A Sweet H2S/H2O2Dual Release System and Specific Protein S-Persulfidation Mediated by Thioglucose/Glucose Oxidase

Article abstract of DOI:10.1021/jacs.1c06372

H2S and H2O2 are two redox regulating molecules that play important roles in many physiological and pathological processes. While each of them has distinct biosynthetic pathways and signaling mechanisms, the crosstalk between these two species is also known to cause critical biological responses such as protein S-persulfidation. So far, many chemical tools for the studies of H2S and H2O2 have been developed, such as the donors and sensors for H2S and H2O2. However, these tools are normally targeting single species (e.g., only H2S or only H2O2). As such, the crosstalk and synergetic effects between H2S and H2O2 have hardly been studied with those tools. In this work, we report a unique H2S/H2O2 dual donor system by employing 1-thio-β-d-glucose and glucose oxidase (GOx) as the substrates. This enzymatic system can simultaneously produce H2S and H2O2 in a slow and controllable fashion, without generating any bio-unfriendly byproducts. This system was demonstrated to cause efficient S-persulfidation on proteins. In addition, we expanded the system to thiolactose and thioglucose-disulfide; therefore, additional factors (β-galactosidase and cellular reductants) could be introduced to further control the release of H2S/H2O2. This dual release system should be useful for future research on H2S and H2O2.

Full text of DOI:10.1021/jacs.1c06372

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A Sweet H S/H O Dual Release System and Specific Protein
2
2 2
S‑Persulfidation Mediated by Thioglucose/Glucose Oxidase
Xiang Ni, Xiaolu Li, Tun-Li Shen, Wei-Jun Qian, and Ming Xian*
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sı Supporting Information
ABSTRACT: H S and H O are two redox regulating molecules that play
2
2
2
important roles in many physiological and pathological processes. While
each of them has distinct biosynthetic pathways and signaling mechanisms,
the crosstalk between these two species is also known to cause critical
biological responses such as protein S-persulfidation. So far, many chemical
tools for the studies of H S and H O have been developed, such as the
2
2
2
donors and sensors for H S and H O . However, these tools are normally
2
2
2
targeting single species (e.g., only H S or only H O ). As such, the crosstalk
2
2
2
and synergetic effects between H S and H O have hardly been studied with
2
2
2
those tools. In this work, we report a unique H S/H O dual donor system
2
2
2
by employing 1-thio-β-D-glucose and glucose oxidase (GOx) as the
substrates. This enzymatic system can simultaneously produce H S and
2
H O in a slow and controllable fashion, without generating any bio-unfriendly byproducts. This system was demonstrated to cause
2
2
efficient S-persulfidation on proteins. In addition, we expanded the system to thiolactose and thioglucose-disulfide; therefore,
additional factors (β-galactosidase and cellular reductants) could be introduced to further control the release of H S/H O . This
2
2
2
dual release system should be useful for future research on H S and H O .
2
2
2
INTRODUCTION
Reactive sulfur species (RSS) and reactive oxygen species
ROS) are two groups of molecules that play regulatory roles
in redox biology. As the most well studied RSS, H S is
endogenously produced by enzymes including cystathionine-β-
synthase (CBS), cystathionine-γ-lyase (CSE), and 3-mercapto-
pyruvate sulfur transferase (3-MST). Dysregulated H S levels
are associated with pathological processes like cancer,
inflammation, hypertension, etc. As the center species of
sulfenic acid (S−OH), which is the oxidation product from
■
(
9
H O . As such, an appropriate level of H O is required to
2
2
2
2
facilitate H S signaling cascades, especially under oxidative
2
stress. In another study H S was found to significantly amplify
2
2
H O -based therapeutic treatment for cancer in a mouse
2
2
10
model. Moreover, the mechanism of acute H S intoxication
2
is due to H S serving as a substrate for complex II of the
2
2
mitochondrial electron transport chain, thereby inducing high
1
,2
11
levels of ROS formation and oxidative stress. These results
ROS, H O is formed from spontaneous dismutation of
indicate that concurrent presence of H S and H O is often
needed to reveal their true biological importance.
