Isolation of proton transfer complexes containing 4-picolinium as cation and pyridine-2,6-dicarboxylate complex as anion: crystallographic and spectral investigations, antioxidant activities and molecular docking studies

Abstract

Three novel complexes with stoichiometry [4-pic-H][M(pda)₂]·2H₂O [M = Cr (1), Fe (2) and Co (3); H₂pda = pyridine-2,6-dicarboxylic acid and 4-pic = 4-picoline] have been prepared. The complexes (1–3) are characterized using elemental analysis, TGA, CV, FTIR, ESI mass, ¹H & ¹³C NMR, EPR, UV Visible, fluorescence, magnetic and X-ray studies. Spectral data ascertained the bonding modes and the geometry of the complexes. Single crystal X-ray data of (2) and (3) revealed the formation of proton transfer complexes in which a proton is transferred from the H₂pda moiety to the pyridine nitrogen of 4-pic. Thermal and ESI mass data confirmed the proposed stoichiometry of the complexes. Cyclic voltammetric (CV) studies confirm the formation of M^II/M^III quasi-reversible redox couples in solution. The antioxidant activity of (3) assessed using DPPH and hydrogen peroxide assays has suggested that the present compounds may be used as potent antioxidants. Molecular docking studies performed for (2) and (3) reveal that the present complexes can efficiently bind with DNA receptor with free energy of binding (FEB) values of −314 (2) and −276.8 kcal mol⁻¹ (3). The molecular docking studies indicated a higher binding ability of (2) to DNA compared to that of (3).

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1. Introduction

The coordinating nature and type of ligands with specific symmetry are key factors to the structures and properties of the final products. Functionalized polycarboxylic acids are among the multidentate ligands which can yield a number of mono and poly metallic coordination complexes. Pyridine-2,6-dicarboxylic acid (H₂pda) has played a key role participating in interesting coordination chemistry.^1,2 The reasons for this interest is the ability of H₂pda to give stable chelates with different versatile flexible [N, O, O] coordination modes with metal ions, the great affinity to form strong hydrogen bonds and its biological activities.^3,4 H₂pda is a planer ligand with a rigid 120° angle between the central pyridine ring and the two carboxylate groups, and therefore could potentially provide various coordination modes to form both discrete and consecutive metal complexes under appropriate synthesis conditions. The most important reactions of H₂pda are proton transfer in acid/base systems with specific interactions such as hydrogen bonding. Their acid–base/proton transfer reactions are important in inorganic chemistry.^5–8 Polycarboxylic acids are most probably used as building blocks for construction of metal coordination complexes by the proton transfer processes. The binding of two or more carboxylic groups in different angles allow the creation of 1D (long chain), 2D (sheet) or 3D (cage) structure.^9–12 Non covalent interaction is backbone of supra molecular and molecular recognition chemistry. Non covalent interactions help to build molecular clusters while a covalent interaction is to form classical molecules.¹³ Non covalent molecular clusters affect the properties of the subsystem, and these changes are important for the detection of cluster formation. The stronger non-covalent interaction causes larger changes in the properties of the subsystem.^14,15 Many proton transfer complexes have been prepared using H₂pda and other ligand like propanol amine.¹⁶ However the chemistry of proton transfer complexes of H₂pda with a heterocyclic moiety has not been reported to the best of our knowledge. We have used H₂pda and 4-picoline (4-pic) in our system. H₂pda behaves as a proton donor and 4-picoline acts as a proton acceptor, which is interacting by van der Waals force as H-bonding. Since such proton transfer complexes also exhibited biological (like SOD) activities, the antioxidant (free radical scavenging) properties are also explored. Moreover, molecular docking studies are performed to investigate the effect of the complexes on DNA receptor.

