An iminocoumarin sulfonamide based turn-on fluorescent probe for the detection of biothiols in aqueous solution

Article abstract of DOI:10.1002/asia.201403056

A new chemodosimeter for the highly selective sensing and imaging of biothiols was designed and realized in phosphate-buffered saline solution at pH 7.4 through a fluorescence "off-on" response. A unique mechanism featuring a two-step cascade (biothiols H

Full text of DOI:10.1002/asia.201403056

DOI: 10.1002/asia.201403056
Full Paper
Biosensors
An Iminocoumarin Sulfonamide Based Turn-On Fluorescent Probe
for the Detection of Biothiols in Aqueous Solution
[
a]
[b]
[a]
[a]
[a]
Yan-Ling Yang, Fu-Ming Zhang, Ya-Wen Wang,* Bao-Xin Zhang, Ran Fang,
[
b]
[b]
Jian-Guo Fang, and Yu Peng*
Abstract: A new chemodosimeter for the highly selective
sensing and imaging of biothiols was designed and realized
in phosphate-buffered saline solution at pH 7.4 through
a fluorescence “off–on” response. A unique mechanism fea-
turing a two-step cascade (biothiols!H O) sequence for this
2
remarkable recognition is disclosed for the first time.
Introduction
covery of the fluorophores. By using this design principle, fluo-
rescent probes for biothiols can achieve high signal-to-noise
[7]
Sensing and signaling of biothiols such as cysteine (Cys), ho-
mocysteine (Hcy), and glutathione (GSH) is a current research
focus owing to the crucial roles of these molecules in main-
taining the appropriate redox status of proteins, cells, and or-
and turn-on responses. However, most probes bearing the
DNBS moiety have worked well only in mixed organic–water
[5b–e,6a,c]
[6d]
media
or in nearly pure water at high pH values, and
this limits their practical application. Therefore, the develop-
ment of other DNBS fluorescent probes for biothiols in aque-
ous buffer solution especially at physiological pH values is still
in high demand.
[
1]
ganisms. Abnormal levels of biothiols are implicated in a vari-
[
2a]
ety of diseases such as cardiovascular disease, neurotoxici-
[
2b]
[2c]
[2d]
ty, Alzheimer’s disease, and cancer. The early diagnosis
and prevention of these diseases could benefit from the detec-
tion of biothiols in living organisms. As a powerful detection
tool, reaction-based small-molecule fluorescent probes have
been used to sense biothiols because of their high selectivity,
In connection with our continuing research on bifunctional
[8]
[9]
fluorescent probes for relay or divergent recognition of bio-
logically and environmentally important species, herein we
report turn-on fluorescent probe 1 based on the iminocoumar-
in fluorophore tethered to a DNBS moiety for the detection of
biothiols in phosphate-buffered saline (PBS)solution at pH 7.4.
Notably, the present recognition event demonstrated an un-
[
3]
sensitivity, versatility, and relatively simple handling. For ex-
ample, various fluorescent probes based on biothiol addition
[
4]
reactions have been described in recent years, in which the
strong nucleophilicity of the mercapto group was utilized. Rel-
atively rare fluorescent probes containing the 2,4-dinitrobenze-
nesulfonyl (DNBS) moiety have also emerged, and biothiol-
mediated cleavage reactions of the corresponding sulfonate
precedented forward cascade sequence triggered by H O as
2
[5,6]
the medium, unlike the usual recycle process.
[
5]
[6]
ester or sulfonamide were instead employed. In particular,
the DNBS moiety is used as an electron-withdrawing group at- Results and Discussion
tached to fluorophores for intramolecular charge-transfer (ICT)
Design and Synthesis of DNBS Probe 1
processes, and it can be removed by cleavage reactions for re-
Designed probe 1 was easily synthesized from 10-(1,3-benzo-
thiazol-2-yl)-2,3,6,7-tetrahydro-1H,5H,11H-[1]benzopyrano[6,7,8-
ij]quinoliz-11-imine (2) and 2,4-dinitrobenzene-1-sulfonyl chlo-
ride (see the Experimental Section for details). Its structure was
[
a] Y.-L. Yang, Prof. Dr. Y.-W. Wang, B.-X. Zhang, Prof. Dr. R. Fang
Key Laboratory of Nonferrous Metal Chemistry and Resources Utilization of
Gansu Province
and College of Chemistry and Chemical Engineering
Lanzhou University
Lanzhou 730000 (P. R. China)
Fax: (+86)931-8912582
E-mail: ywwang@lzu.edu.cn
1
13
determined by H NMR and C NMR spectroscopy and mass
spectrometry (Figures S1–S3, Supporting Information). As
shown in Scheme 1, biothiols promote smooth cleavage of the
[10]
NꢀS bond within 1 to give intermediate 2, the hydrolysis of
which subsequently occurs to generate coumarin derivative 3.
