A fluorescent coumarin-based probe for the fast detection of cysteine with live cell application

Article abstract of DOI:10.3390/molecules22101618

A new coumarin-based fluorescent probe, containing an allylic esters group, has been designed and synthesized for sensing cysteine in physiological pH. In this fluorescent probe, the coumarin was applied as the fluorophore and an allylic esters group was combined as both a fluorescence quencher and a recognition unit. The probe can selectively and sensitively detect cysteine (Cys) over homocysteine, glutathione, and other amino acids, and has a rapid response time of 30 min and a low detection limit of 47.7 nM. In addition, the probe could be applied for cell imaging with low cytotoxicity.

Full text of DOI:10.3390/molecules22101618

molecules
Article
A Fluorescent Coumarin-Based Probe for the Fast
Detection of Cysteine with Live Cell Application
1
,†
2,†
1
1
1
1
Rui-Feng Zeng , Jin-Shuai Lan , Xiao-Die Li , Hui-Fen Liang , Yan Liao , Ying-Jie Lu ,
1
,
2,
Tong Zhang * and Yue Ding *
1
School of Pharmacy, Shanghai University of Traditional Chinese Medicine, Shanghai 201203, China;
ZRF0049@163.com (R.-F.Z.); 18017984650@163.com (X.-D.L.); nuoanuo@126.com (H.-F.L.);
1
5216669831@163.com (Y.L.); fgbddxc@126.com (Y.-J.L.)
2
Experiment Center of Teaching & Learning, Shanghai University of Traditional Chinese Medicine,
Shanghai 201203, China; lanjinshuai_shut@126.com
*
Correspondence: zhangtongshutcm@hotmail.com (T.Z.); dingyue-2001@hotmail.com (Y.D.);
Tel.: +86-021-51322318 (T.Z.)
†
These authors contributed equally to this work.
Received: 23 August 2017; Accepted: 20 September 2017; Published: 26 September 2017
Abstract: A new coumarin-based fluorescent probe, containing an allylic esters group, has been
designed and synthesized for sensing cysteine in physiological pH. In this fluorescent probe, the
coumarin was applied as the fluorophore and an allylic esters group was combined as both a
fluorescence quencher and a recognition unit. The probe can selectively and sensitively detect
cysteine (Cys) over homocysteine, glutathione, and other amino acids, and has a rapid response
time of 30 min and a low detection limit of 47.7 nM. In addition, the probe could be applied for cell
imaging with low cytotoxicity.
Keywords: cysteine; fluorescent probe; coumarin; bioimaging
1
. Introduction
Biothiols, such as cysteine (Cys), homocysteine (Hcy), and glutathione (GSH), are natural
compounds containing sulfydryl, which play crucial roles in the process of intracellular protein
turnover [ ]. Generally, alternations of the level of cellular biothiols lead to many diseases such as
psoriasis, leucocyte loss, cardiovascular disease [ ], acquired immunodeficiency syndrome (AIDS) [ ],
psoriasis-like skin lesions [ ], lethargy, liver damage [ ], muscle weakness, and wasting [ ]. In addition,
GSH is the vastest non-protein thiol in cells [ ], serving as an important regulator of the redox reaction
in vivo and maintaining intracellular redox activities [ ], which is a key indicator of diseases such as
1
2
3
4
5
6
7
8,9
diabetes and Alzheimer’s disease [10]. The level of Hcy has been proved to be connected with neural
tube defects, Alzheimer’s disease, osteoporosis, and inflammatory bowel disease [11]. What is more,
Cys is a semi-essential amino acid that plays a structural role in many proteins, and its concentration
is closely linked with edema, slowed growth [12], and a wide variety of cancers including breast
cancer [13], colorectal cancer [14], nasopharyngeal cancer [15], and lung cancer [16]. Therefore, it is
essential to develop a reliable method for detecting biothiols [17].
Numerous assays have been carried out to detect biothiols including high performance liquid
chromatography [18
,19], mass spectrometry [20,21], colorimetric detection [22], electrochemical
method [23 24], and capillary electrophoresis [25]. However, these studies faced a number of difficulties
,
such as high facility cost, complicated operation, complex preparation, long detecting time, or poor
selectivity. Recently, more attention had been paid to developing fluorescent probes, a cheap and easy
method to distinguish biothiols [26–28]. For example, Tong et al. synthesized a visible blue-to-green
pyrene probe, which could detect polyanion in aqueous media but cannot distinguish Cys from other
Molecules 2017, 22, 1618; doi:10.3390/molecules22101618
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biothiols [29]. Zhao et al. designed a rapid and sensitive probe for Cys, GSH, and Hcy, but it could not
identify them from each other [30]. Only a few reports detected Cys from GSH and Hcy due to their
similar structures containing nucleophilic mercapto groups [31,32]; thus, a new kind of fluorescent
probe with steady, high selectivity and sensitivity is still needed.
Coumarin is a compound containing the parent nucleus of benzopyrone, an economic
chromophore. What is more, coumarin shows high intense fluorescence, good solubility, relatively
high fluorescence quantum yield, ease of production, and molar absorption coefficient if an electron
donating group, such as hydroxy or unalkylated amino group, is substituted at the 7 position [33].
The fluorescence intensity of coumarins has a close relationship with the substituent groups on the ring.
