A fluorescein-based probe with high selectivity to cysteine over homocysteine and glutathione

Article abstract of DOI:10.1039/c2cc33932c

A fluorescent probe based on fluorescein displays excellent selectivity and sensitivity for cysteine and its application for bio-imaging is described.

Full text of DOI:10.1039/c2cc33932c

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A fluorescein-based probe with high selectivity to cysteine over
homocysteine and glutathionewz
Huilin Wang,a Guodong Zhou,a Hongwei Gaib and Xiaoqiang Chen*a
Received 1st June 2012, Accepted 27th June 2012
DOI: 10.1039/c2cc33932c
A fluorescent probe based on fluorescein displays excellent
selectivity and sensitivity for cysteine and its application for
bio-imaging is described.
The Tseng group reported fluorosurfactant-capped gold nano-
particles as a sensor for the highly selective detection of
cysteine.11c Besides, the first fluorescence-enhanced chemo-
dosimeter discriminating Cys from Hcy and GSH was also
reported by Peng et al.11d Most recently, Strongin et al.
developed an ingenious method, where an acrylate moiety
was used to discriminate Cys and Hcy by the different
relative rates in an intramolecular cyclization reaction between
acrylate and thiols.11e Herein, we report a fluorescein-based
probe 1 bearing two acrylate moieties for the selective recogni-
tion of Cys over Hcy and GSH. We speculate that the double
acrylate moieties can effectively enhance the selectivity of the
probe to Cys compared with the single acrylate-containing
analogue.
Intracellular thiols like cysteine (Cys), homocysteine (Hcy)
and glutathione (GSH) play critical roles in the regulation of
important cellular processes.1 For instance, GSH maintains
the reduced state of proteins and protecting the cells against
reactive oxygen species (ROS), drugs or heavy metal ions.2
Hcy, is situated at a critical regulatory branch point in sulfur
metabolism, and can be remethylated to methionine through
methionine synthetase with methylenetetrahydrofolate as a
cofactor in the methionine cycle.2 Cys is a precursor amino
acid of GSH and both are taken up by food or are formed as a
metabolic product of Hcy.3 Alteration of intracellular thiol
concentration results in many diseased states. Elevated levels
of homocysteine and cysteine are considered to be associated
with cardiovascular disease.4 A deficiency of thiols causes
various health problems such as retarded growth, hair depig-
mentation, lethargy, liver damage, muscle and fat loss, and
skin lesions.5 Therefore, the detection and discrimination of
thiol-containing molecules in biological samples are of great
importance. In the past few years, various fluorescent probes
for thiols based on different mechanisms have been exploited,6–10
including Michael addition,7 cyclization reaction with aldehyde,8
cleavage reaction by thiols,9 and others.10
The fluorescein derivates 1 and 2 required for these studies
were synthesized as shown in Scheme 1. Fluorescein was
reacted with acryloyl chloride in the presence of triethylamine
in CH2Cl2 to afford 1 and 2 in 75% and 78% yield, respec-
tively. The experimental details and characterization data for 1
and 2 are given in Electronic Supplementary Information
(ESIz).
Fig. 1 shows the fluorescence and absorbance changes that 1
undergoes upon the addition of various analytes, including
Cys, Hcy, GSH, Gly, Phe, Ser, Glu, Lys, Arg, His, Ala, Gln,
Met and Tyr. After incubation with 10 equiv. of Cys for 10 min,
the probe 1 displays a remarkable fluorescence enhancement at
515 nm and a new absorption band at around 490 nm in
ethanol-phosphate buffer (20 mM, pH 7.4, 2 : 8 v/v). The
analytes without thiols (Phe, Ser, Glu, Arg, Ala, His, Lys,
Gln, Gly, Tyr and Met) induced little changes in fluorescence
intensity and UV-vis spectra under the same conditions.
