Fluorescence sensing and intracellular imaging of Al³⁺ ions by using naphthalene based sulfonamide chemosensor: structure, computation and biological studies

Abstract

A naphthalene based sulfonamide Schiff base, (E)-4-(((2-hydroxynaphthalen-1-yl)methylene)amino)-N-(5-methylisoxazol-3-yl)benzenesulfonamide (HL) has been found to be a flourescence turn-on probe for selective detection of Al³⁺ in aqueous system. Structure of the probe has been established by FTIR, ¹H NMR, mass spectra and X-ray single crystal study. The probe has shown 24 times flourescence enhancement in presence of Al³⁺. The limit of detection (LOD) obtained by 3σ method is 33.2 nM. The probable co-ordination environment of L-Al²⁺ complex has been supported by mass spectral data and DFT computational study. By TD-DFT calculation UV-Vis spectra of HL and L-Al²⁺ complex has been predicted and that has well correlated with experimental data. Cell imaging study reveals that the probe can be used for the intracellular detection of Al³⁺ in cultured Vero cell. Antimicrobial activity of HL has also been evaluated and probable mode of binding inside the DHPS cavity has been predicted by docking study. This sensor is unique with reference to two other predecessors because of its bio-compatibility of sulfonamide derivative and nonmutagenicity.

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Introduction

Aluminium, the third most abundant metal in the earth's crust (8% of its mass), has the remarkable ability to resist corrosion and is a useful structural component of electronic and electrical gadgets, building materials, packaging stuff etc. Elevated aluminium causes neurotoxicity¹ and has been manifested Alzheimer's² and Parkinson's³ diseases, and hampers protein transportation in respiratory system, softening of bone, anemia in living beings. Al³⁺ interferes with the uptake of Ca²⁺ and results the growth retardation of living systems.⁴ Besides, the toxicity of aluminium is a hazard towards aquatic life⁵ and retards agricultural production in acidic soils.⁶ Notwithstanding, the use of Al cannot be avoided in modern life. Therefore, there is a good chance of accumulation of Al³⁺ and toxicity towards human health. As a result, developing of new and practical multi signaling chemosensor for Al³⁺ is very important.^7–9 To date several techniques are available for detection of Al³⁺ including atomic absorption spectra,¹⁰ electrochemical detection,¹¹ mass spectrometry,¹² and ²⁷Al NMR technologies.¹³ Recently fluorescence spectroscopic technique has received worldwide interest because of selective, efficient, time saving, environmentally benign and low cost chemosensing property. Many fluorescent probes have been characterized and some have been used successfully in Al neurochemistry. Most of them have been developed based on quinoline,^14–16 bipyridyl,^17,18 coumarin,^19,20 pyrazoline,^21,22 tripyrrins,¹⁸ BINOL,²³ fluorescein,^20,21 rhodamine^24,25 fluorophores. Functionalized Schiff bases are useful complexing agents and have been designed to incorporate fluorescent moiety for optical sensing of metal ions.^26–28 In this work, a sulfonamide based naphthol Schiff base, (E)-4-(((2-hydroxynaphthalen-1-yl)methylene)amino)-N-(5-methylisoxazol-3-yl)benzene sulfonamide (HL) is structurally characterized which shows high selectivity for Al³⁺ with low detection limit. Sulfonamides and their derivatives have widespread use as antibacterial medicine and several sulfa drugs based receptors are also reported in literature for anion sensing.^29–32 The composition of the complex has been supported by spectroscopic data (IR, Mass, Job's plot, ¹H NMR). The DFT computation of optimized geometry of HL and the complex has been used to explain the electronic spectral properties. The practical applicability of the ligand (HL) has been tested in African green monkey kidney cells (Vero cells, ATCC, Manassas, VA, USA) for the determination of exogenous Al³⁺ ions by fluorescence cell imaging processes and the cell cytotoxicity of the probe has been examined by MTT assay. The antimicrobial activity of HL has also been examined against selected Gram-positive and Gram-negative bacteria which revealed that HL had moderate antibacterial activity (32–64 μg ml⁻¹). The mechanism of antimicrobial activity has been proposed by in silico process of molecular docking of HL in the structure of DHPS protein.

Experimental

Materials and methods

Sulfamethoxazole was purchased from Hi-Media and 2-hydroxy naphthaldehyde was prepared from 2-naphthol following Duff reaction.³³ All other chemicals and solvents were of analytical grades and used without further purification.

Physical measurement

Melting points were determined on a melting point machine apparatus using open capillary. The IR spectra (in KBr pellets) were recorded on a Rx-1 Perkin Elmer spectrophotometer in the range 4000–400 cm⁻¹. The ¹H and ¹³C NMR spectra were recorded in DMSO-d₆ on Bruker 300 MHz FT-NMR spectrometer using TMS as internal standard. The mass spectra were recorded on a Water HRMS model XEVO-G2QTOF#YCA351 spectrometer. UV-Vis spectra were recorded on Lambda 25 Perkin Elmer spectrophotometer and emission spectra were measured in Perkin Elmer LS55 fluorescence spectrophotometer at room temperature (298 K). The fluorescence quantum yield was determined using anthracene as reference with a known quantum yield, ϕ_R = 0.27 in acetonitrile.³⁴ The experimental sample and reference were excited at same wave length, maintaining almost same absorbance and fluorescence were measured. Area of the fluorescence spectra were measured using the software available in the instrument and the quantum yield was calculated by following the formula

ϕ_u/ϕ_R = [A_u/A_R] × [(Abs)_R/(Abs)_u] × [η_u²/η_R²]

where, ϕ_u and ϕ_R are the fluorescence quantum yield of the samples and reference; A_u and A_R are the respective areas under emission spectra of the sample and reference respectively. (Abs)_R, (Abs)_u are the absorbance of sample and reference at the excitation wave length and η_u², η_R² are the refractive index of the solvent used for the sample and the reference.