2. Experimental

2.1. Materials and methods

All reagents of analytical grade were obtained from commercial sources and used without further purification. The infrared (IR) spectrum of the compound was recorded within the range 400–4000 cm⁻¹ using KBr pellets on Perkin Elmer Model spectrum GX spectrophotometer. Melting points was determined by open capillary method and are uncorrected. An electronic spectrum and molar conductivity of 10⁻³ M solution in MeOH was recorded on Perkin Elmer λ-25 UV-visible spectrophotometer, cuvettes of 1 cm path length and Systronics-305 digital conductivity bridge, respectively at room temperature. The elemental C, H and N analyses were obtained from Micro-Analytical Laboratory of Central Drug Research Institute (CDRI), Lucknow, India. ESI mass spectra were recorded on a WATERS Q-TOF Premier mass spectrometer. Fluorescence measurements were determined on Hitachi F-2700 spectrophotometer. ¹H and ¹³C NMR spectra were recorded on a Bruker Avance II 400 NMR spectrometer from Punjab, university Chandigarh, India. The EPR spectrum of the complexes was acquired on a Perkin-Elmer spectrometer using X-band frequency (9.1 GHz) at room temperature in solid. Thermal gravimetric analysis (TGA) data were recorded from room temperature up to 800 °C at a heating rate of 20 °C min⁻¹. The data were obtained using a Shimadzu TGA-50H instrument. Magnetic susceptibility was measured by magnetic susceptibility balance, Sherwood scientific Cambridge U. K. at room temperature. Cyclic voltammetry (CV) was performed on EG&G PAR 273 Potentiostat/Galvanostat an IBM PS2 computer with EG&G M270 software for data acquisition. The three-electrode cell configuration comprised of, a platinum sphere, a platinum plate and Ag(s)/AgNO₃ were used as working, auxiliary and reference electrodes, respectively. The supporting electrolyte used was [ⁿBu₄N]·ClO₄. Platinum sphere electrode was sonicated for 2 min in dilute nitric acid, dilute hydrazine hydrate and then in double distilled water to remove the impurities. The solutions were deoxygenated by bubbling research grade nitrogen gas and an atmosphere of nitrogen was maintained over the solution during measurements.

2.2. Antioxidant (scavenging) activity

2.2.1. DPPH free radical scavenging activity. The antioxidant activity of the complex (3) was determined by DPPH free radical assay.^17,18 The mixture solution (10⁻³ M ethanol) contains 1 mL of sample, 1 mL of DPPH radical solution and 5 mL of absolute ethanol (1 : 1 : 5). On the basis of reaction of antioxidant compounds with 2,2-diphenyl-1-picrylhydrazyl (DPPH), compound donates proton and which is reduced. All the sample solution were incubated at 60 °C on water bath for 100 minutes and the color of the sample solution changes from violet to yellow and absorption band is recorded at 515 nm by using a UV-Vis spectrophotometer (Perkin Elmer, Lambda 40). The mixture of 6 mL ethanol and 1 mL sample solution was used as blank while the control was prepared by mixing ethanol (6 mL) and DPPH radical solution (1 mL). The scavenging activity percentage (AA%) was determined by the following relation:
where A_control = absorbance of DPPH˙ in ethanol without an antioxidant and A_sample = absorbance of DPPH˙ in the presence of an antioxidant.

2.2.2. Hydrogen peroxide scavenging activity. The hydrogen peroxide scavenging ability of the complex (3) was determined by standard method.¹⁹ Hydrogen peroxide solution (2 mM) was prepared in phosphate buffer (50 mM, pH 7.4). The reaction mixture contains 3 mL of sample and 1.8 mL of H₂O₂ solution and phosphate buffer solution without H₂O₂ as a blank. Hydrogen peroxide concentration was determined by UV-Vis spectrophotometer (Perkin Elmer, Lambda 40) at 240 nm as a control. Absorbance of each sample was recorded after 10 minutes against the blank solution. The scavenging ability of hydrogen peroxide was calculated by the following equation:
where A_control = absorbance of H₂O₂ in ethanol without an antioxidant and A_sample = absorbance of H₂O₂ in the presence of an antioxidant.

2.2.3. Molecular docking. The rigid molecular docking studies were performed using HEX 8.0.0 software,²⁰ which is an interactive molecular graphics program for calculating and displaying feasible docking modes of pairs of protein, enzymes and DNA molecule. The structure of the complexes (2 and 3) were sketched by CHEMSKETCH (http://www.acdlabs.com) and converted to pdb format from mol format by OPENBABEL (http://www.vcclab.org/lab/babel/). The crystal structure of the B-DNA dodecamer d(CGCGAATTCGCG)₂ (PDB ID: 1BNA) was downloaded from the protein data bank (http://www.rcsb.org./pdb). Visualization of the docked pose has been done by using CHIMERA (www.cgl.ucsf.edu/chimera) and PyMol (http://pymol.sourceforget.net/) molecular graphics program.

2.3. Synthesis

2.3.1. Synthesis of [4-pic-H][Cr(pda)₂]·2H₂O (1). An ethanolic solution of pyridine-2,6-dicorbaxylic acid (5.0 mmol, 0.84 g) was refluxed with 4-picoline (5.0 mmol, 0.50 mL) for 6 h then cooled at room temperature. An ethanolic solution of CrCl₂·6H₂O (5.0 mmol, 1.18 g) was added dropwise to the above mixture, which produced green color solid. Stirring was continued for 4 h at room temperature. The solid obtained was filtered off, washed with ethanol and dried in vacuum. After repeated recrystallization in various solvents, the solid could not provide single crystals suitable for X-ray studies.

