Design and synthesis of a fluorescent probe with a large stokes shift for detecting thiophenols and its application in water samples and living cells

Article abstract of DOI:10.3390/molecules24020375

A turn-on florescent probe (probe-KCP) was developed for highly selective detection of thiophenols based on a donor-excited photo-induced electron transfer mechanism. Herein, the synthesis of the probe, a chalcone derivative, through a simple straightforward combination of a carbazole-chalcone fluorophore with a 2,4-dinitrophenyl functional group. In a kinetic study of the probe-KCP for thiophenols, the probe displayed a short response time (~30 min) and significant fluorescence enhancement. In selection and competition experiments, the probe-KCP exhibited excellent selectivity for thiophenols over glutathione (GSH), cysteine (Cys), sodium hydrosulfide (NaSH), and ethanethiol (C₂H₅SH) in addition to common anions and metal ions. Using the designed probe, we successfully monitored and quantified thiophenols, which are highly toxic. This turn-on fluorescence probe features a remarkably large Stokes shift (130 nm) and a short response time (30 min), and it is highly selective and sensitive (~160-fold) in the detection of thiophenols, with marked fluorescence in the presence of thiophenols. probe-KCP responds to thiophenols with a good range of linearity (0–15 μM) and a detection limit of 28 nM (R² = 0.9946) over other tested species mentioned including aliphatic thiols, thiophenol analogues, common anions, and metal ions. The potential applications of this carbazole-chalcone fluorescent probe was successfully used to determine of thiophenols in real water samples and living cells with good performance and low cytotoxicity. Therefore, this probe has great potential application in environment and biological samples.

Full text of DOI:10.3390/molecules24020375

molecules
Article
Design and Synthesis of a Fluorescent Probe
with a Large Stokes Shift for Detecting Thiophenols
and Its Application in Water Samples
and Living Cells
Hua Liu 1,2,3,4,†, Chuanlong Guo 1,5,†, Shuju Guo 1,2,3, Lijun Wang 1,2,3,
Dayong Shi 1,2,3,4,*
*
and
1
CAS Key Laboratory of Experimental Marine Biology, Institute of Oceanology, Chinese Academy of Sciences,
Qingdao 266071, China; baihualin55100@sina.cn (H.L.); gcl_cpu@126.com (C.G.); guoshuju@qdio.ac.cn (S.G.)
Laboratory for Marine Drugs and Bioproducts, Qingdao National Laboratory for Marine Science and
Technology, Qingdao 266237, China
Center for Ocean Mega-Science, Chinese Academy of Sciences, 7 Nanhai Road, Qingdao 266071, China
University of Chinese Academy of Sciences, Beijing 100049, China
2
3
4
5
Department of Pharmacy, College of Chemical Engineering, Qingdao University of Science and Technology,
Qingdao 266042, China
*
Correspondence: wanglijun@qdio.ac.cn (L.W.); shidayong@qdio.ac.cn (D.S.);
Tel.: +86-532-8289-8741 (L.W.); +86-532-8289-8719 (D.L.)
†
These authors contributed equally to this work.
ꢀ
ꢁꢂꢀꢃꢄꢅꢆꢇ
ꢀ
ꢁꢂꢃꢄꢅꢆ
Received: 3 January 2019; Accepted: 21 January 2019; Published: 21 January 2019
Abstract: A turn-on florescent probe (probe-KCP) was developed for highly selective detection
of thiophenols based on a donor-excited photo-induced electron transfer mechanism. Herein,
the synthesis of the probe, a chalcone derivative, through a simple straightforward combination of
a carbazole-chalcone fluorophore with a 2,4-dinitrophenyl functional group. In a kinetic study of
the probe-KCP for thiophenols, the probe displayed a short response time (~30 min) and significant
fluorescence enhancement. In selection and competition experiments, the probe-KCP exhibited
excellent selectivity for thiophenols over glutathione (GSH), cysteine (Cys), sodium hydrosulfide
(
NaSH), and ethanethiol (C H SH) in addition to common anions and metal ions. Using the
2 5
designed probe, we successfully monitored and quantified thiophenols, which are highly toxic.
This turn-on fluorescence probe features a remarkably large Stokes shift (130 nm) and a short response
time (30 min), and it is highly selective and sensitive (~160-fold) in the detection of thiophenols,
with marked fluorescence in the presence of thiophenols. probe-KCP responds to thiophenols with
2
a good range of linearity (0–15
µ
M) and a detection limit of 28 nM (R = 0.9946) over other tested
species mentioned including aliphatic thiols, thiophenol analogues, common anions, and metal
ions. The potential applications of this carbazole-chalcone fluorescent probe was successfully
used to determine of thiophenols in real water samples and living cells with good performance
and low cytotoxicity. Therefore, this probe has great potential application in environment and
biological samples.
Keywords: fluorescent probe; thiophenol; water samples; chalcone; living cells
1
. Introduction
Thiophenols are small molecules that serve as essential raw materials in the synthetic
chemical industry. For example: they are used in the preparation of pesticides, polymers,
and pharmaceuticals [1,2]. However, thiophenols are also pollutants which are highly toxic to
Molecules 2019, 24, 375; doi:10.3390/molecules24020375
www.mdpi.com/journal/molecules

