New pyridoxal based chemosensor for selective detection of Zn2+: Application in live cell imaging and phosphatase activity response

Accepted Manuscript Title: New pyridoxal based chemosensor for selective detection of Zn2+ : application in live cell imaging and phosphatase activity response Author: Senjuti Mandal Yeasin Sikdar Dilip K. Maiti Ria Sanyal Debasis Das Abhishek Mukherjee Sushil Kumar Mandal Jayanta Kumar Biswas Antonio Bauz´a Antonio Frontera Sanchita Goswami PII: DOI: Reference:

S1010-6030(16)30120-4 http://dx.doi.org/doi:10.1016/j.jphotochem.2016.10.038 JPC 10422

To appear in:

Journal of Photochemistry and Photobiology A: Chemistry

Received date: Revised date: Accepted date:

18-2-2016 5-10-2016 27-10-2016

Please cite this article as: Senjuti Mandal, Yeasin Sikdar, Dilip K.Maiti, Ria Sanyal, Debasis Das, Abhishek Mukherjee, Sushil Kumar Mandal, Jayanta Kumar Biswas, Antonio Bauz´a, Antonio Frontera, Sanchita Goswami, New pyridoxal based chemosensor for selective detection of Zn2+: application in live cell imaging and phosphatase activity response, Journal of Photochemistry and Photobiology A: Chemistry http://dx.doi.org/10.1016/j.jphotochem.2016.10.038 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. 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.

We reported a pyridoxal-based reversible chemosensor, H4PydChda, exhibiting selective turn-on response for Zn2+. The experimental and theoretical supports are provided to establish the binding mode of H4PydChda to Zn2+. The prob-Zn2+ ensemble is capable of showing appreciable phosphatase activity with NPP substrate.

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A simple Schiff base ligand was synthesized and characterized which shows turn-on response for Zn(II).

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The experimental and theoretical supports in terms of 1H and 13C-NMR spectroscopy and DFT/TDDFT study are provided to establish the binding mode of H4PydChda to Zn2+.

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The resulting Zn(II) complex shows convincing phosphatase activity (kcat = 21.59 s-1).

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New pyridoxal based chemosensor for selective detection of Zn2+: application in live cell imaging and phosphatase activity response Senjuti Mandala, Yeasin Sikdara, Dilip K. Maitia, Ria Sanyala, Debasis Dasa, Abhishek Mukherjeeb, Sushil Kumar Mandalc, Jayanta Kumar Biswasc, Antonio Bauzád, Antonio Fronterad, Sanchita Goswamia,* a

Department of Chemistry, University of Calcutta, 92, A. P. C. Road, Kolkata – 700009, India. E-mail: [email protected] b Drug Development Diagnostic and Biotechnology Division, CSIR-Indian Institute of Chemical Biology, Kolkata – 700032, India. c Department of Ecological Engineering & Environmental Management, University of Kalyani, Kalyani, Nadia-741235, West Bengal, India. d Departament de Química, Universitat de les Illes Balears, Crta. de Valldemossa km 7.5, 07122 Palma de Mallorca (Baleares), SPAIN Abstract Although a variety of fluorescence based chemosensors have been utilized for selective detection of Zn2+, pyridoxal containing simple Schiff bases still remained less explored. Here, we combine pyridoxal hydrochloride and 1,2-diaminocyclohexane to generate a new sensor molecule, H4PydChda [5-Hydroxymethyl-4-({2-[5-hydroxymethyl-2-methylpyridin-3-hydroxy4-ylethylene)-amino]-cyclohexylimino}-methyl)-2-methylpyridin-3-ol].

Chemosensor

H4PydChda exhibits selective turn-on type response in presence of Zn2+ in ethanol-water mixture at physiological pH. Appreciable fluorescence enhancement occurs upon addition of Zn2+ to H4PydChda as a result of inhibited C=N isomerisation and excited state intramolecular proton transfer (ESIPT) leading to efficient chelation enhanced fluorescence (CHEF). The relevant properties, including reversibility, life time measurements and detection limit have been determined for the sensor system. The experimental and theoretical supports in terms of 1H and 13

C-NMR spectroscopy and DFT/TDDFT study are provided to establish the binding mode of

H4PydChda to Zn2+. H4PydChda was employed as a sensor for detection of Zn2+ in Human gastric adenocarcinoma (AGS) cells. Moreover, the resulting probe-Zn2+complex shows convincing phosphatase activity (kcat = 21.59 s-1), opening a promising avenue for further research.

