Development of a reversible fluorescent probe for reactive sulfur species, sulfane sulfur, and its biological application

Article abstract of DOI:10.1039/c6cc08372b

We report a reversible off/on fluorescent probe for monitoring concentration changes of sulfane sulfur by utilizing the unique ability of sulfane sulfur to bind reversibly to other sulfur atoms and the intramolecular spirocyclization reaction of xanthene dyes. It reversibly visualized sulfane sulfur in living A549 cells and primary-cultured hippocampal astrocytes.

Full text of DOI:10.1039/c6cc08372b

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Hanaoka, K. Shimamoto, R. Miyamoto, T. Komatsu, T. Ueno, T. Terai, H. Kimura, T. Nagano and Y. Urano,
Chem. Commun., 2016, DOI: 10.1039/C6CC08372B.
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Marilyn M. Olmstead, Alan L. Balch, Josep M. Poblet, Luis Echegoyenet al.
Reactivity differences of Sc3N@C2n (2n 68 and 80). Synthesis of the
first methanofullerene derivatives of Sc N@D5h-C80
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Development of a Reversible Fluorescent Probe for Reactive
Sulfur Species, Sulfane Sulfur, and its Biological Application
Yoko Takano,a Kenjiro Hanaoka,a,* Kazuhito Shimamoto,a Ryo Miyamoto,b Toru Komatsu,a,c Tasuku
Ueno,a Takuya Terai,a Hideo Kimura,b Tetsuo Naganod and Yasuteru Uranoa,e,f,*
Received 00th January 20xx,
Accepted 00th January 20xx
DOI: 10.1039/x0xx00000x
www.rsc.org/
We report a reversible off/on fluorescent probe for monitoring oxidized by polysulfide, resulting in its inhibition.5 It is also
concentration changes of sulfane sulfur by utilizing the unique reported that sulfane sulfur species are biosynthesized by
ability of sulfane sulfur to bind reversibly to other sulfur atoms three enzymes which have generally been regarded as H2S-
and the intramolecular spirocyclization reaction of xanthene dyes. producers. Akaike et al. demonstrated that cystathionine γ-
It reversibly visualized sulfane sulfur in living A549 cells and lyase (CSE) and cystathionine β-synthase (CBS) are capable of
primary-cultured hippocampal astrocytes.
directly generating cysteine persulfide, Cys-SSH, from cystine,2
and Kimura et al. reported that H2S3 and H2S2, as well as H2S,
are produced from 3-mercaptopyruvate (3MP) by 3-
mercaptopyruvate sulfurtransferase (3MST) in the brain.6
The increasing recognition of the importance of sulfane
sulfur in biological systems has led to the development of
various detection methods for sulfane sulfur. The unique
chemical characteristics of sulfane sulfur species have been
utilized in fluorescent probe design. For example, persulfides
are more acidic and nucleophilic than the corresponding thiols
by 1–2 pKa units due to the α-effect.7,8 On the other hand,
tautomerization of persulfide to thiosulfoxide also generates
reactive species that are very electrophilic and highly reactive
to nucleophiles. Moreover, sulfane sulfur can reversibly bind
to other sulfur atoms. Based on these characteristics,
fluorescent probes such as the SSP, DSP and PSP series have
recently been developed by Xian et al. to detect sulfane sulfur
selectively in terms of a fluorescence increase.9-11 However,
these probes react irreversibly, and therefore they cannot
visualize the dynamics of intracellular sulfane sulfur. So, we set
out to develop a reversible fluorescent probe to monitor the
intracellular dynamics of sulfane sulfur.
