Simultaneous Detection of Glutathione and Hydrogen Polysulfides from Different Emission Channels

Article abstract of DOI:10.1021/acs.analchem.7b04033

Glutathione (GSH) and hydrogen polysulfides (H₂S_n) play crucial roles in many physiological processes. To unravel the complicated interrelationship and cellular cross-talk between GSH and H₂S_n, the development of single-molecule fluorescent probes that can selectively sense GSH and H₂S_n simultaneously from different emission channels is highly desirable. In this report, we have developed the first dual-detection fluorescent probe, ACC-SePh, which responded to GSH with green fluorescence emission, whereas it reacted with H₂S_n and emitted blue fluorescence. The probe exhibited excellent selectivity and sensitivity toward GSH and H₂S_n over other common reactive sulfur species, such as Cys, Hcy and H₂S. Importantly, we also demonstrated that ACC-SePh can be used for dual-channel imaging of endogenous GSH and H₂S_n in living RAW264.7 cells.

Full text of DOI:10.1021/acs.analchem.7b04033

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Article
Simultaneous Detection of Glutathione and Hydrogen
Polysulfides from Different Emission Channels
Wenqiang Chen, Xiuxiu Yue, Hui Zhang, Wenxiu Li, Liangliang
Zhang, Qi Xiao, Chusheng Huang, Jiarong Sheng, and Xiangzhi Song
Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.7b04033 • Publication Date (Web): 03 Nov 2017
Downloaded from http://pubs.acs.org on November 4, 2017
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Simultaneous Detection of Glutathione and Hydrogen Polysulfides
from Different Emission Channels
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Wenqiang Chen,*† Xiuxiu Yue,† Hui Zhang,§ Wenxiu Li,‡ Liangliang Zhang,‡ Qi Xiao,† Chusheng
Huang,† Jiarong Sheng,*†and Xiangzhi Song§
†College of Chemistry and Materials Science, Guangxi Teachers Education University, 530001 Nanning, Guangxi, P. R.
China.
‡State Key Laboratory for the Chemistry and Molecular Engineering of Medicinal Resources of Education Ministry, Guangxi
Normal University, 541004 Guilin, Guangxi, P. R. China.
§College of Chemistry & Chemical Engineering, Central South University, Changsha, Hunan 410083, P. R. China.
*Corresponding author, Eꢀmail: chenwq@csu.edu.cn; Fax: +86ꢀ771ꢀ3908065; Eꢀmail: shengjiarong99@163.com; Fax: +86ꢀ771ꢀ3908018
ABSTRACT: Glutathione (GSH) and hydrogen polysulfides (H2Sn) play crucial roles in many physiological processes. To unravel
the complicated interrelationship and cellular crossꢀtalk between GSH and H2Sn, the development of singleꢀmolecule fluorescent
probes that can selectively sense GSH and H2Sn simultaneously from different emission channels is highly desirable. In this report,
we have developed the first dualꢀdetection fluorescent probe, ACCꢀSePh, which responded to GSH with green fluorescence emisꢀ
sion, whereas it reacted with H2Sn and emitted blue fluorescence. The probe exhibited excellent selectivity and sensitivity toward
GSH and H2Sn over other common reactive sulfur species, such as Cys, Hcy and H2S. Importantly, we also demonstrated that ACCꢀ
SePh can be used for dualꢀchannel imaging of endogenous GSH and H2Sn in living RAW264.7 cells.
specific fluorescent probes for H2Sn were developed based on
the extended version of this strategy.19ꢀ30
INTRODUCTION SECTION
Given that reactive sulfur species (RSS) play crucial roles in
a variety of physiological and pathological processes, an imꢀ
portant and everꢀincreasing research field which focused on
the chemical biology of RSS was emerged in recent decades.1ꢀ3
Among RSS, glutathione (GSH) is most abundant in the bioꢀ
logical system,4 functioning as an essential role in maintaining
redox homeostasis, and defending against free radicals and
toxins.5ꢀ6 Aberrant GSH levels in living systems are closely
associated with various diseases such as cancer, Alzheimer’s
disease, Parkinson disease, AIDS, osteoporosis and cardiovasꢀ
cular disease.7ꢀ8 Very recently, disentangling the redox biology
of hydrogen polysulfides (H2Sn, n≥2) has become the current
intense interest in RSS.9ꢀ12 Increasing evidences suggest that
H2Sn exhibit high potency in modulating various physiological
processes including activating ion channels, transcription facꢀ
tors and tumor suppressors.9ꢀ13
With the inꢀdepth study of the biological pathways of GSH
and H2Sn, growing evidences have indicated that these two
species were interrelated in biological systems. For example,
endogenous H2Sn were produced enzymatically by cystarhioꢀ
nineꢀγꢀlyase (CSE) and cystathionine βꢀsynthase (CBS),31 and
GSH might be the actual sulfur source of H2Sn. Because high
levels of H2Sn precursors, glutathione hydropersulfide (>100
µM), were detected in the plasma, cells, and tissues of mamꢀ
mals.32 To make sense of the mutual relationship and cellular
crossꢀtalk between GSH and H2Sn, the development of fluoꢀ
rescent probes that can sense GSH and H2Sn from distinct
emission channels is highly valuable. To meet the abovemenꢀ
tioned requirements, a simple solution is to use two specific
probes simultaneously in a cell.33ꢀ34 However, just as pointed
out by Suzuki and coꢀworkers, this strategy suffers from limiꢀ
tations such as 1) larger invasive effects, 2) potential crossꢀtalk
between different probes, 3) the complicated scenario caused
by the distinct localization and metabolisms of the probes.35
Therefore, a singleꢀmolecule fluorescent probe that can selecꢀ
tively visualize GSH and H2Sn simultaneously from different
emission channels is desperately needed. To the best of our
knowledge, such a singleꢀmolecule probe has not been reportꢀ
ed yet. Herein, we present the rational design, synthesis and
spectral properties of ACCꢀSePh, the first singleꢀmolecule
fluorescent probe, which can simultaneously detect GSH and
H2Sn from different emission channels. Significantly, with the
help of a laser confocal microscope, we have demonstrated
that ACCꢀSePh was capable of detecting endogenous GSH
Due to the significance of GSH and H2Sn in redox biology,
establishment of convenient detection methods for these speꢀ
cies are highly desirable.14ꢀ16 Unfortunately, the selective deꢀ
tection of GSH over cysteine (Cys) or homocysteine (Hcy)
was a great challenge, and as far as we know, merely few exꢀ
amples were reported. One appealing strategy, reported by
Yang etc., is based on the distinct reaction processes between
GSH, Cys/Hcy and chlorinated BODIPYs, thereby enabling
the highly selective detection of GSH over Cys/Hcy.17 Similarꢀ
ly, fluorescent probes specific for H2Sn over other RSS are
also rare. Pioneered by Xian’s group, the first fluorescent
probe for H2Sn was developed by taking advantage of H2Snꢀ
mediated benzodithione formation.18 From then on, some more
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and H2Sn simultaneously in living RAW264.7 cells through
dualꢀcolor fluorescence imaging.
