Reaction-based fluorescent turn-on probe for selective detection of thiophenols in aqueous solution and living cells

Article abstract of DOI:10.1016/j.dyepig.2017.03.033

A novel turn-on florescent probe based on a nucleophilic substitution reaction was developed for the detection of thiophenols in aqueous solution and in living cells. In this probe, 10-hydroxyl derivative of coumarin 6H was selected as fluorophore, 2, 4-dinitrophenlate moiety acted simultaneously as recognition unit and fluorophore quencher. This probe features a remarkable large Stokes shift (128?nm) and shows a highly selective detection process for thiophenols with significant fluorescence turn-on response. Notably, biothiols, aliphatic thiols, amino acids and reducing anions do not interfere with the sensing of thiophenols. The probe shows good linearity ranges with low detection limit of 36?nM for thiophenols. More importantly, it was successfully applied for practical detection of thiophenols in real water samples with a good recovery and imaging of thiophenols in living cells, demonstrating its practical application in environmental samples and biological systems.

Full text of DOI:10.1016/j.dyepig.2017.03.033

Accepted Manuscript
Reaction-based fluorescent turn-on probe for selective detection of thiophenols in
aqueous solution and living cells
Xiao-Bo Wang, Xue Hao, Datong Zhang, Yan Jiang
PII:
S0143-7208(16)31485-1
10.1016/j.dyepig.2017.03.033
DYPI 5859
DOI:
Reference:
To appear in: Dyes and Pigments
Received Date: 27 December 2016
Revised Date: 15 March 2017
Accepted Date: 15 March 2017
Please cite this article as: Wang X-B, Hao X, Zhang D, Jiang Y, Reaction-based fluorescent turn-on
probe for selective detection of thiophenols in aqueous solution and living cells, Dyes and Pigments
(2017), doi: 10.1016/j.dyepig.2017.03.033.
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ACCEPTED MANUSCRIPT
Graphical abstract

ACCEPTED MANUSCRIPT
Reaction-Based Fluorescent Turn-on Probe for Selective
Detection of Thiophenols in Aqueous Solution and Living Cells
Xiao-Bo Wang, Xue Hao, Datong Zhang*, Yan Jiang
Shandong Provincial Key Laboratory of Fine Chemicals, School of Chemistry and Pharmaceutical
Engineering, Qilu University of Technology, Jinan 250353, Shandong, P. R. China
*
Corresponding author, E-mail: dtzhang@qlu.edu.cn
Abstract: A novel turn-on florescent probe based on a nucleophilic substitution reaction was developed
for the detection of thiophenols in aqueous solution and in living cells. In this probe, 10-hydroxyl
derivative of coumarin 6H was selected as fluorophore, 2, 4-dinitrophenlate moiety acted
simultaneously as recognition unit and fluorophore quencher. This probe features a remarkable large
Stokes shift (128 nm) and shows a highly selective detection process for thiophenols with significant
fluorescence turn-on response. Notably, biothiols, aliphatic thiols, amino acids and reducing anions do
not interfere with the sensing of thiophenols. The probe shows good linearity ranges with low detection
limit of 36 nM for thiophenols. More importantly, it was successfully applied for practical detection of
thiophenols in real water samples with a good recovery and imaging of thiophenols in living cells,
demonstrating its practical application in environmental samples and biological systems.
Keywords: Fluorescent probe; Coumarin 6H; 2, 4-Dinitrophenlate; Thiophenols; Cell imaging
Introduction
Thiols including aliphatic thiols and thiophenols are an important class of organosulfur
compounds in both the biological systems and chemical industry. Aliphatic thiols, such as cysteine
(
Cys), homocysteine (Hcy), glutathione (GSH), et al, play crucial roles in physiological processes [1-3].
Thiophenols have a broad synthetic utility in preparation of pharmaceuticals, pesticides and polymers
4-5]. However, as a class of highly toxic and pollutant compounds, thiophenols have been listed as one
[
of the prioritized pollutants by the United States Environmental Protection Agency (USEPA waste code:
P014) [6]. Due to their high toxicity and ease of entrance into human body by inhalation and skin
absorption, the exposure to thiophenols liquid or vapor may induce grave systemic and central nervous
injuries, even death [7-9]. Existence of thiophenols in soil and water are also reported to cause damage
to natural surroundings [10]. Therefore, it is of great significance to establish a simple, highly sensitive
1

