Two chemodosimeters for fluorescence recognition of biothiols in aqueous solution and their bioimaging application

Article abstract of DOI:10.1016/j.tet.2019.130477

Two fluorescein derivatives containing 2,4-dinitrobenzenesulfonyl group have been developed as fluorescent probes to detect the biothiols (Cys, Hcy and GSH) in aqueous solution. Probes 1 and 2 can distinguish these biothiols in the presence of other amino

Full text of DOI:10.1016/j.tet.2019.130477

Accepted Manuscript
Two chemodosimeters for fluorescence recognition of biothiols in aqueous solution
and their bioimaging application
Wei Du, Rui-Juan Liu, Jianguo Fang, Hong Gao, Ya-Wen Wang, Yu Peng
PII:
S0040-4020(19)30801-4
DOI:
https://doi.org/10.1016/j.tet.2019.130477
Article Number: 130477
Reference:
TET 130477
To appear in: Tetrahedron
Received Date: 20 June 2019
Revised Date: 22 July 2019
Accepted Date: 26 July 2019
Please cite this article as: Du W, Liu R-J, Fang J, Gao H, Wang Y-W, Peng Y, Two chemodosimeters for
fluorescence recognition of biothiols in aqueous solution and their bioimaging application, Tetrahedron
(2019), doi: https://doi.org/10.1016/j.tet.2019.130477.
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Two chemodosimeters for fluorescence
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Wei Du, Rui-Juan Liu, Jianguo Fang, Hong Gao, Ya-Wen Wang, and Yu Peng *

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Tetrahedron
journal homepage: www.elsevier.com
Two chemodosimeters for fluorescence recognition of biothiols in aqueous solution
and their bioimaging application
Wei Du §, Rui-Juan Liu §, Jianguo Fang §, Hong Gao §, Ya-Wen Wang †, and Yu Peng †, §,
*
† School of Life Science and Engineering, Southwest Jiaotong University, Chengdu 610031, China.
§ State Key Laboratory of Applied Organic Chemistry, Lanzhou University, Lanzhou 730000, China.
ARTICLE INFO
ABSTRACT
Article history:
Received
Received in revised form
Accepted
Two fluorescein derivatives containing 2,4-dinitrobenzenesulfonyl group have been developed
as fluorescent probes to detect the biothiols (Cys, Hcy and GSH) in aqueous solution. Probes 1
and 2 can distinguish these biothiols in the presence of other amino acids. While probe 1 can
recognize the biothiols in PBS/DMSO (v:v = 95:5, pH = 7.40) solution, notably probe 2 could be
used in PBS buffer solution (pH = 7.40). The detection limit of Cys for probe 2 reached at 0.021
ꢀM in aqueous solution, which was lower than the intracellular concentration of Cys. In the
recognition process, a reaction between the probes and the biothiols occurred, in which the S−O
Available online
Keywords:
Fluorescence
1
bond was cleaved to remove 2, 4-dinitrobenzenesulfonyl group. The data of H NMR, MS and
Chemodosimeter
Biothiols
DFT/TD-DFT calculation further confirmed the detection mechanism. Moreover, two probes
were successfully applied to the HeLa cell imaging.
2, 4-Dinitrobenzenesulfonyl group
Fluorescein derivatives
2009 Elsevier Ltd. All rights reserved
.
had been used for detecting biothiols,14 because these probes can
react with biothiols specifically. But these probes were rarely
tested in pure water, most of them used mixed water-organic
solution instead.15–17 And the short excitation wavelength was
usually used, resulting in the difficulty of detecting biothiols in
vivo, because the wavelength used is easy to damage cells.17
Therefore, the design of highly selective probe for biothiols with
long excitation wavelength in aqueous solution is still in high
demand.
In connection with our continuing research of fluorescent
probes for the detection of biologically and environmentally
important species,18–21 herein we use fluorescein group as the
skeleton and 2,4-dinitrobenzenesulfonyl group as a switch to
obtain two “turn-on” fluorescent probes. Both of probes have
high selectivity for biothiols with excitation at 491 nm in aqueous
solution.