Molecules 2019, 24, 375
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organisms, including humans, where thiophenol toxicity can have adverse physiological and
psychological effects. Prolonged exposure to thiophenols can cause damage to the central nervous
system, breathing difficulties, muscle weakness, paralysis of the limbs, a coma, and even death [3,4].
Previous research reported that the median lethal dose of thiophenols to fish and mice was
3
0
.01–0.4 mmol/L and 46.2 mg/kg , respectively. Thiophenols are considered a priority pollutant
by the U.S. Environmental Protection Agency (EPA waste code: P014) [5,6]. There is an urgent need
for the development of a rapid, selective, and sensitive method for monitoring of thiophenols in
environmental samples and biological samples.
There are various traditional methods such as high-performance liquid chromatography, gas
chromatography, and nonlinear spectroscopy, as well as nanomaterial-based sensors, available for
the detection of thiophenols [
7–
11]. However, most of these can detect thiophenols only in vitro
.
Fluorescent probe-based methods for the detection of thiophenols have many advantages [12
–
20].
These include high sensitivity and high selectivity, real-time bio-imaging, operational simplicity,
and short response times, all of which are important factors in the detection of thiophenols. The study of
small molecule fluorescent probes has attracted more and more attention [21]., However, many probes
are affected by interference from glutathione (GSH), cysteine (Cys), and homocysteine (Hcy) which
have similar chemical structures with thiophenols, or probes have a narrow Stokes shift (<60 nm) which
is detrimental for biological applications due to excitation backscattering effects [22–27]. Designing a
fluorescent probe that can distinguish between aliphatic thiols and aromatic thiophenols in living cells
is a huge challenge.
The biological activities of chalcone derivatives are well known [28–33]. These derivatives also
exhibit some excellent optical properties. Dyes based on chalcone derivatives are characterized by
large diversity and flexible modes of synthesis, intramolecular charge transfer, large Stoke shifts,
bright fluorescence, and a high quantum yield [34,35]. Therefore, chalcone derivatives have attracted
intense attention in recent years. In the present study, a turn-on fluorescent probe-KCP, based on
a donor-excited photo-induced electron transfer mechanism was designed and applied to detect
thiophenols. The probe-KCP was synthesized through a simple straightforward combination of a
chalcone unit, which served as a fluorescent chromophore, and a 2,4-dinitrophenyl moiety, which acted
as a fluorescent quenching unit. As expected, the designed probe shown pronounced selectivity for
thiophenols over common metal ions, anions, amino acids and aliphatic thiols. The probe-KCP was
substantially nonfluorescent due to the strong quenching effect of the 2,4-dinitrophenyl group. Thus,
the probe-KCP has excellent detection abilities for thiophenols and possesses marked fluorescence
intensity enhancement (~160-fold). It also has a conspicuously large Stokes shift (~130 nm). Therefore,
the probe has been successfully applied for the quantitative detection of thiophenols in real water
samples and fluorescent imaging of thiophenols in living cells.
2
. Results and Discussion
2
.1. Synthesis of the Probe-KCP
The synthesis procedure of the target chalcone fluorescent probe-KCP for the detection of
thiophenols is outlined in Scheme 1. The probe-KCP was synthesized with chalcones as a strong
fluorophore linked to a 2,4-dinitrophenyl group acted simultaneously as recognition unit and a
fluorophore quencher. KCP-OCH was reacted with boron tribromide in dry dichloromethane and
3
◦
cooled to
−
78 C. The crude product was conveniently converted to the probe-KCP in dichloromethane
at room temperature through a straightforward reaction with 2,4-dinitro-fluorobenzene. The structure
of the probe was characterized by 1H-NMR, C-NMR, and MS spectra. The experimental
characterization data are given in the Electronic Supplementary Information.
13

