A novel pyrene-2-(pyridin-2-ylmethylsulfanyl)ethylamine based turn-on dual sensor for Al³⁺: experimental and computational studies

Abstract

A new pyrene based highly sensitive and selective Al³⁺ sensor, pyrene-2-(pyridin-2-ylmethylsulfanyl)-ethylamine (PP), was found to exhibit a turn-on fluorescence enhancement (FE) as high as 7.4 fold with K_d (2.55 ± 0.10) × 10⁻⁴ M and n = 1. This probe binds reversibly with Al³⁺ in the presence of H₂EDTA²⁻, both under intra- and exctracellular conditions. LOD determined by 3σ methods was found to be 15 nM while LOQ = 52.8 nM. The tentative coordination environment in the Al³⁺–PP complex was delineated by DFT calculations both on the free ligand and complex. The TDDFT calculations reveal spectral features comparable to the experimental ones.

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Introduction

Fluorescent chemosensors are a growing field of research and are attracting the interest of chemists, biologists and environmental scientists owing to their potential applications in medicinal and environmental research. Thus, many fluorescent chemosensors for heavy metal ions like Zn²⁺, Cd²⁺ and Hg²⁺, and for many transition metal ions have been developed in recent years. The detection of Al³⁺ has always been a problematic task due to the lack of spectroscopic characteristics and poor coordination ability.¹ In addition, most of the reported Al³⁺ sensors are insoluble in aqueous medium and require complicated synthetic procedures. For practical applications, it is necessary to develop Al³⁺ sensors that are easily prepared and possess selective and sensitive signalling mechanisms. Compared to other metal ions, only a few fluorescent chemosensors have been reported for the detection of Al³⁺.^2–7

Despite of being a non-essential element, the detection of Al³⁺ is of great interest due to its potential toxicity arising out of its widespread application in automobiles, computers, packaging materials, electrical equipment, machinery food additives building construction, clinical drugs and water purification.^8,9 Furthermore, it is well known that 40% of soil acidity is due to aluminium toxicity.¹⁰

Aluminium leaching from soil by acid rain is deadly to growing plants.¹¹ The World Health Organization (WHO) prescribed the average human intake of aluminium is ∼3–10 mg day⁻¹ with a weekly dietary intake of 7 mg day⁻¹ body weight.¹² Aluminium toxicity damages the central nervous system and it is suspected of playing a role in neurodegenerative Alzheimer and Parkinson diseases. It is also responsible for intoxication in haemodialysis patients.¹³ Therefore, detection of Al³⁺ is crucial in controlling its concentration levels in the biosphere and its direct impact on human health.

A number of high-end techniques available for Al³⁺ estimation includes chromatography,¹⁴ accelerator mass spectroscopy (AMS),¹⁵ graphite furnace atomic absorption spectrometry (GFAAS),¹⁶ neutron activation analysis (NAA),¹⁷ inductively coupled plasma-atomic emission spectrometry (ICP-AES),¹⁸ inductively coupled plasma-mass spectrometry (ICP-MS),¹⁹ laser ablation microprobe mass analysis (LAMMA),²⁰ electrothermal atomic absorption spectrometry (ETAAS)²¹ etc. but most of them require sophisticated instruments and time consuming for sample preparation protocols and cost-effective too. On the contrary, fluorescence chemosensors are the best choices, as it is the simplest, sensitive, fast and inexpensive technique²² offering significant advantages over other methods.²³ Hence, recently the design and synthesis of Al³⁺ selective fluorescent probes has received intense attention of the chemists.^24–26

Most of the reported Al³⁺ sensors suffer from interference of Fe³⁺ and Cu²⁺ and require tedious synthetic methodology.²⁷ Al³⁺ being hard acid prefers to bind with hard bases and consequently most of the Al³⁺ probes are found to coordinate with hard donor atoms like N and O. However, the strong binding of a receptor to an analyte may lead to lose selectivity as well as reversibility, which are essential for practical applications. There are a few reports where a soft donor atom like S has been introduced for the recognition of Al³⁺.²⁸ The probes with such soft donor atoms may recognise a hard metal ion with enhanced selectivity over the other competing guests.²⁹ Here we are reporting a highly selective Al³⁺ probe containing N₂S donor atoms that binds selectively to Al³⁺ in presence of other metal ions. The coordination environment around the metal ion in Al³⁺–PP complex was delineated by DFT calculations both on the free ligand and its Al³⁺ complex.

