A novel p-aminophenylthio- and cyano- substituted BODIPY as a fluorescence turn-on probe for distinguishing cysteine and homocysteine from glutathione

Article abstract of DOI:10.1016/j.dyepig.2017.09.020

Biothiols such as cysteine (Cys), homocysteine (Hcy), and glutathione (GSH) play vital roles in various physiological and pathological processes. In this work, a BODIPY-based fluorescent probe XCN was synthesized from multi-step reactions. We first synthe

Full text of DOI:10.1016/j.dyepig.2017.09.020

Accepted Manuscript
A novel p-aminophenylthio- and cyano-substituted BODIPY as a fluorescence turn-on
probe for distinguishing cysteine and homocysteine from glutathione
Qing Wang, Xiaodong Wei, Chengjie Li, Yongshu Xie
PII:
S0143-7208(17)31916-2
10.1016/j.dyepig.2017.09.020
DYPI 6244
DOI:
Reference:
To appear in: Dyes and Pigments
Received Date: 14 August 2017
Revised Date: 8 September 2017
Accepted Date: 9 September 2017
Please cite this article as: Wang Q, Wei X, Li C, Xie Y, A novel p-aminophenylthio- and cyano-
substituted BODIPY as a fluorescence turn-on probe for distinguishing cysteine and homocysteine from
glutathione, Dyes and Pigments (2017), doi: 10.1016/j.dyepig.2017.09.020.
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to
our customers we are providing this early version of the manuscript. The manuscript will undergo
copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please
note that during the production process errors may be discovered which could affect the content, and all
legal disclaimers that apply to the journal pertain.

ACCEPTED MANUSCRIPT
Graphical Abstract
Qing Wang, Xiaodong Wei, Chengjie Li,*and Yongshu Xie*
Key Laboratory for Advanced Materials and Institute of Fine Chemicals, School of Chemistry and
Molecular Engineering, East China University of Science and Technology, Meilong Road 130, Shanghai
2
00237, China
*
Corresponding author: E-mail: yshxie@ecust.edu.cn, chengjie.li@ecust.edu.cn

ACCEPTED MANUSCRIPT
A novel p-aminophenylthio- and cyano- substituted BODIPY as a
fluorescence turn-on probe for distinguishing cysteine and
homocysteine from glutathione
1
2
3
4
Qing Wang, Xiaodong Wei, Chengjie Li,* and Yongshu Xie*
5
6
7
Key Laboratory for Advanced Materials and Institute of Fine Chemicals, School of Chemistry and Molecular Engineering,
East China University of Science and Technology, Meilong Road 130, Shanghai 200237, China
* Corresponding author: E-mail: yshxie@ecust.edu.cn, chengjie.li@ecust.edu.cn
8
9
0
1
2
3
4
5
6
7
8
9
0
Abstract: Biothiols such as cysteine (Cys), homocysteine (Hcy), and glutathione (GSH)
play vital roles in various physiological and pathological processes. In this work, a
BODIPY-based fluorescent probe XCN was synthesized from multi-step reactions. We first
synthesized a BODIPY derivative with a cyano and a bromine moiety attached to the
8-diphenylaminophenyl substituent of BODIPY, followed by the reaction with
p-aminothiophenol under basic condition. Interestingly, compound XCN was successfully
obtained with the p-aminophenylthio moiety introduced into one of the α-positions of the
pyrrolic units. This reaction may compose an efficient approach for synthesizing novel
BODIPY derivatives with substituents attached to the pyrrolic unit without previously
brominating it. XCN can be used as a fluorescence turn-on probe to selectively detect Cys
and Hcy using the cyano group as the recognition site, with the p-aminophenylthio moiety
left unreacted. XCN was found to be nearly nonfluorescent, and it exhibits only slight
1
1
1
1
1
1
1
1
1
1
2
fluorescence enhancement when treated with GSH. However, upon interaction with Cys or
1

ACCEPTED MANUSCRIPT
2
2
2
2
2
1
2
3
4
5
Hcy, the fluorescence was enhanced by 1081 and 1126 folds, respectively. In addition, XCN
exhibits good selectivity and sensitivity towards Cys and Hcy over GSH and other amino
acids in a wide pH range from 2 to 10 in aqueous buffers. Furthermore, XCN was
successfully used for imaging biothiols in living A549 lung cancer cells.
Keywords: Fluorescent probes; Biothiols; BODIPY; Cell imaging.
2
6
1. Introduction
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
In recent years, biothiols like cysteine (Cys), homocysteine (Hcy) and glutathione (GSH)
have attracted extensive interest because of their vital roles in a variety of physiological
processes [1-5]. Abnormal levels of the three species may cause diseases. For example, Cys
deficiency may result in syndromes like liver damage, slower development of children,
detoxification weakening and skin lesions [6-8]. Abnormal concentration of Hcy may be a
sign for cardiovascular diseases [9-10]. Lack of GSH may change intracellular redox state
and lead to severe diseases such as cancer, and Alzheimer’s [11-14]. Hence, it is of great
importance to qualitatively and quantitatively monitor these biothiols. Among various
techniques, fluorescent probes have been demonstrated to be powerful tools with the
advantages of simplicity, high sensitivity and intracellular bioimaging capacity.
So far, a number of fluorescent probes have been designed and synthesized to detect the
three biothiols. These probes are mostly reaction-based, utilizing mechanisms like
nucleophilic substitution, Michael addition, and cyclization reactions with aldehydes and
other functional groups [15-23]. However, it is still a great challenge to discriminate each of
the three biothiols because of their similar structures and reactivity. Only a few reported
2

