Aza-phenol Based Macrocyclic Probes Design for “CHEF-on” Multi Analytes Sensor: Crystal Structure Elucidation and Application in Biological Cell Imaging

Abstract

Metal bound macrocyclic compounds found in biological systems inspired us to design and synthesize two Robson-type macrocyclic Schiff-base chemosensors, H₂L1 (H₂L1=1,11-dimethyl-6,16-dithia-3,9,13,19-tetraaza-1,11(1,3)-dibenzenacycloicosaphane-2,9,12,19-tetraene-1,11-diol) and H₂L2 (H₂L2=1,11-dimethyl-6,16-dioxa-3,9,13,19-tetraaza-1,11(1,3)-dibenzenacycloicosaphane-2,9,12,19-tetraene-1,11-diol). Both the chemosensors have been characterized with different spectroscopic techniques. They act as multianalyte sensor and exhibit “turn-on” fluorescence toward different metal ions in 1X PBS (Phosphate Buffered Saline) solution. In presence of Zn²⁺, Al³⁺, Cr³⁺ and Fe³⁺ ions, H₂L1 exhibits ∼6-fold enhancement of emission intensity, while H₂L2 shows ∼6-fold enhancement of emission intensity in the presence of Zn²⁺, Al³⁺ and Cr³⁺ ions. The interaction between the different metal ion and chemosensor have been examined by absorption, emission, and ¹H NMR spectroscopy as well as by ESI-MS⁺ analysis. We have successfully isolated and solved the crystal structure of the complex [Zn(H₂L1)(NO₃)]NO₃ (1) by X-ray crystallography. The crystal structure of 1 shows 1:1 metal:ligand stoichiometry and helps to understand the observed PET-Off-CHEF-On sensing mechanism. LOD values of H₂L1 and H₂L2 toward metal ions are found to be ∼10^–8 and ∼10^–7 M, respectively. Large Stokes shifts of the probes against analytes (∼100 nm) make them a suitable candidate for biological cell imaging studies. Robson type phenol based macrocyclic fluorescence sensors are very scarce in the literature. Therefore, the tuning of structural parameters as the number and nature of donor atoms, their relative locations and presence of rigid aromatic groups can lead to the design of new chemosensors, which can accommodate different charged/neutral guest(s) inside its cavity. The study of the spectroscopic properties of this type of macrocyclic ligands and their complexes might open a new avenue of chemosensors.

Introduction

Metals are crucial for living systems as they are actively participating in many biological processes. Among them, alkali and alkaline earth metals like Na, K, Ca, and Mg play crucial roles in osmotic regulation, biomineralization, etc., whereas transition metals like Fe, Zn, Cu, Mn, Co, Ni, Mo, and V are important for catalysis, metabolism, and signaling processes.^1,2 Typically, transition metals are present at trace levels in biological systems,¹ and they act as cofactors in diverse enzymes like cytochrome oxidase, histidine ammonia-lyase, glutamate mutase, catalase, etc.,^3,2 due to their electronic structures and redox properties. On the contrary, overdose or deficiency of transition metals are responsible for acute and long-term diseases, including heart disease, cancer and neurodegeneration.⁴ Therefore, the assessment and understanding of the distribution of metal ions in living systems are fundamental for homeostasis and its related diseases.⁴ Among different techniques, fluorescence spectroscopy is recognized as a useful tool to sense biologically important metal ions and, thus, widely used in biology, physiology, pharmacology, and in environmental science also. In this respect, tailored molecular chemosensors can be highly specific and sensitive toward analyte molecules with fast response time.⁵ Chemosensors that can detect more than one analyte simultaneously have attracted great attention in recent years. This not only reduces production costs but also improves analysis time.⁶ However, the development of fluorescence chemosensors that can both recognize multiple analytes and differentiate them still remains a great challenge.⁷

In this context, we are mainly concentrating on chemosensor which can detect Zn²⁺, Al³⁺, Cr³⁺ and Fe³⁺ ions.⁸⁻¹⁶ Iron and zinc are the first and second most abundant transition metals present in human body.¹⁷ In many biochemical processes, both iron and zinc play essential role to complete the biocycle. Iron is indispensable for oxygen storage and transport at the cellular level.¹⁸ Whereas, zinc-containing active sites are present in many metalloenzymes such as carbonic anhydrase, carboxypeptidase A, alcohol dehydrogenase, RNA polymerase, etc.¹⁹ However, the imbalance of both ions is responsible of different neurological disorders as Alzheimer’s disease.²⁰ The presence of chromium decreases uptake of iron and thus reduces oxidative stress, heart disease, cancer, osteoporosis, and arthritis. The scarcity of chromium can raise the risk associated with diabetes and cardiovascular diseases.^21,22 Aluminum is one of the most used metals by our society. Its wide and multidimensional role in industry as well as in daily life provides some comfort, but excess accumulation in the human body causes several serious health issues as Alzheimer’s disease, amyotrophic lateral sclerosis, encephalopathy, Parkinson’s disease, etc.²³⁻²⁵ Fluorometric detection of these metal ions (Al³⁺, Zn²⁺) has been reported by several physicochemical “Turn-On” processes like photo induced electron transfer (PET), chelation induced enhanced fluorescence (CHEF), fluorescence resonance energy transfer (FRET), intermolecular charge transfer (ICT).²⁶⁻³⁰ On the other hand, Cr³⁺ and Fe³⁺, due to their paramagnetic nature of, can act as fluorescence quenchers rather than as activators.⁸⁻¹⁵

Generally, rhodamine, anthraquinone, BODIPY, salicylaldehyde, fluorescein and coumarin based chemosensors are used for trivalent cation sensing,³¹⁻³⁷ while for Zn²⁺ sensing chemosensors are based typically on di-2-picolylamine (DPA),³⁸⁻⁴¹ quinoline,⁴²⁻⁴⁷ bipyridyl,⁴³ etc. units. Therefore, simultaneous sensing of all these cations in a single platform is a challenging task. Several parameters such as avoiding multistep preparations of the probes, solubility in common solvents, high sensing ability, real sample analysis and biological applications (Chart S1, Supporting Information) need to be considered for the preparation of the chemosensors.

