A colorimetric and fluorescent Merocyanine-based probe for biological thiols

Article abstract of DOI:10.1039/b909760k

A new "dual-mode" chromogenic and fluorescent turn-on probe (2) for the selective sensing of biological thiols is reported. In MeOH-H ₂O cosolvent at physiological pH 7.40 (MeOH-H₂O = 3:7), biological thiols cleave the 2,4-dinitroben

Full text of DOI:10.1039/b909760k

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www.rsc.org/obc | Organic & Biomolecular Chemistry
A colorimetric and fluorescent merocyanine-based probe for biological thiols†
Shu-Ping Wang, Wu-Jian Deng, Dan Sun, Min Yan, Hong Zheng* and Jin-Gou Xu
Received 19th May 2009, Accepted 9th July 2009
First published as an Advance Article on the web 10th August 2009
DOI: 10.1039/b909760k
A new “dual-mode” chromogenic and fluorescent turn-on probe (2) for the selective sensing of
biological thiols is reported. In MeOH–H2O cosolvent at physiological pH 7.40 (MeOH–H2O = 3:7),
biological thiols cleave the 2,4-dinitrobenzenesulfonyl group to release the chromo- and fluorophore
merocyanine (3).
and “turn-on” type of fluorescence and absorbance response under
Introduction
physiological conditions.
Recently, particular interest has been focused on the chemosensing
In this context, we have designed a merocyanine-containing
of sulfhydryl-containing amino acids and peptides, as they play
chemodosimeter responsible for the thiol-mediated nucleophilic
many crucial roles in biological systems.1–3 For example, the
thiol group in cysteine (Cys) residues is involved in the three-
dimensional structures of proteins through disulfide bond for-
reaction which involves a cleavage event leading to irreversible and
unique color changes. Merocyanine dyes are conjugate p-electron
systems with “push–pull” substituent pairs. Such molecules show
strong intramolecular charge transfer (ICT),8 and so the merocya-
nine unit is very useful because both its color and fluorescence
changes have a significant response to the electronic effect of
the substrate in the event of the thiol attack. We can envision
that masking the merocyanine chromophore by an electron-
withdrawing group such as the dinitrobenzenesulfonyl group
(DNS) would significantly change its ICT process. Reaction with
this protected cyanine dye would then result in dual-mode signals
(color change and fluorescence variation) that would be convenient
and sensitive enough for practical utilization.
Therefore, a merocyanine chromophore, 3, was first appended
to the DNS group to gain the target probe 2 (Scheme 1), which
“switches off ” the color and fluorescence of the dye. Then the DNS
group can be removed by a well-known thiol-mediated cleavage by
an SNAr process (Scheme 2),6d,7a liberating the free merocyanine
chromophore. This leads to an obvious visual color change as
well as an “off–on” type of fluorescent sensing. To the best of
our knowledge, this is the first report that demonstrates the use of
merocyanine dyes for the detection of biological species.
mation, and plasma levels of glutathione (GSH), cysteine (Cys),
and homocysteine (Hcy) have been linked to various human
diseases.4 Consequently, selective detection and quantification of
the biological thiols is of growing importance.
Classical determination of biological thiols in a chemical
system is generally accomplished by high-performance liquid
chromatography combined with Ellman’s reagent;5 this, however,
is inconvenient and requires expensive equipment. Selective optical
sensing can overcome the drawbacks of HPLC owing to the use
of “low-tech” spectroscopic instrumentation to assay relevant
chemical species in biological and environmental processes. There-
fore, several colorimetric or fluorescent chemosensors for thiol-
containing amino acids have been developed.6 The signal export
of these probes clearly only relates to their fluorescence changes
or UV band shifts upon thiol species introduction. A probe
with a dual-mode sensing behaviors, i.e., a color change together
with fluorescence enhancement, would combine the sensitivity
of fluorescence with the convenience and aesthetic appeal of
a colorimetric assay, but to date, dual-mode probes for thiol-
containing amino acids have been rather rare.7 Therefore, there is
still plenty of room to devise selective and sensitive chemosensors
for thiol-containing amino acids that show a useful color change
Experimental
NMR spectra in DMSO-d6 were recorded on Bruker AV400
NMR spectrophotometer. Mass spectra were measured with
a JEOL JMS-T100LC mass spectrometer (ESI+). Uncorrected
fluorescence emission spectra were conducted on a Hitachi F-4500
luminescence spectrometer. Absorption spectra were determined
on a Beckman DU-7400 spectrophotometer. Doubly distilled
Department of Chemistry and The Key Laboratory of Analytical Sciences of
MOE, College of Chemistry and Chemical Engineering, Xiamen University,
Xiamen, 361005, P. R. China. E-mail: hzheng@xmu.edu.cn
† Electronic supplementary information (ESI) available: UV–vis spectra
of 2 with Hcy, GSH and fluorescence spectra of 2 with Cys and Hcy, and
NMR spectra of compounds 1–3. See DOI: 10.1039/b909760k
Scheme 1 Synthesis of 2.
