Synthesis of a fluorogenic probe for thiols based on a coumarin schiff base copper complex and its use for the detection of glutathione

Article abstract of DOI:10.1016/j.tet.2016.12.012

Glutathione is the most abundant non-protein thiols compound in cells and plays important metabolic roles. Changes in the amount of glutathione or its metabolic dysregulation can lead to a series of diseases. The determination of glutathione levels is very helpful to the diagnosis and treatment of the related diseases. A coumarin schiff base (compound 1) was synthesized from coumarin hydrazide and 2,6-pyridine dicarboxaldehyde and the fluorogenic probe for thiols (compound 1-Cu²⁺) was prepared by coordinating compound 1 with copper ions. Compound 1 showed strong fluorescence, while compound 1-Cu²⁺hardly had fluorescence due to the paramagnetism and/or photoinduced electron transfer of Cu²⁺. However, after the addition of thiols-containing compounds, the fluorescence of compound 1 was restored. The UV–vis absorption and fluorescence spectra indicated that the fluorogenic probe had good thiols selectivity and sensitivity, particularly for glutathione in CH₃CN:HEPES (3:2, v/v) buffer. It was successfully applied to the fluorescence imaging detection of glutathione in human cervical squamous cancer cells (SiHa cells).

Full text of DOI:10.1016/j.tet.2016.12.012

Tetrahedron xxx (2016) 1e6
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Synthesis of a fluorogenic probe for thiols based on a coumarin schiff
base copper complex and its use for the detection of glutathione
Guangjie He*, Jing Li, Zhongquan Wang, Chenxi Liu, Xiangli Liu, Liguo Ji, Chenyan Xie,
Qingzhi Wang**
Department of Forensic Medicine, Xinxiang Medical University, Jinsui Road No. 601, Xinxiang, 453003, PR China
a r t i c l e i n f o
a b s t r a c t
Article history:
Glutathione is the most abundant non-protein thiols compound in cells and plays important metabolic
roles. Changes in the amount of glutathione or its metabolic dysregulation can lead to a series of diseases.
The determination of glutathione levels is very helpful to the diagnosis and treatment of the related
diseases. A coumarin schiff base (compound 1) was synthesized from coumarin hydrazide and 2,6-
pyridine dicarboxaldehyde and the fluorogenic probe for thiols (compound 1-Cu2þ) was prepared by
coordinating compound 1 with copper ions. Compound 1 showed strong fluorescence, while compound
1-Cu2þ hardly had fluorescence due to the paramagnetism and/or photoinduced electron transfer of
Cu2þ. However, after the addition of thiols-containing compounds, the fluorescence of compound 1 was
restored. The UVevis absorption and fluorescence spectra indicated that the fluorogenic probe had good
thiols selectivity and sensitivity, particularly for glutathione in CH3CN:HEPES (3:2, v/v) buffer. It was
successfully applied to the fluorescence imaging detection of glutathione in human cervical squamous
cancer cells (SiHa cells).
Received 1 October 2016
Received in revised form
4 December 2016
Accepted 7 December 2016
Available online xxx
Keywords:
Fluorogenic probe
Coumarin
Thiols
Glutathione
Cell imaging
© 2016 Elsevier Ltd. All rights reserved.
1. Introduction
organisms is helpful to diagnose relevant diseases.5 Therefore, the
development of fast, convenient, accurate and sensitive methods to
The thiols in cells, such as cysteine, homocysteine, and gluta-
thione, play important roles in the regulation of various physio-
logical and pathological processes in humans as vital bioactive
compounds. The endogenous concentrations of these thiols suggest
the functional state of the corresponding enzymes and proteins and
their abnormal levels correlate with diseases.1 Glutathione is the
most abundant non-protein thiols compound in cells, with con-
centration ranges from 1 to 10 mmol/L.2 Glutathione can remove
toxic and hazardous compounds and metabolites, maintain the
completeness of the erythrocyte membrane, reduce DNA damage
and mutation by reducing radical attacks to DNA, participate in the
reduction of methemoglobin, and promote iron absorption.3
Metabolic dysregulation or the change in glutathione levels in or-
ganisms can lead to various diseases including cancers, Alzheimer's
disease, cardiovascular diseases, leukocyte loss, psoriasis, liver
damage and AIDS.4 Measuring the level of glutathione in living
measure physiological and clinical glutathione levels has attracted
wide attention.