Elemental analyses were performed using a Perkin-Elmer 2400 Series-II CHN analyzer, Perkin Elmer, USA elemental analyzer. Cell imaging study were executed using Zeiss Axiovert 40 CFL fluorescence microscope.

Synthesis of (E)-4-(((2-hydroxynaphthalen-1-yl)methylene)amino)-N-(5-methylisoxazol-3-yl)benzenesulfonamide (HL)

2-Hydroxynaphthaldehyde (0.2 g, 1.16 mmol) was dissolved in 15 ml of ethanol followed by the dropwise addition of ethanol solution of sulfamethoxazole (SMX) (0.3 g, 1.18 mmol in 10 ml) with constant stirring for 30 min. The mixture was then refluxed for 2.5 h. The solution was then cooled to room temperature and allowed to evaporate slowly in air; an orange colored crystals of the precipitate of (E)-4-(((2-hydroxynaphthalen-1-yl)methylene)amino)-N-(5-methylisoxazol-3-yl)benzenesulfonamide (HL) deposited on the wall of the beaker. The crystals were filtered and further recrystallized from hot ethanol. The purity of the product was checked by TLC; yield, 0.30 g (63%).

Mp, 244(2) °C. MS: m/z = 408.13; IR: 3458, 3066 (O–H), 2960, 1624 (azomethine, –HC=N–), 1587, 1351, 1170, 1180 (–S=O of SO₂), 1090, 842, 742, 623, 574. ¹H NMR (300 MHz, DMSO-d₆): δ 11.46 (1H, s, OH̲), 9.60 (1H, s, 11-H̲); 8.49 (1H, d, J = 8.4, 3-H̲); 7.96 (1H, s, 8-H̲); 7.92 (3Hs, d, J = 8.73, 14-H̲, 16-H̲, 4-H̲); 7.79 (2Hs, d, J = 8.4, 13-H̲, 17-H̲); 7.56 (1H, t, J = 7.56, 7-H̲); 7.38 (1H, t, J = 7.4, 6-H̲); 7.00 (1H, d, J = 9.2, 5-H̲); 6.17 (1H, s, 19-H̲); 2.29 (3Hs, s, –CH̲₃ at 15-C). ¹³C NMR (300 MHz, DMSO-d₆): δ 170.9 (18-C), 164.4 (2-C), 158.3 (20-C), 157.4 (11-C), 153.8 (15-C), 148.8 (12-C), 138.9 (1-C), 136.8 (3-C), 133.5 (4-C), 132.2 (9-C), 129.5 (10-C), 128.8 (14-C, 16-C), 127.3 (8-C), 124.7 (5-C), 122.5 (7-C), 121.6 (13-C, 17-C), 119.2 (6-C), 95.93 (19-C), 12.52 (CH₃ at 20-C). Anal. calcd for C₂₁H₁₇N₃O₄S (407): C, 61.90; H, 4.21; N, 10.31%. Found: C, 61.30; H, 4.40; N, 10.58%. Figures are given in ESI, Fig. S1 (Mass); Fig. S2 (IR); Fig. S3 (¹H NMR); Fig. S4 (¹³C NMR).†

Synthesis of Al³⁺ complex of HL

To the solution of HL (0.1 g, 0.245 mmol) in 30 ml MeOH, Al(NO₃)₃·9H₂O (0.1 g, 0.267 mmol) in same solvent (20 ml) was added drop wise with constant stirring. The yellow colored solution of HL turned colorless after complete addition of Al(NO₃)₃ and the solution was stirred for further 2 hours. The solution was allowed to evaporate slowly in air and gummy mass was extracted with CH₂Cl₂ and washed with water for several times. A light yellow mass was then dried in vacuuo and purity was checked by TLC prepared in alumina. A single band was eluted by chloroform–ethylacetate (1 : 1, v/v) and it was compared with TLC of HL under similar condition. Yield, 0.09 g (68%).