[Green, yield 65%, m.p. > 300 °C] anal. calcd (%) for C₂₀H₁₄CrN₃O₁₀: C 46.38; H 2.70; N 8.25. Found (%): C 46.87; H 2.65; N 8.32. Molar conductance, Λ_m (10⁻³ M, methanol): 85 Ω⁻¹ cm² mol⁻¹. FT-IR (as KBr disks, cm⁻¹): 1392s ν_sym(COO⁻), 1619s ν_asym(COO⁻), 3250w (N–H), 1583s (C=N), 1436w, 730m, 773s (C=C) pyridine ring stretching vibrations.

2.3.2. Synthesis of [4-pic-H][Fe(pda)₂]·2H₂O (2). Synthetic procedure for (2) was similar to that for (1) except addition of FeCl₃ (5.0 mmol, 0.81 g) instead of CrCl₂·6H₂O. The dark brown solid was obtained which was recrystallized in double distilled water yielding single crystals suitable for X-ray studies.

[Dark brown, yield 60%, m.p. > 300 °C] anal. calcd (%) for C₂₀H₁₄FeN₃O₁₀: C 46.62; H 2.70; N 8.22. Found (%): C 46.96; H 2.70; N 8.35. Molar conductance, Λ_m (10⁻³ M, methanol): 92 Ω⁻¹ cm² mol⁻¹. FT-IR (as KBr disks, cm⁻¹): 1329s ν_sym(COO⁻), 1654s ν_asym(COO⁻), 3305w (N–H), 1591s (C=N), 1428w, 740m, 775s (C=C) pyridine ring stretching vibrations.

2.3.3. Synthesis of [4-pic-H][Co(pda)₂]·2H₂O (3). Synthetic procedure for (3) was similar to that for (1) except addition of CoCl₂·6H₂O (5.0 mmol, 1.18 g) instead of CrCl₂·6H₂O. The pink colour solid was obtained which was recrystallized in double distilled water yielding single crystals suitable for X-ray studies.

[Pink, yield 60%, m.p. > 300 °C] anal. calcd (%) for C₂₀H₁₄CoN₃O₁₀: C 46.58; H 2.72; N 8.15. Found (%): C 46.92; H 2.68; N 8.27. Molar conductance, Λ_m (10⁻³ M, methanol): 90 Ω⁻¹ cm² mol⁻¹. FT-IR (as KBr disks, cm⁻¹): 1386s ν_sym(COO⁻), 1618s ν_asym (COO⁻), 3300w (N–H), 1587s (C=N), 1430w, 730m, 762s (C=C) pyridine ring stretching vibrations.

2.4. Crystallographic data collection and refinements

Single crystal X-ray diffraction of compounds (2 and 3) was performed at 100 K on a Bruker SMART APEX CCD diffractometer, X-ray data was collected using graphite monochromated Mo-Kα radiation (λ = 0.71073 Å). The linear absorption coefficients, scattering factors for the atoms, and the anomalous dispersion corrections were taken from the International Tables for X-ray Crystallography.²¹ The data integration and reduction were processed with SAINT Software.²² An empirical absorption correction was applied to the collected reflections with SADABS²³ and the space group was determined using XPREP.²⁴ Several DIFX command has been given to fix the bond length parameters. The structures were solved by direct methods using SHELXL-97 (ref. 25) and refined on F² by full matrix least squares using the SHELXL-97 program package.²⁶ All non-hydrogen atoms were refined with anisotropic displacement parameters. A summary of the crystallographic data and the structure refinement for the complexes is given in Table 1. CCDC reference numbers for 2 and 3 are 1440315 and 1405365, respectively.

