Rational design and bioimaging applications of highly selective fluorescence probes for hydrogen polysulfides

Article abstract of DOI:10.1021/ja502968x

Reactive sulfur species have received considerable attention due to their various biological functions. Among these molecules, hydrogen polysulfides (H₂S_n, n > 1) are recently suggested to be the actual signaling molecules derived from hydrogen sulfide (H₂S). Hydrogen polysulfides may also have their own biosynthetic pathways. The research on H₂S_n is rapidly growing. However, the detection of H ₂S_n is still challenging. In this work we report a H ₂S_n-mediated benzodithiolone formation under mild conditions. Based on this reaction, specific fluorescent probes for H ₂S_n are prepared and evaluated. The probe DSP-3 shows good selectivity and sensitivity for H₂S_n.

Full text of DOI:10.1021/ja502968x

Communication
pubs.acs.org/JACS
Open Access on 05/08/2015
Rational Design and Bioimaging Applications of Highly Selective
Fluorescence Probes for Hydrogen Polysulfides
Chunrong Liu,†,§ Wei Chen,†,§ Wen Shi,‡ Bo Peng,† Yu Zhao,† Huimin Ma,‡ and Ming Xian*,†
†Department of Chemistry, Washington State University, Pullman, Washington 99164, United States
‡Key Laboratory of Analytical Chemistry for Living Biosystems, Institute of Chemistry, Chinese Academy of Sciences, Beijing 100190,
China
S
* Supporting Information
(SOH) to form S-sulfhydration.12,13 Recently the possibility that
the reaction is caused by H2Sn has been revealed.14−17 From a
ABSTRACT: Reactive sulfur species have received
considerable attention due to their various biological
functions. Among these molecules, hydrogen polysulfides
(H2Sn, n > 1) are recently suggested to be the actual
signaling molecules derived from hydrogen sulfide (H2S).
Hydrogen polysulfides may also have their own bio-
synthetic pathways. The research on H2Sn is rapidly
growing. However, the detection of H2Sn is still
challenging. In this work we report a H2Sn-mediated
benzodithiolone formation under mild conditions. Based
on this reaction, specific fluorescent probes for H2Sn are
prepared and evaluated. The probe DSP-3 shows good
selectivity and sensitivity for H2Sn.
reactivity point-of-view, H2Sn should be much more effective in
S-sulfhydration than H2S. Kimura found that H2Sn were indeed
hundreds times more potent than H2S in inducing Ca2+ influx in
astrocytes via S-sulfhydration on TRPA1 channels.18 He also
found that H2Sn were very effective in S-sulfhydration on Keap1,
the key protein regulating Nrf2 signaling.19 In another report by
Dick and Nagy et al., H2Sn were found to efficiently sulfhydrate
proteins such as roGFP2 and PTEN, while H2S could not cause
sulfhydration in the presence of potassium cyanide, an H2Sn
scavenging reagent.20
In order to better understand the roles of H2Sn and
differentiate H2Sn from H2S, it is important to study the
fundamental chemistry/reactivity of H2Sn and develop new
methods for their detection. The traditional method for
detecting H2Sn is to measure UV absorption peaks at 290−300
eactive sulfur species (RSS) are a family of sulfur-containing
Rmolecules found in biological systems. These molecules and 370 nm, which is not sensitive and applicable for biological
detections.20 In this respect, fluorescence assays may be useful
include thiols, hydrogen sulfide, persulfides, polysulfides, and S-
because of their high sensitivity and spatiotemporal resolution
capability. Unfortunately, there is no report on such fluorescent
probes for H2Sn so far. To this end, we have initiated a program to
study new reactions of H2Sn, aiming at developing new
fluorescent probes based on these reactions. Herein we report
this attempt.
modified cysteine adducts such as S-nitrosothiols and sulfenic
acids. So far many RSS have been demonstrated to exert
interesting biological functions.1−3 Among those, hydrogen
sulfide (H2S) is probably most attractive as this gaseous molecule
has been recently known as a critical cell signaling molecule,
much like nitric oxide. Literature published in the past several
years increasingly suggests that H2S is a mediator of many
physiological and/or pathological processes, especially in
cardiovascular systems.4−7 In contrast, hydrogen polysulfides
(H2Sn, n > 1) have received much less attention. These species
can be considered as oxidized forms of H2S and belong to sulfane
sulfur in RSS family. From a chemistry perspective, H2S and H2Sn
are redox partners and therefore very likely coexist in biological
systems. On the other hand, H2Sn may have their own
biosynthetic pathways or can be generated from H2S. H2Sn
could also be the precursors of H2S through their degradation.