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2
.2. The Absorption and Emission Spectroscopic Properties of the Probe-KCP
To determine the fluorescence properties of the probe-KCP for thiophenols, we first examined
the absorption and fluorescent spectra of probe. The reaction response time of the probe-KCP
for thiophenols was investigated in DMSO/PBS (1:1, v/v, 20 mM, pH = 7.4). As shown
in Figure 1, the absorption spectra of the probe (10
µ
M) solution displayed one band at
06 nm only after the addition of 4-methoxythiophenol (100 M). The absorption maximum
ε = 6.2 × 10 M cm ) displayed a slight red shift 3 nm to 406 nm
4
µ
3
−1
−1
at 403 nm (Φ = 0.027
,
4
−1
−1
(
Φ = 0.19
;
ε = 2.12 × 10 M cm ). In the fluorescence spectra, significant fluorescence enhancement
(~160-fold) was observed at 540 nm after 4-methoxy-thiophenol was added to the DMSO/PBS
◦
solution containing the probe-KCP (10
µM) and incubated for 30 min at 37 C. The solution of the
probe-KCP alone exhibited weak fluorescence intensity, with excitation at 410 nm. After the addition
of 4-methoxythiophenol to the probe-KCP solution, it emitted relatively a strong fluorescent signal
at 540 nm and an absorption maximum at 410 nm. The addition of 4-methoxythiophenol resulted
in obvious green fluorescence from the probe-KCP solution when it was exposed to daylight or to a
hand-held UV lamp. (Figure S8). These results demonstrated that the probe-KCP is a promising fast
(30 min) turn-on fluorescence probe, with a large Stokes shift (130 nm) for the detection of thiophenols.
Figure 1.
(A) Absorption spectra and (B) fluorescence spectra of probe-KCP (10 µM) in the presence
of 100 M 4-methoxythiophenol (red) in DMSO/PBS (1:1, v/v, 20 mM, pH = 7.4). λex = 410 nm and
µ
λem = 540 nm.
To further investigate the sensitivity of the probe-KCP for thiophenols, the fluorescence
properties of the probe in the presence of different concentrations of thiophenols were investigated.
◦
The probe-KCP detected thiophenols in DMSO/PBS (v/v =1:1, 20 mM, pH = 7.4) solvent at 37 C.
The probe-KCP (10 µM) on its own showed a maximum absorption peak at 403 nm and almost no
fluorescence. However, when 4-methoxythiophenol was added to the probe solution, the maximum
absorption peak red-shifted to 406 nm. As shown in Figure 2, the fluorescence intensity was
proportional to the concentration of thiophenol and showed a rapid growth trend. When the
concentration of 4-methoxythiophenol reached 60 µM, the fluorescence intensity was enhanced by
about 160-fold and no change in the fluorescence at subsequent concentrations. An excellent linearly
2
dependent (R = 0.9946) between the enhancement of fluorescence intensity and the concentration of
4-methoxythiophenol (0 µM to 15 µM), thereby indicating that the probe-KCP had the potential to be
used for quantitatively monitoring thiophenols. The detection limit of the samples was calculated
using the following equation:
LD = 3σ/k
(1)
where σ is the standard deviation of a blank measurement, and k is the slope.

Molecules 2019, 24, 375
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The kinetic study showed that the fluorescence intensity of the probe-KCP solution increased
◦
significantly 30 min after adding 4-methoxythiophenol at 37 C and that the fluorescence intensity did
not change substantially after 30 min (Figures S6 and S7). The results pointed to the potential use of
the probe for real-time monitoring of thiophenols.
Figure 2.
(
A
) Fluorescence spectra of 10
M); ( ) The linearity of the probe-KCP fluorescence intensity versus thiophenol concentration
0–15 µM). λex = 410 nm and λem = 540 nm. Relative standard deviation (RSD), n = 3.
µM probe-KCP upon the addition of 4-methoxy thiophenol
(0–100
µ
B
(
2
.3. The Effect of pH on the Probe
The pH of a system can sometimes exert a marked effect on the fluorescence intensity. We tested
the response of the probe-KCP to 4-methoxythiophenol over a wide pH range from 1 to 10.
The influence of pH on the fluorescence spectra of the probe-KCP to 4-methoxythiophenol was
tested. At a pH of <6, no significant changes in the fluorescence intensity of the probe-KCP solution
were observed at 540 nm, and similar results were observed when the pH exceeded 10.0. However, at a
pH of >6, the fluorescence intensity of the probe-KCP (10
-methoxythiophenol at a concentration of 100 M. As shown in Figure S2, the probe-KCP functioned
at 540 nm in a pH range from 7.0 to 10.0. The fluorescent intensity of the probe-KCP (10 M) was stable
µM) decreased significantly in the presence of
4
µ
µ
over a wide pH range, which indicated that the probe was stable in the pH range of physiological
conditions. Thus, in subsequent live cell imaging studies, a pH of 7.4 was selected as optimal for
DMSO/PBS buffer (1:1, v/v, 20 mM).
2
.4. Sensitivity and Selectivity of the Probe for Thiophenols
Next, the spectral responses of the probe-KCP toward various thiophenols, thiols, amino
acids, other potentially interfering substances, common anions and metal ions were investigated by
fluorescence spectroscopy. As shown in Figures S3 and S4, only the introduction of thiophenols such as
p-CH3
−
C H SH, p-NH
−
C H SH, p-F
−
C H SH and p-CH O C H SH to the probe-KCP solution
−
6
4
2
6
4
6
4
3
6 4
induced a significantly enhanced the fluorescence intensity. Reactivity comparison: p-CH O
−
C H SH
6 4
3
>
p-F
−
C H SH > p-NH
−
C H SH > p-CH
−
C H SH. However, the addition of other biologically
6
4
2
6
4
3 6 4
relevant analytes resulted in no change in the fluorescence intensity of the compounds in the probe-KCP
solution. These results clearly suggested that the probe-KCP was particularly selective for thiophenols
over other interfering species. In other words, the probe-KCP displayed high selectivity for thiophenols
(4-fluorothiophenol, 4-toluenethiol, 4-methoxythiophenol, and 4-aminothiophenol). As shown in
Figure 3, it is worth noting that there was no significant fluorescence signal change after the probe
reacted with the other potentially interfering substances interferences (10 eq.), such as mercaptan,
◦
nucleophilic species, and metal ions in the DMSO/PBS buffer (1:1, v/v, 20 mM, pH = 7.4) at 37 C

