyBOP) (71.4 mg, 0.137 mmol, 2 eq) and N,N-diisopropylethylamine
DIPEA) (47.8 μL, 0.274 mmol, 4 eq) were the added to the solution. The
-
1
(
145 μL, 255 U mL ). An aliquot (300 µL) of the test solution was mixed
mixture was stirred for 14 h at room temperature under Ar atmosphere.
The solution was then diluted with water (15 mL) and the solution was
extracted with dichloromethane (15 mL×3). The organic phase was
with a 900 μM catalyst solution (50 μL) in 200 mM phosphate buffer at
pH 7.5 and the phosphate buffer solution (550 µL) in a 1.5 mL microtube.
The resulting solution was immediately followed by addition of 600 μM
bovine pancreatic insulin (ε278 = 6080 cm-1 M-1 at pH 7.0) in the
phosphate buffer solution (100 µL) and mixing by a vigorous vortex for 10
s. The reaction progress was monitored by absorption change at 340 nm
due to consumption of NADPH.
washed with saturated aqueous NH
brine (20 mL×1), dried over MgSO
vacuum. The residual material of crude products was purified by silica gel
column chromatography (EtOAc:n-hexane) to give Boc-protected
4
Cl (20 mL×1), water (20 mL×1), and
4
, filtrated, and concentrated under
a
compound. To deprotect the Boc group, the previous compound was
treated with trifluoroacetic acid (0.5 mL) in dichloromethane (0.6 mL) for
4
h at room temperature.
Hydroperoxidase-like activity assay using DTTred as an activator
This article is protected by copyright. All rights reserved.

Chemistry - A European Journal
10.1002/chem.201902230
FULL PAPER
The solutions of DTTred (4 mM, 400 μL), one of the out the synthesized
compounds 1−4 (0.8 mM, 200 μL), and H O (8 mM, 200 μL) in 100 mM
2 2
Acknowledgements
Tris-HCl buffer at pH 7.5 were mixed in a quartz cell (path length = 10
This work was supported by the JSPS-DST Special Lecture
Tour Program 2014 under the Japan–India Cooperative Science
Program (to M.I.), JST [SAKURA Exchange Program in Science,
no. E20150224001 (to G.C.)], JSPS [KAKENHI Grant Number
17K18123 (to K.A.)], PMAC for Private School of Japan [The
Science Research Promotion Fund (to K.A.)], Tokai University
Supporters [Association Research and Study Grant (to K.A. and
M.I.)] and SERB, New Delhi [J. C. Bose National Fellowship
grant no. SB/S2/JCB-067/2015 (to G.M.)]. We thank Dr. Takeru
Ito and Mr. Toshiyuki Misawa (Tokai University) for supporting X-
ray crystallographic analysis
mm) at 25 °C. The reaction progress was followed by UV absorbance
change at 310 nm due to generation of oxidized DTT (DTTox, ε310 = 110
cm-1 M-1).
Kinetic analysis and determination of reduction potential
The reaction was initiated by mixing of 60 μM DTTred solution (3.5 mL)
and 60 μM selenenyl sulfide (2a, 3a, 2b, or 3b) solution (3.5 mL) in 100
mM Tris-HCl/1 mM EDTA at pH 7.0. The reaction was progressed at 25 ±
0
.1 °C under an argon atmosphere. After certain period of time, the
aliquot of mixture solution (1 mL) was added to aqueous 1 M HCl solution
200 μM) to quench the reaction. The obtained sample solution was
(
immediately injected into a RP HPLC equipped with a 1 mL sample
solution loop. The column was equilibrated with 0.1% TFA in water
Keywords: enzyme model • chalcogen atom • redox control •
antioxidant • medicinal chemistry
-
1
(eluent A) at a flow rate of 1.2 mL min . After injection of the sample
solution, a solvent gradient was applied. For compounds 2a and 3a, a
ratio of and 0.1% TFA in acetonitrile (eluent B) linearly increased from
[
1]
a) D. Mustacich, G. Powis, Biochem. J. 2000, 346, 1–8. b) J. Nordberg,
E. S. J. Arnér, Free Radic. Biol. Med. 2001, 31, 1287–1312. c) E. S. J.
Arnér, Biochim. Biophys. Acta - Gen. Subj. 2009, 1790, 495–526.
a) L. Zhong, E. S. J. Arnér, A. Holmgren, Proc. Natl. Acad. Sci. 2000,
0% to 4% in 0–10 min, from 4% to 7% in 10–20 min. For compounds 2b
and 3b, a ratio of eluent B linearly increased from 0% to 4% in 0–10 min,
from 4% to 7% in 10–20 min, from 7% to 55% in 20–30 min. The
concentration of each species at the equilibrium state was calculated
from the observed peak areas and corresponding calibration curves. The
reduction potential for each compound was calculated by using equation
shown in Ref 13.
