Coumarinyl thioether Schiff base as a turn-on fluorescent Zn(II) sensor and the complex as chemosensor for the selective recognition of ATP, along with its application in whole cell imaging

Abstract

The coumarinyl thioether Schiff base, H₂L, demonstrates turn-on fluorescence sensing towards Zn²⁺ ion with a limit of detection (LOD) of 0.068 μM. Different physicochemical techniques (mass, ¹H NMR, Job's) support the formation of a 1 : 1 metal-to-ligand complex, [ZnL]. The fluorogenic complex [ZnL] recognizes ATP in the presence of all other common anions, inorganic phosphates and biologically important phosphates (nucleosides, nucleotides). The proposed sensor has efficiently been used for ATP sensing with a LOD of 6.7 μM, which is the lowest in literature. Exogenous zinc ions in SCC084 (human oral carcinoma) cells have been checked through fluorescence cell imaging process by adding H₂L in the medium.

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1. Introduction

About one-third of naturally occurring elements are responsible for the origination, growth, reproduction and death of living organisms. Zinc, the second-most abundant (transition) metal following iron in the human body, is an essential nutrient metal ion.¹ Zinc essentiality was established in 1869 for plants, in 1934 for experimental animals and in 1961 for humans.² Zinc deficiency causes mental retardation, digestive dysfunction, loss of brain function and gene transcription, immune deficiency, mammalian reproduction disorder, etc. Biological zinc ions are tightly sequestered by proteins, and the presence of excess “free zinc” may induce pathological diseases such as Alzheimer's and Parkinson's disease.³ Moreover, excess of zinc in the environment may reduce soil microbial activity, which has phytotoxic effects.⁴

Phosphates are among the most significant anions in living cells, playing crucial roles in numerous biological processes, such as universal energy storage, ion-channel regulation, intercellular signalling mediation, protein phosphorylation, enzymatic reactions, DNA replication, etc.^5–8 Among these, ATP is a universal energy transporter in biological systems and contributes to biochemical reactions, active transport, nucleic acid synthesis, muscle activity, and the movements of cells.^9,10 More importantly, deficiency in ATP results in ischemia, Parkinson's disease and hypoglycaemia.¹¹ The screening of ATP concentration levels in different cellular systems, enzymatic processes and even cell apoptosis is a challenging task, and many systems have been devised based on metal–ligand interaction for the selective and sensitive detection of ATP.^12–16

In considering the physiological and biomedical significance of Zn²⁺ ions and ATP, there is overriding interest in the development of selective and sensitive sensors for the simultaneous detection of zinc and ATP. We have engaged in designing Schiff bases that are hexadentate N₂O₄, N₂O₂S₂ donor ligands and are useful as fluorogenic turn-on sensors for Zn²⁺ identification.^17,18 As a continuation of this programme, we have initiated a proposal to use a newly designed, coumarinyl-based thioether Schiff base as a chemosensor for Zn²⁺, and the resulting complex serves as a selective and highly sensitive ATP sensor. Coumarin is a phytochemical, and most of its derivatives show a wide spectrum of pharmacological effects, including antimicrobial, anti-arrhythmic, anti-osteoporosis, anti-HIV, and antitumor activities.^19–23 The incorporation of group(s) alters the property of the parent coumarin and converts it into a more useful product.²⁴ The potent antibiotics, including novobiocin, coumaromycin and chartesium, are coumarin derivatives. Recently, interest in these compounds has been revived owing to their use as fluorescent markers in the biochemical determination of enzymes.²⁵ The ligand in this study selectively binds Zn²⁺ in the presence of other cations and enhances fluorescence intensity. The composition of the complex has been supported by spectroscopic data (mass, Job's plot, ¹H NMR). The DFT computation of the optimised geometry of H₂L and the complex, [ZnL], has been used to explain the electronic properties. The more interesting development is that the complex, [ZnL], shows very high sensitivity to adenosine 5′-triphosphate (ATP) in the presence of all other common anions and biologically important phosphates. The recognition of ATP by metal complexes is currently envisaged.^12–16,26 The practical applicability of the ligand (H₂L) has been checked in SCC084 (human oral carcinoma) for the determination of exogenous zinc ions by fluorescence cell imaging.

