Molecular Engineering of α-Substituted Acrylate Ester Template for Efficient Fluorescence Probe of Hydrogen Polysulfides

Article abstract of DOI:10.1021/acs.analchem.7b03755

In this article, hydrogen polysulfide (H₂S_n)-mediated Michael addition/cyclization cascade reactions toward acrylate ester analogues were exploited and utilized to construct novel and robust H₂S_n-specific fluorescence probe for the first time. Through rational molecular engineering of the α-substituted acrylate ester template, the optimal candidate probe FP-CF₃ containing trifluoromethyl-substituted acrylate ester group as recognition unit and 3-benzothiazol-7-hydroxycoumarin dye BHC as signal reporter can highly selectively detect H₂S_n over other reactive sulfur species, especially biothiols including cysteine (Cys) and homocysteine (Hcy)/glutathione (GSH), with a rapid and significant turn-on fluorescence response (less than 60 s for response time and over 44-fold for signal-to-background ratio). The fast response and high selectivity of FP-CF₃ for H₂S_n could be attributed to a kinetically and spatially favored pentacyclic addition produced by the dual nucleophilic reaction of H₂S_n with the CF₃-substituted acrylate group. The big off-on fluorescence response is due to the pentacyclic intermediate results in the release of the highly fluorescent BHC. Moreover, it has been successfully applied in imaging of endogenous H₂S_n fluctuation in living cells.

Full text of DOI:10.1021/acs.analchem.7b03755

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Article
Molecular Engineering of #-Substituted Acrylate Ester Template
for Efficient Fluorescence Probe of Hydrogen Polysulfides
JingRu Guo, Sheng Yang, Chongchong Guo, Qinghai Zeng,
Zhihe Qing, Zhong Cao, Jishan Li, and Ronghua Yang
Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.7b03755 • Publication Date (Web): 06 Dec 2017
Downloaded from http://pubs.acs.org on December 6, 2017
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Molecular Engineering of αꢀSubstituted Acrylate
Ester Template for Efficient Fluorescence Probe of
Hydrogen Polysulfides
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Jingru Guo,† Sheng Yang,†,* Chongchong Guo,†Qinghai Zeng,§ Zhihe Qing,† Zhong
Cao,† Jishan Li,‡ and Ronghua Yang†,‡,*
†School of Chemistry and Biological Engineering,Hunan Provincial Key Laboratory
of Materials Protection for Electric Power and Transportation, Changsha University
of Science and Technology, Changsha, 410114, P. R. China
‡State Key Laboratory of Chemo/Biosensing and Chemometrics, College of
Chemistry and Chemical Engineering, Hunan University, Changsha, 410082, P. R.
China
§Department of Dermatology, Third Xiangya Hospital, Central South University,
Changsha, 410013, P. R. China
*To whom correspondence should be addressed:
Eꢀmail: yangrh@pku.edu.cn; ysh1127@hnu.edu.cn
Fax: +86ꢀ731ꢀ8882 2523
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ABSTRACT: In this article, hydrogen polysulfide (H2Sn)ꢀmediated Michael
addition/cyclization cascade reactions toward acrylate ester analogues had been
exploited and utilized to construct novel and robust H2Snꢀspecific fluorescence probe
for the first time. Through rational molecular engineering of the αꢀsubstituted acrylate
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ester
template,
the
optimal
candidate
probe
FPꢀCF3
containing
trifluoromethylꢀsubstituted acrylate ester group as recognition unit and
3ꢀbenzothiazolꢀ7ꢀhydroxycoumarin dye BHC as signal reporter can highly selectively
detect H2Sn over other reactive sulfur species, especially biothiols including cysteine
(Cys), homocysteine (Hcy)/glutathione (GSH), with a rapid and significant turnꢀon
fluorescence response (less than 60 secondsfor response time and over 44ꢀfold for
signalꢀtoꢀbackground ratio). The fast response and high selectivity of FPꢀCF3 for
H2Sn could be attributed to a kinetically and spatially favored pentacyclic addition
produced by the dual nucleophilic reaction of H2Sn with the CF3ꢀsubstituted acrylate
group. The big offꢀon fluorescence response is due to the pentacyclic intermediate
results in the release of the highly fluorescent BHC. Moreover, it has been
successfully applied in imaging of endogenous H2Sn fluctuation in living cells.
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INTRODUCTION
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Reactive sulfur species (RSS) are sulfurꢀcontaining molecules that exert crucial
regulatory functions in biological systems.1ꢀ3Among of them, hydrogen sulfide (H2S)
has been deeply studied and recognized as an important endogenous gasotransmitter
in the past decade.4 In contrast, as one member of RSS family and H2S derivates,
hydrogen polysulfides (H2Sn, n>1) have always attracted much less attention.
