Synthesis and application of a “turn on” fluorescent probe for glutathione based on a copper complex of coumarin hydrazide Schiff base derivative

Article abstract of DOI:10.1016/j.bioorg.2019.103176

Discrimination and quantification of intracellular biothiols, such as cysteine (Cys), homocysteine (Hcy), glutathione (GSH) under physiological conditions is significant for academic research and disease diagnosis. A new fluorescent probe (complex 1-Cu²⁺) for discriminate detection of GSH was prepared by copper ions coordinate with coumarin carbohydrazide Schiff base derivative 1. In suitable buffer solution (CH₃CN: HEPES = 3:2, v/v) and under appropriate pH condition (pH = 7.2–7.4), the UV–vis spectroscopy experiments showed that compound 1 and copper ion exhibited a 1:1 ratio binding mode and moderate binding ability. Fluorescence quenching of compound 1 was observed when it complexed with Cu²⁺ ions. An obviously fluorescence restoration appeared after addition of GSH to the solution of probe, which also exhibited a highly selectivity relative to cysteine (Cys) and homocysteine (Hcy) in the amino acid competitive experiments. The minimum detection limit was calculated to 0.12 μM by fluorescent method, which was distinctly below the physiological concentration of GSH in live cells. Its biological application to detect the endogenous GSH was further proved by the HepG2 cell fluorescence image test.

