A simple but effective fluorescent probe with large stokes shift for specific detection of cysteine in living cells

Article abstract of DOI:10.1016/j.jphotochem.2018.05.025

A novel 4′-hydroxybiphenyl-4-carbonitrile-based fluorescent probe, 1, for selective detection of cystein (Cys) over homocystein (Hcy) and glutathione (GSH) was developed. This probe had simple structure and could be easily synthesized with good yield from commercially available materials. Moreover, probe 1 showed a remarkable large Stokes shift (180 nm) and displayed a rapid (5 min) and highly sensitive response (the detection limit was 0.15 μM) for Cys with fluorescence turn-on signal changes (142-fold fluorescence enhancement). Importantly, probe 1 could be used to detect and image both exogenous and endogenous Cys in living A549 cells.

Full text of DOI:10.1016/j.jphotochem.2018.05.025

Accepted Manuscript
Title: A simple but effective fluorescent probe with large
Stokes shift for specific detection of cysteine in living cells
Authors: Song Chen, Peng Hou, Jing Wang, Shuang Fu, Lei
Liu
PII:
S1010-6030(18)30375-7
DOI:
Reference:
https://doi.org/10.1016/j.jphotochem.2018.05.025
JPC 11295
To appear in:
Journal of Photochemistry and Photobiology A: Chemistry
Received date:
Revised date:
Accepted date:
22-3-2018
15-5-2018
18-5-2018
Please cite this article as: Chen S, Hou P, Wang J, Fu S, Liu L, A simple but
effective fluorescent probe with large Stokes shift for specific detection of cysteine
in living cells, Journal of Photochemistry and Photobiology, A: Chemistry (2018),
https://doi.org/10.1016/j.jphotochem.2018.05.025
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A simple but effective fluorescent probe with large Stokes shift for specific
detection of cysteine in living cells
Song Chen,* Peng Hou, Jing Wang, Shuang Fu, Lei Liu
College of Pharmacy, Qiqihar Medical University, 333 Bukui Street, Qiqihar,
Heilongjiang Province, P. R. China, 161006.
* E-mail address: chensongchemistry@163.com
Graphic abstract
1

Highlights
 A 4'-hydroxybiphenyl-4-carbonitrile-based fluorescence probe has been
successfully developed for highly selective, sensitive and rapid detection of
Cys.
 The probe displayed a large Stokes shift (180 nm) in the detection of Cys.
 The probe could be used to detect and image both exogenous and endogenous
Cys in living A549 cells
Abstract
A novel 4'-hydroxybiphenyl-4-carbonitrile-based fluorescent probe, 1, for
selective detection of cystein (Cys) over homocystein (Hcy) and glutathione (GSH)
was developed. This probe had simple structure and could be easily synthesized with
good yield from commercially available materials. Moreover, probe 1 showed a
remarkable large Stokes shift (180 nm) and displayed a rapid (5 min) and highly
sensitive response (the detection limit was 0.15 µM) for Cys with fluorescence
turn-on signal changes (142-fold fluorescence enhancement). Importantly, probe 1
could be used to detect and image both exogenous and endogenous Cys in living
A549 cells.
Keywords: Stokes shift; Fluorescent probe; Cell imaging; Cystein
2

1. Introduction
The small-molecular-weight thiols including cystein (Cys), homocystein (Hcy) and
glutathione (GSH), is essential and the most plentiful amino acid in many
physiological and pathological activities [1-3]. Alterations of the levels of these
specific bio-thiols are implicated in a number of diseases. Although they have the
similar chemistry structures, it is significant different to play fundamental roles in
biological processes [4-8]. Among them, Cys is one of semi-essential amino acid
relevant to amino acid transport, protein synthesis, cellular detoxification and
metabolism in living systems [9-12]. It is reported that Cys deficiency could result in
slow growth, hair pigmentation, edema, lethargy and liver damage. Moreover, the
heighten level of Cys is associated with neurotoxicity [13-17]. Therefore, it is of great
importance to monitor the level change of intracellular Cys for biochemical and clinic
research.
Fluorescent detection using fluorescent probes has been recognized as one of the most
powerful tools for the investigation of reactive species in living systems due to its
simple manipulation, high sensitivity and the capability of the real-time detection
[18-22]. In the past decade, various fluorescence probes have been designed and
synthesized for the detection of biothiols [23-33]. These probes for the Cys sensing
involve mainly the following three strategies: 1) cylization with aldehydes [34-36], 2)
native chemical ligation [37], 3) conjugation addition with α, β–unsaturated carbonyl
3