yellow solid (790 mg, 77% yield). Mp: 180ꢀ182°C. H NMR
(300 MHz, DMSOꢀd6) δ 13.65 (s, 1H), 7.64 (d, J = 9.2 Hz,
1H), 6.86 (dd, J = 9.2, 2.4 Hz, 1H), 6.63 (d, J = 2.4 Hz, 1H),
3.48 (q, J = 7.0 Hz, 4H), 1.14 (t, J = 7.0 Hz, 6H). 13C NMR (75
MHz, DMSOꢀd6) δ 169.06, 162.30, 159.73, 157.27, 150.49,
132.30, 119.41, 115.42, 110.10, 101.55, 49.47, 49.47, 17.48,
17.48. HRMS (ESI) m/z: calcd for C14H14ClNO4Na [M+Na]+,
318.0509; found 318.0509.
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EXPERIMENTAL SECTION
Materials and instruments
Unless otherwise noted, all reagents were obtained from
commercial suppliers and used without further purification.
Solvents were purified by standard methods prior to use.
Twiceꢀdistilled water was used throughout all experiments.
NMR spectra were recorded on a BRUKER 300 and 600 specꢀ
trometer using TMS as an internal standard. All accurate mass
spectrometric experiments were performed on a Xevo G2
QTof MS (Waters, USA). UVꢀVis absorption spectra were
recorded on a TUꢀ1901 (Puxi, P.R. China) spectrophotometer.
Fluorescence spectra were recorded at room temperature using
a HITACHI Fꢀ4600 fluorescence spectrophotometer with both
the excitation and emission slit widths set at 5.0 nm. Cell imꢀ
aging was performed with a Zeiss LSM 710 laser scanning
confocal microscope. TLC analysis was performed on silica
gel plates and column chromatography was conducted using
silica gel (mesh 200ꢀ300), both of which were obtained from
Qingdao Ocean Chemicals, China.
Synthesis of compound 4
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Compound 3 (36 mg, 0.12 mmol) and NaBH4 (6 mg, 0.16
mmol) were dissolved in 0.5 mL ethanol. The mixture was
stirred at room temperature for 10 min under an argon atmosꢀ
phere. The resulting colorless solution was used directly in
next step without further purification.
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Synthesis of compound 5
Compound 2 (60 mg, 0.2 mmol) and Et3N (80 ꢀL, 0.6
mmol) were dissolved in 3.0 mL of anhydrous DMF. Subseꢀ
quently, compound 4 (36 mg, 0.24 mmol) was added. The
reaction mixture was stirred at room temperature for 20 min.
Then the mixture was poured into 100 mL water and extracted
with dichloromethane (3×50 mL). The combined organic layꢀ
ers were washed successively with brine and water, dried over
sodium sulfate, and evaporated in vacuum to leave compound
General procedure for spectral measurements
A stock solution of ACCꢀSePh was prepared at 1 mM in
DMF. Solutions of various testing species were prepared from
Na2S2, glutamate (Glu), proline (Pro), serine (Ser), tyrosine
(Tyr), glutathione (GSH), cysteine (Cys), methionine (Met),
Na2S, NaHSO3, Na2SO4, Na2S2O3, NaClO, H2O2, CaCl2,
MgCl2, KCl and ZnCl2 in twiceꢀdistilled water. Homocysteine
(Hcy) was prepared in PBS buffer (10 mM, pH =7.4). A typiꢀ
cal test solution (10.0 mL) was prepared by placing 0.05 mL
of ACCꢀSePh (1 mM), 5.0 mL of PB buffer (20 mM, pH =
7.4, containing 2 mM CTAB), and an appropriate aliquot of
each analyte stock solution into an appropriate amount of
twiceꢀdistilled water. The resulting solution was shaken well
and kept at room temperature (25°C) for 60 min before recordꢀ
ing its spectra.
1
5 as an orange solid (81 mg, 98%). Mp: 158ꢀ160°C; H NMR
(300 MHz, CDCl3) δ 13.24 (s, 1H), 7.52 (dd, J = 6.4, 3.0 Hz,
2H), 7.38 (d, J = 9.5 Hz, 1H), 7.33 – 7.28 (m, 3H), 6.41 (d, J =
2.6 Hz, 1H), 6.19 (dd, J = 9.5, 2.6 Hz, 1H), 3.39 (q, J = 7.1 Hz,
4H), 1.19 (t, J = 7.1 Hz, 6H). 13C NMR (75 MHz, CDCl3) δ
169.19, 165.84, 163.99, 154.40, 152.47, 134.12, 133.41,
134.41, 133.30, 129.79, 129.79, 128.33, 109.88, 109.37,
106.45, 96.24, 45.02, 45.02, 12.41, 12.41. HRMS (ESI) m/z:
calcd for C20H19NO4SeNa [M+Na]+ 440.0377, found
440.0379.