Full text of DOI:10.1016/j.bioorg.2019.103176

Journal Pre-Proof
Synthesis and application of a “turn on” fluorescent probe for glutathione based
on a copper complex of coumarin hydrazide Schiff base derivative
Guangjie He, Xiaobo Hua, Nan Yang, Linlin Li, Jinhe Xu, Linlin Yang, Qingzhi
Wang, Liguo Ji
PII:
S0045-2068(18)31469-X
DOI:
https://doi.org/10.1016/j.bioorg.2019.103176
YBIOO 103176
Reference:
Bioorganic Chemistry
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Received Date:
Revised Date:
Accepted Date:
4 December 2018
26 July 2019
28 July 2019
Please cite this article as: G. He, X. Hua, N. Yang, L. Li, J. Xu, L. Yang, Q. Wang, L. Ji, Synthesis and application
of a “turn on” fluorescent probe for glutathione based on a copper complex of coumarin hydrazide Schiff base
derivative, Bioorganic Chemistry (2019), doi: https://doi.org/10.1016/j.bioorg.2019.103176
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Synthesis and application of a “turn on” fluorescent probe
for glutathione based on a copper complex of coumarin
hydrazide Schiff base derivative
Guangjie He*, Xiaobo Hua, Nan Yang, Linlin Li, Jinhe Xu, Linlin Yang, Qingzhi Wang*, Liguo
Ji*
Xinxiang Key Laboratory of Forensic Science Evidence, School of Forensic Medicine, Xinxiang
Medical University, Jinsui Road No. 601, Xinxiang, 453003, Henan Province, P. R. China.
* Corresponding author: Tel./Fax: +86-373-3831183
E-mail: guangjiehe@163.com; wqzmgl@126.com; liguoji_academy@126.com
Abstract:
Discrimination and quantification of intracellular biothiols, such as cysteine
(Cys), homocysteine (Hcy), glutathione (GSH) under physiological conditions is
significant for academic research and disease diagnosis. A new fluorescent probe
(complex 1-Cu2+) for discriminate detection of GSH was prepared by copper ions
coordinate with coumarin carbohydrazide schiff base derivative 1. In suitable buffer
solution (CH3CN: HEPES = 3:2, v/v) and under appropriate pH condition (pH = 7.2-
7.4), the UV-vis spectroscopy experiments showed that compound 1 and copper ion
exhibited a 1:1 ratio binding mode and moderate binding ability. Fluorescence
quenching of compound 1 was observed when it complexed with Cu2+ ions. An
obviously fluorescence restoration appeared after addition of GSH to the solution of
probe, which also exhibited a highly selectivity relative to cysteine (Cys) and
homocysteine (Hcy) in the amino acid competitive experiments. The minimum
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detection limit was calculated to 0.12 μM by fluorescent method, which was distinctly
below the physiological concentration of GSH in live cells. Its biological application
to detect the endogenous GSH was further proved by the HepG2 cell fluorescence
image test.
Keywords: fluorescent probe, glutathione probe, coumarin hydrazide Schiff base
derivative, cell imaging
1. Introduction:
Thiols in vivo such as cysteine (Cys), homocysteine (Hcy), and glutathione
(GSH) play crucial roles in maintaining metabolism in biological systems. In
particular, Glutathione as an antioxidant intracellular tripeptide, present in
intracellular compartments such as cell nucleus, mitochondria, and the cytosol, is in
the range of 2-10 mM, whereas it is about 2-20 μM in blood and the extracellular
matrix [1]. It serves many cellular functions, such as maintenance of intracellular
redox activities, xenobiotic metabolism, intracellular signal transduction, and
scavenging free radicals [2,3]. In addition, the occurrence of a variety of human
diseases are related to the alternations in the level of intracellular glutathione, such as
cancer, AIDS, cardiovascular disease, neurodegenerative diseases, Alzheimer's
disease [4,5], etc. Accordingly, it is significant to discriminately detect and quantify
such mercaptan biomolecules under physiological conditions for academic research
and disease diagnosis.
Compared to several of traditional detection techniques [6], optical detections as
a type of convenient method have attracted enormous attention. Especially,
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fluorescent method with the advantage of simplicity, low detection limitation, non-
invasiveness and high spatiotemporal resolution for imaging has made a great
progress [7]. a variety of fluorescent probes based ingenious reactions have been
developed to detect biothiols, such as Michael addition, cyclization with aldehyde,
cleavage of sulfonamide and sulfonate ester by thiols, cleavage of selenium–nitrogen
bond by thiols, cleavage of disulfide by thiols, metal complexes-oxidation–reduction,
metal complexes-displace coordination and so on [8–11]. However, due to the
similarity of the structures and the reaction activities of Cys, Hcy and GSH,
construction of appropriate probes for discriminating one thiol species from another is
still desirable.
Coumarin as fluorophore with the advantage of high fluorescence intensity,
excellent solubility, efficient cell permeation, high quantum yield and ease of
