A selective fluorescent probe for thiols based on α,β- unsaturated acyl sulfonamide

Article abstract of DOI:10.1039/c2cc35513b

We report herein a novel fluorescent probe based on α,β- unsaturated acyl sulfonamide to detect thiols. The probe has good water solubility and reacts with thiols under aqueous conditions. It reacts selectively with cysteine but not with the other natural amino acids. The probe was subsequently applied to detect intracellular thiols.

Full text of DOI:10.1039/c2cc35513b

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A selective fluorescent probe for thiols based on a,b-unsaturated acyl
sulfonamidew
Huatang Zhang, Ping Wang, Yanxin Yang and Hongyan Sun*
Received 8th June 2012, Accepted 7th September 2012
DOI: 10.1039/c2cc35513b
We report herein a novel fluorescent probe based on a,b-unsaturated
acyl sulfonamide to detect thiols. The probe has good water solubility
and reacts with thiols under aqueous conditions. It reacts selectively
with cysteine but not with the other natural amino acids. The probe
was subsequently applied to detect intracellular thiols.
the off–on fluorescent probes based on cleavage of sulfonamide
1
1
or sulfonate ester. Apart from these methods, other novel
approaches have also been developed for the detection of thiol
1
2
species recently. Despite the aforementioned advancement,
further development of synthetically simple and practical
probes for selective detection of thiols is still highly required
because of the broad functional properties of thiols within
complex biological systems.
Small molecule thiols play an important role in maintaining the
1
cellular redox environment. Thiol deficiency has been linked
Michael addition of thiols to a,b-unsaturated ketone has been
widely utilized in organic synthesis. The reaction is usually realized
under harsh conditions such as strong bases, high temperature or
addition of catalysts and organic solvents. Recently, Lin’s group
has reported a fluorescent probe based on a,b-unsaturated ketone
to selectively and sensitively detect thiols. Inspired by this result,
we envisioned that the a,b-unsaturated acyl sulfonamide group
might be used with fluorescent probes for the detection of thiols
under physiological conditions.
with a number of diseases including slow growth in children,
2
liver damage, AIDS, etc. High levels of thiols, on the other
3
hand, could lead to the progression of several other syndromes.
For instance, high concentrations of homocysteine (Hcy) are
associated with cardiovascular disease and Alzheimer’s disease.
It is therefore of growing importance and interest to develop
methods to detect thiols quantitatively and selectively. In
particular, the selectivity criterion for probe design is a critical
factor in detecting thiols within complex biological systems,
considering that there are thousands of other molecules that
might interfere with the biological analysis.
13
Herein we report a new and facile fluorescent probe for selective
detection of thiols. Our probe design employs a reaction unit with
the a,b-unsaturated acyl sulfonamide group. It was shown that
acrylamide reacts slower with thiols than the other electron-
A significant amount of effort has been made in the past to
develop various methods for quantitative measurements of
thiols, such as high performance liquid chromatography
1
4
deficient olefins, such as acrylonitrile and methyl acrylate. The
rate of this Michael-type addition, however, can be enhanced
through the installation of a suitable functional group to the
nitrogen of acrylamide. We reasoned that the reaction group in
our probe might possess improved reactivity towards thiols due to
the strong electron-withdrawing property of the sulfone group.
The design of probe 1 is based on a dansyl fluorophore, which
serves as a reporting unit. A carbon–carbon double bond is
introduced next to the dansyl group as a PET (photo-induced
electron transfer) donor. The fluorescence of probe 1 is effectively
quenched through the PET effect and can be consequently turned
on by reacting with a thiol-containing molecule.
4
5
6
(
HPLC), electrochemical methods, mass spectrometry and
7
capillary electrophoresis. Among all the methods developed,
a fluorescence-based method has emerged as a prominent
approach for quantification of thiols due to its high sensitivity
1
b
and easy implementation. To date, various types of organic
reactions have been employed to design fluorescence-based
probes. For example, cyclization reactions between aldehyde
and thiol groups were utilized by Strongin’s group and others
8
for sensitive detection of Cys and Hcy. Different maleimide-
based probes were employed for thiol detection through
9
Michael addition. Maleimide-based probes are known to
Synthesis of the probe can be easily carried out via two-step
routine synthesis. As depicted in Scheme 1, dansyl amide 2
could be obtained through nucleophilic substitution of the
starting material, dansyl chloride, by ammonia hydroxide.