[38-40]. Although these probes have made a significant contribution to the biothiols,
there are still some limitations existing in the research. On one hand, most of them
have a problem distinguishing Cys from GSH and Hcy because they have similar
structure and reactivity. On the other hand, many of these reported fluorescent probes
also show relatively small Stokes shift (<100 nm). As is well known, with the
exception of an excellent selectivity, a large Stokes shift is also a very important
attribute of a fluorophore because fluorescent dyes with large Stokes shifts can
improve the detection sensitivity by avoiding fluorescence quenching originated from
self-absorption and fluorescence errors [41-42].
On account of above considerations, we presented a novel sensitive and selective
fluorescent probe 1 with a large Stokes shift (180 nm) for the imaging of Cys in living
cells. This probe 1 utilized 4'-hydroxybiphenyl-4-carbonitrile, dye 2, as the
fluorescent platform, and employed methacrylate group as the response site for Cys.
After the treatment of the probe 1 with Cys, an obvious cyan fluorescence signal was
observed. Moreover, the probe 1 exhibited high selectivity to Cys over other
bio-analytes such as GSH and Hcy. In particular, the biological applications were
demonstrated that the probe 1 could be successfully applied for the imaging of Cys in
living A549 cells.
2. Experiment
2.1. Materials and equipment
Unless otherwise stated, all reagents were purchased from commercial suppliers and
4

used as received. All accurate mass spectrometric experiments were performed on a
Waters ® Xevo G2-S QTof™ mass spectrometer (Waters, Milford, MA, USA).
Solvents were purified by standard methods prior to use. Twice-distilled water was
used throughout all experiments. NMR spectras were recorded on a BRUKER 600
spectrometer, using TMS as an internal standard. UV-Vis absorption spectra were
measured using a Shimadzu UV-2450 spectrophotometer. Uncorrected emission
spectra were recorded at room temperature on a Shimadzu RF5301PC. Cell imaging
was performed with a Zeiss LSM710 microscope. TLC analysis was performed on
silica gel plates and column chromatography was conducted over silica gel (mesh
200–300), both of which were obtained from Qingdao Ocean Chemicals. pH
measurements were carried out with a PHS-3C pH meter.
2.2. General procedure for spectral measurements
The stock solution of the probe
1 was prepared at 1 mM in DMSO. The
solutions of various common amino acid (Ala, Gly, Asp, Arg, Gln, Lys, Met,
Phe, Asn, Leu, Ser, His, Glu, IIe, Trp, Pro, Tyr, Val, Thr, Hcy, GSH, Cys) and
ionic salt (NaCl, CaCl2, FeCl3, MgCl2, ZnCl2, NaClO, Na2SO4, NaHSO3,
NaOAc, Na2CO3, NaNO3, Na3PO4) were prepared in twice-distilled water. The
3 mL for test solution was prepared by placing 0.03 mL of probe
1 stock
solution and appropriate testing analyte in 1.8 mL of buffer soultion (7.4, 50
mM PBS). The resulting solution was shaken well and incubated for 5 min at
room temperature before recording the spectra. For fluorescence measurements,
5