Results and discussion

(Pyridin-2-ylmethylsulfanyl)-ethylamine (P) was synthesized as outlined in Scheme 1.³⁰ The probe (PP) was then prepared by a simple Schiff base condensation between P and pyrene-2-aldehyde in EtOH. This ligand is suitable for Al³⁺ recognition and characterized by NMR (Fig. S1–S3†), mass analysis (Fig. S4–S6†) and IR analysis (Fig. S7†).

Scheme 1

The UV-Vis spectrum of sensor PP was recorded in a MeOH–H₂O (9 : 1, v/v) at pH 7.2 (1 mM HEPES buffer) which displayed well-defined bands at 390, 360, 344, 285 and 275 nm typical of pyrene moiety. On gradual addition of Al³⁺ there is an increase in absorbance at all these wavelengths along with the development of a new band at 440 nm (Fig. 1). This indicates a complexation between PP and Al³⁺. However, no such significant change in the absorption spectrum of PP was observed with other tested metal cations.

Fig. 1 (A) Absorption titration of PP (20 μM) with Al³⁺ in MeOH–H₂O (9 : 1, v/v, pH 7.2, HEPES buffer). Inset shows (B) Benesi–Hilderbrand plot. (C) Job's plot.

The composition of complex formed by the reaction between PP and Al³⁺ was determined by absorption titration in MeOH–H₂O (9 : 1, v/v) at pH 7.2 (1 mM HEPES buffer) (Fig. 1). The OD (optical density) at 440 nm increases gradually with the increase in concentration of Al³⁺up to a mole ratio PP : Al³⁺ = 1 : 1 (Fig. 1A) and then becomes saturated. When we plot [(A_max − A_o)/(A − A₀)] vs. 1/[Al³⁺] the slope of such plot gives a value of K_d = (1.94 ± 0.04) × 10⁻⁴ M (Fig. 1B) and n = 1 indicating 1 : 1 binding between PP and Al³⁺. Job's method was further employed to determine the composition of the complex that also indicates a 1 : 1 complexation (Fig. 1C). The probe acts as a naked eye colorimetric sensor for Al³⁺ with the change in colour from colorless to greenish yellow (Fig. S8†).

In the absence of Al³⁺, PP is very weakly fluorescent compared to that of pyrene due to photo-induced electron transfer (PET) from the metal binding site (azomethyne-N) to the fluorescent pyrene unit. The cis–trans isomerisation around the C=N bond of azomethyne group may be neglected by considering the positive dihedral angles (C=N–C–C) which were found to be 125.878 for PP and 117.442 for PP + Al³⁺,³¹ indicating the existence of sole cis-isomer in both cases (vide supra). The fluorescence enhancement observed for compound PP in the presence of Al³⁺ ions was attributed to the chelation enhanced fluorescence (CHEF) effects arising through coordination of azomethine-N along with pyridine-N and S atoms to the metal ion, as a result PET effect is efficiently blocked.

Pyrene seems to be an interesting fluorophore displaying a change in excimer–monomer emission intensities^32–34 due to the formation of self-assembled pyrene-conjugate. In our case we performed DLS (Dynamic Light Scattering) studies to check such possibilities. It was interesting to note that excimer formation takes place through intermolecular π⋯π stacking between pyrene moieties both in absence and presence of Al³⁺. In the DLS spectrum of free ligand the peak corresponding to diameter 1.14 nm may be due to PP monomer while peak corresponding to diameter ∼232 nm is due to excimer. However, such aggregation is much favoured in presence of Al³⁺ as indicated by the increase in particle size in presence of metal ion (Fig. S9 and Table S1†). Here PP–Al³⁺ monomer size is 5.40 nm while the diameter of excimer is 359 nm.

The emission spectra of PP and its fluorescence titration with Al³⁺ were recorded in MeOH–H₂O (9 : 1) at pH 7.2, 1.0 mM HEPES buffer (Fig. 2). The binding between the free probe PP and Al³⁺ leads to chelation enhanced fluorescence (CHEF) effect causing fluorescence enhancement (FE) at 506 nm due to blocking of PET effect. A plot of F.I. vs. [Al³⁺] gives a non-linear curve with decreasing slope at higher concentration of Al³⁺.

