Synthesis of selenocysteine-containing dipeptides modeling the active site of thioredoxin reductase

Article abstract of DOI:10.1080/10426507.2019.1603721

Four cyclic dipeptides modeling the active site of thioredoxin reductase (TrxR), UU, CU, UC, and CC, where U and C represent selenocystine and cystine, respectively, were synthesized and their glutathione peroxidase (GPx)-like catalytic activity was evaluated by the reaction of hydrogen peroxide (H₂O₂) with glutathione (GSH) in the presence of glutathione reductase (GR). Among these, only UC exhibited the significant antioxidant capacity, suggesting that an atomic environment around the Se–S bond is relevant to the reactivity toward a thiol substrate.

Full text of DOI:10.1080/10426507.2019.1603721

Phosphorus, Sulfur, and Silicon and the Related Elements
ISSN: 1042-6507 (Print) 1563-5325 (Online) Journal homepage: https://www.tandfonline.com/loi/gpss20
Synthesis of selenocysteine-containing dipeptides
modeling the active site of thioredoxin reductase
Shingo Shimodaira & Michio Iwaoka
To cite this article: Shingo Shimodaira & Michio Iwaoka (2019): Synthesis of selenocysteine-
containing dipeptides modeling the active site of thioredoxin reductase, Phosphorus, Sulfur, and
Silicon and the Related Elements, DOI: 10.1080/10426507.2019.1603721
To link to this article: https://doi.org/10.1080/10426507.2019.1603721
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PHOSPHORUS, SULFUR, AND SILICON AND THE RELATED ELEMENTS
https://doi.org/10.1080/10426507.2019.1603721
Synthesis of selenocysteine-containing dipeptides modeling the active
site of thioredoxin reductase
Shingo Shimodaira and Michio Iwaoka
Department of Chemistry, School of Science, Tokai University, Kanagawa, Japan
ABSTRACT
ARTICLE HISTORY
Received 28 March 2019
Accepted 1 April 2019
Four cyclic dipeptides modeling the active site of thioredoxin reductase (TrxR), UU, CU, UC,
and CC, where U and C represent selenocystine and cystine, respectively, were synthesized
and their glutathione peroxidase (GPx)-like catalytic activity was evaluated by the reaction of
hydrogen peroxide (H2O2) with glutathione (GSH) in the presence of glutathione reductase
(GR). Among these, only UC exhibited the significant antioxidant capacity, suggesting that an
KEYWORDS
Selenopeptide; selenenyl
sulfide; TrxR mimics;
NHÁÁÁSe hydrogen bond
atomic environment around the Se–S bond is relevant to the reactivity toward
a
thiol substrate.
GRAPHICAL ABSTRACT
Introduction
get information about the importance of CU doublets in the
enzymatic activity.
Thioredoxin reductase (TrxR)[1] is a unique selenoenzyme
that catalyzes reduction of the disulfide (SS) bond of oxi-
dized thioredoxin (Trx) (Figure 1). At the active site, which
is located in the C-terminal domain, juxtaposed cysteine
(Cys, C) and selenocysteine (Sec, U) residues form a
selenenyl sulfide (Se–S) bond in the resting state and are
activated intramolecularly by a dithiol unit of the N-terminal
domain.
Among previous studies dealing with modeling the bio-
logical functions of TrxR,[2] Schneider[3] synthesized a series
of selenopeptides, such as GCUG and GCCG, and investi-
gated the redox potentials using cyclic voltammetry to reveal
the effects of pH and the amino acid sequence on the
enzymatic activity. Separately, Hondal and coworkers[4]
determined a trans/cis conformer ratio of the amide bond
between C and U in similar cyclic tetrapeptides with a Se–S
linkage. However, the effects of the CU amino acid doublets
on the TrxR-like activity have been remained unveiled
till today.
Results and discussion
Synthesis
Cyclic dipeptides, UU (1), CU (2), UC (3), and CC (4),
were synthesized according to Scheme 1. By condensation of
selenocysteine derivative Fmoc-Sec(Bzh)-OtBu (5a) or cyst-
eine derivative Fmoc-Cys(Trt)-OtBu (5b) with Boc-
Sec(Bzh)-OH (6a) or Boc-Cys(Trt)-OH (6b) using diethyl-
amine for deprotection of the Fmoc group and EDCI and
HOBt for activation of the COOH group,[5] four dipeptides
7a-d were obtained in moderate to high yields. According to
Koizumi’s procedure,[6] the protecting groups on the Sec
and Cys side chains were then deprotected with iodine to
afford cyclic dipeptides 8a-d in moderate yields. Finally, the
target dipeptides (1-4) were yielded by the treatment with
trifluoroacetic acid. Relatively low yields in the cyclization
step were due to formation of polymerized products.
In this study, synthesis and structural characterization of
cyclic dipeptides 1-4, which constitute all possible amino
1
Dipeptides 1-4 were characterized by H, 13C, and/or 77Se
acid doublets of C and U, have been performed in order to NMR and ESI-MS spectroscopies.
CONTACT Michio Iwaoka
miwaoka@tokai.ac.jp
Department of Chemistry, School of Science, Tokai University, Kanagawa, Japan.
Color versions of one or more of the figures in the article can be found online at www.tandfonline.com/gpss.
Supplemental data for this article can be accessed on the publisher’s website.
ß 2019 Taylor & Francis Group, LLC