Nature chooses macrocyclic ligands for stability of variety of biological systems which include chlorophyll, hemoglobin and vitamin B12 etc. Macrocyclic Schiff-base ligands have wide applicability in the fields of analytical process, medicinal chemistry, supramolecular chemistry,^48a,48b biochemistry,^48c,48d organic synthesis, materials science,^48e recognition^48f catalysis,^48g etc. Synthetic macrocyclic complexes are much more thermally stable, more resistance toward degradation, chemically unreactive to acids and alkali in comparison with their metal-containing open-chain analogues. Metal coordination gives structural rigidity to the macrocyclic ligand so that the system can emulate active site structure of a metalloprotein.^48h In spite of this, it is important to mention that most of the reported chemosensors for metal ions are of acyclic nature and macrocyclic chemosensors are uncommon.⁴⁹

Cyclodextrin, pillar[n]arene, and metal–organic coordination macrocycles are some interesting macrocyclic compounds which exhibit interesting applications in the fields of molecular sensing, self-assembly, catalysis, molecular machines etc.⁵⁰⁻⁵³ Important structural parameters of Robson-type macrocyclic Schiff-bases⁵⁴ like number and nature of donor atoms, their relative locations, presence of rigid aromatic groups can be tuned to accommodate different charged or neutral guest(s) inside the cavity. O̅kawa and co-workers have made many modifications in the basic structure of phenol based macrocyclic ligands like using of different lateral chains, introduction of an additional donor atom on one lateral chain or made partial or full saturation of azomethine bond. Macrocycles with symmetrical lateral chains give hetero dinuclear Cu(II)M(II) or M(II)Cu(II) [M(II) = Mn, Co, Ni, Cu, Zn] compounds depending on the synthetic procedure and nature of donor sites.^55,56 A. E. Martell et al. have synthesized hydrogenated macrocyclic ligands to study their basicity and their coordination compounds are further studied for phosphate di ester catalytic process.⁵⁷ Some macrocyclic chemosensors are collected in Chart S2. Among them, most of the chemsonsors are selective toward single metal ion. G. Ambrosi et al. have prepared a fluorescent oxadiazole derivative which selectivity detect Zn(II) in alkaline medium. The synthesis involved multistep procedure and presence of alkaline medium restrict cell imaging study of the chemosensor.^58a B. Ghanbari and his group have reported two naphthalene based aza-crown macrocyclic ligands for selective detection of Al(III) ions in ethanol. The LOD values for Al(III) cation by the chemosensors were found to be ∼10^–9 M order.^58b A piperazine linked diimine phenol has been reported by S. Goswami and his group which selectively detect Zn(II) in mixed aquous medium. The Zn-chemosensor complex has been utilized as a receptor for dihydrogen phosphate (DHP) sensing in aqueous medium by metal displacement approach resulting quenching of fluorescence intensity and generating the free chemosensor. The chemosensor further used for sensing of Zn²⁺ and DHP in living cells.^58c Another aza-crown macrocyclic chemosensor, namely, 1-Hydroxy-diaza-15-Crown-4, was used by C. Sinha and co-workers for chromogenic sensing of Al³⁺ in DMSO-water medium. The probe also exhibits fluorescent turn on in the presence of Al³⁺ and Zn²⁺ ions. Furthermore, fluorescence cell imaging study of the probe had been performed in SCC084 (Human Oral carcinoma) cell lines.^58d P. K. Panda and his group have designed a novel meso-expanded calix[4]pyrrolemacrocycle, where a rigid o-phenylene unit is incorporated as a spacer between the α,α′-positions of the tetrapyrrane moiety. It belongs to its smallest expanded analogue. Its selectivity detects fluoride ion via a turn-on response.^58e R. Azadbakh et al. have developed a novel fluorescent nanochemosensor starting from a reduced macrocyclic Schiff Base ligand. It acts as a highly selective and sensitive sensor toward Fe(III) and I^– through a fluorescence “‘on–off”’ process, with a limit of detection in nM range.^58f D. Das et al. have found that an acyclic phenol-based Schiff base compartmental ligand, N,N′-propylene-bis(3-formyl-5-tert-butylsalicylaldimine) upon reaction with Zn(II) produced a dinuclear Zn(II)-cyclic compound. The acyclic compound containing C=N bonds undergone hydrolysis in the presence of Lewis acid ZnCl₂ in organic-aqua medium. Free ligand exhibits yellowish green fluorescence emission at 523 nm when excited at 437 nm in 1:1 water– acetonitrile. In the presence of Zn²⁺, a new fluorescence emission band appears at 481 nm and the intensity enhanced gradually. Thus, the acyclic compound acts as a ratiometric fluorescence chemodosimeter for the selective detection of Zn(II) ions.^58g V. Fusi et al. have synthesized a cyclophane macrocyclic probe containing 1,3-bis(benzo[d]oxazol-2-yl)phenyl fluorophore as sensing unit. It acts as a PET-mediated fluorescent chemosensor, at acidic medium. The macrocyclic probe signals the presence of Zn(II) and Cd(II) metal ions via fluorescence enhancement in a mixed acetonitrile–aqueous medium at physiological pH. Furthermore, experimental and theoretical studies suggested the formation of both mononuclear and dinuclear Zn²⁺-probe complexes, while only the mononuclear complex was found in the case of Cd²⁺.^58h P. Paul and his group have designed a fluorescent compound involving a Re(I)-bipyridine moiety as a fluorogenic unit and amide-incorporated modified calix[4]arene as recognition moiety. It behaves as a dual chemosensor which selectively enables turn-on fluorescence in the presence of Hg²⁺ and naked eye colorimetric detection of Cu²⁺ among different metal ions.⁵⁸ⁱ

Here, we report two aza-phenol based macrocyclic Schiff base ligands, H₂L1 [H₂L1=1,11-dimethyl-6,16-dithia-3,9,13,19-tetraaza-1,11(1,3)-dibenzenacycloicosaphane-2,9,12,19-tetraene-1,11-diol] and H₂L2 as multi analyte chemosensors which fluorimetrically detects different cations (H₂L1: Zn²⁺, Al³⁺, Cr³⁺ and Fe³⁺ and H₂L2: Zn²⁺, Al³⁺ and Cr³⁺) in 1X PBS solution. The interaction of the metal ions with our chemosensors have been examined by absorption, emission and ¹H NMR spectroscopies as well as by elemental and ESI-MS⁺ analysis. The binding mode of Zn²⁺ with H₂L1 (complex 1) has been established by X-ray crystallography. Both the chemosensors exhibit ∼6-fold enhancement of emission intensity in the presence of different metal ions. The LOD values of the probes, H₂L1 and H₂L2 toward these cations are found to be ∼10^–8 and ∼10^–7 M, respectively. Bioimaging studies of H₂L1 and H₂L2 using HepG2 cells along with MTT assays were also performed. The use of aza-phenol based macrocyclic chemosensors for multiple-analyte detection is a unique attempt. The judicious choice of donor centers and ligand topologies results in selective coordination of selected metal ions with high binding constants. The study of the spectroscopic properties of such a type of macrocyclic ligands and of their metal complexes may open new avenues for obtaining multianalyte sensors.