Fig. 2 (A) Fluorescence titration of PP (20 μM) in MeOH–H₂O (9 : 1, v/v, pH 7.2, HEPES buffer) by the gradual addition Al³⁺ with λ_ex = 440 nm, λ_em = 506 nm Inset. (B) Benesi–Hilderbrand plot. (C) Plot of F.I. vs. [Al³⁺].

A Benesi–Hiderband plot of [(F_max − F_o)/(F − F_o)] vs. 1/[Al³⁺] gives straight line with slope K_d =(2.55 ± 0.10) × 10⁻⁴ M with R² = 0.99, indicating 1 : 1 complexation. It is to be mentioned here that there is an excellent agreement between the K_d values obtained from two spectroscopic titrations indicating the self-consistency of our results. The LOD of Al³⁺ calculated by using 3σ method was found to be 15 nM (Fig. S12†).

A K_d value of (2.55 ± 0.10) × 10⁻⁴ M unambiguously demonstrates a moderate binding of PP towards Al³⁺. The stoichiometry of Al³⁺ complex with PP as delineated by UV-VIS and fluorescence titrations was further confirmed by ESI-MS⁺ (m/z) mass spectrometry [Al(PP)(NO₃⁻)(H₂O)]²⁺ (m/z = 245.1001) (Fig. 3) and Job's method. Al³⁺ detection was not perturbed by biologically abundant Na⁺, K⁺, Ca²⁺ etc. metal ions. Several transition metal ions, namely Cr³⁺, Mn²⁺, Fe²⁺, Fe³⁺, Co²⁺, Ni²⁺, Cu²⁺, and heavy metal ions like Cd²⁺, Pb²⁺, and Hg²⁺, also cause no interference (Fig. 4). The sensor was found to bind Al³⁺ reversibly as tested by reacting with excess H₂EDTA²⁻ under both intra- and extracellular conditions (Fig. S13† and 10).

Fig. 3 HRMS spectra of (a) PP and Al³⁺–PP complex in the positive mode.

Fig. 4 Bar chart illustrating fluorescence response of PP at 506 nm towards different cations in MeOH–H₂O (9 : 1, v/v) at pH 7.2. Conditions: PP = 20 μM, Mⁿ⁺ = 100 μM; where, Mⁿ⁺ = Al³⁺, Ca²⁺, Cd²⁺, Co²⁺, Cr³⁺, Cu²⁺, Fe²⁺, Fe³⁺, K⁺, Mg²⁺, Mn²⁺, Na⁺, Pb²⁺, Zn²⁺, Hg²⁺.

In order to support the binding of Al³⁺ with the receptor PP, the ¹H NMR titration was performed in DMSO-d₆ (Fig. 5). The ¹H NMR spectrum of PP contains signals for the HC=N (azomethine, H) at 9.27 ppm and pyridine protons (a, b, c, and d) appear at 7.40(d), 7.71(t), 7.22 (t) and 8.99 (d) ppm, respectively. The pyrene protons appear in the region 8.05–8.48 ppm. The chemical shifts for other protons appear in the usual positions. Addition of 1.2 equivalent of Al(NO₃)₃·9H₂O leads to a down field shift of the azomethine proton (9.32 ppm) and d proton (9.29 ppm) on the pyridine ring. All other protons remain almost invariant in presence of Al³⁺ ion. This clearly indicates the involvement of the azomethine N and pyridine N atoms in bonding with Al³⁺.

Fig. 5 ¹H NMR spectra of PP and Al³⁺–PP complex in DMSO-d₆ on 300 MHz Bruker Instrument.

Geometry optimization and electronic structure

The optimized geometries of PP and its Al³⁺ complex are shown in Fig. 6. The composition of the complex as [[Al(PP)(NO₃)(H₂O)] is based on HRMS studies which displayed the presence of one NO₃⁻ and one water molecules in the molecular fragment. Both PP and Al³⁺–PP (1) complex has C1 point group. The important optimized geometrical parameters of the complex are listed in Table 1. The hexa-coordinated metal centre possesses a distorted octahedral geometry. The calculated Al–N and Al–O bond distances fall in the range 2.020–2.054 and 1.954–2.060 Å, respectively. On complexation, some C–N and C–S bond distances are changed with respect to that in free ligand. Table 2 describes the change in bond lengths in Al³⁺–PP compared to free ligand PP.