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Keywords: Chemosensor; Schiff base;fluorimetry; live cell imaging; Phosphatase activity; DFT 1. Introduction Among many biologically significant metal ions, Zn2+ is the second most abundant metal ions in human body as it is involved in a number of biochemical processes [1].However, both its deficiency and excess can induce human health disorders in the form of Alzheimer’s disease, Parkinson’s disease etc. [2].Therefore, generation of efficient chemosensors specific for Zn2+ detection is the need of the hour. Analytical methods for detection of Zn2+ such as atomic absorption spectrometry[3(a)], inductively coupled plasma mass spectroscopy (ICPMS) [3(b)], inductively coupled plasma-atomic emission spectrometry (ICPAES) [3(c)], and voltammetry [3(d)] require expensive instrumentation and large sample amount. As a result, fluorescence based chemosensors have attracted significant attention due to high sensitivity, easy visualization, short response time for detection and most importantly they can be implemented for real time bio-imaging[4]. In recent years, a large number of probes based on fluorescence detection have been reported for selective detection of Zn2+[5-8]. In an attempt to make the chemosensor design simple and biocompatible, we have undertaken a strategy to generate pyridoxal containing Schiff bases as sensors, coupling the two prerequisites [9]. General signaling mechanism prevalent in such systems is based on chelation enhanced fluorescence (CHEF) induced by restricted C=N isomerisation and excited state intramolecular proton transfer (ESIPT). The binding of analyte disables the non-radiative decay pathways involving C=N isomerisation and ESIPT, thereby restoring the fluorescence (Scheme 1) [9(c)-(e)].In particular, we have demonstrated that the occurrence pyridoxal-3-pyridone tautomerism triggered by Zn2+ binding, imparts additional specificity to the system [9(c), 10, 11].This combined mechanistic rationale has not been explored much in literature (Chart 1). Our goal is to carry out extensive research on pyridoxal Schiff base chemosensors in a systematic and inclusive manner to construct a rich library of biocompatible, simple sensors for selective analyte detection. Thus, based on our previous works, we extended our efforts to explore pyridoxal bearing Schiff base probesby generating a new sensor, H4PydChda, [5−Hydroxymethyl−4−({2−[5hydroxymethyl−2−methylpyridin−3−hydroxy−4−ylethylene)−amino]−cyclohexylimino}−methyl 4

)−2-methylpyridin−3−ol] which serves as an excellent chemosensor for Zn 2+ in ethanol-water at physiological pH and can be used for Zn2+ monitoring in living cells. The NMR spectroscopy and DFT study are utilized to establish the binding mode of H4PydChda to Zn2+ in solution. Again, it is well documented that zinc containing enzymes play an important role in the cleavage of P–OR bond in phosphates [12-14]. In Nature, the enzymes that catalyze phosphate ester hydrolysis are called phosphatase enzymes. As evident from literature reports, a number of mono- and dinuclear Zn2+ complexes were exploited as mimicking phosphatase activity [14]. Building upon these reports, we ventured out to investigate if the generated probe-Zn2+ aggregate is able to exhibit phosphatase activity. 2. Experimental 2.1. Materials and physical methods All reagents were purchased from Sigma-Aldrich and used as received. Solvents were spectroscopic grade and used without purification. Elemental analyses for C, H and N were performed with a Perkin-Elmer CHN analyzer 2400.1H and 13C NMR spectra were recorded with TMS as internal standard on a Bruker, AV300 Supercon Digital NMR system. IR spectra were recorded in the region 400–4000 cm-1 on a Bruker-Optics Alpha–T spectrophotometer with samples as KBr disks. Electronic spectra were obtained by using a Hitachi U-3501 spectrophotometer. The ESI-MS were recorded on Qtof Micro YA263 mass spectrometer. Luminescence property was measured using Hitachi F-7000 fluorescence spectrophotometer at room temperature (298 K) by 1 cm path length quartz cell. Fluorescence lifetimes were obtained by the method of Time Correlated Single-Photon counting (TCSPC) on FluoroCube-01-NL spectrometer (Horiba Jobin Yvon) using a nanoLED as light source (340 nm) and the signals were collected at the magic angle of 54.7° to eliminate any considerable contribution from fluorescence anisotropy decay. The time resolution of our experimental set up is 800 ps. The decays were deconvoluted using DAS-6 decay analysis software. The acceptability of the fits was judged by 2 criteria (fitting analysis having 2 beyond the range 1.20 <2< 1.00 has been neglected) and visual inspection of the residuals of the fitted function to the data. Mean (average) fluorescence lifetimes were calculated using the following equation: τav> =∑ ai τi /∑ ai i i

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in which ai is the pre-exponential factor corresponding to the ith decay time constant, τi. 2.2. Computational details All geometries for H4PydChda and probe-Zn2+ were optimized by density functional theory (DFT) calculations using TURBOMOLE v 7.0 at the BP86-D3/dev2-TZVP level of theory [15]. Vertical electronic excitations based on these optimized geometries were computed using the time-dependent density functional theory (TDDFT) formalism [16] in ethanol using a conductor-like polarizable continuum model (CPCM) [17] by means of the Gaussian-09 program [18]. We have calculated 60 singlet–singlet transition using the ground S0 state geometry at the B3LYP/6-311+G* level of theory. 2.3. Bioimaging study 2.3.1. Reagents for cell study Human gastric adenocarcinoma (AGS) cells were procured from National Center for Cell Science, Pune, India, and used for the experimental studies. Cells were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM; Gibco BRL) supplemented with 10% heat-inactivated fetal bovine serum (FBS, Himedia) and an antibiotic mixture (1%) containing PSN (Himedia) at 37ºC in a humidified incubator with 5% CO2. 2.3.2. Imaging system Bright field and fluorescence images were captured at 20X magnification by a fluorescence microscope (Olympus, 1x70) using Camedia software (Chicago, MI, USA) (E20P 5.0 Megapixel) and were processed using Adobe Photoshop version 10.0. 2.3.3. Cell culture For imaging studies, cells were allowed to grow in 6-well plates at 70-80% confluence. Cells were then incubated in DMEM containing different concentrations of H4PydChda (500 nm, 1 µM and 10 µM) for 30 min at 37 ºC. They were then washed with PBS followed by the addition of 10 µM Zn2+ ions and incubation for 10 min. Bright field and fluorescence images were captured at 20X magnification by a fluorescence microscope (Olympus, 1x70) using Camedia software (Chicago, MI, USA) (E20P 5.0 Megapixel) and were processed using Adobe 6