Synthesis of model compound 6
To a mixture of compound 5 (83 mg, 0.2 mmol), phenol (28
mg, 0.3 mmol), EDCI (28 mg, 0.3mmol) and DMAP (4 mg,
0.03 mmol) was added dry CH2Cl2 (3 mL) at room temperaꢀ
ture. The mixture was stirred at room temperature for 1 hour.
Then solvent was removed under reduced pressure and the
resultant residue was further purified by silica gel chromatogꢀ
raphy to afford the desired product 6 as an orange solid (56
Cell culture and fluorescence imaging
RAW264.7 cells were seeded in a 6ꢀwell plate in Dulbecꢀ
co’s modified Eagle’s medium (DMEM) supplemented with
10% fetal bovine serum and 1% penicillin. The cells were
incubated under an atmosphere of 5% CO2 and 95% air at
37°C for 24 h. Cell imaging was performed with a Zeiss LSM
710 laser scanning confocal microscope. Before each experiꢀ
ment, cells were washed with PBS buffered solution 3 times.
Excitation wavelength: 405 nm. Emissions were collected at
440ꢀ460 nm for blue channel and 520ꢀ560 nm for green chanꢀ
nel.
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mg, 57%). Mp: 155ꢀ156°C; H NMR (600 MHz, CDCl3) δ
7.66 (d, J = 8.9 Hz, 1H), 7.54 (d, J = 3.3 Hz, 2H), 7.38 (t, J =
7.8 Hz, 2H), 7.23 (dd, J = 15.6, 5.3 Hz, 6H), 6.49 – 6.44 (m,
2H), 3.38 (d, J = 7.0 Hz, 4H), 1.18 (t, 6H). 13C NMR (150
MHz, CDCl3) δ 163.68, 157.53, 155.41, 151.61, 150.69,
147.90, 132.47, 131.24, 129.73, 129.60, 129.38, 128.00,
126.09, 121.66, 119.98, 109.43, 97.13, 44.94, 12.41. HRMS
(ESI) m/z: calcd for C26H23NO4SeNa [M+Na]+, 516.084;
found 516.0693.
Synthesis of compound 2
Resorcin (7.609g, 69 mmol) and compound 1 (965 mg, 3.5
mmol) were dissolved in 6 mL of acetonitrile, the mixture was
stirred at 0°C for 5 min. Then, a portion of NaClO2 (1.470 g,
12.25 mmol) and NaH2PO4 (1.418 g, 15.75 mmol) (dissolved
in 1.5 mL water) was added dropwise to the mixture at 0°C.
The reaction mixture was stirred at 0°C for additional 30 min,
poured into ice water (30 mL). After acidification of the mixꢀ
ture by 1M HCl, a large amount of yellow precipitates formed.
The precipitates were collected by filtration and rinsed with
cold water (2×20 mL) and dried under vacuum to afford a
Synthesis of model compound 7
Compound 7 was prepared using the same procedure for
compound 6. Yield: 62%. Mp: 189ꢀ190°C; H NMR (600
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MHz, CDCl3) δ 7.65 (d, J = 9.0 Hz, 1H), 7.53 (dd, J = 6.2, 2.9
Hz, 2H), 7.24 (d, J = 2.7 Hz, 2H), 7.13 (d, J = 9.0 Hz, 2H),
6.88 (d, J = 9.0 Hz, 2H), 6.76 (s, 1H), 6.45 (dd, J = 12.4, 3.1
Hz, 2H), 3.79 (s, 3H), 3.38 (q, J = 7.0 Hz, 4H), 1.18 (t, J = 7.1
Hz, 6H). 13C NMR (150 MHz, CDCl3) δ 164.07, 157.41,
155.40, 151.65, 144.18, 132.46, 131.20, 129.71, 129.62,
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Analytical Chemistry
127.97, 122.41, 115.99, 114.74, 114.37, 109.99, 109.34,
108.43, 97.05, 55.58, 44.87, 12.41. HRMS (ESI) m/z: calcd
for C27H25NO5SeNa [M+Na]+, 546.0796; found 546.0800.
7.1 Hz, 4H), 1.89 (s, 3H), 1.25 (t, J = 5.1 Hz, 6H). HRMS
(ESI) m/z: calcd for C27H30N2O8SNa [M+Na]+ 565.1621;
found 580.1619.
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Synthesis of model compound 8
Compound 12 was obtained in 98% yield. H NMR (300
MHz, CDCl3) δ 8.36 (d, J = 9.1 Hz, 2H), 7.73 (d, J = 9.2 Hz,
1H), 7.55 (d, J = 9.1 Hz, 2H), 6.90 (d, J = 7.1 Hz, 1H), 6.71
(dd, J = 9.2, 2.4 Hz, 1H), 6.53 (d, J = 2.2 Hz, 1H), 4.93 – 4.84
(m, 1H), 3.71 (s, 3H), 3.59 (d, J = 3.9 Hz, 2H), 3.48 (dd, J =
14.1, 7.0 Hz, 4H), 1.91 (s, 3H), 1.29 (s, 6H). HRMS (ESI)
m/z: calcd for C26H27N3O9SNa [M+Na]+ 580.1366; found
580.1373.
Compound 8 was prepared using the same procedure for
compound 6. Yield: 74%. Mp: 156ꢀ157°C; H NMR (600
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MHz, CDCl3) δ 8.25 (d, J = 8.9 Hz, 2H), 7.69 (d, J = 9.1 Hz,
1H), 7.51 (d, J = 3.2 Hz, 2H), 7.32 (d, J = 8.9 Hz, 2H), 7.26 (d,
J = 1.9 Hz, 3H), 6.49 (d, J = 7.3 Hz, 1H), 6.46 (s, 1H), 3.40
(dd, J = 13.8, 6.8 Hz, 4H), 1.20 (t, J = 7.0 Hz, 6H). 13C NMR
(150 MHz, CDCl3) δ 162.74, 157.47, 155.44, 155.38, 152.06,
149.83, 145.45, 132.59, 131.23, 129.82, 129.36, 128.21,
125.11, 122.57, 109.54, 108.46, 97.00, 44.96, 12.42. HRMS
(ESI) m/z: calcd for C26H22N2O6SeNa [M+Na]+, 561.0535;
found 561.0534.