Nevertheless, emerging evidences imply that H2Sn might act as the actual mediators in
some physiological and/or pathological processes associated with H2S.5,6 It was also
reported that H2Snhad higher potency than H2S during activating ion channels, tumor
suppressors, and transcription factors.7 Although the significances of H2Sn molecules
in redox biology have been acknowledged recently, their biological roles and detailed
mechanisms of action are still poorly understood. Therefore, it is critical to develop
effective tools for detection of H2Sn fluctuation in biological systems.
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Due to its advantages of high sensitivity, nonꢀinvasiveness, and spatiotemporal
resolution capability, fluorescence molecular imaging is the promising approach for
real time and in situ monitoring biologically important species of living organisms.8
Since the initial report by Xian et al,9 several fluorescent H2Sn probes have been
designed by exploiting 2ꢀfluoroꢀ5ꢀnitrobenzoic ester template as recognition unit.10ꢀ18
Although these probes proved to be highly selective, relatively high probe loading
may be required during bioimaging applications due to the inevitable probe
consumption by the competing reaction between template and intracellular biothiols.
To address this issue, other H2Sn fluorescence probes have appeared based on some
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H2Snꢀmediated specific reactions, including nucleophilic ringꢀopening reaction and
benzodithiolone formation.19ꢀ23Despite of the fact that considerable efforts have been
made as above, some limitations including low sensitivity, prolonged response times,
and possible toxicity of relatively large recognition moiety are still present, hampering
their further application to tackle biological issues. In response, novel sensing
molecules that use easily accessible and biologically compatible recognition units
with improved sensitivity and response times are needed.
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It is previous reported that acrylateester containing α,βꢀunsaturated carbonyl group
is a powerful recognition template for biothiols Cys and Hcy via the conjugate
addition/cyclization sequence.24 On the basis of this biothiolsꢀtriggered cascade
reaction, numerous Cys fluorescent probes have been developed over the past few
years.25ꢀ32 Inspired by this approach and taking into consideration of bisꢀnucleophilic
feature of H2Sn, we envisioned that acrylate ester analogues should also be able to trap
H2Sn to give out pentacyclic leaving compound, resulting in the release of the masked
fluorophore and then fluorescence recovery as shown in Scheme 1. Moreover, taking
the advantage of steric hindrance and electronic effects, conquest of
biothiolsꢀinterference and improvement of response performances would be achieved
through rational molecular engineering of substituted acrylate ester analogues.
Toward this end, we initiated a program to study the reaction between αꢀsubstituted
acrylate analogues and H2Sn for constructing novel and efficient H2Snꢀspecific
recognition template for the first time. We herein report the design, synthesis, and
evaluation of a series of new H2Sn fluorescent probes, namely FP Series, based on
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hydroxycoumarin fluorophore scaffold decorated with different acrylate derivates as
recognition unit. One of them, FPꢀCF3 bearing trifluoromethylꢀsubstituted acrylate
group,showed selective and fast response toward H2Sn with high sensitivity and was
successfully used for intracellular H2Sn imaging.
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EXPERIMENTAL SECTION
Synthesis of FP Series. The molecular structures and synthetic procedures of FP
Series were depicted in Figure 1. 3ꢀbenzothiazolꢀ7ꢀhydroxycoumarin BHC was firstly
synthesized following the literature method.33Subsequently, these probe molecules
can be readily prepared from BHC with corresponding acrylic acid through only one
step esterification reaction. Details for synthesizes and characterizations are attached
to the Supporting Information.
Spectrophotometric Experiments.Stock solutions of each probe (5 mM) in THF
were prepared in a glovebox. A PB buffer solution (10 mM, pH 7.4) with THF as the
cosolvent (H2O/THF = 1:1, v/v) was used for performing all spectroscopic
measurements.Test solutions were prepared by blendingFP Series and appropriate
analyte stock into a tube and then diluting the solution to 500 ꢁL with the PB buffer
solution.After incubation at room temperature for 5 min, the absorption or
fluorescence spectra measurements were then performed. The fluorescence spectra
were recorded at emission wavelength range from 470 to 580 nm with excitation
wavelength of 460 nm.
Fluorescent Imaging in Living Cells.For imaging of exogenous H2Sn in living cells,
the HeLa cells were stained with 2.0 ꢁM FPꢀH andFPꢀCF3 for 20 min before washed
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three times with PBS, then incubated with 100 ꢁM Na2S4 solution for another 10 min.
In order to monitor the intracellular endogenous H2Sn levels upon lipopolysaccharides
(LPS) stimulation, RAW264.7 cellswere stained with 5.0 ꢁM FPꢀCF3 for 20 min
under different conditions: (a) Cells only (control); (b–e) The cells were induced by
LPS (2 mg/mL) for 0, 4, 8, and 12 h, respectively; (f) The cells were pretreated with
DLꢀpropargylglycine (200 ꢁM) for 30 min, and then treated as (e).After rinsing with
PBS buffer, these cells were exposed to imaging experiment on the twoꢀphoton
confocal microscope. Twoꢀphoton imaging patterns were obtained from the green
channel (480ꢀ560 nm) with twoꢀphoton excitation wavelength of 800 nm.
