A colorimetric and fluorescent chemodosimeter for discriminative and simultaneous quantification of cysteine and homocysteine

Article abstract of DOI:10.1016/j.dyepig.2012.05.006

A highly selective, visible-light-excited and OFF-ON fluorescent chemodosimeter 1 employed conjugate addition/cyclization sequence mechanism, was designed and synthesized to discriminatively detect cysteine (Cys) and homocysteine (Hcy). The addition of Cys and Hcy resulted in the color of the solution of 1 changing from colorless to green under the simulation of physiological condition, and 1 could serve as a naked-eye indicator. Our chemodosimeter can detect Cys and Hcy quantitatively by fluorescence spectrometry method with a detection limit of 0.5 μM (for Cys) and 0.8 μM (for Hcy). To the best of our knowledge, 1 is the first visual and visible-light-excited fluorescent indicator for discriminative and simultaneous detection of Cys and Hcy. Furthermore, the mechanism of the reaction between 1 and Cys was confirmed using ESI-MS and fluorescence spectra.

Full text of DOI:10.1016/j.dyepig.2012.05.006

Dyes and Pigments 95 (2012) 275e279
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Dyes and Pigments
journal homepage: www.elsevier.com/locate/dyepig
A colorimetric and fluorescent chemodosimeter for discriminative and
simultaneous quantification of cysteine and homocysteine
,
a
a
a
b,
**
Liguo Wang a, Qi Zhou a, Baocun Zhu a , Liangguo Yan , Zhenmin Ma , Bin Du , Xiaoling Zhang
*
a School of Resources and Environment, University of Jinan, Jinan 250022, China
b Key Laboratory of Cluster Science of Ministry of Education, Department of Chemistry, School of Chemistry, Beijing Institute of Technology, Beijing 100081, China
a r t i c l e i n f o
a b s t r a c t
Article history:
A highly selective, visible-light-excited and OFFeON fluorescent chemodosimeter 1 employed conjugate
addition/cyclization sequence mechanism, was designed and synthesized to discriminatively detect
cysteine (Cys) and homocysteine (Hcy). The addition of Cys and Hcy resulted in the color of the solution
of 1 changing from colorless to green under the simulation of physiological condition, and 1 could serve
as a “naked-eye” indicator. Our chemodosimeter can detect Cys and Hcy quantitatively by fluorescence
Received 2 April 2012
Received in revised form
7 May 2012
Accepted 10 May 2012
Available online 16 May 2012
spectrometry method with a detection limit of 0.5 mM (for Cys) and 0.8 mM (for Hcy). To the best of our
knowledge, 1 is the first visual and visible-light-excited fluorescent indicator for discriminative and
simultaneous detection of Cys and Hcy. Furthermore, the mechanism of the reaction between 1 and Cys
was confirmed using ESI-MS and fluorescence spectra.
Keywords:
Colorimetric
Fluorescent chemodosimeter
Cysteine
Ó 2012 Elsevier Ltd. All rights reserved.
Homocysteine
Simultaneous detection
Fluorescein
1. Introduction
reported the first conjugate addition/cyclization sequence chemo-
dosimeter for the selective and simultaneous quantitative detec-
Amino acids are of significant importance to the physiological
processes [1]. Among them, the thiol-containing amino acids, such
as cysteine (Cys) and homocysteine (Hcy), play crucial roles in
maintaining the biological redox homeostasis for their participa-
tion in the process of reversible redox reactions, and their levels
have been directly linked to some diseases and cancers [2e6]. For
example, a depressed level of Cys is associated with slowed growth,
hair depigmentation, edema, lethargy, liver damage, muscle and fat
loss, skin lesions, and weakness [7e9], and an elevated level of Hcy
is a risk factor for Alzheimer’s and cardiovascular diseases [10e12].
Thus, the selective and quantitative detection of Cys and Hcy is very
important.
Among the various reported analytical techniques for the
detection of Cys and Hcy, fluorescent chemodosimeters are widely
developed due to operational simplicity and high sensitivity
[13e23]. However, up to now, few reports on the highly selective
fluorescent chemodosimeters for Cys and/or Hcy have been pub-
lished [24e27]. Very recently, based on the kinetic differences in
the intramolecular cyclization reactions, Yang and Strongin
tion of Cys and Hcy [11]. The use of the long excitation and emission
wavelength may eliminate or decrease background interference
from biological samples and its damage to living cells. In addition,
colorimetric chemodosimeters are widely developed because they
have the capability to detect analytes by naked-eye, without the aid
of any advanced instruments [28e34]. Therefore, colorimetric and
long wavelength fluorescent chemodosimeters for the selective and
simultaneous determination of Cys and Hcy become our target.
Herein, we present the design, synthesis and properties of
a highly selective colorimetric and visible-light-excited fluorescent
chemodosimeter for simultaneous determination of Cys and Hcy.
Chemodosimeter 1 is composed of a latent fluorescein fluorophore
and a receptor of acrylate (Scheme 1), and it becomes highly fluo-
rescent upon the spirolactone-opening reaction by Cys and Hcy,
accompanied with the color changing from colorless to green.
2. Material and methods
2.1. General
* Corresponding author. Tel.: þ86 531 82769235; fax: þ86 531 82765969.
** Corresponding author.
All the chemicals used in this paper were commercial products
of analytic grade. 1H NMR and 13C NMR spectra were taken on
E-mail addresses: lcyzbc@163.com (B. Zhu), zhangxl@bit.edu.cn (X. Zhang).
0143-7208/$ e see front matter Ó 2012 Elsevier Ltd. All rights reserved.
doi:10.1016/j.dyepig.2012.05.006

