The Development of Fluorescent Probes for Visualizing Intracellular Hydrogen Polysulfides

Article abstract of DOI:10.1002/anie.201506887

Endogenous hydrogen polysulfides (H₂S_n; n>1) have been recognized as important regulators in sulfur-related redox biology. H₂S_n can activate tumor suppressors, ion channels, and transcription factors with higher potency than H₂S. Although H₂S_n are drawing increasing attention, their exact mechanisms of action are still poorly understood. A major hurdle in this field is the lack of reliable and convenient methods for H₂S_n detection. Herein we report a H₂S_n-mediated benzodithiolone formation under mild conditions. This method takes advantage of the unique dual reactivity of H₂S_n as both a nucleophile and an electrophile. Based on this reaction, three fluorescent probes (PSP-1, PSP-2, and PSP-3) were synthesized and evaluated. Among the probes prepared, PSP-3 showed a desirable off/on fluorescence response to H₂S_n and high specificity. The probe was successfully applied in visualizing intracellular H₂S_n.

Full text of DOI:10.1002/anie.201506887

Angewandte
Chemie
International Edition: DOI: 10.1002/anie.201506887
German Edition: DOI: 10.1002/ange.201506887
Fluorescent Probes
The Development of Fluorescent Probes for Visualizing Intracellular
Hydrogen Polysulfides
Wei Chen, Ethan W. Rosser, Tetsuro Matsunaga, Armando Pacheco, Takaaki Akaike, and
Ming Xian*
Dedicated to Professor Amos B. Smith III on the occasion of his 70th birthday
Abstract: Endogenous hydrogen polysulfides (H2Sn; n > 1)
have been recognized as important regulators in sulfur-related
redox biology. H2Sn can activate tumor suppressors, ion
channels, and transcription factors with higher potency than
H2S. Although H2Sn are drawing increasing attention, their
exact mechanisms of action are still poorly understood. A
major hurdle in this field is the lack of reliable and convenient
methods for H2Sn detection. Herein we report a H2Sn-mediated
benzodithiolone formation under mild conditions. This
method takes advantage of the unique dual reactivity of H2Sn
as both a nucleophile and an electrophile. Based on this
reaction, three fluorescent probes (PSP-1, PSP-2, and PSP-3)
were synthesized and evaluated. Among the probes prepared,
PSP-3 showed a desirable off/on fluorescence response to H2Sn
and high specificity. The probe was successfully applied in
visualizing intracellular H2Sn.
H2Sn from cystine. A series of recent reports have suggested
that H2Sn derivatives might be a new group of signaling
molecules.[2,3c,4] It was found that H2Sn can activate tumor
suppressors, ion channels, and transcription factors with
higher potency than H2S.[4] Some physiological activities
that were originally attributed to being mediated by H2S may
actually be mediated by H2Sn. One such example is S-
sulfhydration.[1a,5,6] This posttranslational modification was
previously thought to be the result of H2S activity. However,
recent results have demonstrated that H2Sn are more effective
than H2S in S-sulfhydration.[2,3b–c]
Although H2Sn molecules have now been recognized as
potent physiological mediators, a number of issues remain to
be clarified, such as the production and degradation pathways
of H2Sn, their regulatory mechanisms, and potential physio-
logical stimuli that induce those regulatory mechanisms. To
address these issues, it is critical to develop effective methods
for H2Sn detection. The traditional method is to measure UV/
Vis absorption bands at l = 290–300 nm and 370 nm, that is, at
the characteristic absorption bands of H2Sn.[2a] However, this
method has low sensitivity and is not applicable for biological
detections. Fluorescence assays could be useful because of
their high sensitivity and spatiotemporal resolution capability.
Although fluorescent probes for H2S have been extensively
studied,[7] the probes for H2Sn are still underdeveloped
because the chemistry of H2Sn is relatively unknown.