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RESULTS AND DISCUSSION
Design Strategy and Mechanism Verification. As we known, coumarin derivates
are frequentlyꢀused fluorophores with excellent characteristics including tunable
emission and easy functionalization and small molecular size,34 from which various
bioimaging probes have been derived over the past years.35ꢀ39 Moreover, it is recently
reported that some coumarin derivates possess admirable twoꢀphoton properties,40ꢀ42
which should be beneficial for bioimaging due to the advantages of twoꢀphoton
microscopy (TPM) such as reduced photoꢀbleaching of fluorophores, minimal
autofluorescence background, improved penetration depth, and less photodamage to
biological samples.43ꢀ45Hence, 3ꢀbenzothiazolꢀ7ꢀhydroxycoumarinBHC was chosen
as the fluorophore scaffold for constructing these designed probes by decorating with
four different acrylate derivates, as depicted in Figure 1A. We anticipated that
incorporation of these strong electronꢀwithdrawing groups with BHC would
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significantly diminish the fluorescence via the PET and ICT mechanisms.46In contrast,
tandem addition of H2Sn into βꢀcarbon and carbonyl position of acrylate template
would generate the twoꢀphoton excitable and highly fluorescent BHC.
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In order to verify our design strategy, photophysical properties of BHC and probe
FPꢀH were firstly explored after synthesis and structural characterization. Indeed, as
expected, FPꢀH is essentially nonꢀfluorescent (ФF = 0.006) in the aqueous buffer
solution, whereas BHC is highly fluorescent (ФF= 0.50) with maximum emission
peak around 490 nm under the same conditions. Significant fluorescence differences
between FPꢀH and BHC imply the possibility of realizing the “switchꢀon” fluorescent
sensing approach. We then proceeded to investigate the response capability of FPꢀH
toward H2Sn. Figure 2A illustrated that treatment of FPꢀH with Na2S4 solutionas H2Sn
donor elicited a dramatic fluorescence enhancement in pH 7.4 buffer solution at room
temperature, accompanied with an obvious green color fluorescence appearance under
a handꢀheld UV lamp. Notably, realꢀtime reaction kinetics of FPꢀH with H2Sn in
Figure 2B revealed that rapidly increased fluorescence intensity of FPꢀH reached a
plateau in less than 1 min upon the addition of Na2S4 solution, indicating that acrylate
ester template possesses the capture ability for H2Sn to quickly release the masked
fluorophore.As a matter of fact, the response time of FPꢀH is shorter than that of most
aforementioned H2Sn probes, which usually fulfill the response in the range of 10ꢀ30
min. It demonstrates that acrylate template would be benefit for the realꢀtime
detection of H2Sn.
In order to provide some insight into the mechanism of H2Snꢀinduced fluorescence
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enhancement of FPꢀH, we studied the reaction product of FPꢀH and Na2S4 by mass
spectrometry analysis. MS data in Figure S1 revealed that new peaks appeared at m/z
119 and 295, indicating that pentacyclic compound 1,2ꢀdithiolanꢀ3ꢀone was generated
through Michael addition/cyclization cascade reaction to afford strongly fluorescent
hydroxycoumarin derivate for a turnꢀon fluorescence response. Furthermore,
superposition of thinꢀlayer chromatography (TLC) spot and the normalized
fluorescence emission spectra of the BHCand FPꢀH + Na2S4 also provide
complementary proofs that the fluorescent product is the same as BHC (Figure
S2,S3).
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Molecular Engineering for Performance Evolution. As mentioned issue that
biothiolsꢀtriggered cascade reaction toward unsubstituted acrylate ester,
unsurprisingly, FPꢀH exhibited poor selectivity toward H2Sn over biothiols, especially
Cys, at their physiologically relevant concentrations (Figure S4), consistent with the
previously reported resultsby Feng et al.26It implies that, although FPꢀH has sensing
capacity for H2Sn, serious interference from intracellular biothiols would be an
annoying shortcoming when bioimaging applications. To deal with such selectivity
drawback, we focused on the substituted influence of αꢀcarbon position on the
acrylate ester template. Expectantly, increasing the steric crowding around αꢀcarbon
by substituted group would hinder the cascade reaction toward sterically demanding
biothiols such as GSH, Cys, and Hcy. Nevertheless, for H2Sn with small molecular
size, such steric hindrance effect would be out of action. In addition, the electronic
perturbation from substituent may also influence response performances of probes by
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changing the electrophilicity of acrylate ester template.