Current methods to detect thiols include high performance
liquid chromatography, capillary electrophoresis, UVevis spec-
trometry, mass spectrometry, electrochemical analysis, and surface
enhanced Raman scattering. Fluorescence detection was consid-
ered to be a convenient method due to its simplicity, sensitivity,
and high efficiency.6 To date, researches on thiols fluorogenic probe
s have shown significant development. Many organic reactions
have been applied to the synthesis of such probes, namely: Michael
addition,7 nucleophilic substitution,8 disulfide exchange reactions,9
cyclization reactions between aldehydes and aminothiols,10 and
demetallation.11 However, due to the fact that most thiols in vivo
have very weak or no fluorescence, a high sensitivity fluorophore is
required for this application. Because of the similarities in the
structures and properties of various thiols compounds, design and
preparation of a probe with high selectivity and sensitivity is still a
great challenge.
Coumarins are often used as chromophores in the preparation of
highly effective fluorogenic probes due to their highly intense
fluorescence, good solubility, ease of preparation, relatively high
* Corresponding author.
** Corresponding author.
E-mail addresses: guangjiehe@163.com (G. He), wqzmgl@126.com (Q. Wang).
http://dx.doi.org/10.1016/j.tet.2016.12.012
0040-4020/© 2016 Elsevier Ltd. All rights reserved.
Please cite this article in press as: He G, et al., Synthesis of a fluorogenic probe for thiols based on a coumarin schiff base copper complex and its
use for the detection of glutathione, Tetrahedron (2016), http://dx.doi.org/10.1016/j.tet.2016.12.012

2
G. He et al. / Tetrahedron xxx (2016) 1e6
molar absorption coefficient and fluorescence quantum yield.12 The
excitation wavelength of coumarins falls within the visible range,
and its derivatization and modification are simple.13 Herein, in this
paper we report a new fluorescence probe 1-Cu2þ, which was
produced by coordination between a coumarin derivate 1 and Cu2þ
ions. The precursor compound 1 was prepared by a simple schiff
base condensation reaction of 7-(diethylamino)-2-oxo-2H-chro-
men-e-3-carbo- hydrazide (2) and pyridine-2,6-dicarbaldehyde.
This probe could be used for rapid, highly selective and sensitive
detection of thiols. The process and mechanism of detection by this
probe were investigated by UVevis, fluorescence spectra titration
and mass spectrometry.
hydroxybenzaldehyde, diethyl malonate, piperidine, hydrazine
hydrate (80%) and the other reagents were purchased from Sino-
pharm Chemical Reagent Co. Ltd. Stock solutions (2.0 ꢀ 10ꢁ2 M) of
the perchlorate Naþ, Mg2þ, Kþ, Ca2þ, Cr3þ, Mn2þ, Fe3þ, Fe2þ, Co2þ
,
Ni2þ, Cu2þ, Zn2þ, Agþ, Cd2þ, Hg2þ, Pb2þ and the amino acids plus
GSH were prepared in aqueous solutions. Stock solutions of com-
pound 1 and compound 1-Cu2þ (10
mM) for spectral measurement
were prepared in CH3CN:HEPES (3:2, v/v) solution. Stock solutions
of compound 1 and compound 1-Cu2þ for fluorescence imaging in
cells were prepared in DMSO solution. DMSO has a better solubility
for compound 1 and compound 1-Cu2þ and the lower cytotoxicity.
Each time a 3 mL compound 1 or compound 1-Cu2þ was filled in a
quartz cell of 1 cm optical path length, and different stock solutions
of metal ions or amino acids were added into the quartz cell
gradually by using a micro-syringe.
2. Experiment section
2.1. Instruments
2.3. Synthetic procedure
1HNMR and 13CNMR spectra were measured on a Bruker
Ascend™ 400 spectrometer with chemical shifts reported as ppm
with TMS as internal standard. Mass spectrometric data were ob-
tained with a Bruker Microtof-QIII spectrometry. UVevis absorption
spectra were recorded with Shimadzu UV2550 spectrophotometer.
Fluorescence spectra were recorded with Edinburgh Instruments
FS-5 fluorescence spectrophotometer. Cell imaging was recorded
with Nikon Eclipse TE2000-S.
The synthetic pathway for compound 1 was shown in Scheme 1.