When the intramolecular charge transfer capability was changed by modifying substituent groups,
the optical properties of the whole molecule were influenced. So, we can control the optical properties
of molecules by adding different substituent groups with pushing or pulling electronics. Moreover,
various mechanisms have been used in the design of Cys fluorescent probes, including the cyclization
reaction with an aldehyde [34–36], Michael addition [37–39], and cleavage reaction [40–42] by thiols.
In this report, a turn-on fluorescence probe based on coumarins was designed for Cys with highly
selectivity and sensitivity. The allylic esters group serves as a blocker of coumarin fluorophore in the
probe, which not only functions as a Michael receptor, but also an electrophile [43]. Because Cys has
lower steric hindrance to conduct Michael additions, the probe can sharply distinguish Cys from other
biothiols within 30 min and has been successfully used in living cell imaging. With the development
of fluorescent probes for the discrimination of Cys from other amino acids and biothiols, real-time
monitoring of the Cys level of cells, tissues, and animals will be possible.
2
. Results and Discussion
2
.1. Characterization of the Probe
The synthesis of the probe is outlined in Scheme 1. Compound 1 was further treated with acryloyl
1
13
chloride to form the probe. The structure of the probe was confirmed by H-NMR, C-NMR, IR,
and MS spectra (Figures S1–S4). The probe was colorless and had two absorption bands centered at
2
78 nm and 312 nm. It showed weak fluorescence (Φ = 0.0194) due to the quencher acrylamide group.
Scheme 1. Synthesis of the probe. Reagents and conditions: (a) trimethylamine, CH Cl , r.t.
2
2
2
.2. UV-Vis Absorption and Fluorescence Spectra
As shown in Figure 1b and Figure S5, the UV-vis absorption spectra of the free probe (10
µM)
showed absorption peaks at 278 nm and 312 nm. Upon the addition of Cys, the absorption at 278 nm
gradually decreased, while the absorption at 312 nm increased and red-shifted to 323 nm. Furthermore,
as shown in Figure 1a, the fluorescence intensity of the probe was enhanced from
Φ = 0.0194 to
Φ = 0.1725 with Cys.
2
.3. Selectivity of the Probe for Cys
The sensing ability of the probe towards various compounds was detected, including cysteine
Cys), homocysteine (Hcy), glutathione (GSH), glycine (Gly), lysine (Lys), histidine (His), alanine (Ala),
tyrosine (Try), cystine (Cys C), valine (Val), isoleucine (Ile), glutamic (Glu), phenylalanine (Phe), serine
(
− −
Ser), threonine (Thr), aspartic (Asp), methionine (Met), proline (Pro), leucine (Leu), CN , SCN ,
(
−
2−
HS , and SO4 in buffered solution (PBS (phosphate buffered solution):DMSO (dimethyl sulfoxide)
6:4, pH = 7.4). In Figure 2, the green fluorescence response was sensed upon the addition of Cys
=

Molecules 2017, 22, 1618
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(50 equiv.) to the probe (10
µM), and no obvious changes in fluorescence emission were observed
in other compounds. The results indicate that Cys is more active in the reaction due to its stronger
nucleophilicity compared with that of GSH and Hcy.
Figure 1.
(a) The fluorescence intensity of the probe and the probe added to Cys-buffered solution (PBS
(phosphate buffered solution):DMSO (dimethyl sulfoxide) = 6:4, pH = 7.4) at room temperature; (
b
) The
absorption spectra of the probe and the probe added to Cys in buffered solution (PBS:DMSO = 6:4
,
pH = 7.4) at room temperature. λex = 325 nm.
Figure 2. Fluorescence intensity of the probe (10 µM) in the presence of different compounds (50 equiv.);
each reaction was in buffered solution (PBS:DMSO = 6:4, pH = 7.4) at room temperature. λex = 325 nm.
In order to ensure an accurate detection of Cys, the probe could not be obstructed by other
compounds. Cys (50 equiv.) and a series of competing compounds (50 equiv.) were added to the
probe (10
µM). As depicted in Figure 3, other compounds hardly had an influence on the fluorescence
emission. The results showed that the probe could sharply distinguish Cys from the other compounds.
Figure 3. Black bar represents the fluorescence response of the probe (10
µM) to various compounds
(50 equiv.). Red bar represents the fluorescence response of the probe (10
µM) to Cys (50 equiv.) in the
presence of other compounds (50 equiv.). Each reaction was in buffered solution (PBS:DMSO = 6:4,
pH = 7.4) at room temperature. The numbers represent analytes: 0. Blank; 1. Cys; 2. Hcy; 3. GSH; 4.
Asp; 5. Val; 6. Glu; 7. Pro; 8. Gly; 9. Phe; 10. Met; 11. Thr; 12. Ser; 13. Ile; 14. His; 15. Phe; 16. Lys; 17.
−
−
−
2−
Try; 18. Leu; 19. Cys C; 20. CN ; 21. SCN ; 22. HS ; 23. SO . λex = 325 nm, λem = 450 nm.
4

Molecules 2017, 22, 1618
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2
.4. Effect of pH on the Fluorescence Response of Probe
To confirm the appropriate pH scope of the probe, the fluorescence intensity of the probe (10
µM)
added to Cys (50 equiv.) with different values of pH (3–10) was detected (Figure 4). The result showed
that the range of pH 7–9 fitted the reaction well. Because pH 7.4 is close to physiological conditions,
we selected a buffered solution at this level (PBS:DMSO = 6:4, pH = 7.4) for subsequent study.