1. Introduction
Biothiols, especially cysteine (Cys), homocysteine (Hcy) and
glutathione (GSH) are important amino acids and play vital
function in vivo. The transport and metabolism of sulfur
compounds in organism are associated with proteins and
enzymes. Abnormal levels of sulfur species in biological systems
are thus related to some diseases. For example, Cys can prevent
and treat radiation damage, prevent senescence, strengthen and
regulate the defense ability of the organism. Lack of Cys can
result in liver damage and slow growth. Abnormal levels of Hcy
may directly or indirectly cause vascular endothelial cell damage,
promote thrombosis, eventually resulting in coronary heart
disease or other cardiovascular and cerebrovascular diseases.1–3
GSH can be combined with free radicals and heavy metals
therefore converting toxic substances into non-toxic substances
and discharging them. GSH can also reduce the prevalence of
cancer, heart disease, diabetes and other chronic diseases.4,5 Due
to the important biochemical properties of biothiols, there were a
lot of methods for detecting biothiols including electrochemical
analysis,6,7 high performance liquid chromatography (HPLC),8
optical technology9 and so on. Among them, fluorescent
technology is especially valued for its high sensitivity and simple
operation.
2. Results and discussion
The probes 1 and 2 were synthesized (Schemes 1 and 2) and
the corresponding compounds were characterized by 1H, 13C
NMR and mass spectra (Figures S1−S12). In order to determine
the fluorescence properties of these probes in different solvents,
DMF, DMSO, THF, Acetone, MeOH, EtOH, CH3CN and EtOAc
were used to study. As shown in Figure S13, when 1 or 2 was
excited at 491 nm, there was weak fluorescence in different
solvents with an emission band of 524 nm or 519 nm. Upon the
addition of Cys to the solution of probes, the fluorescence
Recently, some fluorescent probes were designed and used for
the detection of biothiols.10–13 Among these fluorescent probes, a
chemodosimeter bearing the 2, 4-dinitrobenzenesulfonyl group
———
Corresponding author. E-mail address: ywwang@swjtu.edu.cn (Y.-W. Wang); pengyu@lzu.edu.cn (Y. Peng)

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enhancement was observed. The solution of 1 has the strongest
biologically relevant cations were detected (Figure S15). To
fluorescence in DMSO (Figure S13a). Considering the practical
application, the fluorescence properties of 1 in DMSO with
different proportions of H2O were further examined. The DMSO-
water (5:95, v/v) solution system was selected to study the
fluorescence properties of 1 with Cys in detail. After the addition
of Cys to the solution of 2, the enhancement of fluorescence in
water was obvious (Figure S13b). These results indicated both of
probes could be used to detect Cys in aqueous solution.
The pH effect on the fluorescence intensity changes of probes
and probes + Cys system were shown in Figure S14. The
fluorescence of 1 or 2 was stable in the pH range of 3.00 to
10.00. The fluorescence intensities of probes with Cys exhibited
dramatically enhancement between pH 7.0 and 10.00, which
suggested Cys could react with these two probes.22 In subsequent
experiments, a PBS buffer solution of pH 7.40 was thus used.
validate the selectivity of probes 1 and 2 in practice, the
competition experiments were also measured by the addition of
6.25 equiv of biothiols to the aqueous solutions in the presence of
6.25 equiv of other amino acids as shown in Figures 1c and 1d.
All competitive amino acids had no obvious interference with the
detection of biothiols. These results indicated that both of probes
displayed an excellent selectivity toward biothiols and the system
of probe + biothiols was also hardly affected by these coexistent
amino acids in aqueous solution.
Figure 2. Fluorescent spectra of (a) probe 1 and (b) probe 2
([probe] = 10.0 ꢀM) in the presence of different concentrations of
Cys (0−12.5 equiv).
The fluorescence intensity of 1 was enhanced 52-fold for Cys,
26-fold for Hcy, and 43-fold for GSH in DMSO−PBS (5:95, v/v,
pH = 7.40) solution, respectively, when 12.5 equiv of biothiols
was present (Figures 2a, S16a and S16b). And the corresponding
detection limit25 was found to be 0.052 ꢀM for Cys, 0.059 ꢀM for
Hcy, and 0.065 ꢀM for GSH (Figures S17a, S17b, and S17c),
respectively. After the addition of 12.5 equiv of biothiols to the
solution of 2, the fluorescence intensity was enhanced 43-fold for
Cys, 18-fold for Hcy, and 24-fold for GSH in PBS buffer (pH =
7.40) solution, respectively (Figures 2b, S16c and S16d). The
corresponding detection limit was found to be 0.021 ꢀM for Cys,
0.023 ꢀM for Hcy, and 0.016 ꢀM for GSH, respectively (Figures
S17d, S17e, and S17f).