Interestingly, the addition of Hcy and GSH did not show as
remarkable a fluorescence enhancement and colorimetric
change as those induced by Cys (Fig. S1, see ESIz), even
though mercapto groups were also contained in the two
thiols. By contrast, the solution containing 2, bearing a single
acrylate group, did not show a considerable selectivity to Cys
Owing to the similarity in structure and reactivity of Cys, Hcy
and GSH, fluorescent sensors that could discriminate the three
thiols were surprisingly scarce so far.11 The Strongin group and Li
group provided the pioneer works to specifically detect Hcy.11a,b
a State Key Laboratory of Materials-Oriented Chemical Engineering,
College of Chemistry and Chemical Engineering, Nanjing University
of Technology, Nanjing 210009, China. E-mail: chenxq@njut.edu.cn;
Fax: +86-25-8358-7856; Tel: 86-25-8358-7856
b State Key Laboratory of Chemo/Biosensing and Chemometrics,
Hunan University, Changsha 410082, China
w We note that after our work had been completed, an independent
work entitled ‘‘A colorimetric and fluorescent chemodosimeter for
discriminative and simultaneous quantification of cysteine and homo-
cysteine’’ was published, L. Wang, Q. Zhou, B. Zhu, L. Yan, Z. Ma,
B. Du and X. Zhang, Dyes and Pigments, 2012, 95, 275–279. We were
unaware of this article at the time of submission.
z Electronic supplementary information (ESI) available: experimental
details; synthesis and characterization of 1 and 2; spectroscopic data;
Job’s plots of the reaction between 1 or 2 and Cys; mass spectra of 1 or
2 reacting with Cys. See DOI: 10.1039/c2cc33932c
Scheme 1 Synthesis of sensors 1 and 2.
c
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Chem. Commun., 2012, 48, 8341–8343 8341

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Fig. 2 Top: Fluorescence spectra of 2 (5 mM) upon incubation with
various analytes (50 mM) for 10 min in ethanol-phosphate buffer
(20 mM, pH 7.4, 2 : 8 v/v) (lex = 478 nm, slit: 1.5 nm/1.5 nm).
Bottom: UV/Vis absorption spectra of 2 (10 mM) in ethanol-phosphate
buffer (20 mM, pH 7.4, 2 : 8 v/v) in the presence of 100 mM analytes
including Cys, Hcy, GSH, Phe, Ser, Glu, Arg, Ala, His, Lys, Gln, Gly,
Tyr and Met.
Fig. 1 Top: Fluorescence spectra of 1 (5 mM) upon incubation with
various analytes (50 mM) for 10 min in ethanol-phosphate buffer
(20 mM, pH 7.4, 2 : 8 v/v) (lex = 478 nm, slit: 1.5 nm/1.5 nm).
Bottom: UV/Vis absorption spectra of 1 (10 mM) in ethanol-phosphate
buffer (20 mM, pH 7.4, 2 : 8 v/v) in the presence of 100 mM analytes
including Cys, Hcy, GSH, Phe, Ser, Glu, Arg, Ala, His, Lys, Gln, Gly,
Tyr and Met.
in that Hcy and GSH also induced great changes in the
fluorescence and absorption spectra (Fig. 2). To investigate
detection limit of 1 for Cys, 1 (1 mM) was treated with various
concentrations of Cys (0–500 nM) (Fig. S2a, see ESIz). The
fluorescence intensity at 515 nm was plotted as a function of
the Cys concentration (Fig. S2b, see ESIz). The fluorescence
intensity of 1 was linearly proportional to Cys concentrations
of 0–500 nM, and as low as 77 nM concentration of Cys
was detected by using 1 with a signal-to-noise ratio of 3.
Similarly, the detection limit of 2 for sensing Cys was
evaluated to be 121 nM (Fig. S2c and S2d, see ESIz). The
effect of pH on the fluorescence response of 1 or 2 to Cys was
investigated. The results indicated that both 1 and 2 are useful
for detecting Cys under neutral and basic conditions (Fig. S3,
see ESIz). Time-dependent fluorescence intensity assays show
the reactions between 1 or 2 and Cys (10 equiv.) were almost
complete within 15 min and 10 min, respectively (Fig. S4,
see ESIz).
