A quinoline-based selective ‘turn on’ chemosensor for zinc(II) via quad-core complex, and its application in live cell imaging

Article abstract of DOI:10.1016/j.tet.2019.130710

An efficient quinoline-based fluorescent chemosensor (QLNPY) was successfully developed for the detection of zinc ions (Zn²⁺). This novel chemosensor displayed higher sensitivity and selectivity toward Zn²⁺ over other competitive metal ions accompanying with obvious fluorescence enhancement. The QLNPY-Zn²⁺ complex can be further used as a new fluorescent “turn-off” sensor for pyrophosphate (PPi) and sulfur ion (S^2?) via a Zn²⁺ displacement approach. The limits of detection were calculated to be 3.8 × 10^?8 M for Zn²⁺, 3.7 × 10^?7 M for PPi and 4.9 × 10^?7 M for S^2?. The binding mechanism of QLNPY and Zn²⁺ was investigated through NMR, HR-MS analysis and further studied by crystallographic analysis. Additionally, further application of QLNPY for sequential bioimaging of Zn²⁺ and PPi was studied in HepG2 cells, suggesting that the quinoline-based chemosensor possesses great potential applications for the detection of intracellular Zn²⁺ and PPi in vivo.

Full text of DOI:10.1016/j.tet.2019.130710

Journal Pre-proof
A quinoline-based selective ‘turn on’ chemosensor for zinc(II) via quad-core complex,
and its application in live cell imaging
Haoran Fu, Haiyang Liu, Lei Zhao, Boren Xiao, Tingting Fan, Yuyang Jiang
PII:
S0040-4020(19)31090-7
https://doi.org/10.1016/j.tet.2019.130710
TET 130710
DOI:
Reference:
To appear in: Tetrahedron
Received Date: 5 September 2019
Revised Date: 13 October 2019
Accepted Date: 16 October 2019
Please cite this article as: Fu H, Liu H, Zhao L, Xiao B, Fan T, Jiang Y, A quinoline-based selective ‘turn
on’ chemosensor for zinc(II) via quad-core complex, and its application in live cell imaging, Tetrahedron
(2019), doi: https://doi.org/10.1016/j.tet.2019.130710.
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Graphical Abstract
A quinoline-based selective
‘turn-on’ chemosensor for
zinc(II) via quad-core
complex, and its application
in live cell imaging
a,b,1
b,c,1
a,b
a,b
a,b
Haoran Fu , Haiyang Liu , Lei Zhao , Boren Xiao , Tingting Fan , Yuyang
a,b,d,e*
Jiang
a
Department of Chemistry, Tsinghua University, Beijing, 100084, PR China
b
The State Key Laboratory of Chemical Oncogenomics, Key Laboratory of Chemical
Biology, the Graduate School at Shenzhen, Shenzhen, 518055, PR China
c
Key Lab of Optoelectronics Devices and Systems of Ministry of Education and Guangdong
Province, College of Physics and Optoelectronic Engineering, Shenzhen University,
Shenzhen 518060, P. R. China
d
National & Local United Engineering Lab for Personalized Anti-tumor Drugs, Shenzhen
Kivita Innovative Drug Discovery Institute, the Graduate School at Shenzhen, Tsinghua
University, Shenzhen 518055, China
e
Department of Pharmacology and Pharmaceutical Sciences, School of Medicine, Tsinghua
University, Beijing, 100084, P. R. China.

1
Tetrahedron
journal homepage: www.elsevier.com
A quinoline-based selective ‘turn on’ chemosensor for zinc(II) via quad-core
complex, and its application in live cell imaging
a,b,1
b,c,1
a,b
a,b
a,b
a,b,d,e
Haoran Fu , Haiyang Liu , Lei Zhao , Boren Xiao , Tingting Fan , Yuyang Jiang
*
a
Department of Chemistry, Tsinghua University, Beijing, 100084, PR China
b
The State Key Laboratory of Chemical Oncogenomics, Key Laboratory of Chemical Biology, the Graduate School at Shenzhen, Shenzhen, 518055, PR China
Key Lab of Optoelectronics Devices and Systems of Ministry of Education and Guangdong Province, College of Physics and Optoelectronic Engineering,
Shenzhen University, Shenzhen 518060, P. R. China
National & Local United Engineering Lab for Personalized Anti-tumor Drugs, Shenzhen Kivita Innovative Drug Discovery Institute, the Graduate School at
Shenzhen, Tsinghua University, Shenzhen 518055, China
c
d
e
Department of Pharmacology and Pharmaceutical Sciences, School of Medicine, Tsinghua University, Beijing, 100084, P. R. China.