Our laboratory has initiated a program to study new
chemistry/reactions of H2Sn and to develop reaction-based
fluorescent probes for H2Sn. In 2014, we reported the first
H2Sn-specific probes (DSP; Scheme 1), which employed a 2-
fluoro-5-nitrobenzoic ester template to trap H2Sn and pro-
mote an intramolecular cyclization to release a fluorophore.[8]
DSP probes showed satisfactory sensitivity and selectivity for
H2Sn. Several other groups have adopted the same template to
develop H2Sn probes with interesting properties.[9] However,
a drawback of this probe type is that 2-fluoro-5-nitrobenzoic
esters can also react with biothiols. Although such reactions
do not turn on fluorescence, the consumption of the probes is
a problem. In theory, the reaction product, that is, thioether 1,
can further react with H2Sn to switch on fluorescence
(Scheme 1). We found that the reaction was somewhat slow
and low yielding when the H2Sn concentration was low (at mm
levels). Therefore, a high loading of DSP-type probes may be
needed for biological detection. Recently, we reported
another H2Sn-specific aziridine-opening reaction and devel-
oped a probe (AP) based on this reaction.[10] AP showed
R
eactive sulfur species (RSS) are a group of sulfur-contain-
ing molecules that play regulatory roles in biological systems.
Important RSS include biothiols and S-modified protein
cysteine adducts. RSS also include hydrogen sulfide (H2S) and
sulfane sulfur moieties, such as cysteine persulfides (R-S-SH)
and polysulfides (R-S-Sn-S-R’; n > 0). Understanding the
mechanisms of action of RSS derivatives has become a very
active research area in modern chemical biology, particularly
from the methodological point of view.[1] Among various RSS,
hydrogen polysulfides (H2Sn; n > 1) have attracted particular
attention, mainly because of their involvement in H2S-related
redox biology.[2] H2Sn can be generated from endogenous H2S
upon reaction with reactive oxygen species (ROS).[3] H2S can
also react with other sulfane sulfurs, such as S8, to form
H2Sn.[2d,3c,4] Moreover, H2Sn may have their own biosynthetic
pathways. For example, although CSE-mediated cysteine
metabolism (CSE = cystathionine g-lyase) can produce cys-
teine persulfide,[5] it may also catalyze direct generation of
[*] Dr. W. Chen, E. W. Rosser, A. Pacheco, Prof. Dr. M. Xian
Department of Chemistry, Washington State University
Pullman, WA 99164 (USA)
E-mail: mxian@wsu.edu
Dr. T. Matsunaga, Prof. Dr. T. Akaike
Department of Environmental Health Sciences and Molecular
Toxicology, Tohoku University Graduate School of Medicine
Sendai, 980-8575 (Japan)
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/anie.201506887.
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Scheme 2. The design of fluorescent probes exploiting the dual reac-
tivity of H2Sn.
toward thiols and H2Sn. If H2Sn could selectively react with an
appropriate thioester group, the product, that is, 3, should
further react with H2Sn (now serving as an electrophile) to
form 4. The following spontaneous cyclization should release
the fluorophore. Overall, this process would be specific for
H2Sn.
Scheme 1. Previous designs of H2Sn-specific fluorescent probes.
Based on this idea, we synthesized three probes (PSP-1,
PSP-2, and PSP-3; Scheme 3). Three different R groups (Me,
excellent selectivity for H2Sn and it did not react with biothiols
or H2S under physiological concentrations. However, due to
limited options for aziridine-based fluorophores, the sensi-
tivity of AP for H2Sn was not optimal and attempts to use AP
for imaging H2Sn in cells were not successful. Obviously
further improvement of the probes is needed. Herein, we
report the design, synthesis, and evaluation of a series of new
fluorescent probes (PSP1–3) that showed high sensitivity and
selectivity for H2Sn and can be used for endogenous H2Sn
imaging.
Scheme 3. The structures of PSP probes.
The key to the development of highly efficient fluorescent
probes is to identify a highly specific H2Sn recognition unit.
Ideally, such a recognition unit should only react with H2Sn
and not react with other sulfur species, especially biothiols.
From a chemistry perspective, H2Sn derivatives are quite
different from thiols or H2S. The estimated pKa values of H2Sn
are in the range of 3 to 5.[11] For comparison, the pKa values of
H2S and biothiols are in the range of 7 to 9.2. At physiolo-
gical pH, H2Sn derivatives are expected to be weak acids and
stronger and more reactive nucleophiles than biothiols and
H2S as a result of the alpha effect. Additionally, H2Sn belong
to the sulfane sulfur family. A character of sulfane sulfurs is
that they can function as electrophiles and react with certain
nucleophiles.[12] Overall, H2Sn derivatives have a unique dual
reactivity and can act as both nucleophiles and electrophiles.
Taking advantage of this property, we envisioned that
template 2 (Scheme 2) might be specific for H2Sn derivatives.
