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

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

DOI:

https://doi.org/10.1016/j.tet.2019.130710

Reference:

TET 130710

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. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2019 Published by Elsevier Ltd.

Graphical Abstract A quinoline-based selective ‘turn-on’ chemosensor for zinc(II) via quad-core complex, and its application in live cell imaging Haoran Fua,b,1, Haiyang Liub,c,1, Lei Zhaoa,b, Boren Xiaoa,b, Tingting Fan a,b, Yuyang Jianga,b,d,e* 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.

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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 Haoran Fua,b,1, Haiyang Liub,c,1, Lei Zhaoa,b, Boren Xiaoa,b, Tingting Fana,b, Yuyang Jianga,b,d,e* a

Department of Chemistry, Tsinghua University, Beijing, 100084, PR China 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. b

ARTICLE INFO

ABSTRACT

Article history: Received Received in revised form Accepted Available online

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 (S2-) 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 S2-. 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.

Keywords: Fluorescent chemosensor Quinoline Crystallographic analysis Fluorescence imaging Zinc ion

1. Introduction Zinc(II) ion, ranked as the second-most abundant transition 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 [1-3]. The imbalance of Zn2+ metabolism can lead to various pathophysiology diseases such as Alzheimer's disease, prostate cancer, Parkinson and Amyotrophic lateral sclerosis [4,5]. Simultaneously, excessive Zn2+ 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 recognizing Zn2+ in living organisms and external environment. In recent years, the fluorescence strategy has been proved to be the most effective and economic method to monitor Zn2+ 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 Zn2+ based on exceptional rigid ring structures have been reported, such as schiff base derivatives [10,11], ∗ Corresponding author. Email address: [email protected] (Y. Jiang). 1

The two authors contributed equally to the work.

fluorescein [12,13], cyanine [14,15]，imidazole [16,17] and di(2-pyridylmethyl) amine [5,18,19]. These sensors have superior binding affinity to Zn2+, which can be imaged in cells. However, subject to the unique full shell electronic composition of Zn2+ (3d104s0) bringing about deficiency of spectral properties, the reported Zn2+ fluorescent probes suffered from the shortcomings such as the complex synthetic route, and interference from Cd2+ and Hg2+, which resulted in unsatisfactory recognition and detection performance [13,20]. Therefore, how to develop simple fluorescence chemosensor for the selective recognition of intracellular Zn2+ 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 the recognition of Zn2+. The sensor was facilely synthesized from picolinohydrazide and 2-chloro-N-(quinolin-8-yl) acetamide via the common amination reaction. The non-fluorescence sensor showed high selectivity toward Zn2+ over other competitive metal ions, leading to obvious fluorescence enhancement. When Zn2+ is coordinated with the nitrogen and oxygen atoms of QLNPY to

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Scheme 1 The synthetic route of QLNPY

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 Zn2+-receptor complex was determinated by the Job’s plot, ESImass, NMR and X-ray diffraction measurement. Eventually, the practical application of QLNPY in detecting Zn2+ in HepG2 cells was studied by confocal fluorescence microscopy, indicating the broad development prospect of the fluorescent probe in tracking Zn2+ in vivo. 2. Experimental 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 reports [27,28]. Their structures were confirmed by 1H NMR and 13 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 Na2CO3 (318 mg, 3 mmol) in 10 mL anhydrous acetonitrile was degassed with N2 for five minutes, 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 the resultant yellow solid (196 mg, yield: 61%). 1H NMR (400 MHz, CDCl3) δ 11.06 (s, 1H), 9.66 (s, 1H), 8.90 (dd, J = 4.25, 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, 2H), 7.49 (dd, J = 8.29, 4.23 Hz, 1H), 7.43 (m, 1H), 5.36 (s, 1H), 3.98 (s, 2H). 13C NMR (101 MHz, CDCl3) δ 168.42, 164.11, 148.89, 148.68, 148.34, 138.98, 137.35, 136.36, 134.02, 128.12, 127.34, 126.66, 122.41, 122.06, 121.67, 117.09, 56.29. HRMS m/z calcd for C17H16N5O2 [QLNPY + H]+: 322.1304, Found: 322.1282. 2.2. X-ray diffraction measurement 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 single crystals of QLNPY-Zn2+ complex were obtained by the mixed solution of QLNPY-Zn2+ 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 QLNPY-Zn2+ complex, respectively. (www.ccdc.cam.ac.uk).