2. Experimental

2.1 Material and methods

The reagents and solvents used in this work were commercially available and used as received. All aqueous solutions were prepared using Milli-Q water (Millipore). 2-Aminothiophenol, resorcinol, tetrahydrofuran, 1,2-dibromoethane, and sodium were purchased from E. Merck, Germany. Methanol was purified and dried by the standard method.²⁷ Spectroscopic-grade DMSO was purchased from SRL, India. ZnCl₂ was purchased from Sigma-Aldrich. All other solvents and chemicals were purchased from Merck, India, (AR grade) and used without further purification. ¹H NMR (300 MHz) was recorded from a Bruker (AC) 300 MHz FT-NMR spectrometer using TMS as an internal standard. UV-vis spectra were collected from a PerkinElmer Lambda 25 spectrophotometer, and the fluorescence spectra were obtained from a PerkinElmer spectrofluorimeter model LS55 at room temperature; FT-IR spectra (KBr disk, 4000–400 cm⁻¹) were collected from a PerkinElmer RX-1 FTIR spectrophotometer.

Synthesis of H₂L. 1,2-Bis(2-aminophenylthio)ethane was prepared according to a published method.²⁸ To 1,2-bis(2-aminophenylthio)ethane (0.276 g, 1 mmol) in MeOH (20 mL), 8-formyl-7-hydroxy-4-methylcoumarin (0.408 g, 2 mmol) was added and stirred overnight. The reaction mixture was then evaporated slowly in air, and a yellow crystalline precipitate appeared. The product was then collected by filtration, washed with cold methanol and dried (yield, 70%). The synthetic scheme is given in ESI, Scheme S1.† Melting point > 200 °C. Microanalytical data: C₃₆H₂₈N₂O₆S₂; calcd (found): C, 66.65 (66.58); H, 4.35 (4.27); N, 4.32 (4.40)%. ¹H NMR (300 MHz, CDCl₃): δ 14.87 (s, 2H, OH_a), 9.29 (s, 2H, imine-H, H_b), 6.14 (s, 2H, H_e), 7.58 (d, 2H, J = 8.97, H_f), 6.90–6.95 (m, 2H, H_d), 6.92 (d, 2H, J = 8.91, H_g), 2.42 (s, 6H, –CH₃, H_d), 3.13 (s, 4H, –SCH₂, H_c), 7.38–7.22 (m, 4H, H_h,i,j,k) (ESI, Fig. S1†). Mass: (M⁺ + Na) 670.99 (ESI, Fig. S2†) IR: 3447 cm⁻¹ (phenoloic –O–H), 1718 cm⁻¹ (lactone ring –C=O), 1612 cm⁻¹ (azomethine, –C=N) (ESI, Fig. S3†).

Synthesis of [ZnL] complex. To a methanol solution (30 mL) of Zn(NO₃)₂·6H₂O (0.298 g, 1 mmol), THF solution of H₂L (0.648 g, 1 mmol) was added and stirred for 1 hour; a greenish-yellow precipitate appeared. The solution was slowly evaporated in air, and the crystalline product was filtered. The product was then recrystallized from DMF–CH₃CN mixture. Microanalytical data: C₃₆H₂₈N₂O₆S₂Zn; calcd (found): C, 60.72 (60.81); H, 3.68 (3.61); N, 3.93 (4.02)%. ¹H NMR (300 MHz, CDCl₃): δ 5.98 (s, 2H, H_b), 9.41 (s, 2H, imine-H, H_a), 6.42 (d, 2H, J = 6.9, H_c), 7.37 (d, 2H, J = 6.9, H_d), 2.34 (s, 6H, –CH₃, H_e), 2.95 (s, 4H, –SCH₂, H_f), 7.48–7.57 other aromatic protons (ESI, Fig. S4†). Mass: (M⁺ + Na) 733.03 (ESI, Fig. S5†). IR: 1708 cm⁻¹ (lactone ring –C=O), 1572 cm⁻¹ (azomethine, –C=N), (ESI, Fig. S6†).