In 2, a thioester was employed to trap the nucleophilicity of
H2Sn. One concern with this structure is that the thioester
group could also react with biothiols, seeing as reactions
between thioesters and thiols are known, for example, in
native chemical ligation.[13] However, those reactions are
mostly for synthetic purposes with high concentrations of
reactants and special solvent conditions. The relatively low
concentrations of biothiols in biological systems and the mild,
neutral, and aqueous environment may make the reactions
slow and low yielding. In addition, we expected that the
manipulation of R groups in 2 (in terms of both steric and
electronic effects) could significantly alter its reaction rates
tBu, and Ph) were used to explore the effects of acyl groups on
the reactivity of thioesters toward biothiols. We first tested
their fluorescence properties and responses to H2Sn in PBS
(phosphate-buffered saline). Freshly prepared Na2S2 solu-
tions were used as the equivalents of H2Sn. All three probes
showed almost no fluorescence emission with very low
fluorescence quantum yields (F; FPSP-1 = FPSP-2 = 0.02,
FPSP-3 = 0.01) because of the protection of the two hydroxy
groups of fluorescein. Upon the treatment of the probes with
Na2S2 (5 equiv) for 30 minutes, the fluorescence intensities of
the probes were significantly enhanced (Figure 1A). PSP-
1 and PSP-3 exhibited greater increases in fluorescence
intensity than PSP-2. Presumably the bulky tert-butyl group
of PSP-2 decreased the reactivity toward to H2Sn. We also
measured the time-dependent changes in the fluorescence
Figure 1. A) Fluorescence intensity increases (DIfl) of PSP (10 mm)
upon treatment with Na2S2 (50 mm): 1) PSP-1; 2) PSP-2; 3) PSP-3.
B) Time-dependent fluorescence intensity changes of PSP (10 mm) in
the presence of Na2S2 (50 mm).
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intensities of the probes in the presence of Na2S2 (Figure 1B).
The maximum emission intensities of PSP-1 and PSP-3 were
reached within 2 minutes and 10 minutes respectively, indi-
cating that fluorescence turn-on was fast. The fluorescence
turn-on of PSP-2 was slower (circa 20 min), again suggesting
that steric effects played a role in its reactivity. For the
purpose of reproducibility, a reaction time of 30 minutes was
employed in all of the following experiments.
1 appeared to be labile to biothiols while PSP-2 and PSP-3
were stable. Therefore, the use of PSP-2 and PSP-3 should
not be affected by cellular biothiols.
Having demonstrated the excellent reactivity and stability
of PSP-3, this probe was selected for further studies. We next
validated its selectivity for H2Sn over other common RSS
reagents including homocysteine (Hcy), glutathione disulfide
(GSSG), H2S, SO32À, S2O32À, SO42À, and CH3SSSCH3. As
shown in Figure 3A, no fluorescence increase was observed
Next we studied the stability of the probes in the presence
of biothiols. As shown in Scheme 4, if the probes undergo
Figure 3. A) Fluorescence intensity (Ifl) of PSP-3 (10 mm) at l=515 nm
in the presence of various RSS: 1) probe alone; 2) Hcy (100 mm);
3) GSSG (100 mm); 4) Na2S (200 mm); 5) Na2S2O3 (100 mm); 6) Na2SO3
(100 mm); 7) Na2SO4 (100 mm); 8) CH3SSSCH3 (100 mm); 9) Na2S2
(50 mm); 10) Na2S4 (50 mm). B) Fluorescence intensity of PSP-3
(10 mm) in the presence of ROS (with or without H2S). 1) probe alone;
2) H2O2 (200 mm); 3) ClOÀ (50 mm); 4) O2À (50 mm); 5) COH (50 mm);
6) 1O2 (50 mm); 7) H2O2 (200 mm) + Na2S (50 mm); 8) H2O2 (200 mm)
+ Na2S (100 mm); 9) ClOÀ (50 mm) + Na2S (100 mm); 10) O2À (50 mm)
+ Na2S (100 mm); 11) COH (50 mm) + Na2S (100 mm); 12) 1O2 (50 mm)
+ Na2S (100 mm).
Scheme 4. Possible reactions of PSP probes with biothiols and S8.
R’-SH=Cys or GSH.
thioester exchange with biothiols, the resultant product 5
should be sensitive to sulfane sulfurs, such as elemental sulfur
(S8), and the resultant product should be strongly fluorescent.
Therefore, measuring the fluorescence of the probes in
a mixture of thiols and S8 should be suitable for evaluating
the stability of the probes to thiols.
