A Multi-signal Fluorescent Probe with Multiple Binding Sites for Simultaneous Sensing of Cysteine, Homocysteine, and Glutathione

Article abstract of DOI:10.1002/anie.201800485

A novel fluorescent probe was developed by integrating chlorinated coumarin and benzothiazolylacetonitrile and exploited for simultaneous detection of cysteine (Cys), homocysteine (Hcy), and glutathione (GSH). Featuring four binding sites and different reaction mechanisms for different biothiols, this probe exhibited rapid fluorescence turn-on for distinguishing Cys, Hcy, and GSH with 108-, 128-, 30-fold fluorescence increases at 457, 559, 529 nm, respectively, across different excitation wavelengths. Furthermore, the probe was successfully applied to the fluorescence imaging of endogenous Cys and GSH and exogenous Cys, Hcy, and GSH in living cells.

Full text of DOI:10.1002/anie.201800485

A Journal of the Gesellschaft Deutscher Chemiker
Angewandte
Chemie
International Edition
www.angewandte.org
Accepted Article
Title: A Multi-signal Fluorescent Probe with Multiple Binding Sites for
Simultaneous Sensing of Cys, Hcy and GSH
Authors: Guoxing Yin, Tingting Niu, Yabing Gan, Ting Yu, Peng Yin,
Haimin Chen, Youyu Zhang, Haitao Li, and Shouzhuo Yao
This manuscript has been accepted after peer review and appears as an
Accepted Article online prior to editing, proofing, and formal publication
of the final Version of Record (VoR). This work is currently citable by
using the Digital Object Identifier (DOI) given below. The VoR will be
published online in Early View as soon as possible and may be different
to this Accepted Article as a result of editing. Readers should obtain
the VoR from the journal website shown below when it is published
to ensure accuracy of information. The authors are responsible for the
content of this Accepted Article.
To be cited as: Angew. Chem. Int. Ed. 10.1002/anie.201800485
Angew. Chem. 10.1002/ange.201800485
Link to VoR: http://dx.doi.org/10.1002/anie.201800485
http://dx.doi.org/10.1002/ange.201800485

10.1002/anie.201800485
Angewandte Chemie International Edition
COMMUNICATION
A Multi-signal Fluorescent Probe with Multiple Binding Sites for
Simultaneous Sensing of Cys, Hcy and GSH
Guo-xing Yin, Ting-ting Niu, Ya-bing Gan, Ting Yu, Peng Yin,* Hai-min Chen,* You-yu Zhang, Hai-tao
Li* and Shou-zhuo Yao
Abstract: A novel fluorescent probe by integrating chlorinated
coumarin and benzothiazolylacetonitrile was exploited for
simultaneous detection of Cys, Hcy and GSH. Featured with four
binding sites and different bonding mechanism between probe and
biothiols, this probe exhibited rapid fluorescence turn-on for
distinguishing Cys, Hcy and GSH with 108, 128, 30-fold fluorescence
increase at 457, 559, 529 nm, respectively, through different
excitation wavelengths. Furthermore, the probe was successfully
applied to the fluorescence images of cellular Cys, GSH and
exogenous Cys, Hcy and GSH in living cells.
GSH, several fluorescent probes that allow for selective detection
of two species in one time have been reported.[9] But, it is more
challenging to discriminate the three thiols from each other
simultaneously. Guo and co-workers have developed
a
chlorinated coumarin-hemicyanine probe with three potential
reaction sites for simultaneous detection of Cys and GSH from
different emission channels.[10] Though the signaling promoted by
Hcy was poor and 60 min was needed for the sensing, their work
exhibits an excellent perspective for simultaneous Cys/Hcy/GSH
detection. Liang and co-workers reported a sulfonamide-based
self-quenched fluorescent probe for simultaneously detecting
biothiols.[11] And each product for the probe with Cys/Hcy/GSH
exhibited different emission maxima, but the difference between
the emissions from probe with Cys and Hcy was very small (5 nm)
Small molecule thiols play vital roles in many physiological
processes and are closely related to a lot of diseases.[1] Cysteine
(Cys), homocysteine (Hcy) and glutathione (GSH) are commonly
abundant and the most important biological thiols. Though they
have the similar structures and reactivity, the roles played by Cys,
Hcy and GSH are totally different. Cys is an essential amino acid
for the synthesis of proteins and abnormal level of Cys is related
to edema, slowed growth in children, lethargy, cardiovascular
diseases, liver damage, etc.[2] The role of Hcy in diseases still
remains a highly contentious topic.[3] And its normal concentration
in serum is approximately 5-15 μM and abnormal total
homocysteine might cause cognitive impairment in the elderly.[4]
GSH is well-known to be associated with a series of disorders
including cancer, Alzheimer’s and other ailments.[5] Considering
the important biological roles of biothiols and the relationship of
many diseases with their concentrations’ changes (the
intracellular concentration for Cys: 30-200 μM;[6] for GSH: 1-10
mM[5]), it is of great value to develop effective methods for real-
time simultaneous monitoring of intracellular Cys, Hcy and GSH,
respectively, in biological systems.