Probe 1 was then synthesized through acylation of dansyl amide
using acryloyl chloride in 88% yield. The structural identifi-
possess excellent selectivity towards the thiol group. However,
the hydrolysis of maleimide to unreactive maleamic acid can
compete significantly with thiol addition, especially when pH
1
0
is above 8.0. Maeda, Hilderbrand and other groups designed
1
13
Department of Biology and Chemistry, City University of Hong Kong,
83 Tat Chee Avenue, Kowloon, Hong Kong, P. R. China.
E-mail: hongysun@cityu.edu.hk; Fax: +852 3442 0522;
Tel: +852 3442 9537
cation of the probe was further confirmed by H NMR,
C
NMR and ESI-MS. It is also noted that probe 1 is very stable
and highly soluble in water. It can be stored for several months
in methanol at À20 1C without deterioration. The synthesis of
w Electronic supplementary information (ESI) available. See DOI:
1
5
10.1039/c2cc35513b
probe 1 has been previously reported. However, to our best
1
0672 Chem. Commun., 2012, 48, 10672–10674
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Dilution study with probe 1 also indicated that as low as
À6
0
.625 Â 10 M of probe 1 could be used in the buffer to detect
around 7 fold signal increase compared with the probe itself
Fig. S4, ESIw). These experiments clearly demonstrated that
our probe is quite sensitive to cysteine and can be applied to
(
quantitative measurement of thiol concentration.
To acquire a better understanding of the reaction kinetics of
probe 1, we also carried out time course experiments. Probe 1
(20 mM) was incubated with 10 eq. of cysteine in PBS buffer
Scheme 1 Synthetic scheme of the target probe. (a) NH
h; (b) acryloyl chloride, TEA, DCM, 4 h.
3
ÁH
2
O, reflux,
1
(
pH = 7.4) at 37 1C. The fluorescence spectra of the resulting
knowledge there has been no report that studied its reactivity
towards thiols.
solution were measured at various time intervals. The fluorescence
signal at 520 nm was plotted as a function of time (Fig. S5, ESIw).
The initial rate of the reaction was high. It went through a gradual
increase and finally reached the maximum intensity after
around 5 hours. On the other hand, it was observed that the
fluorescence intensity of the probe without addition of cysteine
displayed no noticeable change under the same conditions.
The selectivity towards the thiol group is one of the most
important criteria for probe design. A probe of biological
importance must be able to discriminate the thiol group from
other nucleophiles under physiological conditions. The selectivity
test was performed by incubating the probe with 20 natural
amino acids (20 eq.) in PBS buffer (pH = 7.4) under the same
conditions. As shown in Fig. 2, a significant increase in fluores-
cence intensity was only observed with the cysteine sample. The
other 19 amino acid samples exhibited no noticeable increase of
the fluorescence signal. These experiments implied that the probe
is highly selective towards the thiol group but not towards other
biologically relevant nucleophiles such as amino and imidazole
groups. We also studied the reactivity of the probe towards other
biothiols such as glutathione (GSH) and homocysteine (Hcy).
Data analysis indicated that the reactivity of probe 1 increases in
In the initial stage of the study, we verified whether the probe
could react with thiol-containing small molecules. b-Mercapto-
ethanol was used in our preliminary experiment. A greenish
solution could be observed by the naked eye after incubating the
probe with b-mercaptoethanol in PBS buffer (Fig. S1, ESIw). In
contrast, the probe without the addition of b-mercaptoethanol
displayed almost no fluorescence under the same conditions.
To further our understanding of the reaction mechanism, we
performed detailed characterization experiments with the reaction
products of the probe and b-mercaptoethanol. Mass spectrum
À
analysis of the product showed peaks at m/z 381.3 [M À H] ,
which corresponds to the molecular weight of the b-mercapto
ethanol adduct. Further H NMR spectrum analysis also
1
revealed the disappearance of the resonance signal after the
probe reacted with b-mercaptoethanol between 5.6 ppm and
6.4 ppm, which is the characteristic peak of carbon–carbon double
bond protons (Fig. S2, ESIw). The results we have obtained clearly
indicate that the fluorescence increase of the probe is attributed to
the formation of thiol adducts via Michael addition.