ARTICLE INFO
ABSTRACT
Article history:
Received
Received in revised form
Accepted
An efficient quinoline-based fluorescent chemosensor (QLNPY) was successfully developed for
2
+
the detection of zinc ions (Zn ). This novel chemosensor displayed higher sensitivity and
2
+
selectivity toward Zn over other competitive metal ions accompanying with obvious
2+
fluorescence enhancement. The QLNPY-Zn complex can be further used as a new fluorescent
2
-
2+
Available online
“turn-off” sensor for pyrophosphate (PPi) and sulfur ion (S ) via a Zn displacement approach.
-8 2+ -7
The limits of detection were calculated to be 3.8 × 10 M for Zn , 3.7 × 10 M for PPi and 4.9
-
7
2-
2+
Keywords:
Fluorescent chemosensor
Quinoline
Crystallographic analysis
Fluorescence imaging
Zinc ion
× 10 M for S . The binding mechanism of QLNPY and Zn was investigated through NMR,
HR-MS analysis and further studied by crystallographic analysis. Additionally, further
2
+
application of QLNPY for sequential bioimaging of Zn and PPi was studied in HepG2 cells,
suggesting that the quinoline-based chemosensor possesses great potential applications for the
detection of intracellular Zn and PPi in vivo.
2
+
1
. Introduction
fluorescein [12,13], cyanine [14,15]，imidazole [16,17] and di-
2-pyridylmethyl) amine [5,18,19]. These sensors have superior
(
Zinc(II) ion, ranked as the second-most abundant transition
2+
binding affinity to Zn , which can be imaged in cells. However,
metal ion in mammalian body, plays a crucial role in various
processes of life including regulation of gene expression, gene
transcription, structural cofactors, and neural signal transmission
2+
subject to the unique full shell electronic composition of Zn
10
0
(
3d 4s ) bringing about deficiency of spectral properties, the
2+
reported Zn fluorescent probes suffered from the shortcomings
2
+
[
1-3]. The imbalance of Zn metabolism can lead to various
2+
such as the complex synthetic route, and interference from Cd
pathophysiology diseases such as Alzheimer's disease, prostate
cancer, Parkinson and Amyotrophic lateral sclerosis [4,5].
2+
and Hg , which resulted in unsatisfactory recognition and
detection performance [13,20]. Therefore, how to develop simple
fluorescence chemosensor for the selective recognition of
2
+
Simultaneously, excessive Zn absorbed by plants will interact
with sulfhydryl part of several enzyme peptide chains in the
chlorophyll biosynthesis pathway resulting in the transform of
absolute configuration change, which finally inhibits the activity
of the enzyme and block chlorophyll synthesis [6,7]. Therefore, it
is extremely important to develop effective methods for
2+
intracellular Zn is quite necessary.
Quinoline-based derivatives are aromatic heterocyclic
compounds which are widely used in the signory of
optoelectronic functional material [21-25]. Owing to their
simplicity to generate π-π* electronic transition and strong
photochemical stability, the fluorescent sensors and metal
complexes displayed significant and specific fluorescence
enhancement [26]. Based on this, we designed and developed
quinoline derivative (QLNPY) as a target fluorescence sensor for
2+
recognizing Zn in living organisms and external environment.
In recent years, the fluorescence strategy has been proved to
2+
be the most effective and economic method to monitor Zn
qualitatively and quantitatively due to the simplicity, high
sensitivity, versatility, fast response time and real-time dynamic
analysis [5,8,9]. Recently, numerous fluorescent chemosensors
for Zn based on exceptional rigid ring structures have been
reported, such as schiff base derivatives [10,11],
2+
the recognition of Zn . The sensor was facilely synthesized from
picolinohydrazide and 2-chloro-N-(quinolin-8-yl) acetamide via
the common amination reaction. The non-fluorescence sensor
2+
2+
showed high selectivity toward Zn over other competitive metal
2
+
ions, leading to obvious fluorescence enhancement. When Zn is
coordinated with the nitrogen and oxygen atoms of QLNPY to
∗
Corresponding author.
Email address: jiangyy@sz.tsinghua.edu.cn (Y. Jiang).
1
The two authors contributed equally to the work.