preparation has been widely used in the design of fluorescence probes [12–18].
Herein we condensed coumarin carbohydrazide and imidazole-2-carbaldehyde to
form a fluorescent Schiff base derivative 1, this compound then coordinated with
copper ions to form a fluorescent probe (complex 1-Cu2+), which have a selectivity to
GSH relative to the Cys and Hcy biothiols. The interaction process and mechanism
were also investigated by UV-Vis spectroscopy, fluorescence spectroscopy and mass
spectrometry and etc.
2. Experimental sections:
2.1 Experimental reagent
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All compounds were analytical grade and directly used as received.
4-
(diethylamino)-2-hydroxybenzaldehyde, diethyl malonate, piperidine, hydrazine
hydrate (80%) and the other chemical reagents were purchased from Energy Chemical
Co. Ltd. Stock perchlorate solutions (2.0×10-2 M) of various metal ions such as Cu2+,
Mn2+, Fe2+, Fe3+, Ni2+, K+, Cd2+, Mg2+, Na+, Cu+, Al3+, Co2+, Pb2+, Hg2+, Cr3+, Ag+,
Ca2+ and Zn2+ were prepared in aqueous solutions. The stock solutions (2.0×10-2 M)
of various amino acid such as GSH, Cys, Hcy, His, Trp, Lys, Phe, Pro, Ser, Thr, Gly
were also produced in aqueous media. In the spectral measurements, the stock
solutions of compound 1 were prepared in CH3CN: HEPES (3:2, v/v, pH = 7.2-7.4)
solutions. A 3 mL solution of compound 1 was filled in a quartz cell of 1 cm optical
path length each time, by using a micro-syringe different stock solutions of metal ions
or amino acid were added into the quartz cell gradually.
2.2 Experimental apparatuses
The NMR spectra data were recorded with chemical shifts as ppm with TMS as
internal standard measured on a Bruker Ascend™ 400 spectrometer. Mass
spectrometric data were measured on a Bruker Microtof-QIII spectrometry. Crystal
diffraction data was collected on Bruker SMART APEX II diffractometer. UV-vis
absorption spectra data were obtained by Shimadzu UV2600 spectrophotometer.
Fluorescence spectra were recorded with Edinburgh Instruments FS-5 fluorescence
spectrophotometer. Cell imaging was recorded with Leica TCS SP8 laser confocal
microscopy.
2.3 Experimental process
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<Scheme 1 Here>
The synthetic procedures were shown in Scheme 1. Following the previous work
[19,20], the coumarin hydrazide (compound 2) and ethyl-7-(diethylamino)-2-oxo-2H-
chromene -3-carboxylate (compound 3) were synthesized. Compound 1 was obtained
conveniently by condensation reaction of compound 2 with 1H-imidazole-2-
1
13
carbaldehyde, its structure was characterized by H NMR, C NMR, and ESI-MS
(Fig. S2, S3, S4). Subsequently, the complex 1-Cu2+ was also prepared by reaction of
compound 1 with Cu(ClO4)2·6H2O under reflux condition for 2 h. Then for revealing
more definite structural information of the compound 1 and complex 1-Cu2+, single
crystal culture experiments were performed. However only a suitable single crystal of
compound 1 (Fig. 1) was obtained by slowly evaporation of its methanol solution in
air at room temperature. In this structure it was seemed that a water molecular was
fixed by four hydrogen bonds formed with two ligand molecules (Fig. S1, Table. S1).
This crystal structure further provided an evidence that the compound 1 was prepared
correctly. More detailed experimental data could be found in supplementary
information.
< Fig. 1 Here>
3. Results and discussions
3.1 The test of the water solubility
Good water solubility is a prerequisite for the application of probes to detect
small biological molecules in vivo, so a test by fluorescent spectrometry was made to
verify the water solubility of the compound 1 (λex = 441nm, λem = 485nm) in different
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ratio solution of CH3CN and HEPES buffer (Fig. S5). The results indicated that the
fluorescent intensity of compound 1 was decreasing obviously along the increasing
ratio of HEPES, for keeping a balance between the fluorescent intensity and water
solubility, the buffer solution (CH3CN: HEPES = 3:2, v/v) was used in the following
experiments.
3.2 The pH dependence of the probe
The stability of the probe is affected obviously by different pH environment, so
the pH dependent experiment of the precursor (compound 1) and probe (compound 1-
Cu2+) were performed in the solutions (CH3CN: HEPES = 3:2, v/v) (Fig. S6). The
results indicated that the fluorescent intensity of both compounds (485nm) was stable
in the pH range from 5.5 to 10. Thus, the pH range in the following solution tests was
controlled in 7.2-7.4 and which also indicated that the probe could be potentially
applied in physiological condition.
3.3 The UV-vis spectral characteristics
For surveying the metal ions selectivity of the compound 1, the UV-Vis
spectrometry experiments were carried on in the solution (10.0 μmol/L, CH3CN:
HEPES = 3:2, v/v) of compound 1 with various 5.0 equivalent metal ions such as
Cu2+, Mn2+, Fe2+, Fe3+, Ni2+, K+, Cd2+, Mg2+, Na+, Al3+, Co2+, Pb2+, Hg2+, Cr3+, Ag+,