So far, many chemical tools have been developed for RSS/
ROS studies. In particular, donor molecules for these reactive
species have received considerable attention. For example, a
2
2
2
2
2
·
−
superoxide (O2 ) or under the catalysis of superoxide
dismutase (SOD). Endogenous H O can also be produced
2
2
by oxidases, such as NADP/H oxidases (NOX), lysyl oxidases
3
,4
(
LOX), xanthine oxidase (XO), amine oxidase (AO), etc.
Aberrant production of H O leads to oxidative stress and
2
2
variety of H S donors have been reported and they can be
2
12−14
damage, which is connected to aging and neurodegenerative
triggered by different cellular factors to release H S.
Figure
2
5
,6
diseases. While H S and H O have their very distinct
2
2
2
1 shows a few examples: GYY4137 and JK-1 are pH-triggered
donors. N-Benzoylthiobenzamides are thiol-triggered donors.
A few enzyme-triggered donors are also reported, including the
esterase-activated donor (HP-101), esterase and carbonic
signaling pathways, they also interact with each other and work
7
collectively in redox signaling. The direct reaction between
H S and H O under physiological conditions is known to be
2
2
2
slow, so their crosstalk is believed to work through indirect
7
effects on enzymes and/or other targets in signaling pathways.
Received: June 20, 2021
Published: August 12, 2021
For example, protein S-persulfidation is an important
posttranslational modification regulated by H S. However,
2
H S cannot directly react with protein thiols to form
2
persulfides and its reaction with disulfides is normally slow
8
,9
and thermodynamically unfavored.
It is likely that
persulfidation is the result of the reaction between H S and
2
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2021 American Chemical Society
https://doi.org/10.1021/jacs.1c06372
J. Am. Chem. Soc. 2021, 143, 13325−13332
1
3325

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Scheme 1. Idea of Using Thioglucose/GOx to Release H S/
2
H O and Induce Protein S-Persulfidation
2
2
which should easily undergo hydrolysis to release H S.
2
Therefore, we envisioned that thioglucose/GOx could be a
unique enzyme triggered H S/H O dual releasing system
2
2
2
Figure 1. Representative examples of synthetic H S and H O donors.
(Scheme 1b). This is attractive, as only nontoxic and
biocompatible gluconic acid would accompany the two species.
We also envisioned that the dual release of H S/H O might
2
2
2
2
2
2
anhydrase (CA)-triggered COS/H S donors, and nitro-
2
be an efficient way to induce protein S-persulfidation.
Furthermore, the thioglucose moiety can be engineered as
stimuli-response prodrugs to deliver H S and H O upon
15−17
reductase-activated donors.
H O donors are much less
2 2
developed, and researchers still tend to use H O directly in
2
2
2
2
2
studies. However, it has been demonstrated that bolus delivery
of H O is often problematic due to rapid consumption of
specific biologically relevant activation.
2
2
With this idea in mind, we first evaluated H S release from
18,19
2
H O in biological systems such as in cells.
This justifies
2
2
thioglucose in the presence of GOx. The standard methylene
the need for slow and continuous H O releasing methods.
2
2
blue (MB) method was used to quantify H S production. As
2
Currently only a few hydroquinone and anthraquinone
shown in Figure 2, thioglucose itself was found to be stable in
20−22
derivatives are reported as H O donors (Figure 1).
