A new pyridoxal based fluorescence chemo-sensor for detection of Zn(II) and its application in bio imaging

Abstract

This paper describes the activity of a Schiff base ligand, derived from pyridoxal, as a promising fluorescence probe for biologically important Zn(II) ion sensing. A physiologically compatible pyridoxal based chemosensor PydDmen was synthesized and evaluated for its fluorescent response towards metal ions. Chemosensor PydDmen exhibits a selective turn-on type response in the presence of Zn²⁺ in ethanol–water mixture. The addition of EDTA quenches the fluorescence of receptor PydDmen-Zn²⁺, making the chemosensor PydDmen reversible. The response is specific for Zn(II) ions, and remains almost unaffected by the presence of alkali and alkaline earth metals but is suppressed to varying degrees by transition metal ions. The selectivity mechanism of PydDmen for Zn²⁺ is the combined effects of proton transfer between the prevailing tautomeric forms, C=N isomerization and CHEF. The DFT optimized structure of the complex is compatible with elemental analysis, mass spectrometry, FT-IR, electronic and NMR spectra. The experimental and theoretical support in terms of NMR spectroscopy and DFT are provided to establish the existence of Zn²⁺ induced transformation of PydDmen to a 3-pyridone tautomeric form.

---

Introduction

The zinc ion (Zn²⁺) is the second most abundant heavy metal ion in the human body and the cellular biochemistry of Zn²⁺ is diverse and far ranging.¹ On the other hand, misregulation of Zn²⁺ is also implicated in human health disorders. It is believed that a lack of zinc ions can result in an increased risk of several diseases such as those effecting stature, mental retardation and digestive dysfunction because the majority of biological zinc ions are tightly sequestered by proteins.² Additionally, the presence of excess “free zinc” in certain cells may be related to severe neurological disorders such as Alzheimer's and Parkinson's diseases.^3a,b Therefore, it is necessary to get an insight into the vital roles of Zn²⁺ in biological processes, resulting in the great demand regarding the design and development of efficient systems that can selectively and sensitively detect Zn²⁺ in living systems. Several analytical methods have played a vital role in the detection of Zn²⁺ including UV-Vis-spectroscopy,^3c potentiometry,^3d flame atomic absorption spectrometry.^3e However, its investigation can be facilitated by the use of fluorescent probes as fluorescence detection, in particular, is considered to be the most effective tool for sensing applications owing to the high sensitivity, easy visualization, short response time for detection and most importantly can be used for real time bio-imaging.⁴ Unfortunately, zinc ions are not intrinsically fluorescent, making direct quantitative detection a difficult task.^5,6 A variety of fluorescent sensors for Zn²⁺ have been reported based on various fluorophores.^7–9 Continuous effort has been dedicated improving the effectiveness of Zn²⁺ sensors.

We are mainly concerned with Schiff bases as probes because Schiff bases are suitable ligands for metal ions. Schiff bases are inherently non-/poorly fluorescent due to conventional modes of non-radiative decay pathways such as, isomerisation of C=N bond in the excited state and ESIPT involving phenolic proton.^10,11 Therefore, it is reasonable to expect that if we limit to simple Schiff bases as prospective probes for metal cations, the probable signaling pathways involve restriction of C=N isomerisation, ESIPT and CHEF (Scheme 1). But, Schiff base related probes can also coordinate strongly other physiologically available metal ions. Therefore, new strategies should be developed to improve Zn²⁺ selectivity of the probe. In order to make analyte binding more specific and favourable, it would be desirable if the binding pattern of the probe with the analyte is unique, i.e. the probe is transformed after binding to the analyte of choice. Such type of phenomena enhances the selectivity manifolds. Tautomerization is an efficient transformable factor in such cases.¹² Spring and co-workers have beautifully and efficiently exploited the imidic acid and amide tautomeric forms for selective binding with metal ions.¹² Till date, reports on tautomerism during analyte binding is relatively scarce in the literature (Chart 1†).¹³

Scheme 1 Probable signaling pathways of probe PydDmen.

In order to enhance the bio-compatibility in bioimaging, we have already started working with pyridoxal¹⁴ containing Schiff bases as chemosensors and reported a chemosensor that selectively detects Cu²⁺ ion.^14,15 The pyridoxal and its derivatives exhibit a wide range biological properties and have been used as substrates in many biological transformations.^16–21 In order to explore further this less ventured path, we herein constructed a novel turn-on sensor for Zn²⁺, which can exhibit emission at longer wavelength (483 nm). The 3-hydroxy pyridine moiety present in the probe permits 3-hydroxy pyridine–3-pyridone tautomeric equilibrium to exist.²² The spectroscopic behaviour of Schiff base and PydDmen [((2-(dimethylamino)ethylimino)methyl)-5-(hydroxymethyl)-2-methylpyridin-3-ol] shows that it is an excellent chemosensor for Zn²⁺ in ethanol–water and can be used for Zn²⁺ monitoring in living cells. The NMR spectroscopy and DFT study are utilized to establish the Zn²⁺ induced transformation of PydDmen to 3-pyridone tautomeric form in solution. To the best of our knowledge, this is the first report that establishes such type of tautomeric transformation as mechanistic rationale by means of ¹H NMR, ¹³C NMR and ¹³C DEPT methods.

Results and discussion

Synthesis and FTIR spectral characterization of PydDmen

The chemosensor PydDmen has been synthesized by condensing pyridoxal hydrochloride with N,N-dimethylethylenediamine under refluxing conditions in ethanol medium (Scheme 2) and the structure of PydDmen was well characterized by ¹H NMR and FTIR spectroscopy (Fig. S1†). The PydDmen-Zn²⁺ complex was characterized by recording FTIR and ESI-MS spectra (Fig. S2†).

Scheme 2 Preparation of chemosensor PydDmen.