Table 1 Crystallographic parameters for complexes (2 & 3)

Parameters	Complex 2	Complex 3
Empirical formula	C20H14FeN3O10	C20H14CoN3O10
Formula weight	512.19	515.27
Temp (K)	296(2)	273(2)
Crystal system	Monoclinic	Monoclinic
Space group	P21/n	P21/n
Unit cell dimensions
a (Å)	15.0469(3)	15.020(7)
b (Å)	9.2868(2)	9.275(4)
c (Å)	15.5703(4)	15.524(7)
α (°)	90.00	90.00
β (°)	94.0110(10)	93.849(7)
γ (°)	90.00	90.00
V (Å^3)	2170.43	2157.8(18)
Z	4	4
ρ (calc) (g cm^−3)	1.567	1.586
F (000)	1044	1048
Crystal size (mm^3)	0.31 × 0.29 × 0.24	0.29 × 0.21 × 0.16
Index ranges	−18 ≤ h ≤ 18	−17 ≤ h ≤ 17
	−11 ≤ k ≤ 11	−11 ≤ k ≤ 11
	−18 ≤ l ≤ 18	−18 ≤ l ≤ 18
No. of independent reflection	4031	2783
No. of reflections collected	26 129	3794
GOF	1.014	1.087
Final R indices [ I > 2σ(I)]	R1 = 0.0489	R1 = 0.0822
	wR2 = 0.1395	wR2 = 0.1903
R indices all data	R2 = 0.0399	R2 = 0.0604
	wR2 = 0.1299	wR2 = 0.1692
μ (mm^−1)	0.758	0.859
Θ range (°)	1.82–25.50	1.18–25.0

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3. Results and discussion

The formation of the compounds can be regarded to involve two step mechanisms (Scheme 1). The first step is the proton transfer process from the dicarboxylic function of the pyridine-2,6-dicorboxylic acid (H₂pda) to 4-picoline (4-pic), which acts as proton acceptor to create the strong H-bonded species [4-Pic-H]⁺ in the solution. The second step involves the in situ complexation reaction of this species with the M³⁺ ion to create the complex anion [M(pda)₂]⁻, which is stabilized by the counter cation [4-pic-H]⁺ to finally give the product [4-pic-H][M(pda)₂]·2H₂O [M = Cr (1), Fe (2) & Co (3)]. The analytical and ESI Mass data are consistent with the proposed stoichiometry of the complexes (1–3). The molar conductance exhibits the 1 : 1 electrolytic nature of the complexes. The structure and bonding features were investigated by spectroscopic & X-ray data.

Scheme 1 Synthetic procedure of the complexes (1–3).

3.1. FT-IR, ¹H and ¹³C NMR spectra

The bonding modes of the compounds (1–3) were observed by FT-IR spectral analysis. IR spectra were nearly the same and show vibrations due to the water and carboxylic acid fragments, ν(M–C), ν(M–O) and ν(M–O–O) frequencies. The FT-IR spectra of the complexes (1–3) contained strong intense bands at the ranges ∼1620 and ∼1340 cm⁻¹, which are characteristic peaks of the ν_asym(COO⁻) and ν_sym(COO⁻) fundamental stretching vibrations, respectively, of the pda²⁻ moiety coordinated to the metal ions. These symmetric and asymmetric vibrational bands are used to diagnose the coordination modes in the carboxylate ligands.²⁷ The separation (Δν) between ν_asym(COO⁻) and ν_sym(COO⁻) stretching vibration bands [i.e., Δν = ν_asym(COO⁻) − ν_sym(COO⁻)] provides ample information and ascertains the binding mode of coordination of the functionalized carboxylate moiety to the metal ions in the complexes (1–3). The carboxylate anion (COO⁻) may bind the metal ion in four possible coordination modes as asymmetric monodentate (A), symmetric bidentate (B) and asymmetric bidentate (C & D) as presented in Scheme 2.

Scheme 2 Important coordination modes of carboxylate (COO⁻) group [i.e., asymmetric monodentate (A), symmetric bidentate (B) and asymmetric bidentate (C & D)].

If the magnitude of Δν lies in the range 250–300 cm⁻¹, the monodentate coordination mode (A) is present.^28,29 However, for the modes depicted in B, C and D the magnitude of Δν is much lower (Δν ≤ 200 cm⁻¹). In the present complexes, the observed magnitude of Δν (∼300 cm⁻¹) suggests that the carboxylate moieties of the pda²⁻ involve the asymmetric monodentate coordination (A). This fact has been further confirmed from the X-ray crystallography of the complexes (1 and 2). The absence of the characteristic bands at around 1700 cm⁻¹ attributed to the carboxyl groups indicates that complete deprotonation of all carboxylate groups in (1–3) has occurred upon reaction with metal ions. The ν(C=N) and ν(C=C) stretching vibration bands in the spectra were indicated in the region 1578–1583 and 1428–1436 cm⁻¹. The observed broad bands in 3492–3456 cm⁻¹ region for the complexes were characteristic of the presence of lattice water molecules in the complex moiety. The appearance of an additional band in the high frequency region cantered at 3305–3311 cm⁻¹ corresponds to the presence of H-bonded, N–H bond^30,31 in the cationic species of [4-Pic-H]. The medium intensity bands appearing at 432–436 and 542–595 cm⁻¹ are characteristic of M–O and M–N bond(s) stretching vibrations, respectively.³²