Sulfane sulfur is one of reactive sulfur species, and is a form
of sulfur with six valence electrons and no charge (S0). Various
sulfane sulfur molecules exist in biological systems, in the
forms of persulfides (R–S–SH), polysulfides (R–S–Sn–S–R),
hydrogen polysulfides (H2Sn), and protein-bound cysteine
persulfide.1 Low-molecular-weight persulfides such as cysteine
and glutathione persulfides exist at μM order concentration
inside cells.2 Persulfides are highly reducing and nucleophilic,
and therefore they are capable of scavenging oxidants and
intracellular electrophiles. Further, some physiological
phenomena that were originally attributed to the bioactivity of
H2S may actually be mediated by sulfane sulfur, though this
remains controversial3; one example is the S-sulfhydration
posttranslational modification, which was considered to be
due to H2S activity.4 However, recent studies have
demonstrated that persulfides are more effective than H2S for
S-sulfhydration; for example, Dick et al. reported that lipid
phosphatase PTEN (phosphatase and tensin homolog) was
For this purpose, we focused on two facts: 1) the unique
ability of sulfane sulfur to reversibly bind to other sulfur atoms
and 2) the intramolecular spirocyclization reaction of xanthene
dyes. As shown in Figure 1a, we anticipated that the
introduction of a thiol group into the benzene moiety of
xanthene dyes would allow persulfide to be generated by
transfer of sulfur from sulfane sulfur species to the thiol group,
and then intramolecular spirocyclization would occur because
of the high nucleophilicity of persulfide. Indeed, we found that
2-thio RB showed an absorbance decrease in the visible region
upon addition of 50 μM Na2S4 (Figure 1b,c). To examine
whether or not this absorbance decrease is associated with
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spirocyclization of 2-thio RB, we synthesized 2-Me, 2-OH, and has a large overlap integral with the absorption spectrum of 2-
2-SMe rhodamine B and examined their reactivity with sulfane thio RB, and thus we expected that energy transfer from
sulfur (Figure S1). These compounds did not show any fluorescein to 2-thio RB would occur efficiently via FRET.14
absorbance decrease. This result suggests that direct
nucleophilic attack of Na2S4 on the xanthene moiety does not
occur. We also measured the ESI-MS spectrum of the reaction
mixture of 2-thio RB and Na2S4, and observed a characteristic
MS peak of the spirocyclized form of 2-thio RB (Figure 1d).
These results confirmed that the absorbance decrease at 560
nm is due to formation of a persulfide group, followed by
intramolecular spirocyclization.
Figure 2. (a) Chemical structure of SSip-1 and reaction
mechanism with sulfane sulfur. (b,c) Changes of absorption (b)
and fluorescence (c) spectra of 1 μM SSip-1 before (blue) and
after (red) reaction with 50 μM Na2S4. Both spectra were
measured in 100 mM sodium phosphate buffer (pH 7.4)
containing 100 μM GSH, 0.1% DMSO as a cosolvent, and 1
mg/mL BSA. (d) Photos showing fluorescence of SSip-1 before
Figure 1. (a) Proposed mechanism of the intramolecular (−) and aꢀer (+) reacꢁon with 50 μM Na2S4 (irradiated at 365
spirocyclization and the ring-opening reaction of 2-thio RB. (b) nm with a handy UV lamp)
Absorption spectra of 1 μM 2-thio RB before (blue) and after
(red) reaction with 50 μM Na2S4. Spectra were measured in
The fluorescence and absorbance spectra of the probe were
100 mM sodium phosphate buffer (pH 7.4) containing 100 μM measured (Figure 2b-d). The probe showed absorption maxima
GSH to reduce the intermolecular disulfide bond of dimerized (λmax) at 480 and 560 nm, and weak fluorescence peaks
2-thio RB. Cosolvent: 0.1% DMSO. (c) Photos of 2-thio RB derived from both fluorescein and rhodamine were also
before (−) and aꢀer (+) reacꢁon with 50 μM Na2S4. (d) ESI-MS observed. Upon addition of 50 μM Na2S4 to the solution, the
spectrum of the reaction mixture of 2-thio RB and Na2S4. Peaks absorbance at 560 nm decreased and the fluorescence at 525
I and II were assigned to 2-thio RB ([M]+) and its spirocyclized nm increased within
1 min. The apparent fluorescence
form ([M+H]+) shown in (a), respectively.