Figure 4. The effect of pH on the fluorescence response of the probe (10
µM) in the presence of Cys
(
500 M) in buffered solution (PBS:DMSO = 6:4, pH = 3–10), pH was adjusted by NaOH and HCl.
µ
λex = 325 nm, λem = 450 nm.
2
.5. Effect of Reaction Time
To track the proper time of the response, we traced the fluorescence intensity of the probe (10 µM)
added to Cys (50 equiv.) while monitoring the time. The fluorescence intensity almost reached the
maximum in 15 min (Figure 5), which was relatively fast. Furthermore, the response obeyed the
pseudo-first-order rate, and the rate constant was calculated according to the following equation [44]:
Ln ((Fmax − F) / Fmax) = −kt,
(1)
where F is the fluorescence intensity at time t, and Fmax is the fluorescence intensity after the reaction
totally completed. The constant k is shown in Figure S6. The value of the rate constant of Cys was
−
1
0
.1618 min
.
Figure 5. Kinetic analysis of the probe towards Cys (10 µM probe with 50 equiv. of Cys) in buffer
solution (DMSO:PBS = 4:6, pH = 7.4). λex = 325 nm, λem = 450 nm.
2
.6. Quantitative Responses of the Probe for Cys
To clarify the sensitivity of the probe, various concentrations of Cys (0–30 equiv.) were added
to the probe solution (10 µM) and the fluorescence intensity at 450 nm was recorded after 50 min.

Molecules 2017, 22, 1618
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The results changed from nearly dark to a strong green fluorescent response as the concentration of
Cys increased. Furthermore, the emission intensity at 450 nm was linearly related to the concentration
2
of Cys from 0 to 50
µ
M (R = 0.9925) (Figure 6), which provided useful conditions for the quantitative
analysis of Cys. And the detection limit of Cys was calculated to be 47.7 nM based on the 3
σ/slope
method [45]. The results indicated that the probe remained very sensitive to Cys.
Figure 6.
(a) Fluorescent spectral changes (
b) The fluorescence intensity at 450 nm of probe (10 µM)
upon addition of Cys (0–300
µM) in buffer solution (DMSO:PBS = 4:6, PH 7.4), incubated 30 min before
detected. λex = 325 nm, λem = 450 nm.
As described above, the probe displayed excellent analytical properties, compared with some
other fluorescent probes of recent reports, for the detection of Cys. The comparison data is listed in
Table 1, indicating that the probe is promising for practical analysis.
Table 1. Comparison of the probe for the detection of thiols.
Reference
Response Time
Stokes Shift
Detection Limit
Selectivity for Cys
Sensors and Actuators B, 2017 [46]
Sensors and Actuators B, 2016 [47]
Biosensors and Bioelectronics, 2017 [48]
Biosensors and Bioelectronics, 2016 [49]
Biosensors and Bioelectronics, 2014 [50]
ChemComm, 2013 [51]
12 min
3 h
20 min
30 min
40 min
2 h
122 nm
110 nm
85 nm
96 nm
55 nm
97 nm
174 nm
125 nm
17.1 nM
192 nM
14 nM
0.874 µM
0.657 µM
-
no
no
yes
no
yes
no
no
Molecules, 2016 [52]
2.5 h
30 min
0.911 µM
47.7 nM
This work
yes
2
.7. Reaction Mechanism
The acrylate group has been applied as a thiols reaction site for detecting thiols [53,54]. Based on
the reported conjugate addition/cyclization sensing mechanism, the mechanism of probe responding
to Cys involved the next two steps: the conjugate addition of Cys to an unsaturated carbonyl moiety
generated thioether (1a), followed by intramolecular cyclization that gave the desired compound 1,

Molecules 2017, 22, 1618
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which was responsible for the enhancement of the fluorescence intensity. To confirm the mechanism,
the probe was incubated with Cys before being isolated by high performance liquid chromatography
(
4
see Figure S7 in the supporting information) and analyzed by mass spectra. In HPLC, a peak at
.54 min corresponded to the compound (coumarin) and a peak at 6.05 min corresponded to the
probe. Furthermore, the probe detected compound (coumarin) after being incubated with Cys for 3 h.
In MS, a peak at 177.0 corresponded to the resulting compound and a peak at 352.1 corresponded
1
1
1
to the additional product compound 1a (see Figure S8 in the supporting information). These data
strongly support the mechanism in Scheme 2.
O
O
Cys
HOOC
S
O
O
O
O
O
O
NH2
Probe
No fluorescence
1a
O
NH
COOH
S
HO
O
O
1
Strong fluorescence
Scheme 2. Proposed reaction mechanism of the probe with Cys.
2
.8. Application of the Probe
An MTS assay with a HepG2 cell line was applied to estimate the cytotoxicity of the probe.
As shown in Figure 7, cellular viability was greater than 80% after 24 h in 27 M (based on
µ
DMSO < 0.1%), which indicated that the probe had low toxicity to HepG2. To investigate the probe’s
◦
capability for cell imaging, HepG2 were incubated with the probe (25
µ
M) in PBS for 2 h at 37 C and
washed three times with PBS. As shown in Figure 8a, green fluorescence response could be observed
inside cells under a confocal fluorescence microscope. This observation shows that the probe can
detect the biothiols in the cells. Furthermore, in Figure 8b, when the cells were preincubated with
Cys (100
µ
M) for 1 h and then incubated with the probe (25
µ
M), the fluorescence response was
M N-ethylmaleimide
NEM), a widely used thiol-blocking agent [55] for 1 h, then incubated with the probe (25 M) for
h. As shown in Figure 8c, the fluorescence response was too slight to be detected, indicating the
partly enhanced. In the control group, the cells were preincubated with 500
(
2
µ
µ
pertinence of Cys. The results obviously indicated that the probe could be used as a probe for both
endogenously and exogenously produced Cys in living cells.