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Synthesis of ACC-SePh
To a solution of compound 5 (80 mg, 0.19 mmol), comꢀ
pound 13 (50 mg, 0.285 mmol), EDCI (56 mg, 0.3 mmol),
DMAP (2.4 mg, 0.02 mmol) was added dry CH2Cl2 (10 mL).
The mixture was stirred for 2 h at room temperature. Then the
solvent was removed under pressure and resultant crude mateꢀ
rial was purified by column chromatography to afford ACCꢀ
SePh as an orange solid (51 mg, 52% yield). Mp: 207ꢀ210°C;
1H NMR (300 MHz, CDCl3) δ 7.74 (d, J = 9.1 Hz, 1H), 7.63
(d, J = 8.7 Hz, 1H), 7.60 – 7.53 (m, 2H), 7.30 (dd, J = 5.1, 1.7
Hz, 3H), 7.24 (dd, J = 8.7, 2.3 Hz, 1H), 7.14 (d, J = 2.2 Hz,
1H), 6.54 (dd, J = 9.1, 2.6 Hz, 1H), 6.49 (d, J = 2.5 Hz, 1H),
6.29 (d, J = 1.2 Hz, 1H), 3.43 (q, J = 7.1 Hz, 4H), 2.45 (d, J =
1.2 Hz, 3H), 1.23 (t, J = 7.1 Hz, 6H). 13C NMR (75 MHz,
CDCl3) δ 163.03, 160.56, 157.51, 155.43, 154.10, 153.10,
151.96, 151.92, 149.43, 132.65, 131.24, 129.85, 129.34,
128.27, 125.33, 118.51, 118.26, 118.04, 114.56, 110.54,
109.65, 108.69, 97.21, 45.04, 18.76, 12.42. HRMS (ESI) m/z:
calcd for C30H25NO6SeNa [M+Na]+ 598.0745; found
598.0751.
Model reaction of compounds 6, 7 and 8 with Na2S2
General procedure: to the solution of compound 6 (30 mg,
0.06 mmol) in CH3CN (1.0 mL) and PBS buffer (1.0 mL, 50
mM, pH 7.4) was added Na2S2 (33 mg, 0.3 mmol). The mixꢀ
ture was stirred for 1 hour at room temperature and then dilutꢀ
ed with ethyl acetate (20 mL). The organic layer was separatꢀ
ed, dried by Na2SO4, and concentrated. Purification by flash
column chromatography afforded compound 9 as a yellow
1
solid (13 mg, 82% yield). Mp: 201ꢀ202°C; H NMR (300
MHz, CDCl3) δ 7.57 (d, J = 9.1 Hz, 1H), 6.67 (dd, J = 9.1, 2.5
Hz, 1H), 6.49 (d, J = 2.5 Hz, 1H), 3.49 (q, J = 7.1 Hz, 4H),
1.27 (t, J = 7.1 Hz, 6H); 13C NMR (75 MHz, CDCl3) δ 170.03,
155.56, 154.81, 153.61, 124.73, 109.77, 106.15, 105.76,
96.56, 45.25, 12.40; HRMS (ESI) m/z: calcd for C14H14NO3S2
[M+H]+ 308.0415, found 308.0414.
In the case of compound 7, compound 9 was obtained in
80% yield.
RESULTS AND DISCUSSION
Rational design of ACC-SePh
In the case of compound 8, compound 9 was obtained in
92% yield.
The design strategy for ACCꢀSePh is depicted in Scheme 1.
Combining the previously reported strategies for GSH
(Scheme 1A) and H2Sn (Scheme 1B) to a singleꢀmolecule
probe through judicious structure modification may provide an
effective approach to achieve the goal of simultaneously deꢀ
tecting GSH and H2Sn from different emission channels. Motiꢀ
vated by this strategy, a novel dualꢀdetection fluorescence
probe, ACCꢀSePh, was rationally designed (Scheme 1C) based
on the following considerations: (1) Two coumarin dyes, 7ꢀ
diethylaminocoumarin and 7ꢀhydroxycoumarin, were chosen
as the fluorophores because they displayed excellent photoꢀ
physical properties. Moreover, their photophysical properties
can be easily tuned through delicate modification at appropriꢀ
ate positions.36 In addition, their emission spectra were wellꢀ
separated, which enable the detection of GSH and H2Sn from
different emission channels. (2) Phenylselenide moiety was
selected as reaction site 1 because it functions not only as a
leaving group in the SNAr substitution reaction, but also as an
effective fluorescence quencher via photoꢀinduced electron
transfer (PET) to ensure a low background signal. Furthermore,
phenylselenide lies in 4ꢀposition of 7ꢀdiethylaminoꢀcoumarin
moiety of ACCꢀSePh, which is doubly activated by two nearꢀ
Model reaction of compounds 6, 7 and 8 with N-acetyl-
cysteine methyl ester
General procedure: To the solution of compound 6 (25 mg,
0.05 mmol) in CH3CN (1.0 mL) and PBS buffer (1.0 mL, 50
mM, pH 7.4) was added Nꢀacetylꢀcysteine methyl ester (45
mg, 0.25 mmol). The mixture was stirred for 1 hour at room
temperature and then diluted with ethyl acetate (10 mL). The
organic layer was separated, washed with brine (3×5 mL),
dried by Na2SO4, and concentrated. Purification by column
chromatography afforded compound 10 (25 mg, 96%). Mp:
1
55ꢀ56 °C; H NMR (300 MHz, CDCl3) δ 7.72 (d, J = 9.1 Hz,
1H), 7.53ꢀ7.43 (m, 2H), 7.37ꢀ7.31 (m, 3H), 7.10 (d, J = 7.5
Hz, 1H), 6.69 (dd, J = 9.2, 2.5 Hz, 1H), 6.52 (d, J = 2.5 Hz,
1H), 4.84ꢀ4.90 (m, 1H), 3.65ꢀ3.60 (m, 1H), 3.55ꢀ3.34 (m, 4H)
1.89 (s, 3H), 1.26 (t, J = 4.9 Hz, 6H) 13C NMR (75 MHz,
CDCl3) δ 170.16, 170.02, 164.48, 157.45, 155.77, 152.01,
150.85, 150.62, 129.57, 128.70, 126.43, 121.66, 118.69,
109.83, 107.19, 97.51, 53.05, 52.75, 45.04, 7.78, 29.70, 12.44.