ACCEPTED MANUSCRIPT
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
sensors can be used to distinguish Cys, Hcy and GSH from one another. In this respect, Yang
and coworkers reported a BODIPY-based ratiometric fluorescent sensor, which could
selectively discriminate Cys and Hcy from GSH taking advantage of the nucleophilic attack
of the thiol moiety followed by the displacement with the amino group to regenerate the thiol
moiety, while the 2nd step was not observed for GSH [24]. Later the same group reported
another probe for selectively detecting Cys over Hcy by means of different rates of the
intramolecular displacement reactions [25]. Yoon and coworkers reported a biothiol probe
based on nitrobenzothiadiazole substituted with a p-aminophenylthio moiety, which also
could selectively detect Cys and Hcy based on their nucleophilicity [26]. Besides, Liang and
coworkers have reported a fluorescent probe, utilizing a cyano group as the recognition
moiety, which could distinguish Cys from the other two [27].
Inspired by the excellent studies mentioned above, we aimed to design and synthesize
fluorescent probes to selectively detect the biothiols. Herein, we report the synthesis of a
fluorescent probe XCN (Scheme 1) by introducing a p-aminophenylthio and a cyano group
into a BODIPY moiety. Interestingly, XCN can be used as a fluorescence turn-on probe to
selectively detect Cys and Hcy over GSH using the cyano group as the recognition site, with
the p-aminophenylthio moiety left unreacted. Furthermore, XCN was successfully used for
imaging biothiols in living A549 lung cancer cells.
6
6
6
0
1
2
2. Experimental section
2.1 Materials and instrumentation
Commercially available solvents and reagents were used as received. Water was used after
3

ACCEPTED MANUSCRIPT
6
6
6
6
6
6
6
7
7
3
4
5
6
7
8
9
0
1
redistillation. Deuterated solvents for NMR measurements were available from Aldrich. UV-
vis absorption spectra were recorded on a Varian Cary 100 spectrophotometer and
fluorescence spectra were recorded on a Varian Cary Eclipse fluorescence spectrophotometer,
1
with a quartz cuvette (path length = 1 cm); both spectrophotometers were standardized. H
1
3
NMR and C NMR spectra were obtained using a Bruker AM 400 spectrometer with
tetramethylsilane (TMS) as the internal standard. High resolution mass spectra (HRMS) were
measured on a Waters LCT Premier XE spectrometer. Confocal laser scanning microscope
(CLSM) images were taken on an inverted fluorescence microscope (Nikon A1R/A1).
CN
Br
CN
CN
H
N
N
N
(
i )
( ii )
( iii )
N
O
O
1
2
3
Br
CN
Br
CN
Br
CN
N
N
N
(
iv )
( v )
( vi )
NH HN
N
F
N
N
N
B
B
F
F
F
S
NH2
4
5
XCN
7
7
7
7
2
3
4
5
(i) 4-Iodobenzonitrile, Pd (dba) , BINAP, t-BuONa, xylene, 120℃ , 60.5%; (ii) POCl , DMF,
ClCH CH Cl, 60℃ , 30.2%; (iii) NBS, CH Cl , 85.0%; (iv) pyrrole, TFA, 57.3%; (v) (a) DDQ;
(b) Et N, BF ·Et O, 63.2%; (vi) p-aminothiophenol, Et N, THF, reflux, 23.6%.
2
3
3
2
2
2
2
3
3
2
3
7
7
6
7
Scheme 1 Synthetic route of probe XCN
7
7
8
8
9
0
2.2 pH influence measurements
pH influence measurements were carried out in the mixtures of DMSO and the following
buffers (2/1, v:v): Na HPO -citric acid buffer (20 mM, pH 2.0, 3.0, 4.0, 5.0),
2
4
4