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and selective method to detect thiophenols in the fields of chemical, environmental and biological
sciences. It is more important to develop efficient techniques to discriminate thiophenols from aliphatic
thiols.
The field of sensing and imaging agents is growing fast, largely driven by the development of
optical techniques. In particular, the method based on fluorescent probe has gained immense attention
due to its high sensitivity, operational simplicity and non-destructive analysis, et al [11-15].
Considerable efforts have been devoted to develop fluorescent probes for thiophenols. Some of them
can discriminate thiophenols from aliphatic thiols. However, among these reported fluorescent
thiophenols probes, some still have several drawbacks including relatively weak fluorescence intensity
and low sensitivity [16-22]. Furthermore, it can be seen from these reports that biothiols, such as Cys,
GSH, and Hcy, which have similar physical and chemical properties with thiophenols, often interfere
with the detection of thiophenols [23-25]. Due to the high level of biothiols in most cells and tissues
(
the intracellular concentration for Cys: 30-200 µM [26]; for GSH: 1-10 mM [27]), it is crucial to
adjust the reactivity of the recognition unit in development of thiophenols selective probes for in vivo
imaging.
A variety of thiophenols fluorescent probes have been developed by utilizing the high nucleophilic
reactivity of thiol groups [28-35]. In many of these probes strongly electro-withdrawing 2,
4
-dinitrobenzene-sulfonyl (DNBS) group has often been used as the recognition unit by getting
attached with the fluorophore [32-35]. Aliphatic thiols are often capable of detaching the DNBS moiety
partially from the fluorophore due to the high lability of DNBS toward -SH group, making
discrimination of thiophenols from aliphatic thiols difficult. Protection of the 10-hydroxyl of coumarin
6
H (HOCOU) with DNBS produced DNBS-HOCOU which showed poor selectivity between
thiophenols and Cys (I/Icys = 1.33) (Figure 1). We envisioned that the introduction of 2, 4-dinitrophenyl
DNP) group with reduced reactivity would be more effective in obtaining higher selectivity towards
(
thiophenols. By masking the HOCOU with DNP group, DNCOU was generated. As expected, there
was almost no fluorescence in the solution of DNCOU after treatment with Cys. In contrast the
solution of DNCOU had a marked fluorescence enhancement (I/Icys = 59) after the addition of
thiophenols (Figure 1). Herein, DNCOU has a promising prospect for highly selective detection of
thiophenols. Encouraged by this observation, we further investigated its optical response towards other
aliphatic thiols, nucleophilic anions, amino acids as well as relevant species. The results suggested that
2