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To verify above hypothesis, three analogues, FPꢀCF3 bearing electronꢀwithdrawing
substituent of trifluoromethyl in the αꢀcarbon position of acrylate ester template,
FPꢀCH3 bearing electronꢀdonating substituent of methyl, andFPꢀPh with phenyl
substituent were synthesized. With theseFP series in hand, their response
performances toward H2Sn over biothiols were firstly evaluated. As can been seen
from Figure 3A that all other nonfluorescent FP series can also be obviously lighted
up after treatment with Na2S4 solution in pH 7.4 PB buffer, despite they exhibited
inferior fluorescence enhancement ratio than unsubstituted FPꢀH. More importantly,
as our expected, the interference from biothols is significantly suppressed under its
biologically relevant concentration for FPꢀCH3,FPꢀCF3, and FPꢀPh, indicating that
substituents play crucial role in improving selectivity of acrylate ester template
toward H2Sn via steric hindrance.
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Realꢀtime reaction kinetics of other FP serieswithH2Sn were then examined by
recording the variations in the emission intensity of wavelength at 490 nm. Figure S5
illustrated that all of them possessed fast response characteristic toward H2Sn similar
as FPꢀH. It is noteworthy that electron effect of substituent exerts a certain degree of
influence on the response rate of acrylate ester template. As depicted in Figure 3B, the
timeꢀdependent processes of their response toward H2Sn follow firstꢀorder kinetics
with different observed rate constant k,47 the relevant observed rate constants of
FPꢀCF3 bearing electronꢀwithdrawing trifluoromethyl substituent (kFPꢀCF3 = 6.0 × 10ꢀ2
sꢀ1) is about 1.5ꢀfold larger than that of unsubstituted FPꢀH (kFPꢀH= 4.0 × 10ꢀ2 sꢀ1),
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whereas FPꢀCH3 bearing electronꢀdonating substituents exhibited inferior response
rate toward H2Sn than FPꢀH. It manifests that electronꢀwithdrawing substituent would
be beneficial for acrylate ester template to capture H2Sn.
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To shed light on these substituent effects from a theoretical basis, computational
calculations for these probes were carried out (Table 1, Figure S6). To our delight, the
introduction of electronꢀwithdrawing trifluoromethyl group increased the positive
charge at the enone βꢀcarbon of template (−0.253 eV of FPꢀCF3 versus −0.371 eV of
FPꢀH), which would make it more electrophilic to handily react with nucleophilic
sulfhydryl of H2Sn. In sharp contrast, electronꢀdonating substituent exhibited opposite
effect on the electrostatic charges of enone of template, indicative of inert reactivity of
FPꢀCH3relative to that of FPꢀH. For FPꢀPh, the electrostatic charge of enone of
template was slight influenced by phenyl substituent due to the mutually offsetting
result between electronꢀdonating and conjugated effect of phenyl, which is in good
agreement the similar reaction kinetics between FPꢀPh and FPꢀH. In addition, the
bond dissociation energies (BDES) of the ester bond gradually magnify from 249.38
to 280.65 kJ molꢀ1 following the order the trend ofFPꢀCH3 < FPꢀPh <FPꢀCF3< FPꢀH,
in accordance with the discrepancy of the fluorescence enhancement ratios of FP
series. Obviously, the cleavage of the ester bond is more effortless as the BDES value
is smaller, indicative of instability and higher background signal of probes. Therefore,
the optimal fluorescence enhancement of FPꢀH upon treatment of H2Sn can be
reasonably explained. In brief, calculated consequences are in accordance with
reaction kinetics and fluorescence enhancement ratios of FP series.
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The above results definitely demonstrate that we can improve the response rate of
the acrylate ester type probes and suppress the severe biothiolsꢀinterference to a
minimum level by rationally tuning steric and electronic effects of the recognition
template. With the optimized probe, we would realize fast and selective response
toward H2Sn, which might contribute to highꢀquality fluorescence imaging for
biological application.
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Response Performances of FPꢀCF3. Taking into account both the H2Sn reaction
kinetics and fluorescence enhancement ability of FP series, FPꢀCF3 was chosen as
the best candidate probe and its capability for quantitative detection of H2Sn was
successively evaluated. As is demonstrated in Figure 4A, the fluorescence emission
spectra of FPꢀCF3 showed dramatic enhancement after the addition of increasing
concentrations of Na2S4 solution (0 to 1000 ꢁM). Observably, a signal to background
ratio S/B= 44.48 (S/B= (FꢀFbuffer)/(F0ꢀFbuffer)),48 was achieved in the presence of 1000
ꢁM Na2S4 (Figure 4B). Where F, Fbuffer, and F0 are fluorescence intensities at 490 nm
of FPꢀCF3 with Na2S4, buffer, and without Na2S4, respectively. Moreover, there was
good linearity between the S/B and the concentrations of Na2S4 in the range 0 to 10
ꢁM with a detection limit (3σ/slope) of 50 nM (Inset of Figure 4B), manifesting that
FPꢀCF3 is potentially adaptive for quantitative determination of trace
Na2S4concentrations.