Light yellow sticky product was dried in vacuuo and MS (m/z) peak appeared at 486.17 (ESI, Fig. S5†); which has supported the composition [AlL(OH)(H₂O)₂](NO₃). The microanalytical data revealed that calcd (%): C₂₁H₂₁AlN₄O₁₀: C, 45.99; H, 3.86; N, 10.22% and found (%), C, 46.08; H, 3.75; N, 10.15%. In ethanol solution the complex shows 1 : 1 conductivity (Λ_M, 106 Ω⁻¹ cm² mol⁻¹). The FTIR spectrum also showed presence of a sharp band corresponding to free ν(NO₃) at 1370 cm⁻¹ along with ν(C=N), ν(SO₂), ν(OH) appear at 1615, 1194 and 2950 cm⁻¹ respectively (ESI, Fig. S6†). The ¹H NMR spectrum of the complex (300 MHz, DMSO-d₆) shows δ 10.05 (1H, s, 11-H̲); 7.03 (1H, b, 3-H̲); 8.19 (1H, s, 8-H̲); 7.61 (3Hs, d, J = 8.33, 14-H̲, 16-H̲, 7-H̲); 6.78 (2Hs, d, J = 8.05, 13-H̲, 17-H̲); 7.46 (1H, t, J = 7.5, 6-H̲); 7.82 (1H, d, J = 8.5, 5-H̲); 6.09 (1H, s, 19-H̲); 2.31 (3Hs, s, –CH̲₃ at 20-C) (b, broad; s, singlet; d, dublet; t, triplet) (ESI, Fig. S7†).

X-ray crystallography

The single crystals of HL (0.16 × 0.12 × 0.09 mm) were obtained by slow diffusion of mixture of dichloromethane with hexane solution (1 : 1, v/v). The X-ray crystal data were collected (Table 1) by Bruker Smart Apex II CCD Area Detector at 296(2) K. Diffraction were recorded with 2θ in the range 1.74 ≤ θ ≤ 25.00° (HL). Fine-focus sealed tube was used as the radiation source of graphite-monochromatized MoKα radiation (λ = 0.71073 Å). Data were corrected for Lorentz and polarization effects and an empirical absorption correction in the hkl range for −9≤ h ≤ 9; −11 ≤ k ≤ 11; −14 ≤ l ≤ 15 for HL. The absorption corrections were done by the multi-scan technique.³⁵ All data were corrected for Lorentz and polarization effects, and the non-hydrogen atoms were refined anisotropically. The structure was solved by direct methods with SHELXL-97 (ref. 36) and refined by full-matrix least-squares techniques on F² using the SHELXS-97 (ref. 36) program with anisotropic displacement parameters for all non-hydrogen atoms. The ORTEP-3 (ref. 37) was used within WinGX³⁸ to prepare figures and tables for publication. Hydrogen atoms were constrained to ride on the respective carbon atoms with isotropic displacement parameters equal to 1.2 times the equivalent isotropic displacement of their parent atom in all cases of aromatic units.

Table 1 Crystallographic data of HL

a R = ∑||Fo| − |Fc||/∑|Fo|.b wR = {∑[w(Fo^2 − Fc^2)^2]/∑[w(Fo^2)^2]}^1/2; w = [σ^2(Fo)^2 + (0.0805P)^2]^−1 where P = (Fo^2 + 2Fc^2)/3.c Goodness-of-fit.
Empirical formula	C21H17N3O4S
Formula weight	407.44
Temperature (K)	296(2)
System	Triclinic
Space group	P1̄
a (Å)	8.3710(5)
b (Å)	10.0074(6)
c (Å)	12.8160(8)
α (Å)	67.264(4)
β (°)	80.722(5)
γ (°)	86.904(5)
V (Å)^3	977.20(10)
Z	2
μ (MoKα) (mm^−1)	0.199
θ range	1.74–25.00
Dcalc (mg m^−3)	1.385
Refine parameters	0.067
Total reflections	13 194
Unique reflections	3316
R1^a [I > 2σ(I)]	0.0602
wR2^b	0.1661
GOF^c	0.989

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Computational details

Electronic structure optimization of the ligand and Al complex were carried out in B3LYP³⁹ using Gaussian09 software⁴⁰ package. The 6-31G(d, p) basis set was assigned for all the elements except sulfur. For sulfur atom 6-31+G(d, p) basis set was used. Theoretical computation for UV-Vis absorption spectral studies were done by time dependent density functional theory (TD-DFT)^41–43 in methanol conductor-like polarizable continuum model (CPCM)^44–46 using the same B3LYP level and basis set. Gauss sum⁴⁷ was used to calculate theoretical electronic spectra and molecular orbital contribution from groups or atoms.

Cell culture and cell imaging study

The Vero cells (African green monkey kidney cells, ATCC, Manassas, VA, USA) was grown and maintained in Dulbecco's modified Eagle's medium (DMEM, Gibco, USA), supplemented with 5–10% fetal bovine serum (FBS, Gibco, USA) in an atmosphere of 5% CO₂ at 37 °C. Vero cells monolayer (1.0 × 10⁶ cells per ml) grown onto 6 well plates at 5% CO₂ for 24 h, were fixed with paraformaldehyde (4%) and blocked with 1% bovine serum albumin (BSA) in 0.1% phosphate buffered saline (PBS, pH 7.2)-triton X100 solution. The cells were washed with PBS, and then permeabilized with 0.1% triton X100 in PBS. The permeabilized Vero cells monolayer treated with the compound (100 μg ml⁻¹) for 1 h at room temperature was then washed twice with PBS to remove the cell debris. After washing with PBS, the cells were added with Al(NO₃)₃·9H₂O for 10 min, washed twice with PBS, and then the cells were observed under Axiovert 40 CFL inverted epifluorescence microscope.⁴⁸

Cell cytotoxicity assay

Cytotoxicity of HL on Vero cells were performed following the MTT ([(3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium)]) assay as per the protocol described earlier.^49,50 The cells seeded in 96 well plates were treated with different concentrations of HL and incubated for 24 hours in 5% CO₂ at 37 °C. For the MTT assay, thiazolyl blue tetrazolium bromide solution (100 μL; 1 mg ml⁻¹) in incomplete medium was added and this mixture incubated for 4 hours. After that, 100 μl of dimethylsulphoxide (DMSO) was added and the plates were rotate for 5 minutes. Optical density was recorded at 550 nm with DMSO as the blank. Percentage of cell viability was plotted against different concentrations of HL and the cells treated without any compound served as control.