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the probe possesses impressively high selectivity towards thiophenols, with low detection limit of 36
nM. Furthermore, DNCOU has been successfully applied for the quantitative detection of thiophenols
in real water samples and fluorescent imaging of thiophenols in living cells.
<
Insert Figure 1>
2
. Experimental
2
.1 Materials and apparatus
All analytical grade chemicals and reagents were purchased from commercial suppliers, and were
used without further purification. Reaction was monitored by thin-layer chromatography (TLC) silica
gel plates (GF-254). Column chromatography was performed on silica gel (200-300 mesh). Absorption
spectra were detected on a T6 New century spectrometer. Fluorescent emission spectra were collected
on a Lengguang F97Pro Fluorospectrophotometer equipped with a xenon lamp as the excitation source.
1
13
The H and C NMR spectra were measured on a Bruker AVANCE II 400 spectrometer using
tetramethylsilane (TMS; δ = 0 ppm) as internal standard. The ESI mass spectra were measured on an
Agilent-6510-Q-TOF spectrometer. All pH measurements were made with a Model PHS-3C pH meter
(
Shanghai, China). The fluorescence images were acquired through Olympus FV1000 laser scanning
microscope.
2
.2 Synthesis of intermediates and DNCOU
The synthesis route of DNCOU is shown in Scheme 1. Compound 1, 2, 3 and 4 were synthesized
according to the reported procedures [36-37], and the detailed data were presented in the Supporting
Information.
<
Insert Scheme 1>
2
.2.1 Synthesis of HOCOU
A suspension of compound 4 (300.0 mg, 1.17 mmol) in 5 mL of aqueous 1M hydrochloric acid
was refluxed under stirring for 4 hours. After cooled and neutralized with ammonium hydroxide (25%),
the reaction mixture was extracted with ethyl acetate. The organic layer was dried with anhydrous
Na SO and evaporated to dryness. The crude product was purified by silica gel chromatography to
2
4
1
give a yellow-green solid (263.6 mg, yield: 87.6%). H NMR (400 MHz, CDCl ): δ 6.92 (s, 1 H), 6.82
3
13
(
s, 1 H), 5.76 (s, 1H), 3.21-3.25 (m, 4 H), 2.78-2.92 (m, 4 H), 2.01-2.05 (m, 4 H). C NMR (100 MHz,
CDCl ): δ 160.79, 146.50, 142.92, 135.46, 123.16, 119.02, 115.78, 108.51, 107.15, 49.49, 49.11, 26.94,
3
+
+
2
1.15, 20.31, 20.02. HRMS (ESI) (C H NO ) m/z: calculated for [M+H] : 258.1130. Found [M+H] :
15 15 3
3

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2
58.1116.
2
.2.2 Synthesis of DNCOU
HOCOU (50.0 mg, 0.2 mmol) was dissolved in dry CH CN (2 mL). 1-Fluoro-2, 4-dinitrobenzene
3
(
55.8 mg, 0.3 mmol) and Et N (30 ꢀL, 0.2 mmol) were added to the solution. The resultant solution
3
was further stirred at room temperature for 6 hours. After the reaction was completed, the mixture was
washed by water and dried with anhydrous Na SO . After removing the solvent under reduced pressure,
2
4
the residue was purified by column chromatography to give the title compound as a bright red solid
1
(
69.3 mg, yield: 80.7 %). H NMR (400 MHz, CDCl ): δ 8.86 (d, J = 2.8 Hz, 1 H), 8.32 (dd, J = 2.4 Hz,
3
J = 9.2. Hz, 1 H), 7.53 (s, 1 H), 7.11 (d, J = 9.2 Hz, 1 H), 6.88 (s, 1 H), 3.29-3.33 (m, 4 H), 2.77-2.90
1
3
(
m, 4 H), 1.99-2.09 (m, 4 H). C NMR (100 MHz, CDCl ): δ 157.13, 155.44, 150.25, 146.30, 141.65,
3
1
2
2
38.64, 133.16, 131.39, 128.69, 125.10, 122.25, 119.76, 117.64, 106.90, 50.05, 49.65, 27.53, 21.25,
+
+
0.36. HRMS (ESI) (C H N O ) m/z: calculated for [M+H] : 424.1145. Found [M+H] : 424.1139.
2
1
17
3
7
.3 Preparation of solutions of DNCOU and analytes
Stock solution of DNCOU (1 mM) was prepared in analytical grade CH CN. Stock solutions of
3
PhSH, p-NH -PhSH, p-CH O-PhSH, CH CH SH, HOOCCH SH and PhOH were prepared in CH CN
2
3
3
2
2
3
(
10 mM, respectively). Other analytes including phenylalanine (Phe), lysine (Lys), serine (Ser),
proline (Pro), alanine (Ala), glycine (Gly), cysteine (Cys), glutathione (GSH), homocysteine (Hcy),
KF, NaCl, KBr, KI, Na CO , AcONa, NaNO , Na S, Na SO , Na SO , KSCN, Na S O , Na S O ,
2
3
3
2
2
3
2
4
2
2
3
2
2
4
NaNO , Co(NO ) , Fe(NO ) , Al(NO ) , Zn(NO ) , Ca(NO ) , Mg(NO ) , and H O were dissolved in
2
3 3
3 3
3 3
3 2
3 2
3 2
2
2
deionized water to afford 10 mM aqueous solution. The stock solutions were used freshly and were
diluted to desired concentrations when needed.
2
.4 Measurements of fluorescence changes of DNCOU upon addition of various analytes
The hybrid solution of various analytes and DNCOU was prepared by adding 100 ꢀL of DNCOU
stock solution and an appropriate volume of each testing species to a 10 mL volumetric flask. PBS
buffer (10 mM, pH 7.4) containing 30% CH CN was added to fill the volumetric flask. All spectra were
3
recorded at room temperature (λ = 395 nm). The incubation time was 50 min.
ex
2
.5 Cell viability study and fluorescence imaging
HepG2 cells were cultured in MEM (minimum essential medium) supplemented with 10% FBS
(
fetal bovine serum). The cells were digested with 0.25% Trypsin-EDTA when their confluence reached
4
9
0% and collected. After resuspended to 5 × 10 cells/mL, 100 ꢀL of cells suspension was added to
4