Figure 1. Fluorescence response of (a) probe 1 in DMSO−PBS
buffer (5: 95 (v/v), pH = 7.40) solution and (b) probe 2 in PBS
buffer (pH = 7.40) solution ([probe] = 10.0 ꢀM, λex = 491 nm)
with different amino acids (62.5 ꢀM) Inset: The photo of probes
with different amino acids under UV light. From left to right:
Cys, Hcy, GSH, Glu, Asn, Leu, Ile, Try, Lys, Pro, Met, Thr, Gly,
The, Arg, Asp, Val, Phe, Gln. Fluorescence responses of (c)
probe 1 in DMSO−PBS buffer (5: 95 (v/v), pH =7.40) and (d)
probe 2 in PBS buffer (pH = 7.40) to various amino acids
([probe] = 10.0 ꢀM, probe 1: λem = 524 nm, probe 2: λem = 519
nm). The black bars represent the emission intensity of probes in
the presence of other amino acids (62.5 ꢀM). The red bars
represent the emission intensity that occurs upon the subsequent
addition of 62.5 ꢀM of Cys to the above solution. From 1 to 18:
GSH, Hcy, Glu, Asn, Leu, Ile, Try, Lys, Pro, Met, Thr, Gly, The,
Arg, Asp, Val, Phe, Gln.
The kinetic studies of the response of 12.5 equiv of Cys, Hcy,
o
and GSH to probes (10.0 ꢀM) in aqueous solution at 37 C were
measured (Figure S18). The pseudo-first-order kinetics
equation26 was used to fit the initial fluorescent intensity changes
of probes. As shown in Figure S18, the observed rate constants
(kobs) were the slopes of the linear functions, which were
calculated to be 0.085 (1 + Cys), 0.18 (1 + Hcy), 0.13 (1 + GSH),
0.12 (2 + Cys), 0.13 (2 + Hcy), and 0.09 (2 + GSH) min-1,
respectively.
In the UV-vis absorption spectra of 1 and 2 (Figure S19), the
absorption bands from 450 to 510 nm were observed only in the
presence of biothiols, and no obvious absorption peaks appeared
with other amino acids. These results suggested ring-opening
structure of the fluorescein derivatives27 were obtained after the
addition of biothiols to the aqueous solution of probes.
As shown in Figure 1, compared to other amino acids
examined, only biothiols caused the fluorescence enhancement of
probes. 1 showed weak fluorescence at 524 nm (Φ = 4.0 %) in
DMSO−PBS (5:95, v/v, pH = 7.40) solution. After the addition
of biothiols to the solution, the emission band at 524 nm was
dramatically increased (Φ = 50.6 % for Cys; 37.6 % for Hcy;
41.4 % for GSH) (Figure 1a). 2 also showed weak fluorescence
at 519 nm (Φ = 7.0 %) in PBS buffer (pH = 7.40) solution. But
the fluorescence intensities were enhanced after the addition of
1
To understand the reaction mechanism, the H NMR spectra
were used to observe the changes of chemical shifts before and
after the addition of Cys to the solution of 1 (Figure 3). In
DMSO-d6/D2O (10/1, v/v) solvent, the chemical shift of the
aldehyde proton (H1) of 1 is at 10.55 ppm, but after the addition
of Cys to the solution the peak disappeared. Meanwhile, the
signals of H2 and H3 protons were observed at about 3.15 and
6.35 ppm, respectively. These results indicated the cyclization
between Cys and the aldehyde group of
1
occurred.28
biothiols to the solution (Φ = 63.9 % for Cys; 51.6 % for Hcy;
Furthermore, the signals of 2, 4-dinitrobenzenesulfonyl group in
1 all disappeared, and the proton signals of quinone form of C1
(about 6.50 ppm) were observed. These results showed that the
reactions occurred between 1 and Cys, resulting in the formation
24
52.5
%
for GSH) (Figure 1b).23,
The corresponding
fluorescence colors of these probes in aqueous solution were
almost nonflurescent, but the colors of probe + biothiols systems
changed to green (Figures 1a and 1b, inset photos). In contrast,
no obvious fluorescence enhancements of two probes were
observed when some anions, reaction oxygen species and some
of
a
five-membered ring and removal of 2, 4-
1
dinitrobenzenesulfonyl groups.28–31 For more clarity, the full H

3
Figure 4. Proposed mechanism of probe 2 with Cys and partial
NMR spectra of Figure 3 and Cys were shown in Figure S20. A
peak at m/z 464.1 was observed in the mass spectrum
corresponding to the compound C1 (Figure S24). The
fluorescence spectrum of C1 in aqueous solution was also
measured (Figure S30a). The emission band at about 524 nm was
in agreement with the fluorescence changes observed in the
fluorescence spectra of 1 upon the addition of Cys. The
corresponding 1H NMR (Figure S22), MS (Figures S25 and S26),
and fluorescence spectra (Figures S30b and S30c) were carried
out for the compounds C2 and C3 obtained by the addition of Hcy
or GSH to the solution of 1.