[2]
97, 5854–5859. b) T. Sandalova, L. Zhong, Y. Lindqvist, A. Holmgren,
G. Schneider, Proc. Natl. Acad. Sci. 2001, 98, 9533–9538.
L. Zhong, A. Holmgren, J. Biol. Chem. 2000, 275, 18121–18128.
M. Björnstedt, M. Hamberg, S. Kumar, J. Xue, A. Holmgren, J. Biol.
Chem. 1995, 270, 11761–11764.
[
3]
4]
[
Cytotoxicity assay using HeLa cell
[
5]
6]
J. M. May, S. Mendiratta, K. E. Hill, R. F. Burk, J. Biol. Chem. 1997,
272, 22607–22610.
HeLa cells were seeded into 96 well-plate (1×10-4 cells/well) in
Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 5%
fetal bovine serum (FBS). After culture in a humidified incubator at 37 °C
[
a) W. Brandt, L. A. Wessjohann, ChemBioChem 2005, 6, 386–394. b)
S. Gromer, L. A. Wessjohann, J. Eubel, W. Brandt, ChemBioChem
2
006, 7, 1649–1652.
Q. Cheng, T. Sandalova, Y. Lindqvist, and E. S. J. Arnér, J. Biol. Chem.
009, 284, 3998–4008.
a) E. E. Alberto, V. do Nascimento, A. L. Braga, J. Braz. Chem. Soc.
with 5% CO
−4 and ebselen solutions in medium (100 μL) without FBS and
incubated for 15 h at 37 °C with 5% CO . The medium was then removed,
2
for 24 h, the cells were treated with 10−50 μM compounds
[
7]
8]
1
2
2
[
and the cells were washed with phosphate buffer saline (PBS). 3-(4,5-
dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) solution (100
μL, 0.5 mg/mL in media) was then added and incubated for 2 h at 37 °C
2
010, 21, 2032–2041. b) K. P. Bhabak, G. Mugesh, Acc. Chem. Res.
010, 43, 1408–1419. c) X. Huang, X. Liu, Q. Luo, J. Liu, J. Shen,
2
Chem. Soc. Rev. 2011, 40, 1171–1184. d) C. Santi, C. Tidei, C.
Scalera, M. Piroddi, F. Galli, Curr. Chem. Biol. 2013, 7, 25–36. e) M.
Iwaoka, in Organoselenium Chemistry: Between Synthesis and
Biochemistry (Ed.: C. Santi), Bentham Science Publishers, 2014, pp.
2
with 5% CO . MTT solution was then removed and DMSO (100 μL) was
added to each well to dissolve the formazan crystals, and the solutions
were incubated at 25 °C with shaking at 350 rpm. Absorbance at 535 nm
was measured using a micro plate reader. The control cells, which were
treated with a media containing no compound, were assumed to be
361–378. f) A. J. Pacuła, F. Mangiavacchi, L. Sancineto, E. J. Lenardão,
J. Ścianowski, C. Santi, Curr. Chem. Biol. 2016, 9, 97–112. g) E. J.
Lenardão, C. Santi, L. Sancineto, in New Frontiers in Organoselenium
Compounds (Eds.: E.J. Lenardão, C. Santi, L. Sancineto), Springer
International Publishing, 2018, pp. 99–143. h) K. N. Sands, T. A. Tuck,
T. G. Back, Chem. – Eur. J. 2018, 24, 9714–9728.
100% viable and the percentage viability of the treated cells was
calculated from eq. (3). The experiment was repeated four times.
…(2)
[9]
a) K. Goto, D. Sonoda, K. Shimada, S. Sase, T. Kawashima, Angew.
Chem. Int. Ed. 2010, 49, 545–547. b) D. Manna, G. Mugesh, Angew.
Chem. Int. Ed. 2010, 49, 9246–9249. c) D. Manna, G. Mugesh, J. Am.
Chem. Soc. 2011, 133, 9980–9983. d) D. Manna, G. Mugesh, J. Am.
Chem. Soc. 2012, 134, 4269–4279.
Antioxidative activity assay using HeLa cell
[
10] N. Metanis, E. Keinan, P. E. Dawson, J. Am. Chem. Soc. 2006, 128,
6684–16691.
4
HeLa cells (1 × 10 ) treated with compounds 1−4 (10 or 20 μM) for 15 h
1
as described above were incubated with H
Balanced Salt Solution (HBSS) containing calcium and magnesium ion
for 3 h in a humidified incubator at 37 °C with 5% CO . The medium was
2 2
O (0.4 mM) in Hanks'
[
11] B. K. Sarma, G. Mugesh, Inorg. Chem. 2006, 45, 5307–5314.