Antimicrobial assay

(E)-4-(((2-Hydroxynaphthalen-1-yl)methylene)amino)-N-(5-methylisoxazol-3-yl)benzene sulfonamide (HL) was tested against Gram-positive (S. aureus ATCC 21737, Enterococcus facelis ATCC 29212) and Gram-negative (E. coli, ATCC 25922, S. typhi MTCC734, K. pneumonia ATCC 714) bacterial strain. Minimum inhibitory concentration (MIC) values of HL and SMX were determined according to CLSI guidelines following broth microdilution method⁵¹ and MIC data were recorded where no visible growth was observed. The bacterial growth and culture conditions were examined as literature report⁵² and all independent experiments were repeated three times.

Result and discussion

Synthesis and formulation of probe

The condensation of 2-hydroxy naphthaldehyde with sulfamethozaxole (SMX) in ethanol under refluxing condition using 1 : 1 molar ratio (ESI, Scheme S1†) has synthesized (E)-4-((2-hydroxynaphthalen-1-yl)methylene)amino)-N-(5-methylisoxazol-3-yl) benzene sulfonamide (HL). The mass spectral peak of HL appears at (m/z) 408.129 (ESI, Fig. S1†) (mol weight, 407.44). The characteristic stretching vibrations of HL are ν(O–H), 3066 cm⁻¹; ν(C=N), 1624 cm⁻¹; ν(S–O), 1170 (sym) and 1351 (asym) cm⁻¹ (ESI, Fig. S2†). The ¹H NMR spectrum of HL (DMSO-d₆) shows two higher frequency resonances at 11.46 ppm (δ(OH)) and 9.6 ppm (δ(CH=N); oxazolyl–Me signal appears at 2.29 ppm, the oxazolyl-H resonates at 6.17 ppm and four phenyl Hs exhibit two doublets at 7.92 ppm (14,16-H, J = 8.73 Hz) and 7.79 ppm (13,17-H, J = 8.70 Hz), naphthyl-Hs are assigned to 8.49–6.90 ppm (ESI, Fig. S3†). The ¹³C NMR spectrum (in DMSO-d₆) of HL is very complex and has been assigned on comparing with literature report⁵³ (ESI, Fig. S4†).

The structure of HL has been confirmed by single crystal X-ray diffraction measurements. The ORTEP plot of HL is shown in Fig. 1 and the selected bond parameters are listed in Table 2. The structure shows that the imine group (–CH=N–) binds naphtholato and SMX. The –SO₂NH– bridges C₆H₄–CH=N– and methyl-oxazolyl units. The S=O bond length (1.423(3), 1.433(3) Å)^53–55 in SMX and imine, –C=N–, bond length, C(11)–N(3) (1.327 Å)⁵⁶ are corroborated with literature data. The bond angle ∠O(2)–S(1)–O(1), 120.31°(17) appropriates to trigonal symmetry. Intramolecular and intermolecular hydrogen bonds such as O(4)–H(4)⋯N(3)–C(8), (H(4)⋯N(3), 1.71(6) Å; O(4)⋯N(3), 2.538(3) Å), ∠O(4)–H(4)⋯N(3), 152(8)°, O(4)–H(4)⋯N(2)–C(4) (H(4)⋯N(2), 2.03(4) Å; O(4)⋯N(2), 2.791(5) Å), ∠O(4)–H(4)⋯N(2), 164(3)°; symmetry, −x, −y, −z give rise to the formation of an 1D tape and π⋯π interaction between sulfonamide phenyl ring and naphthyl ring of neighboring molecule (Cg(2)⋯Cg(3), 3.668(3) Å where Cg(2): C(5)–C(6)–C(7)–C(8)–C(9)–C(10) and Cg(3), C(12)–C(13)–C(14)–C(15)–C(16)–C(21)) generates 2D supramolecular plane (Fig. 2). Relevant H bonds are shown in ESI (Table S1†).

Fig. 1 ORTEP plot of HL with 35% ellipsoidal probability.

Table 2 Selected geometrical parameters of HL

Bond distance (Å)			Bond angle (°)
	X-ray	Calculated		X-ray	Calculated
C(8)–N(3)	1.402(4)	1.408	C(7)–C(8)–N(3)	117.3 (3)	117.45
C(11)–N(3)	1.327(4)	1.316	C(8)–N(3)–C(11)	126.86	122.91
C(11)–C(12)	1.383(5)	1.435	N(3)–C(11)–C(12)	123.3(4)	121.94
C(12)–C(13)	1.450(5)	1.420	C(11)–C(12)–C(13)	119.7(3)	118.92
C(13)–C(14)	1.407(5)	1.419	C(12)–C(13)–O(4)	120.8(4)	121.66
C(13)–O(4)	1.274(4)	1.352	C(14)–C(13)–O(4)	120.7(4)	108.32
O(4)–H(1)	0.899(2)	1.032	O(2)–S(1)–N(2)	105.16(18)	100.67

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Fig. 2 2D supramolecular structure generated by hydrogen bonding and π⋯π interaction in HL.