2
2
While these donor compounds have advanced our under-
standing of H S and H O , limitations still remain: (1) All
2
2
2
these donors are synthetic materials. Inevitably, they will also
produce significant amounts of organic byproducts (in addition
to H S or H O ). This could cause unwanted side effects. (2)
2
2
2
Enzyme-triggered donors (especially H O donors) are still
2
2
very limited. (3) All available donors can only produce one
species (H S or H O ), which can hardly mimic the concurrent
2
2
2
presence of H S and H O or allow the study of their crosstalk
2
2
2
and synergetic effects. Herein, we would like to report a new
H S/H O dual donor system employing thioglucose and
2
2
2
GOx. This can produce H S and H O simultaneously in a
2
2
2
slow and controllable fashion, without generating any bio-
unfriendly organic byproducts. We also demonstrated that this
system can cause efficient S-persulfidation on proteins.
Moreover, this system can be expanded to the disulfides of
thioglucose and thiolactose. As such, additional factors can be
induced to further control the release of H S/H O dual
Figure 2. H S release profile of thioglucose (100 μM) in the presence
2
and absence of GOx (10 μg/mL) in PBS buffer (50 mM, pH 7.4).
The experiments were performed in triplicate, and results are
expressed as mean ± SD (n = 3).
2
2
2
species.
buffers and no obvious H S release was detected in the absence
2
of GOx. However, significant H S release was observed when
2
RESULTS AND DISCUSSION
Glucose oxidase (GOx) is a flavin-containing oxido-reductase
which catalyzes the oxidation of β-D-glucose to D-glucono-δ-
■
GOx was present. The optimized condition was identified as
100 μM thioglucose in PBS buffer (50 mM, pH 7.4)
containing 10 μg/mL GOx. This combination led to slow,
23
lactone by O gas with the production of H O (Scheme 1a).
continuous, and time-dependent H S generation. The
2
2
2
2
Because of this property, the glucose/GOx system has been
used as an alternative for the exogenous addition of H O in
concentration of H S reached the maximum value of ∼28
2
μM in about 2 h and then slowly decreased presumably due to
2
2
19
biological studies. This system can induce hypoxia, oxidation
stress, or enhance acidity in tumor microenvironments. These
have been strategically utilized to achieve multimodal
synergistic cancer therapy by integrating GOx with various
volatilization of H S gas.
2
The H S formation was further verified by fluorescence
2
26
measurements with the H S-specific probe WSP5. As shown
2
in Figure 3, the treatment of WSP5 with thioglucose or GOx
alone did not give any detectable fluorescence. However,
significant fluorescent signals were observed after incubating
the probe (10 μM) with the mixture of thioglucose (100 μM)
24
therapeutic approaches. From a mechanistic point-of-view, if
25
1
-thio-β-D-glucose (thioglucose) is used as the substrate for
GOx, it would produce thio-gluconolactone as the product,
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the reaction between H S and H O is 0.73 M s−1 (pH 7.4,
−
1
2
2
2
3
7 °C), with the formation of a mixture of hydrogen
27
polysulfide (H S ) as the possible products. We also
2
n
wondered if this system could produce H S . To test this, a
2
n
28
specific fluorescent probe PSP-3 was used to monitor the
generation of H S . As shown in Figure 5, negligible
2
n
Figure 3. H S release from thioglucose (100 μM) in the presence of
2
GOx (10 μg/mL) detected by WSP5 (10 μM) in PBS buffer (50 mM,
pH 7.4). (a) WSP5 only, (b) WSP5 + thioglucose, (c) WSP5 + GOx,
(
d) WSP5 + thioglucose + GOx, (e) WSP5 + 100 μM Na S. The
2
experiments were performed in triplicate, and results are expressed as
mean ± SD (n = 3).
Figure 5. Detection of H S produced from thioglucose-GOx by PSP-
2
n
and GOx (10 μg/mL) at room temperature for 1 h, indicating
3
(10 μM). Thioglucose (100 μM) was incubated with GOx (10 μg/
that thioglucose indeed produced H S in the presence of GOx.
