Reaction based fluorescent probes for hydrogen sulfide

Article abstract of DOI:10.1021/ol3008183

A reaction based fluorescence turn-on strategy for hydrogen sulfide (H ₂S) was developed. This strategy was based on a H₂S- specific Michael addition-cyclization sequence. Other biological thiols such as cysteine and glutathione did not pursue the reaction and therefore did not turn on the fluorescence/consume the substrates. The probes showed good selectivity and sensitivity for hydrogen sulfide.

Full text of DOI:10.1021/ol3008183

ORGANIC
LETTERS
2012
Vol. 14, No. 8
2184–2187
Reaction Based Fluorescent Probes for
Hydrogen Sulfide
Chunrong Liu,† Bo Peng,† Sheng Li,‡ Chung-Min Park,† A. Richard Whorton,‡ and
Ming Xian*,†
Department of Chemistry, Washington State University, Pullman, Washington 99164,
United States, and Department of Pharmacology & Cancer Biology, Duke University
Medical Center, Durham, North Carolina 27710, United States
mxian@wsu.edu
Received March 30, 2012
ABSTRACT
A reaction based fluorescence turn-on strategy for hydrogen sulfide (H2S) was developed. This strategy was based on a H2S-specific Michael
additionꢀcyclization sequence. Other biological thiols such as cysteine and glutathione did not pursue the reaction and therefore did not turn on
the fluorescence/consume the substrates. The probes showed good selectivity and sensitivity for hydrogen sulfide.
Hydrogen sulfide (H2S) has traditionally been consid-
ered a highly toxic gas. However, recent studies have dem-
onstrated that H2S is an endogenously generated gaseous
signaling molecule with very potent cytoprotective proper-
ties that are on par with those of two other well-known
endogenous gaseotransmitters: nitric oxide (NO) and car-
bon monoxide (CO).1 To date, hydrogen sulfide’s exact
mechanisms of action are still under active investigation.
Some chemical and biochemical catabolic reactions of H2S
have been reported. For example, H2S can react readily
with methemoglobin toform sulfhemoglobin, which might
act as the metabolic sink for H2S. As a potential reductant,
H2S is likely to be consumed by endogenous oxidant
species such as hydrogen peroxide, superoxide, peroxyni-
trite, etc. H2S can also cause protein S-sulfhydration
(i.e., to form ꢀSꢀSH).2 This process is potentially signifi-
cant because it provides a possible mechanism by which
H2S alters the functions of a wide range of cellular proteins
and enzymes. It is likely that many more important
reactions of H2S are to be discovered. Nevertheless, the
production of endogenous H2S and the exogenous
administration of H2S elicit a wide range of protective
actions including vasodilation, anti-inflammatory, anti-
oxidant, antiapoptotic, and down regulation of cellular
metabolism during stressful states.1 These results strongly
suggest that modulation of H2S levels could have poten-
tial therapeutic values for a number of diseases. It is
important, therefore, to understand the chemistry and
properties of H2S and to appreciate the limitation and
errors that may be generated when measuring H2S in
biological samples.
Currently a critical debate in the field is about the
biologically relevant concentrations of H2S as reports vary
over a 105-fold range.3 Traditional H2S detection methods
† Washington State University.
‡ Duke University Medical Center.
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r
10.1021/ol3008183
Published on Web 04/09/2012
2012 American Chemical Society

