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Modeling the Catalytic Cycle of Glutathione Peroxidase by Nuclear
Magnetic Resonance Spectroscopic Analysis of Selenocysteine
Selenenic Acids

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Ryosuke Masuda, Ryutaro Kimura, Takafumi Karasaki, Shohei Sase,
Kei Goto

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Journal of the American Chemical Society, Vol. 143, issue 17, pp.
6345‒6350

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2021, 4Creative Commons

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Modeling the Catalytic Cycle of Glutathione Peroxidase by Nuclear
Magnetic Resonance Spectroscopic Analysis of Selenocysteine
Selenenic Acids
Ryosuke Masuda, Ryutaro Kimura, Takafumi Karasaki, Shohei Sase, and Kei Goto*

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ABSTRACT: Although selenocysteine selenenic acids (Sec−SeOHs) have been recognized as key intermediates in the catalytic
cycle of glutathione peroxidase (GPx), examples of the direct observation of Sec−SeOH in either protein or small-molecule systems
have remained elusive so far, mostly due to their instability. Here, we report the ﬁrst direct spectroscopic (1H and 77Se NMR)
evidence for the formation of Sec−SeOH in small-molecule selenocysteine and selenopeptide model systems with a cradle-type
protective group. The catalytic cycle of GPx was investigated using NMR-observable Sec−SeOH models. All the hitherto proposed
chemical processes, i.e., not only those of the canonical catalytic cycle but also those involved in the bypass mechanism, including theintramolecular cyclization of Sec−SeOH to the corresponding ﬁve-membered ring selenenyl amide, were examined in a stepwisemanner.

Glutathione peroxidase (GPx)1 is one of the mostimportant selenoenzymes2,3 due to its role in theantioxidant defense in mammalians,4−6 where it catalyzes the
reduction of reactive oxygen species such as H2O2.7 In the
widely accepted catalytic cycle of GPx (Process I in Figure 1a),selenocysteine selenenic acids (Sec−SeOHs),8 generated by
the oxidation of selenocysteine selenols (Sec−SeHs), in the
catalytic site (Step A in Figure 1a), have been proposed as key
intermediates. The Sec−SeH form is assumed to beregenerated by the reaction of the Sec−SeOH intermediatewith glutathione (GSH)9 to produce the selenenyl sulﬁde(Sec−SeSG) form (Step B),10 followed by reduction withanother molecule of GSH (Step C).
However, examples of the direct observation of Sec−SeOHs
in either protein or small-molecule model systems remain
elusive. Even the trapping of Sec−SeOHs in a protein bychemical probes has been limited to very few examples.11−14
One possible reason for this dearth of information is thetendency of Sec−SeOHs to undergo thermal deselenation,
which aﬀords the corresponding dehydroalanines (Step
D).15,16 For example, it has been reported that the Sec
residues in some selenoproteins (including GPx1) areconverted into dehydroalanine in the presence of H2O2.15,17
The mechanism by which GPx maintains its catalytic activity
despite the thermal instability of the Sec−SeOH intermediates
remains to be determined. Flohé et al. have recently postulated
a bypass mechanism to explain the above question (ProcessII).18 They proposed that Sec−SeOHs could undergo
intramolecular cyclization to the corresponding cyclic
selenenyl amides,19−23 with either a ﬁve-membered ring or
an eight-membered ring (Step E), to prevent the thermal
deselenation (Step D) under GSH-deﬁcient conditions. Their
proposal was based on LC-MS/MS analysis of the oxidized
forms of bovine GPx1 and rat GPx4, which was found to be© 2021 The Authors. Published byAmerican Chemical Society

consistent with the formula of the cyclic selenenyl amides, andDFT calculations of Process II using a Sec-Gly-Gly tripeptide
model. Neither the direct detection nor trapping of the Sec−SeOH forms was achieved in their study. For the chemicalelucidation of the mechanistic aspects of the GPx cycle, amodel compound for Sec−SeOH with suﬃcient stability toallow its observation is thus strongly desirable.
In small-molecule model systems, selenenic acids undergo
facile dehydrative self-condensation (Figure 1b).24 Furthermore, selenenic acids generated during the oxidation ofselenols readily form diselenides by the reaction withunreacted selenols.25 Our and other groups have reported on
the observation26−28 and isolation29−35 of nonselenocysteinylderivative selenenic acids by either kinetic or thermodynamic
stabilization techniques. However, these nonselenocysteinyl
derivatives do not adequately reproduce the characteristic
reactivity of Sec−SeOH in proteins.
Recently, we have reported the synthesis of a stable cysteine
sulfenic acid that uses a large molecular cavity as a protective
cradle.36 The molecular cradle is also expected to be eﬀective
for the protection of reactive selenocysteine derivatives, thus
enabling model experiments of reaction processes involving
Sec−SeOHs (Figure 1c). Herein, we report the ﬁrst direct
spectroscopic evidence for Sec−SeOHs that are generated via
the oxidation of a selenocysteine and selenopeptide bearing themolecular cradle by H2O2. The thermal behavior and reactivityof these Sec−SeOHs were investigated using NMR spectrosReceived: March 3, 2021
Published: April 22, 2021