As shown in Fig. S5, QLNPY could combine with Zn2+ accompanied with obvious fluorescence enhancement even in Tris-HCl buffer, but the fluorescence intensity of QLNPY-Zn2+ 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 µM in MeOH-Tris buffer (7/3, v/v, pH 7.3) for fluorescence experiments. Stock solutions (10 mM) of metal salts (Li+, Na+, K+, Mg2+, Ca2+, Ba2+, Zn2+, Mn2+, Cu2+, Ni2+, Fe2+, Sn2+, Pb2+, Hg2+, Cd2+, Fe3+, Al3+ and Cr3+) and sodium salts (HCO3-, SO32-, SO42-, HCOO-, NO2-, OCN-, F-, I-, ClO2-, S2O52-, NO3-, S2O32-, H2PO42−, HPO42-, PO43-, PPi and S2-) were prepared in 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). 2.4. 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 attached at 37 under a humidified atmosphere of 5% CO2 in air. 100 µL fresh culture medium with different QLNPY concentrations (0, 1, 2, 5, 10, 25, 50 and 100 µM) were added and incubated for 24 h. Subsequently, 10 µL 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. 2.5. Cell culture and confocal imaging HepG2 cells were cultured in DMEM medium, supplemented with 10% fetal bovine serum (FBS), 1% antibiotic and antimycotic solution at 37 in a humidified atmosphere with 5% CO2. After 65%-80% confluence, the cells were harvested with 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 µM 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 ZnCl2 (75 µM) for another 2.5 h. After washing the cultured cells with sterilized PBS solution, the HepG2 cells were imaged by confocal fluorescence microscopy (The detailed microscope information in SI section). 3. Results and discussion As shown in Scheme 1, QLNPY was facilely synthesized from 2-chloro-N-(quinol-8-yl) acetamide and picolinohydrazide via the common amination reaction in CH3CN at 60 , and the

Fig. 1 ORTEP drawing of QLNPY with 50% probability ellipsoids.

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Fig. 2 (a) Fluorescence spectra obtained for QLNPY (10 µM) in MeOHTris buffer (7/3, v/v, pH 7.3) after the addition of 5.0 equiv. of various metal ion. (b) The fluorescent intensity at 489 nm of QLNPY (10 µM) with 50 µM of various metal ions, 1, QLNPY; 2, Zn2+ ; 3, Mn2+ ; 4, Fe3+ ; 5, Ba2+ ; 6, Al3+ ; 7, Li+ ; 8, Hg2+ ; 9,Fe2+ ; 10, Sn2+ ; 11, Cr3+ ; 12, Cu2+ ; 13, K+ ; 14, Ni2+ ; 15, Na+ ; 16, Ca2+ ; 17, Pb2+ ;18, Mg2+ ;19 Cd2+.(Inset picture under UV: QLNPY, 10 µM; QLNPY-Zn2+, 10 µM of QLNPY + 50 µM of Zn2+) (λex = 353 nm).

structure was then identified via 1H and 13C 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). 3.1. Spectroscopic studies of QLNPY toward different metals The fluorescence properties of QLNPY (10 µM) toward different metal cations (5 equiv.) including Li+, Na+, K+, Mg2+, Ca2+, Ba2+, Zn2+, Mn2+, Cu2+, Ni2+, Fe2+, Sn2+, Pb2+, Hg2+, Cd2+, Fe3+, Al3+ and Cr3+ 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 under UV illumination (365 nm) when Zn2+ is combined with QLNPY, along with distinctive colorimetric transition from colorless to light green, indicating that QLNPY can indeed serve as a selective ‘naked eye’ sensor for Zn2+ (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 QLNPY (10 µM) with the addition of Zn2+ (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 fluorescence enhancement by Zn2+ 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 PETtype fluorescence quenching largely [31-33]. In practical application, the specificity toward target analyte is one of the most essential features for sensors. To appraise the specificity of QLNPY toward Zn2+, the competitive binding experiment of QLNPY was carried out upon the addition of Zn2+ 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 enhanced when Zn2+ was added to the mixed solution except Cu2+ (Fig. S10). Zn2+ failed to recover the fluorescence of QLNPYCu2+ solution, which can be ascribed to the paramagnetic effect from the spin-orbit coupling of Cu2+ [34,35]. The competitive experiment results indicated that QLNPY could be applied to detect Zn2+ effectively.