2
Tetrahedron
2
.3. Sample preparation and measurements
2+
As shown in Fig. S5, QLNPY could combine with Zn
accompanied with obvious fluorescence enhancement even in
2
+
Tris-HCl buffer, but the fluorescence intensity of QLNPY-Zn
in MeOH was much higher than other water-miscible solvents.
The fluorescent probe QLNPY was completely dissolved in
methanol for a stock solution at 5 mM for confocal imaging and
then diluted to 10 ꢀM in MeOH-Tris buffer (7/3, v/v, pH 7.3) for
fluorescence experiments. Stock solutions (10 mM) of metal salts
Scheme 1 The synthetic route of QLNPY
+
+
+
2+
2+
2+
2+
2+
2+
2+
2+
2+
(
Li , Na , K , Mg , Ca , Ba , Zn , Mn , Cu , Ni , Fe , Sn ,
2+
2+
2+
3+
3+
3+
-
-
compose six-coordinated irregular octahedral structure, the
electron density of the nitrogen is significantly reduced leading to
inhibition of the original unobstructed photoinduced electron
transfer (PET) process. The quad-core spatial structure of the
Pb , Hg , Cd , Fe , Al and Cr ) and sodium salts (HCO
SO
S
3
,
,
2
-
2-
-
-
-
-
-
-
2-
, SO
, HCOO , NO
, OCN , F , I , ClO
, S
O
5
, NO
3
4
2
2
2
3
2
-
2−
2-
3-
2-
O
3
, H
PO
, HPO
, PO
, PPi and S ) were prepared in
2
2
4
4
4
Tris-HCl buffer (20 mM, pH 7.3). All fluorescence spectra were
recorded at room temperature with the excitation wavelength set
at 353 nm (excitation/emission slit width: 5 nm).
2
+
Zn -receptor complex was determinated by the Job’s plot, ESI-
mass, NMR and X-ray diffraction measurement. Eventually, the
2+
practical application of QLNPY in detecting Zn in HepG2 cells
was studied by confocal fluorescence microscopy, indicating the
broad development prospect of the fluorescent probe in tracking
2
.4. Cytotoxicity assay
The cytotoxicity of QLNPY was detected by Cell Counting
Kit-8 experiment. HepG2 cells were seeded into a 96-well plate
at a density of about 7000 cells per well. After the cells were
2+
Zn in vivo.
2
. Experimental
attached at 37 � under a humidified atmosphere of 5% CO in
2
air. 100 ꢀL fresh culture medium with different QLNPY
concentrations (0, 1, 2, 5, 10, 25, 50 and 100 ꢀM) were added
and incubated for 24 h. Subsequently, 10 ꢀL CCK-8 reagent was
added into each well and incubated for another 3 h. TECAN
infinite M 1000 PRO plate reader was used to record absorption
wavelength at 450 nm.
2
.1. Synthesis of QLNPY
The synthetic route of fluorescent sensor QLNPY is shown in
Scheme 1. Picolinohydrazide and 2-chloro-N-(quinolin-8-yl)
acetamide (AQCl) were prepared according to the previous
1
reports [27,28]. Their structures were confirmed by H NMR and
1
3
C NMR spectra (Fig. S1-S4). A mixture composed of AQCl
209.6 mg, 0.95 mmol), picolinohydrazide (137 mg, 1 mmol), KI
157.7 mg, 0.95 mmol) and Na CO (318 mg, 3 mmol) in 10 mL
2
.5. Cell culture and confocal imaging
(
(
2
3
HepG2 cells were cultured in DMEM medium, supplemented
with 10% fetal bovine serum (FBS), 1% antibiotic and
anhydrous acetonitrile was degassed with N for five minutes,
2
Then the reaction was stirred at 60 � for 20 h. The solvent was
evaporated under reduced pressure. The obtained residual
material was purified by flash chromatography over a silica gel
column using dichloromethane/methanol (100:2, v/v) to afford
antimycotic solution at 37 � in a humidified atmosphere with 5%
CO . After 65%-80% confluence, the cells were harvested with
2
trypsin solution in phosphate buffered saline (PBS), and seeded
at a required density in fresh medium. Typically, a subculture
was performed every two days. The cells were adherent cultured
in a confocal 35 mm dish with a glass bottom for 24 h at 37 � .
