Coumarin-based fluorescent chemosensor for the selective quantification of Zn2+ and AcO? in an aqueous solution and living cells

Article abstract of DOI:10.1016/j.tetlet.2017.04.054

In this work, we report a novel fluorescence chemosensor HM based on the coumarin fluorophore for the quantification of Zn²⁺ and AcO^?. HM specifically binds to Zn²⁺ in the presence of other competing cations, and evident c

Full text of DOI:10.1016/j.tetlet.2017.04.054

Accepted Manuscript
Coumarin-based fluorescent chemosensor for the selective quantification of
2
+
−
Zn and AcO in an aqueous solution and living cells
Yan Gao, Huimin Liu, Peng Li, Qinglei Liu, Wei Wang, Bing Zhao
PII:
S0040-4039(17)30494-X
DOI:
http://dx.doi.org/10.1016/j.tetlet.2017.04.054
TETL 48846
Reference:
To appear in:
Tetrahedron Letters
Received Date:
Revised Date:
Accepted Date:
15 February 2017
14 April 2017
18 April 2017
Please cite this article as: Gao, Y., Liu, H., Li, P., Liu, Q., Wang, W., Zhao, B., Coumarin-based fluorescent
2
+
−
chemosensor for the selective quantification of Zn and AcO in an aqueous solution and living cells, Tetrahedron
Letters (2017), doi: http://dx.doi.org/10.1016/j.tetlet.2017.04.054
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2
Coumarin-based fluorescent chemosensor for the selective quantification of Zn and
+
−
AcO in an aqueous solution and living cells
a
b
c
b
b,
d,
Yan Gao , Huimin Liu , Peng Li ,Qinglei Liu , Wei Wang *, Bing Zhao *
a Department of Chemical Engineering, University of Science and Technology Liaoning, Anshan 114051, China
b School of Perfume and Aroma Technology, Shanghai Institute of Technology, Shanghai 201418, China
c School of Petroleum Processing, University of Petroleum and Chemical Engineering Liaoning, Fushun 113001, China
d Chemistry and Chemical Engineering Institute, Qiqihar University, Qiqihar 161006, China
ARTICLE INFO
AB STR ACT
Article history:
Received
In this work, we report a novel fluorescence chemosensor HM based on the coumarin
2
+
−
2+
fluorophore for the quantification of Zn and AcO . HM specifically binds to Zn in the
Received in revised form
Accepted
presence of other competing cations, and evident changes in UV-vis and fluorescence spectra in
2
HEPES buffer are noticed. The in situ generated HM-Zn complex solution exhibit a high
+
−
2+
selectivity toward AcO via Zn displacement approach. The detection limits of HM for Zn
2+
Available online
2
and HM-Zn for AcO were estimated to be 7.24×10 M and 9.41×10 M, respectively. HM
+
−
-8
-8
2
+
and the resultant complex HM-Zn exhibit low cytotoxicity and cell-membrane permeability,
Keywords:
Chemosensor
Coumarin
2
which makes them capable of Zn and AcO imaging in living Hep G2 cells. A B3LYP/6-
+
−
2
+
3
1G(d,p) basis set was employed for optimization of HM and HM-Zn complex.
2013 Elsevier Ltd. All rights reserved.
2
Zn
+
−
AcO
Bioimaging
Introduction
oxidation rate have been frequently used because acetate anion is
considered as an indicator of organic decomposition in marine
Design and synthesis of fluorescent chemosensors capable of
selective recognition of ions and neutral analytes is an active
26, 27
sediments. Because of these significant importance, detection
−
of AcO with the help of easily synthesized receptor and minimal
1-4
field of research in supramolecular chemistry. Among the
metal ions, zinc is a ubiquitous and indispensable element in the
human body and the second most abundant transition metal after
iron, it plays important roles in various biological processes such
as apoptosis, regulators of gene expression, and neural signal
instrumental assistance is desirable towards practical
applications.
28, 29
Recently, one feasible strategy for the design anions sensing
system is by employing the displacement method, in which the
fluorophore-metal “ensemble” is fluorescent because of metal
ion-induced fluorescence enhancement. Further addition of
anions may quench fluorescence. There are several advantages of
this anion sensing method, such as improved water solubility,
5-10
2+
transmitters or modulators.
many diseases ranging from Alzheimer’s disease to prostate
Deficiency of Zn may lead to
1
1-12
cancer and diabetes. Nevertheless, the mechanism of action
of free zinc ion still remains poorly understood.