the excitation wavelength was 340 nm, the excitation slit widths and emission
slit widths were both 5 nm.
2.3. Cell culture and confocal microscopy imaging
A549 cells were incubated in a 6-well plate in Dulbecco’s modified Eagle’s
medium (DMEM) at 5% CO2 and 37
cells were washed with PBS buffer three times. For the endogenous Cys
experiment, A549 cells were incubated with probe (10.0 M) for 30 min at 37
℃ for 24 h. Before the experiments, A549
1
µ
°C, and then fluorescence imaging was performed after washing the cells with
PBS buffer three times. For the exogenous Cys experiment, A549 cells were
pre-incubated with 1.0 mM N-ethylmaleimide (NEM) for 30 min, and then
incubated with probe
1
(10.0
µM) for 30 min at 37 °C. Finally, the cells
incubated with Cys (100.0
µ
M) for 30 min at 37 °C. All the imaging was
performed by using a Zeiss LSM710 microscope.
2.2. Synthesis of probe 1
To a stirred solution of dye
2 (48.8 mg, 0.25 mmol) and triethylamine (10 µL)
in 5 mL dry dichloromethane was added methacryloyl chloride (83 mg, 0.80
mmol). The resulting reaction mixture was allowed to stir at room temperature
for 5 h and subsequently quenched with 20 mL of water. The resulting solution
was extracted twice with 15 mL dichloromethane. After drying the mixture
6

over anhydrous Na2SO4, the drying agent was removed by filtration and the
solvent was removed by distillation. The obtained residue was purified by flash
chromatography on silica gel (CH2Cl2 as eluent) to yield probe
1 (59.9 mg,
1
91.1%). H NMR (600 MHz, DMSO) δ 7.94 (d, J = 8.5 Hz, 2H), 7.91 (d, J =
8.4 Hz, 2H), 7.83 (d, J = 8.6 Hz, 2H), 7.33 (d, J = 8.6 Hz, 2H), 6.32 (s, 1H),
13
5.93 (s, 1H), 2.03 (s, 3H). C NMR (150 MHz, DMSO) δ 165.68, 151.58,
144.22, 136.35, 135.67, 133.36, 128.80, 128.46, 128.04, 123.05, 119.32,
110.56, 18.53. HRMS (EI) m/z calcd for [C17H13NO2 + H]+: 264.1025, Found:
264.1017.
3 Results and discussion
3.1. Design and synthseis of probe 1
Probe 1 was designed to use 4'-hydroxybiphenyl-4-carbonitrile (dye 2) as the
fluorophore and methacrylate group as the response site (Scheme 1). Through
introducing an electron-withdraw group (methacrylate group) which can attenuate the
electron-donating ability of the 4'-hydroxy group, the internal charge transfer (ICT)
effect of the fluorophore was weakened and resulted in the quenching of fluorescence.
On the basis of the Cys-triggered addition-cyclization reaction [43], the methacrylate
moiety can be cleaved to afford the 4'-hydroxy group and restored ICT effect of the
fluorophore (Scheme 2). With these considerations in mind, a novel ICT-based
fluorescent probe 1 for Cys was synthesized via only one step. The structure of probe
1 was confirmed by 1H NMR, 13C NMR and HRMS spectra (Fig. S3-S5).
7

Scheme 1 Synthetic route to probe
1
Scheme 2 The proposed response mechanism of probe
1 to Cys.
3.2. Spectroscopic studies
Initially, we investigated the absorption and emission spectra of dye
probe in 50.0 mM PBS buffer (pH =7.4, containing 40% DMSO) at room
temperature. Under this condition, dye (10.0 µM) exhibited maximum
absorption at 297 nm and strong fluoresces (λem = 477 nm). It’s noteworthy that
dyes featured a large Stokes shift (180 nm), which can reduce the interference
from incident light (Fig. 1). However, probe (10.0 µM) showed an absorption
2 and
1
2
2
1
band with a maximum at 276 nm and almost no fluorescence at 477 nm. When
8

treated with Cys, the solution of probe
which was identical to that of dye (Fig. S1), suggesting that the addition of
Cys resulted in the cleavage of methacrylate moiety in probe
To under the sensing process better, the fluorescence response of probe
1 showed a significant fluorescence
2
1
to released dye
2
.
1
(10.0
µM) to various concentrations of Cys were conducted (Fig. 2). With the
increasing concentration of Cys (from 0.0 to 200.0 µM), the fluorescence
intensity continuously increased. And a maximized enhancement (142-fold)
was obtained when 20.0 equiv. of Cys was added. As displayed in Fig. 3, the
plot between the fluorescence intensity (I477 nm) and the concentration of Cys
(from 0.0 to 10.0 µM) presented a good linearity (R2=0.9974). The limit of
detection (LOD) was measured to be 0.15 µM on the basis of the definition of
three times the deviation of the blank signal (S/N = 3). These results
demonstrated that this turn-on probe
1
had a high sensitivity for Cys detection.
9