¹H NMR spectra of the complexes showed a singlet in the high field side i.e., (at 2.3, 2.0 and 2.1 ppm for (1), (2) and (3), respectively) attributable to aliphatic protons of the –CH₃ group of 4-pic-H⁺ moiety. A multiplet in the low field side (∼6 ppm) corresponds to the pyridine ring protons in the complexes. An important peak characteristic of NH⁺ proton is observed around ∼9 ppm in all the complexes indicating the formation of proton transfer complexes (1–3).^{9,33 13}C NMR spectra of the complexes contained three sets of resonance signals i.e., at 120–150 ppm due to aromatic carbons, at ∼180 ppm due to carboxylate carbon and at ∼25 ppm due to methyl carbon.³³

3.2. Electronic and EPR spectral studies

The electronic absorption spectra of the complexes (1–3) were recorded in methanol solution at room temperature (Fig. 1). The high-energy band at ∼37 500, 30 000 and 26 000 cm⁻¹ can be assigned to n → π*, π → π* and CT as shown in Table 1S (ESI†).³⁴ The charge transfers are LMCT due to M ← pda²⁻ transition, involving oxygen atoms of the ligand. Low energy transitions are d–d bands assignable to high spin ground state of the metal ions i.e., Cr³⁺(t³_2ge⁰_g) with ⁴T₁g(F) ← ⁴A_2g & ⁴T₂g(F) ← ⁴A_2g (1), Fe³⁺ (t³_2ge²_g) with ⁴T₂g ← ⁶A₁g & [⁴T₂g, ⁴T₁g] ← ⁶A₁g (2) and Co³⁺ (t⁴_2ge²_g) with ¹E₁g ← ¹A₁g(F) & ¹A₂g ← ¹A₁g (3), assignments. The electronic spectral data are attributed to hexa-coordinate environment around the metal ions.^35,36

Fig. 1 Absorption spectra for the complexes (1–3).

The solid state X-band EPR spectral data of the complexes (1–3) [Fig. 1S (ESI†)] provide isotropic signal with g = 3.9–4.2 corroborating a distorted octahedral geometry of the complexes as revealed by electronic spectra.

3.3. Luminescence spectra

Fluorescence spectra of the complexes (1–3) were also recorded in methanolic solution at room temperature as shown in Fig. 2. Upon excitation at ∼265 and ∼330 nm, an emission peak at ∼270 and 390 nm, respectively were obtained with moderate intensity in all the complexes. The emission spectral data are indicative of the luminescence properties of the complexes.

Fig. 2 (a) Emission spectra of the complexes (1–3). (b) 3D fluorescence spectra of the complexes (1–3).

3.4. Electrospray ionization mass (ESI-MS) spectra

Electrospray ionization mass (ESI-MS) spectrometry was performed to ascertain the proposed stoichiometry of the complexes (1–3). The complexes exhibited molecular ion peaks at m/z = 478 (1), 475 (2) and 479 (3) assignable to [M(pda)₂][4-pic-H]⁺. All the complexes showed a peak at m/z = 94 (60%) assignable to [4-pic-H]⁺ moiety. The complexes also exhibited peaks at m/z = 392 (1), 389 (2) and 393 (3) characteristic of the [M(pda)₂]⁺ species. The ultimate species [M(pda)]⁺ was formed as observed by peaks at m/z = 225 (1), 222 (2) and 226 (3).

3.5. Description of the crystal structure

X-ray crystal structure analysis for the complexes 2 and 3 reveals that both the complexes have identical molecular formula, i.e., [4-pic-H][M(pda)₂]·2H₂O, where (2) has iron and (3) cobalt ion. Both the compounds crystallize in the monoclinic space group P2₁/n. The complexes contain one [4-pic-H]⁺ cation, one [M(pda)₂]⁻ anion and two uncoordinated water molecules present in lattice (Fig. 3). The metal ion is present in +3 oxidation state and coordinated to two perpendicular tridentate pda²⁻ ligands. The geometry around the M³⁺ ion is distorted octahedral. The axial positions are occupied by pyridine nitrogen atoms N1 and N2, with bond lengths Fe1–N1 = 2.058, Fe1–N2 = 2.060 Å (2) and Co1–N1 = 2.056(3), Co1–N1A = 2.052(3) (3) while the equatorial plane is constructed by four carboxylic groups, with Fe–O bond distances ranging from 2.0030(17) to 2.0092(17) (2) and Co–O, from 1.998(3) to 2.034(3) (3) Å. The presence of picolinium cation [4-pic-H]⁺ shows that the proton transfer process helps the formation of M(III) complex.