Results and Discussion

Synthesis and Characterization

2,6-Diformyl-4-methylphenol (DFP) has been prepared following a standard procedure.⁵⁹ 2,2′-Thiobisethylamine or hydrochloride salt of 2,2′-oxybis(ethan-1-amine) was mixed with DFP in 1:1 molar ratio in a chloroform–methanolic (1:9, v/v) solution under reflux (Scheme S1, Supporting Information) to generate the Schiff base ligands H₂L1 and H₂L2 without further purification. H₂L1 and H₂L2 were thoroughly characterized using different spectroscopic methods (UV–vis, FT-IR, ¹H and ¹³C NMR) and by elemental analysis. In the ESI-MS⁺ analysis, the base peak was found at m/z = 497.18 and 465.23, corresponding to [H₂L1+H]⁺ and [H₂L2+H]⁺, respectively (Figure S1 and Figure S2, Supporting Information). In the FT-IR spectra of the chemosensors a broad band at around ∼3300 cm^–1 indicates the presence of the phenolic–OH groups and the band at ∼1640 cm^–1 is attributed to the C=N (for azomethine) stretching frequency (Figure S3).

H₂L1 and H₂L2 reacts with respective metal ions (H₂L1: Zn²⁺, Al³⁺, Cr³⁺ and Fe³⁺; H₂L2: Zn²⁺, Al³⁺ and Cr³⁺) in 1:1 ratio in methanol to produce the complexes 1–7, respectively (Scheme 1). Complexes were characterized by different spectroscopic techniques, elemental and ESI-MS⁺ analyses. In the FT-IR spectra of all seven complexes, 1–7 show characteristic stretching frequencies at ∼1640 cm^–1 υ(C=N), ∼770 cm^–1 υ(C–H) and ∼1340 cm^–1 (υ(NO₃^®, asymmetric stretch) (Figures S4 and S5).

Scheme 1. Route to the Synthesis of Complexes 1–7.

Crystal Structure Description of [Zn(H₂L1)(NO₃)]NO₃ (1)

We have successfully crystallized [Zn(H₂L1)(NO₃)]NO₃ (1) form slow evaporation of a methanolic solution of zinc nitrate hexahydrate and H₂L1. Crystals of 1 are present in triclinic form with P-1 space group (Table S1, Supporting Information). The crystal structural is shown in Figure 1. Selected bond distances and angles are collected in Table S2. The asymmetric unit consists of the whole molecule, the Zn²⁺ bound chemosensor and two nitrate ions, one bound with the metal ion in the first coordination sphere and the other one acting as a counteranion. In 1, the Zn²⁺ center adopts a distorted octahedral geometry in which the imine nitrogens (N1, N3) are placed in axial positions, while the phenoxido oxygen atoms (O1, O2) and one nitrate anion, coordinated in a bidentate fashion (O6, O7), occupy the equatorial plane. Interestingly, the remaining uncoordinated imine nitrogens (N1, N3) are present in protonated form. Sulfur atoms (S1, S2) also remain uncoordinated. The Zn–N_imino and Zn–O_phenoxido bond distances are 2.096(4)Å (Zn–N1), 2.094(4)Å (Zn–N3), 2.025(3)Å (Zn–O1), and 2.004(3)Å (Zn–O2), respectively. The other Zn–O distances are 2.362(4) Å (Zn–O6) and 2.363(4)Å (Zn–O7), respectively. The equatorial angles vary from 54.01(12)° to 112.54(12)°. The axial N1–Zn–N3 angle is 165.64(14)°. Complex 1 is further stabilized by different supramolecular interactions including π···π (3.792 Å), C–H···π (3.692 Å) and chalcogen interaction (3.206 Å) to form a one-dimensional chain along the “a” axis (Figure S6).

Figure 1.

Crystal structure of the asymmetric unit of 1. Atoms are shown as 30% thermal ellipsoids. H atoms are omitted for clarity.

Importantly, the crystal structure proves the PET-off CHEF-on fluorescence sensing. It also supports the experimentally observed m/z value of 279.96, which corresponds to the molecular ion peak of [Zn(H₂L1)]²⁺ (Figure S7).

NMR Studies

¹H and ¹³C NMR of H₂L1, H₂L2 and of complexes 1, 2, 5, and 6 were recorded in DMSO-d₆ solvent. In ¹H NMR of H₂L1, the imine (H–C=N) protons give a peak at 8.48 ppm. The aromatic protons appear at 7.35 ppm, while the aliphatic protons appear at 3.74 and 2.87 ppm, respectively. Methyl protons are found at 2.19 ppm (Figure S8). Aromatic OH protons give a broad signal at 13.98 ppm. In the ¹³C NMR (Figure S9) the imine carbon atoms appear at 167.10 and 161.94 ppm whereas aromatic carbon atoms appear in the range 139.93–119.42 ppm. Aliphatic carbon atoms appear in the range 55.61–32.38 ppm and methyl carbon atoms appear at 20.34 and 20.06 ppm. Similar type of ¹H and ¹³C NMR spectral pattern were found for H₂L2 also (Figures S10 and S11)

Interaction of Zn²⁺ and Al³⁺ with H₂L1 is well explained with the help of NMR spectroscopy. In the ¹H NMR of 1 (Figure S12), the disappearance of phenolic −OH peaks prove the binding of the phenoxido oxygens to Zn²⁺. The signal of the imine protons splits into two signals at 8.68 and 8.40 ppm, indicating coordinated and uncoordinated imine nitrogens. Aromatic protons appear downfield at 7.78 and 7.53 ppm. In the ¹³C NMR spectrum of 1 (Figure S13), the signals of the imine carbons appear at 170.94 and 169.96 ppm, while the aromatic carbon atoms appear in the range of 147.62–116.55 ppm. Aliphatic carbon atoms are observed in the range of 60.50–30.62 ppm.

In the ¹H NMR of 2 (Figure S14), the disappearances of the phenolic −OH peaks support the binding of the phenoxido oxygens to Al³⁺. The imine protons appear downfield shifted at 8.76 and 8.61 ppm. Both aromatic and aliphatic protons are also undergoing broadening and appear at 7.86, 7.70, 3.95, and 3.00 ppm, respectively. In the ¹³C NMR spectrum of 2 (Figure S15), imine and aromatic carbons appear in the ranges 168.98–167.12 ppm and 146.40–117.30 ppm, respectively. Aliphatic carbon atoms are shown in the range 52.00–28.14 ppm. ¹H and ¹³C NMR spectral data of complexes 5 and 6 are collected in Experimental Section (Figures S16–S19).

We have also performed ¹H NMR titration experiments of H₂L1 and H₂L2 with Zn²⁺ or Al³⁺ in DMSO-d₆ solvent (Figures S20–S23). Upon gradual addition of (0 to 1 equiv) metal ions to one equivalent of chemosensor, disappearance of phenolic −OH protons, significant splitting and downfield shift of imine protons, broadening and splitting of aromatic and aliphatic protons prove strong interaction between chemosensor and corresponding metal ions. The X-ray crystallographic data of complex 1 agrees with the ¹H NMR titration data.