ACCEPTED MANUSCRIPT
8
8
8
8
8
8
8
8
8
9
9
9
9
9
9
9
9
9
9
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
Na HPO -KH PO buffer (20 mM, pH 6.0, 7.0, 7.4), glycine-NaOH buffer (50 mM, pH 9.0,
2
4
2
4
10.0), Na HPO -NaOH buffer (20 mM, pH 12.0).
2
4
2.3 Cell culture
Human lung adenocarcinoma A549 cells were supplied by the Institute of Cell Biology
(Shanghai, China). The cell lines were cultured at 37 ºC under a humidified 5% CO2
atmosphere in the RPMI-1640 medium (GIBCO/Invitrogen, Camarillo, CA, USA)
supplemented with 10% fetal bovine serum (FBS, Biological Industry, Kibbutz Beit Haemek,
Israel) and 1% penicillin-streptomycin (10,000 U/mL penicillin and 10 mg/mL streptomycin,
Solarbio life science, Beijing, China).
2.4 Syntheses of the compounds
2.4.1 Synthesis of compound 1
Diphenylamine (5.42, 32.0 mmol), 4-iodobenzonitrile (7.33, 32.0 mmol), Pd (dba) (660
2
3
mg, 0.720 mmol), BINAP (678 m, 1.09 mmol), t-BuONa (10.8 g, 112 mmol) and xylene (240
mL) were added into a 500 mL three-neck flask. The mixture was stirred for 24 hours at 120 ℃
under nitrogen. Xylene was removed under reduced pressure and the residue was purified on
a silica gel column using CH Cl /PE (1/2, v:v) as the eluent to give a pale solid (5.23 g, yield
2
2
1
60.5%). H NMR (CDCl , 400 MHz, ppm): δ 7.41 (d, J=8.8 Hz, 2H), 7.33 (t, J=8.0 Hz, 4H),
3
7.18-7.19 (m, 6H), 6.96 (d, J=8.8 Hz, 2H).
2.4.2 Synthesis of compound 2
1
1
1
00
01
02
To the solution of compound 1 (5.00 g, 18.5 mmol) in dry 1,2-dichloroethane (300 mL),
was added the Vilsmeier reagent freshly prepared from the reaction of
N,N-dimethylformamide (DMF, 23.0 mL) with POCl (14.0 mL, 185 mmol). The mixture
3
5

ACCEPTED MANUSCRIPT
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
03
04
05
06
07
08
09
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
was stirred at reflux for 24 hours under nitrogen. The reaction mixture was cooled, washed
with water and extracted with CH Cl . The organic solvent was evaporated to dryness and the
2
2
residue was purified on a silica gel column using CH Cl /PE (1/2, v:v) as the eluent to give a
2
2
1
canary yellow solid (1.66 g, yield 30.2%). H NMR (CDCl , 400 MHz, ppm): δ 9.89 (s, 1H,
3
-CHO), 7.77 (d, J=8.8 Hz, 2H, ph-H), 7.52 (d, J=8.8 Hz, 2H, ph-H), 7.40 (t, J=8.0 Hz, 2H,
ph-H), 7.25 (t, J=7.6 Hz, 1H, ph-H), 7.18-7.13 (m, 6H, ph-H).
2.4.3 Synthesis of compound 3
To a solution of compound 2 (248 mg, 0.83 mmol) in dichloromethane (30 mL),
N-bromosuccinimide (NBS, 236 mg, 1.33 mmol) was added gradually. The reaction mixture
was stirred at room temperature for 4 hours. Then, the mixture was washed with water,
extracted with dichloromethane and dried over Na SO . Then the solvent was removed under
2
4
reduced pressure and the residue was purified on a silica gel column using CH Cl /PE (1/1,
2
2
1
v:v) as the eluent to give a brown solid (266 mg, yield 85.0%). H NMR (CDCl , 400 MHz,
3
ppm): δ 9.89 (s, 1H, -CHO), 7.77 (d, J=8.4 Hz, 2H, ph-H), 7.53 (d, J=8.8 Hz, 2H, ph-H), 7.48
(d, J=8.4 Hz, 2H, ph-H), 7.14 (d, J=8.8 Hz, 2H, ph-H), 7.11 (d, J=8.8 Hz, 2H, ph-H), 7.01 (d,
J=8.8 Hz, 2H, ph-H).
2.4.4 Synthesis of compound 4
Compound 3 (262 mg, 0.690 mmol) and pyrrole (5.00 mL, 34.8 mmol) were added into a
100 mL flask. The reaction mixture was stirred at room temperature for 30 min
after trifluoroacetic acid (TFA, 156 µL, 2.00 mmol) was added. Then triethylamine (2.00 mL)
was added into the flask to quench the reaction. Then the solvent was removed under reduced
pressure and the residue was purified on a silica gel column using CH Cl /PE (2/3, v:v) as the
2
2
6

