A turn-on fluorescent sensor for the discrimination of cystein from homocystein and glutathione

Article abstract of DOI:10.1039/c2cc38429a

We report a turn-on fluorescent sensor based on nitrothiophenolate boron dipyrromethene (BODIPY) derivatives for the discrimination of cystein (Cys) from homocystein (Hcy) and glutathione (GSH). The sensor was applied for detection of Cys in living cells.

Full text of DOI:10.1039/c2cc38429a

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A turn-on fluorescent sensor for the discrimination of
cystein from homocystein and glutathione†
Cite this: Chem. Commun., 2013,
4
9, 1294
ab
ab
a
a
a
Li-Ya Niu, Ying-Shi Guan, Yu-Zhe Chen, Li-Zhu Wu, Chen-Ho Tung and
Received 23rd November 2012,
Accepted 19th December 2012
a
Qing-Zheng Yang*
DOI: 10.1039/c2cc38429a
www.rsc.org/chemcomm
We report a turn-on fluorescent sensor based on nitrothiophenolate However, amino-substituted BODIPY exhibited a limited emission
boron dipyrromethene (BODIPY) derivatives for the discrimination shift relative to monochlorinated BODIPY, which is less suitable for
of cystein (Cys) from homocystein (Hcy) and glutathione (GSH). The the detection of Cys and Hcy in biological systems. Herein we report
sensor was applied for detection of Cys in living cells.
a sensor that emits turn-on fluorescence upon addition of thiols. By
means of more rapid intramolecular displacement of sulfur with
Biological thiols, including cystein (Cys), homocystein (Hcy) amino group of Cys than Hcy and GSH, the discrimination of Cys
and glutathione (GSH), play crucial roles in maintaining redox from Hcy and GSH is achieved.
balance of biological systems. Cys deficiency could lead to slow
growth, skin lesions, liver damage, etc. Hcy is associated with a molar absorption coefficients and quantum yields, elevated
BODIPY dyes have many excellent features, such as high
1
variety of diseases such as neutral tube defects, cardiovascular chemical and photostability, which make them good chromo-
2
8
and Alzheimer’s diseases. GSH is a pivotal indicator for the phore candidates as fluorescent sensors in biological systems.
maintenance of xenobiotic metabolism, intracellular signal Furthermore, their spectroscopic and photophysical properties
3
transduction and gene regulation. Accordingly, it is of great can be well tuned by introducing ancillary residues at the
9
importance to detect these biological thiols.
appropriate positions of the dipyrromethene core. By using
A fluorescent sensor is a powerful tool for the detection of 3,5-dichlorinated BODIPY as the precursor, we introduced a
mercapto biological molecules in living systems due to its triethyleneglycol group at the 3-position through Sonogashira
simplicity, high sensitivity and suitability for intracellular coupling followed by click reaction to improve the water-
4
detection. Significant effort has been made to develop fluorescent solubility. The chlorine at the 5-position was displaced with
5
sensors for biothiol detection. However, it still remains a nitrothiophenol or nitrophenol to yield compound 1, which
1
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challenge to discriminate between SH-containing molecules was characterized by H NMR, C NMR, and high-resolution
because of their similarity in structure and reactivity. Only a mass spectrometry (ESI†). A nitrothiophenol or nitrophenol
few sensors discriminating Cys, Hcy and GSH from one another moiety with strong reduction potential attached to a BODIPY
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have been reported so far. Recently we developed the first fluorophore may incur photo-induced electron transfer (PET) to
ratiometric fluorescent sensor for the selective detection of quench the fluorescence.
7
GSH over Cys/Hcy. The chlorine of the monochlorinated boron
In preliminary experiments, we studied the reaction of 1
dipyrromethene (BODIPY) can be rapidly replaced by thiolate. toward methyl mercaptoacetate as a model thiol (Scheme 1).
The amino groups of Cys/Hcy, but not of GSH further replace the The resulting products exhibit strong fluorescence, which were
thiolate to form amino-substituted BODIPY. The significantly identified as compound 2 (Fig. S1 in ESI†). It revealed that
different photophysical properties of sulfur and amino substi- methyl mercaptoacetate substituted the nitrothiophenol or
tuted BODIPY enable the discrimination of GSH over Cys/Hcy. nitrophenol group, which blocked the PET process and led to
a fluorescence enhancement. The observed rate constant (kobs
)
a
for the reaction of 1a and methyl mercaptoacetate is found to
Key Laboratory of Photochemical Conversion and Optoelectronic Materials,
Technical Institute of Physics and Chemistry, Chinese Academy of Sciences,
Beijing, 100190, P. R. China. E-mail: qzyang@mail.ipc.ac.cn;
À2 À1
be 2.0 Â 10
s
(t1/2 = 34.5 s) under a pseudo first order
reaction. 1b showed a slower reactivity (t = 97.4 s), which is
1
/2
Fax: +86 10 6255 4670; Tel: +86 10 8254 3450
probably attributed to the more stable C–O bond compared to
the C–S bond of 1a (Fig. S2 in ESI†). The mild and efficient
reaction for the formation of 2 with significant fluorescence
enhancement showed that 1 might be candidates for turn-on
fluorescent sensors for the detection of thiols.
b
University of Chinese Academy of Sciences, Beijing, 100049, P. R. China
Electronic supplementary information (ESI) available: Synthetic details and
†