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each well of 96-well plate and incubated overnight. Cells were treated with DNCOU (10 ꢀM) for 4 h
and 6 h, respectively. 100 ꢀL of CCK8 was added to each well and incubated at 37°C for 30 min. The
optical absorbance was measured at 450 nm. All the tests were repeated in at least ten independent
experiments.
HepG2 cells were incubated with probe DNCOU (10 ꢀM) for 30 min at 37 °C, washed three
times with PBS buffer, and then imaged. Meanwhile, another portion of the cells were pretreated with
PhSH (10 ꢀM) for 15 min under the same conditions, washed three times with PBS buffer, and then
incubated with DNCOU (10 ꢀM) for 30 min. After incubation, the cells were again washed with PBS
buffer three times and the fluorescence images were obtained using Olympus FV1000 laser scanning
microscope.
3
. Results and discussion
3
.1 Design and synthesis of the probe
In this probe, we chose HOCOU as the fluorophore, which showed good photostability, excellent
solubility, large Stokes shift and high fluorescence intensity. The probe DNCOU was constituted by
masking the free hydroxyl group of HOCOU with DNP, which was chosen as the trigger group for
thiophenols owing to its strong electron withdrawing property. We envisioned that the ether linkage of
DNCOU could be cleaved by thiophenols (pK = 6.5), but not by aliphatic thiols (pK = 8.5) under
a
a
physiological pH. Meanwhile, DNP can fully quench the fluorescence of HOCOU to reduce
background interference. The synthesis of DNCOU is outlined in Scheme 1. The chemical structure of
1
13
the compounds was confirmed by H NMR, C NMR and HRMS spectra (see Supporting
Information).
3
.2 Influences of pH on fluorescence detection of thiophenols
pH of the system is a factor, which can sometimes significantly influence the fluorescence and
molecule interactions. To check the effect of pH on the fluorescence response of DNCOU to
thiophenols, the sensing reaction was done at various pH (2.0-12.0). As shown in Figure 2,
fluorescence intensity changes of DNCOU (10 ꢀM) in the absence and presence of 200 ꢀM PhSH were
examined at different pHs. The fluorescent intensity of DNCOU (10 ꢀM) itself was unaffected over a
wide pH range (2.0-12.0) which indicated that the probe was quite stable. When pH was between 2.0
and 5.0, the solution of DNCOU exhibited very weak response toward PhSH. Similar results were also
observed when pH exceeded 10.0. Fortunately, DNCOU displayed strong fluorescence enhancement in
5