2
mL) and PSP-3 in PBS buffer (50 mM, pH 7.4) for 1 h (white) and 8
h (gray), respectively. Fluorescence responses were recorded at 515
nm. (a) PSP-3 only, (b) PSP-3 + thioglucose, (c) PSP-3 + GOx, (d)
PSP-3 + thioglucose + GOx, (e) PSP-3 + Na S (100 μM) + H O
2
We next monitored the formation of H O by the ferrous
2
2
oxidation−xylenol orange method (FOX 1 assay) under the
optimized conditions described above. As shown in Figure 4, a
2
2
(
100 μM), (f) PSP-3 + Na2S2 (50 μM). The experiments were
performed in triplicate, and results are expressed as mean ± SD (n =
).
3
fluorescence signals were observed after 1 h incubation of
the probe with thioglucose (100 μM) and GOx (10 μg/mL) in
PBS buffer (50 mM, pH 7.4) at rt. However, we did observe
some fluorescence increases upon extending the incubation
time to 8 h. We also compared this enzyme-generation system
with directly mixing H O and Na S. Weak fluorescence signals
2
2
2
were observed with the incubation of a mixture of Na S (100
2
μM) and H O (100 μM) after 1 h while much stronger
2
2
fluorescence was noted after 8 h. These results again suggested
that H S and H O could react to form H S but requires a
2
2
2
2 n
longer reaction time. This should not be a concern when the
thioglucose-GOx system was used in our regular conditions
Figure 4. Time-dependent generation of H O from thioglucose (100
2
2
μM) and GOx (10 μg/mL) in PBS buffer (50 mM, pH 7.4) in the
presence and absence of CAT (50 μg/mL). The experiments were
performed in triplicate, and results are expressed as mean ± SD (n =
(
e.g., ∼1 h).
Our results thus far have demonstrated that the thioglucose-
GOx system under the optimized conditions could slowly and
consistently produce H S and H O under biologically relevant
3).
2
2
2
concentrations. We next wondered if this system could be used
time-dependent H O formation was observed with the peak
to induce protein S-persulfidation, an important posttransla-
2
2
9
,29
concentration of ∼20 μM at about 2 h. Additionally, if catalase
CAT) was present in this system (at 50 μg/mL), the
produced H O was completely scavenged. The H O
tional modification mediated by H S.
This hypothesis was
2
(
based on the knowledge that H S alone can hardly induce
2
protein S-persulfidation (as the reaction between H S and
2
2
2
2
2
formation was also determined by fluorescence measurements
protein disulfides is usually slow and less-productive). H O
2
2
with H O -sensitive Amplex Red/horseradish peroxidase (AP/
can oxidize thiols in human serum albumin (HSA-SH) with a
2
2
−
1
−1
HRP) assay (Figure S5). Interestingly, we noticed that the
second-order rate constant of 2.3−2.7 M
s
(pH 7.4, 37
30
release of H S was not affected by the addition of CAT (Figure
°C), leading to the formation of sulfenic acid HSA-SOH that
can be easily converted to persulfide upon reacting with H S.
2
S2), indicating that thioglucose could be used as a clean H2S
donor if two enzymes (GOx and CAT) were applied.