Fig. 3 ORTEP views of the complexes (a) 2 & (b) 3.

The M–O and M–N bond distances also support the +3 oxidation state of metal ion. In the molecular structure of the complexes, H₂pda are joined to the metal ion centre in its dianionic form (pda²⁻), resulting in monoanionic complexes. The N1–Co1–N1A and N1–Fe1–N2 angle deviate from linear to 169.02 and 168.99(8)°, in 2 and 3, respectively, confirming the coordination geometry around M(III) atom as distorted octahedral (Fig. 4). The value of the O4–Co1–O2, O7–Co1–O5 angles are 151.96 and 151.51°, and the O3–Fe1–O1, O7–Fe1–O5 angles are 151.68(7) and 151.28(7)°, respectively, which are much deviated from the expected (180°) angle for the perfect planarity, indicating that the four oxygen atoms of the carboxylic groups of two pda²⁻ moieties are not planar. In complexes (2& 3), [4-pic-H]⁺ cation, [M(pda)₂]⁻ anion and two uncoordinated H₂O molecules are joined through H-bonds (N–H⋯O and O–H⋯O bond lengths vary from 1.811 to 2.989 Å), which are shown in Fig. 5. The N–H⋯O hydrogen bonds exist between pyridine nitrogen atom (N3) of [4-pic-H]⁺ cation and uncoordinated water molecules. The O–H⋯O hydrogen bonds are located between uncoordinated water molecules as donor atoms and oxygen acceptor atoms from carboxylate groups of [M(pda)₂]⁻ anions or neighbouring water molecules (Fig. 5). Selected bond lengths and angles for 2 and 3 are given in Table 2.

Fig. 4 Selected bond lengths and angle of the complexes (a) 2 and (b) 3.

Fig. 5 The extensive H-bonding (shown by dotted lines) in (a) 2 and (b) 3.

Table 2 Selected bond lengths (Å) and bond angles (°) of the complexes (2 & 3)

Complex (2)		Complex (3)
Bond lengths (Å)
O1–Fe1	2.0347(17)	O2–Co1	1.998(3)
O7–Fe1	2.0092(17)	O4–Co1	2.033(3)
O3–Fe1	2.0030(17)	O5–Co1	2.003(3)
O5–Fe1	2.0403(18)	O7–Co1	2.034(3)
N1–Fe1	2.058(2)	N1–Co1	2.056(3)
N2–Fe1	2.061(2)	N1A–Co1	2.052(3)
Bond angles (°)
O3–Fe1–O7	92.83(8)	C6–N1–Co1	119.6(3)
O3–Fe1–O1	151.68(7)	C1–N1–Co1	118.0(3)
O7–Fe1–O1	95.24(8)	C9–N1A–Co1	118.4(3)
O3–Fe1–O5	92.29(8)	C13–N1A–Co1	118.9(3)
O7–Fe1–O5	151.28(7)	C2–O2–Co1	120.3(3)
O1–Fe1–O5	93.52(8)	O2–Co1–O5	92.82(13)
O3–Fe1–N1	76.46(7)	O2–Co1–O4	151.97(12)
O7–Fe1–N1	114.36(7)	O5–Co1–O4	95.17(13)
O1–Fe1–N1	75.48(7)	O2–Co1–N1A	106.61(12)
O5–Fe1–N2	75.28(7)	O5–Co1–N1A	76.14(13)
N1–Fe1–N2	168.99(8)	O7–Co1–N1A	75.55(13)

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3.6. Thermogravimetric analysis (TGA)

TGA was carried out for polycrystalline samples of complexes (1–3) as shown in Fig. 6 in 25–800 °C temperature range. Thermal stability of the complex has provided enough information regarding stoichiometry. The thermal fragmentation was examined in a nitrogen atmosphere with the heating rate of 20 °C minute⁻¹. The thermograms of the complexes (1–3) shows the release of two water molecules and one 4-pic moiety between the temperature range 25 to 130 °C (found 25%, calculated 26%).³⁷ Further, as the temperature increases above 400 °C, elimination of one H₂pda moiety from the complexes takes place. The final residual product appears to be [M(pda)].^37,38

Fig. 6 Thermogravimetric analysis (TGA) of the complexes (1–3).