In the last decade, numerous scientific endeavors have focused
multiple chemosensor types and straightforward synthesis
30-32
procedures.
properties of Zn ion and the higher affinity of Zn towards the
Considering the fluorescence enhancement
2+
2
+
on the development of fluorescent chemosensors for Zn based
1
2+
3-15
on quinoline, anthracene and coumarin fluorophores.
2
Some of
−
2+
AcO , we may reasonably expect to prepare a novel Zn -
+
them have well overcome the Zn sensor’s limitations such as
low water-solubility, poor selectivity, and/or few with fluorescent
−
fluorophore complex for AcO detection in aqueous and living
system based on displacement approach.
In this work, we report a novel colorimetric and fluorescent
chemosensor HM based on coumarin fluorophore for the
1
6,17
18
enhancements,
Moreover, there has been a challenge to develop chemosensors
while others still confront the problems.
2
+
that can discriminate Zn from Cd because they are in the same
2+
2+
recognition of Zn and AcO in water and excellent cell-
−
1
9
group of the Periodic table and have similar properties. Thus, a
technique to trace and visualize free zinc ions would be highly
demanded.
2+
permeable for the imaging of Zn and AcO in living cells. HM
−
2+
specifically binds to Zn in the presence of other competing
2
0
cations, and drastic fluorescence enhancement and obvious color
change in HEPES buffer are noticed. Based on the displacement
The recognition and sensing of anions by proper design of
anion receptors is currently an expanding research area within the
−
approach, the selective sense AcO with the in situ generated
2
1, 22
field of supramolecular chemistry.
Various kind of anions
4 3
, CH COO , etc. play a major role both
2+
HM-Zn
ensemble gives
a remarkable reversibility of
−
−
−
3−
−
such as F , Cl , I , PO
in environmental and biological systems.
fluorescence and UV-vis absorption spectra. This “off-on-off”
response provided a convenient and practical way to detect both
2
3-25
Among the above
−
anions, AcO received the most attention from chemists because
of its unique properties, For example, acetate production and
2+
Zn and AcO in environmental and biological samples.
−

2
Results and discussion
The studies for the interaction between HM (10µM) and a
series of metal ions such as Ca , Mg , Ni , Co , Cu , Hg ,
2+ 2+ 2+ 2+ 2+ 2+
We chose coumarin as the fluorophore due to its excellent
photophysical properties such as high fluorescence quantum
yield, large Stokes shift, excellent light stability, and low toxicity
2+
3+
3+
+
2+
3+
2+
Ba , Cr , Fe , Ag , Zn , Al and Cd (20µM) were monitored
clearly by the fluorescence spectroscopy in DMF/ H O aqueous
buffer (3:7, v/v, 20mM, pH = 7.2). As presented in Fig. 2a, the
2
3
3
and good cell permeability. The structure of 2 and HM were
confirmed by NMR, high resolution mass spectra and elemental
analysis (Fig.S1-S6). The detailed experimental procedures and
the characterization of the compound HM are described in the
2+
addition of Zn created a remarkable fluorescence enhancement
(
4
9.5-fold) accompanied by a blue shift of 35nm from 510 to
75nm, and a negligible fluorescence response was observed on
the addition of the same amount of other metal ions though there
3
4
experimental section. The spectral data were in agreement with
the desired structures. The coumarin-based Schiff-base
framework is a typical structure utilized for the construction of
3+
2+
were a bit changes for the fluorescence intensity for Fe , Cu
and Cr ions. In agreement to the fluorescent spectra, the
emission color change from colorless to blue can be clearly
3+
2
+
35
Zn fluorescent chemosensors. Accordingly, we believed that
2+
observed under UV light in presence of Zn , which suggested
2+
HM would be an excellent Zn selective fluorescence sensor and
that its resultant complex could be used as a AcO -specific
fluorescence chemosensor in an aqueous solution.
2+
that there would be certain interaction between HM and Zn .
