Trapping hydrogen sulfide (H2S) with diselenides: The application in the design of fluorescent probes

Article abstract of DOI:10.1021/acs.orglett.5b00431

Here we report a unique reaction between phenyl diselenide-ester substrates and H₂S to form 1,2-benzothiaselenol-3-one. This reaction proceeded rapidly under mild conditions. Thiols could also react with the diselenide substrates. However, the resulted S-Se intermediate retained high reactivity toward H₂S and eventually led to the same cyclized product 1,2-benzothiaselenol-3-one. Based on this reaction two fluorescent probes were developed and showed high selectivity and sensitivity for H₂S. The presence of thiols was found not to interfere with the detection process.

Full text of DOI:10.1021/acs.orglett.5b00431

Letter
pubs.acs.org/OrgLett
Trapping Hydrogen Sulfide (H2S) with Diselenides: The Application in
the Design of Fluorescent Probes
Bo Peng,†,∥ Caihong Zhang,†,‡,∥ Eizo Marutani,§ Armando Pacheco,† Wei Chen,† Fumito Ichinose,§
and Ming Xian*,†
†Department of Chemistry, Washington State University, Pullman, Washington 99164, United States
‡School of Chemistry and Chemical Engineering, Center of Environmental Science and Engineering Research, Shanxi University,
Taiyuan, China
§Department of Anesthesia, Critical Care and Pain Medicine, Massachusetts General Hospital and Harvard Medical School, Boston,
Massachusetts 02114, United States
S
* Supporting Information
ABSTRACT: Here we report a unique reaction between phenyl diselenide-ester substrates and H2S to form 1,2-
benzothiaselenol-3-one. This reaction proceeded rapidly under mild conditions. Thiols could also react with the diselenide
substrates. However, the resulted S−Se intermediate retained high reactivity toward H2S and eventually led to the same cyclized
product 1,2-benzothiaselenol-3-one. Based on this reaction two fluorescent probes were developed and showed high selectivity
and sensitivity for H2S. The presence of thiols was found not to interfere with the detection process.
using azide (−N3) substrates;14 (2) H2S-mediated nucleophilic
ydrogen sulfide (H2S), traditionally known as a venom-
reactions;15,16 and (3) H2S-mediated metal−sulfide forma-
Hous gas with a stinky smell, has been recently recognized
as an important signaling molecule.1−5 H2S is generated in
mammalian cells by enzymes including cystathionine β-
synthase (CBS), cystathionine γ-lyase (CSE), and 3-mercapto-
pyruvate sulfurtransferase (3-MST), and cysteine and deriva-
tives serve as the substrates.4−8 Recent studies have revealed a
variety of functions of H2S in physiological and pathological
processes.9−11 However, the mechanisms behind those roles are
still poorly understood. H2S is a highly reactive molecule that
can react with many biological targets such as hemoglobin and
phosphodiesterase.12 These reactions are responsible for the
biological functions of H2S. On the other hand, these
complicated reactions also make the detection of H2S in
biological systems very difficult. Early reports claim physio-
logical H2S concentrations can be as high as 20−80 μM in
plasma. However, it is believed now that the concentrations of
free H2S are low, at submicromolar or nanomolar levels.12b
In the past several years fluorescence based assays have
received considerable attention in this field and a number of
fluorescent probes for H2S have been reported.13 All of these
probes utilize reaction-based fluorescence change strategies, i.e.
using certain H2S-specific reactions to convert nonfluorescent
substrates to materials with strong fluorescence or to change
the fluorescent properties of the probes (for ratiometric
probes). Currently three types of reactions are normally used
in the design of probes: (1) H2S-mediated reductions, often
tion.17 In 2011 our laboratory developed the first nucleophilic
reaction based strategy for the design of probes (i.e., WSP
series).15 The strategy is described in Scheme 1. The probe
(WSP) contains two electrophilic centers. H2S reacts with the
pyridyl disulfide first to give a persulfide intermediate 1, which
precedes a fast cyclization to release the fluorophore and 1,2-
benzodithiol-3-one 2. It is anticipated that the probe can also
react with biothiols to form 3. Theoretically 3 could further
react with H2S to form 1 and then turn on the fluorescence.
However, this reaction is somewhat slow, especially when the
concentration of H2S is low (under physiological conditions18).
