Probing the Formation of a Seleninic Acid in Living Cells by the Fluorescence Switching of a Glutathione Peroxidase Mimetic

Article abstract of DOI:10.1002/anie.201903958

Glutathione peroxidase (GPx) is a selenoenzyme that protects cells against oxidative damage. Although the formation of a seleninic acid (-SeO₂H) by this enzyme during oxidative stress has been proposed, a selenic acid has not been identified in cells. Herein, we report that the formation of a seleninic acid can be monitored in living cells by using a redox-active ebselen analogue with a naphthalimide fluorophore. The probe reacts with H₂O₂ to generate the highly fluorescent seleninic acid. The electron withdrawing nature of the -SeO₂H moiety and strong Se???O interactions, which prevent the photoinduced electron transfer, are responsible for the fluorescence.

Full text of DOI:10.1002/anie.201903958

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Accepted Article
Title: Probing the formation of a Seleninic Acid in Living Cells by a
Fluorescence Switching of a Glutathione Peroxidase Mimetic
Authors: Harinarayana Ungati, Vijayakumar Govindaraj, Megha
Narayanan, and Govindasamy Mugesh
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To be cited as: Angew. Chem. Int. Ed. 10.1002/anie.201903958
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Probing the formation of a Seleninic Acid in Living Cells by a
Fluorescence Switching of a Glutathione Peroxidase Mimetic
Harinarayana Ungati, Vijayakumar Govindaraj, Megha Narayanan and Govindasamy Mugesh*
Abstract: Glutathione peroxidase (GPx) is a selenoenzyme that
protects cells against oxidative damage. Although the formation of a
seleninic acid (-SeO2H) has been proposed for the enzyme and its
mimetics in oxidative stress, such species has not been identified in
the cells. Herein, we report, for the first time, that the formation of
seleninic acid can be monitored in living cells by using a redox active
ebselen analogue having a naphthalimide fluorophore. The probe
reacts with H2O2 to generate a highly fluorescent seleninic acid. The
electron withdrawing nature of the -SeO2H moiety and strong SeO
interactions, which prevent the photoinduced electron transfer, are
responsible for the fluorescence.
that a selenium-based probe that generates a highly fluorescent
seleninic acid can be used to understand the oxidative stress in
the cells during its GPx-like redox regulation.
Hydrogen peroxide (H2O2) plays important roles in redox biology
and cell signalling.[1] However, enhanced levels of H2O2 induce
oxidative stress, resulting in damage to biomolecules such as
DNA, proteins, and lipids.[2] In the long term, these damages
lead to various disorders, such as cardiovascular diseases,
cancer, neurodegeneration, HIV activation, and aging etc.[3]
Glutathione peroxidase (GPx) is a selenoenzyme that protects
the cells from oxidative stress by catalysing the reduction of
H2O2 and organic peroxides using glutathione (GSH) as a
cofactor.[4] The catalytic cycle of GPx involves initial oxidation of
the selenol (E-SeH) by peroxides to produce a selenenic acid
(E-SeOH). Under physiological conditions, the selenenic acid
readily reacts with GSH to generate the enzyme-bound
selenenyl sulfide (E-Se-SG), which upon reaction with a second
GSH, regenerates the selenol with the release of GSSG (Figure
1A).[4] However, the selenenic acid has been shown to undergo
further oxidation to the corresponding seleninic acid (E-SeO2H)
at higher peroxide and lower GSH levels, which represents
severe oxidative stress in the cells.[5]
Figure 1. (A) The proposed mechanism of GPx, indicating the species formed
under normal (cycle 1) and oxidative stress (cycle 2) conditions. (B) Chemical
structures of ebselen (1), proposed GPx-mimetic-based seleninic acids (2-5)
and the ebselen-based fluorescent probe (6) synthesized for this study.
The napthalimide-based ebselen derivative 6 was prepared
from 4-bromo-N-ethyl-1,8-naphthalimide and benzo[d][1,2]
selenazol-3(2H)-one as described in the Supporting Information.