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models, 1a and 1b, are expected to be prevented frombimolecular decomposition (Figure 1b) by the protective
cradle.
The Sec−SeH form of the Sec model (3a) was synthesized
by the reduction of selenocysteine derivative 2 (Scheme 1a).
Scheme 1. Synthesis of the Selenols of the Sec Model (a)and the Sec-Gly-Gly Model (b)The analogous form of the Sec-Gly-Gly model (3b) was
synthesized from selenopeptide 439−41 via the route shown in
Scheme 1b, which involves selenenyl iodide 5 as a synthetic
intermediate.
Selenocysteine-derived selenenyl iodides (Sec−SeIs) havebeen recognized as important intermediates in the thyroid
hormone deiodination by iodothyronine deiodinases,42,43
albeit that examples of the synthesis of a selenenyl iodide
with a selenocysteine backbone remain elusive.44 Sec−SeI 5
was isolated in the form of reddish-purple crystals, and its
structure was determined by a single-crystal X-ray diﬀraction
analysis (Figure 3, Tables S9 and S10).

Figure 1. (a) Proposed catalytic cycle of GPx. (b) Bimolecular
reactions of selenenic acids. (c) Conceptual illustration of this study.

copy, and all hitherto proposed chemical processes in the
catalytic cycle of GPx shown in Figure 1a, including those
involved in the bypass mechanism, were documented in a
stepwise manner.
We designed two model systems. One is the Sec model(Figure 2A), in which a selenocysteine methyl ester isprotected by a cradle-type benzoyl group (henceforth denoted
as Bpsc), and the other is the Sec-Gly-Gly model (Figure 2B),
which corresponds to the generalized form of the tripeptides in
the catalytic site of GPx.37,38 The Sec−SeOH forms of bothFigure 3. Crystal structure of 5 (one of two independent molecules).

Treatment of Sec−SeH 3a with H2O2 (1 equiv) at room
temperature without any additives resulted in the formation of
dehydroalanine 8a in 89% yield together with a small amountof diselenide 9a (Scheme 2). Although the formation of Sec−SeOH 1a was not observed directly in this reaction, its
intermediacy was supported by trapping experiments using
dimedone (10) or acetylacetone (11)33 to produce 12a or 13a,

Figure 2. Cradled selenocysteine (A) and selenopeptide (B) modelcompounds.
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further conﬁrmed the identiﬁcation of 1a as Sec−SeOH. After
1a was generated by the above protocol, diketone 10 or 11 wasadded at −20 °C. 1H NMR monitoring indicated that 1a wasgradually converted to 12a or 13a, respectively, in good
conversion yield (Schemes S18 and S19).
Then, we investigated the thermal deselenation of Sec−SeOH to form a dehydroalanine (Step D). After 1a was
generated by the above protocol, the solution was warmed
from −20 to 15 °C. 1H NMR monitoring conﬁrmed that 84%
of 1a was converted into dehydroalanine 8a (Scheme 3).

Scheme 2. Oxidation of 3a with H2O2 and Trapping
Experiments with 1,3-Diketones

Scheme 3. Thermal Deselenation of 1a to 8a

In contrast, when 1a was generated by the same protocol
and subsequently treated with cysteine thiol 6 (2 equiv) at −20°C, 1a was rapidly converted into selenenyl sulﬁde 7a in 91%
conversion yield, and the formation of 8a was not observed(Scheme 4). These results demonstrate that the reaction of arespectively. It is likely that, in the absence of trapping agents,
the majority of 1a undergoes thermal deselenation to form 8a,while a fraction reacted with the substrate 3a to produce
diselenide 9a. To suppress the formation of 8a and 9a, thefollowing protocol was examined (Figure S1). A THF-d8
solution of 3a was added dropwise to a THF-d8/D2O solutionof H2O2 (10 equiv) and NaOH45 at −65 °C. The mixture wastransferred to a J-Young NMR tube via a tube cooled to thesame temperature, and the 1H and 77Se NMR spectra wererecorded at −20 °C (Figure 4a). The formation of Sec−SeOH