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1H NMR spectra in DMSO-d6/D2O (10/1, v/v), (a) probe 2 + Cys
(12.5 equiv.), (b) probe 2 only.
Compared with 1, there was no an aldehyde group in the
structure of 2. Therefore, only the signals of 2, 4-
dinitrobenzenesulfonyl group (H1, about 9.11 ppm) disappeared
in the 1H NMR spectra (Figure 4), indicating the formation of C4.
1
And no cyclization of Cys with 2 was observed. The full H
NMR spectra of Figure 4 and Cys were shown in Figure S21.
The similar reaction occurred between Hcy or GSH and 2 to
provide the same product C4 (Figure S23). In the Mass spectra of
2 with biothiols in aqueous solution (Figures S27−S29), the same
peak at m/z 358.1 was observed, which was assigned to
compound C4. Moreover, the fluorescence spectrum of C4
(Figure S30d) was in agreement with the fluorescence spectra of
2 upon the addition of biothiols.
To better understand the recognition mechanism of probes 1
and 2 for Cys, Hcy and GSH, the Gaussian 09 program was used
in computational studies. We used the B3LYP/6-31G (d, p) basis
set to optimize the structures, and then used DFT/TD-DFT
calculations32 to study the photophysical properties of two probes
(Figures S31-S36).
Figure 3. Proposed mechanism of probe 1 with Cys and partial
1H NMR spectra in DMSO-d6/D2O (10/1, v/v), (a) probe 1 + Cys
(12.5 equiv.), (b) probe 1 only.
Figure 5. Optimized structures, energy levels, and the molecular
orbital plots of probe 1, C1, C2, C3, probe 2, and C4.
As shown in Figure 5, the lack of overlap between the HOMO
and the LUMO, and the small oscillator strength33, 34 (f = 0.0014
for 1, f = 0.0018 for 2) suggested that S1←S0 transition was
forbidden, which implied the two probes were potentially weak
fluorescent. As for compounds C1, C2, C3, and C4, the main
allowed transition was the HOMO orbital to LUMO orbital. The
small oscillator strength of C1, C2, C3, or C4 (f = 0.266 for C1, f =
0.2357 for C2, f = 0.2667 for C3, f = 0.2439 for C4) was higher
than that of the two probes, which means C1, C2, C3, and C4
would probably fluorescence stronger than that of probes. The
above results were in good agreement with turn-on fluorescence
changes observed of probes 1 and 2 after the addition of
biothiols.
Figure 6. HeLa cell images: (a), (f) bright field and dark field imaging of probe 1 (20.0 ꢀM) with Hela cells for 40 min. (b), (g) bright
field and dark field imaging of Hela cells incubated with NEM (500.0 ꢀM) for 30 min, then incubated with probe 1 (20.0 ꢀM) for 30

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min. (c), (d), (e) bright field and (h), (i), (j) dark field imaging of Hela cells incubated with NEM (500.0 ꢀM) for 30 min, then
incubated with Cys, Hcy, and GSH (125.0 ꢀM) for 40 min, and finally incubated with probe 1 (20.0 ꢀM) for 40 min.