Absorption Spectral Studies

The UV–vis absorption spectrum of both H₂L1 and H₂L2 were studied in 1X PBS solution (pH 7.4). The same buffer medium was chosen for both spectroscopic studies and biological work in order to avoid experimental errors. Characteristic bands around 435 and 265 nm suggest n → π* and π →π* type of transitions within the probes. The gradual addition of a Zn²⁺ (0–20 μM) solution to a 20 μM H₂L1 solution results in a decrease in the intensity of the peak at 435 nm and a concomitant increase in absorbance at around 265 nm (Figure 2). Both the signals at 435 nm and at 265 nm do not further change after the addition of one equivalent of Zn²⁺, suggesting 1:1 binding stoichiometry. A similar situation was observed for the trivalent cations, Al³⁺, Cr³⁺ and Fe³⁺ (Figure 2). In the case of chemosensor H₂L2, upon gradual addition of metal ion (Zn²⁺, Al³⁺ and Cr³⁺ ; 0–20 μM), there is an enhancement of intensity at 440 nm (Figure S24).

Figure 2.

Absorbance titration of H₂L1 (20 μM) with gradual addition of (a) Zn²⁺ (0–20 μM); (b) Al³⁺(0–20 μM); (c) Cr³⁺(0–20 μM), and (d) Fe³⁺(0–20 μM) in 1X PBS solution.

Fluorescence Properties

The fluorescence spectra were recorded in 1X PBS solution at ambient conditions. Upon excitation at 435 nm, H₂L1 exhibits weak fluorescence at 505 nm, probably due to a PET (Photo Induced Electron Transfer) process. Photoinduced intramolecular electron transfer (PET) from the HOMO of the donor imine nitrogen atoms to the excited fluorophoric moiety results in excited state quenching. Addition of Zn²⁺ (0–22 μM, excitation wavelength 435 nm) to a 20 μM solution of H₂L1 produces a gradual increase of the emission intensity and a red shift of the peak at 530 nm (Figure 3), which reaches 6-fold enhancement and a plateau for a ∼1:1 [Zn²⁺]/ [H₂L1] molar ratio (Figure 3). The 1:1 binding stoichiometry has been confirmed by Job’s plot analysis (Figure S25). Coordination of Zn²⁺ to H₂L1, increases the rigidity of the molecule via restriction of free rotation of H₂L1 around the H–C=N bond. Moreover the PET process could not continue further due to donation of the lone pair of electrons by the imine nitrogen. Both these effects are jointly responsible for the fluorescence enhancement (CHEF effect). The apparent binding constant for the 1:1 Zn²⁺/H₂L1 complex was calculated using the Benesi– Hildebrand equation:^60a

1

where K represents the metal to chemosensor binding constant. F_max, F₀, and F_x are fluorescence intensities in the presence of maximum [Zn²⁺] (1 equiv for 1:1 Zn²⁺: H₂L1), free ligand [H₂L1] (1 equiv) and any intermediate Zn²⁺ concentration (0–1.0 equiv), respectively. The binding constant (K) was estimated to be ∼1.67 × 10⁵ M^–1 (Figure S26, Table S3). The stability constant calculated with the program HypSpec ((1.22 ± 0.01) × 10⁵ M^–1) which permits the use of multiple wavelengths to make the fitting, is in good agreement with the value obtained.^60b,60c

Figure 3.

Fluorescence titration of H₂L1 (20 μM) with gradual addition of Zn²⁺ (0–22 μM) in 1X PBS solution and corresponding fluorescence intensity versus molar ratio plot (λ_exc = 435 nm).

Similarly, addition of the trivalent metal ions Al³⁺, Cr³⁺ and Fe³⁺ leads to a red-shift of the H₂L1 band at 505 nm that now appears at 530 nm and to a remarkable ca. 6-fold enhancement of the emission (Figures S27–S29). A plateau of the emission is reached in all cases for 1:1 [M³⁺]:[H₂L1] molar ratio. The 1:1 M³⁺: H₂L1 binding stoichiometry of these trivalent metal ions toward the chemo sensing probe H₂L1 was further confirmed by Job’s plot analysis (Figure S25). The selective response toward Zn²⁺, Al³⁺, Cr³⁺ and Fe³⁺ can be ascribed based on the Pearson principle which allows them to better fit into the macrocyclic coordination framework and coordinate with imine nitrogens and phenoxido oxygens. Again, paramagnetic metal ions like Cr³⁺ or Fe³⁺ are well-known fluorescence quenchers.^61a,61b Here, electron or energy transfer between the metal ions and fluorophores causes very fast and efficient nonradiative decay of the excited states resulting quenching of the fluorescence. An example of fluorescence enhancement in the presence of paramagnetic centers via chelation enhanced fluorescence (CHEF) mechanism is uncommon. Design of chemosensor with suitable multi dentate chelating units, which can effectively coordinate to the metal center resulting CHEF effect depends on the selection of ionophore.^61c−61e Turn-on fluorescent sensors for Cr³⁺ or Fe³⁺ ions are less reported due to the lack of selective ionophores.^61f−61h In presesence of chemosensor H₂L1 and H₂L2, Cr³⁺ (both H₂L1 and H₂L2) and Fe³⁺ (only H₂L1) form stable complexes resulting enhancement of the rigidity of the complex. The 6-fold fluorescence enhancement at 530 nm is a result of chelation-enhanced fluorescence (CHEF).⁶¹ⁱ Further, H₂L1 and H₂L2 are Schiff base ligands where C=N bond undergoes isomerization in the excited state resulting weak fluorescence. Coordination of metal ions with imine nitrogen inhibits C=N isomerization and decreased nonradiative decay of the excited-state, leading to fluorescence enhancement. In the present work, we observed an obvious enhancement of fluorescence intensity in the presence of Zn²⁺ and Al³⁺ as well as paramagnetic metal ions Cr³⁺ and Fe³⁺. Therefore, there is a competition between the fluorescence enhancement due to the inhibition of C=N isomerization and quenching of fluorescence from the metal ion-induced electron or energy transfer processes. Our results shows that the C=N isomerization plays a predominant role.^61j−61l

Interestingly, other metal ions fail to exhibit fluorescence enhancement probably due to several reasons such as the suitable coordination geometry, conformation of the Schiff base sensor, the appropriate ion radius and sufficient binding energy, leading to selective recognition of Zn²⁺, Al³⁺, Cr³⁺ and Fe³⁺. The apparent binding constants for trivalent metal bound chemosensor H₂L1 complexes were calculated using the Benesi–Hildebrand equation.^60a The apparent binding constant (K) values for Al³⁺, Cr³⁺ and Fe³⁺ were estimated to be 1.82 × 10⁵ M^–1, 1.66 × 10⁵ M^–1 and 1.26 × 10⁵ M^–1, respectively (Figures S30–S32, Table S3).