Fig. 6 Optimized geometry of ligand (PP) and [Al(PP)(NO₃)(H₂O)]²⁺.

Table 1 Selected optimized geometrical parameters for 1 in the ground state

Bond lengths (Å)
Al–N1	2.02	Al–O1	2.06
Al–N2	2.05	Al–O2	1.99
Al–S	2.52	Al–O3w	1.95

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Bond angles (°)
N1–Al–S	85.53	N2–Al–O2	95.93
N2–Al–S	78.5	N2–Al–O3w	83.82
N2–Al–Ol	102.31	N1–Al–O1	93.89
O1–Al–S	84.29	N1–Al–O2	107.05
O3w–Al–S	119.77	N2–Al–O3w	88.61
O1–Al–O2	65.73	O2–Al–O3w	90.65

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Table 2 Change in bond lengths for 1 compared to free L in the ground state calculated at B3LYP Levels

Bond lengths (Å)
	Free ligand (L)	Complex 1
N1–C1	1.284	1.327
N1–C2	1.464	1.504
S–C3	1.903	1.908
S–C4	1.916	1.906
N2–C5	1.355	1.374
N2–C6	1.351	1.365

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In case of PP in the ground state, the electron density resides mainly on HOMO − 2, HOMO, LUMO and LUMO + 1 molecular orbitals of the pyrene moiety while in HOMO − 1 and LUMO + 3 molecular orbitals a considerable contribution comes from cystamine moiety along with the contribution from pyridyl moiety with an energy gap between HOMO and LUMO of 3.872 eV (Fig. 7). In case of Al³⁺–PP complex all the HOMO − 2, HOMO − 1 and HOMO molecular orbitals are mainly originating from ligand π and π* orbitals while LUMO + 1 and LUMO + 3 molecular orbitals arise from metal d orbitals along with ligand π orbitals with HOMO–LUMO energy gap of 1.82 eV. These compositions are useful in understanding the nature of transition as well as the absorption spectra of both the ligand and complex.

Fig. 7 Frontier molecular orbital of PP and [Al(PP)(NO₃)(H₂O)]²⁺.

The ligand shows four absorption bands at 343, 286/275, 263/260 and 243/233 nm in a methanolic solution at room temperature and have ILCT character due to the presence of pyrene moiety. These bands are assigned to S₀ → S₂, S₀ → S₇, S₀ → S₁₁ and S₀ → S₁₇ electronic transitions, respectively (Fig. 8). The absorption energies along with their oscillator strengths are given in Table S2.† The Al³⁺–PP complex shows three absorption bands at 390, 360 and 343 nm (Fig. 9) in methanol at room temperature and the corresponding calculated absorption bands are located at 394, 357 and 334 nm which are in excellent agreement with experimental results (Table S3† and 3). These three absorption bands can be assigned to the S₀ → S₆, S₀ → S₈ and S₀ → S₁₁ transitions, respectively originating from an admixture of MLCT and ILCT transitions (Table S2†).

Fig. 8 Frontier molecular orbitals involved in the UV-Vis absorption of ligand (PP).

Fig. 9 Frontier molecular orbitals involved in the UV-Vis absorption of [Al(PP)(NO₃)(H₂O)]²⁺.

Table 3 The comparable calculated optical transitions with Experimental UV/Vis values for the ligand (PP) and complex [Al(PP)(NO₃)(H₂O)]²⁺

Ligand and complex 1	Experimental [λexp (nm)]	Theoretical [λtheo (nm)]	Electronic transition
Ligand	343	339	S0 → S2
Ligand	286/275	291	S0 → S7
Ligand	263/260	266	S0 → S11
Ligand	243/233	246	S0 → S17
Complex 1	390	394	S0 → S6
Complex 1	360	357	S0 → S8
Complex 1	343	334	S0 → S11