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Scheme 2 Cleavage of 2 with thiols.
water was used throughout the experiments. All the materials for
synthesis were purchased from commercial suppliers and used
without further purification. Reactions were monitored by TLC
with visual observation of the dye spots.
tion, and then the solvent was removed under reduced pressure.
The crude product was purified by column chromatography on
silica gel using MeOH/CHCl3 as the mobile phase to give free dye
3 (26.5 mg, 53%).
1H NMR (d6-DMSO, 400 MHz) d 1.770 (s, 6H), 4.081 (s, 3H),
6.96 (d, J = 8.8 Hz, 2H), 7.463 (d, J = 16.4 Hz, 1H), 7.559–
7.634 (m, 2H), 7.827–7.855 (m, 2H), 8.123 (d, J = 8.4 Hz, 2H),
8.363 (d, J = 16 Hz, 1H), 10.822 (s, 1H). 13C NMR (d6-DMSO,
100 MHz) d 26.143 34.522, 52.196, 109.789, 115.134, 116.901,
123.261, 126.477, 129.223, 129.324, 134.060, 142.346, 143.665,
154.215, 163.699, 181.871. ESI MS, m/z: 278.2 (3 - I)+.
Synthesis of 2,4-dinitrophenyl 4-formylphenyl sulfate (1)
A mixture of 4-hydroxybenzaldehyde (1.0 g, 3.75 mmol), 2, 4-
dinitrobenzenesulfonyl chloride (DNSCl, 0.458 g, 3.75 mmol) and
K2CO3 (0.62 g, 4.49 mmol) in dry acetonitrile (12 mL) was stirred
at room temperature for 5.0 hours. The solvent was removed under
reduced pressure, the residue was added with saturated Na2CO3
solution, and the product extracted with ethyl acetate, washed
with brine and dried over anhydrous MgSO4. Evaporation of the
solvents under reduced pressure yielded 0.82 g (60% yield) of
crude product, which was used in the next step without further
purification.
1H NMR: (400 MHz, DMSO-d6, ppm) d 10.01 (s, 1H, -CHO),
9.13 (s, 1H, ArH), 8.61 (d, J = 8.70, 1H, ArH), 8.29 (d, J = 8.68,
1H, ArH), 8.00 (d, J = 7.89, 2H, ArH), 7.44 (d, J = 7.95, 2H, ArH).
13C NMR: (100 MHz, DMSO-d6, ppm) d 191.87, 152.15, 151.55,
148.02, 135.48, 133.56, 131.74, 130.46, 127.53, 122.84, 121.11. ESI
MS, m/z: 352.3 (1).+
General procedure for spectroscopic measurements
A 1.0 ¥ 10-3 M stock solution of compound 2 was prepared in
MeOH. To 10-mL glass tubes containing 1.0 mL of 100 mM
phosphate buffer (pH 7.40) and different amounts of amino acids,
were added redistilled water to achieve a volume of 7.0 mL, then
3.0 mL of MeOH. The resulting solution were mixed thoroughly.
Then 0.10 mL of 1.0 ¥10-3 M stock solution of compound 2 was
added directly with a micropipette, the solutions were mixed again,
and the absorption and fluorescence sensing of aminoacids were
run after 15 minutes.
Synthesis of (E)-2-(4-(2,4-dinitrophenylsulfonyloxy)styryl)-1,3,3-
trimethyl-3H-indolium iodide (2)
Results and discussion
Compound 1 (0.439 g, 1.19 mmol) was dissolved in 10 mL of acetic
anhydride, then 1,2,3,3-tetramethyl-3H-indolium iodide (0.2994 g,
0.0994 mmol) and sodium acetate (0.0816 g, 0.0994 mmol) was
added. Stirring was continued at room temperature for 12 hours,
then 20 mL of diethyl ether was added to the solution, and a
brown precipitate was obtained. The precipitate was filtered and
washed with redistilled water and cold acetonitrile for three times,
respectively. Drying the precipitate in vacuum afforded 0.38 g of
yellow product (50.5%).