o
and it was required 2 h at 37 C for the detection. In 2016, Sun
and partners discovered a commercial fluorescent probe (2,3,5,6-
tetrafluoro-terephthalonitrile) for differentiating Cys, Hcy and GSH
in living cells.[12] Though the probe has four potential reaction sites
for detecting biological thiols, long sensing time (up to 2 h) was
needed to reach an optimal signal and the addition of CTAB (cetyl
trimethylammonium bromide) was required to discriminate Hcy
from GSH, which are significant barriers to the simultaneous
monitoring of Cys, Hcy and GSH in living cells. Simultaneous
analysis of biothiols (Cys/Hcy/GSH) in vivo/vitro from different
emission channels by single molecule fluorescent probe is still
quite rare, which is highly valuable but even more challenging.
Herein, we presented a novel fluorescent probe with four potential
reaction sites for simultaneous and rapid sensing of Cys, Hcy and
GSH from three emission channels, which based on different
reaction mechanisms between probe and biothiols (Scheme 1).
This probe was capable of not only fluorescence imaging of
exogenous Cys, Hcy and GSH but also simultaneous detection of
endogenous Cys and GSH from different emission channels in
living cells. And it has great potential to real-time simultaneous
and quantitative monitoring of cellular Cys, Hcy and GSH, which
will greatly promote the clarification of their complicated
relationship and functions in various physiological processes.
High-performance liquid chromatography (HPLC), mass
spectrometry (MS), capillary electrophoresis and HPLC-MS/MS
have been applied to the detection and determination of specific
biothiols.[7] However, owing to their non-invasiveness, high
selectivity, high sensitivity, relative low cost and operational
simplicity, a large number of fluorescent probes have been
developed to detect and sense biothiols recently.[8] Despite of
similarity among the structures and properties of Cys, Hcy and
[*]
G. Yin, Y. Gan, T. Yu, Dr. P, Yin, Prof. Y. Zhang. Prof. T. Li, Prof. S.
Yao.
Key Laboratory of Chemical Biology and Traditional Chinese
Medicine Research (Ministry of Education), College of Chemistry
and Chemical Engineering, Hunan Normal University, Changsha
410081, China.
Scheme 1. Simultaneous Sensing of Cys, Hcy and GSH Based on Four-Binding
Sites of Probe BCC.
E-mail: yinpeng@hunnu.edu.cn
haitao-li@hunnu.edu.cn
T. Niu, Prof. H. Chen.
Key Laboratory of Marine Biotechnology of Zhejiang Province,
Ningbo University, Ningbo, Zhejiang 315211, PR China
E-mail: chenhaimin@nbu.edu.cn
Probe BCC we have rationally designed has four potential
reaction sites, using coumarin as a fluorophore (Scheme 1). The
chloro atom in the 4-position of the coumarin moiety (site 1) is
Supporting information for this article is given via a link at the end of
the document.
This article is protected by copyright. All rights reserved.