Scheme 4. Reaction of 1a with 6

Sec−SeOH with a thiol to produce a selenenyl sulﬁde (Step B)
is rapid, even at low temperature, where the thermal
deselenation to a dehydroalanine (Step D) does not proceed.
On the basis of these experiments, the reactivity of a Sec−SeOH is summarized as follows: a Sec−SeOH can begenerated as a stable compound by oxidation of a Sec−SeH
with H2O2 at low temperatures (below −20 °C in the presentcase). Under these conditions, a Sec−SeOH readily reacts witha thiol to produce the corresponding selenenyl sulﬁde.
However, in the absence of a thiol, it undergoes thermal
deselenation to a dehydroalanine upon warming; this reaction
can thus be expected to proceed readily at physiologicaltemperature. These properties of a Sec−SeOH strongly suggest
that, in the catalytic cycle of GPx, a protective bypass
mechanism to prevent its inactivation through deselenation
should be operative when the concentration of GSH is
insuﬃcient.
Based on the protocol established using the Sec model(Figure 2A), we then conducted model studies of the bypass
mechanism (Process II in Figure 1a) using the Sec-Gly-Gly
model (Figure 2B). In the oxidation of Sec−SeH 3b with
H2O2 using the above protocol, the formation of Sec−SeOH
1b in 90% NMR yield was observed at −30 °C (Figure 5a and5b), where 1b was found to be stable for at least 24 h. In the77
Se NMR spectrum, the signal of 1b was observed at 1082
ppm (Figure S10). The derivatization of 1b with acetylacetone(11) to produce the corresponding selenide 13b (Scheme
S24) also conﬁrmed the identiﬁcation of 1b as a Sec−SeOH.
To investigate the intramolecular cyclization of a Sec−SeOH

Figure 4. (a) Observation of 1a via the oxidation of 3a. (b) 1H NMR
spectrum (500 MHz) of 1a in THF-d8/D2O at −20 °C. (c) 77Se
NMR spectrum (95 MHz) of 1a in THF-d8/D2O at −20 °C.

1a in 92% NMR yield was observed in the 1H NMR spectrum(Figure 4b).46 In the 77Se NMR spectrum, 1a showed a signal
at 1298 ppm (Figure 4c), which is in good accordance withthose of recently reported arylmethyl-substituted selenenic
acids (Table S4).31,33,47 This is the ﬁrst experimental evidence
for the formation of Sec−SeOHs, as well as the ﬁrst direct
evidence for the oxidation of a Sec−SeH to a Sec−SeOH with
H2O2 (Step A in Figure 1a). Reactions with 1,3-diketones
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Figure 5. (a) Generation of 1b. (b) 1H NMR spectrum (500 MHz) of 1b in THF-d8/D2O at −30 °C. (c) VT NMR spectra (400 MHz, − 20 up to0 °C) for a solution containing 1b in THF-d8/D2O.

to the corresponding cyclic selenenyl amide (Step E), thethermal behavior of 1b was examined by monitoring thetemperature-dependent change in the ratio of 1b to its
products (Figure 5c). When a solution of 1b generated in 87%
NMR yield by the above protocol was warmed from −20 to−10 °C, the formation of cyclic selenenyl amide 1448 with aﬁve-membered ring in 6% yield was observed, while the ratio of
1b decreased from 87% to 79% (Table 1). Notably,
dehydroalanine 8b was not detected at −10 °C. At 0 °C, theratio of 14 increased to 12%, while that of 1b decreased to70%, and only 2% of 8b was detected. These results
demonstrate that a Sec−SeOH undergoes an intramolecularcyclization to the cyclic selenenyl amide (Step E), which
proceeds faster than the thermal deselenation to thedehydroalanine (Step D).
When cysteine thiol 6 (50 equiv) was added at −20 °C to asolution of 1b generated in 75% NMR yield, selenenyl sulﬁde7b was formed in a conversion yield of 83% (Scheme 5). In
this reaction, the generation of 14 or 8b was not detected.
The preference among the three possible reaction processesof a Sec−SeOH, i.e., Steps B, D, and E, is summarized asfollows: Of the two thermal reaction processes, the intra-

Table 1. Intramolecular Cyclization of 1b to 14

NMR yield
temp (°C)