Selectivity of H₂L1 toward Zn²⁺, Al³⁺, Cr³⁺ and Fe³⁺ over other common competitive species was examined by fluorescence titration experiments in the presence of different alkali metals (Na⁺ and K⁺), alkaline-earth metals (Mg²⁺ and Ca²⁺) and various transition-metal ions (Mn²⁺, Fe²⁺, Co²⁺, Ni²⁺, Cu⁺, Cd²⁺ and Hg²⁺) (Figure 4). Upon addition of different common anions like S₂O₃^2–, S^2–, SO₃^2–, HSO₄^–, SO₄^2–, SCN^–, N₃^–, OCN^–, AsO₄^3–, PO₄^3–, ClO₄^–, AcO^–, Cl^–, NO₃^–, P₂O₇^4– (PPi), PF₆^–, F^– and some important bio molecules such as l-Histidine, l-Cysteine, ATP, Glutathione (Figure S33) in 1X PBS solution to the chemosensor no significant fluorescence enhancement was noticed.

Figure 4.

Relative fluorescence intensity profile of H₂L1 in the presence of different cations (1 equiv) in 1X PBS solution [Here, 1 = only H₂L1 (20 μM) and (2–18) = H₂L1 (20 μM) + Mⁿ⁺ (20 μM)].

The competition assay experiments were performed either individually for Zn²⁺ (1.0 equiv) and trivalent metal ions (Al³⁺, Cr³⁺ and Fe³⁺) (1.0 equiv) or in the presence of other metal ions (5.0 equiv) and common anions in 5-fold excess (5.0 equiv) in the same solvent system. Experimental results show that negligible enhancement or quenching occurs either in the presence of common cations (Figure 5, S34–S36) or common anions and some important bio molecules (Figures S37–S40).

Figure 5.

Relative fluorescence intensity profile of [H₂L1+Zn²⁺] system in the presence of different cations in 1X PBS solution [Here, H₂L1 (20 μM); Zn²⁺ (20 μM); other cations (100 μM)].

H₂L1 shows a distinct color change under UV light in the presence of Zn²⁺ and trivalent cations (Al³⁺, Cr³⁺ and Fe³⁺) in 1X PBS solution. H₂L1 shows greenish-yellow fluorescence in the presence of Zn²⁺ and trivalent cations (Al³⁺, Cr³⁺ and Fe³⁺) (Figure 6).

Figure 6.

Visual color changes of chemo sensors (H₂L1) in the presence of Zn²⁺, Al³⁺, Cr³⁺ and Fe³⁺ ions in 1X PBS solution. Here, A = H₂L1 (20 μM), B= H₂L1 (20 μM) + Zn²⁺ (20 μM), C-E = H₂L1 (20 μM) + M³⁺ (M = Al, Cr and Fe) (20 μM), respectively. The upper images were taken under normal light, and the lower images were taken under UV lamp.

Reversibility and regeneration of the free chemosensor are two important aspects for real time applications. In this experiment, the sodium salt of ethylenediaminetetraacetic acid (Na₂EDTA) is used as strong chelating ligand. After the addition of 1 equiv of Na₂EDTA to a solution of Zn²⁺ and H₂L1, the fluorescence changes from greenish-yellow to colorless with obvious decrease in fluorescence intensity, confirming the regeneration of the free probe. Similarly, in the presence of either Al³⁺ or Cr³⁺ or Fe³⁺ and H₂L1, addition of 1 equiv of Na₂EDTA results in a change of the fluorescence from greenish-yellow to colorless indicating again regeneration of the free probe (Figure S41).

The limit of detection (LOD) is an important parameter of chemosensor H₂L1 for real sample analysis. LOD of the chemosensor toward Zn²⁺, Al³⁺, Cr³⁺ and Fe³⁺ were calculated using the 3σ method.⁶² The LODs for the Zn²⁺, Al³⁺, Cr³⁺ and Fe³⁺ are 7.99 × 10^–8 M, 9.65 × 10^–8 M, 8.86 × 10^–8 M and 8.43 × 10^–8 M, respectively, indicating that these metal ions could be detected quantitatively.

Interestingly, chemosensor H₂L2 exhibits similar type of fluorescence properties toward Zn²⁺, Al³⁺ and Cr³⁺ ions. Free chemosensor exhibits weak fluorescence at 540 nm upon excitation at 440 nm. Addition of different metal ions (Zn²⁺, Al³⁺ and Cr³⁺) results in 6-fold enhancement of fluorescence intensity at 530 nm (Figures S42–S44). The 1:1 M²⁺/ M³⁺: H₂L2 binding stoichiometry was also confirmed by Job’s plot analysis (Figure S45). All the relevant results are collected in the Supporting Information (Figures S46–S54). The apparent binding constant (K) values and LOD values toward Zn²⁺, Al³⁺ and Cr³⁺ were estimated to be 1.27 × 10⁵ M^–1, 1.59 × 10⁵ M^–1 and 1.53 × 10⁵ M^–1, respectively and 10^–7 M range (Figures S55–57,Table S3).

Lifetime and Quantum Yield Measurements

Lifetime experiment of H₂L1, H₂L2 and complexes 1–7 were performed at 298 K in 1X PBS solution. The average fluorescence decay lifetimes of all the compounds were determined by using the formula τ_f = a₁τ₁ + a₂τ₂, where a₁ and a₂ are relative amplitude of decay process. The average fluorescence lifetime of H₂L1, H₂L2 and complexes 1–7 are 0.39, 4.58 and 1.30, 1.06, 1.18, 1.47, 4.75, 4.72, and 4.73 ns, respectively (Figure 7, Figure S58 and Table S4)

Figure 7.

Time-resolved fluorescence decay curve (logarithm of normalized intensity vs time in ns) of H₂L1 and H₂L1+Mⁿ⁺ (Mⁿ⁺ = Zn²⁺, Al³⁺, Cr³⁺ and Fe³⁺), respectively.

Fluorescence quantum yields (Φ) of free H₂L1, H₂L2 and complexes 1–7 were calculated using the following formula:

where A is the area under the emission spectral curve, OD is the optical density of the compound at the excitation wavelength and η is the refractive index of the solvent. Here the value of Φ_standard is taken as 0.52 (for Quinine Sulfate). The values of Φ for H₂L1, H₂L2 and complexes 1–7 are found to be 0.04, 0.13 and 0.15, 0.19, 0.13, 0.12, 0.27, 0.25, and 0.23 respectively (Table S4).

Cell Imaging Study

A fluorescence microscopic study was performed to envisage the cellular uptake of both ligands (H₂L1 and H₂L2) (10 μM), Zn²⁺ salt (10 μM), Al³⁺ salt (10 μM),Cr³⁺ salt (10 μM) and Fe³⁺ salt (10 μM), in HepG2 cells. A moderate green signal is observed in the case of the cells treated with the ligand. The intensity of the green signal gets enhanced significantly when the cells were treated with H₂L1 or H₂L2 and respective metal salts. No fluorescence was observed in case of the untreated cells. Thus, we can conclude that the cells readily uptake H₂L1 and H₂L2 as well as its Zn²⁺, Al³⁺, Cr³⁺ and Fe³⁺complexes, which results in prominent green fluorescent signals (Figure 8 and Figure S59).