Ca2+, Zn2+ (as ClO4 salts). The results indicated that the maximum absorption peak of
compound 1 appeared at 441 nm and various degrees of weaken and red shift of the
maximum absorption peak was induced by separately addition of Cu2+, Co2+, Ni2+,
Zn2+ ions (Fig. S7), wherein copper ions induced most changes relative to other three
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metal ions. These results demonstrated that the precursor (compound 1) could be used
to selectively recognize Cu2+ ions. For further surveying the solution properties of
compound 1 with copper ions, the UV-vis spectra titration tests of the compound 1
with Cu2+ ions were also performed (Fig. 2). With the concentration increasing of the
Cu2+ ions, obviously, the sharp decreasing and red shift of the maximum absorption
peak at 441 nm were observed in the titration experiments and approximately 5.0
equivalents Cu2+ ions could induce equilibrium. Meanwhile, a new absorption peak at
500 nm appeared which indicated that a new species was formed in the solution.
For further elucidating the binding behaviors of the compound 1 with the copper
ions in solution, a job’s plot experiment was performed (Fig.S8) in solution (CH3CN:
HEPES = 3:2, v/v). The results indicated that compound 1 and Cu2+ ions exhibited 1:1
binding mode to form complexes in solution. The binding constant was also
calculated from the UV-vis spectra titration data by employing the Benesi–Hildebrand
(B-H) relation [21–24], which could be expressed in terms of absorbance (A) for 1:1
complex as follows:
1
1
1
=
+
2 +
퐴 ‒ 퐴0 퐴1 ‒ 퐴0
(
)
[
퐴1 ‒ 퐴0 퐾 퐶푢
]
Where K is association constant, A0 is the absorbance of compound 1 without
metal ions addition, A and A1 is the absorbance of compound 1 after addition of metal
ion at every successive steps and excess amounts of metal ions at particular
wavelength (441nm), respectively. A straight line was provided by plotting of 1/(A0-
A) vs 1/[Cu2+], which also indicated 1:1 complex formation. The constant value was
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calculated as K = 4.35104 M-1 (R2 = 0.995) (Fig. S9). It was indicated that moderate
strong binding affinity existed between compound 1 and Cu2+ ions.
< Fig. 2 Here>
3.4 The fluorescent spectral characteristics
Subsequently, the fluorescent properties of the precursor (compound 1) was
investigated in solution (CH3CN: HEPES = 3:2, v/v, pH = 7.3). The maximum
fluorescence emission peak of the compound 1 (excited at 441 nm) was observed at
485 nm. Then various metal ions such as Fe2+, Na+, Cu+, Al3+, Mn2+, Pb2+, Mg2+, K+,