◦
Figure 7. MTS assay of HepG2 cells incubated in the presence of the probe (0–27
µ
M) at 37 C for 24 h.

Molecules 2017, 22, 1618
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Figure 8. Fluorescent imaging of HepG2 cells: (Left) fluorescent image; (Middle) bright field image;
and (Right) overlay image. λex = 405 nm, λem = 420–480 nm. ( ) Images of cells incubated with the probe
a
◦
(
25
µ
M) for 2 h at 37 C; (
b
) Images of cells preincubated with Cys (100
) Images of cells preincubated with N-ethylmaleimide (NEM)
500 µM) for 1 h and then incubated with the probe (25 µM) for 2 h at 37 C.
µ
M) for 1 h and then incubated
◦
with the probe (25
(
µ
M) for 2 h at 37 C; (
c
◦
3
. Materials and Methods
3
.1. Materials and Instrumentation
All chemicals (reagent grade) used were purchased from Sino Pharm Chemical Reagent Co., Ltd.
Shanghai, China). Reaction progress was monitored using analytical thin layer chromatography (TLC)
(
on pre-coated silica gel GF254 (Qingdao Haiyang Chemical Plant, Qingdao, China) plates, and spots
were detected under UV light (254 nm). Melting point was measured on an XT-4 micromelting point
instrument and uncorrected. IR (KBr-disc) spectra were recorded by a Bruker Tensor 27 spectrometer
1
13
(
Bruker, German). H-NMR and C-NMR spectra were measured on a BRUKER AVANCE III
◦
spectrometer at 25 C and referenced to tetramethyl silane (TMS) (Bruker, German). Mass spectra were
obtained on an MS Agilent 1100 Series LC/MSD Trap mass spectrometer (Agilent, Santa Clara, CA,
USA). UV-vis spectra were recorded on an Agilent 8454 UV-vis spectrometer (Agilent). Fluorescence
spectra measurements were recorded on an Agilent G9800A fluorescence spectrophotometer (Agilent).
Fluorescence images were obtained on a Leica TCS-SP8 multiphoton; a confocal microscope and a
6
3
×
oil-immersion objective lens was used (Leica, Switzerland). High-resolution EI mass spectra were
recorded on an Agilent 6460 triple quad LC-MS mass spectrometer (Agilent).

Molecules 2017, 22, 1618
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3
.2. Synthesis of the Probe (4-Methyl-2-oxo-2H-chromen-7-yl Acrylate)
A solution of compound 1 (200 mg, 1.14 mmol) and triethylamine (0.12 mL, 0.86 mmol) in dried
◦
CH Cl (10 mL) was added to acryloyl chloride (205.5 mg, 2.27 mmol) at 0 C under nitrogen. After
2
2
stirring for 12 h at room temperature, the reaction mixture was diluted with CH Cl . The CH Cl
2
2
2
2
solution was washed with a saturated aqueous Na CO solution, as well as water, and then dried over
2
3
anhydrous Na SO . The solvent was evaporated under reduced pressure. The crude product was
2
4
purified by column chromatography (SiO , ethyl acetate/hexane = 1/5, v/v) to give the probe as a
2
−
+ 1
1
pale white solid. Yield: 305 mg (80%). IR νmax/cm (KBr) 1721.36, 1616.37 (C=O), 1388.53, 1164.33;
m.p. 151.1–153.4 C; ESI/MS m/z: 231.0 [M + H] ; H-NMR (400 MHz, CDCl )
◦
δ
7.65 (d, J = 8.6 Hz,
3
1
6
H, H coumarin), 7.20 (d, J = 2.2 Hz, 1H, H coumarin), 7.16 (dd, J = 8.6, 2.3 Hz, 1H, H coumarin),
5
8
6
.68 (dd, J = 17.3, 1.1 Hz, 1H, H coumarin), 6.40–6.29 (m, 2H, H propionyl-), 6.11 (dd, J = 10.5, 1.1 Hz,
3
3
13
1
1
H, H propionyl-), 2.47 (d, J = 1.2 Hz, 3H, -CH ). C-NMR (100 MHz, CDCl3) δ 163.8, 160.6, 154.2,
2 3
53.0, 152.0, 133.7, 127.3, 125.4, 118.1, 117.9, 114.6, 110.5, 18.7.
3
.3. Absorption and Fluorescence Spectroscopy
The probe was dissolved in DMSO (1 mM) for a stock solution. The compounds such as cysteine
Cys), homocysteine (Hcy), glutathione (GSH), glycine (Gly), lysine (Lys), histidine (His), alanine (Ala),
tyrosine (Try), cystine (Cys C), valine (Val), isoleucine (Ile), glutamic (Glu), phenylalanine (Phe), serine
(
− − −
Ser), threonine (Thr), aspartic (Asp), methionine (Met), proline (Pro), leucine (Leu), CN , SCN , HS ,
(
and SO42 were all dissolved in purified water (10 mM). Tested solutions were prepared by adding
−
40 µL stock solution and a different analyte solution into the buffer (PBS:DMSO = 6:4, pH = 7.4) to
a confirmed volume of 4 mL. The resulting solutions were mixed well and detected after 30 min at
room temperature.