7-N,N-dimethylamino-2-oxo-2H-3-coumarate (compound 3) and
coumarin hydrazine (compound 2) were prepared according to
previous reports.14 Compound 1 was synthesized from compound 2
and 2,6-pyridine dicarboxaldehyde, and characterized by 1H NMR,
13C NMR, ESI-MS and IR spectra (Figs. S1eS4). Compound 1-Cu2þ
was prepared from compound 1 and Cu(ClO4)2 in ethanol and
characterized by ESI-MS and IR spectra (Figs S5eS7, S11). ESI-MS
showed a 1:1 M ratio between compound 1 and Cu(ClO4)2.
2.2. Reagents
All the chemicals were of analytical grade and used as received.
2,6-pyridine dicarboxaldehyde was purchased from Tianjin
Heowns Biochemical Technology Co., Ltd. 4-(Diethylamino)-2-
2.3.1. Synthesis of ethyl 7-(diethylamino)-2-oxo-2H-chromene-3-
carboxylate14a
Compound 4 (3.8648 g, 0.02 mol), diethyl malonate (6.4067 g,
Scheme 1. Syntheses of compound 1 and compound 1-Cu2þ
.
Please cite this article in press as: He G, et al., Synthesis of a fluorogenic probe for thiols based on a coumarin schiff base copper complex and its
use for the detection of glutathione, Tetrahedron (2016), http://dx.doi.org/10.1016/j.tet.2016.12.012

G. He et al. / Tetrahedron xxx (2016) 1e6
3
0.04 mol) and piperidine (2 mL) were mixed in 60 mL anhydrous
ethanol. The mixture was refluxed for 6 h, cooled to room tem-
perature, and evaporated to give compound 3 as orange oil, which
was used directly for the next step.
dissolved completely in CH3CN:HEPES (3:2, v/v) buffer, as shown in
Fig. S12. Therefore, this ratio is suitable for the spectral
experiments.
3.2. The dependence of pH
2.3.2. Synthesis of 7-(diethylamino)-2-oxo-2H-chromene-3-
carbohydrazide14b
The effect of pH on the fluorescence intensity for compound 1
m
M) treated with Cu2þ (4 equiv.) and GSH (2 equiv.) in
Compound 3 (4.3399 g, 15 mmol) and hydrazine monohydrate
(3.64 mL, 60 mmol, 80%) were added into an ethanol (40 mL) so-
lution. The mixture was stirred for 12 min at room temperature.
After cooling in an ice bath for 15 min, the precipitate was filtered
and purified by chromatography (mobile phase, ethyl acetate) to
give compound 2 as orange needles, mp 161e162 ꢂC. Yield: 83%.
(10
CH3CN:HEPES (3:2, v/v) buffer was showed in Fig. S13. The fluo-
rescence intensity changed less than 10% in the range of 6.0e8.0,
which indicated that compound 1 could be applied in physiological
condition.
3.3. The UVevis absorption and fluorescence spectra responses of
compound 1 to Cu2þ ions
2.3.3. Synthesis of compound 1
Compound 2 (0.3028 g, 1.1 mmol) and 2,6-pyridine dicarbox-
aldehyde (0.0676 g, 0.5 mmol) were dissolved in ethanol (30 mL).
After refluxed for 12 h under nitrogen and cooled to room tem-
perature, the obtained yellow precipitate was filtered, washed and
The UVevis absorption spectrum of compound 1 (10
mM) in
CH3CN:HEPES (3:2, v/v) solution was shown in Fig.1. The maximum
UVevis absorption peak of compound 1 was at 440 nm. Upon
addition of Cu(ClO4)2, the UVevis absorption peak of compound
1 at 440 nm gradually decreased. After the addition of 6 equivalents
of Cu2þ, its peak value reached equilibrium. The UVevis absorption
dried to afford compound 1. 1H NMR (400 MHz, CF3COOD):
d (ppm)
9.53 (s, 2H), 8.95 (s, 2H), 8.92e8.79 (m, 1H), 8.73e8.56 (m, 2H),
8.44e8.31 (m, 2H), 8.04e7.94 (m, 2H), 7.86e7.81 (m, 2H), 3.99 (q,
J ¼ 6.9 Hz, 8H), 1.40 (t, J ¼ 7.0 Hz, 12H); 13C NMR (100 MHz,
spectra of compound 1 (10 m
M) with other metal ions (such as Naþ,
CF3COOD):
d (ppm) 155.1, 152.2, 148.7, 145.9, 142.2, 140.4, 139.9,
Mg2þ, Kþ, Ca2þ, Cr3þ, Mn2þ, Fe3þ, Fe2þ, Co2þ, Ni2þ, Cu2þ, Cuþ, Zn2þ
,
133.8, 127.9, 120.3, 119.7, 117.5, 115.2, 111.7, 55.5, 9.0. ESI-MS: m/z:
Agþ, Cd2þ, Hg2þ, Pb2þ, as ClO4ꢁ salt) were shown in Fig. S14. The
result indicated that Cu2þ showed the most significant response to
compound 1 in the UVevis absorption spectrum, other metal ions
had weak impacts on the absorption.