2.2 DFT computation

Geometry optimization of H₂L and [ZnL] was performed by DFT/B3LYP method using Gaussian 09 software.^29–31 6-311G basis set were used for C, H, N, and O; Lanl2dz basis set was used as effective potential (ECP) set for Zn.^32–34 Vibrational frequency calculations were performed to ensure that the optimized geometries represent the local minima and that there are only positive eigenvalues. Theoretical UV-vis spectra were calculated by time-dependent DFT/B3LYP method in methanol using conductor-like polarizable continuum model (CPCM).^35–37 GAUSSSUM was used to calculate the fractional contributions of various groups to each molecular orbital.³⁸ DFT/B3LYP-excited model was used for triplet-state analyses of the molecules.³⁹

2.3 Cell imaging

The probe H₂L was applied in the detection of SCC084 (human oral carcinoma) to explore its utility in biological systems. SCC084 cells were fixed in paraformaldehyde (4%) and blocked with BSA in PBS-Triton X100 solution. After that, the cells were observed under epifluorescence microscope.⁴⁰ Then, cells were treated with Zn²⁺ solution (30 mM) for a 45 min incubation in buffer and washed again with buffer at pH 7.4 before mounting on a grease-free glass slide. Fluorescence microscope equipped with a UV filter was used to observe the cells after adding H₂L (2 mM). Cells incubated with Zn²⁺ were used as a control. H₂L is easily permeable through tested living cells without any harm (after 30 min of exposure to H₂L at 2 mM). To evaluate the cytotoxicity, MTT assay was done.

3. Results and discussion

3.1 Synthesis and formulation

The condensation reaction of 1,2-bis(2-aminophenylthio)ethane and 8-formyl-7-hydroxy-4-methylcoumarin has isolated the Schiff base, 8,8′-((1Z,1Z′)-(((ethane-1,2-diylbis(sulfanediyl))bis(2,1-phenylene))bis(azanylyldene))bis(methanylylidene))bis(7-hydroxy-4-methyl-2H-chromen-2-one) (H₂L), which in THF solution reacts with Zn(NO₃)₂ in methanol to yield a bright-greenish yellow product, [ZnL], whose composition has been established by microanalytical data. The FTIR spectrum of H₂L shows ν(C=N) at 1612 cm⁻¹, ν(COO) (lactone) at 1718 cm⁻¹ and ν(OH) at 3447 cm⁻¹ (ESI, Fig. S3†) while in the complex [ZnL]; an interesting observation is the elimination of ν(OH) stretching followed by shifting of ν(COO) (lactone) to 1708 cm⁻¹ and ν(C=N) to 1572 cm⁻¹, which undoubtedly support the coordination of the ligand to Zn(II) (ESI, Fig. S6†). The mass ion peak of H₂L at 670.99 corresponds to (M + Na)⁺ (ESI, Fig. S2†), while for the complex, the ion peak at 733.03 corresponds to (M′ + Na)⁺ (ESI, Fig. S5†), which strongly supports the complex formation. The structural assignment was established by ¹H NMR spectral data (in CDCl₃) (ESI, Fig. S1†), which report characteristic signals corresponding to δ(OH) at 14.87 ppm as sharp singlet, imine (–CH=N–) at 9.29 ppm, –S–(CH₂)₂S– at 3.13 ppm, coumarinyl–CH₃ at 2.42 ppm; all other aromatic protons appear at 6.1–7.6 ppm. The ¹H NMR spectral data of [ZnL] (in CDCl₃) (ESI, Fig. S4†) shows the disappearance of the –OH peak with shifting of the imine proton (–CH=N–) to a higher chemical shift (9.41 ppm), which indicates the chelation of phenolato-O and imine-N with Zn(II). The UV-vis spectrum of H₂L in methanol/water (HEPES buffer, pH = 7.4; v/v = 2/1) shows intense absorption at 320 nm corresponding to n–π* transition, which shifts to 343 nm in the complex, and a new peak at 410 nm.