Figure 7. HeLa cell images: (a), (f) bright field and dark field imaging of probe 2 (20.0 ꢀM) with Hela cells for 40 min. (b), (g) bright
field and dark field imaging of Hela cells incubated with NEM (500.0 ꢀM) for 30 min, then incubated with probe 2 (20.0 ꢀM) for 30
min. (c), (d), (e) bright field and (h), (i), (j) dark field imaging of Hela cells incubated with NEM (500.0 ꢀM) for 30 min, then incubated
with Cys, Hcy, and GSH (125.0 ꢀM) for 40 min, and finally incubated with probe 2 (20.0 ꢀM) for 40 min.
Finally, probes 1 and 2 were all used in bioimaging study. As
shown in Figures 6 and 7, when these probes (20.0 ꢀM) were
treated with HeLa cells at 37 C, the green fluorescence was
To a solution of monoaldehyde-functionalized fluorescein37
(180 mg, 0.5 mmol) in 10 mL of dry dichloromethane was added
a solution of 2,4-dinitrobenzenesulfonyl chloride (533.2 mg, 2.0
mmol) under argon. The mixture was stirred at room temperature
for 10 min, and then triethylamine (0.2 mL) was added with a
o
observed (Figures 6f and 7f). In a control experiment, NEM (N-
ethylmaleimide) was used as a thiol-blocking reagent35 to
incubate with HeLa cells first, and probes were then added to the
above cells system, resulting in almost nonfluorescent imaging
(Figures 6g and 7g). After adding the three biothiols to the cells,
the strong green fluorescence was observed again (Figures 6h, 6i,
6j, 7h, 7i, and 7j). These results showed that probes 1 and 2 are
cell membrane permeable and could detect Cys, Hcy and GSH in
living cells. After incubated by NEM, Hela cells still showed
weak fluorescence in the presence of probe 1 (Figure 6g).
Compared with Figures 6h, 6i and 6j, the fluorescence was not
significantly different, so the interference of probe 1 itself could
not be excluded. For probe 2, the significant contrasts of
fluorescence between Figure 7g and 7h, 7i or 7j were observed.
These results indicated probe 2 might be suitable for biological
imaging studies. Further, the cytotoxicity effects of two probes
were studied by MTT assay. As shown in Figures S37 and S38,
probes 1 and 2 at low concentration have almost no influence on
the cells.
o
syringe under ice bath. After stirring at 25 C for 10 h, water (10
mL) was added and extracted with dichloromethane (3 × 30 mL).
The combined organic phase was dried by anhydrous Na2SO4.
The solvent was removed by evaporation, and the residue was
purified by flash column chromatography. Petroleum ether and
ethyl acetate (1:1) were used as eluents to obtain probe 1 as
yellow powder (267 mg, 65%).36 1H NMR (DMSO-d6, 300 MHz)
δ = 10.56 (s, 1H), 9.05 (d, J = 2.4 Hz, 1H), 9.04 (d, J = 2.4 Hz,
1H), 8.67 (dd, J = 2.4, 8.8 Hz, 1H), 8.64 (dd, J = 2.4, 8.8 Hz,
1H), 8.41 (d, J = 1.2 Hz, 1H), 8.39 (d, J = 1.6 Hz, 1H), 8.06 (d, J
= 7.6 Hz, 1H), 7.84 (td, J = 7.6, 1.2 Hz, 1H), 7.77 (td, J = 7.6, 1.2
Hz, 1H), 7.57 (d, J = 2.0 Hz, 1H), 7.42 (d, J = 7.6 Hz, 1H), 7.23
(d, J = 8.8 Hz, 1H), 7.05 (dd, J = 2.4, 8.8 Hz, 1H), 7.02 (d, J =
8.8 Hz, 1H), 6.98 (d, J = 8.8 Hz, 1H) ppm. 13C NMR (DMSO-d6,
100 MHz) δ = 187.0, 186.9, 168.6, 152.5, 152.3, 152.1, 152.0,
150.8, 150.0, 148.7, 148.5, 148.3, 136.8, 135.2, 135.1, 134.1,
132.4, 131.6, 131.5, 130.6, 128.2, 125.8, 125.4, 124.6, 121.7,
121.5, 120.4, 119.6, 119.2, 118.1, 111.9, 111.8, 80.0 ppm.
ESI−MS: m/z 820.9 [M+H]+.