10.1002/anie.201800485
Angewandte Chemie International Edition
COMMUNICATION
reactive and facilitates the thiol-halogen SNAr nucleophilic
aromatic substitution between probe BCC and thiols. And α, β-
unsaturated bond is a well-known Michael acceptor (site 2). The
cyano group could be utilized as an electrophile (site 3), and
under the effect of the cyano group, the unsaturated bond in the
benzothiazolium moiety could be used to another reaction site
(site 4) for discriminating biothiols. On the other hand, the steric
distance between thiol group and amino group in biothiols should
be: Cys < Hcy < GSH (Scheme S1). A probe with four potential
reaction sites in different steric distances would be possible to
discriminate similar biothiols.
the absorption at 391 nm showed a slight decrease and a new
peak at 492 nm increased slowly and reached a plateau after 15
min (details in Figure S2). The absorption at 391 nm would be
assigned to the intermediate, which was further transferred to the
final product BCC-Hcy (QY = 0.474, with Rhodamine B as
reference). The configuration changes (from E to Z) of the
intermediate might happen, which would be favorable for the
addition of free amine group to the cyano group (Scheme S3b).
And supported by the fluorescence color changes and the time-
dependent fluorescence spectra at 360 nm excitation, the slight
emission at 444 nm was decreased slowly after 2 min and
emission at 559 nm was enhanced (Figure S6). More importantly,
a
large fluorescence turn-on at 559 nm with 128-fold
enhancement was observed (λex = 480 nm) (Figures 2c and 2d),
and the fluorescence enhancement at 559 nm (λex = 480 nm) for
Cys (2.4-fold), GSH (13.7-fold) and NAC (0.2-fold) could be
neglected (details in Figure S7).
Figure 1. (a−d) Time-dependent absorption spectra of BCC (10 μM) in the
presence of 10 equiv. of Cys, Hcy, GSH and NAC in DMSO-PBS (pH 7.4, 10
mM, v/v, 6/4) at r.t.
Initially, we synthesized probe BCC (Supporting Information)
and examined its sensing behavior towards Cys/Hcy/GSH by
utilization of time-dependent UV-vis and fluorescence spectra in
DMSO-PBS (pH 7.4, 10 mM, v/v, 6/4) at room temperature. As
shown in Figure 1, the UV−vis spectra of free BCC showed a main
absorption at 500 nm (ε = 3.86 × 104 L mol-1 cm-1, quantum yield
(QY) = 0.003, with Rhodamine B as reference). Upon addition of
Cys, the initial absorption peak at 500 nm decreased dramatically
while a new peak at 374 nm appeared with a 126 nm blue shift
(Figure 1a) (details in Figure S1), which indicated that the
conjugation between the coumarin and benzothiazolylacetonitrile
is broken due to the Michael addition of Cys to site 2 in probe
BCC. The product BCC-Cys with a seven-membered ring (QY =
0.313, with quinine sulfate as reference) might be yielded shortly
(Scheme S3a). Correspondingly, a significant fluorescence turn-
on with 108-fold enhancement at 457 nm was observed in time-
dependent fluorescence spectra at 360 nm excitation within 15
min, but there was only a slight fluorescence enhancement for
Hcy (3.5-fold), GSH (11-fold) and NAC (N-Acetylcysteine) (1.2-
fold) under the same condition (Figures 2a and 2b, details in
Figures S5-S6).
Figure 2. Time-dependent fluorescence spectra of BCC (10 μM) in the
presence of (a) 10 equiv. of Cys excited at 360 nm, (c) 10 equiv. of Hcy excited
at 480 nm, (e) 10 equiv. of GSH excited at 400 nm, respectively. Time-
dependent fluorescence intensity changes toward 10 eq. of biothiols excited at
(b) 360 nm, (d) 480 nm and (f) 400 nm respectively. Condition: DMSO-PBS (pH
7.4, 10 mM, v/v, 6/4) at room temperature. Slit (nm): 2.5/2.5.
We continued to investigate the performance of GSH. As
shown in Figure 1c (details in Figure S3), initial absorption peak
at 500 nm decreased dramatically after the addition of GSH, and
a new absorption peak was found at 432 nm with a 68 nm blue
shift, which indicated that the nucleophilic addition of the amine
group in GSH to site 4 in probe BCC (Scheme S3c). As a model
compound of amino-free thiols, NAC was also tested and the
initial absorption decreased slightly probably due to a thioether
substitution (Figure 1d, Scheme S3d) (details in Figure S4). The
For Hcy, the time-dependent UV-vis spectrum is similar to
Cys in the beginning 2 min, along with the decrease of the initial
absorption peak at 500 nm and a simultaneous increase of a new
broad absorption peak at 391 nm (insert, Figure 1b). After that,
This article is protected by copyright. All rights reserved.