It is of great importance for a bioimaging probe with highly specific response to the
target molecule over other potentially competing species in the complicated
biosystems. Therefore, except for Cys, evaluation on selectivity of FPꢀCF3 was
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extend to various biologically relevant species, including bioꢀrelated metal ions,
reactive oxygen species, reducing agents, small molecule thiols, and other reactive
sulfur species. As shown in Figure S7, besidesNa2S4,other H2Sndonors including
Na2S2,Na2S3, and the mixture of Na2S and NaClOalso induce different degree of
increase in fluorescence emission ofFPꢀCF3.However,Figure 4C depicted that no
obvious variations in S/B were observed in the presence of aforementioned
interference biologically relevant species. These results demonstrate that the probe
FPꢀCF3 shows excellent selectivity for H2Sn and possesses the ability to suitably
detect H2Sn in complicated biological environments. Moreover, FPꢀCF3 is stable and
could respond toward Na2S4 at a biologically relevant pH level (Figure S8), which
promises further application of FPꢀCF3 to imaging of intracellular H2Sn.
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Living Cell Bioimaging. Given that FPꢀCF3 is highly selective, fastꢀresponsive, and
very sensitive under in vitro conditions, we set out to exploreif these merits are
effectual in cellular level. Prior to the imaging experiments, low cytotoxic effect of
this candidate probe had been confirmed by standard MTT assay (Figure S9).
Subsequently, imaging experiments of living HeLa cells were carried out on
twoꢀphoton confocal laser scanning microscopy. One can be found in Figure 5that, the
cells staining with FPꢀCF3 showed no detectable fluorescence signals, yet began to
emit strongly after incubation with Na2S4 solution. Moreover, gradually bright
fluorescence can be visualized along with increasing concentrations of Na2S4 solution
(Figure S10). In addition, timeꢀdependent imaging of H2Sn inside live HeLa cells
suggested that the fluorescence of cells dramatically increased with incubation time
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over only 3 min (Figure S11). These combined results indicate that FPꢀCF3 shows
good cellꢀpermeability and is suitable for rapid visualization of intracellular H2Sn and
monitor its concentration fluctuation. Different from FPꢀCF3, no matter if the
presence of Na2S4, the contrast probe FPꢀH show strong green fluorescence inside
HeLa cells. In contrast, when preꢀtreated with a known thiol trapping reagent
Nꢀethylꢀmaleimide (NEM), no fluorescence was observed for another control cells
staining with FPꢀH, indicating that significant interference from intracellular biothiols
is present in the case of FPꢀH. To our delight, the free cells staining with FPꢀCF3still
keep silent even if treatment with high concentration of Cys (Figure 5), manifesting
that FPꢀCF3 is capable of selectively visualize H2Sn over Cys inside living cells.
It is widely acknowledged that H2Sn could be generated from cystathionine γꢀlyase
(CSE)ꢀmediated cysteine metabolism,49and inflammatory mediators such as
lipopolysaccharides (LPS) can induce the CSE overexpression,50 To demonstrate the
intracellular level of H2Sn is associated with LPS, image of LPSꢀmediated H2Sn
production in RAW264.7 cells using FPꢀCF3 were followingly performed. As
depicted in Figure 6, with the increased stimulation time of LPS (2 ꢁg/mL), cells
stained with FPꢀCF3 displayed a gradually enhanced fluorescence signal. In sharp
contrast, even though in the presence of LPS stimulation, fluorescence change
remained slight when probeꢀloaded RAW264.7 were preincubated with CSE inhibitor
DLꢀpropargylglycine.51 These results indicate that LPS can upregulate the production
of endogenous H2Sn by the CSE overexpression in RAW264.7 cells, and FPꢀCF3is
quite qualified for imaging endogenous H2Sn fluctuation in living cells.
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CONCLUSION. In summary, we found that acrylate ester, which is frequentlyꢀused
as the recognition template of reactionꢀbased Cys fluorescent probe, can also take
action toward H2Sn. To address the biothiolsꢀinterference issue for promising effective
H2Sn detection with acrylate ester template, taking advantage of steric hindrance and
electron effects, rational design and molecular engineering of αꢀsubstituted acrylate
analogues and research on their responses toward H2Sn were carried out for the first
time. After evolution of sensing performances, the optimal probe FPꢀCF3 bearing
trifluoromethyl substitute was proved to possess excellent characters including rapid
response, high sensitivity, positive selectivity over biothiols, and easily staining
ability of living cells, thereby allowing robust visualization of intracellular H2Sn
fluctuation. Above all, we propose a novel approach for designing molecular tool for
exploring H2Sn chemical biology.
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Supporting Information. The supporting information is available free of charge via
the Internet at http://pubs.acs.org. Experimental details, synthesis, NMR and mass
spectra, and additional spectroscopic data as noted in text.