3.2 UV-vis spectroscopic studies: detection of Zn²⁺ ion

The UV-vis spectrum of H₂L in aqueous–methanol buffer solution (0.01 M, pH 7.2, HEPES; v/v = 1/2) shows a high intense band at 320 nm, which is assigned to intraligand charge transfer transition. The titration of H₂L with incremental addition of Zn²⁺ in HEPES buffer (10 mM, pH 7.2) at 25 °C has shown absorption enhancement at 343 nm and 410 nm with isosbestic points at 258, 319 and 455 nm (Fig. 1). Naked-eye colour change of H₂L solution upon addition of Zn²⁺ ion is shown in Fig. S7 (ESI†). The incremental addition of Zn²⁺ shows an increase in absorbance until the ratio of [Zn²⁺] : [H₂L] reaches 1 : 1 and no longer changes with the continuous addition of excess Zn²⁺. The ligand, H₂L, is a dianionic hexadentate N₂O₂S₂ chelating agent and has synthesised the mononuclear complex, [ZnL]. The composition has been checked by Job's plot (ESI, Fig. S8†). The association constant of H₂L with Zn²⁺ is 6.49 × 10⁴ M⁻¹ L (ESI, Fig. S9†). Due to the complexity of the intracellular environment, further examination of the probe was conducted to determine whether other ions were interfering. Selective metal ion assays were performed while keeping the other experimental conditions unchanged.

Fig. 1 Change in absorption spectra of H₂L (50 μM) upon addition of Zn²⁺ in methanol/water (v/v, 2 : 1); nonlinear plot of absorbance (at 343 nm) vs. [Zn²⁺] for the corresponding UV-visible titration is shown inset.

The red shifting of the bands of H₂L upon Zn²⁺ addition is attributed to an intramolecular charge transfer (ICT) through the chelation to Zn²⁺, forming ZnN₂O₂S₂ distorted octahedral coordination with six-membered chelate rings via two nitrogen donor centres of C=N and two oxygen donors of phenolates, along with five-membered chelate rings from two sulphur donor centres of thioether groups (Scheme 1).

Scheme 1 Selective Zn⁺ binding of H₂L and change of emission colour and intensity.

3.3 Fluorescence OFF–ON sensing for Zn²⁺ and ATP

Fluorescence sensors are powerful tools for analytical quantification of small molecules and ions with high precession and accuracy levels. The probe (H₂L) exhibits weak emission at 577 nm, which may be due to ESIPT (Excited-State Intramolecular Proton Transfer) from the phenolic–OH to imine-N when excited at 320 nm in HEPES buffer (10 mM, pH 7.2) at 25 °C in methanol/water (v/v, 2 : 1) (Scheme 2).

Scheme 2 Proposed enol–keto tautomerism and origin of ESIPT.

The addition of Zn²⁺ to the probe solution (H₂L) enhances the emission intensity at 514 nm. Other cations such as Na⁺, K⁺, Ca²⁺, Mg²⁺, Ba²⁺, Hg²⁺, Ni²⁺, Co²⁺, Pb²⁺, Pd²⁺, Cu²⁺, Fe³⁺, Mn²⁺, and Cd²⁺ were completely nonresponsive (Fig. 2), while Al³⁺ showed a very small enhancement of emission. Fluorescence amplification upon addition of Zn²⁺ to the probe may be due to the exclusion of ESIPT via deprotonation of phenolic–OH and also by chelation effect, which improves molecular rigidity; this effect is thus defined as chelation enhancement of fluorescence (CHEF). In order to check the practical utility of Zn²⁺ recognition by H₂L, competitive titrations were carried out in the presence of other biologically and ecologically relevant metal ions. No significant change in emission spectra is observed even after the addition of excess (5 equivalents) metal ions: Na⁺, K⁺, Ca²⁺, Mg²⁺, Mn²⁺, Fe³⁺, Al³⁺, Co²⁺, Ni²⁺, Pd²⁺, Cd²⁺, Hg²⁺, Cu²⁺, Ba²⁺ and Pb²⁺.

Fig. 2 Fluorescence (λ_ex = 320 nm) responses of H₂L upon addition of various metal ions in HEPES buffer (10 mM, pH 7.2) at 25 °C in methanol/water (v/v, 1 : 1) and 8 nm emission slit.