FTIR spectra of PydDmen showed the characteristic band due to ν(C=N) at 1650 cm⁻¹. In the Zn²⁺ complex, the ν(C=N) absorption appears at lower energy (ca. 1631 cm⁻¹) indicating possible coordination to the metal center. The complexes also display broad band of medium intensity around 3400 cm⁻¹ attributable to the –OH stretching vibration of the –CH₂OH group of the pyridoxal part of the chemosensor PydDmen.²³

UV-Vis spectroscopic investigation of PydDmen

In order to ascertain the complexation of Zn²⁺ by PydDmen, absorption titrations were carried out by adding varied concentrations of Zn(NO₃)₂·6H₂O to a fixed concentration of PydDmen. Fig. 1 depicts spectrophotometric changes upon titrating a fixed concentration of PydDmen (5 × 10⁻⁵ M) with incremental additions of Zn(NO₃)₂·6H₂O (55 × 10⁻⁵ M) in EtOH/H₂O (4 : 1, v/v, 25 mM Tris buffer, pH 7.4). The absorption spectrum of PydDmen in same solvent displayed sharp absorption bands centered at 253 nm and 335 nm, which are assigned to the π–π* transitions. The absorption bands at 415 nm is attributed to the n–π* transitions of azomethine group.

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

However, addition of Zn²⁺ induced dramatic modification both in the maxima and shape of the said bands of PydDmen. The band at 253 nm is decreased slightly, accompanied by a blue shift to 246 nm. The band maxima at 335 nm is gradually decreased and a new band appeared at 295 nm. Another broad and moderately intense band around 390 nm could be assigned to O⁻ (phenolate) ↔ Zn²⁺ (LMCT or MLCT). These spectral changes, spanning the 240–390 cm⁻¹ region, are indicative of Zn²⁺ coordination induced perturbation of the –(OH)C=C–C=N– portion of the ligand during the course of the reaction.²⁴ Therefore, these absorption peaks were expected to correspond to coordination of PydDmen with Zn²⁺ generating the PydDmen-Zn²⁺ coordinated species. The accompanying isosbestic points at 268, 302, 356 and 435 nm clearly indicate that the transition between the free and the complexed species occurs and a stable complex resulted at a certain composition. The stoichiometry of the complex formed between PydDmen and Zn²⁺ is 1 : 1 based on Job's plot (Fig. S3†).

Binding behaviour analysed by fluorescence spectroscopy

The chemosensor PydDmen is poorly fluorescent in nature when excited at 411 nm in EtOH/H₂O (4 : 1, v/v, 25 mM Tris buffer, pH 7.4), which may be attributed to the combined effect of C=N isomerisation and ESIPT as commonly encountered in Schiff bases.¹⁰ The fluorescence sensing behaviour of PydDmen towards Zn²⁺ was investigated in buffer solution at physiological pH in EtOH/H₂O (4 : 1, v/v, 25 mM Tris buffer, pH 7.4) using a 5 × 10⁻⁶ M solution (Fig. 2). Zn(NO₃)₂·6H₂O was chosen as the representative Zn²⁺ species in the following experiment. Upon addition of 14 equivalents of Zn(NO₃)₂·6H₂O an enhancement in fluorescence spectrum was observed at 483 nm and maximum emissive wavelength shifts from 477 to 483 nm.

Fig. 2 Fluorescence emission changes of PydDmen (c = 5 × 10⁻⁶ M) upon addition of Zn²⁺ ions (c = 0–70 × 10⁻⁶ M) in EtOH/H₂O (4 : 1, v/v) in Tris buffer at pH 7.4 (λ_ex = 411 nm).

Under the same conditions, the selective sensing behaviour of PydDmen was validated using a variety of other metal ions in place of Zn²⁺, viz., Li⁺, Na⁺, K⁺, Sr²⁺, Ba²⁺, Ca²⁺, Cr³⁺, Mn²⁺, Fe²⁺, Co²⁺, Ni²⁺, Cu²⁺, Hg²⁺, Pb²⁺ and Al³⁺. But they do not show any significant change of fluorescence intensity of PydDmen, whereas Mg²⁺ and Cd²⁺ produce moderate enhancements (Fig. 3). From Fig. 3 it is clear that the Zn²⁺ ion gives rise to the largest fluorescence enhancement among these metal cations.

Fig. 3 Emission spectra of PydDmen (c = 5 × 10⁻⁶ M) in presence of Zn²⁺, Li⁺, Na⁺, K⁺, Ca²⁺, Sr²⁺, Al³⁺, Pb²⁺, Cr³⁺, Mn²⁺, Fe²⁺,Fe^3+, Co²⁺, Ni²⁺, Cu²⁺, Cd²⁺, Hg²⁺ and Mg²⁺(c = 70 × 10⁻⁶ M each metal ion) in EtOH/H₂O (4 : 1, v/v, 25 mM Tris buffer, pH 7.4) (λ_ex = 411 nm).

The competitive studies of PydDmen towards Zn²⁺ over other metal ions are carried out by adding 14 equivalents of Zn²⁺ to the solution of PydDmen (5 × 10⁻⁶ M) in the presence of 14 equivalents of other metal ions, viz., Li⁺, Na⁺, K⁺, Sr²⁺, Ba²⁺, Ca²⁺, Cr³⁺, Mn²⁺, Fe²⁺, Fe³⁺, Co²⁺, Ni²⁺, Cu²⁺, Hg²⁺, Pb²⁺, Al³⁺, Mg²⁺ and Cd²⁺. The competition experiments showed that the emission profile of the PydDmen-Zn²⁺ complex is more or less unaffected in the presence of other cations (Fig. S4†).

Titration of PydDmen with Zn²⁺ (concentration increased from 0–70 μM) revealed enhancement (up to 52 fold enhancement) of fluorescence intensity at 483 nm as a function of the added Zn²⁺ concentration (Fig. 2), suggesting a sensitive and selective recognition of Zn²⁺ by PydDmen. The chelation of Zn²⁺ by PydDmen occurs via N_amine/imine and O_alkoxo giving rigidity to the binding core. Therefore, C=N isomerisation as well as ESIPT is inhibited, reducing the non-radiative decay processes and increasing the possibility of fluorescence emission. This phenomenon results in chelation enhanced fluorescence (CHEF).