HRMS (ESI) m/z: calcd for C26H28N2O7SNa [M+Na]+
535.1515; found 535.1519.
1
Compound 11 was obtained in 95% yield. H NMR (300
by
carbonyl
and
thus
is
reactive.37
(3)
7ꢀ
MHz, CDCl3) δ 7.71 (d, J = 9.1 Hz, 1H), 7.26 (d, J = 9.0 Hz,
2H), 7.10 (d, J = 7.7 Hz, 1H), 6.96 (d, J = 9.1 Hz, 2H), 6.68
(dd, J = 9.2, 2.4 Hz, 1H), 6.51 (d, J = 2.4 Hz, 1H), 4.91 – 4.86
(m, 1H), 3.84 (s, 3H), 3.81 (s, 3H), 3.69 (s, 2H), 3.45 (t, J =
Diethylaminocoumarin and 7ꢀhydroxycoumarin were connectꢀ
ed through simple esterification. The fluorescence of 7ꢀ
hydroxycoumarin would be quenched because of the esterifiꢀ
cation. Notably, the ester group lies in 3ꢀposition (the ortho
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position of phenylselenide) of 7ꢀdiethylaminocoumarin moiety, pound of H2Sn) or Nꢀacetylꢀcysteine methyl ester (as a GSH
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8
and thereby may function as an additional discriminating facꢀ
tor (site 2) to ensure the discrimination of H2Sn from other
RSS.
model) were systematically studied (Scheme 2B). All these
reactions were carried out in PBS buffer (pH 7.4, 10 mM, conꢀ
taining 50% CH3CN, v/v), and the relevant products were anaꢀ
lyzed after 1 h at room temperature. As expected, when comꢀ
pound 6 was treated with 5.0 equivalents of Na2S2, the desired
cyclization product coumarindithiolone (9) was obtained in
good yield (82%). Noteworthy, compound 9 was essentially
nonꢀfluorescent, its quantum yield was determined to be
0.0004 in PBS buffer (containing 1 mM CTAB). Moreover,
both the electronꢀdonating group (ꢀOMe) and electronꢀ
withdrawing group (ꢀNO2) on the substrates (model comꢀ
pounds 7 and 8) caused insignificant influences on this reacꢀ
tion, and the corresponding yields for 9 were identified to be
80% and 92%, respectively. Now, let us turn our attention to
the reactions between model substrates and Nꢀacetylꢀcysteine
methyl ester. After addition of 5.0 equivalents of Nꢀacetylꢀ
cysteine methyl ester to the solution of 6, the expected substiꢀ
tution product 10 was obtained in 96% yield. Similar results
were obtained in the cases of model compounds 7 and 8, the
corresponding yields for substitution products 11 and 12 were
identified to be 95% and 98%, respectively. All these traits
indicated that our proposed design strategy might be useful in
developing dualꢀdetection fluorescent probes which were caꢀ
pable of sensing GSH and H2Sn simultaneously from different
emission channels.
Based on the above design strategy, we anticipate that
ACCꢀSePh could respond to GSH and H2Sn with distinct fluoꢀ
rescence signals. As shown in Scheme 1C, when ACCꢀSePh is
treated with GSH, the phenylselenide moiety would be reꢀ
placed by thiol group of GSH through SNAr substitution reacꢀ
tion to produce ACCꢀGSH. However, the reaction between
H2Sn and ACCꢀSePh is quite different. Initially, similar SNAr
substitution reaction between H2Sn and ACCꢀSePh is expected
to occur to form the corresponding intermediate ACCꢀSSH,
and the following intermolecular cyclization between the thiol
group and the adjacent ester group (site 2) would ultimately
lead to the release of coumarindithiolone (9) and 7ꢀ
hydroxycoumarin (13). Based on the totally different chemical
structures and thus the distinct optical properties of the
corresponding products ACCꢀGSH, 9 and 13, simultaneous
detection of GSH and H2Sn from different emission channels
through a singleꢀmolecule fluorescent probe would be promisꢀ
ing to realize.
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Scheme 1. (A) General idea to discriminate GSH from
Cys/Hcy; (B) General idea to discriminate H2Sn from RSH;
(C) The design rational of novel dual detection fluorescent
probe ACC-SePh for GSH and H2Sn.
Scheme 2. (A) Synthetic route of model compounds 6-8; (B)
Model reaction of the probes with H2Sn and GSH; (C) Con-
trol compound 14-15.
Model reaction studies
To test the aboveꢀmentioned speculation, three 4ꢀphenylꢀ
selenideꢀ7ꢀdiethylaminocoumarin ester derivatives (6ꢀ8) were
synthesized (Scheme 2A) as the model compounds and their
reactions with Na2S2 (H2S2 maybe an active species of H2Sn,
there should be a dynamic equilibrium between H2S2 to other
H2Sn, therefore Na2S2 was selected as the primary model comꢀ
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Analytical Chemistry
compound 14 (Figure S2). In addition, compound 9, 13 and
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the adduct ACCꢀGSH were also observable in the correspondꢀ
ing HRMS titration experiments (Figure S3ꢀS4).
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Scheme 3. Synthetic route of ACC-SePh.