Therefore, the probe recognises Zn²⁺ selectively in the presence of other metal ions (ESI, Fig. S10†), and intensity also enhances with increasing concentration of Zn²⁺ (Fig. 3). The quantum yield of the probe (Φ_H2L, 0.0012) is about forty times lower than that of the complex, [ZnL] (Φ_ZnL, 0.052). The binding constant (K_d, 6.49 × 10⁴ M⁻¹ L) has been determined by fitting fluorescence data as a function of metal ion concentration to a suitable computer-fit nonlinear program (ESI, Fig. S9†). The limit of detection (LOD) of Zn²⁺ has been determined by 3σ/m method and found to be as low as 0.068 μM (ESI, Fig. S11†). The Joint FAO/WHO Expert Committee on Food Additives (JECFA) has proposed a daily dietary requirement for zinc of 0.3 mg kg⁻¹ of body weight and a provisional maximum tolerable daily intake (PMTDI) of 1.0 mg kg⁻¹ of body weight.⁴¹

Fig. 3 Change in fluorescence intensity of H₂L upon addition of 50 μM Zn²⁺ ions in HEPES buffer (10 mM, pH 7.2) at 5 nm emission slit and 25 °C in methanol/water (v/v, 2 : 1).

The DFT-optimized structures of H₂L and [ZnL] have been used to calculate the bonding parameters (bond lengths and bond angles) that are comparable with similar structures of ligand and Zn(II) complexes.^42–46 The metric parameters of H₂L are C=N, 1.27845 Å; C=O, 1.22818 Å; C–O, 1.36650 Å; and C–S, 1.84921 Å; those of [ZnL] are C=N (1.31294, 1.30890 Å), C–O (1.27373, 1.27347 Å), Zn–N (2.09158, 2.08844 Å), Zn–O (2.00109, 1.99587 Å), Zn–S (2.77920, 2.80373 Å), etc., and ∠N–Zn–O, 89.11°; ∠O–Zn–O, 106.81°; ∠N–Zn–N, 173.03°; ∠S–Zn–O, 95.00°; ∠S–Zn–N, 88.75°; ∠S–Zn–S, 76.77°, etc. (ESI, Tables S1 and S2; Fig. S12†). We have not been able to collect diffraction-quality crystals of either probe, H₂L or its complex [ZnL]. On comparing with available structural reports, we have optimized the structures, and the acceptable theoretical model structures were used to generate best molecular functions.^42–46 The subtle change in the electronic properties of the ligand on complexation with Zn²⁺ was examined from the difference in the composition and energy of the MOs (ESI Tables S3–S6†). For brevity, H₂L is distinguished by two parts: coumarinyl (COUM) and phenylthioether (PTE), and MOs are composed of these two parts in different percentages. The HOMO (−5.57 eV) and HOMO−1 (−6.12 eV) have major contribution from PTE (92% and 97%, respectively), while HOMO−2 (−6.31 eV) contains 26% PTE and 74% COUM in the ligand (H₂L). LUMO to LUMO+4 have major COUM (65–89%), and other LUMOs (LUMO+5 and higher) show reverse composition. The calculated HOMO–LUMO energy gap 2.78 eV corresponds to intramolecular charge transfer, which may be assigned to 320 nm in the experimental data for H₂L. In [ZnL], the MOs (occupied and unoccupied) are composed mainly of ligand, >90%. However, the energy of MOs in the complex has been stabilized, such that HOMO in the complex is 0.12 eV lower than that of the probe, H₂L. The HOMO–LUMO energy gap of H₂L is increased from 2.78 eV to 2.83 eV in complex, which significantly supports the red shifting of the absorption band on coordination with Zn²⁺ (320 nm in free H₂L to 343 nm in [ZnL]). The calculated spectral transitions for [ZnL] are at 410, 400 and 341 nm, which are assigned to intraligand charge transfer transitions. The calculated transition in the absorption spectroscopy of [ZnL] are HOMO → LUMO+2 (S₀ → S₇) transition at 340 nm (f, 0.3482) and HOMO → LUMO+1 (S₀ → S₂) at 402 nm (f, 0.1472) (ESI, Table S7†), while the experimental transitions are at 343 and 410 nm, respectively.