The linear relationship of the fluorescence titration showed that PydDmen responded to Zn²⁺ in 1 : 1 stoichiometry as evident from the Job's plot from absorption studies (Fig. S3†). The association constant for Zn²⁺ was estimated to be 1.18 × 10⁴ M⁻¹ by the linear Benesi–Hildebrand equation F₀/(F − F₀) = F₀/[ PydDmen] + F₀/[PydDmen] × K_a × [Zn²⁺]. F is the change in the fluorescence intensity at 483 nm, K_a is the association constant, and [PydDmen] and [Zn²⁺] are the concentration of PydDmen and Zn²⁺ respectively. By plotting F₀/(F − F₀) against the reciprocal of the concentration of Zn²⁺, the association constant value K_a is obtained from the ratio intercept/slope with a good linear correlation coefficient (R² = 0.998) (Fig. 4).

Fig. 4 Benesi–Hildebrand expression fitting of fluorescence titration curve of PydDmen (c = 5 × 10⁻⁶ M) upon addition of Zn²⁺ ions (c = 6, 10, 15, 20, 25, 30 and 35 × 10⁻⁶ M) in EtOH/H₂O (4 : 1, v/v) in Tris buffer at pH 7.4 (λ_ex = 411 nm).

Since many fluorescent probes are sensitive to pH, it is necessary to investigate the pH effect to find the optimal condition when the fluorescence measurements are to be carried out. From the pH dependence of fluorescence study (Fig. S5†), it was found that the fluorescence intensity of PydDmen at 483 nm in EtOH/H₂O remains unaffected at pH 7.4 which makes it suitable for application under physiological conditions. These results indicate that PydDmen can be used as a selective fluorescent probe to recognize and distinguish Zn²⁺ in the presence of various metal ions at pH 7.4.

We have also performed a reversibility experiment (Fig. S6†) which proved that the binding of Zn²⁺ to PydDmen is reversible which is the key requirement of an ideal biologically relevant chemosensor because binding of guest molecule must occur reversibly. In the presence of Na₂EDTA, a strong chelating ligand, due to its strong affinity towards Zn²⁺, decomposition of the PydDmen-Zn²⁺ entity takes place thereby reproducing non fluorescent PydDmen. As shown in Fig. S6† after the addition of Na₂EDTA, the emission intensity of the original ligand was gradually lost. This phenomena certainly gives a tacit support towards the reversible binding of PydDmen with Zn²⁺.

To determine the detection limit, following equation was used. DL = K × S_b/S where K = 2 or 3 (we take 3 in this case), S_b is the standard deviation of the blank solution and S is the slope of the calibration curve.²⁵ Here, the detection limit of PydDmen (Fig. S7†) as a chemosensor for Zn²⁺ was found to be 40.78 × 10⁻⁷ M which is sufficiently low for the detection of submillimolar concentrations of Zn²⁺ ions found in many chemical systems.²⁶

The fluorescence quantum yields (Φ_f)²⁷ of PydDmen and PydDmen-Zn²⁺ states were found to be 0.094 and 0.408 respectively (Table S1†). This substantial increase in the quantum yield of PydDmen in the presence of Zn²⁺ advocates its credibility as an efficient Zn²⁺ sensor.

We have also examined the anion independency of PydDmen by using Cl⁻, Br⁻, I⁻, NO₃⁻, CH₃COO⁻, ClO₄⁻ salts and the spectral output is represented in Fig. S8.†

Time resolved measurement

A picosecond time-resolved fluorescence technique has been used to examine the decay process of free sensor PydDmen and PydDmen-Zn²⁺ in EtOH/H₂O (4 : 1, v/v, 25 mM Tris buffer, pH 7.4, 298 K). According to the equations τ⁻¹ = k_r + k_nr and k_r = Φ_f/τ, the radiative decay rate constant k_r and the total nonradiative decay rate constant k_nr of PydDmen and Zn²⁺-bound species were calculated. The decay curve of the fluorescence intensity of PydDmen and fitting data were shown in Fig. 5 and Table S1.†

Fig. 5 Time resolved fluorescence decay of sensor PydDmen (red) and PydDmen-Zn²⁺ (green) and prompt (black) (λ_ex = 375 nm).

The decay curve and fitting data of PydDmen suggested that there were three main isomeric components of PydDmen to absorb light and emit fluorescence photons of lifetime at 0.821 ns, 3.375 ns and 9.334 ns. The average fluorescence lifetime (τ) of PydDmen was estimated as 3.79 ns. The radiative and nonradiative decay rate constants are calculated to be 2.48 × 10⁷ and 2.39 × 10⁸ s⁻¹ respectively indicating that the nonradiative decay is the predominant process in the excited states.²⁸ In the presence of 70 × 10⁻⁶ M Zn²⁺, the time-resolved fluorescence decay showed significant change, which indicated two components corresponding to PydDmen-Zn²⁺ at 9.078 ns and a small number of isomeric components of PydDmen at 1.479 ns. The lifetime is increased to 8.23 ns, which is longer than that of the free-PydDmen. The radiative and nonradiative decay rate constants changed to 4.96 × 10⁷ and 7.19 × 10⁷ s⁻¹ respectively. This result suggested that both the radiative and nonradiative decay processes became comparative resulting in a strong fluorescence.

¹H and ¹³C NMR titrations and mode of binding present in the 3-pyridone tautomeric form

In order to evaluate the binding mode of PydDmen with Zn²⁺, ¹H and ¹³C NMR titrations, and ¹³C-DEPT NMR experiment were performed by gradual addition of Zn(CH₃COO)₂·2H₂O to the DMSO-d₆ solution of PydDmen. ¹H NMR spectroscopy revealed significant differences as shown in Fig. 6 in the chemical shifts of PydDmen and PydDmen-Zn²⁺, which could be applied for establishing the structure of PydDmen-Zn²⁺. As presumed from the UV-Vis study (Scheme 3), the –(OH)C=C–C=N– region of PydDmen is severely perturbed after addition of Zn²⁺.

Fig. 6 ¹H NMR titration experiment of PydDmen with Zn²⁺.

Scheme 3 Zn²⁺ induced tautomerism in PydDmen (red color indicate the change in molecular fragment due to Zn²⁺ binding).