Photoshop version 10.0. Fluorescent intensity was examined after the addition of N,N,N’,N’Tetrakis(2-pyridylmethyl) ethylenediamine, known as TPEN (50 µM) in the incubation media. 2.3.4. Cell cytotoxicity assay For the assessment of cytotoxicity of H4PydChda, 3-(4,5-dimethylthiazol-2-yl)-2,Sdiphenyltetrazolium bromide (MTT) assay was performed with AGS cells [19]. Following the treatment of overnight culture of AGS cells (103 cells in each well of 96-well plate) with varied concentrations of H4PydChda (0.5, 1, 10 and 100 μM) for 12 h, 10 μl of MTT solution (1mg/ml in PBS) was added in each well and incubated at 37 ºC for 3 h. Culture media were then removed and 100μl of acidic isopropyl alcohol was added into each well. The intracellular formazan crystals (blue-violet) formed in the process were solubilized with 0.04 N acidic isopropyl alcohol and absorbance of the solution was measured at 595 nm wavelength with a microplate reader (Model: THERMO MULTI SCAN EX). The reduction reaction converting MTT to formazan showed high correlation with the cellular health, on the whole. The cell viability was expressed as the optical density ratio of the treatment to control. Values are mean ± standard deviation of three independent experiments. The cell cytotoxicity was calculated as % cell cytotoxicity = 100% - % cell viability. 2.4. Fluorimetric analysis Fluorescence quantum yields (Φ) were estimated by integrating the area under the fluorescence curves with the following equation: Φ sample = (ODstandard/ODsample) × (Asample/Astandard) × Φstandard where, A is the area under the fluorescence spectral curve and OD is the optical density of the compound at the excitation wavelength. The standard used for the measurement of the fluorescence quantum yield was quinine sulphate (Φ = 0.54 in water). 2.5. Kinetic Measurements for the Hydrolysis of 4-NPP in DMF To study the phosphatase activity of our complex, disodium salt of (4nitrophenyl)phosphate hexahydrate (4-NPP), the monophosphate ester substrate was used. The hydrolytic tendency was detected spectrophotometrically by monitoring the time evolution of p7

nitrophenolate (λmax = 425 nm, ε = 18,500 M-1cm-1) through wavelength scan from 200-800 nm in DMF where substrate was in 20 equivalents of the catalyst, till roughly 2% reaction conversion.Kinetic experiments were done both at conditions of excess substrate and excess Zn complex keeping the other constant. Herein we report only the former data. The study comprised 5 sets having the catalyst concentration as 0.05 mmol and substrate as 0.5 (10 eqv.), 0.7 (14 eqv.), 1.0 (20 eqv.), 1.2 (24 eqv.) and 1.5 (30 eqv.) mmol. The reactions were initiated by injecting 0.04 ml of complex (2.5 × 10-3 M) into 1.96 ml of 4-NPP solution and spectrum was recorded only after fully mixing at 25oC. The visible absorption increase was recorded for a total period of 45 minutes at an interval of 5 minutes. All measurements were done in triplicate and the average values were considered. 2.6. Synthesis of H4PydChda and probe-Zn2+ 2.6.1. Synthesis of H4PydChda Pyridoxal hydrochloride (0.406 g, 2 mmol) was dissolved in absolute ethanol (15 mL) in the presence of KOH (0.112 g, 2 mmol) with stirring. After 1 h of stirring, the separated white solid (KCl) was filtered and the obtained clear solution was added to a solution of

1,2-

diaminocyclohexane (0.114 g, 1 mmol) in ethanol (15 mL) with stirring and the resulting reaction mixture was refluxed for 4 h. The completeness of the condensation reaction was checked by performing thin layer chromatography. The solution was evaporated by rotary evaporator and sticky mass obtained was washed by cold ether and dried under vacuum. (Yield: 0.359 g, 87%; Melting point : >250˚C). 1H NMR (300 MHz, DMSO-d6): δ ppm : 8.85(s, 2Ha), 7.81(s, 2Hc), 4.60-4.48 (m, 4He), 3.59 (s, 2Hd), 2.30 (s, 6Hb), 1.93-1.45 (m, 8Hg). Anal.calcd. for C22H28N4O4: C, 64.06; H, 6.84; N, 13.58. Found: C, 63.89; H, 6.53; N, 13.25%.TOF MS ES+, m/z = 413.47 calc. for C22H29N4O4= 413.22. 2.6.2. Synthesis of probe-Zn2+ Pyridoxal hydrochloride (0.406g, 2 mmol) was dissolved in dry methanol. To it, ethanolic solution of Zn(NO3)2.2H2O (0.297g, 1 mmol) was added dropwise under stirring and the solution was stirred for 30 min. Then to this solution, a methanolic solution of 1,2diaminocyclohexane(0.114 g, 1 mmol) was added slowly and the resulting yellowish-orange solution was stirred for 2 h followed by reflux of another 1 h. Then it was evaporated by rotary 8