3. Conclusion
Two fluorescent chemodosimeters
1 and 2 based on
4.2. Synthesis of probe 2
fluorescein skeleton were successfully synthesized. Both of them
displayed high selectivity for the detection of biothiols (Cys, Hcy
and GSH) over other amino acids in aqueous solution. Upon the
addition of biothiols to the solution of probes, the significant
turn-on fluorescence responses were obtained by using 491 nm as
excited wavelength. The origins of the responses were also
revealed by the experiments of 1H NMR, MS and DFT
calculation. Furthermore, probes 1 and 2 were successfully
applied to the HeLa cell imaging.
OH
N
N
CHO
O
OH
HO
NH2OH HCl
EtOH
O
HO
O
HO
O
OH
TsCl, Et3N
CH3CN
O
O
O
O
O
O
O
1'
2'
Cl
S
N
O
O
NO2
NO2
O
S
NO2
O
O
O
S
O
O
NO2
Et3N, DCM
4. Experimental section
O
O2N
NO2
4.1. Synthesis and characterization of probe 1
O
2
Scheme 2. Synthesis of probe 2.
4.2.1 Synthesis and characterization of compound 1′
To a solution of monoaldehyde-functionalized fluorescein
(720 mg, 2.0 mmol) in dry EtOH (20 mL) was added a solution
of hydroxylamine hydrochloride (206 mg, 3 mmol). After stirring
at room temperature for 10 hours, the solvent was removed by
evaporation. The residue was purified by flash column
Scheme 1. Synthesis of probe 1.

5
2. H. Refsum, P. M. Ueland, O. Nygård, S. E. Vollset, Annu. Rev.
chromatography (DCM/EA=7:1,v:v) to obtain compound 1′ as an
orange-yellow powder (465 mg, 62%).38 1H NMR (DMSO-d6,
300 MHz) δ = 11.80 (s, 1H), 11.03 (s, 1H), 10.07 (s, 1H), 8.86 (s,
1H), 8.00 (d, J = 7.6 Hz, 1H), 7.80 (t, J = 7.6 Hz, 1H), 7.72 (t, J =
7.2 Hz, 1H), 7.29 (d, J = 7.6 Hz, 1H), 6.86 (d, J = 1.4 Hz, 1H),
6.70 (d, J = 9.2 Hz, 1H), 6.64 (d, J = 8.8 Hz, 1H), 6.62-6.56 (m,
2H) ppm. 13C NMR (DMSO-d6，100 MHz) δ = 168.4, 159.4,
158.5, 152.1, 151.2, 148.9, 145.3, 135.4, 130.0, 129.5, 128.6,
126.0, 124.5, 123.8, 113.0, 112.5, 109.9, 109.3, 104.9, 102.5,
82.4 ppm. ESI−MS: m/z 376.1 [M+H]+.
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Med. 49 (1998) 31.
3. C. Harris, J. M. Hansen, Methods Mol. Biol. 889 (2012) 325.
4. J. Vacek, B. Klejdus, J. Petrlová, L. Lojková, V. Kubáň, Analyst
131 (2006), 1167.
5. J. S. Stamler, J. Loscalzo, Anal. Chem. 64 (1992) 779.
6. T. Inoue, J. R. Kirchhoff, Anal. Chem. 74 (2002) 1349.
7. B. Seiwert, U. Karst, Anal. Chem. 79 (2007) 7131.
8. T. Matsumoto, Y. Urano, T. Shoda, H. Kojima, T. Nagano, Org.
Lett. 9 (2007) 3375.
9. X. Chen, S.-K. Ko, M. J. Kim, I. Shin, J. Yoon, Chem. Commun.
46 (2010) 2751.
10. H. Lee, H.-J. Kim, Org. Biomol. Chem. 11 (2013) 5012.
11. H. Lee, D. I. Kim, H. Kwon, H.-J. Kim, Sensor Actuat. B-Chem.
209 (2015) 652.
12. Y. Zhou, J. Yoon, Chem. Soc. Rev. 41 (2012) 52.
13. T.-I. Kim, B. Hwang, B. Lee, J. Bae, Y. Kim, J. Am. Chem. Soc.
140 (2018) 11771.
14. H. Maeda, H. Matsuno, M. Ushida. K. Katayama, K.Saeki. N.
Itoh, Angew. Chem. Int. Ed. 44 (2005) 2922.