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the presence of PhSH in the pH range of 6-9, which suggested that it can function well under
physiological conditions.
<
Insert Figure 2>
3
.3 UV–vis absorption and fluorescence spectra
The absorption and emission spectra of DNCOU and reference HOCOU in PBS buffer (10 mM,
pH 7.4) containing 30% CH CN at room temperature were shown in Figure 3. DNCOU exhibited an
3
absorption peak with maximum at 433 nm, which had a 42 nm redshift in comparison with that of
HOCOU. Free DNCOU displayed almost no fluorescence with a quantum yield (Φ) of 0.022 (see
Supporting Information), in sharp contrast to the much higher fluorescence of reference HOCOU (Φ =
0
.296) at 519 nm (Stokes shift = 128 nm). The drastic difference in the emission intensity between
DNCOU and reference HOCOU suggested that DNCOU may be used as a fluorescent turn-on probe
for thiophenols.
<
Insert Figure 3>
3
.4 Time-dependent fluorescence intensity of DNCOU with thiophenols
In order to determine the reaction time for detecting thiophenols with DNCOU, time-dependent
fluorescence experiments were conducted. As shown in Figure 4, various concentrations of PhSH were
incubated with the DNCOU (10 ꢀM) solution and the fluorescence were collected every 2 minutes.
Upon the addition of PhSH (20 equiv.), a strong fluorescence signal was initiated within 2 min (9-fold
enhancement) and reached a stable intensity plateau within 40 min (about 59-fold enhancement). The
increase tendency became slower while treated with the lower concentration of PhSH. Thus, the
detection time point after the addition of thiophenols was selected to be 50 min in the subsequent
experiments.
<
Insert Figure 4>
3
.5 The selectivity and competition of DNCOU for thiophenols
To investigate the selectivity, the probe DNCOU (10 ꢀM) was treated with various species. As
shown in Figure 5, there was no noticeable changes in the emission spectra upon addition of analytes
1.0 mM for GSH; 500 uM for other species), including various anions and metal ions (KF, NaCl, KBr,
KI, Na CO , AcONa, NaNO , NaNO , Co(NO ) , Fe(NO ) , Al(NO ) ), Zn(NO ) , Ca(NO ) ,
(
2
3
3
2
3 3
3 3
3 3
3 2
3 2
Mg(NO ) ,), sulfur species (Na S, Na SO , Na SO , KSCN, Na S O , Na S O ), aliphatic thiols
3
2
2
2
3
2
4
2
2
3
2
2
4
(
CH CH SH, HOOCCH SH, Hcy, Cys, GSH), amino acids (Phe, Lys, Ser, Pro, Ala, Gly), and other
3 2 2
6

ACCEPTED MANUSCRIPT
tested species (H O , PhOH). By contrast, the introduction of thiophenols (200 ꢀM) such as PhSH,
2
2
p-NH -PhSH, and p-CH O-PhSH to the solutions of DNCOU induced a significant enhancement in the
2
3
fluorescent intensity at 519 nm. These data clearly demonstrated that DNCOU is a highly selective
fluorescent probe for discrimination of thiophenols over aliphatic thiols and other relevant species.
<
<
Insert Figure 5>
Insert Figure 6>
To detect thiophenols in an accurate manner, the fluorescence enhancement should not be
interfered by other chemical species. To address this question, competition experiments were carried
out. After the DNCOU (10 µM) solution was pre-treated with competing analyte (1.0 mM for GSH;
5
00 uM for other species), PhSH (final concentration 200 ꢀM) was added. As shown in Figure 6, all the
relevant analytes tested didn’t have influence on the fluorescence intensity, demonstrating the excellent
anti-interference ability of DNCOU for thiophenols recognition.
3
.6 Quantitative responses of DNCOU to thiophenols
To study the sensitivity of DNCOU for detecting thiophenols, series concentrations of PhSH
ranging from 0 to 250 µM were incubated with DNCOU and the emission spectra were recorded 50
min after each input. As shown in Figure 7a the fluorescence intensity increased gradually with the
increasing PhSH concentration. The maximum enhancement of fluorescence intensity was about
5
9-fold in the presence of PhSH. Furthermore, the fluorescence intensity at 519 nm was proportional to
the concentration of PhSH with the linear range from 0 to 50 ꢀM (Figure 7b). From the linear equation
of spectra titration at 519 nm, the detection limit of DNCOU for PhSH was calculated to be 36 nM
based on 3σ/slope method (see Supporting Information).
<
Insert Figure 7>
3
.7 Reaction mechanism
We reasoned that thiophenols could selectively cleave dinitrobenzene moiety in DNCOU via a
SNAr process to generate dye HOCOU which is highly fluorescent upon excited (Scheme 2). To find
evidence supporting our proposed mechanism, we compared the fluorescence spectra of DNCOU
solution (10 ꢀM) incubated with 200 ꢀM PhSH and the solution of HOCOU (10 ꢀM). As shown in
Figure 8, the two solutions exhibited not only the same maximum fluorescence emission wavelength,
but also similar fluorescent intensity at 519 nm. In addition, both the fluorescent product and the
7