Therefore, it may be speculated that addition of Zn²⁺ induces a transformation to PydDmen. Again, it is well documented that 3-hydroxy pyridine-bearing moieties could undergo tautomerism producing the 3-pyridones.¹⁵ If this speculation is true, (Scheme 4), then, it is expected that H_a, H_b, H_d and H_e protons would be upfield shifted because of through bond propagation effect due to tautomerism. Given the greater distance from the perturbed zone, only small differences in the chemical shift values for H_e and H_b are expected. Due to conversion of N_imine to N_amine, H_g would undergo moderate upfield shift. For the H_h protons, downfield shift is expected as the NMe₂ lone pair is used up in the complexation process.

Scheme 4 Pathway of tautomerism in PydDmen.

Analysis of the ¹H NMR spectra after titration revealed that the speculated output were in good agreement with the experiment (Fig. 6). Upon addition of 0.5 equivalent of Zn²⁺ to a solution of PydDmen, the signals for H_a, H_d, H_b, H_g, H_h and H_e significantly broadened indicating incomplete complexation between PydDmen and Zn²⁺. After the addition of one equivalent of Zn²⁺, the resonances of H_a, H_b, H_d, H_e, H_g protons were found to be shielded relative to resonances for PydDmen indicating enhancement of electron density in the associated regions (Table S2†). Deprotonation of –CH₂OH group due to coordination to Zn²⁺, would accumulate negative charge on the oxygen atom resulting in an upfield shift for H_b. But, subsequent complexation process decreases electron density on H_b. Therefore, the extent of upfield shift is less for H_b. The same logic holds true for H_g protons also. The resonances of methylenic protons H_h, α to –NMe₂ fragment, were deshielded and show downfield shift from 2.47 ppm to 2.53 ppm. To understand the fate of the C₃–O₁H_f, a ¹³C NMR experiment was executed (Fig. 7). A new peak appeared at δ 174.0, which was disappeared in the ¹³C-DEPT experiment (Fig. 4). It confirms that C₃–O₁H_f was transformed to corresponding ketone. The ligand after complexation with zinc transformed to corresponding 3-pyridone–Zn²⁺ complex i.e. PydDmen-Zn²⁺ (Scheme 3). The imine bond was subsequently transformed to an amino exocyclic double bond (C₅), which appeared at δ 166.1. There were no further changes when more than one equivalent of Zn²⁺ were added, which was indicative of the 1 : 1 binding ratio between the sensor and Zn²⁺. These features are in accordance with the hypothesis that Zn²⁺-induced tautomerisation of the PydDmen occurs and coordinating environment of Zn²⁺ is composed of two N_amine and one O_alkoxo of the PydDmen. Moreover, the reaction mechanism was also confirmed by mass spectral analysis, and a peak at m/z 386.08 is assignable to the mass of [PydDmen − Me + Zn²⁺ + H₂O + CH₃COO⁻].

Fig. 7 (a) ¹³C NMR of PydDmen and PydDmen-Zn²⁺ in DMSO-d₆. (b) ¹³C DEPT spectral output after adding 1 equivalent of Zn²⁺ in PydDmen in DMSO-d₆.

To further reinforce the Schiff base transformation phenomena and mode of complexation between PydDmen and Zn²⁺, DFT calculations were carried out. Since attempts to isolate single crystals of PydDmen-Zn²⁺ suitable for X-ray diffraction analysis were unsuccessful, the optimized structure of the complex was computed by theoretical methods. The ground state structures of PydDmen and PydDmen-Zn²⁺ were optimized by density functional theory (DFT) as implemented in the Gaussian 09 (B3LYP/6-311G(d,p)) software package.²⁹ Full geometry optimizations were carried out at the UB3LYP level for PydDmen and PydDmen-Zn²⁺, which are shown in Fig. 8.

Fig. 8 B3LYP optimized structure of (a) PydDmen and (b) PydDmen-Zn²⁺.

In the optimized structure of PydDmen-Zn²⁺, it is clear that PydDmen is present in its pyridone tautomeric form and Zn²⁺ is coordinated by means of two N_amine atoms and one O_alkoxo atom. One nitrate anion and two water molecules complete the six coordination geometry around Zn²⁺. The optimized bond parameters are given in Table S3.† Energy of different molecular orbitals of PydDmen-Zn²⁺ were calculated and given in Table S4.† Contour plots of some selected molecular orbitals of PydDmen-Zn²⁺ are given in Fig. S9.† The spatial distributions of Highest Occupied Molecular Orbital (HOMO) and the Lowest Unoccupied Molecular Orbital (LUMO) of PydDmen and PydDmen-Zn²⁺ are presented in Fig. 9. The energy gaps between HOMO and LUMO in PydDmen and PydDmen-Zn²⁺ were 3.80 eV and 3.28 eV respectively. The UV-Vis absorption spectra of PydDmen-Zn²⁺ were calculated with electronic ground and excited states through time dependent density functional theory calculations (TDDFT) using conductor-like polarizable continuum model (CPCM) in ethanol. The calculated singlet–singlet vertical electronic transitions are summarized in Table S5.† The calculated electronic transitions are very close to the experimental electronic bands. All the transitions in PydDmen-Zn²⁺ have intra-ligand charge transfer (ILCT) origin.

Fig. 9 HOMO and LUMO distribution of PydDmen and PydDmen-Zn²⁺.

Biological implications of PydDmen

To demonstrate the potential application of PydDmen, the intracellular Zn²⁺ imaging behaviour of PydDmen was studied on A549, human lung cancer cell lines by fluorescence microscopy. After incubation with PydDmen (10 μM) at 37 °C for 10 min, the cells displayed no intracellular fluorescence (Fig. 10(B)). However, cells displayed light fluorescence with the addition of low concentration of zinc ions (1 μM) (Fig. 10(C)) and exhibited gradually intensive fluorescence when exogenous Zn²⁺ was introduced into the cell via incubation with a zinc nitrate salt solution (Fig. 10(D) and (F)). The intensive fluorescence behaviour was, however, strongly suppressed when TPEN (100 μM) was added to the medium. Since TPEN confers having a strong scavenging action on Zn²⁺ ions, the sensors were competitively inhibited to bind with Zn²⁺ ions, as a result, the intensive fluorescence disappeared (Fig. 10(H)). This presents the confirmatory evidence of the sensor having the selectivity to sense Zn²⁺ ions. The fluorescence responses of PydDmen with various concentrations of added Zn²⁺ proves that such fluorescence intensity can be used as indelible signature of selective sensor response clearly evident from the cellular imaging. Hence, these results indicate that PydDmen is an efficient candidate for monitoring changes in the intracellular Zn²⁺ concentration under biological conditions.