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Fig. 3 Fluorescence intensity recorded for QLNPY (10 µM) at various pH values (adjusted by 1 mol/L HCl or 1 mol/L NaOH) in MeOH-Tris buffer (7/3, v/v) in the absence and presence of 1 equiv. of Zn2+ (λex = 353 nm, λem = 489 nm).

3.2. Effect of pH and response time The effect of pH on the fluorescence response of QLNPY in the presence of Zn2+ was investigated at a variable pH range from 3.0 to 12.0. It was found that the fluorescence intensity of QLNPY-Zn2+ solution changed along with the pH value (Fig. 3). In the pH range of 7.0-9.0, the fluorescence intensity of QLNPY increased obviously after Zn2+ 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], which was not conducive for the formation of the QLNPY-Zn2+ complex. In the alkaline medium (pH ≥ 10), OH- competes with QLNPY to form Zn(II) hydroxide complex [36], decreasing the formation of the QLNPY-Zn2+ complex and quenching the fluorescence emission. The time-dependence response to fluorescence changes was carried out between QLNPY and QLNPY-Zn2+ 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 min). Upon addition of Zn2+ (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 stable QLNPY-Zn2+ complex. The fast response time results indicate that QLNPY could be further used to recognize Zn2+ for practical applications. 3.3. Fluorescence titration study

Fig. 4 (a) Fluorescence emission spectra recorded for QLNPY (10 µM) upon gradual addition of Zn2+ (0-10 equiv.), and (b) The plot of the fluorescence intensity of QLNPY (10 µM) at 489 nm versus the concentration changes of Zn2+ ions (0-10 µM) in MeOH-Tris buffer. (λex = 353 nm). The inset shows the linear range of the curve.

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Fig. 5 ESI mass spectra of QLNPY in the presence of ZnCl2

To further assess the sensing sensitivity of QLNPY toward Zn2+, a fluorescence titration experiment was performed by the continuous variation of concentrations of Zn2+ (0.1-10.0 equiv.). The emission intensity displayed an obvious upward trend until Zn2+ concentration reaches 100 µM (Fig. 4a). The plot of the fluorescence intensity versus Zn2+ concentration in the range of 1-10 µM exhibited a reasonable linear relationship with the R2 value of 0.997 (Fig. 4b). The limit of detection (LOD) of QLNPY for Zn2+ was found to be 3.8 × 10-8 M according to the LOD = 3δ/k equation [37,38]. The obtained detection limit was quite lower than the enforceable guideline for Zn2+ in drinking water (4.6 × 10-5 M) by the World Health Organization (WHO), indicating that QLNPY has excellent sensitivity toward Zn2+ under the current working conditions. 3.4. Binding mechanism study In order to confirm binding stoichiometry and the response mechanism between QLNPY and Zn2+, 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-Zn2+ complex. This was further confirmed by ESI-MS data (Fig. 5). The cluster peak at m/z 322.1290 corresponding to [QLNPY + H+]+, and the cluster peak at m/z 384.0423 corresponding to [QLNPY + Zn2+ - H+]+ and m/z 420.0176 corresponding to [QLNPY + Zn2+ + 2H2O - H+]+ can be clearly observed upon the addition of Zn2+ (5 equiv.) to QLNPY solution. The ESI-mass data also suggested a 1:1 stoichiometry between QLNPY and Zn2+. Based on the Job's plot and ESI-MS data, The related association constant (Ka) of QLNPY-Zn2+ was estimated to be about 6.7 × 105 M-1 via fluorescence titration (Fig. 4b) according to the Benesi-Hildebrand equation [39]. To gain insight into the specific structural aspects between QLNPY and Zn2+, 1H NMR titrations in CD3CN were also performed by incremental addition up to 2.0 equiv. of Zn2+. As shown in Fig. S12, with the addition of Zn2+, the chemical signal of the secondary amine proton H7, H9, and H10 gradually disappeared, revealing that the three N atoms of amino-groups might coordinate to Zn(II). Meanwhile, the chemical shift of the quinoline unit protons (H1, H2, H3, H4, H5, H6) and pyridine unit protons (H11, H12, H13, H14) showed gradual downfield shift. In particular, the chemical shift of the methylene group (-CH2-) protons H8 showed downshift obviously from 3.84 to 4.47, also indicating that the nearby heteroatoms of methylene might be involved in the coordination toward central Zn2+. The structural aspect between QLNPY and Zn2+ was also consistent with the previous literatures [40,41]. To get a further and visualized understanding of the binding model of QLNPY-Zn2+ complex, we tried many methods to grow crystals of the QLNPY-Zn2+ 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 4:4 complex between QLNPY and Zn2+ was confirmed by the