This “OFF-ON” switching mechanism could be attributed to the
−
2+
formation of a rigid system after binding with Zn , causing the
S
37
N
N
N
H
NH2
chelation-enhanced fluorescence (CHEF) effect. In addition. the
high selectivity should be the priority for an excellent probe. So
we explored the selectivity and anti-interference performance of
the probe HM (Fig. 2b) in the presence of cations of interest. The
S
CH
O
3
N
N
CHO
OH H N
N
O
O
C
NHNH2
O
O
OH
2
CH COOH
3
AcOH
HO
O
CH
3
CH
3
2
1
2+
O
addition of Zn (20µM) to the solutions of HM (10µM) with
Br EtOH
Ph
interfering cations (20µM) appeared a significant fluorescence
enhancement which were similar to that caused by Zn only.
N
2
+
N
N
H
S
2
+
These results indicated that the specific response of HM to Zn
was not disturbed by other competitive cations.
O
O
OH
CH
3
HM
Scheme 1. Synthetic route to compound HM.
The general synthetic route of probe HM is given in Scheme 1.
Coumarin aldehyde 1 was prepared by previously reported
3
6
procedures. The receptor HM was synthesized by a three-step
reaction. First, 7-hydroxy-4-formyl coumarin was treated with
hexamethylenetetramine in acetic acid at 95
to form
intermediate 1. Further reaction of 1 with thiosemicarbazide in
acetic acid gave the intermediate 2. Finally, target compound (Z)-
7
-hydroxy-4-methyl-8-((2-(4-phenylthiazol-2-yl)hydrazono)-me-
thyl)-2H-chromen-2-one HM was prepared by the condensation
reaction of 2 with 2-bromoacetophenone in quantitative yield.
The detailed experimental procedures and the characterization of
3
6
the compound HM are described in the section of experimental.
The spectral data were in agreement with the desired structures.
2+
UV-vis and Fluorescence recognition of Zn
HM possessed the multiple N and O atoms in appropriate
position which were already known to be able to bind metal ions,
Fig.2. (a) Changes in the emission spectrum of HM (10µM) in the presence
2
+
the coordination of HM with Zn was first investigated using
UV-vis absorption spectroscopy in HEPES buffer. As shown in
Fig. 1, in the absence of metal ions, HM displays a maximum
absorbance peak at about 324nm, upon addition 2 equiv of
of different metal ions (20µM) in HEPES buffer (DMSO: H O= 3:7, 20mM,
2
2
+
pH=7.2); (b) Fluorescence responses of HM (10µM) to Zn (20µM) in the
presence of other common metal ions (20µM). The green bars represent the
enhancement degree of HM in the presence of cations of interest (all are
20µM). The red bars represent the changes of the emission that occurs upon
2+
2+
2+
2+
2+
2+
2+
different metal ions(Ca , Mg , Ni , Co , Cu , Hg , Ba ,
Cr , Fe , Ag , Al and Cd ), no obvious change of the
2+
the subsequent addition of 20µM of Zn to the above solution.
To get an insight into the sensing properties of HM to Zn ,
3+
3+
+
3+
2+
2+
2+
absorption could be observed. While the addition of Zn to HM
resulted in the enhancement of absorption intensity and a slight
bathochromic shift from 324 nm to 338nm along with obvious
color changes from colorless to orange. These results indicated
fluorescence titration experiments were then performed. As
shown in Fig.3, upon incremental addition of Zn (0-2.0equiv.)
2+
to HM solution, the fluorescence emission was gradually
2+
increased and reached the saturation state when 1.0 equiv. of Zn
2
+
that HM could be used as a potential chemosensor for Zn with
a high selectivity.
ions was employed. The nonlinear fit of the data revealed that the
binding of HM to Zn was most probably of 1:1 stoichiometry.
This binding mode was also supported by the data of Job’s plots
2
+
2+
evaluated from the absorption spectra of HM and Zn with a
38
total concentration of 20µM(Fig.S10). According to linear
Benesi-Hildebrand expression, the measured fluorescence
3
9
2
intensity [1/(F-F0)] varied as a function of 1/[Zn ] in a linear
+
relationship (R2=0.9920), the association constant of HM with
Zn in HEPES buffer was accordingly calculated to be
2
+
5
-1
2+
.687×10 M (Fig.S11). The detection limit for Zn was
1
-8
estimated to be 7.24×10 M based on a 3σ/slope analysis under
these experimental conditions (Fig.S12), which was the average
40
-
8
value of 5 times, the standard deviation was 0.11×10 M. The
detection limit was sufficiently low to detect submicromolar
Fig.1. UV-vis spectra of HM (10µM) before and after adding 2 equiv of
2
various cations in buffered DMSO/H O (v/v = 3:7) solution at pH 7.2.