2
S. SHIMODAIRA AND M. IWAOKA
Scheme 1. Synthesis of cyclic dipeptides 1-4.
1.4
1.2
1
UU (1)
0.8
0.6
0.4
0.2
0
Blank
Figure 1. A catalytic cycle of TrxR.
CU (2)
UC (3)
CC (4)
GPx-like activity
PhSeSePh
TrxR catalyzes the reduction of not only Trx but also
hydroperoxides.[7] Therefore, TrxR-like activity of the
synthesized dipeptides was evaluated conveniently by
application of the protocol for glutathione peroxidase
(GPx) activity assay.[8] In this protocol, hydrogen perox-
ide (H2O2) and glutathione (GSH) were used instead of
oxidized Trx and N-terminal dithiol unit, respectively.
NADPH and glutathione reductase (GR) were added to
reduce oxidized glutathione (GSSG) generated by the
reaction. Progress of the reaction was monitored by UV
absorption at 340 nm due to NADPH.
0
100
200
300
400
500
600
Reaction Time / sec
Figure 2. GPx-like activities of 1-4 and PhSeSePh.
for 1 and 3 are as illustrated in Figure 3. The intermediate
derived from 1 would reversely release RSH through intra-
molecular Se–Se bond formation. On the other hand, the
intermediate derived from 3 would be stabilized by an
NHÁÁÁSe hydrogen bond and readily accept the second RSH
attack to generate an active SeH, SH species. A similar role
As seen in Figure 2, CU (2) with the same amino acid
sequence as native TrxR had only slight GPx-like activity.
Similarly, marginal activities were observed for UU (1) and of an NHÁÁÁSe hydrogen bond has recently been reported in
CC (4). On the other hand, UC (3) with a reverse amino
acid sequence exhibited the significant activity although it
was much less than diphenyl diselenide (PhSeSePh), a well-
known GPx mimic.[9] The results suggested that cyclic pepti-
des with a Se–Se or S–S bond are not good TrxR mimics
and also that the reactivity of a Se–S bond to a thiol sub-
strate (RSH) is changeable depending on the atomic envi-
ronments around the Se atom.
the reaction of the selenenyl sulfide derived from
selenoglutathione.[5]
Conclusions
Cyclic dipeptides 1-4 were synthesized as TrxR active-site
models. The GPx-like activity assay showed that only 3,
which has a UC amino acid sequence with an Se–S linkage,
exhibits the significant activity, suggesting important effects
of an atomic environment around the Se–S bond on the
Wessjohann and coworkers[10] reported based on the X-
ray analysis of TrxR that His existing close to Sec forms a
hydrogen-bonding network at the active site. With consider-
ation of this model, we proposed that the activation steps reactivity toward RSH.

PHOSPHORUS, SULFUR, AND SILICON AND THE RELATED ELEMENTS
3
Figure 3. Plausible activation steps for 1 and 3.
[5] Shimodaira, S.; Asano, Y.; Arai, K.; Iwaoka, M.
Selenoglutathione Diselenide: unique Redox Reactions in the
GPx-like Catalytic Cycle and Repairing of Disulfide Bonds in
Scrambled Protein. Biochemistry 2017, 56, 5644–5653. DOI:
10.1021/acs.biochem.7b00751.
[6] Koizumi, Y.; Nagai, K.; Hasumi, K.; Kuba, K.; Sugiyama, T.
Structure-Activity Relationship of Cyclic Pentapeptide
Malformins as Fibrinolysis Enhancers. Bioorg. Med. Chem. Lett.
2016, 26, 5267–5271. DOI: 10.1016/j.bmcl.2016.09.045.
[7] Zhong, L.; Arner, E. S. J.; Holmgren, A. Structure and
Mechanism of Mammalian Thioredoxin Reductase: The Active
Site Is a Redox-Active Selenolthiol/Selenenylsulfide Formed
from the Conserved Cysteine-Selenocysteine Sequence. Proc.
Natl. Acad. Sci. U.S.A 2000, 97, 5854–5859. DOI: 10.1073/
pnas.100114897.
Acknowledgments
We thank K. Katsumata for assistance of the synthesis of 1-4.
Funding
This work was supported by JSPS KAKENHI Grant Number
JP17K05792 (M. I.).
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