ACCEPTED MANUSCRIPT
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
eluent to give a brown solid (195 mg, yield 57.3%). H NMR (CDCl , 400 MHz, ppm): δ 8.0
3
(s, 2H, -NH), 7.43 (d, J=8.4 Hz, 4H, ph-H), 7.18 (d, J=8.0 Hz, 2H, ph-H), 7.06-6.95 (m, 6H,
ph-H), 6.73 (s, 2H, pyrrolic), 6.17 (s, 2H, pyrrolic), 5.92 (s, 2H, pyrrolic), 5.46 (s, 1H,
1
3
meso-H). C NMR (CDCl , 100 MHz, ppm): δ 157.52, 150.76, 147.43, 145.74, 142.84,
3
138.77, 134.60, 132.61, 132.39, 131.06, 128.33, 126.37, 126.16, 119.41, 118.08, 116.72,
+
115.31, 55.56. HRMS (ESI, m/z): [M+H] calcd for C H BrN : 491.0871, found: 491.0871.
2
8
20
4
2.4.5 Synthesis of compound 5
Compound 4 (195 mg, 0.390 mmol) was added into a 100 mL three-neck flask, followed by
addition of 2,3-dichloro-5,6-dicyano-1,4-benzoquinone (DDQ) (108 mg, 0.470 mmol) under
nitrogen. Then triethylamine (326 µL, 2.34 mmol) was added into the flask and stirred for 5
min at room temperature. Next, BF ·Et O (294 µL, 2.34 mmol) was added, and the reaction
3
2
mixture was stirred for 6 hours. After that, the mixture was washed with sodium bicarbonate
solution and extracted with dichloromethane for three times, followed by washing with water
and extraction with dichloromethane. Then the solvent was removed under reduced pressure
and the residue was purified on a silica gel column using CH Cl /PE (1/1, v:v) as the eluent to
2
2
1
give an orange solid (133 mg, yield 63.2%). H NMR (CDCl , 400 MHz, ppm): δ 7.94 (s, 2H,
3
pyrrolic), 7.56-7.52 (m, 6H, ph-H), 7.22 (d, J=8.4 Hz, 2H, ph-H), 7.15 (d, J=8.4 Hz, 2H,
ph-H), 7.09 (d, J=8.4Hz, 2H, ph-H), 7.01 (d, J=3.6 Hz, 2H, pyrrolic), 6.58 (s, 2H, pyrrolic).
+
HRMS (ESI, m/z): [M+H] calcd for C H BBrF N : 539.0854, found: 539.9557.
2
8
19
2
4
2.4.6 Synthesis of probe XCN
p-Aminothiophenol (250 mg, 2.00 mmol), triethylamine (420 µL) and tetrahydrofuran
(8.00 mL) were added into a 100 mL three-neck flask and stirred for 15 min. Then,
7

ACCEPTED MANUSCRIPT
1
1
1
1
1
1
1
1
1
1
1
1
1
47
48
49
50
51
52
53
54
55
56
57
58
59
compound 5 (539 mg, 1.00 mmol) was added into the flask, and the mixture was refluxed for
16 hours under nitrogen. After that, the solvent was removed under reduced pressure and the
residue was purified on a silica gel column using CH Cl /PE (1/1, v:v) as the eluent to give
2
2
unreacted compound 5 (178 mg) and an amaranthine solid of XCN (92.0 mg, yield 23.6%).
1
H NMR (DMSO-d , 400 MHz, ppm): δ 7.79 (s, 1H, pyrrolic), 7.70 (d, J=8.8 Hz, 2H, ph-H),
6
7.61 (d, J=8.8 Hz, 2H, ph-H), 7.58 (d, J=8.4 Hz, 2H, ph-H), 7.29 (d, J=8.8 Hz, 2H, ph-H),
7.22 (d, J=8.4 Hz, 2H, ph-H), 7.19 (d, J=8.8 Hz, 2H, ph-H), 7.11 (d, J=8.8 Hz, 2H, ph-H),
7.03 (d, J=4.4 Hz, 1H, pyrrolic), 6.82 (d, J=3.6 Hz, 2H, pyrrolic), 6.66 (d, J=8.8 Hz, 2H,
ph-H), 6.55 (dd, J =4.0 Hz, J =2.4 Hz, 1H, pyrrolic), 5.92 (d, J=4.8 Hz, 1H, pyrrolic), 5.79 (s,
1
2
1
3
2H, -NH ). C NMR (CDCl , 100 MHz, ppm): δ 165.22, 150.52, 148.66, 147.73, 144.72,
2
3
140.11, 138.98, 137.17, 137.04, 133.57, 133.41, 133.27, 132.02, 131.63, 131.23, 129.91,
128.04, 126.64, 123.85, 121.92, 119.49, 119.11, 118.90, 116.50, 115.88, 115.45, 115.30,
+
104.99. HRMS (ESI, m/z): [M+H] calcd for C H N SBrF B: 662.0997, found: 662.1005.
3
4
24
5
2
1
60 3. Results and discussion
1
61
3.1 Design and syntheses
1
1
1
1
1
1
62
63
64
65
66
67
As mentioned above, BODIPY has advantages in developing fluorescent probes, and both
the cyano and p-aminophenylthio groups may be utilized as the reaction site to selectively
detect biothiols based on their specific reactions with biothiols. Inspired by the successful
examples [26-27], we herein synthesized a new fluorescent probe for biothiols by introducing
both of the reaction moieties into a BODIPY platform. Thus, we first synthesized a BODIPY
derivative 5 with a cyano and a bromine moiety attached to the 8-diphenylaminophenyl
8