Ca2+, Cr3+, Fe3+, Cd2+, Ag+, Hg2+, Ni2+, Co2+, Cu2+ (as ClO4 salts) were added and the
fluorescent responses were recorded (Fig. 3). The results showed that the most
obvious fluorescence quenching was induced by Cu2+ ions, a slight fluorescence
quenching was observed when Co2+, Ni2+, Hg2+ were added. Other metal ions induced
fluorescence almost no response. The fluorescence quenching mechanism might be
attributed to the photoinduced charge transfer (PCT) mechanism. The Cu2+ cation
binding to the acceptor group of fluorophores 1 enhanced its electron-withdrawing
character, and the interaction between 1 and Cu2+ ion provided an additional
fluorescence quenching pathway, fluorescence of the compound 1 was quenched by
the exciton energy transfer from the ligand to the metal d-orbital or ligand to metal
charge transfer (LMCT) essentially, which had already been confirmed by the
literatures [25–30].
< Fig. 3 Here>
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In addition, a competitive fluorescence experiment of compound 1 (10.0 μmol/L,
CH3CN:HEPES = 3:2, v/v) with copper ions under existence of equivalent other metal
ions such as Fe2+, Na+, Cu+, Al3+, Mn2+, Pb2+, Mg2+, K+, Ca2+, Cr3+, Fe3+, Cd2+, Ag+,
-
Hg2+, Ni2+, Co2+ (as ClO4 salts) was also carried out (Fig. S10). The results
demonstrated that the fluorescence quenching effect of copper ion was almost not
disturbed by various other equivalent metal ions. It was further indicated that
compound 1 could more strongly and selectively bind to copper ions, which was
similar to the results observed in UV-Vis spectroscopy experiments.
Subsequently, a fluorescence titration experiment was also performed to evaluate
the binding behaviors between compound 1 and copper ions (Fig. 4). In the solution
(CH3CN: HEPES = 3:2, v/v), the fluorescence intensity of compound 1 decreased
gradually along with the addition of Cu2+ ions, approximately 5 equivalent copper
ions could induce complete fluorescence quenching although the complex was formed
in the term of 1:1 binding mode. It was attributed that a reaction equilibrium existed
between compound 1 and complex 1-Cu2+, only by excess copper ions the compound
1 could be completely converted into the complex. In addition, the kinetic dynamic
process of compound 1 with the Cu2+ ions in solution was also evaluated by the time-
dependent fluorescence characteristics of the compound 1 after addition of 5
equivalent copper ions (Fig. S14). The results showed that the fluorescence was
quenched rapidly, and the complex was already formed in 20 seconds. The binding
constant was also calculated by the fluorescence titration dates through the Benesi–
Hildebrand (B-H) equation [21–24]. It was evaluated as K = 3.34105 M-1 (R2 = 0.999)
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(Fig. S11), which was similar to the result obtained from the UV-vis spectra titration
dates.
< Fig. 4 Here>
3.5 The fluorescence response of complex 1-Cu2+ to amino acids
For realized the purposes of the probe to be application in biological
environment, in vitro, the fluorescence response of this probe to different amino acids
such as GSH, Cys, Hcy, His, Trp, Lys, Phe, Pro, Ser, Thr, Gly and so on was initially
investigated in solution (CH3CN: HEPES = 3:2, v/v) (Fig. S12). The results indicated
that complex 1-Cu2+ itself was a fluorescence-off probe, the fluorescent intensity of
the probe was very weak. However, varying degrees of fluorescence recovery were
induced after excess different amino acids was added, wherein GSH induce the most
obvious restoration of the fluorescent intensity of the probe. Other biological
molecules including Cys and Hcy performed a little weaker response relative to GSH.
The reasons might attribute to a stronger ability to capture copper metal ions from
complexes 1-Cu2+ by GSH, which resulted in demetallation of the complex 1-Cu2+ to
release the compound 1 and fluorescence restoration [31]. These results provided a
further proof that the complex 1-Cu2+ could be used as a “turn-on” probe to detect
GSH. In addition, a good selectivity of the probe for GSH relative to other amino
acids was also confirmed by competitive experiments (Fig. 5). The results
demonstrated that the fluorescence “turn on” property of the probe by GSH in
solution (CH3CN: HEPES = 3:2, v/v) was not disturbed by equivalent other amino
acids.
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< Fig. 5 Here>
Subsequently, for further clarifying the sensitivity of the probe to GSH by
utilizing the Edinburgh Instruments FS-5 fluorescence spectrophotometer instrument,
the slits were adjusted to 1.5 nm/1.5 nm, a fluorescence titration experiments of the
probe with GSH was performed. As shown in Fig. 6, along with the addition of GSH,
approximately 6.0 equivalent GSH could induce the fluorescence recovery completely.
The detection limit of the probe to GSH was also evaluated by fluorescence method, a
good linear relationship was observed between the fluorescent intensity of probe and
the concentration of GSH in the range of 0 – 30 μM (Fig. S13). The minimum
detection limit was calculated to 0.12 μM, it was distinctly below the physiological
concentration of GSH in live cells, which was inferred that the probe could be used in
physiological conditions.
< Fig. 6 Here>
In addition, the kinetic dynamic process of probe (complex 1-Cu2+) with GSH in
solution was also evaluated by the time-dependent fluorescence variation of the probe
after addition of 6.0 equivalent GSH (Fig. S15). It was demonstrated that the probe
was a rapid response probe, and the fluorescence could be restored to the highest
value in 5 minutes. This feature of quick response to the target was beneficial to their
application in biomolecular detection.
3.6 The cell experiments of probe (complex 1-Cu2+)
In order to realize the biological application of the probe, the fluorescence
imaging test was carried out to detect intracellular glutathione. Leica TCS SP8 laser
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confocal microscopy was used to observe the HepG2 cell fluorescence image (Fig. 7).
Firstly, RPMI-1640 culture medium containing 10% fetal bovine serum was provided
to cultured cells for 24h. Then compound 1 (2.0 μM) was added and incubated for 3h,
rinsed with PBS buffer three times, under laser confocal microscopy a light green
fluorescence was observed. It was demonstrated that precursor (compound 1) could
permeate into the cell (Fig. 7a). However, the fluorescence of precursor was almost
quenched when excess Cu2+ ions (approximately 25 equivalents) were added (Fig. 7e).
The reason might be that the intracellular GSH prior to bind with Cu2+ ions and was
exhausted by the excess copper ions, thus result in no more GSH left to induce the
fluorescence restoration of the formed probe (complex 1-Cu2+), this phenomenon had
been also recorded in the literature [31]. At the meanwhile, the probe (complex 1-
Cu2+, prepared by compound 1 and Cu2+ at 1:5 ratio) (2.0 μM) was added directly and
incubated for 3h, rinsed with PBS buffer three times, under the microscopy, a green
fluorescence was observed again (Fig. 7b). This light green fluorescence might come
from the compound 1 produced by de-metallization of the probe by GSH in the
HepG2 cells. In order to further verify this hypothesis, a control experiment was also
conducted by pretreated the cells with thiol inhibitor (Fig. 7c and 7d). The cells were
prior pretreated by the L-buthionine-S, R-sulfoximine (BSO) (the GSH inhibitor, co-
incubated for 12.0 h), then the probe (complex 1-Cu2+, prepared by compound 1 and
Cu2+ at 1:5 ratio) (2.0 μM) was added directly, this time a very weak fluorescence was
observed (Fig. 7c), subsequently additional GSH (15.0 equivalents, 10.0 minutes) was
added, a green fluorescence was observed again (Fig. 7d). Thus, these results afforded
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a definitely evidence that the probe was suitable to detect the endogenous GSH in
physiological condition.
< Fig. 7 Here>
The cytotoxicity test of the probe (complex 1-Cu2+) was also investigated by
MTT experiment (Fig. S16), the HepG2 cells were cultured and seeded into 96-well
plates, then different concentrations of probes such as 0.0 μM, 2.0 μM, 4.0 μM, 6.0
μM, 8.0 μM and 10.0 μM were added. In each concentration the experiments were
repeated for 5 times, and the cells were co-incubated at the condition (5% CO2, 37 ℃)
for 24 hours, then the survival of cells at different probe concentrations was calculated.
The results demonstrated that the cell viability could still reach to 70.9% in the
concentration of 10.0 μM, which means that the probe was relatively low toxicity to
cells.
4. Conclusion:
Overall, a new fluorescent compound 1 was designed and synthesized by
coumarin hydrazine and imidazole-2-formaldehyde, it selectively coordinated with
Cu2+ ions at 1:1 ratio and formed a new fluorescence probe complex 1-Cu2+. The
fluorescence experiments of the probe to different amino acid indicated that it had
good selectivity and sensitivity to GSH. The minimum detection limit of the probe to
GSH was evaluated to 0.12 μM. The HepG2 cells fluorescence image tests also
demonstrated that it could be used to detect the endogenous GSH under the
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physiological conditions. This probe provided an alternative for the detection of GSH
biological molecular.
Acknowledgements
This work was supported by the National Natural Science Foundation of China
(No. 21371148), Key Scientific and Technological Project of Henan Province (No.
182102210496 and No. 182102310648), Natural Science Foundation of Henan
Province (No. 182300410309), Key Scientific Research Projects of Higher Education
Institutions of Henan Province (No. 19A150007 and No. 18A150044) and the
Doctoral Scientific Research Initiation Foundation of Xinxiang Medical University
(XYBSKYZZ201649).
Appendix: Supplementary data
Supplementary data related to this article can be found at XXX
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Figure captions:
Scheme 1. Synthetic route of compound 1 and the presumable structure of complex 1-
Cu2+.
Fig. 1 The crystal structure of compound 1 containing a water molecule, only three
hydrogen bonds appeared in the structure.
Fig. 2 The UV-vis spectra titration test of compound 1 (10.0 μM) with Cu2+ ions (as