Excited-state calculation was used to explain the spectral emission transitions. The fluorescence emission wavelength calculated with the optimized S₁ state geometry is the energy gap between the S₀ and S₁ state. The fluorescence wavelength was calculated as 482 nm (in MeOH), which is in very good agreement with the experimental value of 514 nm (ESI, Table S8†) (Fig. 4). The geometry relaxation upon photo excitation imparts a remarkable effect on the energy level of the molecular orbitals. In the case of [ZnL] complex, the LUMO is stabilized by 0.12 eV at the S₁ state geometry compared to that at S₀ state geometry, while the HOMO is destabilized by 1.48 eV for S₁ state geometry compared to that at S₀ state geometry. As a result, the energy difference between the HOMO and LUMO is greatly decreased at the S₁ state compared to that at the S₀ state, and this geometry relaxation is the main origin of the large wavelength shift.

Fig. 4 Correlation between HOMO and LUMO functions of H₂L and [ZnL].

The Zn-complex, [ZnL], is optimized at the T₁ state (Fig. 4), and the calculated emission energies, dominant configurations (with larger CI coefficients), transition nature, and the available experimental values are listed in ESI, Table S8.† The calculated emission wavelength for the complex of 543.46 nm also coincides with the experimental value for emission (555 nm).

We examined the reversibility effect of the [ZnL] complex with different anions such as PO₄³⁻, AMP, ATP, P₂O₇⁴⁻, H₂PO₄⁻, C₆H₅O₇³⁻, AsO₄³⁻, AsO₃³⁻, AsO₂⁻, CH₃COO⁻, F⁻, Cl⁻, Br⁻, I⁻, SCN⁻, S₂O₃²⁻, ClO₄⁻, SO₄²⁻, NO₂⁻, N₃⁻, etc. (Fig. 5). Among the aforesaid anions, only ATP has induced a conspicuous change in the absorptive and emissive behaviour of the complex. The UV-vis absorption response of [ZnL] towards ATP is shown in Fig. 6, and it is observed that the bands at 410 and 343 nm characteristic to the [ZnL] complex is decreased, while the band at 320 nm is recovered gradually upon addition of ATP, which is characteristic of the free ligand, H₂L (ESI, Fig. S13†). In the fluorescence spectrum, the band intensity at 514 nm corresponding to [ZnL] is decreased upon addition of ATP (Fig. 7), and the recovery of light emission of the sensor unambiguously suggests interaction of ATP with fluorogenic [ZnL].^12–16

Fig. 5 Fluorescence emission spectra of [ZnL] upon addition of different anions (5 equivalents).

Fig. 6 UV-vis spectra of the of [ZnL] upon addition of different anions (5 equivalents).

Fig. 7 Fluorescence titration spectra of the [ZnL] complex with gradual addition of ATP; non-linear plot of fluorescence intensity (at 514 nm) vs. [Zn²⁺] for the corresponding fluorescence titration (inset).

Fluorescence titrimetric measurement shows that 1 equivalent of ATP per [ZnL] (Fig. 7) is required for completion of the reaction. It is interesting to note that PO₄³⁻, HPO₄²⁻, AMP, H₂PO₄⁻, and P₂O₇⁴⁻ do not affect the emission characteristics of [ZnL] (Fig. 5). This [ZnL] sensor has shown excellent ATP detection, discriminating it from other inorganic and biological phosphates. The proposed sensor shows ATP sensing at very low concentration with LOD of 6.7 μM (ESI; Fig. S14†), which is the lowest in literature for fluorogenic Zn²⁺ complexes.^13–16 Because of solubility problem, the spectra are not very clear. The addition of Na₂ATP hydrogen-bonding interactions may effectively reduce the strength of bonding of phenolato-O and Zn(II), and the ligand is eliminated as proposed in ESI, Scheme S2.†⁴⁷ NMR shows that H₂L is recovered, as evident from the chemical shift position of imine-H in –CH=N–. The aromatic zone of the NMR spectrum of ZnL + ATP is difficult to assign, which may be due to protons from ATP; also, phenolic–OH is not visible (Fig. 8). From mass spectra, a peak at 670.73 is assigned to the ligand (M + Na)⁺, and 570.60 corresponds to [Zn-ATP], along with 593.56 to [Zn-ATP + Na] (ESI, Fig. S15†), which clearly prove that free ligand is produced by the reaction of [ZnL] and Na₂ATP.