3.7. Cyclic voltammetric studies

The cyclic voltammograms of the complexes (1–3) for the electrochemical redox properties in the potential range +1.0 to −1.0 V with reference to Ag/AgCl electrode at 27 °C in the presence of [ⁿBu₄N]·ClO₄ were recorded at 200 V s⁻¹ scan rate to investigate the changes in oxidation state of metal ions in solution. Complexes (1–3) show nearly similar electrochemical behaviour and show two cathodic peaks, E^c_p = −0.07 & 0.7 V (1); 0.24, 0.24 V (2) and −0.25, 0.43 V (3) and one anodic peak, E^a_p = 0.6 V (1), −0.27 V (2) and 0.55 V (3) [Fig. 7(a–c)]. One of the cathodic peaks couples with the anodic peak to form a quasi reversible redox couple at E⁰_1/2 = 0.34 V (1), 0.37 V (2) and 0.33 V (3).³⁹ An additional cathodic peak in all the complexes are consistence with irreversible reaction in solution.^40,41

Fig. 7 (a) Cyclic voltammogram for complex 1. (b) Cyclic voltammogram for complex 2. (c) Cyclic voltammogram for complex 3.

3.8. Magnetic studies

Magnetic study was performed to investigate the paramagnetic behaviour of the complex. Magnetic susceptibility is measured using a very sensitive instrument known as a magnetic susceptibility balance. The balance contains a pair of magnets mounted at opposite ends of the beam, initially in equilibrium. When a sample is introduce into the balance, a disruption of the magnetic field results. A current through a coil located between the poles of the second pair of magnets returns the beam to equilibrium. The current through the coil is measured and transferred into ta numerical reading. Diamagnetic materials are weakly repelled by an external magnetic field, resulting in a negative reading. Paramagnetic materials are attracted to an external magnetic field and give a positive reading.

The magnetic moment of the complexes was calculated using by the formula as follows:

χ_g = LC_bal(R − R_o)/10⁹(m)

L = height of sample in tube in units of centimeters, m = mass of the sample in units of grams, R = reading for tube plus sample, R_o = reading for the empty tube, C_bal = balance calibration constant = 1.0.

The molar magnetic susceptibility is then calculated from the gram magnetic susceptibility using the following equation,

χ_m = χ_g(molar mass),

χ_A = χ_m − diamagnetic correction,

The magnetic susceptibility for a particular substance is not particularly useful in itself. However, the effective magnetic moment for a particular substance can be calculated from the gram magnetic susceptibility using the following equation.

χ_g magnetic susceptibility erg G⁻² g⁻¹, χ_m molar susceptibility erg G⁻² mol⁻¹, and μ_eff effective magnetic moment B. M.

Magnetic data indicate that μ_eff for the complexes 4.1 (1), 6.2 (2) and 5.1 (3) BM is suggestive of highly paramagnetic behaviour of the complexes. It is clear from magnetic data that Cr⁺³ (t³_2ge⁰_g) (1), Fe⁺³ (t³_2ge²_g) (2) and Co⁺³ (t⁴_2ge²_g) (3) are high spin configurations having +3 oxidation state.^42–44

3.9. Biological activities

3.9.1. Antioxidant (free radical scavenging) properties. DPPH free radical scavenging activity is very suitable and significant method for the evaluation of antioxidant activity because DPPH is a stable free radical that accepts a hydrogen free radical and becomes a stable diamagnetic molecule.¹⁸ The reducing properties are associated with the antioxidant activity. After 100 minutes reaction, colour changes from violet to yellow which gives λ_max at 515 nm (absorbance of the sample & control = 1.63 & 2.39, respectively) (Fig. 8), revealing that some antioxidants are present in the solution. The complex (3) exhibited 32% anti-oxidant activity. This activity can be attributed to the fact that chelating agents are effective as secondary antioxidants because they can reduce the redox potential thereby stabilizing the oxidized form of the metal ion.⁴⁵

Fig. 8 DPPH assay of the complex (3).

Additionally, hydrogen peroxide assay was also used to investigate the antioxidant property of the complex (3). The hydrogen peroxide radicals are the most reactive among all oxygen containing reactive species used for the estimation of antioxidant properties of the compound during aerobic metabolism.⁴⁶ The hydrogen peroxide scavenging activity result was in good agreement with DPPH free radical scavenging assay.