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Figure 1. (aꢀb) Timeꢀdependent Uvꢀvis spectra of ACCꢀSePh
(10 µM) in the presence of 10 equiv of Na2S2 in PBS buffer
(10 mM, pH 7.4, containing 1mM CTAB) (a) and the correꢀ
sponding timeꢀdependent absorption intensity changes (b); (cꢀ
d) Timeꢀdependent Uvꢀvis spectra of ACCꢀSePh (10 µM) in
the presence of 10 equiv of GSH in PBS buffer (10 mM, pH
7.4, containing 1mM CTAB) (c) and the corresponding timeꢀ
dependent absorption intensity changes (d).
Synthesize and Uv-vis spectra studies of ACC-SePh
As a proof of concept, the wellꢀdesigned probe, ACCꢀSePh,
was synthesized (Scheme 3) and its structure was fully characꢀ
1
terized by H NMR, 13C NMR and HRMS (see Supporting
Subsequently, the reactivity of ACCꢀSePh toward other bioꢀ
logically abundant RSS, such as Cys, Hcy and H2S, were also
investigated through timeꢀdependent Uvꢀvis spectra in PBS
buffer (10 mM, pH 7.4, containing 1mM CTAB) at room temꢀ
perature. Addition of Cys/Hcy to the solution of ACCꢀSePh,
led to the decrease of the absorption peak at 434 nm, followed
by a simultaneous emergence of a new peak at 375 nm (Figure
S5ꢀS6). The absorption at 375 nm should be assigned to ACCꢀ
NꢀCys/ACCꢀNꢀHcy, which was supported by the control comꢀ
pound 15 (Figure S7ꢀS8). According to the wellꢀestablished
mechanism,17,37ꢀ38 Cys (or Hcy) may react with ACCꢀSePh
through SNAr displacement to form ACCꢀSꢀCys (or ACCꢀSꢀ
Hcy), and the following intermolecular rearrangement would
lead to ACCꢀNꢀCys (or ACCꢀNꢀHcy) (Scheme 4). On the othꢀ
er hand, addition of Na2S to the solution of ACCꢀSePh, led to
the fast decrease of the absorption peak at 434 nm, along with
the simultaneous appearance of a new peak at 365 nm (Figure
S9). We speculate that the absorption peak at 365 nm should
be assigned to ACCꢀSH (Scheme 4). Unfortunately, our atꢀ
tempt to purify ACCꢀSH was unsuccessful, presumably due to
the instability of ACCꢀSH on silica gel. Overall, the above
traits are in good agreement with our proposed reaction mechꢀ
anisms.
Information). Initially, we examined the reactivity of ACCꢀ
SePh toward H2Sn and GSH through timeꢀdependent Uvꢀvis
spectra in PBS buffer (10 mM, pH 7.4, containing 1mM
cetyltrimethyl ammonium bromide (CTAB)) at room temperaꢀ
ture. As shown in Figure 1aꢀb, ACCꢀSePh displayed a main
absorption peak at 434 nm. Upon addition of Na2S2, the abꢀ
sorption peak at 434 nm decreased immediately and a new
peak at 355 nm emerged at the same time; after that, the abꢀ
sorption peak at 434 nm reꢀincreased gradually, along with the
slowly decrease of the absorption peak at 355 nm. According
to the aforementioned speculation, the initial drastic absorpꢀ
tion variation should be assigned to the rapid SNAr displaceꢀ
ment reaction to form the intermediate ACCꢀSSH. The followꢀ
ing sluggish intermolecular cyclization would ultimately lead
to the release of compound 9 and 13 (Scheme 1C), thereby
causing the gradual colorimetric changes at 434 nm and 355
nm. This speculation can be supported by the absorption specꢀ
tra of compound 9 and 13 under the same conditions (Figure
S1). Next, let us turn our attention to GSH. As shown in Figꢀ
ure 1cꢀd, addition of GSH to the solution of ACCꢀSePh led to
a slight increase at 434 nm, which then reached the stable abꢀ
sorption plateau within 2 min. According to the aforemenꢀ
tioned speculation, this new absorption peak should be asꢀ
signed to ACCꢀGSH, which was supported by the control
Scheme 4. Proposed reaction mechanism of ACC-SePh
with H2S and Cys/Hcy.
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Fluorescence spectra studies
The emission behavior of ACCꢀSePh upon addition of H2Sn
and GSH were investigated in PBS buffer (10 mM, pH 7.4,
containing 1mM CTAB). Firstly, we selected the main absorpꢀ
tion peak of 13 at 366 nm as the excitation wavelength to
probe H2Sn. ACCꢀSePh was essentially nonꢀfluorescent due to
the effective PET process induced by phenylselenide moiety.
Upon addition of Na2S2, a strong emission band centered at
465 nm can be observed (Figure S10) and reached a stable
intensity plateau within 60 min (Figure S11). In this case, an
approximate 21ꢀfold increase in fluorescence intensity at 465
nm can be observed. By comparison, GSH and Na2S only elicꢀ
ited a 3ꢀ and 2ꢀfold intensity enhancement at 465 nm, indicatꢀ
ing the potential capability of ACCꢀSePh to detect H2Sn over
GSH and H2S. Secondly, we selected the main absorption peak
of 14 at 430 nm as the excitation wavelength to probe GSH.
Under this excitation wavelength, ACCꢀSePh also displayed
negligible fluorescence. However, upon addition of GSH, the
significant fluorescence enhancement at 540 nm can be obꢀ
served (Figure S12) and the intensity can reached equilibrium
within 5 min (Figure S13). In this case, an approximate 16ꢀ
fold increase in fluorescence intensity at 540 nm can be obꢀ
served. Importantly, addition of Na2S2 or Na2S can hardly
elicited any significant fluorescence changes of ACCꢀSePh,
indicative the strong capability of ACCꢀSePh to detect GSH
over Na2S2 and Na2S.