ACCEPTED MANUSCRIPT
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
68
69
70
71
72
73
74
75
76
77
78
79
80
81
82
83
84
85
86
87
88
substituent of the BODIPY fluorophore (Scheme 1), followed by the reaction with
p-aminothiophenol under basic condition. Interestingly, the p-aminophenylthio moiety was
not attached to the 8-diphenylaminophenyl moiety by replacing the bromine atom. Instead,
compound XCN was obtained with the p-aminophenylthio moiety attached to one of the
α-positions of the pyrrolic units, which can be clearly evidenced by the molecular ion peak at
662.1005 and the isotope pattern characteristic for the presence of the bromine atom in the
1
HRMS (Fig. S11). Consistent to this, one of the two pyrrolic α-protons disappeared in the H
NMR spectrum of XCN, as compared to that of compound 5 (Fig. 1). In addition, the
remaining α-proton of XCN was up-field shifted to δ = 7.79 ppm because of the electron
donating effect of the p-aminophenylthio moiety. The two protons of the amino group exhibit
a singlet peak at 5.79 ppm, which disappears upon treatment with D O (Fig. S12). In addition,
2
from the two-dimensional COSY and NOESY spectra of XCN, five pyrrolic protons can be
identified, and the H1-H6, H6-H7 and H7-H8 couplings are clearly observed (Fig. S13, S14).
All these observations unambiguously indicate that the p-aminophenylthio group was attach
to the α-position of the pyrrole unit.
This reaction may compose an efficient approach for synthesizing novel BODIPY
derivatives with substituents attached to the pyrrolic unit without previously brominating it.
The synthesized compound XCN was found to be nearly nonfluorescent, which is a good
starting point for developing fluorescence turn-on probes. Hence, we continued to check the
biothiol sensing behavior of XCN and elucidate the respective roles of the p-aminophenylthio
and the cyano moieties.
9

ACCEPTED MANUSCRIPT
1
1
89
90
1
Fig. 1 The low field region of the H NMR spectra of 5 and XCN in DMSO-d .
6
1
1
91
92
3.2 Spectroscopic Characteristics
1
1
1
1
1
1
1
2
2
2
2
93
94
95
96
97
98
99
00
01
02
03
After XCN was successfully synthesized, we firstly checked its spectral responses to Cys,
Hcy and GSH. As shown in Fig. 2a, the UV-vis spectrum of free XCN showed an absorption
peak at 546 nm. This band decreased sharply upon gradual addition of Cys, which was
accompanied with the development of a new peak at 480 nm. As shown in Fig. 2b, XCN is
nearly nonfluorescent. Upon adding Cys, a dramatic fluorescence enhancement was observed
with an emission peak at 549 nm. The addition of Hcy to the solution of XCN induced
absorption spectral changes and fluorescence enhancement similar to those observed for Cys
(Fig. S15a, S15b). On the other hand, the treatment of XCN with GSH only caused slight
fluorescence enhancement (Fig. S16). The results indicated that XCN can be used to
discriminate Cys and Hcy from GSH. The kinetics study of XCN in the presence of Cys and
Hcy was carried out at 37 ℃ . As shown in Fig. 2c and S15c, upon addition of Cys and Hcy,
1
0

ACCEPTED MANUSCRIPT
2
2
2
2
2
2
04
05
06
07
08
09
the fluorescence intensity approached their maxima within ca. 5 min, which indicated fast
response of XCN towards Cys and Hcy [28-29]. When the fluorescence emission intensity of
XCN was plotted as a function of Cys and Hcy concentration, good linear relationships were
obtained in the ranges of 0.25-0.50 mM and 0-0.25 mM, respectively (Fig. S18, S19). Thus,
the detection limits for Cys and Hcy were calculated to be 3.1 µM and 1.6 µM, respectively,
according to the 3σ/k formula [30-33].
2
10
2
2
2
2
2
2
2
11
12
13
14
15
16
17
Fig. 2 (a) UV-vis absorption and (b) fluorescence emission spectra of XCN (10 µM) upon
addition of Cys (0-60 eq) in DMSO. λ = 501 nm (the isosbestic point); (c) Kinetics study of
ex
XCN (10 µM) upon interaction with Cys (100 eq) in DMSO. λ =501 nm; (d) pH dependent
ex
fluorescence intensity at 549 nm of XCN (10 µM) in DMSO/buffers (2:1, v/v) upon addition
of Cys (100 eq), λ =501 nm.
ex
2
18
11

ACCEPTED MANUSCRIPT
2
19
2
2
2
2
2
2
2
20
21
22
23
24
25
26
Fig. 3 Fluorescence responses of probe XCN (10 µM) at 550 nm towards various analytes
(Cys and Hcy were used at 100 µM, other analytes were used at 200 µM). Analytes 1–24:
1-blank, 2-Thr, 3-Pro, 4-Ala, 5-Met, 6-Phe, 7-Tyr, 8-Gly, 9-Glu, 10-Val, 11-Gln, 12-Arg,
2
-
13-Ile, 14-His, 15-Trp, 16-Asp, 17-Leu, 18-Ser, 19-Asn, 20-Lys, 21-GSH, 22-S , 23-Cys,
24-Hcy.
2
2
2
2
2
2
27
28
29
30
31
32
Fig. 4 Fluorescence intensity responses of probe XCN (10 µM) at 550 nm for Cys (100 µM)
in the presence of various analytes (200 µM). Black bars represent the addition of a single
analyte. Red bars represent the subsequent addition of Cys to the mixture. Analytes 1–22:
1-none, 2-Thr, 3-Pro, 4-Ala, 5-Met, 6-Phe, 7-Tyr, 8-Gly, 9-Glu, 10-Val, 11-Gln, 12-Arg,
2
-
13-Ile, 14-His, 15-Trp, 16-Asp, 17-Leu, 18-Ser, 19-Asn, 20-Lys, 21-GSH, 22-S .
2
33
2
34
3.3 Selectivity test
2
2
2
2
35
36
37
38
Good selectivity is essential for practical applications of probes. To investigate the
selectivity of XCN towards Cys and Hcy, we checked the fluorescence response of XCN in
the presence of GSH, H S and various amino acids. As shown in Fig. 3, 4 and S20, only the
2
addition of Cys and Hcy caused dramatic fluorescence enhancement, and the coexistence of
1
2