3.9.2. Molecular docking studies. The designing of molecules can lead to the recognition of specific sequences and structures of nucleic acids and plays an important role in the development of new chemotherapeutic drugs. Molecular docking studies have played a very significant role in understanding the drug–DNA interaction in the rational drug design, as well as in the mechanistic study by placing a small molecule into the binding site of the DNA target specific region mainly in a non-covalent fashion, which predicts interactions between the drug molecules and the nucleic acids of DNA. Conformation of docked complexes was analyzed in terms of energy, hydrogen bonding, and hydrophobic interaction between complexes and DNA. Targeting the DNA minor groove for a small molecule has long been regarded as an important pattern in molecular recognition of a specific DNA sequence.⁴⁷

The energetically favourable docked poses obtained from the rigid molecular docking of the complexes (2 and 3) with a DNA duplex of sequence d(CGCGAATTCGCG)₂ dodecamer (PDB ID: 1BNA) are shown in Fig. 9a and b. Obviously, complexes (2 and 3) fit snugly into the curved contour of the DNA target in the minor groove and are situated within the G–C rich region. It is well known that the interactions of chemical species with the minor groove of B-DNA differ from those occurring in the major groove, both in terms of electrostatic potential and steric effects, because of the narrow shape of the former. Small molecules interact with the minor groove, while large molecules tend to recognize the major groove binding site.⁴⁸ The results of molecular docking show that complexes (2 and 3) bind efficiently with the DNA receptor (in minor groove) and exhibit free energy of binding (FEB) values of −314 (2) and −276.8 kcal mol⁻¹ (3). The more negative relative binding energy of complex 2 indicated its strong binding ability to the DNA compared to complex 3.⁴⁹

Fig. 9 (a) Molecular docked model of the complex (2) with DNA (PDB ID: 1BNA). (b). Molecular docked model of the complex (3) with DNA (PDB ID: 1BNA).

The mechanism of binding can also be better understood by the docking results. As illustrated in the Fig. 10(a and b), the carbonyl oxygen and lattice water molecule of the complexes displayed a strong hydrogen-bonding interaction with DNA bases.⁴⁹ The compounds exhibited stabilization through hydrophobic and van der Waals interactions with nearby nucleotides. The results of the docking studies reveal that compounds fit in the minor groove region of B-DNA. The compound 2 forms two hydrogen bonds with B-DNA at G10/G12 (2) [N2(G10)⋯O8 = 3.110 Å, N2(G12)⋯O4 = 2.690 Å], while compound 3 forms only one hydrogen bond at A17/G16 [O3′(G16/A17)⋯OW = 1.993 Å]. This also indicates that 2 has stronger interactions with the B-DNA as compared to 3.

Fig. 10 (a) Molecular docked model showing H-bonding interaction of 2 with B-DNA. (b) Molecular docked model showing H-bonding interaction of 3 with B-DNA.

4. Conclusion

A series of novel proton transfer complexes bearing composition [4-pic-H][M(pda)₂]·2H₂O [M = Cr³⁺ (1), Fe³⁺ (2) and Co³⁺ (3)] using pyridine-2,6-dicarboxylic acid (H₂pda) as a proton donor and 4-picoline (4-pic) as a proton acceptor has been synthesized and characterized using FT-IR, ¹H & ¹³C NMR, ESI Mass, EPR, UV-Visible and fluorescence spectral studies. Single crystal X-ray crystallographic data of 2 and 3 ascertained the structure and binding modes of the ligand. The pda²⁻ ligand binds the metal ion in dianionic tridentate [N, O, O] form resulting in distorted octahedral geometry around the M³⁺ ion. ESI and thermal studies confirmed the presence of cation [4-pic-H], anion [M(pda)₂]⁻ and two lattice water molecules. Cyclic voltammetric studies indicate the presence of quasi-reversible redox couple in solution. DPPH and hydrogen peroxide scavenging study confirmed the antioxidant property of the complexes. The results of molecular docking show that complexes 2 and 3 bind efficiently with the DNA receptor and exhibit free energy of binding (FEB) values of −314 (2) and −276.8 kcal mol⁻¹ (3), respectively. The docking data exhibits that 2 has stronger binding ability to the DNA (1BNA) compared to that of 3.

Acknowledgements

The authors thank Chairman, Department of Chemistry, AMU, Aligarh, for providing necessary research facilities and UGC, New Delhi for financial assistance. M. Shahid acknowledges UGC, New Delhi for Start up Grant [F.30-46/2014(BSR)].

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Footnote

† Electronic supplementary information (ESI) available: Spectral and crystallographic data. CCDC 1440315 and 1405365. For ESI and crystallographic data in CIF or other electronic format see DOI: 10.1039/c5ra25939h

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