ClO4 salts) in solution (CH3CN: HEPS =3:2, v/v). Insert: UV–Vis titration profile of
compound 1 upon addition of Cu2+ ions, the absorption spectra were recorded at 441
nm.
Fig. 3 The fluorescent intensity variation (at 485 nm) of compound 1 in solution (10.0

μM, CH3CN: HEPES = 3:2, v/v) after addition of various metal ions (as ClO4 salts).
Fig. 4 The fluorescence spectra titration of compound 1 (10.0 μM) upon addition of
Cu2+ in solution (CH3CN: HEPES = 3:2, v/v). Insert: fluorescence titration profile at
485 nm upon the addition of Cu2+ (excited at 441 nm).
Fig. 5 Fig. 5 The fluorescence response of the probe (10.0 μM, em = 485 nm) to
different amino acids (6.0 equivalents). The front row represents the fluorescence
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intensity of probe by addition of various amino acids, the back row represents the
fluorescence intensity after further addition of 6.0 equivalents GSH to the front row
solution
Fig. 6 The fluorescent titration experiment of probe (10.0 μM) with GSH in solution
(CH3CN: HEPES = 3:2, v/v), insert: the fluorescence response profile of probe at 485
nm upon addition of GSH (exited at 441 nm).
Fig. 7 the fluorescence images of HepG2 cells incubated with compound 1 (2.0 μM)
(a), the probe (complex 1-Cu2+, prepared by compound 1 and Cu2+ at 1:5 ratio) (2.0
μM) (b), pretreated with L-BSO, then addition of the probe (complex 1-Cu2+, 2.0 μM)
(c), additional GSH (15.0 equivalents) was added into (c) and incubated for another
10.0 minutes (d), incubated with compound 1 (2.0 μM) for 3.0 h then addition of 25.0
equivalents Cu2+ (e), and their corresponding overlay images (f - j) and bright field
images (k - o). All the images were taken after magnifying 40 times.
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Scheme 1. Synthetic route of compound 1 and the presumable structure of complex 1-
Cu2+.
CHO
OH
COOEt
O
CH2(COOEt)2
NH2NH2
piperidine
Et2N
Et2N
O
3
N
O
N
O
O
O
N
N
N
N
NHNH2
H
H
H
(Et)2N
O
O
Et2N
O
2
1
O
N
N
Cu(ClO4)2
Cu2+
N
(Et)2N
O
O
NH
-
ClO4
2+
1
complex -Cu
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Fig. 1 The crystal structure of compound 1 containing a water molecule, only three
hydrogen bonds appeared in the structure.
20

