A novel dual-emission fluorescent probe for the simultaneous detection of H2S and GSH

Article abstract of DOI:10.1039/c6cc00973e

A novel chlorinated coumarin-malononitrile fluorescent probe with three potential reaction sites, which exhibited highly selective, rapid response, low detection limit, and was capable of the simultaneous detection of H₂S (λ_ex/em = 515/564 nm) and GSH (λ_ex/em = 430/517 nm), was first proposed. The probe was successfully applied to dual-channel imaging H₂S and GSH in MCF-7 cells.

Full text of DOI:10.1039/c6cc00973e

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DOI: 10.1039/C6CC00973E
A novel dual-emission fluorescent probe for simultaneous detection
of H2S and GSH
Hongliang Li, Wen Peng, Weipei Feng, Yingxing Wang, Guofeng Chen, Shuxiang Wang,
Shenghui Li, Honfyan Li, Kerang Wang and Jinchao Zhang
A
novel chlorinated coumarin-malonitrile fluorescent probe was synthesized for
simultaneous detection of H2S and GSH from different emission channels.

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A novel dual-emission fluorescent probe for simultaneous
detection of H2S and GSH
H. Li,a W. Peng,a W. Feng,a Y. Wang,a G. Chen,a S. Wang,*a,b S. Li,a H. Li, a K. Wanga and J. Zhang*a,b
Received 00th January 20xx,
Accepted 00th January 20xx
DOI: 10.1039/x0xx00000x
www.rsc.org/
A novel chlorinated coumarin-malonitrile fluorescent probe with cleavage of the selenium-nitrogen bond,19 and GSH-induced
three potential reaction sites, which exhibited highly selective, substitution-cyclizatioin.20 In fact, the determination of H2S
rapid response, low detection limit, and simultaneous detection of and biothiols is important to clarify their complicated
H2S (λex/em= 515/564 nm) and GSH (λex/em= 430/517 nm), was first relationship in various physiological processes. In spite of the
proposed. The probe was successfully applied to dual-channel amazing progress has been made in distinguish H2S or GSH
imaging H2S and GSH in MCF-7 cells.
from other biothiols, to design a single fluorescent probe
displaying a distinct response to H2S and GSH simultaneously
from different emission channels is highly valuable but even
more challenging. In the current study, we present a novel
fluorescent probe, chlorinated coumarin-malonitrile (1), which
could discriminate H2S and GSH in different emission channels.
Particularly, the probe exhibits several meritorious features as
follow: (i) high sensitivity and selectivity to H2S and GSH from
different emission channels; (ii) significant fluorescence
enhancement based on different structures; (iii) rapid
response; (iv) excellent live cell imaging.
Hydrogen sulfide (H2S) and biothiols such as Cysteine (Cys),
homocysteine (Hcy), and glutathione (GSH), play important
roles in many physiological and physiological processes.1 H2S is
involved in various physiological processes, such as
antioxidation,2
ischemia
reperfusion
injury,3
anti-
infammation,4 and apoptosis.5 Cys displays diverse functions in
regulating of matrix degradation and cell motility.6 GSH plays a
central role in combating oxidative stress, maintaining redox
homeostasis, and defending against toxins and free radicals.7
Abnormal levels of H2S or biothiols are associated with various
diseases.8 More importantly, there is a significant correlation
between H2S level and biothiols level in the living systems.9
Hence, the determination of H2S and biothiols in biological
systems is required for better understanding their roles in
biological systems and accurate diagnosis of disease.10 Till
now, a number of fluorescent probes have been developed for
the detection of H2S based on their several significant
characteristic properties: dual-nucleophilicity,11 high binding
affinity towards Cu2+ ion,12 reduce the azide, nitro and
hydroxy-amine groups,13 and thiolysis of dinitrophenyl ether.14
The discrimination of Cys from Hcy/GSH was achieved based
on the Cys-induced SNAr substitution−rearrangement
reaction,15 Michael addition,16 or cyclization reaction with –
CHO.17 And, the discrimination of GSH from Cys/Hcy was
The design rationale is depicted in Fig 1. The probe 1 has
three potential reaction sites, utilizing coumarin as
fluorophore, chloro atom lies in 4-position of coumarin moiety
as a leaving group (Site I), α,β unsaturated malonitrile as a
a
−
Michael acceptor (Site II), and cyano group as an electrophile
(Site III) and a quencher. It displays an extremely low
background signal for the electron withdrawing of
α,β−unsaturated malonitrile owing to intramolecular charge
transfer (ICT) and photoinduced electron transfer (PET)
process. We speculate that probe 1 discriminates H2S from
biothiols and the other biologically related species based on
the dual-nucleophilicity of H2S. Probe 1 reaction with H2S
provided thio-coumarin-malonitrile (1a) by undergoing rapid
thiol-halogen nucleophilic aromatic substitution (SNAr).