276
L. Wang et al. / Dyes and Pigments 95 (2012) 275e279
Scheme 1. Synthesis of chemodosimeter 1.
a Bruker AMX400 spectrometer. Chemical shifts (
d
) were reported
the second-order rate constant for Cys is 25-fold faster than that for
Hcy. The results are in good agreement with the conclusion
reported by Yang and Strongin [11], and these allowed the
discrimination of Cys and Hcy based on their different relative rates
of intramolecular cyclization.
in ppm relative to a Me4Si standard in CDCl3. Electrospray ioniza-
tion (ESI) mass spectra were measured with an LC-MS 2010A
(Shimadzu) instrument. Absorption spectra were recorded on UV-
3101PC spectrophotometer. Fluorescence emission spectra were
measured on PerkineElmer Model LS-55. All pH measurements
were made with a Sartorius basic pH-meter PB-10.
3.3. Quantification of Cys and Hcy
2.2. Synthesis of chemodosimeter 1
The subsequent addition of Cys to the solution of 1 elicited
a progressive increase of fluorescence band centered at around
520 nm. Moreover, there was a good linearity between the
To a solution of fluorescein (332.3 mg, 1 mmol) and triethyl-
amine (303.5 mg, 3 mmol) in anhydrous CH2Cl2 (20 mL), acryloyl
chloride (200.2 mg, 2.2 mmol) mixed with 3 mL anhydrous CH2Cl2
were added dropwise at ꢀ10 ꢁC. And then, the resulting mixture
was allowed to stir at room temperature for 8 h. After removal of
solvent, the residues were purified by silica gel column chroma-
tography using dichloromethane as eluent to afford pure product.
1H NMR (400 MHz, CDCl3)
d
(*10ꢀ6): 6.06(d, J ¼ 10.4 Hz, 2H),
6.28e6.35(m, 2H), 6.63(d, J ¼ 17.2 Hz, 2H), 6.86(t, J ¼ 9.8 Hz, 4H),
7.15(s, 2H), 7.21(d, J ¼ 7.6 Hz, 1H), 7.63e7.72(m, 2H), 8.04(d,
J ¼ 7.6 Hz, 1H). 13C NMR (100 MHz, CDCl3)
d
(*10ꢀ6): 81.66, 110.38,
116.55, 117.72, 124.08, 125.24, 126.13, 127.50, 128.97, 130.07, 133.31,
133.37, 135.30, 151.60, 152.00, 152.93, 163.88, 163.91, 169.11, 169.14.
ESI-MS calcd for C26H17O7 [M þ H]þ 441.1, found 441.1.
3. Results and discussion
3.1. Characteristic spectrum
In this paper, the spectral responses of chemodosimeter 1
(2
mM) toward Cys and Hcy were investigated in a mixture of
ethanol and water (2:3, v/v) solution buffered at pH 7.4 (phosphate
buffer, 20 mM).
As shown in Fig.1, the obvious absorption and fluorescence band
of the solution of free 1 were not observed. Addition of Cys (16
mM)
and Hcy (20 M) to the solution of 1 resulted in a new absorption
m
band centered at around 495 nm, accompanied with the color of
the solution of 1 changing from colorless to green (Fig. 1a). Addi-
tionally, in the fluorescence spectra (Fig. 1b), a remarkable fluo-
rescent enhancement at around 520 nm was observed in the
presence of Cys or Hcy. The results imply that spirolactone-opening
reaction was generated by Cys and Hcy (Scheme 2).
3.2. Effects of reaction time on sensing Cys and Hcy
Reaction time is an important factor for chemodosimeters and
the time required for reaction of 1 with Cys and Hcy at 25 ꢁC were
investigated. As shown in Fig. 2, the fluorescence intensity
increases with reaction time and then almost levels off at reaction
time greater than about 50 min and 400 min for Cys and Hcy,
respectively. So, assay time of 60 min and 420 min were selected for
the quantification of Cys and Hcy. Furthermore, we have investi-
gated the kinetics of fluorescence enhancement for Cys and Hcy at
Fig. 1. Absorbance (a) and fluorescence (b) spectra of 1 (2
mM) upon reaction with Cys
(16 M) and Hcy (20 M) in a mixture of ethanol and water (2:3, v/v) solution buffered
m
m
at pH 7.4 (phosphate buffer, 20 mM). Inset photos are the photographs of the solution
of 1 in the absence (left) and presence (right) of Cys. For Cys, its spectra were acquired
60 min after Cys addition at 25 ꢁC. For Hcy, its spectra were acquired 420 min after Hcy
addition at 25 ꢁC.
different concentrations (5, 10, 15, 20, 25 mM). The result showed