Figure 8.

Bright field, fluorescence and merged microscopic images of untreated HepG2 (Control), cells treated with H₂L1(10 μM), H₂L1(10 μM)+ Zn²⁺(10 μM), H₂L1(10 μM) + Al³⁺(10 μM), H₂L1 (10 μM) + Cr³⁺(10 μM) and H₂L1 (10 μM) + Fe³⁺ (10 μM).

Cell Survivability Assay

The in vitro cytotoxicity of the ligands was estimated for checking the biocompatibility on WI-38 cell lines. The cells were treated with five different concentrations (20, 40, 60, 80, and 100 μg/mL) of ligand for 24 h and followed by MTT assay. It was observed that both the ligands exhibited no significant toxicities even at the highest concentration of 100 μg/mL (Figure 9 and Figure S60). Therefore, the ligands are biocompatible and highly conducive for biological applications.

Figure 9.

Cell survivability of WI-38 cells exposed to the ligand H₂L1.

Summary

In this work, we have successfully developed two aza-phenol based macrocyclic fluorescence probes, H₂L1 and H₂L2 as a “turn-on” fluorescence receptor toward multiple metal ions (H₂L1 for Zn²⁺, Al³⁺, Cr³⁺and Fe³⁺ions; H₂L2 for Zn²⁺, Al³⁺ and Cr³⁺ ions). Fluorescence enhancement originates from a CHEF effect owing to the coordination of metal ions with the macrocyclic ligand through the imine nitrogens and phenoxido oxygens. In presence of above metal ions around 6-fold enhancement of emission intensity in 1X PBS solution is observed.1:1 chemosensor: metal binding stoichiometry both in solid and solution phase has been proven by different techniques viz. X-ray crystallography, ESI-Mass analysis and fluorescence spectroscopy. Important parameters like reversibility and regeneration of the chemosensors have been examined in the presence of Na₂EDTA. The large Stoke shifts of the chemosensors in the presence of metal ions (∼100 nm) and low LOD values make them suitable for bioimaging studies. Indeed, we have successfully performed bioimaging studies of the chemosensors using HepG2 cells along with MTT assay. Therefore, phenol based macrocyclic probes able to behave as multianalyte detectors constitute a unique class of chemosensors. Moreover, the tuning of the macrocyclic framework and donor sites can lead to chemosensors able to accommodate different analytes with varying stoichiometries.

Experimental Section

Materials and Physical Measurements

All reagent or analytical grade chemicals and solvents were purchased from commercial sources and used without further purification. Elemental C, H, and N analysis was carried out using a Perkin–Elmer 240C elemental analyzer. Infrared spectra (400–4000 cm^–1) were recorded from KBr pellets on a Nicolet Magna IR 750 series-II FTIR spectrophotometer. Absorption spectra were measured with a sensitive UV–vis spectrophotometer (UV-2450 spectrophotometer (Shimadzu, Japan)) equipped with double beam light source with a 1 cm-path-length quartz cell. Electron spray ionization mass (ESI-MS⁺) spectra were recorded on a MICROMASS Q-TOF spectrometer. Emission spectra were collected using a Fluoromax-4 spectrofluorometer at room temperature (298 K) under degassed condition. Fluorescence lifetimes were measured using a time-resolved spectrofluorometer from IBH, UK. Measurements of ¹H and ¹³C NMR spectra were conducted using a BRUKER 300 spectrometer.

X-ray Crystallography

Single crystal X-ray data of 1was collected on a Bruker SMART APEX-II CCD diffractometer using graphite monochromated MoKα radiation (λ = 0.71073 Å) at room temperature. Data processing, structure solution, and refinement were performed using the Bruker Apex-II suite program. All available reflections in 2θ_max range were harvested and corrected for Lorentz and polarization factors with Bruker SAINT plus.⁶³ Reflections were then corrected for absorption, interframe scaling, and other systematic errors with SADABS.⁶⁴ The structures were solved by direct methods and refined by means of full matrix least-squares technique based on F² with SHELX-2018/3 software package.⁶⁵ All the non hydrogen atoms were refined with anisotropic thermal parameters. C–H hydrogen atoms were inserted at geometrical positions with U_iso = 1/2U_eq to those they are attached. Crystal data and details of data collection and refinement for complex 1 are summarized in Table S1.

Synthesis of 2,6-Diformyl-4-methylphenol (DFP)

2,6-Diformyl-4-methylphenol (DFP) was prepared following a standard literature procedure.⁵⁹

Synthesis of Chemosensor H₂L1 [H₂L1 = 1,11-Dimethyl-6,16-dithia-3,9,13,19-tetraaza-1,11(1,3)-dibenzenacycloicosaphane-2,9,12,19-tetraene-1,11-diol]

A mixture of 2,6-diformyl-4-methylphenol (2.0 mmol, 0.3283 g) and 2,2-thiobis(ethylamine) (2.0 mmol, 0.2404 g) was stirred (∼30 min.) and then heated to reflux for 4 h in a chloroform–methanol (1:9 v/v) mixture. An orange colored gummy mass was obtained after evaporation of the solvent.

Yield: 0.427g (86%). Anal. Calc. for C₂₆H₃₂N₄O₂S₂: C 60.40%; H 6.26%; N 9.99%. Found: C 60.19%; H 6.17%; N 10.02%. IR (cm^–1, KBr): υ(C=N) 1634s; ν(O–H) 3363s. ESI-MS⁺ in MeOH: The base peak was detected at m/z = 497.18, corresponding to [H₂L1+H]⁺. UV–vis, λ_max (nm), (ε (dm³ mol^–1cm^–1)) in 1X PBS solution: 435 (6450), 265(5850).

¹H NMR (DMSO-d₆, 300 MHz) δ ppm: 2.19 (Ar–CH₃) (s, 6H), 2.87 (−CH₂) (t, 8H, J₁= J₂= 6 Hz), 3.74 (−CH₂) (t, 8H, J₁ = J₂ = 6 Hz), 7. 35 (Ar–H) (s, 4H), 8.48 (−CH=N) (s, 4H), 13.98(Ar–OH) (s, 2H).

¹³C NMR (DMSO-d₆, 75 MHz) δ ppm: 20.06, 20.34, 32.38, 33.49, 52.89, 55.61, 119.42,121.08, 124.77, 125.49, 126.62, 132.83, 135.14, 139.93, 161.94,167.10.

Synthesis of Chemosensor H₂L2 [H₂L2 =1,11-Dimethyl-6,16-dioxa-3,9,13,19-tetraaza-1,11(1,3)-dibenzenacycloicosaphane-2,9,12,19-tetraene-1,11-diol]

A mixture of hydrochloride salt of 2,2′-oxybis(ethan-1-amine) (2.0 mmol, 0.354 g) and sodium acetate (6.0 mmol, 0.492 g) was stirred for ∼30 min in methanol solvent. A clear solution was found. After that 2,6-diformyl-4-methylphenol (2.0 mmol, 0.328 g) was added to this solution and heated to reflux for 4 h in a chloroform–methanol (1:9 v/v) mixture. Deep yellow colored solid mass was obtained after evaporation of the solvent.