Molecules 2019, 24, 375
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for 30 min. These results showed that the probe-KCP could be used to monitor thiophenols in the
presence of competitive interference from other substances.
Figure 3. The fluorescence intensity at 540 nm (λex = 410 nm) of probe-KCP (10
µ
◦
M) in the presence of
1
00
µM of various analytes in DMSO/PBS buffer (1:1, v/v, 20 mM, pH 7.4) at 37 C prior to (black bars)
and after (red bars) addition of 100
µM 4-methoxythiophenol to the individual probe/analyte solution.
(
(
(
1) blank, (2) GSH,(3) Cys, (4) Hcy, (5) Ala, (6) Ser, (7) NaSH, (8) C H SH, (9) Ph-OH, (10) Ph-NH ,
2
5
2
11) NaCl, (12) FeCl , (13) KCl, (14) CaCl , (15) FeCl , (16) MgSO , (17) NaNO , (18) KF, (19) NaHCO ,
2
2
3
4
3
3
20) 4-toluenethiol, (21) 4-methoxythiophenol, (22) 4-aminothiophenol, (23) 4-fluorothiophenol. Data
were recorded 30 min after addition of analytes/ thiophenols with excitation at 410 nm and emission at
40 nm. Error bars are relative standard deviations (RSD), n=3.
5
2
.5. Plausible Mechanism of Detection of Thiophenols
To determine a plausible mechanism for the detection of thiophenols, H-NMR, 13C-NMR,
and mass spectrometry (Figures S1 and S9) were employed to monitor the reaction of the probe-KCP
1
with 4-methoxythiophenol. Two products (probe-OH and S-NO ) were isolated and their structural
2
characterizations are shown in Figure S1.
To better understand the reaction mechanism of the probe-KCP with thiophenols, density
functional theory was applied using the Gaussian 09 program (Figure 4 and Figure S10). As shown in
Figure 4A, for the probe-OH moiety, electron clouds on both HOMO and LUMO essentially resided
on its entire skeleton. By contrast, in the case of the probe-KCP (Figure 4B), the electron clouds on
the HOMO and LUMO+2 were mainly distributed in the electron-donating moiety of the probe-OH.
However, those on the LUMO and LUMO+1 were primarily located on the electron-withdrawing
2,4-dinitrophenyl group. These findings indicated that the probe-KCP could bear efficient electron
transfer from the moiety of the probe-OH to the 2,4-dinitrophenyl moiety, resulting in weak or no
fluorescence of the probe. After the addition of 4-methoxythiophenol, the electron transfer from
the moiety of the probe-OH to the 2,4-dinitrophenyl was blocked following the elimination of the
2,4-dinitrophenyl group. The resulting almost complete overlap of electrons on the transition orbitals
may induce strong fluorescence emission from the moiety of the probe-OH.

Molecules 2019, 24, 375
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Figure 4. (A) Frontier molecular orbital plots of dye probe-OH (CPCM model); (B) Frontier molecular
orbital plots of probe-KCP (CPCM model). The fluorescence emission of probe-KCP moieties is
quenched by d-PET.
2
.6. Measurements of Thiophenols in Water Samples
To evaluate whether the probe could directly detect thiophenols in a real environmental sample,
the probe was tested in experiments involving deionized water, artificial seawater, and tap water
samples. The water samples were spiked with different concentrations of 4-methoxythiophenol and
◦
then incubated with the probe for 30 min at 37 C.
As shown in Figure 5, the experimental results showed that the fluorescence intensity of the probe
increased in proportion to the concentration of 4-methoxythiophenol added and that the recovery of
4-methoxythiophenol was 92–107%. This finding indicated that the probe was capable of detecting
thiophenols in real water samples. The results shown in Table 1 was reported as the mean
deviation of triplicate experiments for thiophenol spiked from 0 to 10 µM.
±
standard
Figure 5. A linear calibration curve between the fluorescence intensities of probe-KCP and
concentration of 4-methoxythiophenol in the range of 0–10 M in real water samples. ( ) Fluorescent
detection of different concentrations of 4-methoxythiophenol in “tap water”, “deionized water”,
and “artificial water” by probe-KCP (10 M) in PBS buffer (20 mM) with 50% DMSO; ( Linear of
fluorescence intensity changes of probe-KCP against the of 4-methoxythiophenol for real water sample.
µ
A
µ
B–D