ACKNOWLEDGMENTS
This work was financially supported by the National Natural Science Foundation of
China (21505006, 21735001, 21575018, 21605008, 31527803, 21545010), Hunan
Provincial Natural Science Foundation of China (2017JJ3332), the Scientific
Research Fund of Hunan Provincial Education Department (16C0033), the Open
Fund of State Key Laboratory of Chemo/Biosensing and Chemometrics of Hunan
University(2015011), and the Open Fund of Hunan Provincial Key Laboratory of
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Materials Protection for Electric Power and Transportation(Changsha University of
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REFERENCES
10
11
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18
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24
25
26
27
28
29
30
31
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35
36
37
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(1) Giles, G. I.; Tasker, K. M.; Jacob, C. Free Radical Biol. Med. 2001, 31,
1279−1283.
(2) Giles, G. I.; Jacob, C. Biol. Chem. 2002, 383, 375−388.
(3) Gruhlke, M. C. H.; Slusarenko, A. J. Plant Physiol. Biochem. 2012, 59, 98−107.
(4) Wang, R. Antioxid. Redox Signaling 2010, 12, 1061−1064.
(5) Greiner, R.; Pálinkás, Z.; Bäsell, K.; Becher, D.; Antelmann, H.; Nagy, P.; Dick, T.
P. Antioxid. Redox Signaling 2013, 19, 1749−1756.
(6) Kimura, Y.; Mikami, Y.; Osumi, K.; Tsugane, M.; Oka, J.; Kimura, H. FASEB J.
2013, 27, 2451–2457.
(7) Kimura, H. Proc. Jpn. Acad. Ser. B 2015, 91, 131−159.
(8) Gao, M.; Yu, F. B.; Lv, C. J.; Choo, J.; Chen, L. X. Chem. Soc. Rev. 2017,
46,2237–2271
(9) Liu, C.; Chen, W.; Shi, W.; Peng, B.; Zhao, Y.; Ma, H.; Xian, M. J. Am. Chem. Soc.
2014, 136, 7257−7260.
(10)Zeng, L.; Chen, S.; Xia, T.; Hu, W.; Li, C.; Liu, Z.Anal. Chem. 2015, 87,
3004−3010.
(11)Gao, M.; Yu, F.; Chen, H.; Chen, L. Anal. Chem. 2015, 87, 3631−3638.
(12)Yu, F.; Gao, M.; Li, M.; Chen, L. X.Biomaterials2015, 63, 93–101.
(13)Shang, H.; Chen, H.; Tang, Y.; Guo, R.; Lin, W. Sens. Actuators, B 2016, 230,
ACS Paragon Plus Environment

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Page 16 of 28
1
2
3
4
773−778.
5
6
7
8
9
(14)Huang, Y.; Yu, F.; Wang, J.; Chen, L. Anal. Chem. 2016, 88, 4122−4129.
(15)Han, Q.; Mou, Z.; Wang, H.; Tang, X.; Dong, Z.; Wang, L.;Dong, X.; Liu, W.
Anal. Chem. 2016, 88, 7206−7212.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
(16)Zhang, J.; Zhu, X. Y.; Hu, X. X.; Liu, H. W.; Li, J.; Feng, L. L.; Yin, X.; Zhang, X.
B.; Tan, W. Anal. Chem. 2016, 88, 11892−11899.
(17)Chen, W.; Pacheco, A.; Takano, Y.; Day, J. J.; Hanaoka, K.; Xian, M. Angew.
Chem., Int. Ed. 2016, 55, 9993ꢀ9996.
(18)Ma, J.; Fan, J.; Li, H.; Yao, Q.; Xu, F.; Wang, J.; Peng, X. J. Mater. Chem. B.
2017, 5, 2574−2579.
(19)Chen, W.; Rosser, E. W.; Zhang, D.; Shi, W.; Li, Y.; Dong, W.ꢀJ.;Ma, H.; Hu, D.;
Xian, M. Org. Lett.2015, 17, 2776−2779.
(20)Hou, Y.; Yang, X. F.; Zhong, Y.; Li, Z. Sens. Actuators, B 2016, 232, 531−537.
(21)Chen, W.; Rosser, E. W.; Matsunaga, T.; Pacheco, A.; Akaike, T.; Xian, M. Angew.
Chem., Int. Ed. 2015, 54, 13961−13965.
(22)Chen, W.; Pacheco, A.; Takano, Y.; Day, J. J.; Hanaoka, K.; Xian, M. Angew.
Chem., Int. Ed. 2016, 55, 9993ꢀ9996.
(23) Fan, Y.; Chen, W.; Shi, W.; Li, H.; Xian, M.; Ma, H. Chem. Commun. 2017, 53,
8759−8762.
(24)Yang, X.; Guo, Y.; Strongin, R. M. Angew. Chem., Int. Ed. 2011, 50,
10690−10693.