The photophysical properties of 6 were studied in 100 mM
phosphate buffer at pH 7.4. A strong UV-Vis absorption band at
350 nm was observed, which shifted by ≈25 nm higher
wavelength upon treatment with H2O2, indicating a facile
reaction of 6 with H2O2. The fluorescence measurements
indicated that 6 is essentially non-fluorescent in aqueous
solution (Figure 2A), probably due to quenching of the
napthalimide fluorescence by the cyclic selenazole moiety. To
understand whether a photoinduced electron-transfer (PET)
mechanism is responsible for its non-fluorescent nature, density
functional theory (DFT) calculations were carried out by using
B3LYP/6-311+G(d,p) basis set to determine the orbital energy
levels.[7] The model compounds 7 and 8 were used as acceptor
(A) and donor (D), respectively (Figure 2B). The highest
occupied molecular orbital (HOMO) energy of the donor (-6.12
eV) falls between the HOMO and LUMO energy levels of the
acceptor, which allows an efficient PET process to occur,
leading to quenching of the fluorescence. Interestingly, a strong
concentration-dependent fluorescence was observed at 465 nm
upon addition of various amounts of H2O2 (0-12 µM, Figure 2A).
A linear increase in the fluorescence was observed even at
lower concentrations of H2O2 (50-500 nM, Figure 2C), indicating
that the selenium moiety in 6 is highly redox sensitive. It is
known that the selenium centre in ebselen (1) undergoes
oxidation not only with H2O2, but also with other reactive oxygen
species (ROS) such as superoxide (O2•−) and peroxynitrite
As the redox chemistry of selenium in GPx plays a key role
in the cytoprotection, synthetic selenium compounds that
functionally mimic the natural enzyme have been reported.[5]
These mimetics include the well-known ebselen (1, Figure 1B)
and its analogues, camphor-based selenenyl amide, various
diselenides, azaselenonium chloride, allyl and alkyl selenides,
and a cyclic selenelate ester.[6] They catalytically reduce H2O2
and organic peroxides in the presence of GSH or aromatic thiols.
Although the formation of seleninic acid such as 2-5 has been
observed for ebselen and other related compounds under
different experimental conditions (Figure 1B),[5,6] it is not clear
whether such species is produced in cells, particularly under
oxidative stress conditions. Herein, we report for the first time
[*]
H. Ungati, Dr. V. Govindaraj M. Narayanan and Prof. Dr. G. Mugesh
Department of Inorganic and Physical Chemistry
Indian Institute of Science, Bangalore 560012, India
E-mail: mugesh@iisc.ac.in
Supporting information and the ORCID identification number(s) for
the author(s) of this article can be found under:
https://doi.org/10.1002/anie.2018xxxx.
•−
(ONOO−).[5] When 6 was treated with O2 and ONOO−, an
increase in the fluorescence was observed, although the
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10.1002/anie.201903958
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formation of the seleninic acid 10, which was the only major
species in the reaction mixture even at higher concentrations (up
to 30 equiv.) of H2O2. The rapid formation of 10 should occur
through the initial oxidation of 6 by H2O2, followed by the
hydrolysis of the resulting selenoxide 9, as observed for ebselen.
While the formation of the selenenic acid 13 was not observed
due to its rapid cyclization to 6,[8,9] only a small amount of the
overoxidized selenonic acid 14 was observed when a large
excess of H2O2 (more than 30 equiv.) was used. The reaction of
6 with GSH readily generated the selenenyl sulfide 11 (HPLC:
12.6 min). However, the reaction of 11 with GSH to generate the
selenol 12 was very slow (Figure S17, SI). Interestingly, a
remarkably higher fluorescence was observed for the seleninic
acid 10 as compared to that of 6 and 11. When GSH was added,
the regeneration of 6 was observed by HPLC (Figure S18A, SI),
confirming that 6 can act as an ON/OFF fluorescence probe.
Crucially, the reactions of 6 with O2•− and ONOO− also produced
10 (Figure S18B, SI), indicating that the fluorescence increase
•−
upon treatment of 6 with O2 and ONOO− (Figure 2D,E) is due
to the formation of the seleninic acid 10, which is in agreement
with the reactivity of ebselen towards these species.[5]
Figure 2. (A) Enhancement of fluorescence upon treatment of 6 (λex = 350 nm
λem = 475 nm) with H2O2 (2-12 µM) in phosphate buffer at pH 7.4. (B) The
HOMO-LUMO energy levels of 7 (acceptor, A), and 8 (donor, D) obtained by
DFT calculations. (C) A linear increase in the fluorescence observed for 6 at
low concentrations (nM) of H2O2. (D) The fluorescence behaviour of 6 in the
presence of various ROS after 10 min. (E) Fluorescence response of 6 for
various biologically relevant species after 10 min. (F) Effect of GSH on the
fluorescence bahaviour of 6 (10 µM) after treatment with H2O2.
increase was much lower than that observed for H2O2 within the
given time. In contrast, no enhancement in the fluorescence was
observed when 6 was treated with •OH, L-cysteine, GSH,
cysteine proteins (bovine and human serum albumins, BSA and
HSA, respectively), divalent metal ions (Fe2+, Cu2+, Zn2+, Cd2+
−
−
and Hg2+), nitric oxide (NO), nitrite (NO2 ), nitrate (NO3 ) and
singlet oxygen (1O2) (Figure 2E). A rapid decrease in the
fluorescence was observed when GSH was added to 6 after
treatment with H2O2 (Figure 2F), indicating that the oxidized
selenium species generated by treatment of 6 with H2O2 (Figure
2B) is converted to a non-fluorescent species by GSH.