Molecules 2019, 24, 375
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Table 1. Determination of thiophenol concentrations in water samples.
Thiophenol Spiked
Thiophenol Recovered
Sample
Recovery (%)
(
µM)
(µM)
0
2
4
6
8
not detected
1.99 ± 0.334
3.79 ± 0.5484
5.63 ± 0.560
8.41 ± 0.617
10.12 ± 0.777
not detected
1.84 ± 0.105
4.19 ± 0.411
6.12 ± 0.397
8.57 ± 0.464
9.99 ± 0.405
not detected
2.11 ± 0.304
3.80 ± 0.453
6.21 ± 0.3555
8.11 ± 0.605
9.62 ± 0.592
100%
95%
94%
105%
101%
Artificial seawater
1
0
0
2
4
6
8
92%
105%
102%
107%
100%
Tap water
1
0
0
2
4
6
8
106%
95%
104%
101%
96%
Deionized water
1
0
2
.7. Determination of the Potential Cytotoxicity of the Probe
To evaluate the potential cytotoxicity of the probe before applying it in live cell imaging,
we performed an MTT assay in A549 cells. Different concentrations of the probe-KCP (0, 2.5, 5, 10, 20,
and 50 M) were added to the A549 cells and incubated for 24 h. As shown in Figure S5, the probe-KCP
µ
was nontoxic to the cultured cells under the experimental conditions, which demonstrated that the
probe was safe and suitable for cell imaging.
2
.8. Cell Imaging
Given the favorable properties of the probe-KCP (e.g., high selectivity, excellent sensitivity, a short
response time, and lack of cytotoxicity) in terms of the detection of thiophenols in vitro in DMSO/PBS
buffer (1:1, v/v, 20 mM, pH = 7.4), we tested the potential applications of the probe-KCP in live cell
imaging assays. The A549 cells were treated with the probe-KCP and then observed under a confocal
fluorescence microscope. As shown in Figure 6, the probe-KCP (10
cells for 30 min at 37 C. Almost no intracellular fluorescence signals were observed in the green
µ
M) was incubated with the A549
◦
channel by CLSM. However, following the addition of 300 µM 4-methoxythiophenol to the probe-KCP
solution and incubation with the A549 cells for 30 min, green fluorescence signals were observed in the
green channel. These findings indicated that the probe-KCP could be employed to detect thiophenols
in A549 cells via CLSM. Thus, the designed probe-KCP is cell membrane permeable and can detect
thiophenols with obvious fluorescence signals in living cells.

Molecules 2019, 24, 375
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Figure 6. Imaging of 4-methoxythiophenol in living A549 cells using the probe-KCP. The A549 cells
◦
were pretreated with 4-methoxythiophenol (300
probe-KCP (10 µM) for 30 min at 37 C.
µ
M) for 30 min at 37 C and then incubated with the
◦
3
. Experimental
3
.1. Starting Materials and Instruments
Carbazole (CAS No.
86-74-8, 98%), bromoethane (CAS No.
74-96-4, 98%),
0
4
-methoxy-acetophenone (CAS No. 100-06-1, 99%), 2,4-dinitrofluorobenzene (CAS No. 70-34-8, 99%),
boron tribromide (CAS No. 10294-33-4), and cesium carbonate (CAS No. 534-17-8, 99%) were obtained
commercially and used without further purification. Other reagents (phosphorus oxychloride,
ethanol, ethyl acetate, petroleum ether, dichloromethane, sodium bicarbonate, and anhydrous sodium
sulfate) were of commercial analytical grade and were used without further purification. Silica gel
(
200 300 mesh, Qingdao Makall Group Co., Ltd., Qingdao, China) was used in silica gel column
−
chromatography. The fins salt was obtained commercially (Qingdao Haike General Sea Salt Co.,
Ltd., Qingdao, China). The pH was determined by a pH meter (LEICI, Shanghai, China). UV-vis
spectra were measured on a UV-Vis spectrophotometer (Agilent Technologies, Waldbronn, Germany).
Fluorescence spectra were recorded on a Tecan Infinite M1000 Pro Microplate Reader (Tecan,
1
13
TM
Männedorf, Switzerland). H-NMR and C-NMR spectra were recorded on a Bruker Avance III
00 MHz NB Digital NMR spectrometer (Bruker-Biospin, Billerica, MA, USA) in CDCl or DMSO-d
5
3
6
with TMS as an internal standard. High-resolution mass spectra (HRMS) were obtained with a
MALDI SYNAPT G2 HDMS system (Waters, Milford, MA, USA). In general, the designed probe-KCP
compounds were synthesized as depicted in Scheme 1, and the experimental details are shown in
Scheme 1.