L. Wang et al. / Dyes and Pigments 95 (2012) 275e279
277
Scheme 2. A proposed reaction mechanism of chemodosimeter 1 with Cys and Hcy.
fluorescence intensity and the concentrations of Cys in the range of
2e25 M with a detection limit of 0.5 M (Fig. 3a). Similarly, the
observed fluorescence intensity is proportion to the concentrations
of Hcy in the range of 0e20 M with a detection limit of 0.8 mM
m
m
m
(Fig. 3b). These results implied that chemodosimeter 1 might
detect Cys and Hcy qualitatively and quantitatively by the fluores-
cence spectrometry method.
To discriminate Cys and Hcy, the fluorescence spectra were
determined after the addition of Cys and Hcy for 60 min and
420 min, respectively. The results show that chemodosimeter 1
could allow the discrimination and quantification of Cys and Hcy
(Fig. 3c).
3.4. Selectivity to Cys and Hcy
The effects of some relative amino acids, such as Cys, Hcy,
glutathione (GSH), glycine (Gly), leucine (Leu), alanine (Ala),
glutamine (Glu), lysine (Lys), threonine (Thr), valine (Val), and
praline (Pro), on fluorescence spectra of 1 were investigated.
Remarkable changes were observed for Cys and Hcy (Fig. 4). Also,
the effects of interference of other relative amino acids on moni-
toring Cys and Hcy were studied (Fig. 4). GSH obviously disturb the
detection of Cys and Hcy for its addition reaction with
carbonecarbon double bond of chemodosimeter 1. Further, the
effects of GSH on monitoring Cys and Hcy in the presence of excess
chemodosimeter 1 were investigated. The primary experimental
results showed that the interference of GSH may be ignored.
Therefore, chemodosimeter 1 possesses high selectivity toward Cys
and Hcy in the presence of other amino acids.
3.5. Mechanism of 1 in sensing Cys and Hcy
To confirm the reaction of 1 with Cys and Hcy by conjugate
addition/cyclization sequence, a mixture of N-ethylmaleimide
(NEM, a known thiol-blocking agent) and Cys was added to the
solution of 1, no obvious change in the fluorescence spectra was
observed (Fig. 5), implying the addition reaction of 1 with thiol of
Cys. Next, to demonstrate the reaction of 1 with Cys by the
subsequent cyclization reaction, addition of thioglycolic acid (TA)
did not elicit a remarkable fluorescence change. However, addition
Fig. 2. The fluorescence spectra of 1 (2
Hcy (25 M) (b) during different time in a mixture of ethanol and water (2:3, v/v)
solution buffered at pH 7.4 (phosphate buffer, 20 mM), lex ¼ 450 nm.
mM) after the addition of Cys (25 mM) (a) and
m