15. Y. Yang, H.Wang, Y.-L. Wei, J. Zhou, J.-F. Zhang, Y. Zhou, Chin.
Chem. Lett. 28 (2017) 2023.
16. K.-H. Hong, S.-Y. Lim, M.-Y. Yun, J.-W. Lim, J.-H. Woo, H.
Kwon, H.-J. Kim, Tetrahedron Lett. 54 (2013) 3003.
17. W. Lin, L. Long, L. Yuan, Z. Cao, B. Chen, W. Tan, Org. Lett. 10
(2008) 5577.
18. Z.-H. Fu, X. Han, Y. Shao, J. Fang, Z.-H. Zhang, Y.-W. Wang, Y.
Peng, Anal. Chem. 89 (2017) 1937.
19. Z.-H. Fu, L.-B. Yan, X. Zhang, F.-F. Zhu, X.-L. Han, J. Fang, Y.-
W. Wang, Y. Peng, Org. Biomol. Chem. 15 (2017) 4115.
20. Y.-W. Wang, Y.-X. Hua, H.-H. Wu, X. Sun, Y. Peng, Chin. Chem.
Lett. 28 (2017) 1994.
21. A.-M. Luo, Y. Shao, K.-J. Zhang, Y.-W. Wang, Y. Peng, Chin.
Chem. Lett. 28 (2017) 2009.
22. J. Han, K. Burgess, Chem. Rev. 110 (2010) 2709.
23. K. Komatsu, Y. Urano, H. Kojima, T. Nagano, J. Am. Chem. Soc.
129 (2007) 13447.
24. W. H. Melhuish, J. Phys. Chem. 65 (1961) 229.
25. Y.-Q. Sun, M. Chen, J. Liu, X. Lv, J.-F. Li, W. Guo, Chem.
Commun. 47 (2011) 11029.
26. D. Lee, G. Kim, J. Yin, J. Yoon, Chem. Commun. 51 (2015) 6518.
27. N. Klonis, W. H. Sawyer, J. Fluoresc. 6 (1996) 147.
28. Y.-W. Wang, S.-B. Liu, W.-J. Ling, Y. Peng, Chem. Commun. 52
(2016) 827.
29. J. Zhang, A. Shibata, M. Ito, S. Shuto, Y. Ito, B. Mannervik, H.
Abe, R. Morgenstern, J. Am. Chem. Soc. 133 (2011) 14109.
30. J. Yin, Y. Kwon, D. Kim, D. Lee, G. Kim, Y. Hu, J.-H. Ryu, J.
Yoon, J. Am. Chem. Soc. 136 (2014) 5351.
4.2.2 Synthesis and characterization of compound 2′
To a solution of compound 1′ (200 mg, 0.53 mmol) and p-
toluenesulfonyl chloride (152 mg, 0.8 mmol) in dry acetonitrile
(5 mL) was added a solution of triethylamine (147 ꢀL, 1.06
mmol). After stirring at room temperature for 8 hours, the solvent
was removed by evaporation, and the residue was purified by
flash column chromatography (DCM/EA = 25:1, v:v) to obtain
compound 2′ as a yellow powder (63 mg, 33%).39 1H NMR
(DMSO-d6，300 MHz) δ = 10.33 (s, 1H), 9.68 (s, 1H), 8.18 (d, J
= 7.6 Hz, 1H), 7.92 (t, J = 7.2 Hz, 1H), 7.87 (t, J = 7.6 Hz, 1H),
7.61 (d, J = 8.8 Hz, 1H), 7.43 (d, J = 7.6 Hz, 1H), 7.15 (d, J = 8.8
Hz, 1H), 7.00 (s, 1H), 6.81 (s, 2H) ppm. 13C NMR (DMSO-d6,
100 MHz) δ = 169.0, 164.0, 160.2, 153.3, 151.4, 145.4, 145.1,
136.3, 131.1, 130.8, 129.7, 126.3, 125.3, 124.5, 114.3, 113.5,
111.7, 109.9, 106.5, 103.0, 82.4 ppm. ESI−MS: m/z 358.2
[M+H]+.