Molecules 2019, 24, 375
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Scheme 1. The synthetic routes and the chemical structures of the probe-KCP. Reagents and
◦
(a) NaH, DMF, bromoethane; (b) POCl , DMF, 90 C; (c) C H OH, C H ONa,
3 2 5 2 5
conditions:
4
0
◦ ◦
78 C; (e) 2,4-dinitrofluorobenzene, Cs CO , DCM, 30 C.
2 3
-methoxy-acetophenone; (
d
) BBr , DCM,
−
3
3
.2. Synthesis of KCP-OCH3
The synthesis of carbazole compound
2
was performed in accordance with methods described in
the literature [36
Compound
,
37]. The synthesis of the compound KCP-OCH was also based on the literature [38].
3
0
2
(1 mmol) was added to a solution of 4 -methoxyacetophenone (1 mmol) in ethanol
(
10 mL), and fresh sodium ethoxide (4 mmol) solution (5 mL) was then added slowly. The mixture was
stirred at room temperature overnight. The resulting precipitate was filtered and recrystallized from
ethanol to give (E)-3-(9-ethyl-9H-carbazol-3-yl)-1-(4-methoxyphenyl)-prop-2-en-1-one (KCP-OCH )
3
1
as a faint yellow solid in 85% yield: H-NMR (500 MHz, CDCl )
δ 8.36 (s, 1H), 8.14 (d, J = 7.7 Hz,
H
3
1
7
4
H), 8.10 (d, J = 8.5 Hz, 2H), 8.06 (d, J = 15.5 Hz, 1H), 7.77 (d, J = 8.4 Hz, 1H), 7.60 (d, J = 15.5 Hz, 1H),
.50 (t, J = 7.6 Hz, 1H), 7.40 (dd, J = 13.6, 8.3 Hz, 2H), 7.29 (t, J = 7.4 Hz, 1H), 7.00 (d, J = 8.5 Hz, 2H),
.34 (q, J = 7.1 Hz, 2H), 3.89 (s, 3H), 1.44 (t, J = 7.2 Hz, 3H); 13C-NMR (125 MHz, CDCl3)
δ
188.73,
C
1
1
63.08, 145.52, 141.25, 140.36, 131.53, 130.62, 126.20, 126.00, 123.35, 122.78, 121.40, 120.56, 119.57, 118.69,
+
13.68, 108.79, 77.00, 55.40, 37.67, and 13.78. The HRMS (ESI) m/z was calculated for C H NO
2
24
22
+
[M + H] : 356.1651, Found 356.1641.
3
.3. Synthesis of the probe-KCP
Boron tribromide (as a 1.0 M solution in dichloromethane; 2 mL, 2 mmol) was added dropwise
over a 10 min period to a suspension of KCP-OCH (0.5 mmol) in dry dichloromethane (15 mL
3
◦
◦
cooled to 0 C). The resulting mixture was stirred at 0 C to room temperature. Water and
dichloromethane were added. The organic layer was separated and dried with anhydrous Na SO .
2
4
After concentrate under reduced pressure, the crude product was used for the next step, without
separation or purification. Then, Cs CO (Equation (2)) was added and stirred at room temperature
2
3
for 15 min. Subsequently, 2,4-dinitrofluorobenzene was added and stirred at room temperature for 2 h.
The resulting precipitate was filtered and recrystallized from ethyl acetate: petroleum ether (v:v = 2:8)
to (E)-1-(4-(2,4-dinitrophenoxy)phenyl)-3-(9-ethyl-9H-carbazol-3-yl)prop-2-en-1-one (probe-KCP) as
a give yellow solid (yield 76%): 1H-NMR (500 MHz, CDCl3)
δ
8.84 (s, 1H), 8.43–8.28 (m, 2H),
H
8
7
.22–8.02 (m, 4H), 7.78 (d, J = 8.3 Hz, 1H), 7.59–7.47 (m, 2H), 7.42 (t, J = 8.5 Hz, 2H), 7.32–7.18 (m, 3H),
.12 (d, J = 9.0 Hz, 1H), 4.37 (d, J = 6.9 Hz, 2H), 1.45 (t, J = 6.8 Hz, 3H). 13C-NMR (125 MHz, CDCl3)
δ
C
188.63, 156.93, 154.91, 147.21, 142.02, 141.55, 140.41, 136.66, 131.10, 128.85, 126.41, 125.54, 123.43, 122.70,
1
22.09, 121.75, 120.56, 119.83 (d, J = 11.5 Hz), 119.55, 118.10, 108.92, 77.00, 37.74, and 13.80. The HRMS
+
+
(ESI) m/z was calculated for C H NO [M + H] : 508.1509, Found 508.1503.
29 22 2