Because of these properties, some biological mechanisms that
were originally attributed to H2S may actually be mediated by
H2Sn. For instance, one of the most interesting reactions of H2S is
S-sulfhydration, i.e., converting protein cysteines (-SH) to
persulfides (-S-SH). This reaction is significant because it
provides a possible mechanism by which H2S alters the functions
of a wide range of cellular proteins and enzymes.8−11 However,
how this reaction proceeds is still unclear. Theoretically H2S itself
can hardly react with protein cysteine residues or disulfides to
form S-sulfhydration. It is possible that H2S reacts with modified
cysteines such as S-nitrosothiols (SNO) or S-sulfenic acids
H2Sn is a combination of polysulfide species. The dissolution
of any polysulfide salts should result in similar distribution of
these species (this will depend on the relative ratios of sulfide vs
the oxidizing equivalents and the applied pH).14 Hydrogen
disulfide (H2S2) may be an active species of H2Sn, and there
should be a dynamic equilibrium between H2S2 and other H2Sn.18
Therefore, our focus has been put on the chemistry of H2S2.
Taking the advantage of two -SH groups in H2S2, we envisioned
that compounds containing bis-electrophilc groups should be
able to selectively capture H2S2 (Scheme 1). If one of the
electrophilic groups is a latent fluorophore and can be released
under nucleophilic reactions (such as A in Scheme 1), the
strategy may be suitable in the development of fluorescent
probes for H2S2. It is possible that biothiols, i.e., cysteine (Cys)
and glutathione (GSH), may compete with H2S2 in reacting with
probe A. However, product B should not turn-on the
fluorescence. Moreover, upon manipulating electronic properties
of the probe, H2Sn/H2S2 may further react with B (via the SN2Ar
Received: March 28, 2014
Published: May 8, 2014
© 2014 American Chemical Society
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Communication
may also react with the probes that are designed for trapping
H2Sn, leading to the consumption of the probes. To address this
concern, we tested the reaction of 2 with a biothiol model 7. The
substitution product 8 was obtained in 40% yield under the same
conditions. This suggests that biothiols are less reactive (than
H2Sn) toward the substrate. Moreover, compound 8 was able to
further react with H2Sn to give the cyclization product 5. These
results indicate that biothiols would not interfere with the
detection of H2Sn.
Scheme 1. Proposed Strategy for Capturing and Visualizing
H2Sn
The reaction shown in Scheme 2 provides a possible
application in developing fluorescent probes for H2Sn. It is
known that hydroxyl (-OH) protection (e.g., acylation or
alkylation) of many fluorophores can result in fluorescence
quenching, and deprotection can restore the fluorescence.21−28 If
-OH sensitive fluorophores are introduced to the benzoiate of 2,
the resultant compounds would be specific probes for H2Sn as
they may react with H2Sn to release the fluorophores. Based on
this strategy, three probes (DSP-1, DSP-2, and DSP-3) are
synthesized (Scheme 3). Detailed synthetic protocols and
structure characterizations are provided in the Supporting
Information.
reaction) to switch the thioether and turn on the fluorescence
(vide infra).
With this idea in mind, three 2-fluorobenzoiate derivatives (1−
3) were prepared and studied in the reactions of H2S2 (Scheme
2). In this study H2S2 was always used as the primary model
Scheme 3. Structures of New H2Sn Fluorescent Probes
Scheme 2. Model Reactions of the Probes with H2Sn
Next we tested the probes’ fluorescence properties and
responses to H2Sn. We first studied the detection conditions and
found that PBS buffer (50 mM, pH 7.4) containing 25 μM
cetrimonium bromide (CTAB) was the optimum system (Figure
S1). DSP-1 and DSP-3 showed almost no fluorescence emission
at 515 nm due to the protection of the two hydroxyl groups of
fluorescein, but DSP-2 showed some background fluorescence
due to the protection of only one hydroxyl group of fluorescein.