The concurrent formation of H S and H O in this system
2
However, this method needs very high concentrations of H O
2
2
31
(4 mM) and H S (2 mM). We expected that our slow and
2
2
2
2
could lead to a concern that H S and H O might react with
continuous H S/H O generation under low concentrations
2
2
2
2
2
2
each other, so the release of H S will not be efficient. However,
would have some advantages in inducing protein S-
persulfidation. Before we tested this in proteins, we decided
to test if thioglucose-GOx would induce S-persulfidation on
low molecular weight (LMW) biothiols (such as Cys and
2
the results shown in Figures 2 and 4 clearly suggested this
should not present a major problem due to the slow reaction
between them, especially under biologically relevant concen-
trations. It is reported that the second-order rate constant of
32
GSH). SSP4, a persulfide sensitive fluorescent probe, was
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used to measure persulfidation. Fluorescence intensities were
measured after incubation of the probe with each analyte in
PBS buffer (50 mM, pH 7.4) at rt. As shown in Figure 6, SSP-4
Figure 7. Thioglucose-GOx induced BSA S-persulfidation detected by
SSP4. In these studies, 30 μM reduced BSA and 10 μM SSP4 were
used. Fluorescence response was recorded at 515 nm. (a) SSP4 only,
(
+
b) SSP4 + BSA, (c) SSP4 + BSA + Na S (60 μM), (d) SSP4 + BSA
H O (60 μM) + CAT (25 μg/mL), (e) SSP4 + BSA + Na S (60
2 2 2
2
Figure 6. Fluorescence responses of SSP4 toward various low
molecular weight biothiols in the presence of thioglucose-GOx. SSP4
μM) + H O (60 μM) + CAT (25 μg/mL) (f) SSP4 + BSA +
2
2
thioglucose (200 μM), (g) SSP4 + BSA + GOx (10 μg/mL), (h)
SSP4 + BSA + thioglucose (200 μM) + GOx (10 μg/mL) + CAT (25
μg/mL), (i) BSA (600 μM) + H O (4 mM) + CAT (25 μg/mL) +
(10 μM) was incubated with different substrates in PBS buffer (50
mM, pH 7.4) for 1 h, then the fluorescence responses were recorded
at 515 nm. (1) SSP4 only, (2) SSP4 + thioglucose (100 μM), (3)
SSP4 + GOx (10 μg/mL), (4) SSP4 + Cys (100 μM), (5) SSP4 +
GSH (1 mM), (6) SSP4 + thioglucose (100 μM) + Cys(100 μM),
2
2
Na S (2 mM) + SSP4. The experiments were performed in triplicate,
2
and results are expressed as mean ± SD (n = 3). Statistical analysis
was performed using one-way ANOVA. ***P < 0.001.
(
(
7) SSP4 + thioglucose (100 μM) + GSH (1 mM), (8) SSP4 + GOx
10 μg/mL) + Cys(100 μM), (9) SSP4 + GOx (10 μg/mL) + GSH(1
mM), (10) SSP4+ thioglucose (100 μM) + GOx (10 μg/mL), (11)
H O and H S were similar to those of thioglucose-GOx
2
2
2
SSP4 + Na S (30 μM) + H O (20 μM), (12) SSP4+ thioglucose
2
2
2
system (e.g., 60 μM H O and 60 μM Na S, shown in column
2 2 2
(
100 μM) + GOx (10 μg/mL) + Cys(100 μM), (13) SSP4+
e), we only observe weak fluorescence. When much higher
concentrations of the reagents were used (600 μM BSA with 4
mM H O and 2 mM Na S) we were able to observe strong
thioglucose (100 μM) + GOx (10 μg/mL) + GSH(1 mM), (14)
SSP4+ thioglucose (100 μM) + Na S (30 μM) + H O (20 μM),
2
2
2
2
2
2
(
(
15) SSP4 + GOx (10 μg/mL)+ Na S (30 μM) + H O (20 μM),
2 2 2
fluorescent signals (column i). It should be noted that these
high concentrations are unrealistic for real biological
applications. Therefore, our results indicated that thioglu-
cose-GOx is an efficient method to cause protein persulfidation
under physiologically relevant H O /H S concentrations and
16) SSP4+ Cys(100 μM) + Na S (30 μM) + H O (20 μM), (17)
2
2
2
SSP4 + GSH (1 mM) + Na S (30 μM) + H O (20 μM), (18) SSP4
2
2
2
+
Na S (50 μM). The experiments were performed in triplicate, and
2 2
results are expressed as mean ± SD (n = 3).