(25)Guo, Z.; Nam, S.; Park, S.; Yoon, J. Chem. Sci. 2012, 3, 2760ꢀ2765.
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(26)Zhang, Q.; Yu, D.; Ding, S.; Feng, G. Chem. Commun. 2014, 50, 14002−14005.
(27)Lee, Y. H.; Ren, W. X.; Han, J.; Sunwoo, K.; Lim, J. Y.; Kim, J. H.; Kim, J. S.
Chem. Commun. 2015, 51, 14401−14404.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
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52
53
54
55
56
57
58
59
60
(28)Niu, W.; Guo, L.; Li, Y.; Shuang, S.; Dong, C.; Wong, M. S. Anal. Chem. 2016,
88, 1908−1914.
(29)Ali, F.; Kushwaha, S.; Taye, N.; Chattopadhyay, S.; Das, A. Anal. Chem. 2016, 88,
12161−12168.
(30)Yue, Y. K.; Huo, F. J.; Li, X. Q.; Wen, Y.; Yi, T.; Salamanca, J.; Escobedo, J. O.;
Strongin, R. M.; Yin, C. X. Org. Lett. 2017, 19, 82−85.
(31)Fu, Z. H.; Han, X.; Shao, Y.; Fang, J.; Zhang, Z. H.; Wang, Y. W.; Peng, Y. Anal.
Chem. 2017, 89, 1937−1944.
(32)Nawimanage, R. R.; Prasai, B.; Hettiarachchi, S. U.; McCarley, R. L. Anal. Chem.
2017, 89, 6886−6892.
(33)Lin, W. Y.; Long, L. L.; Tan, W. Chem. Commun. 2010, 46, 1503−1505.
(34)Lavis, L. D.; Raines, R. T. ACS Chem. Biol. 2008, 3, 142−155.
(35)Li, Y.; Wang, H.; Li, J.; Zheng, J.; Xu, X.; Yang, R. Anal. Chem. 2011, 83,
1268−1274.
(36)Wu, Z.; Li, Z.; Yang, L.; Han, J.; Han, S. Chem. Commun. 2012, 48,
10120−10122.
(37)Yuan, L.; Lin, W.; Xie, Y.; Chen, B.; Zhu, S. J. Am. Chem. Soc. 2012, 134,
1305−1315.
(38)Sun, Q.; Li, J.; Liu, W. N.; Dong, Q. J.; Yang, W. C.; Yang, G. F. Anal. Chem.
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2013, 85, 11304−11311.
5
6
7
8
9
(39)Mao, G. J.; Zhang, X. B.; Shi, X. L.; Liu, H. W.; Wu, Y. X.; Zhou, L. Y.; Tan, W.
H.; Yu, R. Q. Chem. Commun. 2014, 50, 5790−5792.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
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60
(40)Yuan, L.; Lin, W.; Chen, H.; Zhu, S.; He, L. Angew. Chem., Int. Ed. 2013, 52,
10018−10022.
(41)Chen, Q.; Yang, J. F.; Li, Y. H.; Zheng, J.; Yang, R. H. Anal. Chim. Acta. 2015,
896, 128−136.
(42)Cheng, D.; Pan, Y.; Wang, L.; Zeng, Z. B.; Yuan, L.; Zhang, X. B.; Chang, Y. T. J.
Am. Chem. Soc. 2017, 139, 285−292.
(43)Kim, H. M.; Cho, B. R. Chem. Rev. 2015, 115, 5014−5055.
(44)Sarkar, A. R.; Heo, C. H.; Kim, E.; Lee, H. W.; Singh, H.; Kim, J. J.; Kang, H.;
Kang, C.; Kim, H. M.Chem. Commun., 2015, 51, 2407–2410
(45)Bae, S. K.; Heo, C. H.; Choi, D. J.; Sen, D.; Joe, E. H.; Cho, B. R.; Kim, H. M. J.
Am. Chem. Soc.2013, 135, 9915–9923.
(46)Li, X.; Zheng, Y.; Tong, H.; Qian, R.; Zhou, L.; Liu, G.; Tang, Y.; Li, H.; Lou, K.;
Wang, W. Chem. Eur. J. 2016, 22, 9247−9256.
(47)Dale, T. J.; Rebek, J. J. Am. Chem. Soc.2006, 128, 4500–4501.
(48)Zhao, W. J.; Li, Y. H.; Yang, S.; Chen, Y.; Zheng, J.; Liu, C. H.; Qing, Z. H.; Li, J.
S.; Yang, R. H. Anal. Chem. 2016, 88, 4833−4840.
(49)Ishigami, M.; Hiraki, K.; Umemura, K.; Ogasawara, Y.; Ishii, K.;Kimura, H.
Antioxid. Redox Signaling2009, 11, 205−214.