2
+
2+
To further understand the Zn displacement approach,
concentration of the Zn , which belongs to the range found in
many chemical and biological systems, implying that probe HM
holds great potential for the development of sensor materials for
2+
fluorescence emission changes of HM-Zn in the absence and
−
presence of different concentrations of AcO were also
investigated. As shown in Fig.5, the addition of increasing
2
+
Zn .
−
concentrations of AcO (0-4equiv.), the fluorescence emission of
2
+
the HM-Zn system was gradually quenched and became
essentially identical with the emission wavelength of free HM.
The detection limit of HM-Zn for AcO was evaluated to be
2+
−
-
8
9
the average value of 5 times, the standard deviation was 0.16×10
.41×10 M based on a 3σ/slope analysis (Fig. S14), which was
-
8
M.
Fig. 3. Fluorescence spectra of HM (10µM, pH 7.2) in buffered aqueous
+
2
phase upon addition of increasing concentrations of Zn (0-2equiv) with an
excitation wavelength at 398nm.
For many biological applications, it is very important that the
sensor can be suitable for measuring specific cation in the
physiological pH range. Therefore, we also measured the
2
+
fluorescence intensity of HM in absence and presence of Zn ion
at various pH values (Fig. S13). It has been observed that the
fluorescence intensity of HM is stable at pH 4-7.0 without
obvious florescence responses. When the pH is more than 7.5,
the enhancement in the fluorescence intensity at 450 nm was
evidently enhanced and reaches its maximum at pH around 9.
Meanwhile, it can be seen that the fluorescence intensity of HM-
2
+
Fig.5 Fluorescence spectra of HM-Zn (10µM) in HEPES aqueous buffer
(DMF/H O=3:7, 20mM, pH=7.2) in the presence of different amounts of
2
−
2+
AcO (0-40µM). Inset: normalized fluorescence intensities of HM-Zn
−
(
10µM) as a function of AcO (0-40µM).
In addition, to evaluate the utility of HM-Zn as a fluorescent
2+
−
chemosensor for the detection of AcO in complicated
2
+
Zn is evidently enhanced when the pH is more than 7 and
reaches its maximum at pH 9.5. Which attributed to the
deprotonation of the hydroxyl group was favorite to the chelation
2+
environment, a HM-Zn HPEES solution was also treated with
−
AcO in the presence of different anions and polyphosphate
2
+
species. As shown in Fig.6, all of the chosen competitive anions
showed minor influence on the fluorescence detection of AcO ,
of HM to Zn . However, concentrated alkaline would lead to the
precipitation of Zn . Thus, the fluorescence intensity of HM-
−
2+
2+
−
suggesting that HM-Zn is an AcO -specific fluorescence
2
Zn solution decreased when the pH value of the solution was
+
chemosensor even in the presence of competing anions.
above 9.5. The results indicated that the HM can be used as a
2
+
chemosensor for Zn detection under physiological conditions.
−
Spectral recognition of AcO by HM-Zn
2+
2+
−
Considering the fact that Zn can coordinate with AcO
anions to form a highly stable zinc acetate, we are encouraged to
2
+
envision that the obtained fluorescence sluggish HM-Zn
system could be employed as a potential ensemble for
− 2+
fluorescence “ON-OFF” detection of AcO via the Zn
displacement approach. The HM-Zn solution was prepared in
2+
2
+
situ by the addition of 1.0equiv. Zn to HM (10µM) in HEPES
buffer. Subsequently, the fluorescence responses of HM-Zn to
different biological important anions were investigated. As
2
+
2
+
2
shown in Fig.4, HM-Zn showed negligible fluorescent response
+
Fig. 6. Normalized fluorescence responses of HM-Zn (10µM) in HEPES
(
DMSO:H
2
O = 3:7, 20mM, pH=7.2) in the presence of various analytes
−
−
upon addition of diverse anions, such as F , Cl , Br , SCN ,
−
−
−
−
−
2−
−
−
−
, 7. SCN , 8. NO
−
(
CO3 ,10. SO4 , 11, PO4 , 12. HPO4 , 13. H PO 14. AcO ,
20µM):1. F , 2. Cl , 3. Br , 4. S , 5. NO
3
, 6. HSO
−
4
2
, 9.