Figure 3. (A) Chemical structures of 9-14. (B and C) HPLC chromatograms
and fluorescence spectra of 6, 10 and 11 (20 µM, λex = 350 nm, λem = 475 nm).
(D) HPLC chromatograms of 6 with various amounts of GSH. (E) Chemical
structures of 15-20 used for the computational studies. (F) The HOMO-LUMO
energy levels of 7 (acceptor, A), 17 and 19 (donor, D).
We next investigated the nature of species produced in the
reactions of 6 with H2O2 and GSH. It is known that ebselen (1)
reacts with H2O2 to produce the corresponding selenoxide,
which undergoes a spontaneous hydrolysis to generate the
seleninic acid (2).[8] The crystal structure of GPx4 also revealed
the formation of a seleninic acid.[5c] When the reaction of 6 with
H2O2 (1 equiv) was followed by HPLC, a single peak at 14.1 min
was observed (Figure 3B). The isolation and characterization by
77Se NMR spectroscopy and mass spectrometry confirmed the
To understand the HOMO-LUMO energy levels of the
intermediates, the model compound 7 was used as acceptor (A)
and 15-20 were used as donors (D) (Figure 3E,F and Figure
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10.1002/anie.201903958
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S29, SI). As the HOMO energy levels for 19 and 20 (D) were
lower than that of 7 (A) (Figure 3F), the introduction of seleninic
and selenonic acid moieties can block the PET, leading to
fluorescence enhancement. Therefore, we carried out structure
optimization and Natural Bond Orbital (NBO) analysis on 10 and
14.[10] These calculations indicate that the carbonyl oxygen of
the amide moiety interacts strongly with selenium in 10 (Figure
4A). Although the SeO interaction in 10 (ESeO: 5.4 kcal.mol-1)
is stronger than that of 14 (1.8 kcal.mol-1), the cooperative effect
of the SeO interaction and the electron-withdrawing nature of
the seleninic and selenonic acid moiety can prevent the PET-
mediated fluorescence quenching in 10 and 14 by decreasing
the electron donation from the donor to the acceptor
(naphthalimide) moiety. As the experimental results indicate that
the oxidation of seleninic acid 10 to the selenonic acid 14 is not
a favoured process, the increase in the fluorescence upon
reaction with H2O2 can be ascribed to the seleninic acid (10)
(Figure 4B).
5C), indicating that 6 can enter cultured normal mammalian cells.
The increase in the fluorescence can be ascribed to the reaction
of 6 with H2O2 as shown in Figure 2A,C-E. To confirm the nature
of selenium species produced in the cells, we analysed the cell
lysates by HPLC. Interestingly, the treatment of 6 with cells pre-
treated with H2O2 indicated the formation of the seleninic acid 10
as the major product along with small amounts of 6 and the
selenenyl sulfide 11 (Figure 5D), suggesting that the formation
of 10 can be observed under physiological conditions. As the
formation of the selenonic acid 14 was not observed, the
increase in the fluorescence upon inducing oxidative stress in
the cells can be ascribed entirely to the formation of seleninic
acid (10).
Figure 4. (A) Optimized structures of 10 and 14, showing the non-covalent
SeO interactions. (B) The reversible generation of the highly fluorescent
seleninic acid 10.
Figure 5. (A) The fluorescence measured by a plate reader after 30 min of
treatment of HepG2 cells with 6 at various concentrations in the absence and
presence of 100 M of H2O2. *p < 0.05 compared with the untreated control
cells; # p < 0.05 compared with cells treated with H2O2 and compd. 6. (B,C)
The fluorescence measured by a plate reader (B) and confocal microscopy
images (C) after 30 min of treatment of cells with 6 and various substrates and
inhibitors. (D) HPLC analysis of the intracellular forms of the probe 6. The
identity of each peak was confirmed by co-eluting the sample with authentic
samples of 6, 10 and 11 (Fig. S19).