2
2
2
was quite stable upon treatment with a series of substrates
alone including thioglucose, GOx, Cys, GSH, as well as the
mixtures of thioglucose/Cys, thioglucose/GSH, GOx/Cys,
GOx/GSH (columns 1−9). No fluorescence was noted in
these studies. In addition, negligible responses were observed
in the mixture of thioglucose/GOx (column 10) and the direct
mixture of Na S/H O under similar concentrations of the
SSP4 is suitable for the detection of protein S-persulfidation.
The formation of BSA persulfide was further confirmed by
liquid chromatography-tandem mass spectrometry (LC-MS/
MS) analysis. Briefly, BSA was treated with thioglucose-GOx as
described above. The resulted protein was then incubated with
β-(4-hydroxyphenyl)ethyl iodoacetamide (HPE-IAM, 10 mM)
to block the -SH and -SSH. Controls using untreated BSA were
also performed. Proteins were then digested with trypsin and
subjected to LC-MS/MS. As shown in Figure 8, in
thioglucose/GOx treated samples the extracted ion chromato-
gram (XIC) signals clearly showed a substantial level of
persulfide (-SSH) adduct on peptide GLVLIAFSQYLQQCPF-
DEHVK (Figure 8C). The site of persulfidation (Cys34) was
confirmed by higher-energy collision dissociation (HCD) MS/
MS (Figure 8D). Instead, the main observed peptide species
containing Cys34 in untreated BSA was the Cys34-thiol (-SH)
alkylated form (Figure 8A/B). In addition to BSA we also
tested this thioglucose/GOx method with two other proteins-
papain and GAPDH. Effective persulfidation was obtained on
both proteins (Figures S8 and S9 in Supporting Information).
It was interesting to discover that the slow and steady
production of H S and H O from thioglucose/GOx acted as a
2
2
2
enzyme-promoted system (column 11). Furthermore, exposure
of biothiols to thioglucose-GOx or the corresponding amounts
of Na S/H O showed no fluorescence increase (columns 12−
2
2
2
1
7). As a positive control, SSP4 showed high fluorescence
response to Na S (column 18) a persulfide standard. These
2
2
results clearly demonstrated that the thioglucose-GOx system
does not induce effective persulfide formation on small
molecular thiols.
Next, we tested if the combination of thioglucose and GOx
could lead to the formation of protein persulfides. In this study,
reduced BSA (30 μM) was incubated with thioglucose (200
μM) and GOx (10 μg/mL) in PBS at room temperature for 1
h. CAT (25 μg/mL) was then added to remove excess H O .
2
2
SSP4 (10 μM) was next applied to measure persulfide
3
3
formation. As demonstrated in Figure 7, this treatment
column h) led to an obvious increase in fluorescence,
2
2
2
(
more efficient protein persulfidation system than the direct
indicating the desired persulfide formation on BSA. Control
experiments, e.g. BSA treated with each individual reagent used
in the study (columns a-d, f, g), did not give significant
fluorescence. We also tested a known protein persulfidation
method using H O and H S. When the concentrations of
addition of H S and H O even at similar concentrations.
2
2
2,
While the detailed mechanism is still unclear, we propose the
following explanations (Scheme 2): In previous studies,
protein persulfidation was normally achieved by sequential
treatments with high concentrations of H O and then H S. A
2
2
2
2
2
2
1
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Scheme 2. Proposed Explanations on High Efficiency of
Protein Persulfidation by the Thioglucose/GOx System
problem with this method is overoxidation in the first step,
which produces protein sulfinic acid (P-SO H) or sulfonic acid
2
(
P-SO H). These species cannot be converted to P-SSH by
2
3
H S. This overoxidation may exist when BSA is treated with
bolus additions of H O /H S pair, which diminishes the
2
2
2
efficiency of persulfidation. Another possibility is that excess
H O would always exist in bolus additions and it could rapidly
2
2
react with the newly formed protein persulfides and therefore,
decrease persulfidation. It is known that persulfides (RSSH)
34,35
are highly reactive to H O to form RSSO H.