After being incubated with 25 ꢀM QLNPY for 4h or 6 h, the cells
were washed 4 times with sterilized PBS solution to remove
extracellular QLNPY. Finally, the cells were incubated with
1
the resultant yellow solid (196 mg, yield: 61%). H NMR (400
MHz, CDCl ) δ 11.06 (s, 1H), 9.66 (s, 1H), 8.90 (dd, J = 4.25,
3
1
.70 Hz, 1H), 8.80 (dd, J = 5.43, 3.57 Hz, 1H), 8.49 (d, J = 4.10
Hz, 1H), 8.18 (m, 2H), 7.85 (td, J = 7.73, 1.75 Hz, 1H), 7.57 (m,
2
3
1
1
H), 7.49 (dd, J = 8.29, 4.23 Hz, 1H), 7.43 (m, 1H), 5.36 (s, 1H),
13
.98 (s, 2H). C NMR (101 MHz, CDCl ) δ 168.42, 164.11,
3
ZnCl (75 ꢀM) for another 2.5 h. After washing the cultured cells
2
48.89, 148.68, 148.34, 138.98, 137.35, 136.36, 134.02, 128.12,
with sterilized PBS solution, the HepG2 cells were imaged by
confocal fluorescence microscopy (The detailed microscope
information in SI section).
27.34, 126.66, 122.41, 122.06, 121.67, 117.09, 56.29. HRMS
+
m/z calcd for C H N O [QLNPY + H] : 322.1304, Found:
17
16
5
2
3
22.1282.
3
. Results and discussion
2
.2. X-ray diffraction measurement
As shown in Scheme 1, QLNPY was facilely synthesized
from 2-chloro-N-(quinol-8-yl) acetamide and picolinohydrazide
The single crystals of QLNPY were obtained by the evenly
mixed solution of QLNPY in dichloromethane/n-hexane (1/2,
v/v) after three days on slow evaporation of the solvent. The
via the common amination reaction in CH CN at 60 � , and the
3
2+
single crystals of QLNPY-Zn complex were obtained by the
2+
mixed
solution
of
QLNPY-Zn
complex
in
methanol/dichloromethane (3/1, v/v) after six days on slow
evaporation of the solvent. The crystal data collection details are
summarized in Table S1 and Table S2. The X-ray
crystallographic data were collected on a Rigaku Oxford
Diffraction Supernova Dual Source. Data integration and
correction were executed by applying Olex2. The structures
composed of non-hydrogen atoms were solved and refined on F2
with the SHELXL-201825 [29]. Crystallographic data for the
structures in this report have been deposited at the Cambridge
Crystallographic Data Center with the deposition numbers:
CCDC-1922449 and CCDC-1922450 for compound QLNPY and
Fig. 1 ORTEP drawing of QLNPY with 50% probability ellipsoids.
2+
QLNPY-Zn complex, respectively. (www.ccdc.cam.ac.uk).

Tetrahedron
3
Fig. 2 (a) Fluorescence spectra obtained for QLNPY (10 ꢀM) in MeOH-
Tris buffer (7/3, v/v, pH 7.3) after the addition of 5.0 equiv. of various
Fig. 3 Fluorescence intensity recorded for QLNPY (10 ꢀM) at various pH
metal ion. (b) The fluorescent intensity at 489 nm of QLNPY (10 ꢀM)
2+
2+
3+
values (adjusted by 1 mol/L HCl or 1 mol/L NaOH) in MeOH-Tris buffer
with 50 ꢀM of various metal ions, 1, QLNPY; 2, Zn ; 3, Mn ; 4, Fe
;
;
2+
2
+
3+
+
2+
2+
2+
3+
2+
(7/3, v/v) in the absence and presence of 1 equiv. of Zn (λex = 353 nm, λem
89 nm).
=
5
1
, Ba ; 6, Al ; 7, Li ; 8, Hg ; 9,Fe ; 10, Sn ; 11, Cr ; 12, Cu
+
2+
+
2+
2+
2+
2+
4
3, K ; 14, Ni ; 15, Na ; 16, Ca ; 17, Pb ;18, Mg ;19 Cd .(Inset
2+
picture under UV: QLNPY, 10 ꢀM; QLNPY-Zn , 10 ꢀM of QLNPY
+
2
+
5
0 ꢀM of Zn ) (λex = 353 nm).
3
.2. Effect of pH and response time
The effect of pH on the fluorescence response of QLNPY in
1
13
structure was then identified via H and C NMR, and ESI-mass
measurements (Fig. S6-S8). The spatial structure of QLNPY was
also confirmed by single crystal X-ray diffraction study (Fig. 1).
2+
the presence of Zn was investigated at a variable pH range from
.0 to 12.0. It was found that the fluorescence intensity of
3
2+
QLNPY-Zn solution changed along with the pH value (Fig. 3).