−
4
−
−
2−
, S , CO
2−
2−
3−
2−
2
−
2−
3−
2−
−
HSO
, NO
−
3
, NO
2
3
, SO
4
, PO
4
, HPO
4
2+
and
2
4
2+
H
dramatically quenched with the addition of AcO . The quenching
2
PO
4
.
By contrast, the fluorescence of HM-Zn was
We also examined the UV-vis changes of the HM-Zn
−
ensemble with AcO . Interestingly, the UV-Vis absorption
−
−
of fluorescence is due to the strong affinity of AcO for the Zn ,
2+
2+
spectrum of the HM-Zn system was unperturbed in the
presence of important physiological and environmental anions.
2
+
which resulted in decomplexation of the HM-Zn complex.
2
These results demonstrated that HM-Zn has an excellent
+
−
However, the addition of AcO resulted in an obvious blue shifts
−
2+
selectivity toward AcO and the proposed Zn displacement
occurred.
from 324nm to 338nm, which corresponds to the free
chemosensor HM (Fig.7).
2
+
2+
Fig.7. UV-vis absorption spectra of HM-Zn (10µM) in HEPES
Fig.4. Fluorescence responses of HM-Zn (10µM) in HEPES
(
DMF/H
2
O=3:7, 20mM, pH=7.2) in the presence of all kinds of analytes
2−
(DMF/H
2
O=3:7, 20mM, pH = 7.2) in the presence of all types of analytes
−
−
−
−
−
−
−
, 8. SCN , 9.
−
−
−
−
2−
−
−
−
, SCN , NO
−
2−
2−
4
, SO ,
(
NO
20µM): 1. AcO , 2. F , 3. Cl , 4. Br , 5. S , 6. NO
3
, 7. HSO
−
4
(20µM): AcO , F , Cl , Br , S , NO
3
, HSO
4
2
, CO
3
−
2−
2−
3−
2−
3−
2−
−
2
, 10. CO
3
,11. SO
4
, 12, PO
4
, 13. HPO
4
, 14. H
2
PO
4
4 4 2 4
PO , HPO , H PO

4
2+
The reversibility of the recognition process of HM-Zn was
−
performed by adding the AcO . As shown in Fig.8, the addition
system. Thus, these results showed a favorable complexation
according to proposed coordination.
− 2+
of AcO to a mixture of HM-Zn resulted in the decrease of the
fluorescence intensity, which indicated the regeneration of the
2
+
free sensor HM. Therefore, it meant that the receptor HM-Zn
could be used as a selective fluorescent sensor for detection and
-2.35324
−
recognition of AcO in such fields of environmental analysis
-2.97447
2
+
HM lomo
E=3.14781
HM-Zn
lomo
E=3.44742
-5.50105
-6.42189
2+
HM homo
HM-Zn
homo
Fig. 9. HOMO and LUMO energy levels and the molecular orbital plots of
2
HM and HM-Zn
+
Fig.8. Reversible changes in fluorescence intensity of HM (10µM) after the
sequential addition of Zn and AcO
2
+
−
2+
−
MTT assay
The recognition mechanism of HM toward Zn and AcO
To explore the mechanism underlying the detection of Zn
2
+
The cytotoxicity of HM toward the HeLa cells was
investigated by the reduction activity of the methyl thiazolyl
tetrazolium (MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetr-
azolium bromide]) assay. As shown in Fig. S15, following
incubation of HM (2-16µM) with HeLa cells for 24 h at 37 ℃,
no significant change in the proliferation of HeLa cells was
observed. Even after incubation with 10µM HM for 24 h, the
cellular viability remained above 89%, indicating that HM is
biocompatible and suitable for fluorescence imaging in living
cells. The results revealed that HM is low cytotoxicity both at
low and high concentration and will not kill the HeLa cells being
probed.