10.1002/anie.201800485
Angewandte Chemie International Edition
COMMUNICATION
time-dependent fluorescence spectra for probe with GSH (λex
=
there compounds with different photophysical properties would be
yielded after the reactions between probe BCC with Cys, Hcy and
GSH respectively. Further mechanism studies are still in progress
in our lab.
360 and 480 nm) exhibited weak fluorescence (Figures S5 and
S7), but a 30-fold fluorescence enhancement at 529 nm was
achieved (λex = 400 nm) (QY = 0.097, with quinine sulfate as
reference). While, Cys, Hcy and NAC displayed less than 5-fold
fluorescence enhancement at 529 nm (Figures 2e and 2f, details
in Figure S8). Furthermore, probe BCC exhibited little
fluorescence (λex = 360, 400 and 480 nm, respectively) and was
stable for a long time (Figure S9).
Taken together, the results from spectral studies
demonstrated that probe BCC could distinguish Cys, Hcy and
GSH by different fluorescence signals, which showed excellent
performances for sensing biothiols (Table S1). Encouraging by
these results, we further evaluated the selectivity of probe BCC
over other biologically related species, including various amino
acids and representative anions (Figures S10-S12). Only Cys,
Hcy and GSH could promote obvious fluorescence intensity
enhancement at 457, 559 and 529 nm (λex = 360, 480 and 400
nm, respectively) with short time (15 min), confirming excellent
selectivity of probe BCC for Cys, Hcy and GSH. Furthermore,
results from the detection of Cys, Hcy and GSH in the mixture of
the three biothiols demonstrated that probe BCC could be applied
to simultaneously sensing of Cys, Hcy and GSH in complex
biological environments (Figures S16-S23).
Subsequently, the capabilities of probe BCC to selectively
sense exogenous and intercellular Cys, Hcy and GSH were
evaluated. Firstly, the results from MTT assays showed that probe
BCC has good cell permeability and low cytotoxicity to living cells
within a short time (Figure S26). For exogenous biothiols, the cells
were firstly pretreated with NEM and cellular biothiols and SH-
containing proteins were deactivated. After incubation with probe
BCC, almost no fluorescence could be observed (Figure S27, A1-
A3). While, after subsequently treated with Cys, Hcy and GSH
respectively, blue, red and green fluorescence were observed
from three different emission channels in living cells with high
selectivity (Figure 3, C1-C3; Figure S27). Fluorescence images
for BEL-7402 cells with different concentrations of exogenous Cys
(or GSH) were also conducted, the differences in fluorescence
intensities indicated the different concentrations of Cys/GSH in
living cells (Figure S28).
In addition, the fluorescence titration experiments under the
same conditions were conducted. As shown in Figures S13-S15,
an excellent linear from 0-3 equiv. of Cys was obtained and the
detection limit was calculated to be as low as 0.5 nM based on
S/N = 3. The fluorescence intensities were linearly proportional to
the amount of Hcy and GSH from 0 to 2 equiv., and the detection
limits were calculated to be as 3.6 nM and 6.9 nM, respectively.
In view of these results, probe BCC could be applied to sense
intracellular biothiols.
Figure 3. Confocal fluorescence images of Cys, GSH and Hcy in BEL-7402
cells. (A1−A3) Cells were incubated for 30 min, then imaged. (B1−B3) Cells
were incubated with probe BCC (2.5 μM) for 30 min, then imaged. (C1−C3)
Cells were pretreated with NEM (0.5 mM, 30min), subsequently incubated with
Cys/GSH/Hcy (500 μM, 30 min) and probe BCC (2.5 μM, 30 min), then imaged
(λex = 405 nm, λem = 421-475 nm for the blue channel; λex = 458 nm, λem = 500-
550 nm for the green channel; and λex = 543 nm, λem = 552-617 nm for the red
channel). Scale bar: 20 μm.
Scheme 2. Proposed mechanisms of probe BCC with Cys, Hcy and GSH.