(colorimetric/electrochemical assays, gas chromatography,
and sulfide precipitation)4 often require complicated post-
mortem processing and/or destruction of tissues or cell
lysates. Given the high reactivity of H2S in biological
environments, these methods could yield inconsistent re-
sults. Fluorescence based assays could be useful in this field
due to the high sensitivity and convenience. Fluores-
cence methods are suitable for nondestructive detection
of biotargets in live cells or tissues with readily available
instruments. Since 2011, several fluorescent probes which
can potentially be used for H2S detection in living systems
have been reported.5 The fluorescence turn-on strate-
gies of these probes were based on several different
H2S-specific reactions.5 Among these methods, the
strategy developed by our group utilized the unique
dual nucleophilicity of H2S.5b In our first generation
probes (Scheme 1), 2-pyridinyl disulfide was employed
as the H2S trap to initiate the tandem reaction to release
the fluorophore. Such probes showed high selectivity
for H2S and potential applications in monitoring H2S
in living cells.5b However, the 2-pyridinyl disulfide
could also react with biological thiols. Although the
reaction will not lead to fluorescence turn-on, a rela-
tively high probe loading may be necessary. To solve
this problem, here we report a new generation of probes
which only react with H2S while not interfering with
other thiols.
The design of the second generation probes is shown
in Scheme 1. Experiments by Holmes et al. revealed that
simple thiols reacted readily with some Michael accep-
tors at physiological pH, but the products could not be
obtained or identified.6 This is possibly due to the fact
that the Michael addition was a rapid equilibrium
process and no stable covalent product was formed.
Based on Holmes’ results, we envisioned that certain
Michael acceptors might be useful to differentiate H2S
from biological thiols. It is also known that H2S in
aqueous solution has a pKa of 7.0 while thiols have
higher pKa values of ∼8.5. So, H2S should be a better
nucleophile than thiols in physiological media. If Mi-
chael acceptors are employed in the probes, they should
be able to react with H2S and promote the intramolec-
ular cyclization to release the fluorophore. The reac-
tions between Michael acceptors and biological thiols
such as cysteine and glutathione, however, should be
reversible and therefore should not consume the
probes.
Scheme 1. Reaction-Based Fluorescence Turn-on Strategies
With this idea in mind, two model compounds 1 and 2
were prepared (see Supporting Information (SI) for the
synthesis). When 1 was treated with H2S (using NaHS as
the equivalent) for 1 h at rt, the desired cyclization product
3 was obtained in 31% yield (Scheme 2). The remaining
material was unreacted 1. Compound 2 proved to be more
reactive in this reaction. Under the same conditions,
cyclization product 4 was obtained in 91% yield. As ex-
pected, when both compounds were treated with cysteine or
glutathione, no Michael addition products were isolated.
Only the starting materials were recovered. In addition, 1
and 2 showed no reaction toward primary amines and
ammonia (see SI). These results confirmed our hypothesis
that certain Michael acceptors can be used to selectively trap
H2S and they are not consumed by thiols.
Scheme 2. Model Reactions
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Berkman, C. E.; Whorton, A. R.; Xian, M. Angew. Chem., Int. Ed. 2011,
50, 10327–10329. (c) Peng, H.; Cheng, Y.; Dai, C.; King, A. L.;
Predmore, B. L.; Lefer, D. J.; Wang, B. H. Angew. Chem., Int. Ed.
2011, 50, 9672–9675. (d) Qian, Y.; Karpus, J.; Kabil, O.; Zhang, S.; Zhu,
H.; Banerjee, R.; Zhao, J.; He, C. Nat. Commun. 2011, 2, 495–501. (e)
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We then prepared two Michael acceptor-based fluorescent
probes (5 and 6, Figure 1) and tested their fluorescence
(6) Pritchard, R. B.; Lough, C. E.; Currie, D. J.; Holmes, H. L. Can.
J. Chem. 1968, 46, 775–781.
Org. Lett., Vol. 14, No. 8, 2012
2185