The structural parameters calculated from optimized structure using DFT computation technique are marginally elongated by 0.006–0.13 Å (bond lengths) and 0.5–2° (bond angles) (Table 2) from that of X-ray crystallographic data. Thus the optimized functions are useful to explain the electronic structure and electronic properties of the compound (vide infra).

Absorption spectroscopic study and Al³⁺ sensing

The ligand, HL exhibits well-defined absorption bands at 450, 370, 317 and 267 nm in MeOH–H₂O (1 : 5, v/v) (HEPES buffer, pH = 7.4). Aqueous solution of different metal ions (Na⁺, K⁺, Ca²⁺, Mg²⁺, Ba²⁺, Hg²⁺, Ni²⁺, Co²⁺, Pb²⁺, Pd²⁺, Mn²⁺, Cd²⁺, Cu²⁺, Fe³⁺, Fe²⁺ and Zn²⁺) has been added to methanol solution of HL maintained at pH, 7.4 and the absorption spectra are recorded. The spectral pattern of HL does not show any significant change on adding majority of metal ions except Al³⁺ (Fig. 3). The interaction of HL with Al³⁺ has been examined by spectrophotometric titration at 25 °C in MeOH–water (1 : 5, v/v, pH = 7.4) (Fig. 3). Intense absorption of HL at 450 nm has been blue shifted to 360 nm upon addition of Al³⁺ solution and naked eye observation is the changes of colour from bright yellow to colorless. The bright yellow color has remained unchanged in presence of other metal ions (ESI, Fig. S8†). The spectral observation undoubtedly suggests the conversion of free HL to the corresponding Al³⁺ complex. The appearance of isosbestic point (298 nm) also clearly indicates that the reaction is clean and straightforward. The change of absorbance is linear until the molar ratio [Al³⁺] : [HL] reaches 1 : 1, and no longer changes with increase in [Al³⁺]. It suggests that the stoichiometry between HL and Al³⁺ is 1 : 1 and the association constant (K_a) is 1.38(1) × 10⁴ M⁻¹ l (ESI, Fig. S9†). Preference of HL to Al³⁺ has been examined by solution absorption spectral presentation by adding O-donor ligands such as Na₂EDTA, citrate, oxalate, acetate to the solution. The spectral pattern and symmetry remains undisturbed only the absorption intensity of L-Al²⁺ is increased slightly upon addition of O-donor ligands in the ratio Al³⁺ : HL : O-donor ligands 1 : 1 : 1 (ESI, Fig. S12†). However, increase concentration of O-donor ligands even upto 1 : 1 : 6 does not show any change. This implies higher preference of HL to Al³⁺.

Fig. 3 Change in absorption spectra of HL on gradual addition of Al³⁺.

Fluorescence spectroscopic study

The ligand, HL, emits weakly at 520 nm when excited at 370 nm and the fluorescence quantum yield is (ϕ_HL) 0.0057. The excitation at 450 nm does not show any emission while excitation of other absorption bands at 317 and 267 nm also show emission at 520 nm. On gradual addition of Al³⁺ the emission band of HL (520 nm) is blue shifted to 425 nm. The fluorescence spectrum of HL with other cations (Na⁺, K⁺, Ca²⁺, Mg²⁺, Ba²⁺, Hg²⁺, Ni²⁺, Co²⁺, Pb²⁺, Pd²⁺, Mn²⁺, Cd²⁺, Cu²⁺, Fe³⁺ and Zn²⁺) using their acetate salts in MeOH–water (1 : 5, v/v, pH = 7.4) solution have been examined on excitation at 370 nm (Fig. 4) and the turn-on emission is observed in presence of Al³⁺ at room temperature (25 °C). On increasing [Al³⁺] to the solution of HL the fluorescence intensity increases and becomes saturated when reached at 1 : 1 molar ratio which results enhancement of quantum yield (ϕ, 0.137, 24 fold compared to ligand) (Fig. 4). Addition of excess of [Al³⁺] has no effect on the emission intensity of the mixture. Such an enhancement in fluorescence intensity is very selective for Al³⁺ (Fig. 5). The increment in fluorescence intensity for HL + Al³⁺ may arise from the elimination of photoinduced electron transfer (PET) in free HL and chelation enhancement effect (CHEF) through the co-ordination of azomethine-N and phenolic-O to metal ion (Scheme 1). To get further insight about the complexation reaction of HL and Al³⁺ fluorometric titration has been done in MeOH–H₂O (1 : 5, v/v, pH = 7.4) (Fig. 6c). [(F_max − F₀)/(F − F₀)] vs. 1/[Al³⁺] has been plotted following Benesi–Hildebrand equation (Fig. 6b) and a straight line is obtained. From the slope of the best fit line binding constant [K_d, (1.04 ± 0.01) × 10⁴] has been calculated. The limit of detection (LOD) of Al³⁺ has been calculated 33.2 nM following the 3σ method (ESI, Fig. S10†). The fluorescence enhancement of L-Al²⁺ complex has been persisted in existence of other metal ions. Hence, HL can detect Al³⁺ selectively in biological or environmental samples even in presence of other metal ions. Naphthyl appended fluorophores are efficient detector of Al³⁺ at very low concentration level. Singh et al.⁵⁷ reported lowest LOD of Al³⁺ (1.35 nM) using (E)-N-[(2-hydroxy-naphthalen-1-yl)methylene]thiophene-2-carbohydrazide and second lowest LOD was obtained by Das et al.⁵⁸ (10 nM) with 2-((naphthalen-6-yl)methylthio)ethanol. In this work we report third lowest LOD of Al³⁺ (33.2 nM). Other reports show LOD of Al³⁺ higher than 50 nM.¹⁸ Although present report is in third position in the detection efficiency series but its advantage is the use of biocompatible sulfonamide derivative. Sulfonamides are established drug; hence they are biofriendly and nontoxic (see below MTT assay, ADMET prediction). Effect of pH variation on fluorescence intensity of HL and L-Al²⁺ complex has been studied; it has observed that there is no significant fluorescence emission of HL at the pH range of 4 to 12 but in presence of Al³⁺ the ligand emits in the pH range between 4.0 to 8.5 (Fig. 7). This finding indicates that HL is useful for detection of Al³⁺ in biological pH. To establish the binding stoichiometry of HL and Al³⁺ Job's plot has been generated by plotting fluorescence intensity against different mole fractions of Al³⁺ while volume of solution has remained fixed (ESI, Fig. S11†). Maxima in this plot has been obtained at ∼0.5 mole fraction, which suggests about 1 : 1 complex formation of HL and Al³⁺. Upon addition of one equivalent of O-donor ligands such as Na₂EDTA, citrate, oxalate, acetate to the solution of L-Al²⁺ the fluorescence intensity is decreased by 20% (Na₂EDTA), 36% (oxalate, acetate) and 44% (citrate); however, complete recovery of HL has not been successful even in presence of large excess (>10 times) of O-donor ligands (ESI, Fig. S13†). This is supportive to absorption spectroscopic data of preference of HL to Al³⁺.