In the pH range of 7.0-9.0, the fluorescence intensity of QLNPY
3
.1. Spectroscopic studies of QLNPY toward different metals
2+
increased obviously after Zn addition, and kept subtle in this
condition, which indicates QLNPY could be used as a suitable
and efficient sensor under common environmental and
physiological conditions. In the acidic conditions (pH < 6.0), the
fluorescence intensity decreased significantly owing to the
protonation of multiple nitrogen atoms in the chemosensor [32],
The fluorescence properties of QLNPY (10 ꢀM) toward
+
+
+
2+
different metal cations (5 equiv.) including Li , Na , K , Mg ,
2
+
2+
2+
2+
2+
2+
2+
2+
2+
2+
2+
Ca , Ba , Zn , Mn , Cu , Ni , Fe , Sn , Pb , Hg , Cd ,
3+
3+
3+
Fe , Al and Cr were primarily investigated via fluorescence
spectra in MeOH-Tris buffer (7/3, v/v, pH 7.3), and the results
are shown in Fig. 2. In the fluorescence testing, significant
fluorescence enhancement ''turn-on'' was intuitively observed
2
+
which was not conducive for the formation of the QLNPY-Zn
-
complex. In the alkaline medium (pH ≥ 10), OH competes with
2+
under UV illumination (365 nm) when Zn is combined with
QLNPY, along with distinctive colorimetric transition from
colorless to light green, indicating that QLNPY can indeed serve
QLNPY to form Zn(II) hydroxide complex [36], decreasing the
2+
formation of the QLNPY-Zn complex and quenching the
fluorescence emission.
2+
as a selective ‘naked eye’ sensor for Zn (Fig. 2). Free QLNPY
displayed almost no fluorescence owing to the PET process
involving the non-bonding electron pairs of nitrogen atoms of
picolinohydrazide moiety, which transfer electrons to the excited
fluorophore leading to fluorescence quenching [30]. An obvious
fluorescence enhancement was noticed in the emission profile of
The time-dependence response to fluorescence changes was
2+
carried out between QLNPY and QLNPY-Zn solutions. As
shown in Fig. S11 (Line B), the fluorescence intensity of QLNPY
basically did not vary with the increasing time (in a period of 30
2+
min). Upon addition of Zn (5 equiv.), the fluorescence intensity
of the solution increased rapidly within 40 s and kept almost
constant in 30 min (Line A), due to the gradual formation of the
2+
QLNPY (10 ꢀM) with the addition of Zn (5 equiv.) and the
emission intensity was enhanced by about 52 folds, while the
addition of other common metal ions did not arouse any
noticeable fluorescence intensity increase. The significant
2+
stable QLNPY-Zn complex. The fast response time results
2
+
indicate that QLNPY could be further used to recognize Zn for
practical applications.
2+
fluorescence enhancement by Zn addition can be ascribed to the
inhibition of the PET effect from electron donating
picolinohydrazide moiety to electron-receptor 8-aminoquinoline
moiety [31]. The electron density of the heteroatoms in the
picolinohydrazide moiety will reduce, causing the receptor to
become a less efficient electron donor, which inhibits the PET-
type fluorescence quenching largely [31-33].
3
.3. Fluorescence titration study
In practical application, the specificity toward target analyte is
one of the most essential features for sensors. To appraise the
2+
specificity of QLNPY toward Zn , the competitive binding
2+
experiment of QLNPY was carried out upon the addition of Zn
mixed with other excessive metal cations in the testing solution.
The addition of various competitive metal cations into the
QLNPY solution could not cause significant fluorescence
enhancement, but the fluorescence intensity was dramatically
Fig. 4 (a) Fluorescence emission spectra recorded for QLNPY (10 ꢀM) upon
2+
gradual addition of Zn (0-10 equiv.), and (b) The plot of the fluorescence
2+
2+
intensity of QLNPY (10 ꢀM) at 489 nm versus the concentration changes of
enhanced when Zn was added to the mixed solution except Cu
2
+
2+
Zn ions (0-10 ꢀM) in MeOH-Tris buffer. (λex = 353 nm). The inset shows
(
Fig. S10). Zn failed to recover the fluorescence of QLNPY-
2+
the linear range of the curve.
Cu solution, which can be ascribed to the paramagnetic effect
from the spin-orbit coupling of Cu [34,35]. The competitive
experiment results indicated that QLNPY could be applied to
2+
2+
detect Zn effectively.