650.2672, [compound 1 þ H]þ; 672.2513, [compound 1 þ Na]þ.
2.3.4. Synthesis of probe compound 1-Cu2þ and other complexes
Compound 1 (0.0065 g, 0.01 mmol) and excess Cu(ClO4)2$6H2O
(0.0074 g, 0.02 mmol) were dissolved in ethanol (20 mL). After
refluxed for 30 min, the obtained precipitate was filtered, washed
and dried to afford compound 1-Cu2þ. Yield: 79%. ESI-MS: m/z:
711.1933. In order to investigate the behaviors of compound 1 with
other metal ions, complexes such as compound 1-Co2þ, compound
1-Zn2þ, and compound 1-Mg2þ were also prepared in a similar
method, and further characterized by ESI-MS (Figs. S8eS10).
The fluorometric titration experiment of compound 1 (10 mM)
with Cu2þ was proceeded in CH3CN:HEPES (3:2, v/v) solution
(Fig. 2). compound 1 exhibited a strong fluorescence emission peak
at 485 nm (excited at 440 nm). Upon addition of Cu(ClO4)2, the
fluorescence intensity of compound 1 gradually decreased. After
the addition of 4 equivalents of Cu2þ, the peak intensity reached
equilibrium. The low fluorescence intensity of the compound 1-
Cu2þ might be due to the quenching effect resulted from the
paramagnetism and photoinduced electron transfer of the copper
ion.15 As a comparison, the fluorescence responses of compound 1
3. Results and discussions
to the other metal ions (such as Naþ, Mg2þ, Kþ, Ca2þ, Cr3þ, Mn2þ
,
Fe3þ, Fe2þ, Co2þ, Ni2þ, Cu2þ, Cuþ, Zn2þ, Agþ, Cd2þ, Hg2þ, Pb2þ, as
ClOꢁ4 salt.) were also investigated (Fig. S15), the results were
3.1. The experiment of water solubility
Compound 1 has a relatively high fluorescence intensity and
Fig. 1. The UVevis spectra titration of compound 1 (10
m
M) upon addition of Cu2þ in
Fig. 2. The fluorescence spectra titration of compound 1 (10 mM) upon addition of
CH3CN:HEPES (3:2, v/v) solution. Inset: UVevis titration profile of compounds 1 upon
Cu2þ in CH3CN:HEPES (3:2, v/v) solution. Insert: fluorescence titration profile at
addition of Cu2þ, the absorption spectra were recorded at 440 nm.
485 nm upon the addition of Cu2þ (excited at 440 nm).
Please cite this article in press as: He G, et al., Synthesis of a fluorogenic probe for thiols based on a coumarin schiff base copper complex and its
use for the detection of glutathione, Tetrahedron (2016), http://dx.doi.org/10.1016/j.tet.2016.12.012

4
G. He et al. / Tetrahedron xxx (2016) 1e6
consistent with that observed in UVevis titration experiments, in
which Cu2þ showed the most significant quenching effect to
compound 1. Other metal ions especially Cuþ had no influence to
compound 1, and Co2þ ion had relative weak influence than Cu2þ
ion.
probability that the -C¼N- group participated in the coordination
while the carbonyl groups of the -CO-NH- and coumarin units were
not involved in the coordination.17
3.4. The fluorescence spectra response of the probe (compound 1-
Furthermore, the solid form of compound 1-Cu2þ was prepared
in ethanol and characterized by ESI-MS and IR spectra. The evi-
dence of complexation behavior of compound 1 (C35H35N7O6) with
Cu2þ came directly from ESI-MS spectrum. As shown in Fig. S5, the
predominated peak at m/z ¼ 711.1933 corresponding to [(com-
pound 1)þCu2þ-Hþ] ¼ C35H34CuN7Oþ6 was observed in ESI-MS
spectrum. The experimental isotopic patterns (Fig. S6) fit well
with the theoretical simulation result (the predominated peak at
m/z ¼ 711.1966, Fig. S7) calculated by using the IsoPro 3.0 program.