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The intracellular imaging behaviours of PP on HepG2 cells with the aid of fluorescence microscopy displayed weak intracellular fluorescence when treated with 10 μM PP (Fig. 10). This weak fluorescence response towards the probe was due to the presence of intracellular Al³⁺, though may be in a very low concentration; but very low detection limit (semi-ppb level, vide infra) of this probe for Al³⁺ enables us to visualize the presence of intracellular Al³⁺. Fluorescence images of HepG2 cells were taken separately for another set of experiment where cells incubated with 10 μM PP + 10 μM Al³⁺ for 30 min where bright fluorescence was observed. Similarly, in another set of experiment, cells were incubated with 10 μM PP + 10 μM Al³⁺ for 30 min followed by addition of 100 μM H₂EDTA²⁻ for another 30 min and fluorescence images were taken. In this case, HepG2 cells showed almost complete quenching of fluorescence due to removal of Al³⁺ from the Al³⁺–PP complex by H₂EDTA²⁻ which manifests a reversible binding of PP towards Al³⁺, satisfying one of the crucial requirements for in vivo monitoring of a chemical species.

Fig. 10 The phase contrast and fluorescence images of HepG2 cells were capture after incubated with PP, PP + Al³⁺, for 30 min at 37 °C and followed by addition of 100 μM H₂EDTA²⁻ after cells being pre incubated with PP + Al³⁺ for 30 min at 37 °C.

For convenient biological application of this probe under physiological conditions it is necessary to verify its pH-stability. There is no obvious fluorescence emission of PP between pH 4 and 12, which suggests that PP is stable over this wide range of pH and can work well under physiological conditions (Fig. 11). However, in presence of selective guest like Al³⁺ ion it fluoresces effectively in the range pH 4.0–8.0 with almost constant intensity which clearly indicates the compatibility of the probe for biological applications under physiological conditions. At pH ≥ 8.0 there is gradual decrease in FI may be due to the formation of Al(OH)₂⁺ Al(OH)₃ and Al(OH)₄⁻ depending on pH of the medium.

Fig. 11 pH study of PP and corresponding Al³⁺ complex.

A comparison of our probe, with the previously reported pyrene based analogous systems is shown in the Chart 1. A quick inspection of these studies reveal that all these are turn-on sensor towards different metal ions with moderate LOD values.

Chart 1 Comparison of some previously reported systems (a–c) with our probe (d).

Conclusion

In summary, we have synthesized a novel pyrene based N₂S donor probe for selective recognition of Al³⁺, in presence of large number of background metal ions, which was found to undergo 1 : 1 complexation in MeOH–H₂O (9 : 1, v/v, pH 7.2, HEPES buffer). The probe itself is very weakly fluorescent due to combined PET effect arising out of the transfer of electron from azomethine-N to the pyrine moiety in the excited state. It becomes fluorescent only in presence of Al³⁺ which causes PET blocking through selective coordination to Al³⁺. The complexation of PP to Al³⁺ was confirmed by ¹H NMR and ESI-MS⁺ studies and further strengthened by DFT calculations on both the free ligand and its Al³⁺ complex. The UV-Vis and Fluorescence titrations help us to delineate K_d values (1.94 × 10⁻⁴ M and 2.55 × 10⁻⁴ M, respectively) which are in excellent agreement manifesting the self-consistency of our results. The reversible binding of Al³⁺ to PP was confirmed by reacting with excess H₂EDTA²⁻ both in extra- and intracellular conditions.

Experimental

Materials and methods

The starting materials, such as, cystamine hydrochloride (Sigma Aldrich), 2-chloromethyl pyridine (Sigma Aldrich) were used for the preparation of amine and then used to condense with pyrene carboxyaldehyde (Sigma Aldrich) for the preparation of ligand PP. Al(NO₃)₃·9H₂O (Sigma Aldrich) was used to prepare Al³⁺ complex. Solvents like MeOH, MeCN etc. (Merck, India) were of reagent grade.

Physical measurements

Elemental analyses were carried out using a Perkin-Elmer 240 elemental analyzer. Infrared spectra (400–4000 cm⁻¹) were recorded from KBr pellets on a Nickolet Magna IR 750 series-II FTIR spectrophotometers. ¹H-NMR was recorded in CDCl₃ and DMSO-d₆ on a Bruker 300 MHz NMR spectrometer using tetramethylsilane (δ = 0) as an internal standard. UV-Vis spectra were recorded on an Agilent diode-array spectrophotometer (Model, Agilent 8453), steady-state Fluorescence were recorded on a PTI spectrofluorimeter (Model QM-40.), ESI-MS⁺ (m/z) of the amine, ligand (PP) and Al³⁺–PP complex were recorded on a HRMS spectrometer (Model: QTOF Micro YA263).