Scheme 2 A plausible mechanism for the reaction between 1 and Cys.
respectively (Fig. S5, See ESIz). Mass spectrometry analysis of
a product obtained from the reaction of 1 or 2 with Cys in
CH3CN also supports the formation of fluorescein and a
cyclized product 3. A peak at 331.1 corresponding to the
resulting fluorescein and a peak at 174.0 corresponding to
the cyclization product 3 were clearly observed in the mass
spectra (Fig. S6, see ESIz). Compared to 2, the higher selec-
tivity of 1 to Cys over Hcy and GSH should be attributed to
the dual addition-cleavage processes in the reaction between 1
and Cys.
For the detection mechanism, the addition of thiol to the
acryloyl group in the probe followed by the cleavage of an
ester bond to form the fluorescein is likely to be responsible for
the fluorescence enhancement and UV-vis spectral change
(Scheme 2). To examine this plausible mechanism, the
stoichiometry of a binding event between thiol and 1 or 2
was initially determined. The results obtained from Job’s plots
show the 1 : 2 stoichiometry for the reaction between 1 and Cys
and 1 : 1 stoichiometry for the reaction between 2 and Cys,
In order to further demonstrate that the permeability
and the monitoring of thiols in living cells, confocal micro-
scopy experiments were carried out. When PC-12 cells were
c
8342 Chem. Commun., 2012, 48, 8341–8343
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R. A. M. Dhonukshe-Rutten, S. M. F. Pluijm, M. van der Klift,
R. de Jonge, J. Lindemans, L. G. M. de Groot, A. Hofman,
J. C. C. P. M. Witteman, J. P. T.M. van Leeuwen, M. M. B.
Breteler, P. Lips, H. A. P. Pols and A. G. Uitterlinden, N. Engl. J.
Med., 2004, 350, 2033.
5 S. Shahrokhian, Anal. Chem., 2001, 73, 5972.
6 (a) X. Chen, Y. Zhou, X. Peng and J. Yoon, Chem. Soc. Rev., 2010,
39, 2120; (b) Y. Zhou and J. Yoon, Chem. Soc. Rev., 2012, 41, 52;
(c) X. Chen, T. Pradhan, F. Wang, J. S. Kim and J. Yoon, Chem.
Rev., 2012, 112, 1910.
7 (a) L. Yi, H. Li, L. Sun, L. Liu, C. Zhang and Z. Xi, Angew. Chem.,
Int. Ed., 2009, 48, 4034; (b) H. S. Hewage and E. V. Anslyn, J. Am.
Chem. Soc., 2009, 131, 13099; (c) V. Hong, A. A. Kislukhin and
M. G. Finn, J. Am. Chem. Soc., 2009, 131, 9986; (d) X. Chen,
S.-K. Ko, M. J. Kim, I. Shin and J. Yoon, Chem. Commun., 2010,
46, 2751; (e) Y.-Q. Sun, M. Chen, J. Liu, X. Lv, J.-f. Li and
W. Guo, Chem. Commun., 2011, 47, 11029; (f) D. Kand,
A. M. Kalle, S. J. Varma and P. Talukdar, Chem. Commun.,
2012, 48, 2722; (g) H. Kwon, K. Lee and H.-J. Kim, Chem.
Commun., 2011, 47, 1773; (h) H. S. Jung, K. C. Ko, G.-H. Kim,
A.-R. Lee, Y.-C. Na, C. Kang, J. Y. Lee and J. S. Kim, Org. Lett.,
2011, 13, 1498; (i) G.-J. Kim, K. Lee, H. Kwon and H.-J. Kim,
Org. Lett., 2011, 13, 2799.