Fluorescent quantum yields were determined using Quinine sulphate (Φs = 0.54 in 0.1 M of
H SO solution) as a standard, according to a published method [56]. The fluorescent quantum yield
2
4
was calculated based on the following equation:
2
2
Φx = Φs × (Fx/Fs) × (As/Ax) × (ηx /ηs )
(2)
where
Φ
is the fluorescent quantum yield, Φs = 0.54 in 0.1 M of H SO solution, F
x
and F
s
are the
2
4
integrated fluorescence intensities of the sample and the standard, respectively, at the same excitation
wavelength, A and A are the absorbance at the excitation wavelength, and is the refractive index of
the respective solvent.
x
s
η
3
.4. Cell Culture for HepG2
HepG2 cells were provided by Chinese Academy of Sciences. HepG2 cells were cultured
in minimum essential medium (MEM) blended with 10% fetal bovine serum (FBS) and 1%
◦
penicillin-streptomycin solution, (100
×
) at 37 C under an atmosphere of 5% CO . Cells were
2
transplanted on confocal dishes and adhered for 24 h before use in an experiment.
3
.5. Fluorescence Imaging of Cys in Living Cells
Experiments to evaluate the sense ability of the probe for biothiols were executed in culture
◦
medium (90% MEM, 10% FBS). The cells were incubated in the probe (25
µ
M) for 2 h at 37 C and
washed with 0.1 M PBS (0.6 mL
cells were treated with Cys (100
the cells were incubated in the probe (25
×
3) before observation. To detect exogenously produced biothiols,
◦
µ
M) at 37 C for 2 h and washed with 0.1 M PBS (0.6 mL
×
3). Then,
3)
◦
µM) for 2 h at 37 C and washed with 0.1 M PBS (0.6 mL
×
◦
before observation. For the control experiment, cells were treated with 100
µ
M NEM for 1 h at 37 C
3). Then, the cells were incubated in the probe (25 M) for 2 h
3) before observation. For the positive experiment,
and washed with 0.1 M PBS (0.6 mL
×
µ
◦
at 37 C and washed with 0.1 M PBS (0.6 mL
×
◦
cells were treated with coumarin chromophore (25 µM) for 2 h at 37 C and washed with 0.1 M

Molecules 2017, 22, 1618
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PBS (0.6 mL × 3) before observation. A fluorescence microscope and a 63
×
oil-immersion objective
lens was used. The cells were excited with UV light below 405 nm, and emission was collected at
4
50 ± 40 nm.
4
. Conclusions
The probe showed a stable, highly selective and sensitive fluorescence response towards Cys over
GSH, Hcy, and other compounds. The main mechanism of the pertinence of Cys could be attributed to
the nucleophilic addition and its lower steric hindrance. Confocal fluorescence microscopy imaging
using HepG2 cells indicated that the probe can be applied for the detection of Cys in living cells.
Supplementary Materials: The supplementary materials are available online.
Acknowledgments: This work was supported by the programs of the National Natural Science Foundation of
China (grant numbers 81403175, 81274200); Project from Shanghai Committee of Science and Technology (grant
number 13401900301); Youth Talent Sail Plan from Shanghai Committee of Science and Technology (grant number
1
4YF1411300); Project from Shanghai Municipal Commission of Health and Family planning (grant number
2
0134Y053, 2017YQ072, 201740152), Research Fund for the Doctoral Program of Shanghai (grant number B201703)
and project of Xinglin young talent training system.
Author Contributions: Conceived and designed the experiments: Y.D. and T.Z. Performed research and analyzed
the data: J.-S.L., R.-F.Z., X.-D.L., H.-F.L., Y.L. and Y.-J.L.; wrote the paper: J.-S.L. and R.-F.Z. All authors read and
approved the final manuscript.
Conflicts of Interest: The authors declare no conflict of interest.
References
1
.
Bragoszewski, P.; Wasilewski, M.; Sakowska, P.; Gornicka, A.; Bottinger, L.; Qiu, J.; Wiedemann, N.;
Chacinska, A. Retro-translocation of mitochondrial intermembrane space proteins. Proc. Natl. Acad. Sci. USA
2
015, 112, 7713–7718. [CrossRef] [PubMed]
Abby, S.L.; Harris, I.M.; Harris, K.M. Homocysteine and cardiovascular disease. Curr. Atheroscler. Rep. 2004
, 101–106. [CrossRef]
2
3
4
.
.
.
,
6
Chen, X.; Zhou, Y.; Peng, X.; Yoon, J. Fluorescent and colorimetric probes for detection of thiols.
Chem. Soc. Rev. 2010, 39, 2120–2135. [CrossRef] [PubMed]
Hirai, T.; Kanda, T.; Sato, K.; Takaishi, M.; Nakajima, K.; Yamamoto, M.; Kamijima, R.; Digiovanni, J.;
Sano, S. Cathepsin K is involved in development of psoriasis-like skin lesions through TLR-dependent Th17
activation. J. Immunol. 2013, 190, 4805–4811. [CrossRef] [PubMed]
5.
6.
7.
8.