Subsequently, the intramolecular nucleophilic-addition adduct
(iminocoumarin, 1b, Fig 1A) was constructed by intramolecular
nucleophilic attack of the thiophenol to site 3,21 which resulted
in blocking the ICT and PET process, and increased the π-
conjugation system. Now, let us turn our attention to the
reaction of 1 with GSH. Similar initial thio-halogen SNAr would
lead to thio-coumarin-malononitrile 2a (Fig 1B). However, due
to the unstable 10-membered macrocyclic transition state, it is
difficult for 2a to undergo the ring intramolecular
proposed
based
on
deprotection
of
2,4-
dinitrobenzenesulfonyl-conjugated fluorophore,18 selective
a.
Key Laboratory of Chemical Biology of Hebei province, College of Chemistry and
Environmental Science, Hebei University, Baoding 071002, China.
b.
Key Laboratory of Medicinal Chemistry and Molecular Diagnosis of Ministry of
Education, Baoding 071002, China. Email: jczhang6970@163.com;
wsx@hbu.edu.cn
† Electronic supplementary information (ESI) available: Experimental details. See
DOI: 10.1039/x0xx00000x
This journal is © The Royal Society of Chemistry 20xx
J. Name., 2013, 00, 1-3 | 1
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rearrangement to generate amino-coumarin-malononitrile.16e probe 1 could reaction with H2S and GSH, offered different
Alternatively, it is possible that the free thiol group in 2a would products and exhibited different optical properties.
attack site 2 or 3 to produce the 12- or 14-membered ring Furthermore, the absolute fluorescence quantum yield of 1b
product glutathione-coumarin-malononitrile 2b or 2c. In fact, and 2b was determined to be 23.84% and 5.94% at the
the speculation could be partially supported by recent concentration of 3×10-6 M in PBS at 515 and 430 nm
publications on large ring products.16e,16f, 20 To confirm the final excitations. These results indicate that 1b possesses high
product (2b), we choose DODT as a model compound. 1H NMR fluorescence properties in water at low concentrations. So,
(Fig. S12, ESI†) of product obtained by adding DODT to 1 probe 1 could selectively detect H2S and GSH in the respective
indicated that the free thiol group attacked site 3 to produce detection channels.
the corresponding ring product (Fig. S8E, ESI†). Overall, if our
500
B
hypothesis is rational, it would be promising to realize the
A
600
400
probe
selective discrimination of H2S and GSH in terms of the
probe
probe
probe
probe
+
+
+
+
H2S
probe+H
S
2
500
400
300
200
100
0
Cys
probe+Cys
300
200
100
0
Hcy
GSH
different chemical structures and photophysical properties of
the corresponding 1b and 2b. As for Cys/Hcy (Fig S8C and S8D,
ESI†), site 1 of 1 was attacked by thiol group of Cys/Hcy to
produce thio-coumarin-malononitrile 3a/4a, and the following
rearrangement would lead to generate amino-coumarin-
malononitrile 3b/4b, and the following Michael addition
reaction of the thiol group with site 2 form a seven/eight-
membered cyclized adduct 3c/4c. Meanwhile, the site 3
reactions with excessive Cys/Hcy generates condensation
product 3d/4d with similar photophysical properties. So, it is
difficult to discriminate between Cys and Hcy with different
fluorescence signals.