evaporator and resulting orange colored sticky mass was washed by cold ether and dried under vacuum. (Yield: 0.304g, 64%). Anal.calcd. for [probe-Zn2+]: C, 55.53; H, 5.51; N, 11.77. Found: C, 55.04; H, 5.19; N, 11.31%. TOF MS ES+, m/z = 475.29 calc. for C22H27N4O4Zn= 475.13. 3. Results and discussion 3.1. Synthesis and FTIR spectral characterization of H4PydChda The current chemosensor, H4PydChda, is straightforwardly synthesized in one step, by condensing pyridoxal hydrochloride with 1, 2−diaminocyclohexane under refluxing conditions in ethanol medium and the synthetic pathway is depicted in Scheme 2. The structure of H4PydChda was well characterized by 1H NMR and FTIR spectroscopy (Fig. S1). The probeZn2+ complex was characterized by FTIR and ESI-MS spectroscopy (Fig. S2). FTIR spectra of H4PydChda showed the characteristic band due to ν(C=N) at 1625 cm-1. In the Zn2+ complex, the ν(C=N) absorption appears at higher energy (ca. 1634cm-1) indicating possible back donation of electrons from Zn2+ to the ligand center. The complex also displays broad band of medium intensity around 3253 cm-1 attributable to the -OH stretching vibration of the -CH2OH group of the pyridoxal part of the chemosensor [20]. 3.2. UV-Vis spectroscopic investigation of H4PydChda In order to ascertain the binding of Zn2+ by H4PydChda, absorption titrations were carried out by adding varied concentrations of Zn(NO3)2.2H2Oto a fixed concentration of H4PydChda. Fig. 1 depicts spectrophotometric changes upon titrating H4PydChda (10-5 M) with incremental additions of Zn(NO3)2 (50 × 10-5 M) in EtOH/H2O (4:1, v/v, 25 mM Tris buffer, pH 7.4). The absorption spectrum of H4PydChda in same solvent displayed sharp absorption bands centered at 252 nm and 336 nm, which are assigned to the π–π* transitions. The absorption bands at 416 nm is attributed to the n–π* transitions of azomethine group. However, addition of Zn2+ induced dramatic modification both in the maxima and shape of the said bands of H4PydChda. The band at 252 nm is decreased and accompanied by a blue shift to 222 nm. The band maxima at 336 nm is gradually decreased. Another broad and moderately intense band around 375 nm could be assigned to O-(phenolate) ↔ Zn2+ (LMCT or MLCT). The accompanying isosbestic points at 292, 346 and 426 nm clearly indicate that the transition 9

between the free and the complexed species occurs and a stable complex resulted at a certain composition. The stoichiometry of the complex formed between H4PydChda and Zn2+ is 1:1 based on Job’s plot (Fig. S3). Absorptive response of H4PydChda towards Zn2+ with respect to Li+, Na+, K+, Ca2+, Sr2+, Al3+, Pb2+, Cr3+, Mn2+, Fe2+, Fe3+, Co2+, Ni2+, Cu2+, Cd2+, Hg2+ and Mg2+ ions is also depicted in Fig. S4. 3.3. Binding behavior analyzed by fluorescence spectroscopy With regard to the emission spectra (λex = 416 nm), H4PydChda emission was poorly perceptible (Φf = 0.0279). Upon addition of 40 equivalents of Zn(NO3)2, drastic enhancement in fluorescence intensity was observed at 475 nm and maximum emissive wavelength shifts from 479 to 475 nm with Φf = 0.196. (Table S1). This substantial increase in the quantum yield of H4PydChda in the presence of Zn2+ advocates its credibility as an efficient Zn2+ sensor. Moreover, H4PydChda exhibited high selectivity for Zn2+ compared to various biologically relevant species such as, Li+, K+, Na+, Ba2+, Mg2+, Ca2+, Sr2+, Cr3+, Mn2+, Fe2+, Fe3+, Co2+, Ni2+, Cu2+, Hg2+, Pb2+ and Al3+ in EtOH/ H2O (4:1, v/v) in HEPES buffer solution at pH 7.4. There were no evident fluorescence changes for the rest of the cations whereas Cd2+ exhibited only slight emission enhancement (Fig. 2). As an important characteristic of fluorescence probe, the selectivity of H4PydChda towards different metal cations was examined by mixing H4PydChda (5 μM) with Zn2+ and other relevant cations, Li+, Na+, K+, Sr2+, Ba2+, Ca2+, Cr3+, Mn2+, Fe2+, Fe3+, Co2+, Ni2+, Cu2+, Hg2+, Pb2+, Al3+, Mg2+ andCd2+ in EtOH/H2O (4:1, v/v) in 25 mM HEPES buffer at pH 7.4. From the bar diagrams, we can conclude that except for Al3+, Co2+, Ni2+ and Cu2+, background of competing metal ions showed no interference with the detection of Zn2+ ion in EtOH/H2O (4:1, v/v) in 25 mM HEPES buffer at pH 7.4 (Fig. S5). Fluorescence titration experiments of H4PydChda with Zn2+ in EtOH/ H2O (4:1, v/v) in HEPES buffer at pH 7.4 were performed to assess the practical parameters like stoichiometry, binding constant and detection limit. As shown in Fig. 3, the addition of Zn2+ with increasing concentrations from 0 to 200 μM, elicited a gradual enhancement of the emission at 476 nm. The fluorescence enhancement reached a maximum (50 fold) in the presence of 200 μM Zn2+. Moreover, there was an excellent linear correlation between F/F0 and Zn2+ concentration in the 10