Fig. 10 (A) Phase contrast, (B) fluorescence image of A549 cells incubated with 10 μM PydDmen for 10 min at 37 °C. PydDmen (10 μM) incubated cells were washed with PBS and were exposed to the presence of sequentially increased concentrations of added extracellular Zn²⁺ ion as (C) 1 μM, (D) 10 μM, (E) 20 μM and (F) 50 μM. (G) Represent the merge image of phase contrast and fluorescence image. (H) Represent disappearance of fluorescence intensity in the A549 cells treated with the PydDmen and Zn²⁺ ion after further addition of 100 μM TPEN. For all imaging, the samples were excited at 410 nm.

The cytotoxicity study (MTT assay) in human lung cancer cells treated with various concentrations of PydDmen for up to 12 h as shown in Fig. 11 showed that PydDmen concentrations up to 10 μM did not show significant cytotoxic effects on human lung cancer cells for at least up to 12 h of its treatment. The study suggests that PydDmen can be readily used as an efficient, selective and sensitive tool for bioimaging at the indicated doses and incubation time without cytotoxic effects. Thus the intensity based Zn-sensors can offer promising potential to probe physiological and biochemical consequences of metal dynamics with wide metabolic spectrum in cellular environment with appreciable fidelity.

Fig. 11 % Cell viability of A549 cells treated with different concentrations (1–100 μM) of PydDmen for 12 h determined by MTT assay. Results are expressed as mean ± S.D of three independent experiments.

Conclusion

In conclusion, we have synthesized and characterized a new pyridoxal containing Schiff base turn-on Zn²⁺ probe, PydDmen. The poorly fluorescent probe responds giving a strong selective, fast and specific fluorescence signal in presence of Zn²⁺ ions, i.e. there is a zinc triggered fluorescence switching. 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, ESI-MS, and NMR studies, which show formation of an 1 : 1 PydDmen-Zn²⁺ complex. The chelating agent EDTA can switch off the fluorescence signal by coordinating with the zinc ion, releasing the chemosensor to the solution. ¹H, ¹³C NMR and DEPT analysis indicates Zn²⁺ induced transformation of the chemosensor to 3-pyridone tautomeric form. The DFT/TDDFT calculation was carried out to demonstrate the electronic properties of the chemosensor and PydDmen-Zn²⁺ and it supports the prevailing 3-pyridone tautomeric form. Furthermore, we have demonstrated that the probe is applicable for Zn²⁺ imaging in the living cells. We believe that the development of a new turn-on Zn²⁺ probe, its outstanding fluorescence enhancement, spectroscopic and DFT studies, and cell imaging will find considerable application in the chemical science and its allied branches.

Experimental section

General information and materials

All reagents were purchased from Sigma-Aldrich and used as received. Solvents were spectroscopic grade and used without purification. Elemental analyses (carbon, hydrogen and nitrogen) were carried out with a Perkin-Elmer CHN analyzer 2400. The ¹H and ¹³C NMR spectra were measured on Bruker-300 MHz spectrometer. 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. Luminescence property was measured using LS-55 PerkinElmer 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 typical 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 χ² criteria (fitting analysis having χ² beyond the range 1.20 < χ² < 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:
in which a_i is the pre-exponential factor corresponding to the i^th decay time constant, τ_i.

Reagents for cell study

A549, human lung cancer cell lines were collected from National Center for Cell Science, Pune, India, and used throughout the experiments. Cells were grown in DMEM (Himedia) supplemented with 10% FBS (Himedia), and an antibiotic mixture (1%) containing PSN (Himedia) at 37 °C in a humidified incubator with 5% CO₂ and cells were grown to 80–90% confluence, harvested with 0.025% trypsin (Himedia) and in phosphate-buffered saline (PBS), plated at the desired cell concentration and allowed to grow overnight before any treatment.

Imaging system

Fluorescence images of A549 cells were taken by a fluorescence microscope (Model: LEICA DM4000B, Germany) with an objective lens of 20× magnification.

Cell culture

Cells were rinsed with PBS and incubated with DMEM containing PydDmen making the final concentration up to 10 μM in DMEM [the stock solution (3 mmol) was prepared by dissolving PydDmen into ethanol] for 10 min at 37 °C. After incubation, bright field and fluorescence images of A549 cells were taken by a fluorescence microscope (Model: LEICA DM4000B, Germany) with an objective lens of 20× magnification. Similarly, fluorescence images of A549 cells (pre-incubated with 10 μM PydDmen) were taken with addition of different concentrations (1–50 μM) of zinc nitrate salt at 10 minutes interval. A merged image between phase contrast and fluorescent images at 50 μM salt concentration were taken and consequently fluorescence images were taken after further addition of TPEN (100 μM).

Cell cytotoxicity assay

In order to test the cytotoxicity of PydDmen, 3-(4, 5-dimethylthiazol-2-yl)-2,S-diphenyltetrazolium bromide (MTT) assay was performed in A549 cells according to standard procedure.³⁰ Briefly, after treatment of overnight culture of A549 cells (10³ cells in each well of 96-well plate) with PydDmen (1, 10, 20, 50 and 100 μM) for 12 h, 10 μL of a MTT solution (1 mg mL⁻¹ in PBS) was added in each well and incubated at 37 °C continuously for 3 h. All media were removed from wells and 100 μL of acidic isopropyl alcohol was added into each well. The intracellular formazan crystals (blue-violet) formed 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 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.

Computational studies

All geometries for PydDmen and PydDmen-Zn²⁺ were optimized by density functional theory (DFT) calculations using Gaussian 09 (B3LYP/6-311G(d,p)) software package.³¹ The vibrational frequency calculations were performed to ensure that the optimized geometries represent the local minima and that there is only positive Eigen values. Vertical electronic excitations based on B3LYP optimized geometries were computed using the time-dependent density functional theory (TDDFT) formalism³² in ethanol using a conductor-like polarizable continuum model (CPCM).³³

Fluorimetric analysis

Fluorescence quantum yields (Φ) were estimated by integrating the area under the fluorescence curves with the following equation:

Φ_sample = (OD_standard/OD_sample) × (A_sample/A_standard) × Φ_standard

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).