The interesting fluorescent properties of 6 in the presence of
H2O2 prompted us to investigate its cytotoxicity and fluorescence
response in HepG2 (human liver carcinoma) cells and HUVEC
(human umbilical vein endothelial cells). The cell viability was
determined by using the standard MTT assay. Interestingly, 6
did not exhibit any cytotoxicity up to 50 M concentration in
HepG2 cells (Figure S30, SI), indicating that the low toxicity of
the selenazole group present in ebselen was retained upon
functionalization with a naphthalimide moiety. To understand the
redox activity of 6 in cells, we studied the fluorescence
behaviour of 6 in both HepG2 cells and HUVEC by using laser
scanning microscopy and fluorescence microplate reader
techniques. When the HepG2 cells were treated with 100 nM of
6, very weak fluorescence was observed even in the presence
of 100 µM of H2O2. A slight increase in the fluorescence was
observed at 0.5 and 1 µM concentrations, and a significant
increase was observed when 10 µM of 6 was used (Figure 5A-
C). An increase in the fluorescence was also observed in
primary HUVECs upon addition of 50 or 100 µM H2O2 (Figure
It is known that ebselen and other selenium compounds
utilize cellular GSH for their GPx-like activity. To understand the
redox response of 6 upon reduction in the GSH level, the cells
were treated with buthionine sulfoximine (BSO), which is known
to block the GSH biosynthesis by inhibiting γ-glutamylcysteine
synthetase.[11] The HepG2 cells were treated with 50 µM BSO
prior to treatment with 6. As shown in Figure 5B and C, a
significant enhancement in the fluorescence was observed upon
depletion of GSH, indicating that 6 responds well to the increase
in the oxidative stress. The HPLC analysis indicated the
formation of the seleninic acid 10 under this condition (Figure
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5D). The increase in the fluorescence due to a decrease in the
GSH:H2O2 ratio can be brought back to the control levels by
treating the cells with GSH. When the cells were treated with
GSH before addition of H2O2, no enhancement in the
fluorescence was observed (Figure 5B and C). The HPLC
analysis indicated that the selenenyl sulfide 11 and seleninic
acid 10 are the major species produced when the GSH:H2O2
ratio are 1:1 and 1:3, respectively (Figure S21). These
observations indicate that 6 undergoes redox reactions with
H2O2 and GSH and the fluorescence intensity depends on the
relative ratio of these two species in the cells.
for her help with the cell culture work. G. M. thanks the SERB for
the award of J. C. Bose fellowship (SB/S2/JCB-067/2015).
Keywords: antioxidant
• ebselen • fluorescent probe •
glutathione peroxidase • selenium
[1]
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cells upon inhibition of CAT was also confirmed independently
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cells. Interestingly, 6 is also sensitive to the superoxide level in
the cells. A significant increase in the fluorescence was
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•−
observed for 6, when the O2 concentration was increased
intracellularly by inhibiting superoxide dismutase (SOD) using
diethyldithiocarbamate (DDC). The increase in the O2•− level was
confirmed further by treating the cells with superoxide-specific
dihydroethidium (DHE) (Figure 5C).
[8]
[9]
In summary, we synthesized a redox active naphthalimide -
•−
based selenazole, which can react with H2O2, O2
or
peroxynitrite to produce a highly fluorescent seleninic acid. The
interesting fluorescence behavior of the seleninic acid can be
ascribed to the electron withdrawing nature of the -SeO2H
moiety and strong SeO interactions, which together prevent
the photoinduced electron transfer (PET) by lowering the HOMO
energy level. The reversible, redox active and self-switching
fluorescent probe reported in this paper is useful not only for
controlling the level of ROS in mammalian cells, but also for
designing fluorescent probes to understand the nature of
species produced during the GPx-like activity of selenium-based
compounds in cellular environment.
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Acknowledgements
This study was supported by the Science and Engineering
Research Board (SERB, EMR/IISc-01/2016), DST, New Delhi. H.
U. and V. G. acknowledge the IISc, Bangalore, and SERB,
respectively, for a fellowship. We thank Mrs Mary Nirmala Sarkar
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10.1002/anie.201903958
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Entry for the Table of Contents
COMMUNICATION
A redox active ebselen analogue
having a naphthalimide moiety
can be used as a fluorescence
probe to understand the formation
of a seleninic acid in living cells.
The fluorescent property of the
seleninic acid can be ascribed to
the strong SeO noncovalent
interactions and electron
Harinarayana Ungati,
Vijayakumar Govindaraj, Megha
Narayanan and Govindasamy
Mugesh*
Page No. – Page No.
Probing the formation of a
Seleninic Acid in Living Cells
by a Fluorescence Switching of
a Glutathione Peroxidase
Mimetic
withdrawing nature of the
seleninic acid moiety, which
prevent the PET by lowering the
HOMO energy level.
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