12] A. P. Lothrop, G. W. Snider, E. L. Ruggles, A. S. Patel, W. J. Lees, R. J.
Hondal, Biochemistry 2014, 53, 654–663.
[
2
then removed, and the cells were washed with PBS. The viabilities of
cells were estimated by MTT assay as described above. The experiment
was repeated four times.
[
13] K. Arai, H. Ueno, Y. Asano, G. Chakrabarty, S. Shimodaira, G. Mugesh,
M. Iwaoka, ChemBioChem 2018, 19, 207–211.
[
14] H. F. Gilbert, Methods Enzymol. 1998, 290, 26–50.
15] S. R. Wilson., P. A. Zucker., R.-R. C. Huang, A. Spector, J. Am. Chem.
Soc. 1986, 116, 2557.
[
[16] B. K. Sarma, G. Mugesh, J. Am. Chem. Soc. 2005, 127, 11477–11485.
This article is protected by copyright. All rights reserved.

Chemistry - A European Journal
10.1002/chem.201902230
FULL PAPER
[
[
[
[
[
18] S. Shimodaira, Y. Asano, K. Arai, M. Iwaoka, Biochemistry 2017, 56,
644–5653.
19] S. Shimodaora, M. Iwaoka, Phosph. Sulf. Silic. Rel. Elem., 2019, doi:
0.1080/10426507.2019.1603721.
[23] a) A. Müller, E. Cadenas, P. Graf, H. Sies, Biochem. Pharmacol. 1984,
33, 3235–3239. b) A. Wendel, M. Fausel, H. Safayhi, G. Tiegs, R. Otter,
Biochem. Pharmacol. 1984, 33, 3241–3245.
5
1
[24] N. V. Barbosa, C. W. Nogueira, P. A. Nogara, A. F. de Bem, M.
Aschner and J. B. T. Rocha, Metallomics 2017, 9, 1703–1734.
[25] a) T. D. Ashton, K. M. Aumann, S. P. Baker, C. H. Schiesser, P. J.
Scammells, Bioorg. Med. Chem. Lett. 2007, 17, 6779–6784. b) J. C.
Lukesh, M. J. Palte, R. T. Raines, J. Am. Chem. Soc. 2012, 134, 4057–
4059.
20] F. Lia, P. B. Lutz, Y. Pepelyayeva, E. S. J. Arnér, C. A. Bayse, S.
Rozovsky, Proc. Natl. Acad. Sci. USA 2014, 111, 6976–6981.
21] T. Harada, H. Yoshida, K. Ohno, H. Matsuura, J. Zhang, M. Iwaoka, S.
Tomoda, J. Phys. Chem. A 2001, 105, 4517–4523.
22] S. Gromer, L. Johansson, H. Bauer, L. D. Arscott, S. Rauch, D. P.
Ballou, C. H. Williams Jr., R. H. Schirmer, and E. S. J. Arnér, Proc. Natl.
Acad. Sci. USA 2003, 100, 12618–12623.
[26] T. W. Bruice, G. L. Kenyon, J. Protein Chem. 1982, 1, 47–58.
[27] K. Arai, T. Takei, R. Shinozaki, M. Noguchi, S. Fujisawa, H. Katayama,
L. Moroder, S. Ando, M. Okumura, K. Inaba, et al., Communs. Chem.
2018, 1, 26.
This article is protected by copyright. All rights reserved.

Chemistry - A European Journal
10.1002/chem.201902230
FULL PAPER
Entry for the Table of Contents (Please choose one layout)
Layout 1:
FULL PAPER
Thioredoxin
reductase
(TrxR)
Kenta Arai, Takahiko Matsunaga,
model: Model study using synthetic
selenenyl sulfides as a mimic of the
TrxR active site comprising C497,
U498, and H472 suggested that the
basic H472 would accelerate the
reduction of the Se-S bond in the
catalytic cycle. Furthermore, the
Haruhito Ueno, Nozomi Akahoshi,
Yuumi Sato, Gaurango Chakrabarty,
Govindasamy Mugesh,* Michio Iwaoka*
Se
S
Page No. – Page No.
(
(Insert TOC Graphic here: max.
width: 5.5 cm; max. height: 5.0 cm))
Modeling Thioredoxin Reductase-like
Activity Using Cyclic Selenenyl
Sulfides: Participation of an NH•••Se
Hydrogen Bond through Stabilization
of the Mixed Se–S Intermediate
N
N
S
compounds exhibited
a
defensive
S
ability against H -induced oxidative
2
O
2
stress in cultured cells, suggesting the
possibility for medicinal applications to
control a redox balance in cells.
Active site of thioredoxin reductase
This article is protected by copyright. All rights reserved.