Fig. 1 UV-vis absorption (black) and fluorescence (red) spectra of dye 2 in PBS
buffer.
Fig. 2 Fluorescence response of probe 1 (10.0 μM) upon the addition of Cys
(0.0–200.0 μM) in PBS buffer. Inset: the corresponding fluorescence images of probe
1 (10.0 μM) in the absence (left) and presences (right) of Cys (200.0 μM) under a 365
nm UV lamp.
10

Fig. 3 Fluorescence intensity of probe 1 (10.0 μM) at 477 nm as a function of Cys
concentration (0.0–200.0 μM) in PBS buffer. Inset: the linear relationship between
fluorescence intensity and Cys at low concentrations.
3.3. Selectivity studies
In order to examine whether probe
physiological amino acids, bio-relevant anions and ions, we then investigated
the fluorescence spectra of probe in the presence of other species (Fig. 4, Fig.
1 can selectively detect Cys among other
1
S2). The addition of 20.0 equiv. of physiological amino acids (Ala, Gly, Asp,
Arg, Gln, Lys, Met, Phe, Asn, Leu, Ser, His, Glu, IIe, Trp, Pro, Tyr, Val, Thr,
2-
-
2-
-
3-
Hcy, GSH), anions (ClO-, SO4 , HSO3 , AcO-, CO3 , NO3 , PO4 ) and ions
(Na+, Ca2+, Fe3+, Mg2+, Zn2+) did not significantly change the fluorescence of
probe 1. In contrast, when probe 1 was treated with 20.0 equiv. of Cys, a
dramatic fluorescence response was observed. Moreover, competitive
experiment revealed that the coexistence of these relevant species almost had
no effects on the performance of probe
1
in the detection of Cys. From the
showed excellent selectivity
above results, it could be concluded that probe
towards Cys over other species.
1
11

Fig. 4 The fluorescence intensity at 477 nm of probe 1 (10.0 μM) upon the addition of
the various common amino acid (a-y: 200.0 μM for Ala, Gly, Asp, Arg, Gln, Lys,
Met, Phe, Asn, Leu, Ser, His, Glu, IIe, Trp, Pro, Tyr, Val, Thr, Hcy, GSH, Cys)
(black). The fluorescence intensity at 477 nm of probe 1 (10.0 μM) to Cys (200.0 μM)
with the competition amino acid in PBS buffer (a-x: 200 μM for Ala, Gly, Asp, Arg,
Gln, Lys, Met, Phe, Asn, Leu, Ser, His, Glu, IIe, Trp, Pro, Tyr, Val, Thr, Hcy, GSH)
(red).
3.4. Effect of reaction time
The time-dependent fluorescence spectra of probe
1 (10.0 µM) in the presence
of different amounts of Cys (0.0 µM, 6.0 µM, 20.0 µM, 50.0 µM, 100.0 µM,
200.0 µM) were studied by measuring the fluorescence intensity at 477 nm
along with the time. As depicted in Fig. 5, upon the treatment with 20.0 equiv.
12

of Cys, the fluorescence intensity increased immediately and leveled off with 5
min. However, negligible changes in the fluorescence intensity at 477 nm were
detected in the absence of Cys for the same interval of time. These findings
indicated that this probe
to detect Cys.
1 could be acted as a rapid response fluorescent probe
Fig. 5 Time-dependent fluorescence intensity of probe 1 (10.0 μM) at 477 nm reacted
with the different concentration of Cys (0.0, 6.0, 20.0, 50.0, 100.0, 200.0 μM) in PBS
buffer.
3.5. Effect of pH
The pH effect on the fluorescence spectra of probe
(200.0 µM) was also investigated in aqueous solution. As shown in Fig. 6, the
fluorescence intensity of probe remained constant while the pH changes from
2 to 12, which indicated that probe
1 (10.0 µM) toward Cys
1
1
itself was pH-insensitive. On the contrary,
13