Syntheses

Preparation of (2-(pyridin-2′-ylmethylsulfanyl) ethanamine) (P). 2-Aminoethanethiol hydrochloride (12.2 mmol) and the 2-(chloromethyl)pyridine hydrochloride (6.1 mmol) were added to a solution of NaOH (24.4 mmol) in EtOH (20 mL) in ice bath. The reaction mixture was stirred for 30 min before the ice bath was removed and then the mixture was stirred at an ambient temperature for 2.5 h. The EtOH was removed under reduced pressure and water (25 mL) was added to the resulting residue. The aqueous solution was extracted with CH₂Cl₂ (3 × 25 mL) and the combined organic layer was washed with brine (20 mL), dried with K₂CO₃, filtered and solvent was removed in vacuo to afford a crude oil. It was further purified by column chromatography (silica, CH₂Cl₂/MeOH/NH₃ = 9/2/0.04) to give 1.78 g as yellow liquid in 89% yield. Anal. calcd for C₈H₁₂N₂S : C, 57.11; H, 7.19; N, 16.65. Found: C, 57.55; H, 7.33; N, 16.07.¹H NMR (300 MHz, CDCl₃) (δ, ppm): 2.01(b, 2H, NH₂), 2.77 (t, 2H, CH₂), 2.55 (t, 2H, CH₂), 3.77 (s, 2H, CH₂), 7.30 (d, 1H, ArH), 7.58 (t, 1H, ArH), 7.11 (t, 1H, ArH), 8.46 (d, 1H, ArH) (Fig. S1†). Mass spectrum: m/z = 169.1076 (P + H⁺), 191.0693 (P + Na⁺) (Fig. S4†).

Pyrene-2-(pyridin-2-ylmethylsulfanyl)-ethylamine (PP). Pyrenecarboxaldehyde (0.460 g, 2 mmol) was dissolved in 25 mL MeOH. To this solution was added P (0.336 g, 2 mmol) drop wise and stirred at room temperature for 2 h and then the reaction mixture was filtered out and evaporated to dryness in rota-evaporator. After that liquid product was collected and purified by column chromatography. (Neutral allumina, CH₂Cl₂/MeOH = 8/2) (yield, 80.34%). Anal. calcd for C₂₅H₂₀N₂S : C, 78.91; H, 5.30; N, 7.36%. Found: C, 78.55; H, 5.53; N, 7.24%. ¹H-NMR (in DMSO-d₆) (δ, ppm): 2.88 (t, 2H, CH₂), 3.94(4H, CH₂, CH₂), 7.40 (d, 1H, ArH), 7.71 (t, 1H, ArH), 7.22 (t, 1H, ArH), 8.99 (d, 1H, ArH), 9.27(s, 1H, azomethane) and 8.05–8.48 (9H, pyrene protons) (Fig. S2†). ESI-MS⁺ (m/z): 381.1424 (PP + H⁺) (Fig. S5†).

Synthesis of complex

To a solution of PP (0.76 g, 2 mmol) in 30 mL MeOH was added Al(NO₃)₃·9H₂O (0.75 g, 2 mmol) in 20 mL MeOH drop wise with continuous stirring. After 1 hour the solution was filtered and kept aside undisturbed. After one day crystalline complex was precipitated out. It was collected by filtration, washed several times with MeOH and dried in air. It was recrystallized from methanol. Several trials to grow single crystals were failed. Elemental analysis for [Al(PP)(NO₃)(H₂O)](NO₃)₂(C₂₅H₂₂N₅O₁₀SAl): calculated: C, 49.10; H, 3.63; N, 11.45%; found: C, 49.25; H, 3.53; N, 11.30%. ¹H-NMR (in DMSO-d₆) (δ, ppm): 2.90 (t, 2H, CH₂), 3.96 (4H, CH₂, CH₂), 7.43 (d, 1H, ArH), 7.71 (t, 1H, ArH), 7.24 (t, 1H, ArH), 9.28 (d, 1H, ArH), 9.32 (s, 1H, azomethane) and 8.06–8.97 (9H, pyrene protons) (Fig. S3†). ESI-MS⁺ (m/z): 245.1001 ([Al(PP)(NO₃⁻)(H₂O)]²⁺) (Fig. S6†).