range 5 to 45 μM with a detection limit 5.9 μM (Fig. S6) [21]. The fluorescence titration data reinforces the 1:1 stoichiometry as evident from the Job’s plot from absorption studies (Fig. S3). The association constant for Zn2+ was calculated to be 9.6 × 103 M−1 by the linear BenesiHildebrand equation (Fig. 4). The emission (476 nm) intensity of H4PydChda can be reversibly switched by alternating addition of Zn2+/Na2H2EDTA, enabling H4PydChda a reversible Zn2+ sensor (Fig. S7). We have also examined the anion independency of H4PydChda by using NO3-, CH3COO-, ClO4-, Cl-, Br-, I- salts and the spectral output is represented in Fig. S8. In order to investigate the response of H4PydChda to Zn2+ in acidic and basic pH, fluorescence pH titrations were carried out in EtOH/ H2O (4:1, v/v) in HEPES buffer solution at various pH values in the presence and absence of Zn2+ (Fig. S9). The low fluorescence intensity in acidic condition may be triggered by protonation of the coordinating atoms of H4PydChda. The maximum fluorescence intensity occurs in the pH range 5.0 to 8.0 due to favorable deprotonation of H4PydChda leading to a stable Zn2+-probe aggregate. Therefore, the study reaffirms that H4PydChda can sense Zn2+ at the physiological pH = 7.4 which prompted us to explore its application in live cell imaging. 3.4. Time resolved measurement In accordance with the previous studies, H4PydChda lifetime is significantly affected after chelation with Zn2+. A picosecond time-resolved fluorescence technique has been used to examine the decay processes of free sensor H4PydChda and probe-Zn2+in EtOH/H2O (4:1, v/v, 25 mM HEPES buffer, pH 7.4) (Fig. 5) and the lifetime data are summarized in Table S1. The quantum yield, lifetime and decay rate constants are correlated according to the following equations, τ-1 =kr + knr and kr=Φf/τ, where, kr is the radiative decay rate constant and knr is the total non-radiative decay rate constant. The decay curve and fitting data of H4PydChda suggested that there were three main isomeric components of H4PydChda to absorb light and emit fluorescence photons of lifetime at 0.18 ns, 1.57 ns and 4.89 ns. The average fluorescence lifetime (τ) of H4PydChdawas estimated 11

to be 1.65 ns. The radiative and non-radiative decay rate constants are calculated to be 0.0168× 109 and 0.5866× 109 sec-1 respectively indicating that the non-radiative decay is the predominant process in the excited state of H4PydChda[22]. In the presence of 200 × 10-6 M Zn2+, the timeresolved fluorescence decay showed substantial change, which indicated three components corresponding to probe-Zn2+ at 7.84 ns and two other components of H4PydChda at 2.08 ns and 0.39 ns. The lifetime is increased to 3.67 ns, which is longer than that of the free-H4PydChda. The radiative and non-radiative decay rate constants changed to 0.0534× 109 and 0.2186× 109 s-1 respectively. This result suggested that both the radiative and non-radiative decay processes became comparative resulting in fluorescence enhancement. The reason for non-exponential nature of the curve may originate from some solvent dependent dynamics, presence of isomeric species etc. 3.5. NMR based binding experiment The 1H and 13C-NMR titrations were performed to get information regarding the binding mode of H4PydChda with Zn2+ by gradual addition of Zn(CH3COO)2.2H2O to the DMSO-d6 solution of H4PydChda (Fig. 6). Upon addition of 0.5 equivalent of Zn2+ to H4PydChda, Ha, Hc and Hd showed significant shifts in the 1H-NMR output. In

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C NMR experiment (Fig. 7), C3,

C4, C5 and C9 recorded appreciable shifts along with generation of a C=Oacetate peak. These changes in chemical shifts indicated the involvement of phenolate oxygen and imine nitrogen of H4PydChda in complexation with Zn2+. Moreover, the reaction composition was also confirmed by mass spectral analysis, and a peak at m/z 475.29 is assignable to [H2PydChda + Zn2+ + H+]. 3.6. Theoretical study 3.6.1 Geometric and energetic features of H4PydChda and probe-Zn2+ To further reinforce the Schiff base transformation phenomena and mode of complexation between H4PydChda and Zn2+, DFT calculations were carried out. Since attempts to isolate single crystals of probe-Zn2+ suitable for X-ray diffraction analysis were unsuccessful, the optimized structure of the complex was computed by theoretical methods. The geometries of H4PydChda and probe-Zn2+ were optimized by at the BP86-D3/def2-TZVP level of theory. The resulting geometric and energetic features are shown in Fig. 8. For the free ligand we have optimized three possible keto-enol tautomers and compared their relative energies. The results 12