Fig. 4 The receptor (HL) shows strong emission in presence of Al³⁺ at 450 nm on excitation at 360 nm.

Fig. 5 Bar chart presenting fluorescence response of HL in presence of different metal ions.

Scheme 1 Schematic representation of enhancement of fluorescence of HL in presence of Al³⁺.

Fig. 6 (a) Fluorescence titration of HL in MeOH–water (1 : 5, v/v, pH 7.4) by the gradual addition of Al³⁺ with λ_ex = 370 nm, (b) Benesi–Hilderman plot, and (c) plot of fluorescence intensity vs. [Al³⁺].

Fig. 7 Fluorescence intensity of HL and L-Al²⁺ complex at different pH.

Time resolved flourescence measurement of HL in presence and absence of Al³⁺ shows that complexation enhances life time (τ_complex, 0.9 ns) than the free ligand (τ_HL, 0.05 ns). The decay profile of HL and of its L-Al²⁺ complex is shown in Fig. 8. Lifetime data were taken on excitation at 370 nm. The fluorescence decay fits bi-exponential nature. The radiative and non-radiative rate constants (k_r and k_nr) were calculated and the data show k_nr values, which are unusually higher with respect to the k_r ones (ESI, Table S2†).

Fig. 8 Decay profile of HL and L-Al²⁺ complex.

Stoichiometric study by NMR titration

To elucidate the nature of the binding of HL to Al³⁺ the ¹H NMR spectrum of HL has been investigated in absence and in presence of Al³⁺ in CD₃OD (Fig. 9). The ¹H NMR spectrum of HL shows signals for HC=N (azomethine) at 9.64 ppm and other aromatic-Hs appear at 6.97 to 8.32 ppm (ESI, Fig. S3†) and in Al-L²⁺ complex the naphtholato-Hs (3-H to 8-H; Δδ = 0.053 (3-H), 0.015 (4-H), 0.158 (5-H), 0.108 (6-H), 0.035 (7-H), 0.351 (8-H) ppm respectively), imine-H (11-H, Δδ = 0.41 ppm) undergo downfield shifting while aromatic/heterocyclic ring(s) of sulfonamide, 13-H to 19-H shift to lower δ (Δδ = 0.369 for 14, 16-H ppm; 0.777 ppm for 13, 17-H; 0.166 ppm for 19-H). Addition of 0.5 equivalents of Al(NO₃)₃ leads to a downfield shift of the signal corresponding to the CH=N proton (Δδ = 0.506 ppm) (Fig. 9). The co-ordination through phenolic oxygen atoms confirmed by NMR titration as the signal of phenolic-OH signal (δ, 11.46 ppm) is vanished after addition of one equivalent Al(NO₃)₃. Thus, according to the ¹H NMR titration data, interaction of HL with Al³⁺ leads to the formation of a [Al-L]²⁺ structure. The stoichiometry of L-Al²⁺ complex has been further established by means of mass spectroscopic data. ESI-MS data of HL and Al-L²⁺ gives a peak at m/z 408.13 and 486.17 (ESI, Fig. S1 and S5†) respectively which corresponds to [Al(L)(OH)(H₂O)₂]⁺ and signifies the 1 : 1 complexation.

Fig. 9 ¹H NMR titration of HL by Al³⁺ in CD₃OD at 294 K.