In addition, H2S may react with both sulfurs of the disulfide of
3, which makes this pathway not productive. It should be noted
that the reaction between WSP and biothiols cannot turn the
fluorescence on; therefore, the selectivity of WSP is found to be
excellent. We were also able to optimize the detection
conditions and found fluorescence “turn-on” could be achieved
in a few minutes. Such a fast reaction allows effective detection
of H2S even when high concentrations of biothiols are
presented, albeit the fluorescence signals are significantly
decreased.15b
Received: February 10, 2015
© XXXX American Chemical Society
A
DOI: 10.1021/acs.orglett.5b00431
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Letter
(RSH). Such reactions should lead to two products: 6 and a S−
Se conjugate 8. Again, 6 should be oxidized to regenerate the
probe. As for 8, previous studies revealed that nucleophilic
attack by thiols at selenium is both kinetically much faster and
thermodynamically more favorable.19 As such, the reaction
between 8 and other biothiols should not change the probe’s
structural template (i.e., maintaining the −S−Se− conjugation).
However, if H2S is present, the reaction should lead to
intermediate 5, and the following cyclization should produce 7
and the fluorophore. Overall we expected the probe would
specifically react with H2S to release the fluorophore and the
presence of biothiols should not interfere with the process.
With this idea in mind, a model compound 9 was prepared
and tested. As shown in Scheme 2, the reaction between
Scheme 1. Design of the Fluorescent Probes for H2S
Detection
Scheme 2. Model Reactions between Compound 9 and H2S
compound 9 and H2S (5 equiv, using Na2S as the equivalent)
was found to be fast, which completed in 10 min. The desired
products 7 and phenol were obtained in excellent yields. The
yield of 7 was calculated based on two selenium moieties in the
starting material. We did not observe the formation of the
benzeneselenol product in the reaction. When Cys (10 equiv)
coexisted with H2S (5 equiv) in the reaction, we obtained the
same products in high yields. More interesting, even if Cys (5
equiv) was treated with 9 first for 1 h, the addition of H2S (2.5
equiv) still provided the desired products in similar yields.
These results supported our hypothesis that diselenide-based
substrates could selectively and effectively react with H2S to
form the cyclized product 7 and this reaction was not affected
by thiols.
As described above, the possible consumption of WSP
probes by biothiols is a weakness. To solve this problem two
strategies may be applied: (1) a H2S-specific “trapper” should
be used to replace the pyridyl-disulfide. Such a trapper should
only react with H2S, not react with biothiols. This might be
difficult as intracellular concentrations of biothiols are much
higher than H2S; (2) a nonconsumptive trapper with biothiols
should be identified and used. Such a trapper may react with
biothiols, but the reaction should not lead to the release of the
fluorophore. Moreover, ideally the reaction product or
intermediate should maintain high reactivity toward H2S
which therefore can lead to fluorescence “turn-on” by H2S.
Herein we report our progress in pursuing the latter strategy.
Diselenide-based substrates were found to be suitable for this
goal.
Based on this unique reaction we synthesized two fluorescent
probes SeP1 and SeP2 (shown in Figure 1). 7-Hydroxycou-
Our design of the diselenide-based strategy for the selective
trapping of H2S (not consumed by biothiols) is shown in
Scheme 1. It is known that diselenide bonds can be cleaved by
sulfur-based nucleophiles very effectively (about 5 orders of
magnitude faster than disulfide bonds).19,20 It is also known
that diselenides can facilitate disulfide formation from thiols.21
H2S (pKa 6.8) is a stronger nucleophile than common biothiols
such as Cys and GSH. Therefore, we expect diselenide-based
reagents such as 4 should react with H2S very effectively. As
such two products should be formed: a thio-benzeneselenol
derivative 5 and a benzeneselenol derivative 6. As an extremely
unstable intermediate, benzeneselenol 6 should be rapidly
oxidized to reform the probe 4.22 In contrast, 5 should undergo
a fast and spontaneous cyclization to form 1,2-benzothia-
selenol-3-one 7 and release the fluorophore. We also expected
the diselenide bond should be quite reactive to biothiols
Figure 1. Structures of diselenide-based probes.
marin and 2-methyl TokyoGreen were selected as the
fluorophores, as they have excellent fluorescence properties
and their fluorescence can be easily quenched upon acylation
on hydroxy groups.
With these two probes in hand, we tested their fluorescent
properties. Both probes exhibited very weak fluorescence with
low quantum yields (Φ < 0.1), due to esterification of the
fluorophores. We then tested the probes’ fluorescence
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DOI: 10.1021/acs.orglett.5b00431
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responses to H2S in different solvent systems, and a mixed
CH3CN/PBS buffer (10 mM, pH 7.4, 1:1) solution was found
to be the optimum system for the measurement. In this system
the fluorescence intensity of the probe (10 μM) could reach the
maximum in less than 2 min upon treatment of H2S (using 50
μM Na2S) as shown in Figure 2, demonstrating this was a fast
Figure 2. Time-dependent fluorescence changes of the probes (10
μM) in the presence of Na2S (50 μM). The reactions were carried out
for 30 min at room temperature in CH3CN/PBS buffer (10 mM, pH
7.4, 1:1, v/v). Data were acquired at 455 nm with excitation at 340 nm
Figure 4. Fluorescence intensity of the probes (10 μM) in the
presence of various reactive sulfur species: (1) control; (2) 50 μM
Na2S; (3) 200 μM Na2SO3; (4) 200 μM Na2S2O3; (5) 200 μM
Na2SO4; (6) 1 mM Cys; (7) 1 mM GSH; (8) 50 μM Na2S + 1 mM
Cys; (9) 50 μM Na2S + 1 mM GSH. SeP1 (A), SeP2 (B).