are shown in Fig. 8a-c. It can be observed that all tautomeric forms present a remarkable geometry where the aromatic rings form a parallel displaced π-stacking interaction that is reinforced by two strong OH···Npy intramolecular hydrogen bonds (highlighted in Fig. 8). The OH···N distance is 2.02 Å as indicated in the perspective view, where the antiparallel arrangement of both aromatic rings can be clearly observed. We have explored other possible conformations for the ligand varying several dihedral angles; however this arrangement is the lowest in energy for all tautomeric forms due to the formation of the H-bonds. The energetic difference between the three forms is very small, being the keto form the most stable. All three forms likely coexist at room temperature. For the probe-Zn2+ complex, we have considered several coordination modes and the global minimum corresponds to the complex shown in Fig. 8d where the Zn2+ metal center exhibits a pseudo-tetrahedral coordination. It is bonded to two phenoxide O atoms and two N atoms belonging to the imine functional groups. This coordination mode strongly agrees with the experimental structure of a similar complex (CSD code AGUJOC) [23] which was solved by Xray analysis via its hydrochloride form (Fig. 8c), giving reliability to the proposed geometry. Interestingly the geometric features of the DFT optimized probe-Zn2+ complex and the X-ray structure are in excellent agreement. The coordination bonds are slightly longer in the X-ray structure likely due to the presence of a chlorido ligand coordinated to the Zn. Further support to the proposed geometry of the DFT optimized complex is that the theoretical UV spectrum presents very similar bands compared to the experimental one, as further discussed below. 3.6.2 TDDFT study In the ground state (S0) of the probe-Zn2+ complex the HOMO and HOMO–1 are almost isoenergetic (–5.925 and –5.927 eV, respectively) due to the approximate C2 symmetry of the optimized geometry. They are basically composed by the aromatic moieties, the phenoxide atoms and the conjugated C=N bond (Fig. 9). Similarly the LUMO and LUMO+1 are also close in energy and basically composed by the aromatic moieties and the conjugated C=N bond. The energy difference between the HOMO and LUMO is 3.683 eV. To get better insight into the experimental absorption values TDDFT calculations were done for the complex on the basis of the optimized geometry at the B3LYP/6-311+G* level of theory. The calculated absorption energies associated with their oscillator strengths, the main configurations and their assignments 13

of the complex are given in Table 1. Here the lowest lying distinguishable singlet → singlet absorption band at λexp =375 nm can be mainly attributed to one excitation. It occurs at 3.3815 eV (λ = 366 nm and f = 0.1342) due to an HOMO → LUMO+1 transition with ILCT character, which can be assigned to a π(L) → π*(L) transition (Fig. 9, highlighted in blue). The following absorption band (λexp = 222 nm) consists of two excitations. The first one (5.4701 eV, 226 nm and f = 0.2511) that is attributed to several transitions (HOMO–1 → LUMO+6, HOMO → LUMO+4 and HOMO → LUMO+7) which can be assigned to π(L) → π*(L) transitions with basically ILCT character (plots of LUMO+4 and LUMO+6 are shown in Fig. S10 and LUMO+7 in Fig. 9). The HOMO–1 → LUMO+6 transition exhibits some ligand-to metal charge transfer (LMCT) character. The second excitation (5.5419 eV, 223 nm, f = 0.2835) is represented in Fig. 9 (highlighted in red). It is also attributed to several transitions. Interestingly the HOMO → LUMO+5 and HOMO → LUMO+7 transitions can be assigned to π(L) → π*(L) and the HOMO → LUMO+9 presents some mixing character (ILCT and LMCT). The experimental values are in excellent agreement with the theoretical ones, giving reliability to the proposed geometry for the probe-Zn2+ complex and the level of theory. 3.8. Application of H4PydChda in cellular imaging H4PydChda was investigated for the ability to respond to Zn2+ in living cells. To demonstrate the potential application of H4PydChda, the intracellular Zn2+ imaging behaviour ofH4PydChda was studied on Human gastric adenocarcinoma (AGS) cells by fluorescence microscopy. The results of the MTT assay clearly indicated that H4PydChda at different working concentrations showed no significant cytotoxicity in AGS cells. Even 100 µM concentration of the sensor was unable to produce any significant toxicity outcomes (Fig. 10). In fluorescence imaging studies, the H4PydChda alone was unable to produce any fluorescence in absence of Zn2+ (Figure B-I, II & III). However, addition of Zn2+ ions to the cells showed blue fluorescence (Figure C-I, II & III). TPEN addition resulted in disappearance of the blue fluorescence (Figure D-I, II & III). The intracellular Zn2+ imaging behaviour of H4PydChda on AGS cells as demonstrated by fluorescence microscopy revealed that incubation with H4PydChdaalone could not offer any recognizable fluorescence signal from the cells. However, when aided with added Zn2+ ions, the cells presented significant fluorescence, the intensity of which amplified with increase in the H4PydChda concentration. The intensive fluorescence behaviour was suppressed and obliterated 14

with the addition of TPEN. As TPEN is an efficient cell-permeable zinc-targeted chelator, it confers proficient scavenging action on Zn2+ ions competitively inhibiting H4PydChda in binding with Zn2+ ions. As a consequence of such cellular quenching, the fluorescence disappears. Hence, TPEN reliant reduction in signal intensity is a positive proof of the principle that variation in fluorescence intensity caused by concentration dependent differential probeZn2+ binding which gets quenched with TPEN treatment. This also proves the sensor’s selective and sensitive zinc-reporting efficiency par excellence. The fluorescence signaling of H4PydChda in presence of Zn2+ may be utilized as indelible signature of selective sensor response. Hence, these results indicate that H4PydChda is an efficient tool for monitoring changes in the intracellular Zn2+ concentration under biological conditions. The MTT assay results confirm that H4PydChda has no significant cytotoxic effects on AGS cells up to 12 h of treatment. Endowed with attributes like high binding affinity, specificity and biostability H4PydChda has the promising potential of being a tool for probing biochemical dynamics of Zn in the cellular environment with appreciable sensitivity and fidelity.