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non-fluorescent product (compound 5) of the sensing reaction were successfully isolated by column
1
chromatography. H NMR spectral analysis of the isolated fluorescent product was carried out, as
1
shown in Figure 9. As expected, the H NMR spectrum of the isolated fluorescent product is
1
13
consistent with that of HOCOU. Furthermore, the H NMR and C NMR spectra of non-fluorescent
product (compound 5) are in conformity with the previous research (see Supporting Information).
These results are indicative of the cleavage of the DNP group and the production of fluorophore
HOCOU in the sensing reaction.
<
<
<
Insert Scheme 2>
Insert Figure 8>
Insert Figure 9>
3
.8 Measurements of thiophenols in water samples
To assess the utility of DNCOU, we applied it to the determination of thiophenols in real
environmental samples in a standard addition method [22, 38]. The crude water samples from the
Heihu Spring and Lake of Yuanboyuan in Jinan City were passed through a microfiltration membrane
and then adjusted to pH 7.4 PBS buffer (10 mM) before use. Aliquots (7 mL) water samples were taken
and then spiked with different concentrations of PhSH that had been accurately prepared. The resulting
samples were then treated with DNCOU in CH CN (3 mL). The final mixtures (10 mL) contained
3
DNCOU (10 µM) and PhSH (0, 2, 3, 5, 8, 10, 15 µM). The solution was incubated for 50 min at room
temperature, and the fluorescence was measured at 519 nm. From the results in Table 1, we can see that
thiophenols in real water samples could be accurately measured with good recovery (from 99.5% to
1
05.5%). This result shows that DNCOU has potential application for quantitative detection of
thiophenols in water samples.
<
Insert Table 1>
3
.9 Cell imaging
Inspired by the above performance in aqueous media, we sought out to investigate the ability of
DNCOU for visualizing thiophenols in living cells. It’s known that fluorescent probe with low
cytotoxicity was more desirable for fluorescence detection in living cells. Thus, we performed CCK8
assays on HepG2 cells to evaluate the cytotoxicity of DNCOU. The results indicated that this probe
had low cytotoxicity for living cells (see Supporting Information).
To explore the practical application in biological systems, the potential of DNCOU for imaging
8