The whole cascade represents a new pathway of sensing bio-
thiols in aqueous solution.
[
b] F.-M. Zhang, Prof. Dr. J.-G. Fang, Prof. Dr. Y. Peng
State Key Laboratory of Applied Organic Chemistry
Lanzhou University
Lanzhou 730000 (P. R. China)
E-mail: pengyu@lzu.edu.cn
Supporting Information for this article is available on the WWW under
http://dx.doi.org/10.1002/asia.201403056.
Chem. Asian J. 2014, 9, 1 – 6
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ure 1a). Relative to the fluores-
cence intensity of other amino
acids examined, that of 1 was
dramatically increased only in
the presence of biothiols (e.g.,
Cys/Hcy/GSH) with a clear batho-
chromic shift from 524 to
545 nm. In addition, the fluores-
cence color changed from
almost nonfluorescent to bright
Scheme 1. Tandem mechanism of sensing biothiols.
green with the addition of these
biothiols (Figure 1d, see also Fig-
Fluorescence responses
ure S4). To validate the selectivity of 1 in practice, competition
experiments were also measured by the addition of biothiols
to the PBS solutions of 1 in the presence of other amino acids
(Figure 1b; see also Figures S5 and S6). Pleasingly, amino acids
such as Ala, Arg, Asp, Gly, Glu, His, Leu, Ile, Met, Phe, Pro, Ser,
Thr, and Val had no clear interference with the detection of
these biothiols. These results suggested that 1 can function as
a fluorescent probe for biothiols through a “turn-on” response
in PBS solution.
Considering potential application in bioimaging, the fluores-
cence of probe 1 was investigated in PBS solution (pH 7.4). As
shown in Figure 1, 1 shows a very weak emission band at lem
ꢁ
524 nm upon excitation at l =486 nm, which is in the
ex
The fluorescence titrations of Cys were then conducted by
using a 20.0 mm solution of 1 in PBS (pH 7.4) solution (Fig-
ure 1c). Upon the addition of Cys to the solution, a significant
increase in the fluorescence emission band centered at lem
=
5
45 nm was observed upon excitation at l =486 nm. The en-
ex
hancement in the total fluorescence at l=545 nm was deter-
mined to be 185-fold. The fluorescence titrations of Hcy and
GSH were also investigated under the same conditions (Figur-
es S7 and S8). The total fluorescence intensity of 1 at l
=
em
[11]
5
45 nm increased 26- and 137-fold for Hcy and GSH in PBS
[12]
solution, respectively. The fluorescence quantum yields (F)
of 1 at lem =545 nm increased from 0.11% to 11.22, 1.59, or
8
.57% in the presence of Cys, Hcy, or GSH in PBS solution, re-
[13]
spectively. The corresponding detection limits were found to
be 5.0, 10.0, and 5.0 mm for Cys, Hcy, and GSH, respectively
(Figures S9–S11). Kinetic studies of the response of biothiols
Figure 1. a) Fluorescence spectra of 1 (20.0 mm) with various amino acids
80.0 equiv.) in PBS (pH 7.4) solution (lex =486 nm). b) The selectivity of
(20.0 mm) for Cys. The black bars represent the emission intensity of 1 in
the presence of other amino acids (1.6 mm); The gray bars represent the
emission intensity that occurs upon the subsequent addition of Cys
(
1
(
1.6 mm) to probe 1 (20.0 mm) in PBS solution at 378C were
measured (Figures S12–S14). The observed rate constants (kobs)
ꢀ
4
ꢀ4
were estimated to be 5.38ꢁ10 , 4.19ꢁ10 , and 4.88ꢁ
ꢀ
4
ꢀ1
(1.6 mm) to the above solution. From 1 to 17: none, Ala, Arg, Asp, Gly, Glu,
10
s
for Cys, Hcy, and GSH, respectively, by fitting the initial
His, Leu, Ile, Met, Phe, Pro, Ser, Thr, Val, Hcy, and GSH (lem =545 nm). c) Fluo-
rescence spectra of 1 (20.0 mm) upon the addition of Cys (0–16.0 mm)
(lex =486 nm) Inset: Fluorescence intensity at l=545 nm as a function of
Cys concentration. d) Fluorescence color change of 1. Left to right: 1 only,
Ala, Leu, Phe, Ile, Pro, Lys, GSH, Cys, and Hcy. For a colored version, see the
Supporting Information.