Molecules 2019, 24, 375
10 of 14
3
.4. Preparation of the Test Solutions
The fluorescence properties of the probe-KCP for thiophenols were evaluated in DMSO/PBS
buffer (1:1, v/v, 20 mM, pH = 7.4). A stock solution of the probe-KCP was prepared at a concentration
of 2.5 mM in 5 mL of DMSO and then diluted to the desired concentration with PBS (20 mM pH = 7.4
,
,
containing 50% DMSO). Stock solutions of thiophenols and other analytes, in addition to those of
common anions and metal ions, were prepared at a concentration of 5 mM in distilled water and then
diluted to the desired concentrations with PBS (20 mM, pH = 7.4, containing 50% DMSO). Absorbance
and fluorescence spectral data were recorded 30 min after the addition of the analytes. For fluorescence
measurements, the excitation wavelength was 410 nm, and emissions were acquired from 450 nm
to 800 nm.
3
.5. General Procedure for Analysis
Stock solutions (100 M) of thiophenols (4-toluenethiol, 4-fluorothiophenol, 4-methoxy-
thiophenol, and 4-aminothiophenol) and other analytes, including glutathione (GSH), cysteine (Cys),
and homocysteine (Hcy); alanine (Ala), serine (Ser), aniline (Ph-NH ), phenol (Ph-OH), sodium
µ
2
hydrosulfide (NaSH) and ethanethiol (C H SH), sodium chloride (NaCl), Ferrous chloride (FeCl ),
2
5
2
Potassium chloride (KCl), calcium chloride (CaCl ), ferric chloride (FeCl ), magnesium sulfate (MgSO ),
2
3
4
Sodium nitrate (NaNO ), Potassium fluoride (KF) and Sodium bicarbonate (NaHCO ) were prepared
3
3
◦
with PBS buffer (20 mM, pH = 7.4, containing 50% DMSO). The resulting solution was kept at 37 C
for 30 min before recording its absorption and fluorescence spectra.
3
.6. Kinetic Study
To determine the reaction time of the probe-KCP, the increase in fluorescence intensity upon
the addition of thiophenols (100
µM) to the probe solution (10
µM) in DMSO/PBS buffer (1:1, v/v,
2
4
3
0 mM, pH = 7.4) was investigated using a microplate reader. As shown in Figure S1, the addition of
-methoxythiophenol immediately led to significant fluorescence enhancement at 540 nm for up to
0 min, followed by steady-state fluorescence. Taken together, these data indicated that the reaction
time for the detection of thiophenols was 30 min. Thus, 30 min was considered an appropriate reaction
time in subsequent experiments. The kinetic study indicated that the probe-KCP had a rapid response
time, with high sensitivity for the detection of thiophenols.
3
.7. Fluorescence Quantum Yield
The fluorescence quantum yield of the probe-KCP was determined according to methods
described in the literature [39] using rhodamine 6G (
Φ = 0.95, λex = 530 nm, in ethanol solutions) as a
fluorescence standard. The u values of the samples were calculated using the following equation:
2
Fu
Fs
As
Au
n
n
u
φu =
×
×
× φs
(2)
2
s
where the subscripts u and s denote the test and standard respectively,
Φ
is the fluorescence quantum
yield, A is the absorbance at the excitation wavelength, F is the area under the corrected emission
curve, and n is the refractive index of the solvent used. The subscripts s and u refer to the standard
and the sample.
3
.8. Reaction Mechanism
A possible reaction mechanism of the probe-KCP with thiophenols was proposed based upon
studies of the UV-vis spectra, the fluorescence intensity and other experiments. The reaction products
1
13
were investigated by H-NMR, C-NMR, and mass spectrometry. The likely interaction mechanism
of the probe-KCP with thiophenols is shown in Scheme 2. The probe-KCP (Equation (1)) and
4-methoxythiophenol (Equation (1)) was dissolved in the DMSO/PBS (20 mM, pH = 7.4) solution,

Molecules 2019, 24, 375
11 of 14
followed by stirring at room temperature for 2 h. The mixture was diluted with 10 mL of water and
extracted by ethyl acetate (3 10 mL). The organic layer was evaporated under reduced pressure and
purified by silica gel column chromatography to give probe-OH and S-NO . The characterization data
×
2
for the probe-OH and S-NO are given in the Electronic Supplementary Information (Figure S1).
2
Scheme 2. Proposed thiophenol detection mechanism of the probe.
3
.9. Detection of Thiophenols in Water Samples
Deionized water, artificial sea water, and tap water were filtered using a microfiltration membrane
◦
before use. The studied water samples were kept at 4 C before use. The water samples were spiked
with different concentrations of thiophenol (0, 2.0, 4.0, 6.0, 8.0, 10.0
µM). The solutions were incubated
for 30 min before measurement and the fluorescence intensity was observed at 540 nm.
3
.10. Cell Culture
A human lung cancer cell line, A549, was purchased from the Cell Bank, Chinese Academy
of Sciences (Shanghai, China). The cells were maintained in F-12K, supplemented with 10% FBS,
◦ ◦
µg/mL of streptomycin at 37 C. The cells were cultured at 37 C in a
100 U/mL of penicillin, and 100
humidified CO (5%) atmosphere.
2
3
.11. Determination of Cell Viability
Cell viability was determined using a 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl tetrazolium
bromide (MTT) assay. Cells in growth phase were plated into a 96-well culture plate at a density of
3
3
× 10 cells per well and incubated for 24 h. The cells were then exposed to 2.5, 5, 10, 20, and 50
µM
of the probe-KCP for 24 h. Following this treatment, the cells were further incubated with MTT for 4 h.
The medium in each well was carefully removed, and DMSO (150 L) was then added to each well.
The samples were thoroughly agitated for 10 min on a shaker. Finally, the absorbance of the samples at
µ
4
90 nm was measured using a microplate reader.
3
.12. Fluorescence Microscopic Cell Imaging
The A549 cells were seeded on a cover glass and incubated overnight. The probe-KCP dissolved
in DMSO was added to the cell medium at a final concentration of 10 µM for 30 min. The medium was
then removed, and the cells were washed with PBS three times. For comparison purposes, the cells
◦
were pretreated with 4-methoxythiophenol (300
µ
M) at 37 C for 30 min, followed by washing three
times. Subsequently, the cells were treated with the probe-KCP for 30 min. The cells were imaged on a
FV1000 confocal laser scanning microscope (CLSM, Olympus, Tokyo, Japan).
4
. Conclusions
In summary, we described the synthesis of a safe and efficient chalcone fluorescent probe-KCP for
the detection of thiophenols. The probe-KCP responded selectively to thiophenols over other amino
acids and common metal ions and to potential interference from other substances in DMSO/PBS
2
(
1:1, v/v, 20 mM, pH = 7.4). The detection limit of the probe was 28 nM (R = 0.9946). Thus, we conclude
that the probe-KCP exhibits high selectivity toward thiophenols. We used the probe to successfully
monitor and quantify highly toxic thiophenols. This turn-on fluorescence probe features a remarkably
large Stokes shift (130 nm) and a fast response time (30 min). It is highly selective and sensitive in terms