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Fig. 2 The UV-vis spectra titration test of compound 1 (10.0 μM) with Cu2+ ions (as

ClO4 salts) in solution (CH3CN: HEPS =3:2, v/v). Insert: UV–Vis titration profile of
compound 1 upon addition of Cu2+ ions, the absorption spectra were recorded at 441
nm.
21

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Fig. 3 The fluorescent intensity variation (at 485 nm) of compound 1 in solution (10.0

μM, CH3CN: HEPES = 3:2, v/v) after addition of various metal ions (as ClO4 salts).
22

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Fig. 4 The fluorescence spectra titration of compound 1 (10.0 μM) upon addition of
Cu2+ in solution (CH3CN: HEPES = 3:2, v/v). Insert: fluorescence titration profile at
485 nm upon the addition of Cu2+ (excited at 441 nm).
23

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Fig. 5 The fluorescence response of the probe (10.0 μM, em = 485 nm) to different
amino acids (6.0 equivalents). The front row represents the fluorescence intensity of
probe by addition of various amino acid, the back row represents the fluorescence
intensity after further addition of 6.0 equivalents GSH to the front row solution.
24

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Fig. 6 The fluorescent titration experiment of probe (10.0 μM) with GSH in solution
(CH3CN: HEPES = 3:2, v/v), insert: the fluorescence response profile of probe at 485
nm upon addition of GSH (exited at 441 nm).
25

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Fig. 7 the fluorescence images of HepG2 cells incubated with compound 1 (2.0 μM)
(a), the probe (complex 1-Cu2+, prepared by compound 1 and Cu2+ at 1:5 ratio) (2.0
μM) (b), pretreated with L-BSO, then addition of the probe (complex 1-Cu2+, 2.0 μM)
(c), additional GSH (15.0 equivalents) was added into (c) and incubated for another
10.0 minutes (d), incubated with compound 1 (2.0 μM) for 3.0 h then addition of 25.0
equivalents Cu2+ (e), and their corresponding overlay images (f - j) and bright field
images (k - o). All the images were taken after magnifying 40 times.
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a) a novel fluorescent probe for detection of endogenous GSH was produced based
on the coumarin hydrazide Schiff base derivative.
b) The probe exhibited high selectivity for GSH relative to Cys and Hcy, the
detection limit was evaluated to 0.12 μM, which is lower than the phycological
condition concentration.
c) The fluorescence quenching of precursor 1 by Cu2+ ions can be explained by PCT
mechanism, and the fluorescence “turn on” of the probe is depended on de-
metallization process.
d) the probe can be used to detect the endogenous GSH, which was further proven
by the HepG2 cells fluorescence image experiments.
27