4
Tetrahedron
Fig. 5 ESI mass spectra of QLNPY in the presence of ZnCl
2
To further assess the sensing sensitivity of QLNPY toward
2+
Zn , a fluorescence titration experiment was performed by the
2+
continuous variation of concentrations of Zn (0.1-10.0 equiv.).
The emission intensity displayed an obvious upward trend until
2+
Zn concentration reaches 100 ꢀM (Fig. 4a). The plot of the
2
+
2+
Fig. 6 (a) ORTEP drawing of QLNPY-Zn with 50% probability ellipsoids.
fluorescence intensity versus Zn concentration in the range of2
-10 ꢀM exhibited a reasonable linear relationship with the R
value of 0.997 (Fig. 4b). The limit of detection (LOD) of QLNPY
2+
(b) QLNPY-Zn planar structure (c) Ball and stick representation of single
1
2+
crystal X-ray structure of QLNPY-Zn tetramer
2+
-8
for Zn was found to be 3.8 × 10 M according to the LOD =
δ/k equation [37,38]. The obtained detection limit was quite
3
crystal structure analysis. Good quality single crystal could be
obtained from the reaction of QLNPY and equimolar amounts of
Zn(OAc) (We also tried to obtain the crystal of QLNPY-Zn by
2+
lower than the enforceable guideline for Zn in drinking water
-
5
2
+
(4.6 × 10 M) by the World Health Organization (WHO),
2
2+
indicating that QLNPY has excellent sensitivity toward Zn
under the current working conditions.
using the same amount of QLNPY and ZnCl as reactants, but
2
failed.)
2
+
3
.4. Binding mechanism study
In order to confirm binding stoichiometry and the response
A perspective view of QLNPY-Zn complex is shown in Fig.
. The crystal unit cell consists of four QLNPY molecules and
6
2+
four zinc(II). It should be noted that the zinc(II) atoms are
coordinated to N (quinoline unit), N (-NH- of aminoquinoline
mechanism between QLNPY and Zn , a continuous variation
method by fluorescence titration known as Job’s plot analysis
was carried out (Fig. S13). The plot exhibited the maximum at an
inflection point of 0.5, indicating a 1:1 stoichiometric ratio for
QLNPY-Zn complex. This was further confirmed by ESI-MS
data (Fig. 5). The cluster peak at m/z 322.1290 corresponding to
1
2
unit), N (-CH NH- unit), N (-NHCO- of the picolinohydrazide
unit of an adjacent QLNPY), N (pyridine unit of an adjacent
QLNPY), and O (-NHCO- of the picolinohydrazide unit of an
adjacent QLNPY) to compose of stereoscopically six-coordinated
irregular octahedral structure. The bond lengths between the
donor (N and O) and the central Zn(II) and the donor-metal-
donor bond angles are shown in Table S3 and Table S4. The
donor-Zn(II)-donor bond angles fall in the usual ranges, and the
3
2
4
5
2+
3
+
+
[
QLNPY + H ] , and the cluster peak at m/z 384.0423
2+ + +
corresponding to [QLNPY + Zn - H ] and m/z 420.0176
corresponding to [QLNPY + Zn + 2H O - H ] can be clearly
observed upon the addition of Zn (5 equiv.) to QLNPY
solution. The ESI-mass data also suggested a 1:1 stoichiometry
between QLNPY and Zn . Based on the Job's plot and ESI-MS
data, The related association constant (K ) of QLNPY-Zn was
estimated to be about 6.7 × 10 M via fluorescence titration (Fig.
4
2+
+ +
2
2+
Zn(II)-N and Zn(II)-O bond lengths (Zn-N : 2.146
Å
, Zn-N2:
, Zn-N : 2.206 , Zn-
) are similar to the Zn-ligand complex reported by
Guo et al [42].
1
2+
2.078
Å
, Zn-N : 2.250
Å
, Zn-N : 2.105
Å
Å
3
4
5
2+
O : 2.103 Å
3
a
5
-1
2-
b) according to the Benesi-Hildebrand equation [39].