278
L. Wang et al. / Dyes and Pigments 95 (2012) 275e279
Fig. 4. The fluorescence responses of 1 (2
mM) toward Cys (a) and Hcy (25
mM) (b) in
the absence and presence of other amino acids (final concentration: 25
mM, GSH: 5
m
M)
in a mixture of ethanol and water (2:3, v/v) solution buffered at pH 7.4 (phosphate
buffer, 20 mM), lex ¼ 450 nm.
of 2-aminoethanethiol (TEA), as expected, resulted in a clear fluo-
rescence enhancement. Furthermore, the reaction products of 1
with Cys were subjected to electrospray ionization mass spectral
analyses. The peak at m/z 331 corresponding to the fluorescein was
observed. Therefore, a proposed mechanism was proposed as
shown in Scheme 2.
Fig. 3. (a) The fluorescence responses of 1 (2
mM) toward different concentrations of
Cys (final concentration: 2, 4, 6, 8, 10, 14, 20, 25
mM) in a mixture of ethanol and water
(2:3, v/v) solution buffered at pH 7.4 (phosphate buffer, 20 mM), lex ¼ 450 nm. Inset is
the plot of fluorescence intensity at 520 nm vs concentration of Cys. Each spectrum was
acquired 60 min after Cys addition at 25 ꢁC. (b) The fluorescence responses of 1 (2
m
M)
toward different concentrations of Hcy (final concentration: 0, 2, 6, 8, 10, 12, 14, 18,
20 M) in a mixture of ethanol and water (2:3, v/v) solution buffered at pH 7.4
m
(phosphate buffer, 20 mM), lex ¼ 450 nm. Inset is the plot of fluorescence intensity at
520 nm vs concentration of Hcy. Each spectrum was acquired 420 min after Hcy
addition at 25 ꢁC. (c) The fluorescence responses of 1 (40
mM) in the presence of 25
mM
Cys toward different concentrations of Hcy (final concentration: 2, 4, 8, 10, 12 M) in
m
a mixture of ethanol and water (2:3, v/v) solution buffered at pH 7.4 (phosphate buffer,
20 mM), lex ¼ 450 nm. The plot of the changes of fluorescence intensity at 520 nm
from 60 min to 420 min vs concentration of Hcy.
Fig. 5. The fluorescence spectra of 1 (2
thioglycolic acid (TA) (25 M), 2-aminoethanethiol (TEA) (25
solution of Cys (25 M) with NEM (1 mM) in a mixture of ethanol and water (2:3, v/v)
solution buffered at pH 7.4 (phosphate buffer, 20 mM), lex ¼ 450 nm.
m
M) in the absence and presence of Cys (25
mM)
m
m
M), and a pretreated
m

L. Wang et al. / Dyes and Pigments 95 (2012) 275e279
279
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4. Conclusions
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In summary, we have presented the synthesis and properties of
a highly selective visible-light-excited fluorescent chemodosimeter
1 for discrimination and simultaneous quantification of Cys and
Hcy. The color of the solution of 1 changing from colorless to green
and remarkably enhanced fluorescence upon reaction of Cys and
Hcy were observed, and 1 could serve as a “naked-eye” indicator for
Cys and Hcy. Furthermore, 1 can detect Cys and Hcy quantitatively
in the range of 2e25 mM (for Cys) and in the range of 0e20 mM (for
Hcy). Finally, the mechanism of 1 on sensing Cys and Hcy was
confirmed using ESI-MS and fluorescence spectra.
Acknowledgments
We thank the National Natural Science Foundation of China (No.
21107029, 21175057, 21075052, 20975012, and 40672158), National
Major Projects on Water Pollution Control and Management
Technology (2008ZX07422) and National Science and Technology
Major Project (2009ZX07212-003) for financial support.
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