We then tested the fluorescence responses of the probes
(10 mm) to Cys (1 mm) and glutathione (GSH; 5 mm) in the
absence or presence of S8 (50 mm). As shown in Figure 2, Cys,
GSH, or S8 alone did not induce any response. The mixtures
of biothiols (GSH or Cys) and S8 produced strong fluores-
cence for PSP-1. The same mixtures led to almost no increase
in fluorescence intensity for PSP-2 and PSP-3. As a positive
control, the thioester exchange product 5 was strongly
fluorescent in the presence of S8 under the same conditions.
These results confirmed our hypothesis that the acyl group of
the thioester could affect its reactivity towards thiols. PSP-
for these RSS (columns 2–8). Only Na2S2 and Na2S4 triggered
significant increases in fluorescent intensity (columns 9
and 10). Additionally, it is known that H2Sn could be
generated from the reactions between H2S and ROS. There-
fore, we wondered if the probe could sense in situ H2Sn
formation from H2S and ROS. As shown in Figure 3B, the
responses of PSP-3 to a series of ROS including hydrogen
À
peroxide (H2O2), hypochlorite (ClOÀ), superoxide (O2 ),
hydroxyl radical (COH), and singlet oxygen (1O2) were first
tested. These ROS did not induce any fluorescence enhance-
ments (columns 2–6). When H2S (in the form of Na2S) was
added to these ROS, a strong fluorescence signal was
observed from ClOÀ (column 9), but not from other ROS.
These results correspond to previous reports that H2Sn can be
efficiently derived from H2S and ClOÀ, but other ROS are not
effective for H2Sn generation under these concentrations.[3,8–10]
The responses of PSP-3 in the presence of other representa-
tive amino acids were also evaluated and no responses were
observed (see Figure S1 in the Supporting Information).
To further evaluate the sensitivity of PSP-3 for H2Sn,
a series of varied concentrations of Na2S2 (0–50 mm) were
tested. The fluorescence intensities were linearly related to
the concentrations of Na2S2 employed in the range of 0–20 mm
(Figure S2). The detection limit was calculated to be 3 nm.
This is the most sensitive probe reported to date for H2Sn. To
clarify the mechanism of fluorescence turn-on, we studied the
reactions between the probes and Na2S2. All of the reactions
gave fluorescein and benzodithiolone as the isolated products
in good yields (80–90%; Figure S3). As such, we believe that
Figure 2. Increases in the fluorescence intensities (DIfl) of each probe
(10 mm) in the presence of biothiols and S8: a) Probe + GSH (5 mm);
b) Probe + Cys (1 mm); c) Probe + S8 (50 mm); d) Probe +
GSH (5 mm) + S8 (50 mm); e) Probe + Cys (1 mm) + S8 (50 mm);
f) Compound 5 (10 mm) + S8 (50 mm).
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fluorescence turn-on is initiated by H2Sn-mediated thioester
cleavage. The resulting thiolate further reacts with H2Sn and
promotes cyclization to form benzodithiolone and release the
fluorophore.
Next we wondered if PSP-3 could be used for imaging
H2Sn in cells. We first validated the capability of PSP-3 in the
visualization of exogenous H2Sn in cells. As shown in Figure 4,
Having demonstrated the capability of PSP-3 in detecting
exogenous H2Sn in cells, we then sought to use PSP-3 to
monitor endogenous H2Sn formation. Our recent studies
found that CSE overexpression causes significant elevation of
persulfide and polysulfide levels in cells.[5] Therefore, cells
which overexpress CSE provide a suitable system for
endogenous H2Sn production. As shown in Figure 5a–d,
normal and CSE-overexpressed[5] COS-7 cells were sepa-
rately treated with PSP-3 for 30 minutes. As expected,
significant fluorescent enhancement was detected for CSE-
overexpressed cells as compared to normal cells. In fact, an
appreciable amount of H2Sn was directly generated from
a recombinant CSE in a cell-free reaction mixture containing
cystine as a substrate (Figure S6). These results demonstrate
that PSP-3 is suitable for detecting endogenous H2Sn. Addi-
tionally, Kimura et al. recently described a new H2S biosyn-
thetic pathway from d-Cys in mammalian cells, which occurs
predominantly in the kidney and the cerebellum.[14] This
process seems to be regulated by 3-mercaptopyruvate sulfur-
transferase (3MST) and d-amino acid oxidase (DAO). It was
suggested that H2S produced from d-Cys might be stored as
bound sulfane sulfur and the levels of sulfane sulfur can be
significantly enhanced by d-Cys. In theory, H2Sn could be
produced from the reactions between H2S and sulfane sulfurs.