3
.5. Fluorescence ON–OFF sensing of PPi or S
To gain insight into the specific structural aspects between
In addition to the above properties of QLNPY for the
2+
1
QLNPY and Zn , H NMR titrations in CD CN were also
3
recognition of cations, the complex was also tried to be used as a
metal-based sensor to recognize anions. The fluorescence
2+
performed by incremental addition up to 2.0 equiv. of Zn . As
2+
2+
shown in Fig. S12, with the addition of Zn , the chemical signal
of the secondary amine proton H , H , and H gradually
response of QLNPY-Zn was subsequently investigated toward
7
9
10
disappeared, revealing that the three N atoms of amino-groups
might coordinate to Zn(II). Meanwhile, the chemical shift of the
quinoline unit protons (H , H , H , H , H , H ) and pyridine unit
1
2
3
4
5
6
protons (H , H , H , H ) showed gradual downfield shift.
11
12
13
14
In particular, the chemical shift of the methylene group (-CH -)
2
protons H showed downshift obviously from 3.84 to 4.47, also
8
indicating that the nearby heteroatoms of methylene might be
2+
involved in the coordination toward central Zn . The structural
2+
aspect between QLNPY and Zn was also consistent with the
previous literatures [40,41].
Fig. 7 (a) Fluorescence emission spectra obtained for QLNPY (10 ꢀM) in the
2+
presence of Zn (10 ꢀM ) in MeOH-Tris buffer after the addition of 50 ꢀM
2+
To get a further and visualized understanding of the binding
other anions. (b) The fluorescent intensity at 489 nm of QLNPY-Zn with
2+
-
2-
2-
model of QLNPY-Zn complex, we tried many methods to grow
2
various anions, 1, QLNPY; 2, ZnCl ; 3, HCO3 ; 4, SO3 ; 5, SO4 ; 6,
2+
-
-
-
-
-
-
2-
-
crystals of the QLNPY-Zn complex. Fortunately, the crystal of
this complex was eventually obtained, and the details of the
crystal data are summarized in Fig 6 and Table S2. Formation of
2 2 2 5
HCOO ; 7, NO ; 8, OCN ; 9, F ; 10, I ; 11, ClO ; 12, S O ; 13, NO3 ; 14,
2-
2−
2-
3-
2-
S2O3 ; 15, H PO4 ; 16, HPO4 ; 17, PO4 , 18, PPi, 19, S ; (Inset picture
2
2+
2+
under UV, from left to right: QLNPY; QLNPY-Zn ; QLNPY-Zn and
addition of 5.0 equiv. of PPi) (λex = 353 nm).
2+
4
:4 complex between QLNPY and Zn was confirmed by the

Tetrahedron
5
2
Fig. 8 (a) Fluorescence emission spectra recorded for QLNPY-Zn + (10 ꢀM)
upon gradual addition of PPi (0-5 equiv.), and (b) The plot of the
fluorescence intensity of QLNPY (10 ꢀM) at 489 nm versus the
concentration changes of PPi (0-10ꢀM) in MeOH-Tris buffer. (c)
Fig. 9 Confocal fluorescence microscopy images of HepG2 cells. (a) HepG2
cells were incubated with 25 ꢀM QLNPY for 6 h; (b) HepG2 cells were
2+
incubated with 25 ꢀM QLNPY for 4 h and then 75 ꢀM Zn for 2.5 h; (c)
HepG2 cells were incubated with 25 ꢀM QLNPY for 6 h and then 75 ꢀM
2
Fluorescence emission spectra recorded for QLNPY-Zn + (10 ꢀM) upon
2+
Zn for 2.5 h (d) HepG2 cells were incubated with 25 ꢀM QLNPY for 6 h,
2
-
gradual addition of S (0-5 equiv.), and (b) The plot of the fluorescence
2+
7
5 ꢀM Zn for 2.5 h and then 125 ꢀM PPi for 2 h.
intensity of QLNPY (10 ꢀM) at 489 nm versus the concentration changes of
2
-
S
(0-10ꢀM) in MeOH-Tris buffer. The inset shows the linear range of the
To investigate the potential application of QLNPY in the
curve. (λex = 353 nm).
2+
biosystem, the intracellular Zn imaging of HepG2 was
examined by addition of the chemosensor. For this purpose, the
cells were incubated with QLNPY for 4h and 6h. After flushing
different anions into the complex solution (Fig. 7). The
fluorescence behavior of QLNPY-Zn showed only slight
2
+
2+
four times with PBS buffer, Zn (75 ꢀM) was added to the
confocal dish. Under confocal fluorescence microscopy, it was
found that QLNPY showed no fluorescence in cells (Fig. 8a), but
obvious fluorescence enhancement could be observed after the
-
2-
2-
-
-
change after addition of HCO ; SO ; SO ; HCOO ; NO ;
3
3
4
2
-
-
-
-
2-
-
2-
2−
2-
3-
OCN ; F ; I ; ClO ; S O ; NO ; S O ; H PO ; HPO ; PO
2
2
5
3
2
3
2
4
4
4
(
5.0 equiv.) in the optimized buffer solution. But after addition of
2-
2+
PPi or S (5.0 equiv.), an obvious fluorescence quenching was
observed. It was attributed to the competitive coordination of PPi
or S with the Zn from QLNPY-Zn complex, resulting in the
release of Zn from QLNPY-Zn and consequently turning on
the PET process of free QLNPY [23,43].