8 (a) K.-S. Lee, T.-K. Kim, J. H. Lee, H.-J. Kim and J.-I. Hong,
Chem. Commun., 2008, 6173; (b) X. Zhang, X. Ren, Q.-H. Xu,
K. P. Loh and Z.-K. Chen, Org. Lett., 2009, 11, 1257; (c) W. Lin,
L. Long, L. Yuan, Z. Cao, B. Chen and W. Tan, Org. Lett., 2008,
10, 5577; (d) P. Wang, J. Liu, X. Lv, Y. Liu, Y. Zhao and W. Guo,
Org. Lett., 2012, 14, 520; (e) M. Hu, J. Fan, H. Li, K. Song,
S. Wang, G. Cheng and X. Peng, Org. Biomol. Chem., 2011, 9, 980;
(f) X. Liu, N. Xi, S. Liu, Y. Ma, H. Yang, H. Li, J. He, Q. Zhao,
F. Li and W. Huang, J. Mater. Chem., 2012, 22, 7894; (g) L. Yuan,
W. Lin and Y. Yang, Chem. Commun., 2011, 47, 6275; (h) S. Lim,
J. O. Escobedo, M. Lowry, X. Xu and R. Strongin, Chem.
Commun., 2010, 46, 5707.
9 (a) M. M. Pires and J. Chmielewski, Org. Lett., 2008, 10, 837;
(b) B. Tang, L. Yin, X. Wang, Z. Chen, L. Tong and K. Xu, Chem.
Commun., 2009, 5293; (c) B. Tang, Y. Xing, P. Li, N. Zhang, F. Yu
and G. Yang, J. Am. Chem. Soc., 2007, 129, 11666; (d) W. Jiang,
Q. Fu, H. Fan, J. Ho and W. Wang, Angew. Chem., Int. Ed., 2007,
46, 8445; (e) H. Maeda, H. Matsuno, M. Ushida, K. Katayama,
K. Saeki and N. Itoh, Angew. Chem., Int. Ed., 2005, 44, 2922;
(f) S. Ji, J. Yang, Q. Yang, S. Liu, M. Chen and J. Zhao, J. Org.
Chem., 2009, 74, 4855; (g) J. Bouffard, Y. Kim, T. M. Swager,
R. Weissleder and S. A. Hilderbrand, Org. Lett., 2008, 10, 37;
(h) X. Cao, W. Lin and Q. Yu, J. Org. Chem., 2011, 76, 7423;
(i) M. H. Lee, J. H. Han, P.-S. Kwon, S. Bhuniya, J. Y. Kim,
J. L. Sessler, C. Kang and J. S. Kim, J. Am. Chem. Soc., 2012,
134, 1316; (j) L. Long, W. Lin, B. Chen, W. Gao and L. Yuan,
Chem. Commun., 2011, 47, 893; (k) S. Ji, H. Guo, X. Yuan, X. Li,
H. Ding, P. Gao, C. Zhao, W. Wu, W. Wu and J. Zhao, Org. Lett.,
2010, 12, 2876; (l) C. S. Lim, G. Masanta, H. J. Kim, J. H. Han,
H. M. Kim and B. R. Cho, J. Am. Chem. Soc., 2011, 133, 11132;
(m) H.-Y. Shiu, M.-K. Wong and C.-M. Che, Chem. Commun.,
2011, 47, 4367.
Fig. 3 Phase contrast and fluorescence images of PC-12 cells. Top:
PC-12 cells were treated with 50 mM of 1 for 30 min; bottom: PC-12
cells were pre-incubated with 500 mM NEM for 50 min and then
treated with 50 mM of 1 for 30 min (a and d, phase contrast images;
b and e, fluorescence images; c and f, merged images).
incubated with 1 (50 mM), strong fluorescence was exhibited
inside the cells (Fig. 3, top). However, in a control experiment,
cells were pre-treated with an excess (0.5 mM) of the thiol-
reactive N-ethylmaleimide (NEM), a trapping reagent for thiol
species, followed by treatment with 1. The confocal micro-
scopic images did not show a significant fluorescence signal
(Fig. 3, bottom). This confirms the specificity of 1 for thiols
over other analytes in living cells.