Therefore, d-Cys stimulation in Vero cells may be a useful
system for endogenous H2Sn generation and this system was
used to further validate the probeꢀs sensitivity for endogenous
H2Sn. As shown in Figure 5e–h, the Vero cells were incubated
with PSP-3 (5 mm) for 25 minutes at 378C. The control
(Figure 5e) showed almost no fluorescence. When cells were
treated with d-Cys (30 mm), a modest but obvious fluores-
cence signal was observed (Figure 5 f). When the concentra-
tion of d-Cys was increased to 1 mm, we observed strong
fluorescence (Figure 5g). Moreover, when the cells were first
treated with sodium benzoate (a DAO inhibitor), the
stimulation by d-Cys (1 mm) led to significantly weakened
fluorescence. Taken together, these results indicate that
d-Cys and DAO may be responsible for cellular H2Sn
production.
Figure 4. Fluorescence images of exogenous H2Sn in HeLa (a–c),
RAW264.7 (d–f), and Vero (g–i) cells. Cells were incubated with PSP-3
(5 mm) for 25 min, then washed and subjected to different treatments.
a,d,g) Controls (no added Na2S2 or Na2S4); b,e,h) cells treated with
Na2S2 (30 mm); c,f,i) cells treated with Na2S4 (30 mm).
HeLa, RAW264.7, and Vero cells were respectively incubated
with PSP-3 (5 mm) for 25 minutes. Any extracellular probe
was washed off, and no significant fluorescence was observed
within the cells (Figure 4a,d,g). However, when cells were
treated with Na2S2 or Na2S4 (30 mm) for 20 minutes, strong
fluorescence was observed. These results demonstrated that
PSP-3 has good cell permeability and could
be used to monitor H2Sn in cells. Additionally,
a cell viability assay demonstrated that PSP-3
has no cytotoxicity to cells (Figure S4).
H2Sn derivatives are short-lived species
and can readily decompose in buffers. With
this in mind, in situ generation of H2Sn from
H2S and ClOÀ provides a more reliable and
sustainable system for H2Sn production.
Herein PSP-3 was used to monitor the
in situ generation of H2Sn in cells. As shown
in Figure S5, neither ClOÀ nor H2S gave
noticeable fluorescence responses whereas
the mixture of H2S/ClOÀ led to a strong
fluorescence enhancement, which was even
brighter than those obtained with exogenous
H2Sn. These results confirmed that in situ
generation of H2Sn by H2S and ClOÀ is a more
effective system than simply using Na2S2 (or
Na2S4) to maintain H2Sn levels in cells.
Figure 5. Fluorescence images of endogenous H2Sn in COS-7 (a–d) and Vero (e–h) cells.
a) Normal cells not treated with PSP-3; b) Normal cells treated with PSP-3; c) CSE-
overexpressed cells treated with PSP-3; d) normal cells treated with Na2S2 (20 mm) and
PSP-3. e–g) Cells were incubated with PSP-3 (5 mm) for 25 min, washed, and subjected to
different treatments. e) Cells were incubated with FBS-free media (FBS=fetal bovine
serum) for 30 min; f) cells were incubated with d-Cys (30 mm) for 30 min; g) cells were
incubated with d-Cys (1 mm) for 30 min; h) cells were pretreated with sodium benzoate
(250 mm) for 30 min, then washed and incubated with PSP-3 (5 mm) for 25 min, and then
washed and incubated with d-Cys (1 mm) for 30 min.
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In summary, we report here the development of three new
fluorescent probes (PSP-1, PSP-2, PSP-3) which exploit the
unique dual reactivity of H2Sn as both a nucleophile and an
electrophile. Among the probes prepared, PSP-3 showed an
off/on fluorescence response to H2Sn as well as high specific-
ity. PSP-3 was also successfully applied in the visualization of
both exogenous and endogenous H2Sn in cells. This novel
probe overcomes the major limitations of previously reported
H2Sn probes and it is expected to serve as a useful tool in
understanding the physiological functions of H2Sn.
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Acknowledgements
This work was supported by an ACS Teva Scholar Grant and
the NIH (R01HL116571). This work was also supported in
part by Grants-in-Aid for Scientific Research and Grants-in-
Aid for Scientific Research on Innovative Areas from MEXT
of Japan.
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Received: July 24, 2015
Published online: September 18, 2015
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