Such results indicated that the composition of compound 1-Cu2þ is
Cu2þ) to thiols
The fluorometric titration experiment of probe (compound 1-
Cu2þ) with glutathione was proceeded in CH3CN:HEPES (3:2, v/v)
solution. As shown in Fig. 3, along with glutathione added, the
fluorescence intensity of this probe (compound 1-Cu2þ) was
gradually restored, which may caused by demetallation effect of the
probe to released compound 1 into the solution. After 2 equivalents
of glutathione added, the fluorescence intensity of the solution
reached equilibrium. Therefore, compound 1-Cu2þ could be used
for the detection of glutathione with fluorescence response “turn-
on” manner.
1: 1 M ratio between compound 1 and Cu2þ
.
IR spectrum of compound 1-Cu2þ revealed the possible coor-
dination model of compound 1 with copper ions (Scheme 1). IR
spectrum of compound 1 exhibited characteristic peaks of certain
groups such as -C¼N- group (1582.63 cmꢁ1), the carbonyl group of
-CO-NH- unit (1618.05 cmꢁ1) and the carbonyl group of the
coumarin unit (1700.47 cmꢁ1).11a,16 After coordinated with copper
ions, the characteristic stretching band of the -C¼N- group was
shifted to lower value (1573.50 cmꢁ1) while the carbonyl stretching
bands of the -CO-NH- unit (1617.82 cmꢁ1) and coumarin unit
To further investigate the selectivity of the probe 1-Cu2þ for
thiols, the fluorescence responses of the probe to different amino
acids were measured (Fig. 4). The fluorescence intensity of the
thiols probe itself was very weak. When 2 equivalents of thiols-
containing amino acids (excess), such as N-acetyl-
L-cysteine, DL-
homocystine, -homocysteine, -cysteine and GSH, were added
L
L
respectively, the fluorescence intensity increased. Among these
thiols-containing compounds, GSH exhibited the most obvious
enhancement. The reason might be that GSH has the propensity of
generating CueS bond, which may grab the Cu2þ ions from probe
and lead to a demetallation reaction of the probe, however Cys and
(1701.39 cmꢁ1
) only shifted slightly. It was assumed in all
Fig. 3. The fluorescence responses of compound 1-Cu2þ (10
mM) upon addition of GSH
in CH3CN:HEPES (3:2, v/v) solution upon the addition of increasing GSH. Insert:
fluorescence titration profile of compound 1-Cu2þ at 485 nm upon the addition of GSH
(excited at 440 nm).
Fig. 5. Fluorescence intensity (485 nm) of compound 1-Cu2þ (1
(3:2, v/v) solution upon additions of GSH with an excitation wavelength at 440 nm.
mM) in CH3CN:HEPES
Fig. 4. The fluorescence intensities of compound 1-Cu2þ (10
equivalents of different amino acids; the second row showed the fluorescence intensities after subsequent addition of 2 equivalents of GSH to the non-sulfhydryl amino acids
m
M) to different amino acids (lem ¼ 485 nm). The first row showed the fluorescence intensities upon addition of 2
solution, respectively.
Please cite this article in press as: He G, et al., Synthesis of a fluorogenic probe for thiols based on a coumarin schiff base copper complex and its
use for the detection of glutathione, Tetrahedron (2016), http://dx.doi.org/10.1016/j.tet.2016.12.012

G. He et al. / Tetrahedron xxx (2016) 1e6
5
Fig. 6. The fluorescence images of SiHa cells incubated with 1 (a), SiHa cells incubated with probe (b), SiHa cells pretreated by erlotinib then incubated with probe (c) and their
corresponding bright field images.
Hcy tend to form CueN bond, which is less stable than CueS bond
formed by GSH.18 Thus GSH manifested a higher fluorescence
response than Cys/Hcy. Meanwhile, as a control group, addition of 2
equivalents of non-sulfhydryl amino acid did not result in obvious
change of the fluorescence intensity. Subsequently 2 equivalents of
GSH were added, the fluorescence intensity significantly increased,
which indicated that compound 1-Cu2þ had a particular response
to GSH, and the response was not disrupted by other competitive
non-thiols amino acids. The above experiments demonstrated that
compound 1-Cu2þ had good selectivity for GSH.