characterizations of compounds 1a and 1b; fluorescent response of 1a in the
presence of Hcy or GSH; emission spectra of 1b in the presence of Cys, Hcy or
GSH. See DOI: 10.1039/c2cc38429a
1
294 Chem. Commun., 2013, 49, 1294--1296
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The time-dependent fluorescence response of 1a in the
presence of Cys was investigated at 37 1C in aqueous HEPES
buffer (20 mM, pH 7.4) containing 20% acetonitrile (Fig. 1a).
Upon addition of Cys, the emission band centered at 588 nm
increased in the initial 3 minutes, indicating that the thiolate
displaced the nitrothiophenol group and turned off the PET
quenching. The emission band then shifted to 564 nm and
increased gradually as a result of the formation of amino-
substituted product. We studied the fluorescence response of
1
(
a with Hcy and GSH under otherwise identical conditions
Fig. S3 in ESI†). In the presence of Hcy, the sulfur-substituted
product was formed initially as expected. Due to the relatively
Scheme 1 Reaction of 1 with methyl mercaptoacetate.
7
Based on our previous work, we hypothesized the reaction slow rate of further intramolecular displacement with amino
mechanism of 1 with Cys, Hcy and GSH as illustrated in group, the emission of 588 nm increased steadily and main-
Scheme 2. Initially, the thiolate can replace the nitrothiophenyl tained its strong fluorescence for a few hours. In the presence
or nitrophenyl group of 1 to generate compound 3, which is of GSH, one can only observe the fluorescence enhancement at
attributed to a kinetics controlled reaction. Once 3 is formed, 588 nm since intramolecular cyclization cannot happen. There-
the distance between the amine group and the active site of fore, the discrimination of Cys from Hcy and GSH was achieved
BODIPY is getting close. Driven by thermodynamics, the amino (Fig. 1b).
group in compound 3 can further attack the carbon attached to
We also measured the fluorescence response of 1b toward
the sulfur by a five- or six-membered cyclic transition state 4, Cys, Hcy and GSH. Similar but relatively slow sensing behavior
followed by the formation of the thermodynamically more stable was observed (Fig. S4 in ESI†).
C–N bond and the cleavage of the C–S bond to yield compound 5.
Fig. 2 showed the absorption and emission changes of 1a
In the case of GSH, after sulfur is substituted for chlorine, with different concentrations of Cys in acetonitrile/HEPES
intramolecular substitution is difficult to happen since there is buffer (2 : 8, v/v, 20 mM, pH 7.4) at 37 1C. With increasing
no adjacent amine group to form a favored transition state in amounts of Cys, the absorption band centered at 571 nm
GSH, and it will result in the stable product 6.
gradually decreased with concomitant growth of a new band
at 495 nm. When excited at the isosbestic point of 528 nm,
Fig. 1 (a) Time-dependent fluorescence spectra of 1a (10 mM) with 100 equiv.
of Cys. (b) Fluorescence spectra of 1a (10 mM) after 1 h addition of 100 equiv. of
GSH, Cys and Hcy in acetonitrile/HEPES buffer (2 : 8, v/v, 20 mM, pH 7.4) at 37 1C.
l
ex = 528 nm.
Fig. 2 (a) Absorption and (b) fluorescence spectral changes of 1a (10 mM) upon
addition of Cys (0, 10, 20, 40, 60, 80, 100, 200, 300 and 500 mM). Inset: the
fluorescence intensity at 564 nm as a function of the concentrations of Cys. lex
28 nm. Each spectrum was recorded 40 min after Cys addition in acetonitrile/
HEPES buffer (2 : 8, v/v, 20 mM, pH 7.4) at 37 1C.
=
5
Scheme 2 Proposed mechanism for the reaction of 1 with Cys, Hcy and GSH.
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fluorescence. The result exhibits that 1a is cell-permeable and it
can make turn-on detection for Cys in living cells possible.
In conclusion, we have developed a highly sensitive and
selective turn-on fluorescent sensor for the discrimination of
Cys from Hcy and GSH. The nitrothiophenol or nitrophenol
group can be substituted by thiolate and result in turn-on
fluorescence. The discrimination of Cys from Hcy and GSH is
achieved through more rapid intramolecular displacement of
sulfur with the amino group of Cys rather than that of Hcy and
GSH. The interesting reaction mechanism may provide new
insight for designing highly selective and sensitive fluorescent
sensors for the detection of Cys.
Fig. 3 Emission response at 564 nm of 1a (10 mM) upon addition of 100 equiv.
of various amino acids and GSH. Each data point was acquired 2 h after addition
of different amino acids in acetonitrile/HEPES buffer (2 : 8, v/v, 20 mM, pH 7.4)
at 37 1C.
We are grateful for financial support by 973 Program
(2013CB933800), the National Natural Science Foundation
of China (21222210, 21102155, 21072202), and the Chinese
Academy of Sciences (100 Talents Program). We thank Prof.
P. Wang (TIPC, CAS) for providing HeLa cells.
the emission at 564 nm increased significantly upon addition
of Cys. The emission intensity at 564 nm showed good linear
relationship with the Cys concentration (0–100 mM). The
À7
detection limit was determined to be 2.12 Â 10 M (S/N = 3). Notes and references
The recognition of GSH and Hcy were also investigated (Fig. S5
1
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in ESI†).
To examine the selectivity, the fluorescence responses of 1a
to various amino acids were measured under physiological
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1
296 Chem. Commun., 2013, 49, 1294--1296
This journal is c The Royal Society of Chemistry 2013