Compound 2 was synthesized easily by a two-step reaction
(Scheme 1). The intermediate compound 1 was synthesized from
4-hydroxybenzaldehyde with DNS-Cl, and 2 was prepared by
coupling 1,2,3,3-tetramethyl-3H-indolium iodide with 1 in 50.5%
yield.
For fully dissolved probe 2 in aqueous solutions, an methanol–
water (3:7, v/v) with pH 7.40 phosphate buffer solution (PBS)
was optimal for the spectroscopic investigations. The reaction of 2
with Cys was first studied by UV-Vis spectroscopic titration. The
spectral changes during the titration of Cys are shown in Fig. 1.
Compound 2 exhibits an absorption maximum at 380 nm
(e ª 21000 M-1 cm-1), which is responsible for the slight yellow
color of the solution. In the presence of Cys, the absorbance
at 380 nm decreased dramatically while a new band at 530 nm
developed with the absorbance increasing linearly with increasing
1H NMR: (400 MHz, DMSO-d6, ppm) d 9.15 (d, J = 2.0 Hz,
=
1H), 8.63 (dd, J = 8.8 Hz, 2.4, 1H), 8.40 (d, J = 16.4, 1H, -CH CH-
), 8.31–8.29 (m, 3H), 7.95–7.89 (m, 2H), 7.70 (d, J = 16.4 Hz, 1H,
=
-CH CH-), 7.67–7.64 (m, 2H), 7.430 (d, J = 8.8 Hz, 2H), 4.17 (s,
3H, -CH3), 1.78 (s, 6H, -CH3). 13C NMR: (100 MHz, DMSO-d6,
ppm) d 181.72, 151.47, 150.90, 150.26, 148.04, 143.68, 141.68,
134.34, 133.57, 132.33, 130.50, 129.67, 128.94, 127.51, 122.78,
121.11, 115.44, 114.70, 52.33, 34.87, 24.92. ESI MS, m/z: 508.3
(2 - I)+.
the concentration of Cys from 1.0 ¥ 10-7 M to 8.0 ¥ 10-5
M
(linear correlation coefficient of 0.9976) (Fig. 1), the relatively
large molar absorptivity at 530 nm (e ª 78000 M-1 cm-1) may be
responsible for this high spectrophotometric detective sensitivity.
Such a huge red-shift of 150 nm in absorption behavior changes
the color of the resultant solution from slight yellow into orange,
allowing “naked-eye” detection. Similar experimental phenomena
were also observed when we added Hcy or GSH into the solution
of 2 (Fig. S1 and S2†).
Synthesis of 3 by reaction of 2 with thioglycolic acid
To a stirred solution of 2 (50 mg, 0.18 mmol) in MeOH (2.0 mL)
were added HSCH2CO2H (21 mg, 0.23 mmol) and Et3N (35 mg,
0.36 mmol). Stirring was continued at room temperature for
2–3 h. Concentrated HCl (0.3 mL) was added to acidify the solu-
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Fig. 2 Absorbance spectra of 2 in pH 7.40 of phosphate buffer solution
(MeOH–H2O = 3:7, v/v) in the presence of 0.8 equiv. of Hcy, 0.4 equiv. of
Cys, GSH and other amino acids. Amino acids containing: Gly, Ser, Asp,
Met, Asn, Lys, Tyr, Trp, Arg, and His. [2] = 2.0 ¥ 10-5 M.
Fig. 1 UV–Vis spectra of 2 in pH 7.40 of phosphate buffer solution
(MeOH–H2O = 3:7, v/v) in the presence of Cys. Inset: plot of absorbance
at 530 nm vs. concentration of Cys. [2] = 2.0 ¥ 10-5 M. [Cys] = 0, 0.1, 0.2,
0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 2.0, 3.0, 4.0, 5.0, 6.0, 7.0 and 8.0 mM,
respectively.