Figure 2. (a) Fluorescence spectra of ACCꢀSePh (10 µM)after
addition of various concentrations of Na2S2 (0, 2, 4, 6, 8, 10, 12,
14, 16, 18, 20, 24, 26, 28, 30, 32, 34, 36, 40, 45, 50, 55, 60 µM)
and (b) the corresponding linear relationship between F465 and
Na2S2 concentrations (0ꢀ40 µM) (λex = 366 nm, slits 5/5 nm); (c)
Fluorescence spectra of ACCꢀSePh after addition of various conꢀ
centrations of GSH (0, 1, 2, 3, 4, 5, 6, 7, 9, 10, 14, 18, 20, 25, 30,
40, 50, 60 µM) and (d) the corresponding linear relationship beꢀ
tween F540 and GSH concentrations (0ꢀ18 µM) (λex = 430 nm, slits
5/5 nm).
As GSH and H2Sn may coꢀexist in biological systems, we
wondered if the probe ACCꢀSePh could give effective reꢀ
sponses when GSH and H2Sn coꢀexist. As a result, the fluoresꢀ
cence spectra of ACCꢀSePh versus varying H2Sn/GSH mixture
(total sulfur concentration was fixed to be 50 µM) were studꢀ
ied. As the concentration of GSH seems much higher than
H2Sn in biological systems, the corresponding [H2Sn]/[GSH]
ratios were installed from 0 to 1. As shown in Figure 3, folꢀ
lowing the increases of [H2Sn]/[GSH] ratios, the emission at
465 nm increased when excited at 366 nm. On the contrary, at
430 nm excitation, the emission at 540 nm decreased upon the
increasing of [H2Sn]/[GSH] ratios. In addition, both F465 and
F540 changed linearly with [H2Sn]/[GSH] ratios in the range of
0 to 0.42 (Figure S15ꢀS16). These traits indicated that ACCꢀ
SePh could be used for the determination of relative H2Sn and
GSH concentrations when they coꢀexist.
Next, we evaluated the changes in the emission spectra of
ACCꢀSePh upon addition of incremental amounts of Na2S2 or
GSH in PBS buffer (10 mM, pH 7.4, containing 1mM CTAB).
As shown in Figure 2a, addition of incremental amounts of
Na2S2 to the solution of ACCꢀSePh led to a gradual increase of
the fluorescence intensity at 465 nm. Moreover, the plot of the
fluorescence intensity at 465 nm versus the concentrations of
Na2S2 exhibited good linearity (R = 0.9914) in the range of 0ꢀ
40 µM (Figure 2b). The detection limit (S/N = 3) for Na2S2
was determined to be 40 nM. Noteworthy, other H2Sn species,
such as Na2S3 or Na2S4, exhibited similar reactivity toward
ACCꢀSePh (Figure S14). In the case of GSH, the fluorescence
intensity at 540 nm increased linearly as the concentration of
GSH up to 20 µM (Figure 2c). The corresponding detection
limit was determined to be 56 nM (S/N = 3) (Figure 2d). Takꢀ
ing together, these experimental results demonstrate high senꢀ
sitivity of ACCꢀSePh for H2Sn and GSH, suggesting that
ACCꢀSePh may has the potential to be used for intracellular
H2Sn or GSH monitoring.
Figure 3. Fluorescence spectra of ACCꢀSePh (10 µM) with varꢀ
ing [H2Sn]/[GSH] mixture solution ([H2Sn]/[GSH] ratios were 0,
0.11, 0.25, 0.42, 0.66, 0.81, 1). (a) Excited at 366 nm, slits 5/5
nm); (b) Excited at 430 nm, slits 5/5 nm.
Selectivity and effect of pH
To evaluate the selectivity of ACCꢀSePh, the fluorescence
spectra of ACCꢀSePh toward various biologically relevant
species, such as KCl, CaCl2, ZnCl2, MgCl2, NaClO, LꢀGlu, Lꢀ
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Ser, DLꢀTyr, DꢀPro, Cys, DLꢀMet, NaHSO3, Na2S2O3, Na2SO4, permeable and can be used for sensing intracellular H2Sn and
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H2O2, Na2S, Hcy, GSH, Na2S2, were investigated. At 366 nm
excitation, ACCꢀSePh exhibited a limited fluorescence enꢀ
hancement for various species; only Na2S2 elicited a signifiꢀ
cant fluorescence enhancement at 465 nm (Figure 4a, Figure
S17). Noteworthy, biothiols (Cys, Hcy and GSH) also trigꢀ
gered a slight fluorescence enhancement of ACCꢀSePh at 465
nm due to the formation of blue emissive amino substituted
products. These observations were consistent with the aboveꢀ
mentioned Uvꢀvis spectra results. At 430 nm excitation, only
GSH caused a significant fluorescence turnꢀon at 540 nm,
other biological species, including Cys and Hcy, triggered
almost no significant fluorescence enhancement (Figure 4b,
Figure S18ꢀS19). Overall, these results demonstrate that ACCꢀ
SePh is highly selective for H2Sn and GSH over other competiꢀ
tive species at different excitations.
GSH simultaneously from different emission channels. In adꢀ
dition, the MTT assay for ACCꢀSePh was also conducted, and
the results showed that ACCꢀSePh has minimal cytotoxicity
(Figure S22), thereby holding great potential for biological
applications.
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Figure 5. Confocal microscopic images of GSH and H2Sn in livꢀ
ing RAW264.7 cells. (A1ꢀA4) RAW264.7 cells incubated with 5
µM ACCꢀSePh and CTAB (0.5 mM); (B1ꢀB4) RAW264.7 cells
pretreated with 25 µM Na2S2 and then incubated with 5 µM ACCꢀ
SePh and CTAB (0.5 mM); (C1ꢀC4) RAW264.7 cells pretreated
with 1mM NEM and then incubated with 5 µM ACCꢀSePh and
CTAB (0.5 mM). Excitation wavelength: 405 nm. Emissions were
collected at 450ꢀ470 nm for blue channel and 540ꢀ560 nm for
green channel. Scale bar: 10 µm.