Computational details

Ground state electronic structure calculations in methanol solution of both the ligand and complex have been carried out using DFT method³⁵ associated with the conductor-like polarizable continuum model (CPCM).³⁶ Becke's hybrid function³⁷ with the Lee–Yang–Parr (LYP) correlation function³⁸ was used throughout the study. The geometry of the ligand and complex were fully optimized without any symmetry constraints. On the basis of the optimized ground state geometry, the absorption spectral properties PP and Al³⁺–PP in methanol were calculated by time-dependent density functional theory (TDDFT)³⁹ associated with the conductor-like polarizable continuum model (CPCM).³⁶ We have computed the lowest 40 singlet–singlet transitions and the presence of electronic correlation in the TDDFT (B3LYP) method⁴⁰ enables to get accurate electronic excitation energies.

For H atoms we used 6-31G basis set; for C, N, O and Al atoms we employed 6-31G and for S atom we adopt 6-31 + G(d, p) basis sets for the optimization of the ground state. The calculated electronic density plots for frontier molecular orbitals were prepared by using Gauss View 5.1 software. All the calculations were performed with the Gaussian 09W software package.⁴¹ Gauss Sum 2.1 program⁴² was used to calculate the molecular orbital contributions from groups or atoms.

Cell culture

HepG2 cell line, Human hepatocellular liver carcinoma cells, were procured from National Center for Cell Science, Pune, India, and used throughout the study. Cells were cultured in DMEM (Gibco BRL) supplemented with 10% FBS (Gibco BRL), and a 1% antibiotic mixture containing Penicillin, Streptomycin and Gentamicin (Gibco BRL) at 37 °C in a humidified incubator with 5% CO₂ and cells were grown to 60–80% confluence, harvested with 0.025% trypsin (Gibco BRL) and 0.52 mM H₂EDTA²⁻ (Gibco BRL) in phosphate-buffered saline (PBS), plated at the desired cell concentration and allowed to re-equilibrate for 24 h before any treatment.

Cell cytotoxicity assay

To test the cytotoxicity of PP assay was performed as per the procedure described earlier.⁴³ After treatment with PP at different doses of 1, 10, 20, 50 and 100 μM, respectively, for 12 h, 10 μL of MTT solution (10 mg mL⁻¹ PBS) was added to each well of a 96-well culture plate and again incubated continuously at 37 °C for a period of 3 h. All media were removed from wells and 100 μL 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 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 were expressed as mean ± standard errors of three independent experiments. The cell cytotoxicity was calculated as % cell cytotoxicity = 100% − % cell viability (Fig. S14†).

Cell imaging study

HepG2 Cells were incubated with 10 μM PP [the stock solution (1 mM) was prepared by dissolving PP to the mixed solvent [DMSO–water = 1 : 9 (v/v)] in the culture medium, allowed to incubate for 30 min at 37 °C. After incubation, cells were washed twice with phosphate-buffered saline (PBS). Bright field and fluorescence images of HepG2 cells were taken by a fluorescence microscope (Leica DM3000, Germany) with an objective lens of 40× magnification. Fluorescence images of HepG2 cells were taken separately from another set of experiment where cells incubated with 10 μM PP + 10 μM Al³⁺ for 30 min. Similarly, in another set of experiment, cells were incubated with 10 μM PP + 10 μM Al³⁺ for 30 min followed by addition of 100 μM H₂EDTA²⁻ for another 30 min and fluorescence images were taken. HepG2 cells showed almost complete quenching of fluorescence due to removal of Al³⁺ from the complex.

Acknowledgements

Financial support from DST (Ref. SR/S1/IC-20/2012) New Delhi is gratefully acknowledged. We are also thankful to Professor Ajay Kumar Mishra, Department of Chemistry and Chemical Technology, Vidyasagar University for computational help.

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Footnote

† Electronic supplementary information (ESI) available: See DOI: 10.1039/c4ra06018k

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