9.
Shahrokhian, S. Lead phthalocyanine as a selective carrier for preparation of a cysteine-selective electrode.
Anal. Chem. 2001, 73, 5972–5978. [CrossRef] [PubMed]
Michel, L.Y.M.; Hoenderop, J.G.J.; Bindels, R.J.M. Calpain-3-mediated regulation of the Na -Ca exchanger
isoform 3. Pflugers Arch. 2016, 468, 243–255. [CrossRef] [PubMed]
Yin, C.; Huo, F.; Zhang, J.; Martínez-Máñez, R.; Yang, Y.; Lv, H.; Li, S. Thiol-addition reactions and their
applications in thiol recognition. Chem. Soc. Rev. 2013, 42, 6032. [CrossRef] [PubMed]
Dalton, T.P.; Shertzer, H.G.; Puga, A. Regulation of gene expression by reactive oxygen. Annu. Rev.
Pharmacol. Toxicol. 1999, 39, 67–101. [CrossRef] [PubMed]
Yang, Y.; Huo, F.; Yin, C.; Zheng, A.; Chao, J.; Li, Y.; Nie, Z.; Martinez-Manez, R.; Liu, D. Thiol-chromene
click chemistry: A coumarin-based derivative and its use as regenerable thiol probe and in bioimaging
applications. Biosens. Bioelectron. 2013, 47, 300–306. [CrossRef] [PubMed]
+
2+
1
1
0. Harfield, J.C.; Batchelor-Mcauley, C.; Compton, R.G. Electrochemical determination of glutathione: A review.
Analyst 2012, 137, 2285–2296. [CrossRef] [PubMed]
1. Seshadri, S.; Beiser, A.; Selhub, J.; Jacques, P.F.; Rosenberg, I.H.; D’Agostino, R.B.; Wilson, P.W.; Wolf, P.A.
Plasma homocysteine as a risk factor for dementia and Alzheimer’s disease. ACC Curr. J. Rev. 2002, 11, 30.
[CrossRef]
1
2. Xue, S.; Ding, S.; Zhai, Q.; Zhang, H.; Feng, G. A readily available colorimetric and near-infrared fluorescent
turn-on probe for rapid and selective detection of cysteine in living cells. Biosens. Bioelectron. 2015, 68, 316.
[CrossRef] [PubMed]

Molecules 2017, 22, 1618
10 of 12
1
1
1
3. Lin, W.; Tang, L.Y.; Cen, Y.L.; Lin, Y.; Su, F.X.; Jia, W.H.; Ren, Z.F. Interaction of body mass index and a
polymorphism in gene of catalytic subunit of glutamate-cysteine ligase on breast cancer risk among Chinese
women. Zhonghua Liu Xing Bing Xue Za Zhi 2013, 34, 1115. [PubMed]
4. Roy, N.; Nazeem, P.A.; Babu, T.D.; Abida, P.S.; Narayanankutty, A.; Valsalan, R.; Valsala, P.A.;
Raghavamenon, A.C. EGFR gene regulation in colorectal cancer cells by garlic phytocompounds with
special emphasis on S-Allyl-L-Cysteine Sulfoxide. Interdiscip. Sci. 2017, 1–8. [CrossRef] [PubMed]
5. Li, X.; Liu, X.; Li, C.Y.; Ding, Y.; Chau, D.; Li, G.; Kung, H.F.; Lin, M.C.; Peng, Y. Recombinant adeno-associated
virus mediated RNA interference inhibits metastasis of nasopharyngeal cancer cells in vivo and in vitro by
suppression of Epstein-Barr virus encoded LMP-1. Int. J. Oncol. 2006, 4, 595–603. [CrossRef]
6. Geng, P.; Yao, J.; Zhu, Y. Hogg1 Ser326Cys polymorphism and lung cancer susceptibility: A meta-analysis.
Mol. Biol. Rep. 2014, 41, 2299. [CrossRef] [PubMed]
1
1
7. Chen, Y.H.; Tsai, J.C.; Cheng, T.H.; Yuan, S.S.; Wang, Y.M. Sensitivity evaluation of NBD-SCN towards
cysteine/homocysteine and its bioimaging applications. Biosens. Bioelectron. 2014, 56, 117–123. [CrossRef]
[PubMed]
1
1
8. Guo, X.F.; Zhu, H.; Wang, H.; Zhang, H.S. Determination of thiol compounds by HPLC and fluorescence
detection with 1,3,5,7-tetramethyl-8-bromomethyl-difluoroboradiaza-S-indacene. J. Sep. Sci. 2013, 36,
6
58–664. [CrossRef] [PubMed]
9. Kono, Y.; Iizuka, H.; Isokawa, M.; Tsunoda, M.; Ichiba, H.; Sadamoto, K.; Fukushima, T. HPLC-fluorescence
determination of thiol compounds in the serum of human male and female subjects using HILIC-mode
column. Biomed. Chromatogr. 2014, 28, 589–593. [CrossRef] [PubMed]
2
2
0. Capehart, S.L.; Carlson, E.E. Mass spectrometry-based assay for the rapid detection of thiol-containing
natural products. Chem. Commun. 2016, 52, 13229. [CrossRef] [PubMed]
1. García-Santamarina, S.; Boronat, S.; Domènech, A.; Ayté, J.; Molina, H.; Hidalgo, E. Monitoring in vivo
reversible cysteine oxidation in proteins using ICAT and mass spectrometry. Nat. Protoc. 2014, 9, 1131–1145.