■
●
for SeP1 ( ) and at 521 nm with excitation at 498 nm for SeP2 ( ).
process. Fluorescence increases were also found to be
significant. Intensities increased 35- and 14-fold for probe
SeP1 and SeP2 respectively. When a series of different
concentrations of Na2S were treated with the probes we
observed fluorescence intensities increased in almost a linear
fashion in the range 0−15 μM. Data obtained with SeP2 are
shown in Figure 3. The detection limit of SeP2 was calculated
to be 0.06 μM. Data of SeP1 are shown in Figure S1 in the
Supporting Information.
concentrations of biothiols were present (at 25−55% levels of
the signal of H2S only).15b
Next we tested SeP2 in imaging H2S in cells. Freshly
cultured HeLa cells were first incubated with SeP2 (50 μM) for
30 min and then washed with DMEM to remove excess probe.
We did not observe significant fluorescent cells (Figure 5).
However, strong fluorescence in the cells was observed after
treating with Na2S (100 μM) for 30 min. These results
demonstrated that SeP2 could be used for cell imaging.
Finally we wondered if SeP2 could be used to measure
endogenous H2S concentrations changes. To this end, human
neuroblastoma cells (SH-SY5Y) were separately treated with L-
and D-cysteine (which are H2S biosynthestic substrates), S-
Figure 3. Fluorescence emission spectra of SeP2 (10 μM) in the
presence of varied concentrations of Na2S (0, 0.5, 1, 2, 3, 4, 5, 6, 7, 8,
9, 10, 15 μM). The reactions were carried out for 5 min at room
temperature in CH3CN/PBS buffer (10 mM, pH 7.4, 1:1, v/v). Data
were acquired with excitation at 498 nm for SeP2.
We also examined the selectivity of the probes for H2S over
other reactive sulfur species, including cysteine (Cys),
glutathione (GSH), sulfite (SO32−), sulfate (SO42−), and
thiosulfate (S2O32−). As shown in Figure 4, all of these species
did not show a significant fluorescence increase even under
much higher concentrations (up to mM). In addition, when
H2S (50 μM) coexisted with Cys or GSH (1 mM), we observed
strong fluorescence responses that were comparable (at ∼90%
levels) to the signals obtained with H2S only. This was a
significant improvement from our pyridyl disulfide based
probes, which gave much decreased fluorescence when high
Figure 5. Fluorescence images of H2S in HeLa cells using SeP2. Cells
were incubated with the probe (50 μM) for 30 min, then washed, and
subjected to different treatments. (a and b) Control (no Na2S was
added); (c and d) treated with 100 μM Na2S (scale bar: 100 nm).
C
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protocols in the Supporting Information). Fluorescence
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treated with H2S substrates or CBS activator showed clearly
enhanced fluorescence while cells treated with CBS inhibitor
showed decreased fluorescence. Not surprisingly cells treated
with Na2S showed the strongest fluorescence. These results
suggest that SeP2 can be used in determining endogenous H2S
changes.
In conclusion, we reported herein a unique reaction between
phenyl diselenide and H2S to form 1,2-benzothiaselenol-3-one.
The presence of thiols did not affect this process. Based on this
reaction two fluorescent probes for the detection of H2S were
prepared and evaluated. The probes showed excellent
sensitivity and selectivity.
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ASSOCIATED CONTENT
* Supporting Information
■
(17) (a) Sasakura, K.; Hanaoka, K.; Shibuya, N.; Mikami, Y.; Kimura,
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S
Detailed synthetic procedures, characteristic data, and exper-
imental procedures. This material is available free of charge via
the Internet at http://pubs.acs.org.
AUTHOR INFORMATION
Corresponding Author
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*E-mail: mxian@wsu.edu.
Author Contributions
∥B.P. and C.Z. contributed equally to this work.
Notes
The authors declare no competing financial interest.
(22) Rasmussen, B.; Sørensen, A.; Gotfredsen, H.; Pittelkow, M.
Chem. Commun. 2014, 50, 3716.
ACKNOWLEDGMENTS
■
This work is supported by an American Chemical Society Teva
USA Scholar Grant and the NIH (R01HL101930 and
R01HL116571).
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DOI: 10.1021/acs.orglett.5b00431
Org. Lett. XXXX, XXX, XXX−XXX