ACCEPTED MANUSCRIPT
the interfering species did not seriously disturb the response of XCN towards Cys and Hcy.
All these results indicated that probe XCN exhibited excellent selectivity towards Cys and
Hcy.
2
2
2
39
40
41
2
42
3.4 pH dependence
2
2
2
2
43
44
45
46
To identify the pH influence on the sensing behavior, pH influence experiments were
carried out at different pH within the range from 2 to 12. As shown in Fig. 2d and S15d,
apparent fluorescence enhancement could be observed upon addition of Cys and Hcy within a
wide pH range of 2−10, which is favorable for the practical applications of XCN.
2
47
3.5 Sensing mechanism study
2
2
2
2
2
2
2
2
2
2
2
48
49
50
51
52
53
54
55
56
57
58
Researches on the sensing mechanism of probe XCN were also carried out. Consistent with
the reported results [26], the detection of Cys may be achieved by the nucleophilic addition of
the thiol group at the cyano moiety followed by the formation of a five-membered
heterocycle (Scheme S1), which may be evidenced by the absorption and fluorescence
spectra of the main reaction product (Fig. S21), as well as the observed m/z of 779.0920,
-
corresponding to the hypothesized product [XCN-Cys-H] (calcd.779.0881 for
-
C H BBrF N O S ) (Fig. S22). Similarly, the m/z peak of the product [XCN-Hcy-H] can
37
27
2
6
2 2
also be observed in the HRMS (Fig. S23), indicative of a similar reaction mechanism. The
reaction of the biothiols at the cyano moiety rather than the p-aminophenylthio moiety may
be ascribed to the more electron deficient character of the cyano group as compared with the
p-aminophenylthio moiety.
2
59
1
3

ACCEPTED MANUSCRIPT
2
60
2
2
2
2
2
2
2
61
62
63
64
65
66
67
Fig. 5 (A-D) Confocal fluorescence and bright-field images of A549 cells incubated with
probe XCN (5 µM) for 40 min after preincubation with Cys (200 µM) for 30 min. (E-H)
Confocal fluorescence and bright-field images of A549 cells incubated with probe XCN (5
µM) for 40 min after preincubation with NEM (1 mM) for 40 min. (I-L) Confocal
fluorescence and bright-field images of A549 cells incubated with probe XCN (5 µM) for 40
min. (A, E, I ) bright-field images; (B, F, J) red channel images; (C, G, K) green channel
images; (D, H, L) overlay.
2
2
2
2
2
2
2
2
2
2
2
68
69
70
71
72
73
74
75
76
77
78
3.6 Cell imaging
Encouraged by the above results, we continued to investigate the application of XCN in
biosystems. Thus, the fluorescent imaging behavior was checked in living A549 lung cancer
cells (Fig. 5). When A549 cells were incubated with XCN (5 µM) after preincubation with
Cys (200 µM), bright fluorescence in green and red channels were observed simultaneously
(Fig. 5B, 5C). However, when the cells were pretreated with 1 mM N-ethylmaleimide (NEM),
a well-known thiol-blocking agent, before incubation with probe XCN (5 µM), only very
weak fluorescence was observed (Fig. 5F, 5G). These results indicated the good
membrane-permeability, biocompatibility and ability of XCN to detect exogenous biothiols.
1
4

ACCEPTED MANUSCRIPT
2
2
2
2
2
79
80
81
82
83
Then, detection of endogenous biothiols was also checked. Apparent fluorescence was
observed both in red and green channels upon incubation of probe XCN without
preincubation of Cys (Fig. 5J, 5K). These results indicate that probe XCN can detect not only
exogenous biothiols, but also endogenous biothiols. The excellent performance of XCN in
cell imaging revealed that it may be practically used in bioimaging.
2
84 4. Conclusion
2
2
2
2
2
2
2
2
2
2
2
2
85
86
87
88
89
90
91
92
93
94
95
96
In summary, we herein report a new fluorescent probe XCN based on BODIPY. We first
synthesized a BODIPY derivative with a cyano and a bromine moiety attached to the
8-diphenylaminophenyl substituent of BODIPY, followed by the reaction with
p-aminothiophenol. Interestingly, XCN was obtained with the p-aminophenylthio moiety
attached to one of the α-positions of the pyrrolic units. This reaction may compose an
efficient approach for synthesizing novel BODIPY derivatives with functionalities attached to
the pyrrolic unit without previously brominating it. XCN can be used as a fluorescence
turn-on probe to selectively detect Cys and Hcy over GSH and other amino acids using the
cyano group as the recognition site, with the p-aminophenylthio moiety left unreacted. The
detection works well in a wide pH range from 2 to 10 in aqueous buffers. Furthermore, XCN
was successfully used for imaging biothiols in living cells. These results provide further
insight into developing novel fluorescent probes based on BODIPY.
2
97 Acknowledgment
2
2
98
99
This work was financially supported by NSFC (21472047, 21772041, 21702062), the
Program for Professor of Special Appointment (Eastern Scholar, GZ2016006) at Shanghai
1
5