Next, to further investigate the reaction mechanism between
probe BCC and Cys/Hcy/GSH, products of BCC-Cys, BCC-Hcy
and BCC-GSH were observed in the corresponding HRMS
titration experiments (Figures S38-S40). Though the isolation of
product BCC-Cys and BCC-GSH was failed, the product BCC-
Hcy between probe BCC and Hcy was successfully obtained,
which was well characterized. So we could propose the reaction
mechanism between probe BCC with biothiols (Scheme 2, details
in Scheme S3), which was based on spectral studies and NMR
analyses (Figures S24-S25; S36-S47). As four binding sites in the
probe and the differences from the structure of Cys, Hcy and GSH,
For the images of cellular biothiols, cells incubated without
probe BCC exhibited almost no fluorescence (Figure 3, A1-A3).
After incubation with probe BCC for 30 min, BEL-7402 cells
exhibited different blue fluorescence (from blue channel) for Cys
detection and green fluorescence (from green channel) for GSH
detection. And almost no fluorescence (from red channel) was
This article is protected by copyright. All rights reserved.

10.1002/anie.201800485
Angewandte Chemie International Edition
COMMUNICATION
[1]
[2]
(a) Z. A. Wood, E. Schroder, J. Robin Harris, L. B. Poole, Trends
Biochem. Sci. 2003, 28, 32-40; (b) T. V. Mishanina, M. Libiad, R.
Banerjee, Nat. Chem. Biol. 2015, 11, 457-464; (c) S. Zhang, C.-N.
Ong, H.-M. Shen, Cancer Lett. 2004, 208, 143-153.
(a) Y. Zhang, X. Shao, Y. Wang, F. Pan, R. Kang, F. Peng, Z.
Huang, W. Zhang, W. Zhao, Chem. Commun. 2015, 51, 4245-
4248; (b) S. Shahrokhian, Anal. Chem. 2001, 73, 5972-5978.
B. Debreceni, L. Debreceni, Cardiovasc. Ther. 2014, 32, 130-138.
H. Y. Lee, Y. P. Choi, S. Kim, T. Yoon, Z. Guo, S. Lee, K. M. K.
Swamy, G. Kim, J. Y. Lee, I. Shin, J. Yoon, Chem. Commun. 2014,
50, 6967-6969.
C. Hwang, A. Sinskey, H. Lodish, Science 1992, 257, 1496-1502.
Y. Yue, F. Huo, P. Ning, Y. Zhang, J. Chao, X. Meng, C. Yin, J.
Am. Chem. Soc. 2017, 139, 3181-3185.
(a) J. Vacek, B. Klejdus, J. Petrlova, L. Lojkova, V. Kuban, Analyst
2006, 131, 1167-1174; (b) T. Inoue, J. R. Kirchhoff, Anal. Chem.
2002, 74, 1349-1354; (c) M. J. MacCoss, N. K. Fukagawa, D. E.
Matthews, Anal. Chem. 1999, 71, 4527-4533; (d) B. Seiwert, U.
Karst, Anal. Chem. 2007, 79, 7131-7138.
(a) X. Chen, Y. Zhou, X. Peng, J. Yoon, Chem. Soc. Rev. 2010,
39, 2120-2135; (b) J. Chan, S. C. Dodani, C. J. Chang, Nat. Chem.
2012, 4, 973-984; (c) H. S. Jung, X. Chen, J. S. Kim, J. Yoon,
Chem. Soc. Rev. 2013, 42, 6019-6031; (d) L.-Y. Niu, Y.-Z. Chen,
H.-R. Zheng, L.-Z. Wu, C.-H. Tung, Q.-Z. Yang, Chem. Soc. Rev.
2015, 44, 6143-6160; (e) C.-X. Yin, K.-M. Xiong, F.-J. Huo, J. C.
Salamanca, R. M. Strongin, Angew. Chem., Int. Ed. 2017, 56,
13188-13198.
(a) O. Rusin, N. N. St. Luce, R. A. Agbaria, J. O. Escobedo, S.
Jiang, I. M. Warner, F. B. Dawan, K. Lian, R. M. Strongin, J. Am.
Chem. Soc. 2004, 126, 438-439; (b) X. Yang, Y. Guo, R. M.