excitated with blue light (Fig. 6a), which indicated that compound 1
had strong cell permeability. The probe (compound 1-Cu2þ) was
used directly to stain the SiHa cells (Fig. 6b), and the significant
intracellular green fluorescence in SiHa cells was also observed,
which indicated that the fluorescence of probe could be restored by
GSH in SiHa cells. Another well was pretreated by erlotinib (Fig. 6c)
(an anticancer drug) for 1 h. The intracellular green fluorescence
intensity is relative weak than that without pretreated by erlotinib,
which might be due to treatment by erlotinib could create a redox
stress environment and decrease the intracellular GSH content.21
The results above proved that the probe (compound 1-Cu2þ
)
3.5. The detection limit of the probe (compound 1-Cu2þ) for GSH
could determine the intracellular GSH.
Then MTT experiments (Fig. S16, Table 1) were proceeded, and
the results demonstrated that the cells were visible and showed no
significant toxicity or side effects when the probe concentration
To further investigate the sensitivity of the probe 1-Cu2þ for
GSH, the detection limit experiment for GSH was performed. When
was less than 10 mM (viability was higher than 75%, approximately
the probe was employed at 1
mM and the slits were adjusted to 1.5
16 h). The fluorescent imaging experiment indicated that the thiols
fluorogenic probe (compound 1-Cu2þ) could be used for the
detection of GSH in living cells.
nm/1.5 nm, the fluorescence intensity at 485 nm was linearly
proportional to the concentration of GSH in the range of 0.0e1.8 mM
(Fig. 5), indicating the potential capability of compound 1-Cu2þ for
quantitative detection for GSH. The detection limit for GSH was
determined to be 0.2
mM, which was significantly below the
4. Conclusion
physiological levels of GSH in live cells (1e10 mM).19 In vivo ex-
periments, we utilize the 10ꢁ7 order of magnitude probe to incu-
bate the SiHa cells at 37 ꢂC for 60 min. Under Nikon Eclipse TE2000-
S fluorescent inverted microscope, green fluorescence could still be
observed. The result indicated that in vivo the probe still keep very
sensitive to GSH (Fig. S17).
In summary, a new thiols fluorogenic probe (compound 1-Cu2þ
was prepared. The fluorescence spectra indicated that the probe
had relatively good selectivity and sensitivity to GSH in
CH3CN:HEPES (3:2, v/v) solution. And the detection limit for GSH
)
was determined as 0.2 mM, suggesting that the probe is sensitive
enough for the determination of GSH. On the other hand, the flu-
orogenic probe at pH 6.0e8.0 conditions was stable, indicating that
the probe could be used under physiological condition. In addition,
the probe showed good cell membrane permeability and low
toxicity to cells and could be used for the detection of GSH in living
cells.
3.6. The fluorescence imaging of the probe (compound 1-Cu2þ) for
GSH
Cell imaging with a fluorogenic probe had important signifi-
cance and application.20 The fluorescence imaging of the probe
(compound 1-Cu2þ) for GSH was performed in cervical cancer SiHa
cells. Before the fluorescence imaging, SiHa cells were cultured in a
12-well cell culture plate for 24 h, incubated with compound 1
Acknowledgment
(3
mM) and the probe (3
m
M) at 37 ꢂC for 60 min respectively, and
This work was financially supported by the National Natural
Science Foundation (No.21371148), the financial support from Key
Scientific and Technological Project of Henan Province
(No.122102310196), the financial support from Education Project of
Henan Province (No. 12A150019), and the support project for the
subsequently washed twice with PBS solution. Under Nikon Eclipse
TE2000-S fluorescent inverted microscope, the images of the SiHa
cells were shown in Fig. 6 (Fig. 6a as control). Compound 1 showed
significant intracellular green fluorescence in SiHa cells when
Please cite this article in press as: He G, et al., Synthesis of a fluorogenic probe for thiols based on a coumarin schiff base copper complex and its
use for the detection of glutathione, Tetrahedron (2016), http://dx.doi.org/10.1016/j.tet.2016.12.012

6
G. He et al. / Tetrahedron xxx (2016) 1e6
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Appendix A. Supplementary data
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.tet.2016.12.012.
(c) Chowdhury A, Howlader P, Mukherjee PS. Chem
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Please cite this article in press as: He G, et al., Synthesis of a fluorogenic probe for thiols based on a coumarin schiff base copper complex and its
use for the detection of glutathione, Tetrahedron (2016), http://dx.doi.org/10.1016/j.tet.2016.12.012