quantum yield increased from 0.015 to 0.20 (for details, see
Supporting Information). The fluorescence intensity was well
Next, we examined whether this compound shows the same correlated to the concentration of added Na2S4 in the range of
absorbance decrease in the presence of 5 mM GSH, because 0.5-20 μM in the absence of GSH (Figure 3a). The detection
GSH is reported to be present at the concentration of 0.5-10 limit was found to be 0.42 μM.
mM inside cells.12 The absorbance decrease upon addition of
We next examined whether this probe can reversibly
Na2S4 occurred even in the presence of 5 mM GSH, but the respond to sulfane sulfur by addition of 5 mM GSH (Figure 3b).
absorbance gradually recovered thereafter (Figure S2a). This Treatment of SSip-1 with 50 μM Na2S4 resulted in 15-fold
recovery was not observed in the absence of 5 mM GSH fluorescence increase due to intramolecular spirocyclization of
(Figure S2b). So, we think that GSH reduced the intramolecular the FRET acceptor (2-thio RB moiety). Subsequently, the
disulfide bond of the spirocycle, regenerating the open form of intramolecular disulfide bond of the spirocycle was slowly
2-thio RB, as shown in Figure 1a, and thereby resulting in reduced by GSH, causing the fluorescence at 525 nm to
recovery of the absorbance. Further, since 2-thio RB is weakly decrease due to the restoration of FRET between fluorescein
fluorescent, probably due to photoinduced electron transfer and 2-thio RB. The fluorescence intensity values were well
(PeT),13 we expected that it would act as a fluorescence correlated to the amount of added Na2S4 even in the presence
quencher. Therefore, we considered that 2-thio RB would be of 5 mM GSH (Figure S3). The fluorescence intensity probably
available as the reversible off/on switching moiety of a depends upon the equilibrium between sulfane sulfur and GSH, and
fluorescent probe for sulfane sulfur.
therefore this probe can monitor the concentration change of
Accordingly, we designed and synthesized
a
novel sulfane sulfur. But, to measure the exact concentration of sulfane
fluorescent probe for sulfane sulfur, SSip-1, in which 2-thio RB sulfur in equilibrium with GSH, we would need to prepare a
serves as the Förster resonance energy transfer (FRET) standard curve using other methods, such as LC-MS analysis. The
acceptor (fluorescence quencher) and fluorescein as the FRET fluorescence decrease was also correlated to the amount of
donor (Figure 2a). The fluorescence spectrum of fluorescein added GSH (Figure S4). Polysulfides such as Na2S4 are rapidly
2 |Chem. Commun., 2012, 00, 1-3
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decomposed by GSH through thiol−disulfide exchange, hydrophilicity derived from the carboxyl group and the thiol
releasing H2S.15 We found that the reversible response to group, we synthesized diacetylated and thiol-protected SSip-1
sulfane sulfur could be repeated at least four times with no (SSip-1 DA) to improve its membrane permeability (Figure 4a).
loss of dynamic range of the probe, i.e., the fluorescence Indeed, this compound proved to be a membrane-permeable
intensity maximum (Figure S5). As the addition of Na2S4 was precursor that is converted to SSip-1 by intracellular esterases
repeated, we interestingly found that the fluorescence and reductive conditions probably related to GSH (Figure S7).
intensity did not decrease completely to the original level even Therefore, A549 cells were incubated with 10 μM SSip-1 DA
at 60 min after addition of Na2S4. We confirmed that this result DMEM for 1 h and then washed with HBSS. Upon addition of
was not due to decomposition of the probe by measuring the 10 μM Na2S4, a large intracellular fluorescence enhancement
fluorescence increase upon addition of SSip-1 to premixed was observed, while almost no fluorescence was observed in
solutions of various concentrations of Na2S4 and 5 mM GSH the absence of Na2S4 (Figure S8). This fluorescence increase
(Figure S6). We consider that sulfane sulfur may be gradually was directly related to the concentration of added Na2S4
stored in the reaction mixture due to stabilization by GSH, i.e., (Figure S9). The result of CCK-8 assay also showed that SSip-1
sulfane sulfur may exist in the form of glutathione persulfides, DA exhibited no cytotoxicity at concentrations up to 100 μM
etc. We also examined the selectivity of SSip-1 for sulfane (Figure S10). Then, we investigated whether this probe can
sulfur. As shown in Figure 3c, the fluorescence enhancement reversibly detect sulfane sulfur in living cells. A549 cells were
of SSip-1 occurred only upon addition of polysulfides. No loaded with 10 μM SSip-1 DA for 1 h; almost no fluorescence
fluorescence increase was observed in the presence of other was observed (Figure 4c). However, addition of 10 μM Na2S4 to
biothiols, inorganic sulfur compounds, or H2S. Thus, SSip-1 the extracellular medium resulted in a substantial fluorescence
shows high selectivity for sulfane sulfur.