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thiophenols in living cells was investigated next. Cell images were obtained using a confocal
fluorescence microscope. When HepG2 cells were incubated with DNCOU (10 µM) for 30 min at
3
7 °C, only slight fluorescence was observed (Figure 10 A). However, when HepG2 cells were
pre-incubated with 10 µM PhSH for 15 min before incubating with 10 µM DNCOU under the same
conditions, there was remarkable intracellular fluorescence (Figure 10 B). An overlay of fluorescence
and bright field images showed that the fluorescence signals were localized in the intracellular area,
indicating that DNCOU has good cell membrane permeability and is able to provide information on the
intracellular existence of thiophenols
<
Insert Figure 10>
4
Conclusions
In conclusion, we presented the design and synthesis of the 2, 4-dinitrophenyl ether of 10-hydroxy
coumarin 6H derivative, DNCOU, as a turn-on fluorescent probe for the detection of thiophenols. This
probe exhibited a high selectivity toward thiophenols against aliphatic thiols and other biologically
relevant species with a detection limit of 36 nM. This probe featured a 59-fold fluorescence
enhancement with a 128 nm large Stokes shift in response to thiophenols. DNCOU has been
successfully applied for quantitative detection of thiophenols in real water samples. Furthermore, we
demonstrated that this probe was useful for sensing thiophenols in living cells. All these features show
that DNCOU is promising for practical application in environmental and biological systems.
Acknowledgement
This work was supported by Program for Scientific Research Innovation Team in Colleges and
Universities of Shandong Province.
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Table 1 Determination of thiophenols concentrations in real water samples.
Samples
Thiophenols spiked (M)
0
Thiophenols recovered (M)
not detected
Recovery (%)
Heihu Spring water
-
-
-
-
6
6
6
6
-6
2
3
5
8
×10
×10
×10
×10
(1.99 ± 0.08) ×10
99.5
102.3
102.8
101.4
98.1
-6
(3.07 ± 0.12) ×10
-6
(5.14 ± 0.11) ×10
-6
(8.11 ± 0.15) ×10
-6
-6
1
1
0×10
5×10
0
(9.81 ± 0.09) ×10
-6
-6
(15.60 ± 0.15) ×10
104.6
Yuanboyuan Lake water
not detected
-
-
-
-
6
6
6
6
-6
2
×10
×10
×10
×10
(2.11 ± 0.05) ×10
105.5
99.7
-6
3
5
8
(2.99 ± 0.09) ×10
-6
(5.03 ± 0.08) ×10
100.6
101.6
101.1
102.4
-6
(8.13 ± 0.12) ×10
-6
-6
1
1
0×10
5×10
(10.11 ± 0.11) ×10
-6
-6
(15.36 ± 0.13) ×10

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Figures and Schemes
Figure 1 Fluorescence intensity of DNBS-HOCOU (10 µM) and DNCOU (10 µM) after treatment
with thiophenols (200 ꢀM) or Cys (200 ꢀM) for 50 min in PBS buffer (pH = 7.4, 10 mM) solutions
containing 30% CH CN. I and I
are the integrated fluorescence intensities after addition of
Cys
3
thiophenols (200 ꢀM) and Cys (200 ꢀM).
Figure 2 Fluorescence responses of DNCOU (10 ꢀM) in the absence and presence of 200 ꢀM PhSH in
1
0 mM PBS buffer solutions containing 30% CH CN as a co-solvent at different pHs.
3
Figure 3 The absorption (▼) and emission (●) spectra of HOCOU and DNCOU in PBS buffer (10.0
mM, pH = 7.4) containing 30% CH CN.
3
Figure 4 Time-dependent fluorescence intensity changes of DNCOU (10 ꢀM) at 519 nm upon addition
of varied concentrations of PhSH in PBS buffer (10.0 mM, pH = 7.4) containing 30% CH CN at room
3
temperature.
Figure 5 Fluorescence spectra changes of DNCOU (10 µM) towards 200 µM thiophenol analogues
(
PhSH, p-NH -PhSH, p-CH O-PhSH) and other common analytes (1.0 mM for GSH; 500 uM for other
2 3
species) in PBS buffer (pH = 7.4, 10 mM) solutions containing 30% CH CN as a co-solvent.
3
Figure 6 Fluorescence response of DNCOU (10 µM) at 519 nm toward thiophenols (200 ꢀM) in the
presence of other common analytes (1.0 mM for GSH; 500 uM for other species) in PBS buffer (pH =
7
.4, 10 mM) containing 30% CH CN. Black bar represents the fluorescence intensity of only a single
3
analyte with DNCOU. Red bar represents the fluorescence intensity of a mixture of analyte and PhSH
with DNCOU. [Legend: (1) DNCOU only, (2) KF, (3) NaCl, (4) KBr, (5) KI, (6) Na CO , (7) AcONa,
2
3
(
(
8) NaNO , (9) Na S, (10) Na SO , (11) Na SO , (12) KSCN, (13) Na S O , (14) Na S O , (15) NaNO ,
3 2 2 3 2 4 2 2 3 2 2 4 2
16) Co(NO ) , (17) Fe(NO ) , (18) Al(NO ) , (19) Zn(NO ) , (20) Ca(NO ) , (21) Mg(NO ) , (22)
3
3
3 3
3 3
3 2
3 2
3 2
H O , (23) PhOH, (24) HOOCCH SH, (25) CH CH SH, (26) Phe, (27) Lys, (28) Ser, (29) Pro, (30) Ala,
2
2
2
3
2
1