addition of Zn . Futhermore, it can be find that the fluorescence
enhanced as the contacting time of QLNPY with the cells
increased from 4h to 6h (Fig. 8b and 8c). Then PPi (125 ꢀM) was
added to the above growth media, remarkable intracellular
fluorescence quenching could be found due to the reduction of
2-
2+
2+
2+
2+
2+
2
+
QLNPY-Zn complex concentration and the combination of a
The interference experiment data of QLNPY-Zn toward PPi
2+
2-
more stable complex between PPi and Zn (Fig. 8d). The
imaging experiments demonstrated that QLNPY has potential
or S clearly displayed that the addition of common anions did
not show any obvious fluorescence changes (Fig. S14),
2+
2
+
applications for sequential recognition of Zn and PPi in the
biosystem.
suggesting QLNPY-Zn has great potential in detecting PPi and
2-
S . In the fluorescence titration experiment, the fluorescence
2
+
intensity of QLNPY-Zn decreased gradually with the addition
4
. Conclusion
2-
of PPi or S (Fig. 8), and the limits of detection were calculated
-
7
-7
2-
In summary, a simple quinoline-based chemosensor QLNPY
to be 3.7 × 10 M for PPi and 4.9 × 10 M for S , respectively,
was synthesized and developed as a selectivity and sensitivity
using the equation LOD = 3δ/k. As shown in Fig S15, the
quenching (95% quenching) time of QLNPY-Zn toward PPi
2+
2
+
sensor for the recognition of Zn accompanying with distinct
2
+
2+
2-
fluorescence enhancement. The obtained complex QLNPY-Zn
can be further used as a new cascade chemosensor for sequential
(
(
80s) was quite shorter than that of QLNPY-Zn toward S
260s). The different response time results indicated that QLNPY
2+
2-
2
+
detection of PPi or S leading to fluorescence OFF–ON-OFF
switching. The binding mechanism of unique quad-core QLNPY-
could be used to recognize Zn and the obtained QLNPY-Zn
complex could be used as a cascade chemosensor for the
2+
2
-
Zn complex was also explained in detail based on NMR, HR-
MS and the crystal structure analysis. The detection limits were
detection of PPi or S .
.6. Cytotoxicity and cell imaging
The potential utility of QLNPY for sensing Zn and PPi in
-
8
2+
-7
3
calculated to be 3.8 × 10 M for Zn , 3.7 × 10 for PPi and 4.9 ×
-
7
2-
2
+
10 M for S , respectively. Moreover, QLNPY was also evaluated
to be used for bioimaging recognition of zinc(II) and PPi in
living cells, suggesting that this chemosensor could be potentially
applied in biological systems.
living cells was further evaluated. Before the cell imaging, the
cytotoxicity of the fluorescent probe was tested by a CCK-8
assay in HepG2 cells incubated with different concentrations of
QLNPY (0, 1, 2, 5, 10, 25, 50 and 100 ꢀM). Fig. S16 displayed
that cellular viability was apparently greater than 95% with the
QLNPY concentration ranged 0-10 µM and was about 82% with
the concentration of 25 µM, indicating that QLNPY had no
obvious cytotoxicity and good biocompatibility.
Acknowledgements
We appreciate the financial supports from Shenzhen
Development and Reform Committee (No. 20151961 and
No.2019156) and Department of Science and Technology of

6
Tetrahedron
(
2015) 41905-41913.
Guangdong Province (No. 2017B030314083). H. Y. Liu thanks
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Highlights
2
+
1.
2.
3.
4.
5.
The quinoline-based fluorescent sensor displayed high sensitivity toward Zn .
The stoichiometric binding mode was confirmed by a crystallographic study.
2
-
The obtained complex can recognize PPi and S by fluorescence quenching.
2
-
Different fluorescence quenching times were used to distinguish PPi and S .
2
+
The probe was used for tracking Zn and PPi in cell by fluorescence imaging.