3.9. Phosphatase activity of probe-Zn2+ The efficiency of P-O bond cleavage of the mononuclear Zn2+ complex was screened completely by monitoring the spectral change in the wavelength scan of a catalytic solution for 2 h where complex to phosphomonoester is maintained at 1:20 stoichiometrically. The electronically silent substrate 4-NPP (1 × 10-3 M) leads to a band maxima at 425 nm characteristic of p-nitrophenolate ion owing to the hydrolytic action of Zn2+ complex (5 × 10-5 M) in DMF. The change in spectral behaviour for the complex is depicted in Fig. 11 and its absorbance profile at 425 nm in Fig. S11. The initial first-order rate constants, V (s−1), for the cleavage of 4-NPP were obtained directly from the plot of log[A∞/(A∞ − At)] values versus time (through origin) which were linear with R2 ≥ 0.96 (Fig. S12). As can be observed, initially the cleavage rate increases linearly with the increase of 4-NPP concentration but deviates gradually from linearity and finally tends toward a saturation curve (Fig. 13A). In other words, it is a first order kinetics at lower concentration which gradually differs from unity at higher concentration. The kinetic parameters (kcat, Vmax, kM, kcat) for the catalyzed reactions were determined from the linear plots of 1/V versus 1/[NPP] values (Lineweaver-Burk plot) as per the Michaelis-Menten 15

approach of enzymatic kinetics (Table 2). The results of calculation remained the same when the experiment was done by varying the [probe-Zn2+] under investigation at constant [NPP]. The observed kcatvalue (21.59 s-1) displayed the second highest phosphatase activity in comparison to those reported earlier as depicted in Table. S2. To correct for the spontaneous cleavage of 4-NPP, each reaction was measured against a reference cell that was identical to the sample cell in composition except for the absence of Zn complex. The spontaneous hydrolysis of 4-NPP had a much lower reaction rate when compared to the metal catalyzed reaction and hence the rate of spontaneous hydrolysis was not separately determined [14(m),(n)]. To confirm that the synthesized ligand, H4PydChda, and the zinc-salt used does not participate in the catalytic reaction, rigorous control experiments have been performed to intake their role if any (Fig. S13). The results indicated no appreciable change. 4. Conclusion Pyridoxal containing simple Schiff base, H4PydChda, represent a potential reversible chemosensor for selective detection of Zn2+ in aqueous ethanol medium at physiological pH. The C=N isomerisation and ESIPT are inhibited upon binding with Zn2+ ions, which causes CHEF effect, inducing an enhancement in the fluorescence intensity of the chemosensor. The complex formation, stoichiometry, and binding mode have been thoroughly examined by UV-Vis, ESIMS, and NMR studies, which show formation of a 1:1 probe-Zn2+ complex. The DFT/TDDFT calculation was carried out to further demonstrate the electronic properties of the probe-Zn2+ aggregate. Biological application of H4PydChda was also evaluated on Human gastric adenocarcinoma (AGS) cells. In addition, we have demonstrated that the probe-Zn2+ ensemble is capable of showing appreciable phosphatase activity (Kcat = 21.59 s-1) with NPP substrate. Notably, probe-Zn2+complex turns out to be the second most effective phosphatase mimicking entity among the reported mono- and dinuclear Zn2+ complexes. In view of these merits, we anticipate that pyridoxal containing Schiff base probes and their resultant Zn2+ complexes would open up a new direction for the development of multifunctional molecular entities for bioanalytical and biomimetic applications. Acknowledgement

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Financial support from the University Grants Commission for senior research fellowship to S. Mandal [Sanction No. UGC/847/Jr. Fellow (Upgradation)] and from the DST to Y. Sikdar (Sanction no. SR/FT/CS-107/2011) are gratefully acknowledged. A.B. and A.F. thank DGICYT of Spain (projects CTQ2014-57393-C2-1-P and CONSOLIDER INGENIO CSD2010-00065, FEDER funds) for funding. We thank the CTI (UIB) for free allocation of computer time. The authors gratefully acknowledge Ashis Kundu, Deborin Ghosh and Ramij R. Mondal for helpful discussions. References 1 (a) E.L. Que, D.W. Domaille, C. J. Chang, Metals in Neurobiology: Probing Their Chemistry and Biology with Molecular Imaging, Chem. Rev. 108 (2008) 1517–1549;(b) P. Jiang, Z. Guo, Fluorescent detection of zinc in biological systems: recent development on the design of chemosensors and biosensors, Coord. Chem. Rev. 248 (2004) 205– 229; (c) J.M. Berg, Y. Shi, The galvanization of biology: A growing appreciation for the roles of zinc, Science 271 (1996) 1081–1085; (d) X. Xie, T.G. Smart, A physiological role for endogenous zinc in rat hippocampal synaptic neurotransmission, Nature 349 (1991) 521–524; (e) B.L. Vallee, K.H. Falchuk, The biochemical basis of zinc physiology, Physiol. Rev. 73 (1993) 79–118 ; (f) K. H. Falchuk,The molecular basis for the role of zinc in developmental biology, Mol. Cell. Biochem. 188 (1998) 41–48; (g) F.d. Silva, J.J.R. Williams, R.J.P. In The Biological Chemistry of the Elements; Oxford University Press: New York, 2001, 315−335; (h) M. Dhanasekaran, S. Negi, Y. Sugiura, Designer zinc finger proteins: tools for creating artificial DNA-binding functional proteins, Acc. Chem. Res.39 (2006) 45−52; (i) S.F. Sousa, P.A. Fernandes, M.J. Ramos, The carboxylate shift in zinc enzymes: a computational study, J. Am. Chem. Soc. 129 (2007) 1378−1385. 2 (a) M.d. Onis, E.A. Frongillo, M. Blcssner, Is malnutrition declining? An analysis of changes in levels of child malnutrition since 1980, Bull. W. H. O. 78 (2000) 1222-1233; (b) A.I. Bush, 17

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Fig. 1. UV-Vis spectral changes of sensor H4PydChda(c = 10-5 M) in EtOH/H2O (4:1, v/v, 25 mM HEPES buffer, pH 7.4) solutions upon addition of Zn2+ ions (0-50 equivalent) (c = 0-55 × 10-5M).