Upon treatment with Na2S2, both DSP-1 and DSP-3 gave
significant fluorescence enhancements (Figure 1), whereas DSP-
2 did not, which may be attributed to its strong background
compound of H2Sn. We expected that the activated fluoroben-
zoiates should undergo nucleophilic aromatic substitution with
H2S2 to form the corresponding persulfide intermediates I, which
in turn undergoes a cyclization to form benzodithiolone products
and release phenol. In these experiments freshly prepared
solutions of Na2S2 were used as the equivalent of H2S2. The
reactions were carried out in a mixed solution of CH3CN/PBS
(pH 7.4, 1:1 v/v). The products were analyzed after 1 h at room
temperature. As expected, when the parent compound 1 was
treated with H2S2, the desired cyclization product 4 was obtained
in low yield (7%). The substrates with electron-withdrawing
groups (-CN and -NO2) showed much improved reactivity, and
the corresponding cyclization products (5 and 6) were obtained
in modest to good yields. As the nitro-substitution (compound
2) was found to be most effective, this compound was selected
for further studies.
We then tested the reaction between 2 and another hydrogen
polysulfide model compound (Na2S4). The reaction worked
well, and the desired cyclization product was obtained in good
yield (85%). This result confirms that H2S2 may be the major
component of H2Sn or that there is a fast equilibrium between
H2Sn and H2S2.18 It therefore suggests that compounds like 2 are
suitable for capturing H2Sn. Another concern is that biothiols
Figure 1. Fluorescence enhancements (F/F0) of probe (10 μM) (1)
DSP-1; (2) DSP-2; and (3) DSP-3 with Na2S2 (50 μM) in PBS buffer
(50 mM, pH 7.4) containing 25 μM CTAB. Reactions were carried out
for 20 min at room temperature. Data were acquired at 515 nm with
excitation at 490 nm.
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Communication
fluorescence. As DSP-3 exhibited a much stronger fluorescence
response than DSP-1 (137 vs 57 fold), this probe was selected for
further evaluation.
Figure 2 shows the time-dependent fluorescence changes of
DSP-3 (10 μM) in the presence of Na2S2 (50 μM). The
DSP-3 for H2S2 and hydrogen polysulfides, suggesting that DSP-
3 may be useful for monitoring of H2Sn in biological systems.
To demonstrate the efficiency of this probe in the measure-
ment of H2Sn, varying concentrations of Na2S2 (0.5−50 μM)
were added to the solutions of DSP-3 (10 μM). The fluorescence
intensities were linearly related to the concentrations of Na2S2 in
the range of 0.5−15 μM (Figure 4). The detection limit30,31 was
calculated to be around 71 nM, indicating a high sensitivity.
Figure 2. Time-dependent fluorescence intensity changes of DSP-3 (10
μM) in the presence of Na2S2 (50 μM). Reactions were monitored for
40 min at room temperature.
maximum emission intensity at 515 nm was reached within 5
min, indicating a fast reaction. For the purpose of reproducibility,
a reaction time of 20 min was employed in all other experiments.
The effects of pH in this reaction were also investigated, and
DSP-3 was found to work effectively in neutral to weak basic pH
range of 7−8 (Figure S2).
To test the selectivity of the probe for H2Sn, DSP-3 was treated
with a series of RSS including GSH, Cys, Hcy, GSSG, H2S,
SO32−, S2O32−, CH3SSSCH3, and S8. As shown in Figure 3A, no
Figure 4. Fluorescence emission spectra of DSP-3 (10 μM) with varied
concentrations of Na2S2 (0, 0.5, 1, 3, 6, 10, 15, 20, 30, 40, 50 μM for
curves 1−11, respectively). Reactions were carried out for 20 min at
room temperature.