probe+Hcy
probe+GSH
probe
450
500
550
600
650
700
540 560 580 600 620 640 660 680 700 720
Wavelength
(
nm
)
Wavelength (nm)
Fig. 2 Fluorescence response of probe (5 μM in PBS buffer) to 10
equiv. of H2S (A, λex= 515 nm, Slits: 5/5 nm), Cys, Hcy and GSH (B,
λex=430 nm, Slits: 10/10 nm) at r. t.
To apply this probe in more complicated systems, we also
tested the effect of pH on the fluorescence response of 1 to H2S and
GSH (Fig. S5, ESI†). It was found that this probe is stable enough in
pH range of 6-9, and displayed the obvious response for H2S and
GSH in the neutral or weak base condition at 515 and 430 nm
excitation. Due to the neutral or weak base condition enhancement
the nucleophilic property of H2S and GSH that led to speed up the
thio-chlorin SNAr and intramolecular nucleophilic addition of 1 with
H2S and GSH. The stable fluorescence of 1 at neutral condition is
favourable for the selective detection of H2S and GSH in biological
samples.
Subsequently, we examined the emission behaves of 1 towards
the mixture of H2S, Cys, Hcy and GSH (Fig. S3, ESI†). Meanwhile, the
time-dependent fluorescence response of 1 to Cys and Hcy in the
same conditions was investigated (Fig. S4, ESI†). It was found that
Cys/Hcy/GSH hardly elicited any significant fluorescence changes of
Fig. 1 Sensing scheme of 1 with H2S and GSH
Initially, we synthesized probe 1 (Scheme S1, ESI†)
，and
1
response to H2S. As well, H2S/Cys/Hcy hardly elicited any
examined the optical sensing behaviour by utilization time-
dependent UV−vis spectrum of 1 reaction with H2S or GSH and
Cys/Hcy in DMF/phosphate buffer (V/V: 3/7, 10 mM, pH 7.4) at
r.t. (Fig. S1, ESI†). Upon addition of H2S or GSH/Cys/Hcy to
probe 1, UV-vis spectra are dramatically changed, a peak at
λmax= 505 nm disappeared while new peaks around 350 to 470
nm appeared. The profound blue-shift indicated that the
conjugation between the coumarin and the malonitrile is
broken due to the Michael addition of GSH/Cys/Hcy to 1.
Treated with 10 equiv of H2S in PBS buffer (10 mM, pH 7.4) at
25 °C, the product mixture exhibited a significant fluorescence
enhancement at λex/em= 515/564 nm (Fig. 2A). As a result, the
new emission peak appeared and reached a plateau after 6
min, indicating the completion of the reaction. In this case, an
approximate 16-fold increase in fluorescence signal could be
observed. As shown in Fig. 2B, upon addition of GSH to the
solution of 1 leaded to a fluorescence enhancement of 7.5-fold
at 517 nm (λex = 430 nm). These results indicated that the
significant fluorescence changes of 1 response to GSH. The above
results confirmed that probe 1 exhibited the capability of highly
selective detection of H2S and GSH from different emission channels.
Further, we evaluated the changes in the emission spectra of
probe upon addition of different concentrations of H2S and GSH
were in DMF/phosphate buffer (3/7: V/V, 10 mM, pH 7.4) solution
at room temperature. As show in Fig 3, it was found that
approximate 2 equiv of H2S (equiv of GSH) could complete the
reactions. In addition, the fluorescent intensity was linearly
proportional to the amount of them from 0 to 2 equiv and the
linear correlations (R2 = 0.99788 for H2S, and 0.99577 for GSH) were
found between the observed fluorescence intensity and
concentrations. The detection limits (DL, S/N= 3) for H2S and GSH
were determined to be 42 and 87 nM, respectively. In addition, the
sensing reactions can be completed within 6 min (Fig. S2, ESI†).