Density functional theory calculation

Geometry optimization of HL and [Al(L)(OH)(H₂O)₂]⁺ has been performed using DFT calculation with B3LYP method and 6-31G(d, p) basis set. Based on mass spectral data composition of the complex ion has been assigned as [Al(L)(OH)(H₂O)₂]⁺. According to DFT optimized structure Al is penta-coordinated and distorted square pyramid in geometry (Fig. 10). HL acts as mono anionic N, O chelator to Al³⁺; theoretically calculated Al–N(imine) and Al–O(phenolato) distances are 1.946 Å and 1.827 Å, respectively and have been comparable with similar structure.^59,60 Due to complex formation the C(8)–N(3), C(11)–N(3), C(13)–O(4) bond distances have been significantly elongated compared to free ligand (ESI, Tables S3 and S4†). The result shows that electron density in HOMO of HL is mainly distributed on the naphthyl ring and LUMO is distributed between naphthyl and sulfonamide moiety. For HOMO of [Al(L)(OH)(H₂O)₂]⁺, the π electron density is allocated mainly on naphthyl ring but the LUMO is spread over azomethine, sulfonamide and naphthyl moiety. Contour plots of the selected orbitals of the compounds have been given in ESI, Fig. S14 and S15.† The HOMO–LUMO gap in HL (3.43 eV) has slightly increased in [Al(L)(OH)(H₂O)₂]⁺ (3.64 eV) which supports the blue shift in UV-Vis spectra (Fig. 11).

Fig. 10 Optimized structure of HL and L-Al²⁺ by DFT/B3LYP 6-311g(d) method.

Fig. 11 Frontier molecular orbital of HL and Al-L²⁺.

To interpret electronic spectra of HL and [Al(L)(OH)(H₂O)₂]⁺ the TD-DFT calculation has been executed by DFT/B3LYP method using 6-31G(d, p) basis set in MeOH. The ligand exhibit four electronic transitions corresponding to 450, 370, 317, 267 nm and they are assigned for S₀ → S₁, S₀ → S₃, S₀ → S₁₁, S₀ → S₂₀ (Fig. 12). In the UV-Vis spectra of HL there is a strong band at visible region (450 nm) which is assigned to HOMO → LUMO, intramolecular charge transfer π(naphthyl ring) → π*(naphthyl/sulphonamide ring), and it is blue shifted upon addition of Al³⁺ giving rise to 360 nm (Fig. 14). The complex shows three bands at 360, 318 and 267 nm in UV-Vis spectrum (Fig. 3) who are assigned to S₀ → S₁, S₁ → S₄ and S₀ → S₁₁ transitions (Fig. 13). Absorption energies of HL and its Al³⁺ complex along with their oscillator strength are given in ESI, Table S5.†

Fig. 12 Frontier molecular orbitals involved in electronic transition in UV-Vis region of HL.

Fig. 13 Frontier molecular orbitals involved in electronic transition in UV-Vis region of L-Al²⁺.

Biological application

Cell imaging. The application of HL as fluorescent probe for in vitro living cell imaging and detection of intracellular Al³⁺ ions have been studied on African green monkey kidney cells (Vero cells, ATCC, Manassas, VA, USA) using fluorescence microscope. The fluorescence imaging of intracellular Al³⁺ in living cells is shown in Fig. 14. Vero cells fixed in paraformaldehyde (4%) and blocked with BSA in PBS-triton X100 solution. Then, the cells were observed under epifluorescence microscope.⁴⁸ Then, the cells were treated with Al³⁺ solution (10 μM HL + 10 μM Al³⁺) for 30 min in buffer for incubation, washed again with buffer at pH 7.4 and mounted on a grease free glass slide. Cells were observed under a fluorescence microscope equipped with a UV filter after adding HL (2 mM). Cells incubated only with Al³⁺ were used as a control. HL can permeate easily through tested living cells without any harm (as the cells remain alive even after 30 min of exposure to HL at 2 mM). The MTT assay has been applied for labeling dead cells to evaluate cytotoxicity of the probe and is shown in Fig. 15. The study shows that HL has no cytotoxicity towards cells upto 100 μg ml⁻¹ (approx. 250 μM). These results indicate that the probe has a huge potential in both in vitro and in vivo applications as Al³⁺ sensor and in live cell imaging. ADMET (absorption, distribution, metabolism, excretion and toxicity) prediction of HL (ESI, Table S6†) says that HL has good aqueous solubility and absorption, so it might have chemical activity for detection of Al³⁺ within living cells. These results in contrast to two other ligands of lower LOD;^57,58 the ligand, (E)-N-[(2-hydroxy-naphthalen-1-yl)methylene]thiophene-2-carbohydrazide shows LOD to Al³⁺ 1.35 nM but no cell imaging or cytotoxicity studies are reported. Although 2-((naphthalen-6-yl)methylthio)ethanol (LOD to Al³⁺, 10 nM)⁵⁸ shows cell signaling activity but no cytotoxicity studies is reported. We have checked the mutagenic activity by using ADMET filtration and it is computed that 2-((naphthalen-6-yl)methylthio)ethanol is mutagenic. Although present report of HL shows one order lower LOD to Al³⁺ but biologically more friendly and nonmutagenic.