It should be noted that the biosynthetic pathways of H2Sn are
still unclear. Recent studies suggested that they may come from
H2S in the presence of reactive oxygen species
(ROS).12,14,15,17,18,20,32 We then applied DSP-3 in detecting in
situ generated H2Sn from H2S and ROS. As shown in Figure 5, the
Figure 3. (A) Fluorescence enhancements (F/F0) of DSP-3 (10 μM) in
the presence of various RSS. (1) probe alone; (2) 8 mM GSH; (3) 500
μM Cys; (4) 100 μM Hcy; (5) 100 μM GSSG; (6) 100 μM Na2S; (7)
100 μM Na2S2O3; (8) 100 μM Na2SO3; (9) 100 μM Na2SO4; (10) 100
μM CH3SSSCH3; (11) 100 μM S8; (12) 50 μM Na2S2; (13) 50 μM
Na2S4. (B) Fluorescence enhancements (F/F0) of DSP-3 (10 μM) to
the mixture of various RSS with 50 μM Na2S2. (1) 1 mM GSH; (2) 500
μM Cys; (3) 100 μM Hcy; (4) 100 μM GSSG; (5) 100 μM Na2S; (6)
100 μM Na2S2O3; (7) 100 μM Na2SO3; (8) 100 μM Na2SO4; (9) 100
μM CH3SSSCH3; (10) 100 μM S8; (11) 50 μM Na2S2.
Figure 5. Fluorescence enhancements (F/F0) of DSP-3 (10 μM) in the
presence of various reactive oxygen species (with or without H2S).
Reactions were carried out for 20 min at room temperature. (1) 50 μM
−
H2O2; (2) 200 μM H2O2; (3) 50 μM ClO−; (4) 50 μM O2 ; (5) 50 μM ·
OH; (6) 50 μM 1O2; (7) 50 μM H2O2 + 50 μM Na2S; (8) 200 μM H2O2
+ 50 μM Na2S; (9) 50 μM ClO− + 50 μM Na2S; (10) 50 μM O2− + 50
μM Na2S; (11) 50 μM •OH + 50 μM Na2S; (12) 50 μM 1O2 + 50 μM
Na2S.
significant fluorescence increase was observed for any of these
compounds (columns 2−11). Only Na2S2 and Na2S4 gave strong
fluorescence increase (columns 12 and 13). We also tested the
responses of DSP-3 to other representative amino acids and
found no responses (Figure S3). Moreover, when Na2S2 (50
μM) and other RSS coexisted, we still observed obvious
fluorescence enhancements (Figure 3B). Compared to the
results of Na2S2 only, almost the same levels of fluorescence turn-
on responses (without any loss) were observed for most of these
compounds. GSH, Cys, and Hcy did cause some fluorescence
decrease, presumably due to the reaction between H2S2 and
thiols, leading to the decreased concentrations of H2S2 in
solutions.18−20,29 These results demonstrate good selectivity of
probe did not give any response to commonly existing ROS
including hydrogen peroxide (H2O2), hypochlorite (ClO−),
−
superoxide (O2 ), hydroxyl radical (•OH), and singlet oxygen
(1O2) (columns 1−6). However, when H2S was premixed with
ROS (columns 7−12), significant fluorescence signals were
obtained, suggesting the formation of H2Sn in these systems.
Apparently H2S together with ClO− gave the strongest signals
(column 9), indicating that ClO− is the most effective ROS
converting H2S to H2Sn in our in vitro testing systems. This result
confirms the discovery by Nagy et al. that hypochlorous acid can
rapidly react with H2S to form hydrogen polysulfides.32
Finally the application of DSP-3 in monitoring H2Sn in
cultured cells was tested. As shown in Figure 6, HeLa cells were
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biothiols and H2S. Based on this reaction, a fluorescent probe,
DSP-3, was developed for sensitive and selective detection of
H2Sn/H2S2 in aqueous buffers as well as in cells. With probe
DSP-3, we also confirm the possibility of H2Sn formation from
the reaction of H2S with ROS such as ClO−. We are now utilizing
these probes to study the contributions of hydrogen polysulfides
to physiological and pathological processes. It should be noted
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polysulfides may be needed, and our present design approach
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ASSOCIATED CONTENT
* Supporting Information
Experimental details and characterization data. This material is
available free of charge via the Internet at http://pubs.acs.org
■
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AUTHOR INFORMATION
Corresponding Author
mxian@wsu.edu
■
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Author Contributions
§These authors contributed equally.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
This work is supported by NIH (R01HL116571) and the ACS-
Teva U.S.A. Scholar Grant.
■
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