On the other
2
2
n
hand, the slow and steady H O /H S production from
2
2
2
thioglucose/GOx may be able to avoid the presence of excess
H O in the system, thus preventing the overoxidation
2
2
reactions.
It was also interesting to see that thioglucose/GOx induced
persulfidation worked effectively on proteins but not on small
molecules. This could be attributed to two reasons: (1) H O -
2
2
oxidation works less effectively on small molecule thiols as
compared to proteins. We analyzed the total − SH contents in
the reaction between Cys and thioglucose/GOx. As shown in
Figure S7, only a minor thiol concentration decrease in this
reaction was observed, suggesting Cys oxidation in this system
was slow. (2) Even small molecule thiols react with H O ; the
2
2
resultant sulfenic acids (RSOH) are much more unstable than
protein-derived sulfenic acids. They should further react with
another molecule of thiol to form a disulfide. As such,
persulfide formation on small molecules in this system will not
be feasible.
Having demonstrated that thioglucose-GOx is a controllable
platform for H S/H O dual release under biologically friendly
2
2
2
environments, we wondered if we could further tune the
releasability with a dual enzyme system or multistimuli
response. In this strategy, thioglucose could be considered as
a caged H S/H O vehicle. Engineering this motif with another
2
2
2
triggering group would enable their generation in response to
specific biological stimuli. To test this hypothesis, we prepared
1
-thio-β-D-lactose (thiolactose) which could be hydrolyzed by
β-galactosidase (β-Gal) to form galactose and thioglucose. As
such, only under the dual-enzyme catalysis (β-Gal and GOx)
will it produce H S and H O . As shown in Figure 9,
2
2
2
thiolactose was stable in PBS buffers. It did not release H2S
under the treatment of either β-Gal or GOx. However, in the
presence of both β-Gal and GOx, a time-dependent H2S
Figure 8. Extracted ion chromatograms (XIC) and MS/MS spectra of
the HPE-IAM labeled Cys34 containing peptide (GLVLIAFS-
QYLQQCPFDEHVK) from BSA, with or without the treatment of
thioglucose/GOx. (A) XIC of the −SH peptide, (B) MS/MS
spectrum of the −SH peptide, (C) XIC of the −SSH peptide, (D)
MS/MS spectrum of the −SSH peptide.
formation was observed with the peak concentration of ∼16
μM in about 2 h. The capability of H O delivery from this
2
2
thiolactose-dual enzyme system was also confirmed by AP/
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Figure 9. H S release profile of thiolactose (100 μM) in the presence
2
of β-Gal (10 U/mL) and GOx (10 μg/mL) in PBS buffer (50 mM,
pH 7.4). The experiments were performed in triplicate, and results are
expressed as mean ± SD (n = 3).
Figure 10. H S release profile of thioglucose disulfide (50 μM) in the
2
presence of GSH (0 μM, 50 μM, 250 μM, 500 μM and 1000 μM) and
GOx (10 μg/mL) in PBS buffer (50 mM, pH 7.4). The experiments
were performed in triplicate, and results are expressed as mean ± SD
(
n = 3).
HRP assay under the same conditions (Figure S10). It is worth
noting that elevated lysosomal β-Gal activity has been well-
known as an important biomarker for senescent cells and
3
6,37
primary ovarian cancers.
Also H S exhibits protective
2
38,39
demonstrated that thioglucose combined with GOx can serve
as a new platform for controlled H S/H O dual release with
effects against cellular senescence and certain cancers.
2
2
2
Thus, β-Gal activated H S donors like thiolactose might be
2
no bio-unfriendly byproducts. The generation of H O in this
2 2
explored as potential antiaging or anticancer agents.
system did not affect H S release. This thioglucose-GOx
2
To further demonstrate the tunability of the thioglucose-
GOx platform, we next synthesized thioglucose disulfide. This
symmetrical glucosyl disulfide is expected to undergo disulfide-
bond cleavage induced by biothiols to form thioglucose, which
could subsequently release H S and H O in the presence of
system was used to effectively induce protein S-persulfidation.