Figure 4. Fluorescence enhancement (F/F0) of ACCꢀSePh (10 µM)
in the presence of 100 µM various biological related species in
PBS buffer (10 mM, pH 7.4, containing 1 mM CTAB). (a) F/F0 at
465 nm, excited at 366 nm; (b) F/F0 at 540 nm, excited at 430 nm.
The effect of pH on the response of ACCꢀSePh and its reꢀ
sponse to Na2S2 and GSH were also studied. As shown in Figꢀ
ure S20ꢀS21, free ACCꢀSePh exhibited no significant fluoresꢀ
cence enhancement at 465 nm or 540 nm in broad pH range
(1.0ꢀ9.0), indicating that ACCꢀSePh was stable at physiologiꢀ
cal conditions. However, under strong basic conditions (pH >
10.0), strong fluorescence at 465 nm was observed, presumaꢀ
bly due to the fact that the ester group in ACCꢀSePh was hyꢀ
drolyzed under these conditions to release the compound 13.
Meanwhile, at 366 nm excitation, addition of Na2S2 induced
remarkable fluorescence enhancement at 465 nm in the pH
range 6.0ꢀ9.0. At 430 nm excitation, addition of GSH induced
significant fluorescence enhancement at 540 nm in the pH
range 7.0ꢀ11.0. All these results suggested that ACCꢀSePh can
function properly in biological environments.
For a further biological study, we investigate whether ACCꢀ
SePh can be used for the detection of endogenously produced
H2Sn. According to the literature, endogenous H2Sn can be
biosynthesized from cystine through cystarhionineꢀγꢀlyase
(CSE).10 Therefore we sought to determine whether ACCꢀ
SePh could detect endogenous H2Sn that was derived from
cystine and CSE in living RAW264.7 cells. Initially,
RAW264.7 cells were stimulated with 1 µg/mL lipopolysacꢀ
charide (LPS) (a compound can induce the CSE mRNA in
RAW264.7 cells overexpressed40) for 8 hours. Then, cells
were incubated with 1 mM NEM for 15 min to diminish the
interference caused by intracellular GSH. Next, cells were
incubated with 200 µM cystine for 30 min. Finally, cells were
incubated with ACCꢀSePh for additional 30 min. As shown in
Figure 6A1ꢀA2, strong fluorescence in blue channel and weak
fluorescence in green channel can be observed. In control exꢀ
periment, cells were incubated with NEM (1 mM), cystine
(200 µM), and ACCꢀSePh successively, only weak fluoresꢀ
cence in both blue channel and green channel can be observed
(Figure 6B1ꢀB2). These results demonstrated that ACCꢀSePh
was capable of detecting endogenous H2Sn in RAW264.7 cells.
Cell imaging
Encouraged by the excellent sensing properties of the probe
in aqueous solution, we further evaluated the capability of
ACCꢀSePh to selectively sense H2Sn and GSH in living cells.
As shown in Figure 5, when RAW264.7 cells were incubated
with ACCꢀSePh for 30 min, they gave weak fluorescence in
blue channel and strong fluorescence in green channel (Figure
5A1ꢀA2), indicating that ACCꢀSePh is responsive to intracelꢀ
lular GSH. When RAW264.7 cells were pretreated with 50
µM Na2S2 for 15 min and then further incubated with ACCꢀ
SePh, a significant increase in blue channel and a marked deꢀ
crease in green channel can be observed (Figure 5B1ꢀB2). In
control experiments, cells were pretreated with 1 mM Nꢀ
ethylmaleimide (NEM, a scavenger of GSH)39 for 15 min and
then incubated with ACCꢀSePh for 30 min, no obvious fluoꢀ
rescence in both blue and green channels were observed (Figꢀ
ure 5C1ꢀC2). These results indicate that ACCꢀSePh is cellꢀ
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Figure 6. Confocal microscopic images of endogeneously proꢀ
duced H2Sn in living RAW264.7 cells. (A1ꢀA4) RAW264.7 cells
incubated with 1 µg/mL LPS for 8 hours and then 1 mM NEM for
15 min, 200 µM cystine for 30 min, and finally incubated with 5
µM ACCꢀSePh and CTAB (0.5 mM) for additional 30 min; (B1ꢀ
B4) RAW264.7 cells pretreated with 1 mM NEM for 15 min, 200
µM cystine for 30 min and then incubated with 5 µM ACCꢀSePh
and CTAB (0.5 mM) for additional 30 min. Excitation wavelength:
405 nm. Emissions were collected at 450ꢀ470 nm for blue channel
and 540ꢀ560 nm for green channel. Scale bar: 10 µm.
CONCLUSIONS
In summary, we have presented in this study the rational deꢀ
sign, synthesis, and evaluation of the first dualꢀdetection fluoꢀ
rescent probe ACCꢀSePh that can selectively sense H2Sn and
GSH from different emission channels (H2Sn: λex/em = 366/465
nm with an approximate 21ꢀfold increase in fluorescence inꢀ
tensity at 465 nm; GSH: λex/em = 430/540 nm with an approxiꢀ
mate 16ꢀfold increase in fluorescence intensity at 540 nm)
based on the different reaction processes between ACCꢀSePh
and common RSS (e.g. H2S, H2Sn, Cys/Hcy, and GSH). Preꢀ
liminary biological experiments have indicated ACCꢀSePh
could simultaneously monitor intracellular H2Sn and GSH in
living RAW264.7 cells. In addition, ACCꢀSePh can also be
used for sensing endogenously produced H2Sn. We hope this
novel strategy could open up an avenue to develop new sysꢀ
tems for RSS discrimination.
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ASSOCIATED CONTENT
Supporting Information
Experimental details and additional spectroscopy data
1H NMR, 13C NMR and HRMS spectra of the products
The Supporting Information is available free of charge on the
ACS Publications website.
AUTHOR INFORMATION
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENT
This work was supported by the Guangxi Natural Science
Foundation (2016GXNSFBA380229), “BAGUI Scholar” proꢀ
gram of Guangxi Province of China and Graduate Innovation
Training Program of Guangxi Province (YCSW2017189).
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