increase in the intracellular region within 1 min (Figure 4d).
During further incubation for 30 min, the intracellular
fluorescence decreased to the basal level (Figure 4e). Second
addition of 10 μM Na2S4 to the medium resulted in a
fluorescence increase in the intracellular region to the same
level as that after the first addition of 10 μM Na2S4 (Figure 4f).
The fluorescence intensity again decreased to the original level
when the cells were incubated for another 30 min (Figure
4b,g). These results confirm that SSip-1 can reversibly detect
sulfane sulfur in living cells. The detection cycle could be
repeated at least three times (data not shown).
5 mM
GSH
a
b
20 µM
Na2S4
800
600
400
200
0
800
600
400
200
0
0
5
10
15
20
0
15
30
Na2S4 (µM)
Time (min)
c
20
15
10
5
0
1
2
3
4
5
6
7
8
9
10 11
Figure 3. (a) Fluorescence intensity of SSip-1 (1 μM) upon
addition of various concentrations of Na2S4 (0, 2.5, 5, 7.5, 10,
12.5, 15, 17.5, 20 μM). Fluorescence intensity was measured
60 min after addition of Na2S4. (b) Fluorescence intensity
change of SSip-1 after addition of 20 μM Na2S4 followed by 5
mM GSH. The fluorescence intensity (525 nm) of the reaction
mixture was monitored. λex = 470 nm. SSip-1 was treated with
Figure 4. (a) Chemical structure of SSip-1 DA. (b) Fluorescence
intensity change of A549 cells loaded with SSip-1 DA upon
repeated additions of 10 μM Na2S4. Data represent the mean ±
SD (n = 5). The mean fluorescence intensity of 5 cells is shown
in the graph. (c-g) Fluorescence confocal microscopic images of
A549 cells loaded with SSip-1 DA upon repeated additions of
10 μM Na2S4. Scale bar: 75 μm.
20 μM Na2S4 and then reduced with
5 mM GSH. (c)
Fluorescence enhancement of 1 μM SSip-1 measured in 100
mM sodium phosphate buffer (pH 7.4) with 10 μM TCEP (to
reduce the intermolecular disulfide bond of dimerized SSip-1)
upon addition of various thiol species. Bars represent the
relative fluorescence intensities 1 min after addition of various
thiol species. The reactions of 1 μM SSip-1 with (1) 5 mM GSH,
(2) 1 mM L-Cys, (3) 1 mM L-Hcys, (4) 1 mM GSSG, (5) 100 μM
Na2S2O3, (6) 100 μM Na2SO3, (7) 100 μM Na2SO4, (8) 50 μM
Na2S, (9) 50 μM Na2S2, (10) 50 μM Na2S3 and (11) 50 μM Na2S4
were examined.