Fig. 6 (a) ORTEP drawing of QLNPY-Zn2+ with 50% probability ellipsoids. (b) QLNPY-Zn2+ planar structure (c) Ball and stick representation of single crystal X-ray structure of QLNPY-Zn2+ tetramer

crystal structure analysis. Good quality single crystal could be obtained from the reaction of QLNPY and equimolar amounts of Zn(OAc)2 (We also tried to obtain the crystal of QLNPY-Zn2+ by using the same amount of QLNPY and ZnCl2 as reactants, but failed.) A perspective view of QLNPY-Zn2+ complex is shown in Fig. 6. The crystal unit cell consists of four QLNPY molecules and four zinc(II). It should be noted that the zinc(II) atoms are coordinated to N1 (quinoline unit), N2 (-NH- of aminoquinoline unit), N3 (-CH2NH- unit), N4 (-NHCO- of the picolinohydrazide unit of an adjacent QLNPY), N5 (pyridine unit of an adjacent QLNPY), and O3 (-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-metaldonor bond angles are shown in Table S3 and Table S4. The donor-Zn(II)-donor bond angles fall in the usual ranges, and the Zn(II)-N and Zn(II)-O bond lengths (Zn-N1: 2.146 Å, Zn-N2: 2.078 Å, Zn-N3: 2.250 Å, Zn-N4: 2.105 Å, Zn-N5: 2.206 Å, ZnO3: 2.103 Å) are similar to the Zn-ligand complex reported by Guo et al [42]. 3.5. Fluorescence ON–OFF sensing of PPi or S2In addition to the above properties of QLNPY for the recognition of cations, the complex was also tried to be used as a metal-based sensor to recognize anions. The fluorescence response of QLNPY-Zn2+ was subsequently investigated toward

Fig. 7 (a) Fluorescence emission spectra obtained for QLNPY (10 µM) in the presence of Zn2+ (10 µM ) in MeOH-Tris buffer after the addition of 50 µM other anions. (b) The fluorescent intensity at 489 nm of QLNPY-Zn2+ with various anions, 1, QLNPY; 2, ZnCl2 ; 3, HCO3- ; 4, SO32-; 5, SO42- ; 6, HCOO- ; 7, NO2-; 8, OCN- ; 9, F-; 10, I- ; 11, ClO2-; 12, S2O52- ; 13, NO3- ; 14, S2O32- ; 15, H2PO42− ; 16, HPO42-; 17, PO43-, 18, PPi, 19, S2-; (Inset picture under UV, from left to right: QLNPY; QLNPY-Zn2+; QLNPY-Zn2+ and addition of 5.0 equiv. of PPi) (λex = 353 nm).

Tetrahedron

Fig. 8 (a) Fluorescence emission spectra recorded for QLNPY-Zn2+ (10 µM) upon gradual addition of PPi (0-5 equiv.), and (b) The plot of the fluorescence intensity of QLNPY (10 µM) at 489 nm versus the concentration changes of PPi (0-10µM) in MeOH-Tris buffer. (c) Fluorescence emission spectra recorded for QLNPY-Zn2+ (10 µM) upon gradual addition of S2- (0-5 equiv.), and (b) The plot of the fluorescence intensity of QLNPY (10 µM) at 489 nm versus the concentration changes of S2- (0-10µM) in MeOH-Tris buffer. The inset shows the linear range of the curve. (λex = 353 nm).