35

Fig. 2. Emission spectra of H4PydChda (c=5×10-6 M) in presence of Zn2+, Li+, Na+, K+, Ca2+, Sr2+, Al3+, Pb2+ , Cr3+, Mn2+, Fe2+, Fe3+, Co2+, Ni2+, Cu2+, Cd2+, Hg2+ and Mg2+(c=200x10-6 M each metal ) in EtOH/H2O (4:1, v/v, 25 mM HEPES buffer, pH 7.4) (λex=416 nm).

36

Fig. 3. Fluorescence emission changes of H4PydChda (c = 5×10-6 M) upon addition of Zn2+ ions (c = 0-200× 10-6 M) in EtOH/ H2O (4:1, v/v) in HEPES buffer at pH 7.4 (λex=416 nm).

37

Fig. 4. Benesi-Hildebrand expression fitting of fluorescence titration curve of H4PydChda(c = 5× 10-6 M) upon addition of Zn2+ ions in EtOH/ H2O (4:1, v/v) in HEPES buffer at pH 7.4 (λex=416 nm).

38

Fig. 5. Time resolved fluorescence decay of sensor H4PydChda (red) and probe-Zn2+ (blue) and prompt (black) (λex=375nm).

39

Fig. 6.1H NMR titration experiment of H4PydChda with Zn2+.

40

41

Fig. 7.(a) 13C NMR titration experiment of H4PydChda with Zn2+; (b) 13C DEPT spectral output after addition of 1 equivalent of Zn2+ to H4PydChdain DMSO-d6.

42

Fig. 8. (a-c) BP86-D3/def2-TZVP optimized geometries and relative energies of the ligand in its keto, enol and mixed keto-enol form. (d) Optimized geometry of the probe-Zn2+ complex. (e) Xray geometry of a related complex retrieved from the literature. Distances in Å.

43

Fig. 9. Frontier molecular orbitals involved in two observable UV-vis absorption bands of the probe-Zn2+ complex.

44

Fig. 10. (A-I), (A-II) & (A-III) are the bright field images of 500 nM, 1µM & 10 µM H4PydChda treated cells respectively. (B-I), (B-II) & (B-III) represent fluorescence images treated with different concentrations of H4PydChda in absence of Zn2+ ions. (C-I), (C-II) & (CIII) indicate fluorescence images treated with different concentrations of H4PydChda in addition of 10 µM Zn2+ ions. Fluorescence was disappeared by further addition of TPEN (50 µM) represented by (D-I), (D-II) & (D-III) respectively. For all imaging, the samples were excited at 415 nm.

45

105

Viable cells (%)

100 95 90 85 80 0

0.5 1 10 Sensor concentration (µM)

100

Fig. 11. Denotes % cell viability of AGS cells treated with different concentrations (0.5 µM-100 µM) of H4PydChda for 12 h determined by MTT assay. Results are expressed as mean  S.D. of three independent experiments.

Fig. 12. Wavelength scan for the hydrolysis of 4-NPP in the absence and presence of Zn2+ complex (substrate:catalyst = 20:1) in DMF recorded at 298 K at an interval of 5 minutes for 2 h. [4-NPP]=1 × 10-3(M), [probe-Zn2+]=0.05 ×10-3(M). Arrow shows the change in absorbance with reaction time. 46

Fig. 13. (A) Plot of enzymatic kinetics for Zn2+ complex(V vs S); (B) Line-weaver Burk plot (1/V vs 1/S) having intercept=15.441 (error=1.993), slope=0.019 (error=0.001) and R2=0.990, Standard deviation = 1.671.

47

Scheme 1: Probable signalling pathways of probe H4PydChda.

48

Scheme 2: Preparation of chemosensor H4PydChda.

49

Table 1. Selected parameters for the vertical excitation (UV-vis absorptions) of 1, electronic excitation energies (eV) and oscillator strengths (f), configurations of the low-lying excited states of 1; calculation of the S0–Sn energy gaps based on optimized ground-state geometries (UV-vis absorption) (EtOH used as solvent). Only those excitations that contribute higher than 12% to each electronic transition are listed. Process Absortion Absortion

Electronic transitions S0 → S4 S0 → S27 S0 → S31

Composition HOMO → LUMO+1 HOMO–1 →LUMO+6 HOMO → LUMO+4 HOMO → LUMO+7 HOMO →LUMO+5 HOMO → LUMO+7 HOMO → LUMO+9

Excitation energy 3.3815 eV (366 nm) 5.4701 eV (226 nm)

Oscillator strength (f) 0.1342 0.2511

5.5419 eV (223 nm)

0.2831

Contribution 100% 43% 24% 16% 20% 40% 16%

λexp (nm) 375

222

50

Table 2. Kinetic parameters of phosphatase activity of Zn2+ complex.

103 Vmax (Ms-1)

1.08

103 Std.

kcat

103kM

err.

(s-1)

(M)

8.32

21.59

0.021

103

10-3

Std.

kassoc

err.

(M-1)

0.27

47.73

103kcat/kM (M-1s-1)

0.45

51