Synthesis of the chemosensor PydDmen

The chemosensor molecule PydDmen was synthesized by following procedure. 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 N,N-dimethylethylenediamine (0.176 g, 2 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: 0.355 g, 0.75%). ¹H NMR (300 MHz, DMSO-d₆): δ 8.797 (s, H_a), 7.762 (s, H_d), 4.548 (s, H_b), 3.672 (t, H_g), 2.471 (t, H_h), 2.271 (s, H_e), 2.10 (s, H_i). Anal. calc. for C₁₂H₁₉N₃O₂: C, 60.74; H, 8.07; N, 17.71. Found: C, 59.97; H, 7.79; N, 17.05%.

Synthesis of PydDmen-Zn²⁺

Pyridoxal hydrochloride (0.203 g, 1 mmol) was dissolved in absolute ethanol. To it, ethanolic solution of Zn(NO₃)₂·2H₂O (0.297 g, 1 mmol) was added dropwise under stirring and the solution was stirred for 15 min. Then to this solution, N,N-dimethylethylenediamine (0.088 g, 1 mmol) was added slowly and the resulting yellowish-orange solution was stirred for 2 h. Then it was evaporated by rotary evaporator and resulting sticky mass was washed by cold ether and dried under vacuum (yield: 0.211 g, 0.53%). Anal. calc. for [Zn(PydDmen)(NO₃)(H₂O)₂]: C, 36.06; H, 5.55; N, 14.02. Found: C, 35.54; H, 5.14; N, 13.87%.

Acknowledgements

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) is gratefully acknowledged. Dr Guru Prasad Maiti, Research Associate, is grateful to DST-PURSE Programme at University of Kalyani for financial support to complete a part of this work. Dr Sushil Kumar Mandal is grateful to DST-PURSE PROGRAMME for partial financial support of the biological work. The authors gratefully acknowledge Saikat Khamarui and Deborin Ghosh for helpful discussions.