Because the intracellular concentration of H2S and GSH are in the
range of l0-l00 μM 22, 23 and 1–10 mM,24 respectively. So, probe 1 is
sensitive enough to image H2S and GSH in cells.
2 | J. Name., 2012, 00, 1-3
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To evaluate the specific nature of 1 for sensing of H2S and GSH, at m/z: 545.1520 attributing to [4d+H]+ (calcd. 545.1528 for
we then examined the fluorescence enhancement of 1 incubated C25H29N4O6S2, Fig. S16, ESI†). These results are in good agreement
with various species (Fig. S6, ESI†), all of which are biologically with the designed strategy. Above all results would be promising
related. At 515 nm excitation, this probe exhibited a very limited the selective discrimination of H2S and GSH in terms of the different
fluorescence enhancement to various amino acids (eg. Ala, Val, Leu, photophysical properties.
Ile, Pro, Phe, Trp, Met, Gly, Ser, Thr, Glu, Lys, Arg, Tyr, His, Asp, Glu,
Finally, we investigated the potential use of probe 1 in live
and Asn), glucose, ascorbic acid, and thiols (Cys, Hcy, and GSH); only cell imaging. When MCF-7 cells were incubated with it for 30
H2S elicited significant fluorescence turn-on of probe 1 at 564 nm. min, bright fluorescence in blue and red channels was
At 430 nm excitation, various amino acids, glucose, ascorbic acid, observed simultaneously (Fig. 4). Furthermore, when the cells
H2S, Cys, and Hcy triggered almost no fluorescence intensity were pre-treated with N-ethylmaleimide (NEM, a scavenger of
changes, and only GSH caused significant fluorescence turn-on at H2S and biothiols) and then treated with 1, the fluorescence in
517 nm. These results demonstrate that this probe is high selective the red and blue channels was both decreased dramatically,
for H2S and GSH over other competitive species at different conforming that probe 1 is a specific tool for the detection of
excitation.
endogenous H2S and GSH in actual living cells. These results
showed that probe 1 not only discriminates H2S and GSH at the
blue and red channels, but also can be employed to monitor
endogenous H2S and GSH level in living cells.
A1
0
600
0.5 ꢀꢀ
M
ꢀꢀ
M
600
500
400
300
200
100
0
550
500
450
400
350
300
250
200
150
100
50
A2
20
ꢀM
1
2
3
4
5
6
7
8
9
10 ꢀꢀ
12 ꢀꢀ
14 ꢀꢀ
16 ꢀꢀ
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M
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ꢀꢀ
M
Equation
y
=
a
+
b*x
0.99788
Adj. R-Square
M
Value
Standard Error
M
Intercept
Slope
34.1869
44.48514
3.50603
0.61878
B
B
18 ꢀꢀ
20 ꢀꢀ
M
M
0
540 560 580 600 620 640 660 680 700 720
0
5
10
15
20
H
S
(
ꢀ
M
)
Wavelength
(
nm)
2
500
400
300
200
100
0
B1
0
ꢀꢀΜΜ
ꢀꢀΜΜ
ꢀꢀΜΜ
ꢀꢀΜΜ
ꢀꢀΜΜ
B2
450
400
350
300
250
200
150
100
50
0.5 ꢀꢀΜΜ
1
2
3
4
5
6
7
8
12 ꢀΜ
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0
ꢀꢀΜΜ
ꢀꢀΜΜ
ꢀꢀΜΜ
ꢀꢀΜΜ
ꢀꢀΜΜ
ꢀꢀΜΜ
10 ꢀꢀΜΜ
ꢀꢀΜΜ
12 ꢀꢀΜΜ
y
=
a
+
b*x
ꢀꢀΜΜ
Equation
Pearson's
0.99831
0.99577
r
Adj. R-Square
Value
Standard Error
5.40286
2.40623
Intercept
Slope
61.12187
82.57531
B
B
450
500
550
600
650
700
0
1
2
3
4
5
6
7
8 9 10 11 12 13
GSH (ꢀΜ)
GSH (ꢀΜ
)
Fig. 3 Fluorescence spectra of 1 (5 μM in PBS buffer) upon addition
of varied concentrations of H2S (A, λex= 515 nm, Slits: 5/5 nm), and
GSH (B, λex=430 nm, Slits: 10/10 nm) at r. t.