Yield: 0.394g (85%). Anal. Calc. for C₂₆H₃₂N₄O₄: C 67.22%; H 6.94%; N 12.06%. Found: C 67.19%; H 6.91%; N 12.02%. IR (cm^–1, KBr): υ(C=N) 1637s; ν(O–H) 3392. ESI-MS⁺ in MeOH: The base peak was detected at m/z = 465.23, corresponding to [H₂L2+H]⁺. UV–vis, λ_max (nm), (ε (dm³ mol^–1cm^–1)) in 1X PBS solution: 440 (2975), 348(2350).

¹H NMR (DMSO-d₆, 300 MHz) δ ppm: 2.13 (Ar–CH₃) (s, 6H), 3.55(−CH₂) (s, 8H) 3.67 (−CH₂) (t, 8H, J₁ = J₂ = 3 Hz), 7.21 (Ar–H) (s, 4H), 8.36 (−CH=N) (s, 4H), 13.82(Ar–OH) (s, 2H).

¹³C NMR (DMSO-d₆, 75 MHz) δ ppm: 20.36, 20.44, 58.89, 59.35, 59.16, 69.89, 121.12, 126.20, 126.47, 132.10, 154.42, 159.48, 160.13, 162.40, 164.00, 170.50.

Synthesis of Complex (1) {[Zn(H₂L1)(NO₃)]NO₃}

A 5 mL methanolic solution of zinc nitrate hexahydrate (1.0 mmol, 0.297 g) was added dropwise to a 20 mL methanolic solution of H₂L1 (1.0 mmol, 0.497 g). The resultant reaction mixture was stirred for ∼4 h. Yellow colored block shape crystals were obtained after few days.

Yield: 0.514g (75%). Anal. Calc. for C₂₆H₃₂N₆O₈S₂Zn: C 45.52%; H 4.70%; N 12.25%. Found: C 45.44%; H 4.62%; N 12.19%. IR (cm^–1, KBr): υ(C=N) 1638s; υ(NO₃^–)1363s; ν(C–H) 769 s. ESI-MS⁺ in MeOH: The base peak was detected at m/z = 279.96, corresponding to [Zn (H₂L1)]²⁺. UV–vis, λ_max (nm), (ε (dm³ mol^–1cm^–1)) in 1X PBS solution: 435(3900), 255(11500).

¹H NMR (DMSO-d₆, 300 MHz) δ ppm: 2.31 (−CH₃) (s, 6H), 3.01 (s, 2H), 3.07 (s, 2H), 3.71 (s, 2H), 3.93 (−CH₂) (s, 2H), 7.53 (Ar–H) (s, 2H), 7.78 (Ar–H) (s, 2H,), 8.40 (−CH=N) (s, 2H), 8.68 (−CH=N) (s, 2H,).

¹³C NMR (DMSO-d₆, 300 MHz) δ ppm: 19.63, 19.84, 30.62, 34.50, 55.05, 60.50, 116.55, 121.20, 122.38, 123.78, 125.49, 141.18, 141.82, 147.62, 169.96, 170.94.

Synthesis of Complex (2) {[Al(H₂L1)(NO₃)](NO₃)₂}

A 5 mL methanolic solution of aluminum nitrate nonahydrate (1.0 mmol, 0.375 g) was added dropwise to a 20 mL methanolic solution of H₂L1 (1.0 mmol, 0.497 g). The resultant reaction mixture was stirred for ∼4 h. A greenish-yellow solid mass was obtained after evaporation of the solvent.

Yield: 0.517 g (73%). Anal. Calc. for C₂₆H₃₂N₇O₁₁S₂Al: C 44.00%; H 4.55%; N 13.82%. Found: C 43.98%; H 4.50%; N 13.76%. IR (cm^–1, KBr): υ(C=N) 1638s; υ(NO₃^–)1339s; ν(C–H) 772 s. ESI-MS⁺ in MeOH: The base peak was detected at m/z = 174.85, corresponding to [Al(H₂L1)]³⁺ (Figure S61). UV–vis, λ_max (nm), (ε (dm³ mol^–1cm^–1)) in 1X PBS solution: 435 (5350), 255 (10300).

¹H NMR (DMSO-d₆, 300 MHz) δ ppm: 2.26 ppm (−CH₃) (s, 6H), 3.00 (s, 4H), 3.95 (−CH₂) (s, 4H), 7.70 (Ar–H) (d, 2H, J = 9 Hz), 7.86 (Ar–H) (s, 2H), 8.61 (−CH=N) (d, 2H, J = 12 Hz), 8.76 (−CH=N) (s, 2H).

¹³C NMR (DMSO-d₆, 300 MHz) δ ppm: 20.00, 20.07, 28.14, 28.64, 30.35, 30.94, 31.72, 32.12, 50.75, 52.00, 117.30, 117.38, 119.44, 123.06, 123.74, 124.73, 125.73, 129.76, 132.87, 137.84,139.89, 146.40, 167.12, 167.17, 168.81, 168.98.

Synthesis of Complex (3) {[Cr(H₂L1)(NO₃)](NO₃)₂}

A 5 mL methanolic solution of chromium nitrate nonahydrate (1.0 mmol, 0.400 g) was added dropwise to a 20 mL methanolic solution of H₂L1 (1.0 mmol, 0.497 g).The resultant reaction mixture was stirred for ∼4 h. A wine red colored solid mass was obtained after evaporation of the solvent.

Yield: 0.543g (74%). Anal. Calc. for C₂₆H₃₂N₇O₁₁S₂Cr: C 42.51%; H 4.39%; N 13.35%. Found: C 42.49%; H 4.35%; N 13.30%. IR (cm^–1, KBr): υ(C=N) 1634s; υ(NO₃^–)1305s; ν(C–H) 818s.UV–vis, λ_max (nm), (ε (dm³ mol^–1cm^–1)) in 1X PBS solution: 435 (5495), 255 (10000).

Synthesis of Complex (4) {[Fe(H₂L1)(NO₃)](NO₃)₂}

A 5 mL methanolic solution of iron nitrate nonahydrate (1.0 mmol, 0.404 g) was added dropwise to a 20 mL methanolic solution of H₂L1 (1.0 mmol, 0.497 g). The resultant reaction mixture was stirred for ∼4 h. A dark brown colored solid mass was obtained after evaporation of the solvent.

Yield: 0.568g (77%). Anal. Calc. for C₂₆H₃₂N₇O₁₁S₂Fe: C 42.28%; H 4.37%; N 13.28%. Found: C 42.24%; H 4.32%; N 13.19%. IR (cm^–1, KBr): υ(C=N) 1632s; υ(NO₃^–)1332s; ν(C–H) 762s.UV–vis, λ_max (nm), (ε (dm³ mol^–1cm^–1)) in 1X PBS solution: 435 (7000), 260 (12450).