References

1. (a) A. I. Bush, W. H. Pettingell, G. Multhaup, M. Paradis, J. P. Vonsattel, J. F. Gusella, K. Beyreuther, C. L. Masters and R. E. Tanzi, Science, 1994, 265, 1464–1465; (b) C. J. Frederickson, J. Y. Koh and A. I. Bush, Nat. Rev. Neurosci., 2005, 6, 449–452; (c) D. D. Mott, M. Benveniste and R. J. Dingledine, J. Neurosci., 2008, 28, 1659–1671; (d) J. M. Berg and Y. Shi, Science, 1996, 271, 1081–1085.
2. A. Krężel and W. Maret, J. Biol. Inorg. Chem., 2006, 11, 1049–1062.
3. (a) A. I. Bush, Trends Neurosci., 2003, 26, 207–214; (b) D. Noy, I. Solomonov, O. Sinkevich, T. Arad, K. Kjaer and I. Sagi, J. Am. Chem. Soc., 2008, 130, 1376–1383; (c) C. V. Banks and R. E. Bisque, Anal. Chem., 1957, 29, 522–526; (d) A. R. Fakhari, M. Shamsipur and K. H. Ghanbari, Anal. Chim. Acta, 2002, 460, 177–183; (e) Q. Li, X. H. Zhao, Q. Z. Lv and G. G. Liu, Sep. Purif. Technol., 2007, 55, 76–81.
4. (a) R. Y. Tsien, Fluorescent and Photochemical Probes of Dynamic Biochemical Signals inside Living Cells, ed. A. W. Czarnik, American Chemical Society, Washington, DC, 1993, pp. 130–146; (b) Y. Xiang, A. J. Tong, P. Y. Jin and Y. Ju, Org. Lett., 2006, 8, 2863–2866.
5. (a) K. Kikuchi, K. Komatsu and T. Nagano, Curr. Opin. Chem. Biol., 2004, 8, 182–191; (b) E. L. Que, D. W. Domaille and C. J. Chang, Chem. Rev., 2008, 108, 1517–1549; (c) N. C. Lim, H. C. Freake and C. Bruckner, Chem.–Eur. J., 2005, 11, 38–49; (d) Z. C. Xu, J. Y. Yoon and D. R. Spring, Chem. Soc. Rev., 2010, 39, 1996–2006.
6. (a) Y. H. Lau, P. J. Rutledge, M. Watkinson and M. H. Todd, Chem. Soc. Rev., 2011, 40, 2848–2866; (b) D. Maity and T. Govindaraju, Chem. Commun., 2010, 46, 4499–4501; (c) K. Jobe, C. H. Brennan, M. Motevalli, S. M. Goldup and M. Wakinson, Chem. Commun., 2011, 47, 6036–6038; (d) T. L. Mindt, H. Struthers, L. Brans, T. Anguelov, C. Schweinsberg, V. Maes, D. Tourwe and B. Schibli, J. Am. Chem. Soc., 2006, 128, 15096–15097; (e) C. Ornelas, J. R. Aranzaes, E. Cloutet, S. Alves and D. Astruc, Angew. Chem., Int. Ed., 2007, 46, 872–877; (f) K.-C. Chang, I.-H. Su, A. Senthilvelan and W.-S. Chung, Org. Lett., 2007, 9, 3363–3366; (g) B. Colasson, M. Save, P. Milko, J. Roithova, D. Schroder and O. Reinaud, Org. Lett., 2007, 9, 4987–4990; (h) S. Huang, R. J. Clark and L. Zhu, Org. Lett., 2007, 9, 4999–5002; (i) K.-C. Chang, I.-H. Su, G.-H. Lee and W.-S. Chung, Tetrahedron Lett., 2007, 48, 7274–7278; (j) R. M. Meudtner, M. Ostermeier, R. Goddard, C. Limberg and S. Hecht, Chem.–Eur. J., 2007, 13, 9834–9840; (k) S. Y. Park, J. H. Yoon, C. S. Hong, R. Souane, J. S. Kim, S. E. Matthews and J. Vicens, J. Org. Chem., 2008, 73, 8212–8218; (l) D. Schweinfurth, K. I. Hardcastle and U. H. F. Bunz, Chem. Commun., 2008, 2203–2205; (m) M. Juricek, P. H. J. Kouwer, J. Rehak, J. Sly and A. E. Rowan, J. Org. Chem., 2009, 74, 21–25; (n) J. Camponovo, J. Ruiz, E. Cloutet and D. Astruc, Chem.–Eur. J., 2009, 15, 2990–3002; (o) R. K. Pathak, V. K. Hinge, M. Mondal and C. P. Rao, J. Org. Chem., 2011, 76, 10039–10049.
7. (a) K. K. Upadhyay, A. Kumar, J. Zhao and R. K. Mishra, Talanta, 2010, 81, 714–721; (b) J. F. Zhang, S. Kim, J. H. Han, S. J. Lee and J. S. Kim, Org. Lett., 2011, 13, 5294–5297; (c) Y. Xu, J. Meng, L. X. Meng, Y. Dong, Y. X. Cheng and C. J. Zhu, Chem.–Eur. J., 2010, 16, 12898–12903; (d) Q. H. You, P. S. Chan, W. H. Chan, N. K. Mak and R. N. S. Wong, RSC Adv., 2012, 2, 11078–11083; (e) H. Y. Lin, P. Y. Cheng, C. F. Wan and A. T. Wu, Analyst, 2012, 137, 4415–4417.
8. (a) S. Comby, S. A. Tuck, L. K. Truman, O. Kotova and T. Gunnlaugsson, Inorg. Chem., 2012, 51, 10158–10168; (b) J. Jia, Q. C. Xu, R. C. Li, X. Tang, Y. F. He, M. Y. Zhang, Y. Zhang and G. W. Xing, Org. Biomol. Chem., 2012, 10, 6279–6286; (c) X. Meng, S. Wang, Y. Li, M. Zhu and Q. Guo, Chem. Commun., 2012, 48, 4196–4198; (d) T. Mukherjee, J. C. Pessoa, A. Kumar and A. R. Sarkar, Dalton Trans., 2012, 5260–5271; (e) S. H. Mashraqui, R. Betkar, S. Ghorpade, S. Tripathi and S. Britto, Sens. Actuators, B, 2012, 174, 299–305.
9. (a) G. Mandal, M. Darragh, Y. A. Wang and C. D. Heyes, Chem. Commun., 2013, 49, 624–626; (b) G. Sivaraman, T. Anand and D. Chellappa, Analyst, 2012, 137, 5881–5884; (c) L. J. Liang, S. J. Zhen, X. J. Zhao and C. Z. Huang, Analyst, 2012, 137, 5291–5294; (d) P. G. Sutariya, N. R. Modi, A. Pandya, B. K. Joshi, K. V. Joshi and S. K. Menon, Analyst, 2012, 137, 5491–5494; (e) Y. W. Choi, G. J. Park, Y. J. Na, H. Y. Jo, S. A. Lee, G. R. You and C. Kim, Sens. Actuators, B, 2014, 194, 343–352; (f) E. J. Song, H. Kim, I. H. Hwang, K. B. Kim, A. R. Kim, I. Noh and C. Kim, Sens. Actuators, B, 2014, 195, 36–43; (g) G. J. Park, H. Kim, J. J. Lee, Y. S. Kim, S. Y. Lee, S. Lee, I. Noh and C. Kim, Sens. Actuators, B, 2015, 215, 568–576; (h) J. J. Lee, S. A. Lee, H. Kim, L. T. Nguyen, I. Noh and C. Kim, RSC Adv., 2015, 5, 41905–41913; (i) A. K. Bhanja, C. Patra, S. Mondal, D. Ojha, D. Chattopadhyay and C. Sinha, RSC Adv., 2015, 5, 48997–49005; (j) C.-X. Yin, L.-J. Qu and F.-J. Huo, Chin. Chem. Lett., 2014, 25, 1230–1234.
10. J. Wu, W. Liu, X. Zhuang, F. Wang, P. Wang, S. Tao, X. Zhang, S. Wu and S. T. Lee, Org. Lett., 2007, 9, 33–36.