We noticed that the final absorption spectra of the reaction
system composed of Cys and 2, and that of free merocyanine 3 in
the same buffer solution, were essentially identical, indicating the
release of 3 in the reaction (Fig. S3). Meanwhile, in the titration
traces, a clear isosbestic point at 436 nm was observed, which
was attributed to the formation of the 3 in equilibrium with
2. Moreover, the validity of the proposed product was further
confirmed by the synthesis of the free dye 3 by reaction of 2 with
thioglycolic acid. These facts supported the proposed reaction
shown in Scheme 2.
Fig. 3 The color change of 2 in pH 7.40 of phosphate buffer solution
(MeOH–H2O = 3:7, v/v) in the presence of 10 equiv. of certain amino
acids. From left to right: none, Tyr, Glu, Met, Lys, His, Cys, Hcy and
GSH. [2] = 2.0 ¥ 10-5 M.
Since the design of 2 is based on a classical donor–p-acceptor ar-
chitecture, this obvious red shift of absorption can be explained by
the thiol-mediated cleavage of the electron-withdrawing sulfonyl
group from 2, releasing an ionized hydroxyl group of 3 (Scheme 2).
This increases the ICT character of merocyanine chromophore,
resulting in a large bathochromic shift in its absorption spectrum.
An important feature of compound 2 is its high selectivity
toward the analyte over other competitive species. Fig. 2 shows a
representative chromogenic behavior of 2 toward a variety of tested
aminoacids. Only thiol-containing amino acids such as Cys, Hcy
and GSH led to a prominent decrease in the absorbance at 380 nm
and the development of a new absorption band at 530 nm. A minor
change in the absorbance of 2 was found upon addition of Gly,
Ser, Asp, Met, Asn, Lys, Tyr, Trp, Arg, and His, confirming 2 as a
highly selective colorimetric chemodosimeter for thiol-containing
amino acids. Furthermore, as shown in Fig. 3, 2 also displayed in
its color response an excellent selectivity to Cys, Hcy and GSH.
We also noticed that the reaction of 2 with GSH produced
an “off–on” type of fluorescence emission at 553 nm with
maximum excitation at 490 nm. Therefore, a fluorescence titration
of GSH was conducted (Fig. 4). As shown in Fig. 4, 2 alone
showed minimized fluorescence at 553 nm, addition of GSH
induced prominent fluorescence enhancement. The fluorescence
intensity of 2 solution at 553 nm increases linearly with the
increasing concentration of GSH in the range of 1 ¥ 10-7 mol/L
– 5 ¥ 10-6 mol/L (R = 0.9959) with a detection limit of about
2.4 ¥ 10-8 mol/L. The similar experimental phenomena were
Fig. 4 Fluorescence spectra of 2 in pH 7.40 of phosphate buffer(0.01 M)
solution (MeOH–H2O = 3:7, v/v) in the presence of GSH with an exitation
at 490 nm. Inset: fluorescence intensity at 553 nm vs GSH concentration.
[2] = 1.0 ¥ 10-5 M, [GSH] = 0, 0.1, 0.3, 0.5, 0.7, 0.9, 1.0, 2.0, 3.0, 4.0 and
5.0 mM, respectively.
also observed when added Cys or Hcy into the solution of 2
(Fig. S4 ~ S5).
The changes in the fluorescence properties of 2 solution caused
by different amino acids, including Gly, Ser, Asp, Met, Asn, Lys,
Tyr, Trp, Arg, and His, were also measured, and they induced no
observable fluorescence enhancement, whereas the fluorescence
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enhanced markedly with the addition of Cys, Hcy and GSH. These
results show that compound 2 is remarkably selective for thiol-
containing amino acids (Fig. 5).
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Fig. 5 Fluorescence response of 2 in pH 7.40 of phosphate buffer solution
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Asn, Lys, Tyr, Trp, Arg, and His. [2] = 1.0 ¥ 10-5 M.
Conclusions
In conclusion, we have developed a rapid and simple method
for recognition and quantification of biological thiols by using
a merocyanine-based chemodosimeter that exhibits a dual-mode
of optical signal output. The recognition of biological thiols gave
obvious color changes from light yellow to orange, which was
clearly visible to the naked eye, while it also showed a “turn-
on” type of absorption and fluorescence response. Furthermore,
both the colorimetric and fluorescent response exhibit excellent
selectivity and sensitivity toward sulfhydryl-containing amino
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Acknowledgements
This work was supported by the National Natural Science
Foundation of China (No. 20675067, No. 20835005) and NCET
of Fujian Province of China.
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