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(
31) Gly, (32) Cys, (33) GSH, (34) Hcy, (35) PhSH, (36) p-CH O-PhSH, (37) p-NH -PhSH]
3
2
Figure 7 (A) Fluorescent spectra changes of DNCOU (10 ꢀM) upon addition of different
concentrations of PhSH (0-250 ꢀM). Inset: The fluorescence emission color changes of DNCOU after
treatment with thiophenols, photo a (10 ꢀM DNCOU) and photo b (10 ꢀM DNCOU with 200 ꢀM
PhSH) were taken under a UV lamp with λ = 365 nm. (B) Fluorescence intensity changes of DNCOU
ex
at 519 nm against PhSH concentrations from 0 ꢀM to 250 ꢀM. Inset: The linear relationship between
fluorescent intensity and PhSH concentration (0-50 ꢀM).
Figure 8 Fluorescence response of 10 ꢀM HOCOU (a), 10 ꢀM DNCOU with 200 ꢀM PhSH (b), and
1
0 ꢀM DNCOU (c) in 10 mM PBS buffer solutions containing 30% CH CN.
3
1
Figure 9 H NMR spectra of the isolated fluorescent product of DNCOU with PhSH (a), HOCOU (b),
and DNCOU (c) in CDCl3.
Figure 10 Confocal fluorescence imagines of HepG2 cells: (a) Bright field, (b) fluorescence, and (c)
overlay image of cells incubated with only DNCOU (10 µM) for 15 min. (d) Bright field, (e)
fluorescence, and (f) overlay image of cells pre-treated with PhSH (10 µM) for 15 min, then incubated
with DNCOU (10 µM) for 15 min.
Scheme 1 The synthesis route of DNCOU.
Scheme 2 Diagram of the reaction mechanism for thiophenols detection.
2

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Figure 1
Figure 2
3

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Figure 3
Figure 4
4

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Figure 5
Figure 6
5

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Figure 7
Figure 8
6

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Figure 9
Figure 10
7

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Scheme 1
CHO
OH
H
2
N
OH
Cl
Br
POCl3
DMF
N
OH
N
NaHCO , DMF
3
1
2
NO
2
NH2
O
O
2
N
COOEt
SnCl
2
2
2H O
N
O
O
N
O
n-BuOH
37% HCl
3
4
NO
2
O N
2
NO2
OH
O
O
NO
2
HCl
F
N
O
O
reflux
Et N, CH CN
N
O
3
3
HOCOU
DNCOU
Scheme 2
8

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Highlights:
ꢀ
A new turn-on fluorescent probe was exploited with a large Stokes shift (128 nm).
The probe showed high selectivity toward thiophenols with detection limit of 36
nM.
ꢀ
ꢀ
ꢀ
The probe was applied for detection of thiophenols in real water samples.
The probe can be used in imaging thiophenols in living cells.