in the presence of Cys, the fluorescence intensity at 477 nm was intensively
increased with the pH changes from 6 to 9. These results suggested that probe
had potential to be used in physiological environment.
1
Fig. 6 pH effect on the fluorescence intensity of only probe 1 (10.0 μM) (black) and in
the presence (red) of Cys (200.0 μM) at 477 nm.
3.6. Reaction mechanism
Based on our experimental results and reported literatures [44-46], the reaction
mechanism of probe
Cys into the assay system, the ester bond cleaved by Cys turned probe
dye . This was evidenced by HPLC (Fig. 7). The HPLC chromatogram of
probe showed a single peak with a retention time at 1.89 min and it was 0.53
min for dye . Once probe was treated with 3.0 equiv. of Cys, the peak at 1.89
1
toward Cys was proposed as follows: upon addition of
1
into
2
1
2
1
min decreased and a new peak at 0.53 min occurred. The addition of 20.0 equiv.
14

of Cys to the solution of probe
min, indicating that probe was completely converted into dye
support for the reaction mechanism comes from the results of a HRMS study.
The mass spectral peak of the fluorescent product ( +Cys) at m/z 196.0753
correspond to [M+H+]+ for dye
(Fig. S6). All the above results strongly
1
induced the disappearance of the peak at 1.89
1
2
. Additional
1
2
support the proposed reaction mechanism depicted in Scheme 2.
Fig. 7 The HPLC chromatograms: (black) Probe 1; (red) Probe 1 with 3.0 equiv. of
Cys incubated for 10 min; (green) Probe 1 with 20.0 equiv. of Cys incubated for 10
min; (blue) Dye 2. Condition: eluent, H2O/CH3CN (v/v, 5/5), flow rate, 1.0 mL/min;
temperature, 25℃; injection volume, 20.0 µL.
3.7. Fluorescent imaging in living A549 cells
15

To further demonstrate the practical applicability of probe
systems, we carried out to investigate the ability of probe to visualize Cys in
living A549 cells (Fig. 8). As expected, Cyan fluorescence was observed inside
the cells when living A549 cells were incubated with probe (10.0 µM) for 30
min at 37 , indicating that probe had permeated into cells and reacted with
1 in biological
1
1
℃
1
endogenous Cys to produce discernible fluorescence. However, there was
almost no fluorescence when A549 cells were pretreated with 1.0 mM
N-ethylmaleimide (NEM) and then incubated with probe
1 (10.0 µM) for
additional 30 min. When these cells were further incubated with Cys (100.0 µM)
for 30 min, cyan fluorescence inside the cells appared again. These
fluorescence imaging experiments showed that probe
biological applications.
1 had great potential for
16

Fig. 8 Confocal fluorescence contrast images (A1) bright image (B1) fluorescence
image and (C1) overlay image of living A549 cells only incubated with 10.0 µM
probe 1 for 30 min at 37℃; (A2) bright image (B2) fluorescence image and (C2)
overlay image of living A549 cells pretreated with 1.0 mM NEM and then incubated
with 10.0 µM Probe 1; (A3) bright image (B3) fluorescence image and (C3) overlay
image of living A549 cells pretreated with 1.0 mM NEM and then incubated with
10.0 µM Probe 1 and 100.0 µM Cys.
4. Conclusions
In conclusion, we presented the design, synthesis and preliminary evaluation of the
methacrylate of 4'-hydroxybiphenyl-4-carbonitrile, probe 1, as a turn-on fluorescent
probe for the detection of Cys in aqueous solution. The reaction mechanism was
confirmed by means of HPLC and HRMS. The large Stokes shift (180 nm), fast
response (5 min) and high sensitivity (the detection limit was 0.15 µM) of probe 1
make it a practical and reliable method for fluorescence imaging. Furthermore, the
practical application of probe 1 for the selective detection of Cys was successfully
demonstrated in living A549 cells.
Acknowledgments
17

The research was supported by University Nursing Program for Young
Scholars with Creative Talents in Heilongjiang Province (No.
UNPYSCT-2017167), China.
18

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