(50)Oh, G. S.; Pae, H. O.; Lee, B. S.; Kim, B. N.; Kim, J. M.; Kim, H. R.; Jeon, S. B.;
Jeon, W. K.; Chae, H. J.; Chung, H. T. Free Radical Biol. Med.2001, 41, 106–109.
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(51)Lin, V. S.; Lippert, A. R.; Chang, C. J. Proc. Natl. Acad. Sci. U. S. A.2013, 110,
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Scheme 1. Response Mechanism of αꢀSubstituted Acrylate Ester Template toward
H2Sn.
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H
CH3
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CF3
O
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O
O
N
O
O
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O
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S
FP-Ph
FP-CF3
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OH
HO
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R1=H
O
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O
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R3=Ph
R4=CF3
S
S
BHC
FP Series
Figure 1. Molecular structures and synthetic procedures of FP series.
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510
540
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Time/s
Wavelength/nm
Figure 2. (A) Fluorescence emission spectra of 0.2 ꢁM FPꢀH (a) in buffer solution
(50% THF, pH 7.4) and its reaction with 1 mMNa2S4 solution (b), λex = 460 nm. Inset:
the corresponding fluorescent color change (UV lamp, 365 nm). (B) Realꢀtime
fluorescence intensity records of 0.2 ꢁM FPꢀH in buffer solution (50% THF, pH 7.4)
upon additions of 1 mM Na2S4 solution. λex/λem = 460 nm/490 nm.
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Probe only
Probe + GSH
Probe + Hcy
Probe + Cys
Probe + Na2S4
-3
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F
C
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H
h
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F
Figure 3. (A) Fluorescence enhancement ratio (F/F0) of 0.2 ꢁMFP series reaction
with GSH, Hcy, Cys, and Na2S4respectively. λex/λem = 460 nm/490 nm. (B) Kinetic
plot of reaction of 0.2 ꢁM FP series with 200 ꢁM Na2S4. Inset: variations of the
observed k′ versus kinds of FP series.
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Probe
Series
Electrostatic
Charges, eV
Bond Dissociation
Energies, kJꢁmolꢀ1
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FPꢀH
FPꢀCF3
FPꢀCH3
FPꢀPh
280.65
278.23
249.38
270.23
−0.371
−0.253
−0.447
−0.397
Table 1.Calculated Electrostatic Charges at the Enone βꢀcarbonandBond Dissociation
Energies (BDES) at the Ester of FP Series.
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.5
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C
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Competing species
Competing species + Na2S4
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10
Figure 4. (A) Fluorescence response of 0.2 ꢁMFPꢀCF3 to Na2S4 (0ꢀ1000 ꢁM) in PB
buffer (pH = 7.4, containing 50% THF). λex = 460 nm. (B) Signalꢀtoꢀbackground
ratios (S/B) of FPꢀCF3 as a function of Na2S4 concentrations (0ꢀ1000 ꢁM) in PB
buffer (pH = 7.4, containing 50% THF). Inset: linear responses of S/B of FPꢀCF3 to
changing Na2S4 concentrations (0−10 ꢁM). (C) Fluorescence changes of FPꢀCF3 for
Na2S4 in the presence of other common species in PB (pH = 7.4, containing 50%
THF). The concentrations of Na2S4 and other species were 100 µM and 200 µM,
respectively.
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B1
A2
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D
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B3
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+Na2S4
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+Na2S4
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Control
2
+Cys
+NEM
Control
FP-CF3
FP-H
Figure 5. (Upper row of images) Confocal fluorescence images of HeLa cells using
2.0 ꢁM FPꢀCF3 (A) and FPꢀH (B) under different conditions. Left: loading with
probe (A1, B1); middle: treatment with 500 ꢁM Cys (A2), pretreated with 200 ꢁM
NEM (B2); right: in the presence of 100 ꢁM Na2S4 solution (A3, B3). The images
were collected at 480ꢀ560 nm upon excitation at 800 nm. Inset: the corresponding
bright fields of cells. Scale bar: 30 ꢁm. (Lower row of images) Quantification and
comparison of mean pixel intensities obtained from the corresponding fluorescence
images of FPꢀCF3 (C) and FPꢀH (D).
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g
1.0
.8
.6
.4
.2
c
d
e
f
a
b
Figure 6. Effects of the LPS stimulation on the endogenous H2Sn levels in RAW264.7
cells. Confocal fluorescence images: (a) Cells only (control); (b–e) The cells were
induced by LPS (1 mg/mL) for 0, 4, 8, and 12 h, respectively, and then incubated with
5ꢁMFPꢀCF3 for 20 min; (f) The cells were pretreated with DLꢀpropargylglycine (100
ꢁM) for 30 min, and then treated as (e).The images were collected at 480ꢀ560 nm
upon excitation at 800 nm. Inset: the corresponding bright fields of cells. Scale bar: 30
ꢁm. (g) relative pixel intensities obtained from the corresponding fluorescence
images.
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