[CrossRef] [PubMed]
2
2
2. Huo, F.-J.; Sun, Y.-Q.; Su, J.; Chao, J.-B.; Zhi, H.-J.; Yin, C.-X. Colorimetric detection of thiols using a chromene
molecule. Org. Lett. 2009, 11, 4918–4921. [CrossRef] [PubMed]
3. Kruusma, J.; Benham, A.M.; Williams, J.A.; Kataky, R. An introduction to thiol redox proteins in the
endoplasmic reticulum and a review of current electrochemical methods of detection of thiols. Analyst 2006
,
1
31, 459–473. [CrossRef] [PubMed]
2
4. Vandeberg, P.J.; Johnson, D.C. Pulsed electrochemical detection of cysteine, cystine, methionine, and
glutathione at gold electrodes following their separation by liquid chromatography. Anal. Chem. 2002, 65,
2
713–2718. [CrossRef]
2
2
2
5. Chen, G.; Zhang, L.; Wang, J. Miniaturized capillary electrophoresis system with a carbon nanotube
microelectrode for rapid separation and detection of thiols. Talanta 2004, 64, 1018–1023. [CrossRef] [PubMed]
6. Guo, Z.; Nam, S.W.; Park, S.; Yoon, J. A highly selective ratiometric near-infrared fluorescent cyanine sensor
for cysteine with remarkable shift and its application in bioimaging. Chem. Sci. 2012, 3, 2760–2765. [CrossRef]
7. Yuan, L.; Lin, W.; Zhao, S.; Gao, W.; Chen, B.; He, L.; Zhu, S. A unique approach to development of
near-infrared fluorescent sensors for in vivo imaging. J. Am. Chem. Soc. 2012, 134, 13510. [CrossRef]
[PubMed]
2
2
3
3
8. Tang, B.; Xing, Y.; Li, P.; Zhang, N.; Yu, F.; Yang, G. A rhodamine-based fluorescent probe containing a
Se-N bond for detecting thiols and its application in living cells. J. Am. Chem. Soc. 2007, 129, 11666–11667.
[CrossRef] [PubMed]
9. Tong, D.; Duan, H.; Wang, J.; Yang, Z.; Lin, Y. Aggregation-enhanced excimer emission (AEEE) based on
pyrenylchalcone and 2-to-4 molecular decoder by biothiols and polyanions in aqueous media. Sens. Actuators
B Chem. 2014, 195, 80–84. [CrossRef]
0. Zhao, W.; Sun, M.; Lei, T.; Liu, X.; Zhang, Q.; Zong, C. An indicator-displacement assay based on the
Murexide-Hg2+ system for fluorescence turn-on detection of biothiols in biological fluids. Sens. Actuators
B Chem. 2017, 249, 90–95. [CrossRef]
1. Mei, J.; Wang, Y.; Tong, J.; Wang, J.; Qin, A.; Sun, J.Z.; Tang, B.Z. Discriminatory detection of cysteine and
homocysteine based on dialdehyde-functionalized aggregation-induced emission fluorophores. Chemistry
2
013, 19, 613–620. [CrossRef] [PubMed]

Molecules 2017, 22, 1618
11 of 12
3
3
3
3
3
3
3
2. Song, L.; Sun, Q.; Wang, N.; Chen, Z.; Zhang, W.; Qian, J. A fluorescent probe for the discrimination between
Cys and GSH. Anal. Methods 2015, 7, 10371–10375. [CrossRef]
3. Li, J.; Zhang, C.F.; Yang, S.H.; Yang, W.C.; Yang, G.F. A coumarin-based fluorescent probe for selective and
sensitive detection of thiophenols and its application. Anal. Chem. 2014, 86, 3037–3042. [CrossRef] [PubMed]
4. Lin, W.; Long, L.; Yuan, L.; Cao, Z.; Chen, B.; Tan, W. A ratiometric fluorescent probe for cysteine and
homocysteine displaying a large emission shift. Org. Lett. 2008, 10, 5577. [CrossRef] [PubMed]
5. Kim, T.K.; Lee, D.N.; Kim, H.J. Highly selective fluorescent sensor for homocysteine and cysteine.
Tetrahedron Lett. 2008, 49, 4879–4881. [CrossRef]
6. Lee, K.S.; Kim, T.K.; Lee, J.H.; Kim, H.J.; Hong, J.I. Fluorescence turn-on probe for homocysteine and cysteine
in water. Chem. Commun. 2008, 46, 6173. [CrossRef] [PubMed]
7. Jung, H.S.; Pradhan, T.; Han, J.H.; Heo, K.J.; Lee, J.H.; Kang, C.; Kim, J.S. Molecular modulated
cysteine-selective fluorescent probe. Biomaterials 2012, 33, 8495–8502. [CrossRef] [PubMed]
8. Jung, H.S.; Han, J.H.; Pradhan, T.; Kim, S.; Lee, S.W.; Sessler, J.L.; Kim, T.W.; Kang, C.; Kim, J.S. A
cysteine-selective fluorescent probe for the cellular detection of cysteine. Biomaterials 2012, 33, 945–953.