Encouraged by these results, we moved forward to study the
effect of thiol concentration on the fluorescence signal. Different
concentrations of cysteine ranging from 0 eq. to 20 eq. were
incubated with the probe. As shown in Fig. 1, a gradual increase
of the fluorescence signal was observed with increasing concentra-
tions of cysteine. A strong emission peak at 520 nm was observed
when the reaction mixture was excited at 350 nm. Around 30-fold
fluorescence enhancement could be detected when the reaction was
complete. Subsequent data analysis revealed an excellent linear
relationship (R = 0.9945) between the normalized fluorescence
signal at 520 nm and the cysteine concentration. The detection limit
for cysteine was determined to be 1.67 Â 10À6 M (Fig. S3, ESIw).
the following sequence: Hcy o GSH o Cys. There has been
16
2 2
strong interest recently in developing probes to detect H S. H S
is a better nucleophile than thiols under physiological conditions.
In our study, we use Na S or NaHS to examine whether probe 1
2
could react with H S. We observed a fluorescence increment of
2
around 3-fold after 12-hour incubation. It is quite low compared
with the cysteine sample (Fig. S6, ESIw).
Many probes can only function properly within a narrow
pH window. It is therefore also important to investigate the
12
appropriate pH range for the probe to function. For this purpose,
we performed the reaction using a series of buffers with
different pH values. The results indicated that the response
Fig. 1 Fluorescence emission spectra of probe 1 in PBS buffer (pH = 7.4)
with addition of increasing concentrations of cysteine ([probe 1] = 20 mM,
Fig. 2 Selectivity experiments of 20 mM probe 1 assayed with 20
natural amino acids, GSH and Hcy in PBS buffer (pH = 7.4).
l
Ex = 350 nm, lEm = 450–650 nm).
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The reaction can take place in aqueous buffer without using
organic solvents and additional catalysts. More importantly,
the probe could selectively detect thiols rather than other strong
nucleophiles such as amines. We also proved that the probe could
be applied to detect thiols in live cells. On account of the above
experimental results, we believe that this probe would provide a
useful tool for quantifying thiols for biomedical analysis.
The author would like to thank for the financial support from
the City University of Hong Kong Start-up Grant (7200258).
Notes and references
Fig. 3 pH dependent experiments of probe 1. The reaction of probe 1
20 mM) with different amino acids (200 mM) was performed with
buffers at different pH values. The fluorescent intensity was measured
after overnight incubation.
(
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Fig. 4 Fluorescence microscopy experiments to detect thiol in A549
cells. Fluorescence image obtained (a) immediately after addition of
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maximum fluorescence signal was observed in the pH range
of 6 to 10 (Fig. S7, ESIw). There was no reaction spotted with
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the pH interval 6–10 (Fig. 3). From these results, it is evident
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To evaluate the function of the probe for biological applica-
tions, we performed an assay to detect intracellular thiols in
live cells. A549 cells were used in our study. As shown in
Fig. 4, there was no fluorescence detected with A549 cells upon
addition of the probe (Fig. 4a). After incubating with probe 1
for different time intervals, increasing levels of green fluores-
cence could be observed inside the cells under a fluorescence
microscope. The images also indicated that probe 1 was
mainly localized in the cytoplasm. Similar observations were
1
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(Fig. S8, ESIw). The method described here might provide
a simple way to monitor alterations of thiol concentration
1
1
5 S. Takahashi, Japanese Patent JP02003448, 1990.
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in live cells.
In summary, we have developed a new fluorescent probe for
selective detection of thiols based on 1,4-Michael addition.
17 H. Kwon, K. Lee and H. J. Kim, Chem. Commun., 2011, 47, 1773.
This journal is c The Royal Society of Chemistry 2012
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0674 Chem. Commun., 2012, 48, 10672–10674