Synthesis of Complex (5) [Zn(L2)]

A 5 mL methanolic solution of zinc nitrate hexahydrate (1.0 mmol, 0.297 g) was added dropwise to a 20 mL methanolic solution of H₂L2 (1.0 mmol, 0.464 g). The resultant reaction mixture was stirred for ∼4 h. Yellow colored solid mass was obtained after evaporation of the solvent.

Yield: 0.410g (78%). Anal. Calc. for C₂₆H₃₀N₄O₄Zn: C 59.15%; H 5.73%; N 10.61%. Found: C 59.10%; H 5.69%; N 10.58%. IR (cm^–1, KBr): υ(C=N) 1661s. ESI-MS⁺ in MeOH: The base peak was detected at m/z = 527.13, corresponding to [Zn(L2)+H]⁺(Figure S62). UV–vis, λ_max (nm), (ε (dm³ mol^–1cm^–1)) in 1X PBS solution: 435(6850), 348(3900).

¹H NMR (DMSO-d₆, 300 MHz) δ ppm: 2.12 (−CH₃) (s, 6H), 3.77 (−CH₂) (t, 8H, J₁ = 6 Hz & J₂ = 3 Hz), 3.84 (s, 8H), 7.42 (Ar–H) (s, 4H), 8.54 (−CH=N) (s, 4H).

¹³C NMR (DMSO-d₆, 300 MHz) δ ppm: 19.81, 20.10, 46.23, 48.10, 73.10, 73.69, 121.38, 124.09, 126.31, 136.52, 141.09, 141.37, 148.89, 155.79, 165.52, 172.80.

Synthesis of Complex (6) {[Al(L2)](NO₃)}

A 5 mL methanolic solution of aluminum nitrate nonahydrate (1.0 mmol, 0.375 g) was added dropwise to a 20 mL methanolic solution of H₂L2 (1.0 mmol, 0.464 g). The resultant reaction mixture was stirred for ∼4 h. An orange colored solid mass was obtained after evaporation of the solvent.

Yield: 0.372 g (76%). Anal. Calc. for C₂₆H₃₀N₅O₇Al: C 56.62%; H 5.48%; N 12.70%. Found: C 56.58%; H 5.46%; N 12.65%. IR (cm^–1, KBr): υ(C=N) 1637s; υ(NO₃^–)1328s. ESI-MS⁺ in MeOH: The base peak was detected at m/z = 539.50, corresponding to [Al(L2)(H₂O) (CH₃OH)]⁺ (Figure S63). UV–vis, λ_max (nm), (ε (dm³ mol^–1cm^–1)) in 1X PBS solution: 435 (6900), 348 (3400).

¹H NMR (DMSO-d₆, 300 MHz) δ ppm: 2.12 (−CH₃) (s, 6H), 3.60 (t, 8H, J₁ = J₂ = 3 Hz), 3.77 (−CH₂) (t, 4H, J₁ = J₂ = 3 Hz), 3.88(−CH₂) (s, 4H), 7.37 (Ar–H) (s, 2H), 7.77 (Ar–H) (s, 2H), 8.48 (−CH=N) (d, 4H, J = 9 Hz).

¹³C NMR (DMSO-d₆, 300 MHz) δ ppm: 19.97, 21.55, 51.50, 55.68, 66.75, 67.07, 116.74, 119.26, 122.09, 125.25, 129.76, 137.90, 146.03, 154.54, 168.71, 172.55.

Synthesis of Complex (7) {[Cr(L2)](NO₃)}

A 5 mL methanolic solution of chromium nitrate nonahydrate (1.0 mmol, 0.400 g) was added dropwise to a 20 mL methanolic solution of H₂L2 (1.0 mmol, 0.464 g). The resultant reaction mixture was stirred for ∼4 h. A dark green colored solid mass was obtained after evaporation of the solvent.

Yield: 0.416g (81%). Anal. Calc. for C₂₆H₃₀N₅O₇Cr: C 54.16%; H 5.24%; N 12.15%. Found: C 54.13%; H 5.22%; N 12.11%. IR (cm^–1, KBr): υ(C=N) 1639s; υ(NO₃^–)1330s. UV–vis, λ_max (nm), (ε (dm³ mol^–1cm^–1)) in 1X PBS solution: 435 (7150), 348 (3400).

UV–visible and Fluorescence Spectroscopic Studies

Stock solutions of the various ions (1 × 10^–3 M) were prepared in deionized water. A stock solution of H₂L1 and H₂L2 (1 × 10^–3 M) were prepared in MeOH medium. All the spectroscopic experiments including competitive assays of various cations and anions were performed in 1X PBS solution. In titration experiments, a 60 μL solution of (1 × 10^–3 M) H₂L1 and H₂L2 was taken for 3000 μL in a quartz optical cell of 1.0 cm optical path length, and the corresponding metal stock solutions of the metal ions were gradually added to it, respectively.

Cell Culture

The HepG2 cells and WI38 were obtained from the National Center for Cell Science (NCCS) Pune, India. The cells were grown in DMEM with 10% FBS (Fetal Bovine Serum), penicillin/streptomycin (100 units/ml) at 37 °C and 5% CO₂. All the treatments were conducted at 37 °C and at a cell density allowing exponential growth.

Cell Imaging

The HepG2 cells were grown in coverslips for 24 h. Then the cells were either mock-treated or treated with 10 μM of ligand H₂L1 and H₂L2, Zn²⁺ salt (10 μM), Al³⁺ salt (10 μM), Cr³⁺ salt (10 μM) and Fe³⁺ salt (10 μM) for 24 h at 37 °C. The cells were washed with 1X PBS. Then they were mounted on a glass slide and observed under fluorescence microscope (Leica) with a filter having excitation of 450–500 nm (blue) and an emission of 500–570 nm (green).

Cell Survivability Assay

Cell survivability of H₂L1 and H₂L2 were studied for WI-38 (noncancerous cells), following reported procedure.⁶⁶ In brief, viability of these cells after exposure to various concentrations of ligand was assessed by MTT assay. The cells were seeded in 96-well plates at 1 × 10⁴ cells per well and exposed to ligand at concentrations of 0 μM, 20 μM, 40 μM, 60 μM, 80 μM, 100 μM for 24 h. The resulting formazan crystals were dissolved in an MTT solubilization buffer and the absorbance was measured at 570 nm by using a spectrophotometer (BioTek) and the value was compared with control cells. The cell cytotoxicity of the complexes toward the WI-38 cell was envisaged following the above-mentioned MTT assay protocol.

Supporting Information Available

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acsomega.2c06549.

• Crystallographic data for complex 1 (CIF)

• Additional supplementary data as mentioned in the text (PDF)

The authors declare no competing financial interest.