Fig. 8 ¹H NMR spectra recorded in DMSO-d₆ of H₂L, [ZnL] and [ZnL] + ATP in DMSO-d₆ + D₂O.

3.4 Whole cell imaging

The fluorescence imaging of intracellular Zn²⁺ ion in whole cells of SCC084 (human oral carcinoma) has been examined by adding the probe, H₂L (Fig. 9). The cells were fixed in paraformaldehyde (4%) and blocked with BSA in PBS-Triton X100 solution. Then, the cells were observed under epifluorescence microscope. Then, the cells were incubated with Zn²⁺ solution (30 mM) for 45 min in buffer, washed again with buffer at pH 7.4, and mounted on a grease-free glass slide. Fluorescence microscope was used to observe the control cells. H₂L is easily permeable through tested living cells without any harm (after 30 min of exposure to H₂L at 2 mM). To evaluate cytotoxicity, MTT assay was done Fig. 10. There was no cytotoxicity for up to 200 μg mL⁻¹ H₂L. These results indicate that H₂L has a huge potential in both in vitro and in vivo as a Zn²⁺ sensor and in whole cell imaging.

Fig. 9 Fluorescence microscopy images of SCC084 (human oral carcinoma): (A) control (phase contrast); (B) control (bright field); (C) cells incubated with Zn²⁺ solution; (D) cells incubated with H₂L; (E) cells incubated with Zn²⁺ solution and H₂L.

Fig. 10 MTT assay for cell viability test of H₂L.

4. Conclusion

In this article, a coumarinyl thioether Schiff base, H₂L, a N₂O₂S₂ hexadentate probe, shows highly selective Zn²⁺ binding property with a limit of detection (LOD) of 0.068 μM. The absorption band of the H₂L probe at 320 nm shifts to 343 nm along with an additional band at 410 nm upon addition of the Zn²⁺ ion. The reaction of Zn²⁺ with H₂L may prohibit PET and ESIPT and induce rigidity in the resulting molecule, which reasonably produces a large chelation-enhanced fluorescence (CHEF) effect. ¹H NMR, mass spectral analysis and Job's plot have supported the 1 : 1 metal-to-ligand complex formation. The new fluorescent chemosensor has been used for imaging and sensing Zn²⁺ ions in cultured cells as evident from the study of SCC084 (human oral carcinoma) cells. The fluorogenic complex [ZnL] recognizes ATP in the presence of all other common anions, inorganic phosphates and biologically important phosphates (nucleosides, nucleotides).

Acknowledgements

Financial support from the West Bengal Department of Science & Technology, Kolkata, India (Sanction No. 228/1(10)/(Sanc.)/ST/P/S&T/9G-16/2012) and the Council of Scientific and Industrial Research (CSIR, Sanction No. 01(2731)/13/EMR-II), New Delhi, India are gratefully acknowledged. One of us (C. Patra) thanks University Grants Commission, New Delhi for the fellowship.

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Footnote

† Electronic supplementary information (ESI) available: The synthesis (Scheme S1), spectral data of H₂L (¹H NMR, Fig. S1; mass, Fig. S2; FT-IR, Fig. S3), spectral data of ZnL (¹H NMR, Fig. S4; mass, Fig. S5; FT-IR, Fig. S6), comparison of emission of H₂L and [ZnL] under illumination in UV chamber (Fig. S7), Job's plot (Fig. S8), Benesi-Hildebrand plot (Fig. S9), fluorescence intensity comparison plot of different metal ions (Fig. S10), limit of optical detection of zinc (LOD) (Fig. S11), DFT-optimized structures of H₂L and its Zn(II) complex (Fig. S12), absorption spectra of L–Zn complex upon addition of ATP (Fig. S13), LOD of ATP (Fig. S14), mass spectrum of [ZnL] + ATP (Fig. S15), calculated bond parameters of H₂L (Table S1) and [ZnL] (Table S2), composition of frontier molecular orbitals of H₂L (Table S3) and [ZnL] (Tables S4–S6), calculated spectral transitions for [ZnL] (Table S7), ground state and triplet excited state of [ZnL] in MeOH using DFT and TD-DFT computation of CPCM model (Table S8), and a schematic proposal of dechelation (Scheme S2) are included. See DOI: 10.1039/c6ra12369d

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