4.2.3 Synthesis and characterization of compound 2
The synthesis procedure was similar to that of probe 1. A
yellow powder of probe 2 was obtained (80 mg, 70%).36 1H NMR
(DMSO-d6, 300 MHz) δ = 9.11 (d, J = 2.0 Hz, 1H), 9.05 (d, J =
2.0 Hz, 1H), 8.69 (dd, J = 2.4, 8.8 Hz, 1H), 8.64 (dd, J = 2.0, 8.4
Hz, 1H), 8.47 (d, J = 8.8 Hz, 1H), 8.42 (d, J = 8.4 Hz, 1H), 8.06
(d, J = 7.6 Hz, 1H), 7.83 (td, J = 7.2, 1.2 Hz, 1H), 7.77 (td, J =
7.2, 1.2 Hz, 1H), 7.54 (d, J = 2.4 Hz, 1H), 7.44 (d, J = 7.6 Hz,
1H), 7.34 (d, J = 8.8 Hz, 1H), 7.16 (d, J = 8.8 Hz, 1H), 7.08 (dd,
J = 2.4, 8.8 Hz, 1H), 7.03 (d, J = 8.8 Hz, 1H) ppm. 13C NMR
(DMSO-d6，100 MHz) δ = 167.8, 152.0, 151.7, 151.6, 151.4,
150.3, 149.7, 149.3, 147.9, 136.1, 135.1, 135.0, 133.7, 133.4,
130.9, 130.8, 130.1, 127.8, 127.5, 125.1, 124.5, 124.0, 121.1,
121.0, 119.7, 119.1, 118.7, 118.5, 111.0, 111.0, 110.4, 96.6, 78.8
ppm. ESI−MS: m/z 817.9 [M+H]+.
31. J. Mei, Y. Wang, J. Tong, J. Wang, A. Qin, J. Z. Sun, B. Z. Tang,
Chem. –Eur. J. 19 (2013) 613.
32. M. J. Frisch, G. W. Trucks, H. B. Schlegel, G. E. Scuseria, M. A.
Robb, J. R. Cheeseman, G. Scalmani, V. Barone, B. Mennucci, G.
A. Petersson, H. Nakatsuji, M. Caricato, X. Li, H. P. Hratchian, A.
F. Lzmayloy, J. Bloino, G. Zheng, J. L. Sonnenberg, GAUSSIAN
09 (Revision A.1), Gaussian. Inc., Wallingford CT, 2009.
33. Y.-L. Yang, F.-M. Zhang, Y.-W. Wang, B.-X. Zhang, R. Fang, J.-
G. Fang, Y. Peng, Chem.–Asian J. 10 (2015) 422.
34. X. Zhang, L. Chi, S. Ji, Y. Wu, P. Song, K, Han, H, Guo, T. D.
James, J. Zhao, J. Am. Chem. Soc. 131 (2009) 17452.
35. C. R. Yellaturu, M. Bhanoori, I. Neeli, G. N. Rao, J. Biol.
Chem. 277 (2002) 40148.
36. F. Y. Thanzeel, C. Wolf, Angew. Chem. Int. Ed. 56 (2017) 7276.
37. W. Wang, O. Rusin, X. Xu, K. K. Kim, J. O. Escobedo, S. O.
Fakayode, K. A. Fletcher, M. Lowry, C. N. Schowalter, C. M.
Lawrence, F. R. Fronczek, I. M. Warner, R. M. Strongin, J. Am.
Chem. Soc. 127 (2005) 15949.
Acknowledgments
This work was supported by the National Natural Science
Foundation of China (Nos. 21572091 and 21772078), the
Fundamental Research Funds for the Central Universities (Nos.
2682019CX70 and 2682019CX71). We also thank MSc. Bao-Jun
Wang for his assistance.
Supplementary data
Supplementary data related to this article can be found online at
doi:
38. X. Cheng, H. Jia, T. Long, J. Feng, J. Qin, Z. Li, Chem. Commun.
47 (2011) 11978.
39. H. Lee, H.-J. Kim, Tetrahedron 70 (2014) 2966.
References and notes
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1. S. Seshadri, A. Beiser, J. Selhub, P. F. Jacques, I. H. Rosenberg,
R. B. D'agostino, P. A. Wolf, New Engl. J. Med. 346 (2002) 476.

ACCEPTED MANUSCRIPT
Two fluorescein derivatives containing 2,4-dinitrobenzenesulfonyl group
have been developed as fluorescent probes to detect the biothiols in
aqueous solution, and were further applied to the HeLa cell imaging.