Molecules 2019, 24, 375
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of the detection of thiophenols, with significant fluorescence enhancement. In addition, this probe
can be successfully applied to detect thiophenol in real water samples and in living cells. Therefore,
this work provides a chalcone fluorescent probe (probe-KCP) for the detection of the highly toxic
thiophenols found in environmental and biological samples.
Supplementary Materials: The following are available online, Figure S1. date for investigation of the sensing
mechanism. Figure S2. The effect of pH on the fluorescence intensity (λem = 540 nm) of probe-KCP (10
µ
M,
M 4-methoxy thiophenol after
M) to thiophenol and
λex = 410 nm) in DMSO/PBS buffer (1:1, v/v, 20 mM) upon addition of 100
µ
◦
incubation at 37 C for 20 min. Figure S3. Fluorescence responses of probe-KCP (10
µ
other various analytes (100
Enhanced fluorescence response at 540 nm of the probe-KCP (10
100 µM) in PBS buffer solution (20 mM, pH = 7.4) containing 50 % DMSO. Figure S5. Percentage of viable A549
cells after treatment with different concentrations of the probe-KCP after 24 h using an MTT assay. Figure S6.
A)Time-dependent the fluorescence response of probe-KCP (10 M) in the absence (blank) and presence of
-methoxythiophenol, GSH, Cys, Hcy, NaSH or C H SH (10 equiv) in PBS buffer solution (20 mM, pH = 7.4)
µ
M) in PBS buffer solution (20 mM, pH = 7.4) containing 50 % DMSO. Figure S4.
µM) to thiophenol and other various analytes
(
(
4
µ
2
5
containing 50 % DMSO. (B) Time-dependent the fluorescence response of probe-KCP (10
µM) in the absence
(
blank) and presence of 4-methoxythiophenol, GSH, Cys, Hcy, NaSH or C H SH (10 equiv) at 0 min, 10 min,
2
5
2
0 min, 30 min. Figure S7. Time-dependent the fluorescence response of probe-KCP (10
µM) in the presence of
4
-methoxythiophenol (10 equiv) in PBS/ DMSO solution (v:v = 1:1, 20 mM, pH = 7.4). Figure S8. Photograph
of probe-KCP solutions (10
irradiation (365 nm).
µM) in the presence of 4-methoxythiophenol (10 equiv) under natrual light and UV
Author Contributions: H.L. performed probe-KCP synthesis and characterization and data analysis, drafted the
manuscript and finalized the manuscript; C.G. participated cell culture, determination of cell viability, fluorescence
microscopic cell imaging; S.G. helped on probe-KCP synthesis and data analysis; L.W. helped on data analysis;
D.S. guided the experiments.
Funding: This work was supported by the National Natural Science Foundation of China (No. 81773586, 81703354,
8
1600782), and Shandong Provincial Natural Science Foundation for Distinguished Young Scholars (JQ201722),
and Key Research Program of Frontier Sciences, CAS (QYZDB-SSW-DQC014), and the Project of Discovery,
Evaluation and Transformation of Active Natural Com-pounds, Strategic Biological Resources Service Network
Program of Chinese Academy of Sciences (ZSTH-026) and National Program for Support of Top-notch Young
Professionals, and Taishan scholar Youth Project of Shandong province and Key research and development project
of Shandong province (2018GSF118223) and Natural Science Foundation of Shandong Province (ZR2016DQ07).
Conflicts of Interest: The authors declare no conflict of interest.
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Sample Availability: Samples of the compounds probe-KCP is available from the authors.
©
2019 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access
article distributed under the terms and conditions of the Creative Commons Attribution
CC BY) license (http://creativecommons.org/licenses/by/4.0/).
(