fluorescent intensity changes according to a pseudo-first-order
kinetics equation. These results indicated that the response of
probe 1 to biothiols were not so fast, probably because of the
two-step mechanism. In the UV/Vis spectra of 1 in PBS solu-
tion, there were almost no changes observed upon the addi-
tion of some amino acids, including the biothiols (Figure S15).
range of visible light, and this diminishes damage upon intra-
cellular processes. The weak emission is due to the ICT effect
with the iminocoumarin fluorophore as an electron donor and
the DNBS unit as an electron acceptor. This behavior was fur-
ther revealed by time-dependent density functional theory
Rationalization of the Recognition of Biothiols
To verify the interaction of biothiols with probe 1, a preparative
experiment was performed (see the Supporting Information).
[14]
Notably, coumarin 3 instead of iminocoumarin 2 was ob-
1
13
(TD-DFT) calculations (see below, see also Figure 3). Next, Ala,
tained, and it was characterized by H NMR and C NMR spec-
[15]
Arg, Asp, Gly, Glu, His, Leu, Ile, Met, Phe, Pro, Ser, Thr, Val, Cys,
Hcy, and GSH were used to investigate the selectivity of
troscopy, mass spectrometry, and X-ray diffraction analysis
(Figures S16–S18 and Table S1; see also Figure 2). As expected,
1
(20.0 mm) in PBS solution by fluorescence spectroscopy (Fig-
biothiol-triggered cleavage of the NꢀS bond of probe 1 to re-
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moiety; this is clear indication of a ICT process. As for 3, the
S !S transition with an oscillator strength (f) of 1.0467 is
0
1
a fully allowed transition, which suggests overlap of the HOMO
and LUMO. This result also shows that the S !S transition is
1
0
[6c]
fully allowed, and therefore, coumarin 3 is potentially fluo-
rescent. Moreover, the electron densities of the HOMO and
LUMO of 3 are located over the whole molecule, which is indi-
cative of the strong fluorescence character of 3. These results
are in agreement with turn-on changes observed in the fluo-
rescence spectra of 1 upon the addition of biothiols.
3
Figure 2. X-ray structure of 3 (crystalline solvent CH CN is omitted for clari-
ty).
Practical Applications
lease 2; this species quickly underwent hydrolysis to generate
3
in aqueous solution. Notably, a similar hydrolysis from Nile
To explore the practical application of probe 1, we also evalu-
ated the effect of human blood serum on the fluorescence re-
sponse of probe 1 to biothiols. As shown in Figure 4, probe
1 exhibited a dramatic turn-on response to human blood
[10]
blue A to Nile red was reported by Lewis very recently.
Moreover, 3 was proven to be responsible for the fluorescence
emission band at lem =545 nm in PBS solution (Figure S19).
Notably, the cascade reaction leading to 3 is distinctively differ-
[5,6]
ent from simple deprotection of previous DNBS probes.
To further understand the relationship between the structur-
al changes of 1, 3, and the respective fluorescence response,
TD-DFT calculations with the B3LYP/6-31G(d) basis set were
performed by using the Gaussian 09 program. As shown in Fig-
ure S20, the optimized structure of probe 1 has a dihedral
angle of approximately 43.378 between the iminocoumarin
ring and the benzothiazole moiety. By contrast, the coumarin
and benzothiazole moieties of 3 are essentially planar in the
optimized structure, with a dihedral angle of 0.588, and this re-
sults in the formation of delocalized p bonds (Figure S21).
Quantum calculations of probe 1 and 3 are summarized in
Figure 3 and Table S2. The main contribution transition of
probe 1 for the S !S energy state comes from HOMO!
Figure 4. Fluorescence response of 1 with the addition of human blood
serum in PBS buffer (pH 7.4) solution (lex =486 nm).
serum in PBS (pH 7.4) solution upon excitation at l=486 nm,
which indicates that probe 1 is potentially useful for the detec-
tion of biothiols in human blood serum.
0
2
LUMO+1 and HOMO!LUMO+2. As shown in Figure 3, the
electron density of the HOMO is mainly localized on the imino-
coumarin and benzothiazole moieties, whereas the electron
density of LUMO+1 is localized on the iminocoumarin ring
and the electron density of LUMO+2 is localized on the DNBS
Finally, the application of probe 1 in the bioimaging of bio-
thiols in living cells was investigated (Figure 5; see also Fig-
ure S22). HeLa cells were incubated in PBS (pH 7.4) solution for
1
h at 378C, and no fluorescence was observed (Figure 5b). If
the HeLa cells were incubated with 1 (20.0 mm) in culture
Figure 5. Images of HeLa cells: a) Bright field and b) fluorescence images of
HeLa cells. c) Bright field and d) fluorescence images of HeLa cells incubated
with 1 (20.0 mm) for 1 h. e) Bright field and f) fluorescence images of HeLa
cells pretreated with 500.0 mm of NEM for 0.5 h and then incubated with
1 (20.0 mm) for 1 h. For a color version, see the Supporting Information.