Fig. 14 (a) Cell control, (b) cell control in fluorescence microscope, (c) cell in presence of HL, (d) cell in presence of L-Al²⁺ complex.

Fig. 15 Cell viability data of HL.

Antibacterial and molecular docking studies. The fluorescent probe HL is a Schiff base derivative of sulfamethoxazole, so antimicrobial activity of HL against several Gram-positive (S. aureus ATCC 21737, Enterococcus facelis ATCC 29212) and Gram-negative (E. coli, ATCC 25922, S. typhi MTCC734, K. pneumonia ATCC 714) bacterial strain. The Schiff base has shown moderate antimicrobial activity (MIC 32–64 μg ml⁻¹) (ESI, Table S7†).

The molecular docking of HL has been carried out with DHPS (dihydropteroate synthase) to find the probable mode of binding using CDOCKER module of receptor–ligand interaction section of Discovery Studio Client 4.1. It has been observed that there are 15 amino acid residues (Asn22, Ser27, Phe28, Ser61, Thr62, Arg63, Pro64, Asp96, Gln149, Gly189, Phe190, Lys221, Ser222, Gln226, Arg255) (Fig. 16) close to the ligand in DHPS-HL complex and HL forms three H-bonds with Ser61 (2.51 Å, ∠D–H–A (Ser OH⋯N oxalyl) 160°), Arg63 (2.93 Å, ∠D–H–A (Arg NH⋯N oxalyl) 116°) and Ser222 (2.83 Å, ∠D–H–A (Ser HO⋯HO phenol) 103°) (Fig. 17). In the best docked pose of L the binding energy (E_binding = E_complex − E_ligand − E_receptor) is −53.7 kcal mol⁻¹ (ESI, Table S8†).

Fig. 16 Best docked pose (2D) of HL inside the cavity of DHPS.

Fig. 17 Best docked pose (3D) of HL inside the cavity of DHPS.

Conclusion

In summary, naphthyl attached to sulfonamide by –C=N– has been successfully prepared and serves as selective fluorogenic probe to Al³⁺ ion in presence of large number of other metal ions. The receptor itself is weakly fluorescent while Al³⁺ has enhanced its emission by 24 fold and limit-of-detection (LOD), 33.2 nM, is third lowest in the series of naphthyl based sensor. The binding composition of receptor and Al³⁺ is 1 : 1 and that has been established by ¹H NMR, MS studies and further supported by DFT computation. Cell imaging experiment and MTT assay reveals that the new ligand can be efficient probe to detect Al³⁺ in the biological samples. The advantage of present sensor over other reported molecules^57,58 are [a] least interference by other metal ions, [b] experiment requires less organic solvents, [c] it is non mutagenic, less toxic and compatible in biological pH, [d] it is the 1^st reported molecule from sulfonamide family to detect Al³⁺ cation. Besides, sulfonamide Schiff base antibacterial activity against several Gram-positive (S. Aureus ATCC 21737, Enterococcus facelis ATCC 29212) and Gram-negative (E. coli, ATCC 25922, S. typhi MTCC734, K. pneumonia ATCC 714) bacterial strain of MIC, 32–64 μg ml⁻¹.

Acknowledgements

Financial support from West Bengal DST (228/1(10)/(Sanc.)/ST/P/S&T/9G-16/2012), Kolkata and University Grants Commission (F.42-333/2013(SR)), New Delhi are gratefully acknowledged. S. Mondal is thankful to UGC, New Delhi, India for fellowship. We are thankful to Mr Kumer Saurav Keshri an M.Sc student of Indian School of Mines, Dhanbad, for his initial contribution in the synthesis and some characterisation part of this work.

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Footnote

† Electronic supplementary information (ESI) available: Reaction detail in Scheme S1; the spectral data of HL (mass, Fig. S1; FT-IR, Fig. S2; ¹H NMR, Fig. S3; ¹³C NMR, Fig. S4), MS spectrum of [L-Al(OH)(H₂O)₂]⁺ (Fig. S5); IR spectrum of [L-Al(OH)(H₂O)₂]⁺ (Fig. S6); ¹H NMR spectrum of the complex (Fig. S7); naked eye observation of colour change of HL solution by adding different cation (Fig. S8); Benesi–Hildebrand plots (Fig. S9), limit of detection (LOD), (Fig. S10); Job's plot (Fig. S11); effect of O-donor chelating agents on absorption spectrum of L-Al²⁺ (Fig. S12); effect of O-donor chelating agents on fluorescence spectrum of L-Al²⁺ (Fig. S13); contour plots of HL (Fig. S14); contour plots of [L-Al(OH)(H₂O)₂]⁺ (Fig. S15). Hydrogen bonds for HL (Table S1); life time data of L and L-Al²⁺ complex (Table S2); selected geometrical parameter (calculated) of L-Al²⁺ in the ground state (Table S3); change in bond length (calculated) in (Al-L²⁺) compared to free ligand (HL) (Table S4); calculated transitions and their assignment for ligand (HL) and Al³⁺-complex (Table S5); ADMET prediction data (Table S6); antimicrobial results (MIC) of HL (Table S7); docking score of HL with DHPS protein (downloaded from PDB) (Table S8) CCDC 1405369. For ESI and crystallographic data in CIF or other electronic format see DOI: 10.1039/c5ra11548e

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