In summary, we have developed a new fluorescein-based
fluorescent probe for the detection of cysteine with high
selectivity and sensitivity. The fluorescence enhancement and
UV-vis spectral change are attributed to the addition of
thiol to acryloyl group in the probe followed by the cleavage
of ester bond to form the fluorescein. Compared with the
single acrylate-containing fluorescein derivate 2, the double
acrylate-containing sensor 1 indicated a remarkable enhance-
ment in selectivity to Cys over Hcy and GSH, owing to the
dual addition-cleavage processes in the reaction between 1
and Cys.
This work was supported by grants from NSFY of
China (21002049), the Natural Science Foundation of the
Jiangsu Higher Education Institutions of China (Grant
No. 10KJB150005), and the Opening Project of State Key
Laboratory of Chemo/Biosensing and Chemometrics of
Hunan University.
10 (a) B. Han, J. Yuan and E. Wang, Anal. Chem., 2009, 81, 5569;
(b) M. Zhang, M. Yu, F. Li, M. Zhu, M. Li, Y. Gao, L. Li, Z. Liu,
J. Zhang, D. Zhang, T. Yi and C. Huang, J. Am. Chem. Soc., 2007,
129, 10322; (c) Y. Fujikawa, Y. Urano, T. Komatsu, K. Hanaoka,
H. Kojima, T. Terai, H. Inoue and T. Nagano, J. Am. Chem. Soc.,
2008, 130, 14533; (d) H. S. Jung, J. H. Han, Y. Habata, C. Kang
and J. S. Kim, Chem. Commun., 2011, 47, 5142; (e) C. Luo,
Q. Zhou, B. Zhang and X. Wang, New J. Chem., 2011, 35, 45;
(f) L. Zhou, Y. Lin, Z. Huang, J. Ren and X. Qu, Chem. Commun.,
2012, 48, 1147.
11 (a) W. Wang, J. O. Escobedo, C. M. Lawrence and R. M. Strongin,
J. Am. Chem. Soc., 2004, 126, 3400; (b) H. Chen, Q. Zhao, Y. Wu,
F. Li, H. Yang, T. Yi and C. Huang, Inorg. Chem., 2007, 46, 11075;
(c) H.-P. Wu, C.-C. Huang, T.-L. Cheng and W.-L. Tseng,
Talanta, 2008, 76, 347; (d) H. Li, J. Fan, J. Wang, M. Tian,
J. Du, S. Sun, P. Sun and X. Peng, Chem. Commun., 2009, 5904;
(e) X. Yang, Y. Guo and R. M. Strongin, Angew. Chem., Int. Ed.,
2011, 50, 10690.
Notes and references
1 (a) J. B. Schulz, J. Lindenau, J. Seyfried and J. Dichgans, Eur. J.
Biochem., 2000, 267, 4904; (b) S. Seshadri, A. Beiser, J. Selhub,
P. F. Jacques, I. H. Rosenberg, R. B. D’Agostino, P. W. F. Wilson
and P. A. Wolf, N. Engl. J. Med., 2002, 346, 476; (c) R. O. Ball,
G. Courtney-Martin and P. B. Pencharz, J. Nutr., 2006,
136, 1682S; (d) R. Hong, G. Han, J. M. Fernandez, B.-J. Kim,
N. S. Forbes and V. M. Rotello, J. Am. Chem. Soc., 2006,
128, 1078.
2 I. Morgenstern, M. T. M. Raijmakers, W. H. M. Peters,
H. Hoensch and W. Kirch, Dig. Dis. Sci., 2003, 48, 2083.
3 I. M. W. Ebisch, W. H. M. Peters, C. M. G. Thomas, A. M. M.
Wetzels, P. G. M. Peer and R. P. M. Steegers-Theunissen, Hum.
Reprod., 2006, 21, 1725.
4 (a) H. Refsum, P. M. Ueland, O. Nygard and S. E. Vollset,
Annu. Rev. Med., 1998, 49, 31; (b) J. B. J. van Meurs,
c
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