Figure 1. Structures of Michael acceptor-based fluorescent
probes.
properties in aqueous buffers. Both compounds exhibited no
absorption features in the visible region (fluorescence
quantum yield of 5: Φ = 0.0002; 6: Φ = 0.0008). As
shown in the images in Figure 2, the reactions of 5 and 6
with H2S (using NaHS as the equivalent) yielded signifi-
cant fluorescence signals. Control experiments using cy-
steine or glutathione did not lead to any fluorescence
increase. As expected, when H2S and thiols coexisted, we
still observed a strong fluorescence change. In addition,
we noticed that probe 6 apparently showed much better
responses than probe 5. This was consistent with the fact
observedinScheme 2 thatthe cyanoacrylate derivativehad
better reactivity with H2S than the benzylidenemalonate
derivative.
Figure 2. Fluorescence images of probe 5 (top row) and probe 6
(bottom row) in pH 7.4 phosphate buffer. (a) Probe only (5 μM);
(b) probe þ NaHS (100 μM); (c) probe þ cysteine (1 mM);
(d) probe þ glutathione (1 mM), (e) probe þ cysteine (1 mM) þ
NaHS (100 μM), (f) probe þ glutathione (1 mM) þ NaHS
(100 μM).
The turn-on responses of the probes were also measured
by a spectrofluorometer. As shown in Figure 3, upon
treatment of 5 μM probes 5 or 6 with 100 μM NaHS,
significant increases in fluorescence intensity were ob-
served. Within a 30 min reaction time under these condi-
tions, the benzylidenemalonate probe 5 produced an 11-
fold turn-on response. Again, the cyanoacrylate probe 6
proved to be more sensitive to H2S, and it led to a 160-fold
turn-on response (Figure 3). The analysis of reaction
products confirmed the production of the correspond-
ing fluorescein dye. When the probes were treated with
abundant biologically relevant thiols, including 1 mM
cysteine and 1 mM glutathione, no significant fluores-
cence increase was observed. Moreover, when H2S
(100 μM) and thiols (1 mM) coexisted, we still observed
strong fluorescence enhancements using both probes.
These results demonstrated that the turn-on responses
of probes 5 and 6 were selective for H2S and the probes
could be used for the detection of H2S in the presence of
a high concentration of biological thiols.
Figure 3. Fluorescence responses of (A) 5 μM probe 5 and (B)
5 μM probe 6 to H2S and biologically relevant thiols: (a) probe
only, (b) probe þ NaHS (100 μM); (c) probe þ cysteine (1 mM);
(d) probe þ glutathione (1 mM), (e) probe þ cysteine (1 mM) þ
NaHS (100 μM), (f) probe þ glutathione (1 mM) þ NaHS
(100 μM). Data were acquired in phosphate buffer (pH 7.4,
10 mM) with excitation at λex 465 nm, 25 °C.
To demonstrate the efficiency of the probes in the mea-
surement of H2S concentration, varying concentrations of
NaHS (1ꢀ100 μM) were added to the solutions of 5 and 6
(5 μM). The fluorescence intensities were indeed linearly
related to the concentrations of NaHS in such concentration
ranges (Figures 4 and S2 in SI). The detection limit for H2S
using these probes was found to be ∼1 μM.
fluorescene intensity of both probes did not show a
significant increase when they were treated with ester-
ase for 2 h. After that, NaHS was added and a fluores-
cence increase was immediately observed. These results
We recognized that probes 5 and 6 contained ester
groups which could potentially be hydrolyzed by cel-
lular esterase. We then tested the stability of probes
in the presence of esterase. As shown in Figure 5, the
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Org. Lett., Vol. 14, No. 8, 2012

Figure 4. Fluorescence spectra of probe 5 (A) and probe 6 (B)
in phosphate buffer (pH 7.4, 10 mM). Probe concentration was
5 μM. Spectra were recorded after incubation with different
concentrations of NaHS for 30 min.
Figure 5. Fluorescence spectra of probe 5 (A) and probe 6 (B) in
the presence of esterase. Probe concentration: 5 μM. Esterase
concentration: 0.025 g/mL. Spectra were recorded after incuba-
tion for different times.
suggested that the probes were quite stable toward
esterase.
Since probe 6 was identified as a better probe than
compound 5, due to its higher sensitivity and faster
reaction with H2S, this probe was selected for monitor-
ing H2S in living cells. Briefly, COS7 cells were incu-
bated with compound 6 for 10 min and then washed
twice with phosphate buffer (Figure 6). We did not
observe any significant fluorescent cells. However,
strong fluorescence in the cells was observed after
treatment with sodium sulfide for 10 min. Thus we
conclude that probe 6 can be used for the detection of
H2S in cultured cells.
In summary, we reported in this study an improved
fluorescent probe for H2S detection. The design was
based on a Michael addition of H2S followed by an
intramolecular cyclization to release the fluorophore.
This process proved to be selective for H2S. Other
biological thiols reacted with the Michael acceptors in
a reversible way and did not lead to covalent products.
Therefore, thiols could not turn on the fluorescence and
did not consume the probe. Compared to other reaction
based H2S probes,5 our design has some advantages.
Our probes contain three structural subunits: a H2S
trapper, linker, and fluorophore. Many chemical mod-
ifications can be carried out to improve the efficiency of
the probes. We believe this strategy will be useful for the
design of more sensitive and selective probes for the
detection of H2S in biological systems.
Figure 6. Fluorescence images of H2S detection in COS7 cells
using probe 6. COS7 cells on glass coverslips were incubated
with 6 (100 μM) for 10 min, washed, and then subjected to
different treatments. Left was control (no sodium sulfide was
added); right was treated with sodium sulfide (200 μM).
Acknowledgment. This work is supported in part by a
CAREER award from the NSF (0844931) to M.X. and
NIH (R01HL088559) to A.R.W.
Supporting Information Available. Spectroscopic and
analytical data and selected experimental procedures.
This material is available free of charge via the Internet
at http://pubs.acs.org
The authors declare no competing financial interest.
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