Finally, we applied SSip-1 DA to primary-cultured
hippocampal astrocytes. It is reported that polysulfides induce
Ca2+ influx in rat astrocytes by activating transient receptor
potential (TRP) A1 channels through modification of N-
terminal cysteine residues.16,17 However, it has not been
established whether the activation of TRPA1 channels occurs
simultaneously with the increase in intracellular concentration
of polysulfides. So, we attempted to visualize the
We next examined the suitability of SSip-1 for fluorescence
imaging of intracellular sulfane sulfur. Since this probe was
expected to be membrane-impermeable because of its
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concentration change of intracellular polysulfide in primary-
In summary, we designed a reversible off/on fluorescent
cultured hippocampal astrocytes and also to monitor probe for sulfane sulfur based on the findings that 2-thio RB
activation of TRPA1 channels induced by Na2S3 by fluorescence reacts with sulfane sulfur to afford an intramolecular
imaging of intracellular Ca2+. In this experiment, we used Na2S3 spirocycle, and this spirocycle is cleaved in the presence of 5
as a sulfane sulfur donor because H2S3 is a dominant form of mM GSH (a typical intracellular concentration). The
H2Sn produced by 3MST6; Na2S3 is known to possess almost the spirocyclization is accompanied with a marked decrease of
same properties as Na2S4 as
a sulfane sulfur donor. absorbance at 560 nm. Since 2-thio RB is weakly fluorescent,
Hippocampi dissected from embryonic rats were dispersed, we considered that it could act as a quencher, and could serve
and the cells were cultured for 14 days. The cultured a reversible fluorescence off/on switching moiety. Therefore,
astrocytes were loaded with either 10 µM SSip-1 DA or 2 µM we synthesized SSip-1, in which 2-thio RB serves as a FRET
Fluo-4 AM (Ca2+ indicator) and then Na2S3 was applied. acceptor and fluorescein as a FRET donor. We found that SSip-
Changes in sulfane sulfur and Ca2+ concentrations were 1 reacts with sulfane sulfur to afford a highly fluorescent,
monitored as changes of fluorescence intensity relative to the intramolecularly spirocyclized structure, which is subsequently
corresponding control image acquired before stimulation cleaved by intracellular GSH to regenerate the weakly
(Figure 5). SSip-1 DA successfully visualized the increase of fluorescent open form. This probe successfully and repeatedly
sulfane sulfur concentration in astrocytes, which was visualized concentration changes (both increase and decrease)
dependent on the amount of added Na2S3 (Figure 5a,b). of intracellular sulfane sulfur in living cells. We expect that this
Reversibility of SSip-1 was also confirmed, i.e., the probe will be a useful tool for exploring the physiological
fluorescence intensity increased after addition of Na2S3, then functions of sulfane sulfur.
decreased upon wash-out of the extracellular medium. This
procedure was repeated three times with different amounts of
added Na2S3. The intracellular Ca2+ concentrations also
changed, dependently on the amount of added Na2S3 (Figure
5c,d). These results indicate that the extent of Ca2+ influx was
closely related to the intracellular concentration of polysulfide
in the astrocytes.
Notes and references
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*
6
5
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3
2
1
0
a
b
SSip-1
3
2
1
0
*
5
6
7
8
Na2S3 (ꢀM)
30
0.1
1
10
25
0.1
1
10
0
5
10
15
20
Na2S3 (µM)
Time (min)
4
5
4
3
2
1
0
c
n.s.
Fluo-4
d
3
2
1
0
*
9
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Na2S3 (ꢀM)
0.1
10
1
0
5
10
15
20
25
30
0.1
1
10
Time (min)
Na2S3 (µM)
Figure 5. Changes in intracellular polysulfide and Ca2+ levels
induced by Na2S3 in cultures of hippocampal astrocytes. (a)
Increases in intracellular polysulfide levels induced by addition
of Na2S3 to the extracellular medium. Na2S3 (0.1, 1 or 10 µM)
was applied to astrocytes pre-loaded with 10 µM SSip-1 DA for
60 min at 37°C. (b) Graphic representation of (a). (c) Increases
in intracellular Ca2+ levels induced by addition of Na2S3 to the
extracellular medium. Na2S3 was applied to astrocytes pre-
loaded with 2 µM Fluo-4 AM for 30 min at room temperature.
(d) Graphic representation of (c). All data represent the mean
± SEM (n = 10). *: p<0.05
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We developed a reversible fluorescent probe for sulfane sulfur.
This probe enabled us to monitor concentration changes of
sulfane sulfur.
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