[
CrossRef] [PubMed]
9. Huo, F.J.; Sun, Y.Q.; Su, J.; Yang, Y.T.; Yin, C.X.; Chao, J.B. Chromene “lock”, thiol “key”, and mercury(II) ion
hand”: A single molecular machine recognition system. Org. Lett. 2010, 12, 4756–4759. [CrossRef] [PubMed]
0. Pires, M.M.; Hrycyna, C.A.; Chmielewski, J. Bivalent probes of the human multidrug transporter
P-glycoprotein. Biochemistry 2006, 45, 11695–11702. [CrossRef] [PubMed]
3
4
4
“
1. Maeda, H.; Matsuno, H.; Ushida, M.; Katayama, K.; Saeki, K.; Itoh, N. 2,4-Dinitrobenzenesulfonyl fluoresceins
as fluorescent alternatives to Ellman’s reagent in thiol-quantification enzyme assays. Angew. Chem. 2005, 44,
2
922–2925. [CrossRef] [PubMed]
2. Jiang, W.; Fu, Q.; Fan, H.; Ho, J.; Wang, W. A highly selective fluorescent probe for thiophenols. Angew. Chem.
007, 46, 8445–8448. [CrossRef] [PubMed]
4
4
2
3. Yang, X.; Guo, Y.; Strongin, R.M. Conjugate addition/cyclization sequence enables selective and simultaneous
fluorescence detection of cysteine and homocysteine. Angew. Chem. 2011, 50, 10690–10693. [CrossRef]
[PubMed]
4
4. Ma, W.-W.; Wang, M.-Y.; Yin, D.; Zhang, X. Facile preparation of naphthol AS-based fluorescent probe
for highly selective detection of cysteine in aqueous solution and its imaging application in living cells.
Sens. Actuators B Chem. 2017, 248, 332–337. [CrossRef]
4
4
4
4
5. Liu, Y.; Xiang, K.; Tian, B.; Zhang, J. A fluorescein-based fluorescence probe for the fast detection of thiol.
Tetrahedron Lett. 2016, 57, 2478–2483. [CrossRef]
6. Dai, X.; Zhang, T.; Liu, Y.Z.; Yan, T.; Li, Y.; Miao, J.Y.; Zhao, B.X. A ratiometric fluorescent probe for cysteine
and its application in living cells. Sens. Actuators B Chem. 2015, 207, 872–877. [CrossRef]
7. Liao, Y.C.; Venkatesan, P.; Wei, L.F.; Wu, S.P. A coumarin-based fluorescent probe for thiols and its application
in cell imaging. Sens. Actuators B Chem. 2016, 232, 732–737. [CrossRef]
8. Chen, Z.; Sun, Q.; Yao, Y.; Fan, X.; Zhang, W.; Qian, J. Highly sensitive detection of cysteine over
glutathione and homo-cysteine: New insight into the Michael addition of mercapto group to maleimide.
Biosens. Bioelectron. 2017, 91, 553–559. [CrossRef] [PubMed]
4
5
5
9. Chen, C.; Liu, W.; Xu, C.; Liu, W. A colorimetric and fluorescent probe for detecting intracellular biothiols.
Biosens. Bioelectron. 2016, 85, 46–52. [CrossRef] [PubMed]
0. Xu, C.; Li, H.; Yin, B. A colorimetric and ratiometric fluorescent probe for selective detection and cellular
imaging of glutathione. Biosens. Bioelectron. 2015, 72, 275–281. [CrossRef] [PubMed]
1. Wei, M.; Yin, P.; Shen, Y.; Zhang, L.; Deng, J.; Xue, S.; Li, H.; Guo, B.; Zhang, Y.; Yao, S. A new turn-on
fluorescent probe for selective detection of glutathione and cysteine in living cells. Chem. Commun. 2013, 49,
4
640–4642. [CrossRef] [PubMed]
2. Na, R.; Zhu, M.; Fan, S.; Wang, Z.; Wu, X.; Tang, J.; Liu, J.; Wang, Y.; Hua, R. A simple and effective ratiometric
fluorescent probe for the selective detection of cysteine and homocysteine in aqueous media. Molecules 2016
1, 1023. [CrossRef] [PubMed]
5
5
,
2
3. Wei, L.F.; Thirumalaivasan, N.; Liao, Y.C.; Wu, S.P. Fluorescent coumarin-based probe for cysteine and
homocysteine with live cell application. Spectrochim. Acta A Mol. Biomol. Spectrosc. 2017, 183, 204–208.
[CrossRef] [PubMed]

Molecules 2017, 22, 1618
12 of 12
5
4. Yang, J.; Yu, Y.; Wang, B.; Jiang, Y. A sensitive fluorescent probe based on coumarin for detection of cysteine
in living cells. J. Photochem. Photobiol. A Chem. 2017, 338, 178–182. [CrossRef]
5
5. Wang, P.; Wang, Y.; Li, N.; Huang, J.; Wang, Q.; Gu, Y. A novel DCM-NBD conjugate fluorescent probe
for discrimination of Cys/Hcy from GSH and its bioimaging applications in living cells and animals.
Sens. Actuators B Chem. 2017, 245, 297–304. [CrossRef]
5
6. Nagaraja, D.; Melavanki, R.M.; Patil, N.R.; Kusanur, R.A. Solvent effect on the relative quantum yield and
fluorescence quenching of 2DAM. Spectrochim. Acta A Mol. Biomol. Spectrosc. 2014, 130, 122–128. [CrossRef]
[PubMed]
Sample Availability: Samples of the compounds are not available from the authors.
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