2+
1
by sensor HM, the HM-Zn complex was prepared and the H
NMR change of complex as shown in Fig. S5, upon
2
+
complexation of HM with Zn caused the signals of the proton
peaks all to broaden, the proton signals (Ha) underwent a upfield
shift from 12.559 to 12.011ppm, the hydroxyl proton signals
(
Hb) disappeared due to chelation process, and the imino proton
signals (Hc) underwent a upfield shift from 8.698 to 8.603ppm,
other aromatic H experienced the change in varying degrees. The
c
b
results showed that the N atoms of HC=N and O atoms of O-H ,
N atoms of thiazole group participated in the chelation to zinc
ion.
−
Living-cell fluorescence imaging studies
The proposed displacement strategy for the AcO detection
was further supported by the results of mass spectra studies. As
2+
−
To access the feasibility of Zn /AcO detection in living cells,
the fluorescence imaging was recorded using confocal
fluorescence microscopy. The cells were incubated with a
HEPES buffer solution containing 1µM of compound HM for 30
shown in Fig. S5-S7, FT-MS spectra of HM showed a molecular-
+
+
2+
ion peak [HM+H ] at m/z 378.09254(Fig. S7). When the Zn
ion was added into the solution of HM, the peak at m/z
2
+
–
14.38559 is assignable to [HM+Zn +2Cl +H ] species (Fig.
+ +
min at 37°C in a 5% CO -95% air incubator. After washed with
2
5
S8). This result confirmed that 1:1 stoichiometry complex
HEPES for three times, the cells were subjected to microscopy
imaging analysis. As shown in Fig. 10 (A), No intracellular
fluorescence of Hep G2 cells after staining with HM was
2+
−
between HM and Zn . In the presence of AcO , FT-MS spectra
displayed a molecular-ion peak at m/z 378.09055 again (Fig. S9),
2
+
2
+
detected. On further incubation with 10µM Zn , the strong blue
fluorescence of the system appeared (Fig. 10 (B)), suggesting
indicating that the decomplexing of Zn ion in the presence of
AcO led to the liberation of fluorophore, HM (scheme 2).
−
2+
that the sensor HM could be used to detect Zn in the living cells
through its fluorescence behavior. After treating the cells with
sodium acetate (20µM) for another 20min, the intense blue
fluorescence of the cells was quenched (Fig. 10 (C)). These
results indicate that HM could be potentially used to monitor
S
N
HN
Zn2+
N
N
N
H
S
N
2+
Zn
O
O
O
O
OH
O
2+
2+
AcO-
intracellular Zn and the resulting HM-Zn ensemble can be
used as an imaging agent for AcO in living cells.
−
Zn(AcO)
2
CH
3
CH3
λex=398nm
λex=398nm
2
Scheme 2. The proposed mechanism for Zn and AcO detection by HM
+
−
2
+
To better understand the nature of the coordination of Zn
with HM, energy-optimized structures of HM and its
corresponding metal complexes were obtained by density
functional theory (DFT) calculations at the B3LYP/6-31G(d)
level using Lanl2dz basis set for simple receptor HM and HM-
2
+
Zn complex. The spatial distributions and orbital energies of the
highest occupied molecular orbital (HOMO) and the lowest
unoccupied molecular orbital (LUMO) of HM and its
corresponding metal complexes were also calculated. As shown
in Fig.9, the π electrons of HOMO is distributed on the
Fig. 10. Confocal bright-field (top) and fluorescence (bottom) imaging of
2
+
−
Zn and AcO in living Hep G2 cells. (A) Cells stained with HM (1µM) at
2
+
3
2
7
in a 5% CO
2
incubator for 30min, (B) and treated with Zn (10µM) for
−
0 min, (C) then the cells incubated with AcO (20µM) for another 20 min.
2+
substituted thiazole ring in HM and HM-Zn , whereas LUMO is
Conclusion
2+
distributed on the coumarin ring in HM and HM-Zn . The
energy gaps between the HOMO and LUMO in the HM and
In conclusion, we have developed a novel coumarin-based
2+
2+
fluorescent chemosensor HM for Zn detection in mixed
aqueous medium (DMF/H O=3:7, HEPES buffered, pH=7.2).