11. (a) X. Zhang, L. Guo, F. Y. Wu and Y. B. Jiang, Org. Lett., 2003, 5, 2667–2670; (b) M. Royzen, A. Durandin, V. G. Young, N. E.Geacintov and J. W. Canary, J. Am. Chem. Soc., 2006, 128, 3854–3855.
12. (a) Z. Xu, K.-H. Baek, H. N. Kim, J. Cui, X. Qian, D. R. Spring, I. Shin and J. Yoon, J. Am. Chem. Soc., 2010, 132, 601–610; (b) Z. Xu, X. Liu, J. Pan and D. R. Spring, Chem. Commun., 2012, 48, 4764–4766.
13. (a) V. Bhalla, Roopa and M. Kumar, Dalton Trans., 2013, 975–980; (b) V. Bhalla, Roopa and M. Kumar, Org. Lett., 2012, 14, 2802–2805; (c) A. Satheshkumar, E. H. El-Mossalamy, R. Manivannan, C. Parthiban, L. M. Al-Harbi, S. Kosa and K. P. Elango, Spectrochim. Acta, Part A, 2014, 128, 798–805; (d) M. J. Kim, K. Kaur, N. Singh and D. O. Jang, Tetrahedron, 2012, 68, 5429–5433; (e) K. Kaur, V. K. Bhardwaj, N. Kaur and N. Singh, Inorg. Chem. Commun., 2012, 26, 31–36; (f) B. Babur, N. Seferoğlu and Z. Seferoğlu, Tetrahedron Lett., 2015, 56, 2149–2154; (g) A. D. Dubonosov, V. I. Minkin, V. A. Bren, E. N. Shepelenko, A. V. Tsukanov, A. G. Starikov and G. S. Borodkin, Tetrahedron, 2008, 64, 3160–3167; (h) A. Misra and M. Shahid, J. Phys. Chem. C, 2010, 114, 16726–16739; (i) S. Mukherjee, A. K. Paul and H. S. Evans, Sens. Actuators, B, 2014, 202, 1190–1199; (j) A. Samanta, S. Dalapati and N. Guchhait, J. Photochem. Photobiol., A, 2012, 232, 64–72; (k) Z.-H. Pan, J.-W. Zhou and G.-G. Luo, Phys. Chem. Chem. Phys., 2014, 16, 16290–16301; (l) B. Liu, H. Wang, T. Wang, Y. Bao, F. Du, J. Tian, Q. Li and R. Bai, Chem. Commun., 2012, 48, 2867–2869.
14. S. Mandal, R. Modak and S. Goswami, J. Mol. Struct., 2013, 1037, 352–360.
15. T. Mukherjee, J. C. Pessoa, A. Kumar and A. R. Sarkar, Dalton Trans., 2012, 5260–5271.
16. A. C. Eliot and J. F. Kirsch, Annu. Rev. Biochem., 2004, 73, 383–415.
17. R. A. John, Biochim. Biophys. Acta, 1995, 1248, 81–96.
18. M. D. Toney, Arch. Biochem. Biophys., 2005, 433, 279–287.
19. P. Christen and D. E. Metzler, Transaminases, Wiley, New York, 1985.
20. H. Hayashi, H. Mizuguchi, I. Miyahara, M. M. Islam, H. Ikushiro, Y. Nakajima, K. Hirotsu and H. Kagamiyama, Biochim. Biophys. Acta, 2003, 1647, 103–109.
21. W. Liu, P. E. Peterson, J. A. Langston, X. Jin, X. Zhou, A. J. Fisher and M. D. Toney, Biochemistry, 2005, 44, 2982–2992.
22. O. K. Abou-Ziad and O. I. K. Al-Shini, Phys. Chem. Chem. Phys., 2009, 11, 5377–5383.
23. K. Nakamoto, Infrared Spectra of Inorganic Compounds, Wiley, New York, 1970.
24. L. Wang, W. Qin, X. Tang, W. Dou and W. Liu, J. Phys. Chem. A, 2011, 115, 1609–1616.
25. (a) L. Long, D. Zhang, X. Li, J. Zhang, C. Zhang and L. Zhou, Anal. Chim. Acta, 2013, 775, 100–105; (b) M. Zhu, M. Yuan, X. Liu, J. Xu, J. Lv, C. Huang, H. Liu, Y. Li, S. Wang and D. Zhu, Org. Lett., 2008, 10, 1481–1484; (c) C. Kar, M. D. Adhikari, A. Ramesh and G. Das, Inorg. Chem., 2013, 52, 743–752.
26. G. L. Long and J. D. Winefordner, Anal. Chem., 1983, 55, 712A–724A.
27. B. K. Paul, S. Kar and N. Guchhait, J. Photochem. Photobiol., A, 2011, 220, 153–163.
28. B. Valeur, Molecular Fluorescence: Principles and Applications, Wiley-VCH, Weinheim, Germany, 2002.
29. (a) A. D. Becke, J. Chem. Phys., 1993, 98, 5648–5652; (b) C. Lee, W. Yang and R. G. Parr, Phys. Rev. B: Condens. Matter Mater. Phys., 1988, 37, 785–789.
30. T. Mossman, J. Immunol. Methods, 1983, 65, 55–63.
31. M. J. Frisch, G. W. Trucks, H. B. Schlegel, G. E. Scuseria, M. A. Robb, J. R. Cheeseman, G. Scalmani, V. Barone, B. Mennucci, G. A. Petersson, H. Nakatsuji, M. Caricato, X. Li, H. P. Hratchian, A. F. Izmaylov, J. Bloino, G. Zheng, J. L. Sonnenberg, M. Hada, M. Ehara, K. Toyota, R. Fukuda, J. Hasegawa, M. Ishida, T. Nakajima, Y. Honda, O. Kitao, H. Nakai, T. Vreven, J. A. Montgomery Jr, J. E. Peralta, F. Ogliaro, M. Bearpark, J. J. Heyd, E. Brothers, K. N. Kudin, V. N. Staroverov, R. Kobayashi, J. Normand, K. Raghavachari, A. Rendell, J. C. Burant, S. S. Iyengar, J. Tomasi, M. Cossi, N. Rega, J. M. Millam, M. Klene, J. E. Knox, J. B. Cross, V. Bakken, C. Adamo, J. Jaramillo, R. Gomperts, R. E. Stratmann, O. Yazyev, A. J. Austin, R. Cammi, C. Pomelli, J. W. Ochterski, R. L. Martin, K. Morokuma, V. G. Zakrzewski, G. A. Voth, P. Salvador, J. J. Dannenberg, S. Dapprich, A. D. Daniels, Ö. Farkas, J. B. Foresman, J. V. Ortiz, J. Cioslowski and D. J. Fox, Gaussian 09, Revision D.01, Gaussian, Inc., Wallingford CT, 2009.
32. (a) R. Bauernschmitt and R. Ahlrichs, Chem. Phys. Lett., 1996, 256, 454–464; (b) R. E. Stratmann, G. E. Scuseria and M. J. Frisch, J. Chem. Phys., 1998, 109, 8218–8224; (c) M. E. Casida, C. Jamorski, K. C. Casida and D. R. Salahub, J. Chem. Phys., 1998, 108, 4439–4449.
33. (a) V. Barone and M. Cossi, J. Phys. Chem. A, 1998, 102, 1995–2001; (b) M. Cossi and V. Barone, J. Chem. Phys., 2001, 115, 4708–4717; (c) M. Cossi, N. Rega, G. Scalmani and V. Barone, J. Comput. Chem., 2003, 24, 669–681.

---

Footnote

† Electronic supplementary information (ESI) available: NMR & IR of the ligand, IR & ESI-MS of the complex, Job's plot, EDTA reversibility plot, detection limit plot, table of fluorescence lifetime of ligand and complex, tale of theoretical bond length and bond angle of complex, table of energy of selected molecular orbitals of complex, tables for calculated vertical electronic transitions of complex. See DOI: 10.1039/c5ra15353k

---