Figure 3. HOMO and LUMO orbital plots of 1 and 3.
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medium for 1 h at 378C, strong green fluorescence from the
intracellular area was observed (Figure 5d). In a control experi-
ment, the HeLa cells were pretreated with N-ethylmaleimide
The X-ray diffraction data of 3 was obtained with a Bruker SMART
Apex CCD area detector diffractometer with a graphite-monochro-
mated MoKa radiation source (l=0.71073 ꢂ). Lorentz-polarization
and absorption corrections were applied for the compound. The
structure was solved with direct methods and was refined with
[
16]
(NEM, as a thiol-blocking reagent) for 0.5 h and they were
further incubated with probe 1 for 1 h, which resulted in very
weak fluorescence (Figure 5 f). These results suggest that
probe 1 is cell-membrane permeable and also that it works
well in living cells.
2
full-matrix least-squares on F by using the SHELXL-97 program
[17]
package. All non-hydrogen atoms were subjected to anisotropic
refinement, and all hydrogen atoms were added in idealized posi-
tions and were refined isotropically.
The HeLa cells were grown in H-DMEM (Dulbecco’s modified
Eagle’s medium, high glucose) supplemented with 10% FBS (fetal
Conclusions
bovine serum) under an atmosphere of 5% CO and 95% air at
2
4
3
78C. Cells were seeded in a six-well plate at a density of 10 cells
In summary, we reported chemodosimeter 1 for biothiols on
the basis of a novel two-step cascade mechanism: removal of
the 2,4-dinitrobenzenesulfonyl moiety promoted by biothiols
and facile hydrolysis of the resulting iminocoumarin in phos-
phate-buffered saline (pH 7.4) solution. Probe 1 displayed
almost no fluorescence because of an intramolecular charge-
transfer process; upon the addition of biothiols, a significant
fluorescence enhancement with a color change from dark to
bright green was observed owing to the formation of a strong
fluorescent coumarin derivative. The origin of this recognition
was further disclosed by time-dependent DFT calculations. Fur-
thermore, this probe was able to detect biothiols in human
blood serum and was applied to chemoselective bioimaging in
HeLa cells. The present work enriches our continuing investiga-
tion into bifunctional fluorescent probes for diverse combina-
per well in culture media. Immediately before the experiments, the
cells were washed with PBS buffer, and then cells were treated
with 20.0 mm of 1 in culture media for 1 h at 378C in a humidified
incubator. For the control experiment, the cells were treated with
5
00.0 mm of N-ethylmaleimide (NEM) in culture media for 30 min at
378C in a humidified incubator. After washing with PBS, the cells
were further incubated with 20.0 mm of 1 in culture media for 1 h.
Synthesis and characterization of probe 1
Iminocoumarin 2 (49.7 mg, 0.13 mmol) was added to dry pyridine
(4 mL) under an atmosphere of nitrogen, and the resulting solution
was stirred for 30 min at 08C. 2,4-Dinitrobenzenesulfonyl chloride
(286.1 mg, 1.07 mmol) was then added to the above solution por-
tionwise, and the mixture was stirred for 30 min at 08C and for
1
4 h at room temperature. The solvent was evaporated, and the
[
8,9]
residue was extracted with ethyl acetate (3ꢁ15 mL). The combined
tions of analytes.
organic layer was washed with saturated NaHCO (20 mL), water
3
(
10 mL), and brine (10 mL), and it was then dried with Na SO . The
2 4
solvent was removed under reduced pressure, and the residue was
Experimental Section
purified by flash column chromatography (CHCl ) on silica gel to
3
afford probe 1 (38.1 mg, 47%) as a dark red solid (see Scheme 2).
General methods
1
M.p. 258–2608C; H NMR (400 MHz, [D ]DMSO): d=9.10 (s, 1H),
6
Commercially available chemicals were used without further purifi-
cation. All of the solvents used were analytical-reagent grade. Melt-
1
ing points were determined with a Kofler apparatus. H NMR and
1
3
C NMR spectra were recorded with a Bruker 400 MHz or Varian
INOVA 600 MHz instrument by using tetramethylsilane as an inter-
nal standard. Mass spectra were obtained with a Bruker microTOF
mass spectrometer. Fluorescence spectra were determined with
a Hitachi F-7000 spectrophotometer. UV/Vis absorption spectra
were determined with a Varian UV-Cary100 spectrophotometer. All
pH measurements were made with a pH-10C digital pH meter. In-
verted microscope fluorescence imaging was performed with
a Leica DMI4000B equipped with a charge coupled device camera.