Interaction of HM with Zn led to complete enhancement of
HM-Zn were calculated to be 3.14781eV and 3.44742eV,
2
+
respectively, indicating that the binding of Zn to HM increases
the HOMO-LUMO energy gap of the complex and stabilizes the
2
2+

fluorescence intensity through a 1:1 binding mode. The detection
limit of HM for Zn was estimated to be 7.24×10 M. The
A
mixture of coumarin aldehyde 1(5mmol), thiosemicarbazide
5mmol) were magnetically stirred in glacial acetic acid (3ml) and
2
+
-8
(
2
+
ethanol (20ml) at refluxing until the reactions were completed
(monitored by TLC), the result precipitate was collected and the
crude product was purified by column chromatography (silica gel,
petroleum ether/ethyl acetate, v/v=90:10) to afford compound 2 as a
enhancement fluorescence of the in situ generated HM-Zn
−
ensemble could be quenched upon the addition of AcO , realizing
− 2+
the detection of AcO by utilizing the Zn displacement
-
8
1
approach with a detection limit of 9.41×10 M. The MTT assay
determined that HM exhibits low cytotoxicity toward living Hep
G2 cells. Confocal microscopy imaging suggested that HM has
yellow solid in 74% yield. H NMR (400 MHz, DMSO) δ: 11.65 (s,
1
H, NH), 10.47 (s, 1H, OH), 8.74 (s,1H, CH=N), 8.30 (s, 1H, NH
2
),
2
7.95 (s, 1H, NH ), 7.70 (d, J=8.8Hz, 1H, Ar), 6.95 (d, J=8.8Hz, 1H,
Ar), 6.25(s, 1H, Ar), 2.46 (s, 3H, CH
1
3
2+
−
3
). C NMR (125 MHz,
potential as a powerful tool for the imaging of Zn and AcO in
living cells. A B3LYP/6-31G (d,p) basis set was employed for
DMSO)δ: 159.70, 154.09, 139.50, 128.14, 113.75, 111.09, 106.76,
+
8.80. FT-MS, m/z: 278.11927(M+H );Anal.Calcd for C12
1
C, 51.98; H, 4.00; N, 15.15. Found: C, 52.05; H, 3.98; N, 15.11.
11 3 3
H N O S:
2
+
optimization of HM and HM-Zn complex.
Synthesis of (Z)-7-hydroxy-4-methyl-8-((2-(4-phenylthiazol-2-yl)
hydrazono) methyl)-2H-chromen-2-one (HM)
Acknowledgements
A
mixture of 2(5mmol), 2-bromoacetophenone(5mmol) were
This work was supported by National Natural Science
Foundation of China (21506106) and the Key Laboratory Project
of Liaoning Province (2008S127)
magnetically stirred in 30ml of acetic acid at refluxing until the
reactions were completed (monitored by TLC). A orange solid
appeared which was then filtered from solution, the result
precipitate was collected and the crude product was purified by
column chromatography (silica gel, petroleum ether/ethyl acetate,
References and notes
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1
5
1
9%). H NMR (500 MHz, DMSO) δ: 12.559 (s, 1H, NH), 11.371 (s,
H, OH), 8.698(s, 1H, CH=N), 7.870 (d, J=6.5 Hz, 2H, Ar ), 7.689
1
2
3
4
.
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3
2
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1
3
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Synthesis of 2

6
Highlights
1
2
3
. A novel coumarin derivative HM was
2
prepared for the detection of Zn and
+
−
AcO detection.
. Chemosensor HM displays highly
sensitive fluorescence “OFF-ON-OFF”
2
response to Zn and AcO .
+
−
2+
. The detection limits of HM for Zn and
2
HM-Zn for AcO were 7.24×10 M and
+
−
-8
-
.41×10 M.
8
9
4
5
. This chemosensor HM was applied to
living cells imaging.
. A B3LYP/6-31G(d,p) basis set was
employed for optimization of HM and
2
+
HM-Zn complex.

Graphical abstract
S
N
HN
Zn2+
AcO-
N
N
N
H
S
N
2+
Zn
O
O
O
O
OH
O
Zn(AcO)
2
CH
3
CH3
λex=398nm
λex=398nm
A novel fluorescence probe HM based on the
coumarin was prepared. Which displayed highly
2
sensitive “OFF-ON-OFF” response to Zn and
+
−
AcO in aqueous solution. Moreover, an imaging
2
+
−
application of HM for Zn and AcO in live cells
was developed by fluorescence microscopy. A
B3LYP/6-31G(d,p) basis set was employed for
2
optimization of HM and HM-Zn complex.
+