ACCEPTED MANUSCRIPT
Institutions of Higher Learning, and the Fundamental Research Funds for the Central
Universities (WK1616004, 222201717003).
3
3
00
01
3
02 References
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
03
04
05
06
07
08
09
10
11
12
13
14
15
16
17
18
19
20
[1] X.Q. Chen, Y. Zhou, X.J. Peng, J. Yoon, Fluorescent and colorimetric probes for detection
of thiols. J. Chem. Soc. Rev. 39 (2010) 2120-2135.
[2] S.Y. Zhang, C.N. Ong, H.M. Shen, Critical roles of intracellular thiols and calcium in
parthenolide-induced apoptosis in human colorectal cancer cells, Cancer Lett. 208 (2004)
143-153.
[3] K.G Reddie, K.S Carroll, Expanding the functional diversity of proteins through cysteine
oxidation, Curr. Opin. Chem. Biol. 12 (2008) 746-754.
[4] W. Sun, J. Li, W.H. Li, L.J. Sun, L.P. Du, M.Y. Li, Design of OFF/ON fluorescent thiol
probes based on coumarin fluorophore, Sci. China Chem. 55 (2012) 1776-1780.
[5] C.M. Han, H.R. Yang, M. Chen, Q.Q. Su, W. Feng and F.Y. Li, Mitochondria-targeted
near-infrared fluorescent off-on probe for selective detection of cysteine in living cells
and in vivo, ACS Appl. Mater. Interfaces 7 (2015) 27968-27975.
[6] W. Dröge, H.P. Eck, S. Mihm, HIV-induced cysteine deficiency and T-cell dysfunction-a
rationale for treatment with N-acetylcysteine, Immunol. Today 13 (1992) 211-214.
[7] M.W. Lieberman, A.L. Wiseman, Z.Z. Shi, B.Z. Carter, et al., Growth retardation and
cysteine deficiency in γ-glutamyl transpeptidase-deficient mice, Proc. Natl. Acad. Sci. U
S A. 93 (1996) 7923-7926.
[8] S. Shahrokhian, Lead phthalocyanine as a selective carrier for preparation of a
1
6

ACCEPTED MANUSCRIPT
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
cysteine-selective electrode, Anal. Chem. 73 (2001) 5972-5978.
[9] H. Refsum, P. M. Ueland, O. Nygard and S. E. Vollset, Homocysteine and cardiovascular
diease, Annu. Rev. Medicine 49 (1998) 31–62.
[10] G.G. Klee, Cobalamin and folate evaluation: measurement of methylmalonic acid and
homocysteine vs vitamin B12 and folate, Clin. Chem. 46 (2000) 1277-1283.
[11] R. Janáky, V. Varga, A. Hermann, P. Saransaari, S.S. Oja, Mechanisms of L-Cysteine
Neurotoxicity, Neurochem. Res. 25 (2000) 1397-1405.
[12] D.M. Townsend, K.D. Tew and H. Tapiero, The importance of glutathione in human
disease, Biomed. Pharmacother. 57 (3003) 145-155.
[13] X.F. Wang, M.S. Cynader, Pyruvate released by astrocytes protects neurons from
copper-catalyzed cysteine neurotoxicity, J. Neurosci. 21 (2001) 3322-3331.
[14] M. Li, X.M. Wu, Y. Wang, Y.S. Li, W.H. Zhu and T.D. James, A near-infrared
colorimetric fluorescent chemodosimeter for the detection of glutathione in living cells,
Chem. Commun., 50 (2014) 1751-1753.
[15] X.M. Wu, A.D. Shao, S.Q. Zhu, Z.Q. Guo, W.H. Zhu, A novel colorimetric and
ratiometric NIR fluorescent sensor for glutathione based on dicyanomethylene-4H-pyran
in living cells, Sci. China Chem. 59 (2016) 62-69.
[16] Y.Q. Sun, M.L. Chen, J. Liu, X. Lv, J.F. Li, W. Guo, Nitroolefin-based coumarin as a
colorimetric and fluorescent dual probe for biothiols, Chem. Commun. 47 (2011)
11029-11031.
[17] J. Guy, K. Caron, S. Dufresne, S.W. Michnick, W.G. Skene, J.W. Keillor, Convergent
Preparation and Photophysical Characterization of Dimaleimide Dansyl Fluorogens:
1
7