different anions into the complex solution (Fig. 7). The fluorescence behavior of QLNPY-Zn2+ showed only slight change after addition of HCO3-; SO32-; SO42-; HCOO-; NO2-; OCN-; F-; I-; ClO2-; S2O52-; NO3-; S2O32-; H2PO42−; HPO42-; PO43(5.0 equiv.) in the optimized buffer solution. But after addition of PPi or S2- (5.0 equiv.), an obvious fluorescence quenching was observed. It was attributed to the competitive coordination of PPi or S2- with the Zn2+ from QLNPY-Zn2+ complex, resulting in the release of Zn2+ from QLNPY-Zn2+ and consequently turning on the PET process of free QLNPY [23,43]. The interference experiment data of QLNPY-Zn2+ toward PPi or S2- clearly displayed that the addition of common anions did not show any obvious fluorescence changes (Fig. S14), suggesting QLNPY-Zn2+ has great potential in detecting PPi and S2-. In the fluorescence titration experiment, the fluorescence intensity of QLNPY-Zn2+ decreased gradually with the addition of PPi or S2- (Fig. 8), and the limits of detection were calculated to be 3.7 × 10-7 M for PPi and 4.9 × 10-7 M for S2-, respectively, using the equation LOD = 3δ/k. As shown in Fig S15, the quenching (95% quenching) time of QLNPY-Zn2+ toward PPi (80s) was quite shorter than that of QLNPY-Zn2+ toward S2(260s). The different response time results indicated that QLNPY could be used to recognize Zn2+ and the obtained QLNPY-Zn2+ complex could be used as a cascade chemosensor for the detection of PPi or S2-. 3.6. Cytotoxicity and cell imaging The potential utility of QLNPY for sensing Zn2+ and PPi in 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 µM). 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.

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Fig. 9 Confocal fluorescence microscopy images of HepG2 cells. (a) HepG2 cells were incubated with 25 µM QLNPY for 6 h; (b) HepG2 cells were incubated with 25 µM QLNPY for 4 h and then 75 µM Zn2+ for 2.5 h; (c) HepG2 cells were incubated with 25 µM QLNPY for 6 h and then 75 µM Zn2+ for 2.5 h (d) HepG2 cells were incubated with 25 µM QLNPY for 6 h, 75 µM Zn2+ for 2.5 h and then 125 µM PPi for 2 h.

To investigate the potential application of QLNPY in the biosystem, the intracellular Zn2+ 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 four times with PBS buffer, Zn2+ (75 µM) 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 addition of Zn2+. 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 µM) was added to the above growth media, remarkable intracellular fluorescence quenching could be found due to the reduction of QLNPY-Zn2+ complex concentration and the combination of a more stable complex between PPi and Zn2+ (Fig. 8d). The imaging experiments demonstrated that QLNPY has potential applications for sequential recognition of Zn2+ and PPi in the biosystem. 4. Conclusion In summary, a simple quinoline-based chemosensor QLNPY was synthesized and developed as a selectivity and sensitivity sensor for the recognition of Zn2+ accompanying with distinct fluorescence enhancement. The obtained complex QLNPY-Zn2+ can be further used as a new cascade chemosensor for sequential detection of PPi or S2- leading to fluorescence OFF–ON-OFF switching. The binding mechanism of unique quad-core QLNPYZn2+ complex was also explained in detail based on NMR, HRMS and the crystal structure analysis. The detection limits were calculated to be 3.8 × 10-8 M for Zn2+, 3.7 × 10-7 for PPi and 4.9 × 10-7 M for S2-, 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. Acknowledgements We appreciate the financial supports from Shenzhen Development and Reform Committee (No. 20151961 and No.2019156) and Department of Science and Technology of

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Guangdong Province (No. 2017B030314083). H. Y. Liu thanks the China Postdoctoral Science Foundation Funded Project (2018M643146). References [1] [2]

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Highlights 1.

The quinoline-based fluorescent sensor displayed high sensitivity toward Zn2+.

2.

The stoichiometric binding mode was confirmed by a crystallographic study.

3.

The obtained complex can recognize PPi and S2- by fluorescence quenching.

4.

Different fluorescence quenching times were used to distinguish PPi and S2-.

5.

The probe was used for tracking Zn2+ and PPi in cell by fluorescence imaging.