Scheme 2. The synthesis of 1.
Various stock solutions of the amino acids (100.0 mm) of l-Ala, l-
Arg, l-Asp, l-Gly, d-Glu, l-His, l-Leu, l-Ile, l-Met, d-Phe, l-Pro, l-Ser,
l-Thr, l-Val, l-Cys, dl-Hcy, and GSH in deionized water were pre-
pared. Stock solution of 1 (2.0 mm) was also prepared in DMF. Test
solutions were prepared by placing an aliquot (2.0 mL) of the probe
stock solution into a test tube, and it was then diluted to 2.0 mL
with deionized water; this was followed by the addition of an ap-
propriate aliquot of each amino acid stock solution. For all meas-
urements, fluorescence spectra were obtained by excitation at l=
8
.90 (s, 1H), 8.65 (d, J=8.4 Hz, 1H), 8.51 (d, J=8.8 Hz, 1H), 8.10 (d,
J=8.0 Hz, 1H), 7.99 (d, J=8.0 Hz, 1H), 7.52 (d, J=7.2 Hz, 1H), 7.50
s, 1H), 7.40 (t, J=7.6 Hz, 1H), 3.41–3.60 (m, 4H), 2.75 (t, J=5.6 Hz,
(
2
13
H), 2.57 (t, J=6.0 Hz, 2H), 1.90–1.84 ppm (m, 4H); C NMR
(150 MHz, [D ]DMSO): d=160.2, 157.5, 151.8, 150.7, 149.5, 149.4,
6
1
1
1
47.3, 143.7, 139.3, 135.8, 131.0, 127.7, 127.2, 126.3, 124.6, 122.6,
21.9, 120.0, 109.5, 109.0, 108.8, 105.0, 49.9, 49.3, 29.8, 26.9, 20.1,
+
9.0 ppm. MS (TOF): m/z: 604.0972 [M+H] .
4
86 nm. Both the excitation and emission slit widths were 2.5 nm.
Fluorescence quantum yields were determined in solution by
using fluorescein (F=0.85 in 0.1m NaOH) as a standard. For the
detection of biothiols in human blood serum, a pure human blood
serum sample (2.0 mL) was added to a cuvette, and the fluores-
cence emission spectrum was recorded per 10 min at room tem-
perature.
Acknowledgements
This work was supported by the National Natural Science
Foundation of China (NNSFC) (Nos. 21172096, 21472075, and
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Chem. Asian J. 2014, 9, 1 – 6
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ꢀ 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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Full Paper
J1103307), the Fundamental Research Funds for the Central
Universities (Nos. lzujbky-2014-59, lzujbky-2014-66, and lzujb-
ky-2013-ct02), the Program for Changjiang Scholars and Inno-
vative Research Team in University (IRT1138), and the “111”
Project of Ministry of Education.
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Keywords: biothiols · domino reactions · chemodosimeter ·
fluorescence · molecular recognition
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3
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4
0148–40155. The control experiments suggested that NEM cannot
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[
6] (HEPES/CH
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CN=9:1 v/v, pH 7.4) a) J. Bouffard, Y. Kim, T. M. Swager, R.
Received: September 9, 2014
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Published online on && &&, 0000
Chem. Asian J. 2014, 9, 1 – 6
www.chemasianj.org
5
ꢀ 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
&
&
These are not the final page numbers! ÞÞ

Full Paper
FULL PAPER
&
Biosensors
Y.-L. Yang, F.-M. Zhang, Y.-W. Wang,*
B.-X. Zhang, R. Fang, J.-G. Fang, Y. Peng*
&& – &&
An Iminocoumarin Sulfonamide Based
Turn-On Fluorescent Probe for the
Detection of Biothiols in Aqueous
Solution
The sixth sense: A new chemodosime-
ter for the selective sensing of biothiols
in phosphate-buffered saline solution at
pH 7.4 by using a fluorescence “off–on”
response is developed. An unprecedent-
ed cascade mechanism featuring a two-
step cascade (biothiols!H O) sequence
2
for this remarkable recognition is note-
worthy.
&
&
Chem. Asian J. 2014, 9, 1 – 6
www.chemasianj.org
6
ꢀ 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
ÝÝ These are not the final page numbers!