1b

14

8b

−20
−10
0

87
79
70

n.d.
6
12

n.d.
n.d.
2

molecular cyclization (Step E) proceeds faster than thedeselenation (Step D). In the presence of a suﬃcientconcentration of thiol, the reaction of a Sec−SeOH with thethiol (Step B) proceeds faster than Steps D and E. These
results are consistent with the bypass mechanism as proposedby Flohé et al.18
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Authors

Scheme 5. Reaction of 1b with 6

Ryosuke Masuda − Department of Chemistry, School of
Science, Tokyo Institute of Technology, Tokyo 152-8551,
Japan
Ryutaro Kimura − Department of Chemistry, School of
Science, Tokyo Institute of Technology, Tokyo 152-8551,
Japan
Takafumi Karasaki − Department of Chemistry, School of
Science, Tokyo Institute of Technology, Tokyo 152-8551,
Japan
Shohei Sase − Department of Chemistry, School of Science,
Tokyo Institute of Technology, Tokyo 152-8551, Japan

Cyclic selenenyl amide 14 was converted into selenenyl
sulﬁde 7b by treatment with thiol 6 in 86% yield (Scheme 6),

Complete contact information is available at:https://pubs.acs.org/10.1021/jacs.1c02383

Scheme 6. Reaction of 14 with 6 Followed by Reduction
with DTT

Notes

The authors declare no competing ﬁnancial interest.

■

ACKNOWLEDGMENTS
This work was partly supported by Grants-in-Aid for ScientiﬁcResearch (B) (No. 15H03776 and 19H02698) from the
Ministry of Education, Culture, Sports, Science, andTechnology (Japan).

■

indicating that the cyclic selenenyl amide form can bereintegrated into the canonical catalytic cycle by reaction
with a thiol (Step F). The reduction of 7b with DTT aﬀorded
Sec−SeH 3b, which corresponds to Step C. Thus, using the
Sec-Gly-Gly model, all the chemical processes proposed for thecatalytic cycle of GPx, i.e., Steps A−C (Process I) and Steps E
and F (Process II), have been experimentally documented in a
stepwise manner.
In conclusion, we have presented the ﬁrst direct observationof selenocysteine selenenic acids (Sec−SeOHs) using cradledselenocysteine and selenopeptide models. The reactivity of the
Sec−SeOHs was experimentally examined by 1H and 77Se
NMR monitoring, and all chemical processes that havepreviously been proposed for the catalytic cycle of GPx,
including the bypass mechanism, were documented andconﬁrmed.

■

ASSOCIATED CONTENT

sı Supporting Information*

The Supporting Information is available free of charge athttps://pubs.acs.org/doi/10.1021/jacs.1c02383.
Experimental procedures and spectral data (PDF)
Accession Codes

CCDC 2063205 contains the supplementary crystallographic
data for this paper. These data can be obtained free of charge
via www.ccdc.cam.ac.uk/data_request/cif, or by emailing
data_request@ccdc.cam.ac.uk, or by contacting The Cambridge Crystallographic Data Centre, 12 Union Road,
Cambridge CB2 1EZ, UK; fax: +44 1223 336033.

■

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Corresponding Author

Kei Goto − Department of Chemistry, School of Science, Tokyo
Institute of Technology, Tokyo 152-8551, Japan;
orcid.org/0000-0002-5422-1074; Email: goto@
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and Iodine: Influence of Solution Equilibria Between Covalent
Compounds and Charge − Transfer Complexes. Phosphorus, Sulfur
Silicon Relat. Elem. 1992, 67, 67−71.
(45) To accelerate the rate of oxidation of Sec−SeH, a base wasneeded.
(46) Sec−SeOH 1a exhibited a singlet corresponding to the OH
proton at 5.35 ppm.
(47) 77Se NMR chemical shifts of previously reported arylmethylsubstituted selenenic acids: S12 (1243 ppm) and S13 (1261 ppm);
for details, see Table S4.
(48) Cyclic selenenyl amide 14 was also obtained by the
intramolecular cyclization of Sec−SeI 5 in the presence of NaOH
(Scheme S26). The ﬁve-membered ring structure of 14 wasdetermined by 2D NMR spectroscopy (Figure S12). Compound 14
was found to be less susceptible to oxidative deselenation than Sec−SeH 3b (Scheme S30, Tables S7 and S8).

■NOTE ADDED AFTER ISSUE PUBLICATIONThis article was initially published with an incorrect copyrightstatement and was corrected on or around May 28, 2021.

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https://doi.org/10.1021/jacs.1c02383
J. Am. Chem. Soc. 2021, 143, 6345−6350