Moreover, thioglucose is a highly tunable motif and can be
readily modified to introduce additional release-control factors
so other biologically relevant stimuli can be used to regulate
the delivery of H S/H O . We expect this thiosugar-GOx
2
2
2
2
2
2
GOx. To test this idea, thioglucose disulfide (50 μM) was
platform will be a useful tool for elucidating the mechanisms of
H S/H O signaling and promoting H S based therapeutic
incubated with GSH (0−1000 μM) in PBS (50 mM, pH 7.4)
2
2
2
2
containing GOx (10 μg/mL), and its H S production was
2
applications.
monitored by MB assay. As shown in Figure 10, thioglucose
disulfide did not produce detectable H S in the absence of
GSH. However, in the presence of GSH, thioglucose disulfide
2
ASSOCIATED CONTENT
sı Supporting Information
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/jacs.1c06372.
■
*
exhibited dose- and time-dependent H S-releasing. The
2
formation of H O was also confirmed using AP/HRP assay
2
2
(
Figure S11). These results indicate that thioglucose disulfide
Compound characterizations, experimental protocols,
additional experimental data (PDF)
is an efficient thiol-activated H S/H O donor catalyzed by
2
2
2
GOx.
CONCLUSIONS
■
AUTHOR INFORMATION
Corresponding Author
■
Compounds that can release redox regulating molecules such
as H S and H O are not only useful research tools but also
2
2
2
Ming Xian − Department of Chemistry, Brown University,
Providence, Rhode Island 02912, United States;
orcid.org/0000-0002-7902-2987; Email: ming_xian@
brown.edu
potential therapeutic agents. While many such compounds
have been reported, a common problem is that they also
produce organic byproducts that could cause unwanted side
effects. This problem should be considered in the development
of next generation donor templates. In addition, current efforts
in this field tend to focus on individual regulating species,
which could miss the important crosstalk and synergistic effects
between multiple species. Nevertheless, the coexistence of
these species is real in nature but donors that can release
multiple ROS/RSS are still unavailable. In this work, we
attempted to solve the aforementioned challenges by
developing novel dual-releasing donor templates. We have
Authors
Xiang Ni − Department of Chemistry, Brown University,
Providence, Rhode Island 02912, United States
Xiaolu Li − Biological Sciences Division, Pacific Northwest
National Laboratory, Richland, Washington 99352, United
States
Tun-Li Shen − Department of Chemistry, Brown University,
Providence, Rhode Island 02912, United States
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Article
Wei-Jun Qian − Biological Sciences Division, Pacific Northwest
National Laboratory, Richland, Washington 99352, United
States; orcid.org/0000-0002-5393-2827
(17) Shukla, P.; Khodade, V. S.; SharathChandra, M.; Chauhan, P.;
Mishra, S.; Siddaramappa, S.; Pradeep, B. E.; Singh, A.; Chakrapani,
H. ″On demand″ redox buffering by H2S contributes to antibiotic
resistance revealed by a bacteria-specific H2S donor. Chem. Sci. 2017,
Complete contact information is available at:
https://pubs.acs.org/10.1021/jacs.1c06372
8
, 4967−4972.
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Notes
The authors declare no competing financial interest.
(19) Marinho, H. S.; Cyrne, L.; Cadenas, E.; Antunes, F.Chapter
Ten - H2O2 Delivery to Cells: Steady-State Versus Bolus Addition. In
Methods in Enzymology, Cadenas, E.; Packer, L., Eds. Academic Press:
ACKNOWLEDGMENTS
This work was supported by NIH (R01GM125968) and NSF
2100870). The mass spectrometry experiments described
herein were performed in the Environmental Molecular
Sciences Laboratory, Pacific Northwest National Laboratory,
a national scientific user facility sponsored by the Department
of Energy under Contract DE-AC05-76RL0 1830.
■
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