Cytotoxicity is an important indicator for applicability of probe
in biological systems. Therefore, the effect of 1 on cell proliferation
was determined by MTT assay in MCF-7 cells. Probe 1 showed no
cytotoxicity at a detection range concentration (0.1-10 μM) (Fig. S7,
ESI†). These results implied that this probe was suitable for H2S and Fig. 4 Imaging H2S and GSH in MCF-7 cells by probe 1 from different
GSH detection in live cells.
emission channels, emission was collected at 450–510 nm for green
channel assigned to GSH (λex=405 nm), and 550–610 nm for red
channel assigned to H2S (λex= 515 nm).
Furthermore, the products of probe 1 with H2S and GSH were
evidenced by HRMS. The mixture of H2S and probe 1 in CH3CN-H2O
(1/1, V/V) was separated, and showed a major signal at m/z
348.07791 (calcd. 348.07772; Fig. S14, ESI†) assigned to [1b+Na]+
(C17H15N3NaO2S). The formation of 1b was also evidenced by 1H
NMR study. Upon addition of NaHS to 1, an imine proton (Ha) was
appearance at 11.1 ppm, and the vinyl proton (Hb) was obvious
upfield shifts, as well as the benzol protons (Hc, Hd) (Fig. S11, ESI†).
The product mixture of GSH with probe 1 in CH3CN-H2O (1/1, V/V)
solution, exhibits a peak at m/z: 597.1784 correspond to [2c-H]-
(calcd. 597.1768 for C27H29N6O8S, Fig. S17, ESI†). In addition, the
products of probe 1 with Cys and Hcy were also investigated. A
prominent peak at m/z: 517.1217 corresponding to [3d+H]+ (calcd
517.1215 for C23H25N4O6S2) was clearly observed in the HRMS data
of reaction probe 1 with Cys in CH3CN-H2O (1/1, V/V) solution (Fig.
S15, ESI†). A similar result of probe 1 with Hcy was observed, a peak
Cells in A and B were incubated with 10 μM probe 1 for 30 min.
Cells in C and D were first incubated with NEM for 30 min, and then
were incubated with 10 μM probe for 30 min.
In summary, we presented a novel chlorinated coumarin-
malononitrile fluorescence probe with three potential reaction
sites for discrimination H2S and GSH from different emission
channels (H2S: λex/em= 515/564 nm with 16-fold increase in
fluorescence, GSH: λex/em= 430/517 nm with 7.5-fold increase
in fluorescence) based on different chemical structures with
distinct photophysical properties. This probe exhibits a high
selectivity for H2S and GSH, low limit of detections (DL for H2S
and GSH were 42 and 87 nM), and fast response speeds. And,
This journal is © The Royal Society of Chemistry 20xx
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Wang, S. Cui, S. Sun and X. Peng, Chem. Commun., 2013, 49
,
the probe could simultaneously monitoring endogenous H2S
and GSH in MCF-7 cells with only minimal cytotoxicity. We
hope that the novel strategy could inspire the exploration of
new systems to reveal H2S and GSH levels in biological systems.
This work was supported by the Key Basic Research Special
Foundation of Science Technology Ministry of Hebei Province
(Grant No. 12966418D), the Key Research Project Foundation
of Departement of Education of Hebei Province (Grant No.
ZH2012041), the Natural Science Foundation of Hebei Province
(No. B2015201213).
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