Strongin, Angew. Chem., Int. Ed. 2011, 50, 10690-10693; (c) Y.
Yue, F. Huo, X. Li, Y. Wen, T. Yi, J. Salamanca, J. O. Escobedo,
R. M. Strongin, C. Yin, Org. Lett. 2017, 19, 82-85; (d) L.-Y. Niu, Y.-
S. Guan, Y.-Z. Chen, L.-Z. Wu, C.-H. Tung, Q.-Z. Yang, J. Am.
Chem. Soc. 2012, 134, 18928-18931; (e) S.-Y. Lim, K.-H. Hong, D.
I. Kim, H. Kwon, H.-J. Kim, J. Am. Chem. Soc. 2014, 136, 7018-
7025; (f) W. Chen, H. Luo, X. Liu, J. W. Foley, X. Song, Anal.
Chem. 2016, 88, 3638-3646; (g) Q. Hu, C. Yu, X. Xia, F. Zeng, S.
Wu, Biosens. Bioelectron. 2016, 81, 341-348; (h) L. He, X. Yang,
K. Xu, X. Kong, W. Lin, Chem. Sci. 2017, 8, 6257-6265.
J. Liu, Y.-Q. Sun, Y. Huo, H. Zhang, L. Wang, P. Zhang, D. Song,
Y. Shi, W. Guo, J. Am. Chem. Soc. 2014, 136, 574-577.
Q. Miao, Q. Li, Q. Yuan, L. Li, Z. Hai, S. Liu, G. Liang, Anal. Chem.
2015, 87, 3460-3466.
observed for Hcy, which may be due to the concentration of Hcy
in living cells is very small (Figure 3, B1-B3). Fluorescence
images for cellular Cys and GSH in different kinds of cells were
further performed (Figure S29). Thus, this probe could be further
applied to the real-time quantitative monitoring of cellular Cys and
GSH.
[3]
[4]
In summary, we have designed and synthesized a novel
chlorinated coumarin-benzothiazolium fluorescent probe BCC
with four binding sites, which could simultaneously and selectively
detect Cys, Hcy and GSH from three different emission channels.
The fluorescence increments of probe BCC for Cys, Hcy and GSH
at 457, 559 and 529 nm are 108, 128 and 30-fold increase,
respectively (λex = 360, 480, and 400 nm) based on different
bonding mechanism, with high selectivity and high sensitivity.
Furthermore, fluorescence imaging studies in different living cells
demonstrated that probe BCC is able to simultaneously monitor
endogenous Cys and GSH and exogeneous Cys, Hcy and GSH
via multicolor imaging. This study would strongly improve the
further exploration about biothiols’ dynamics and their function in
biological systems and promote the research of biothiols in
biomedicine and diagnostics.
[5]
[6]
[7]
[8]
[9]
Acknowledgements
This work was supported by the funding from the National Natural
Science Foundation of China (Grant No. 21405043, 21675051
and 81370532), K.C. Wong Magna Fund in Ningbo University,
LiDakSum Marine Biopharmaceutical Development Fund and
Opening Fund of Key Laboratory of Chemical Biology and
Traditional Chinese Medicine Research (Ministry of Education of
China), Hunan Normal University.
[10]
[11]
[12]
H. Zhang, R. Liu, J. Liu, L. Li, P. Wang, S. Q. Yao, Z. Xu, H. Sun,
Chem. Sci. 2016, 7, 256-260.
Keywords: multi-signal • multiple binding sites • fluorescent
probe • simultaneous sensing • biothiols
This article is protected by copyright. All rights reserved.

10.1002/anie.201800485
Angewandte Chemie International Edition
COMMUNICATION
COMMUNICATION
Guoxing Yin, Tingting Niu, Yabing Gan,
Ting Yu, Peng Yin,* Haimin Chen,*
Youyu Zhang, Haitao Li* and Shouzhuo
Yao
Page No. – Page No.
A Multi-signal Fluorescent Probe with
Multiple Binding Sites for
Simultaneous Sensing of Cys, Hcy
and GSH
A multi-signal fluorescent probe was exploited for simultaneously distinguishing and
sensing of cysteine, homocysteine and glutathione in living cells.
This article is protected by copyright. All rights reserved.