ACCEPTED MANUSCRIPT
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
61
62
63
64
Elucidation of the Maleimide Fluorescence Quenching Mechanism, J. Am. Chem. Soc.
129 (2007) 11969-11977.
[18] W.H. Wang, O. Rusin, X.Y. Xu, K.K. Kim, et al., Detection of homocysteine and
cysteine, J. Am. Chem. Soc. 127 (2005) 15949-15958.
[19] L.L. Bu, J.Q. Chen, X.D. Wei, X. Li, H. Ågren, Y.S. Xie, An AIE and ICT based NIR
fluorescent probe for cysteine and homocysteine, Dyes and Pigments 136 (2017)
724-731
[20] M.J. Cao, H.Y. Chen, D. Chen, Z.Q. Xu, S.H. Liu, X.Q. Chen, J. Yin,
Naphthalimide-based fluorescent probe for selectively and specifically detecting
glutathione in the lysosomes of living cells, Chem. Commun. 52 (2016) 721-724.
[21] N. Zhao, Q. Gong, R.X. Zhang, J. Yang, Z.Y. Huang, N. Li, B.Z. Tang, A fluorescent
probe with aggregation-induced emission characteristics for distinguishing
homocysteine over cysteine and glutathione, J. Mater. Chem. C 3 (2015) 8397-8402.
[22] H.J. Xiang, H.P. Tham, M.D. Nguyen, J.G. Liu, et al., An aza-BODIPY based
near-infrared fluorescent probe for sensitive discrimination of cysteine/homocysteine
and glutathione in living cells, Chem. Commun. 53 (2017) 5220-5223.
[23] Q.Q. Li, Z. Li, AIE probes towards biomolecules: the improved selectivity with the aid
of graphene oxide, Sci. China Chem. 58 (2015) 1800-1809.
[24] L.Y. Niu, Y.S. Guan, Y.Z. Chen, L.Z. Wu, Q.Z. Yang, et al., BODIPY-based ratiometric
fluorescent sensor for highly selective detection of glutathione over cysteine and
homocysteine, J. Am. Chem. Soc. 134 (2012) 18928-18931.
[25] L.Y. Niu, Y.S. Guan, Y.Z. Chen, L.Z. Wu, C.H. Tung, Q.Z. Yang, A turn-on fluorescent
1
8

ACCEPTED MANUSCRIPT
sensor for the discrimination of cysteine from homocysteine and glutathione, Chem.
Commun. 49 (2013) 1294-1296.
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
65
66
67
68
69
70
71
72
73
74
75
76
77
78
79
80
81
82
83
84
85
[26] D. Lee, G. Kim, J. Yin and J. Yoon, An aryl-thioether substitute nitrobenzothiadiazole
probe for the selective detection of cysteine and homocysteine, Chem. Commun. 51
(2015) 6518-6520.
[27] Q.Q. Miao, Q. Li, Q.P. Yuan, L.L. Li, Z.J. Hai, S. Liu, G.L. Liang, Discriminative
fluorescence sensing of biothiols in vitro and in living cells, Anal. Chem. 87 (2015)
3460-3466.
[28] X.H. Lu, W. Wang, Q. Dong, X.L. Bao, X.C. Dong, W.L. Zhao, et al. A multi-functional
probe to discriminate Lys, Arg, His, Cys, Hcy and GSH from common amino acids.
Chem. Commun. 51 (2015) 1498-1501.
[29] C.P. Ge, H. Wang, B.X. Zhang, J. Yao, X.M. Li, J.G. Fang, et al., A thiol-thiosulfonate
reaction providing a novel strategy for turn-on thiol sensing. Chem. Commun. 51 (2015)
14913-14926.
[30] Y.B. Ding, T. Li, X. Li, Y.S. Xie, From nonconjugation to conjugation: novel meso-OH
2
+
substituted dipyrromethanes as fluorescence turn-on Zn probes, Org. Biomol. Chem.
11 (2013) 2685-2692.
[31] Y.S. Xie, Y.B. Ding, X. Li, C. Wang, J. P. Hill, K. Ariga, W. B. Zhang, W.H. Zhu,
Selective, sensitive and reversible ‘‘turn-on’’ fluorescent cyanide probes based on
2
+
2,2’-dipyridylaminoanthracene-Cu
ensembles, Chem. Commun. 48 (2012)
11513-11515.
1
9

ACCEPTED MANUSCRIPT
3
3
3
3
3
3
86
87
88
89
90
91
[32] J.C. Ge, Q.Y. Jia, W.M. Liu, L. Guo, Q.Y. Liu, M.H. Lan, H.Y. Zhang, X.M. Meng, P.F.
Wang, Red-emissive carbon dots for fluorescent, photoacoustic, and thermal theranostics
in living mice, Adv. Mater. 27 (2015) 4169-4177.
[33] X.D. Wei, L.L. Bu, W.Q. Tang, S.L. Zhao and Y.S. Xie, Selective and sensitive
2
+
fluorescence “turn-on” Zn probes based on combination of anthracene, diphenylamine
and dipyrrin, Sci. China Chem. 60 (2017) 1212-1218.
2
0

ACCEPTED MANUSCRIPT
Highlights
1
. A novel fluorescence turn-on probe based on a BODIPY moiety and a cyano group for
discriminating Cys and Hcy from GSH
. p-aminophenylthio moiety successfully introduced into one of the α-positions of BODIPY
without previously brominating it
. A novel biothiol probe based on the cyclization reaction between the cyano group and
biothiols
2
3