Synthesis, characterization, anti-microbial, DNA binding and cleavage studies of Schiff base metal complexes

2.1 Materials and reagents

All the chemicals used were of analytical reagent grade and the solvents were dried and distilled before use according to a standard procedure (Vogel, 1989). Butanedione monoxime and 3,3′-diaminobenzidine were purchased from Aldrich and were used as received.

2.2 Physical measurements (apparatus and experimental condition)

C, H and N contents were determined by Perkin Elmer CHN 2400 elemental analyzer, and IR Spectra were recorded in the range 4000 cm⁻¹–100 cm⁻¹ with a Bruker IFS66V in KBr and polyethylene medium for all complexes. The molar conductance of the complexes in DMF (10⁻³ M) solution was measured at 27 ± 3 °C with an Elico model conductivity meter. UV–visible spectra were recorded in DMF with Perkin–Elmer Lambda 35 spectrophotometer in the range of 200–800 nm. ¹H NMR spectra were recorded on Bruker 300 Hz spectrophotometer using DMSO d₆ as solvent. Chemical shifts are reported in ppm relative to tetramethylsilane, using the solvent signal as internal reference. EPR spectra were recorded at room temperature on JEOL JESTE100 ESR spectrometer. The spectrometer was operated at X-band (8–12 GHz) with microwave power of 1 mW. The room temperature magnetic moments were measured on a PAR vibrating sample magnetometer (Model-155). The TGA and DTA curves of the complexes were recorded on NETZSCH-STA 409PC thermal analyzer in heating rate of 10 K/min with the range of 50 °C to 900 °C

2.3 Anti-microbial activity

The Schiff base ligand and its complexes were investigated for anti-bacterial and anti-fungal against Staphylococcus aureus and Streptococcus pyogenes as gram positive bacteria and Escherichia coli and Klebsiella pneumoniae as Gram-negative and the fungi Fusarium oxysporum and Aspergillus fumigatus by using disc-agar diffusion method. All complexes exhibit anti-bacterial and anti-fungal activities against these organisms and are found to be more effective than the free ligand.

The anti-microbial activity was carried out at Progen Lab at Salem, Tamilnadu (India). The standard disc-agar diffusion method (Gross and De vay, 1977) was followed to determine the activity of the synthesized compounds against the sensitive organism S. aureus and S. pyogenes as gram positive bacteria and Escherichia coli and K. pneumoniae as Gram-negative and the fungi F. oxysporum and A. fumigatus. The antibiotic chloramphenicol was used as standard reference in the case of Gram-negative bacteria, tetracycline was used as standard reference in case of Gram-positive bacteria and clotrimazole was used as standard anti-fungal reference. The tested compounds were dissolved in DMF (which have no inhibition activity), to get concentration of 100 μg/mL. The test was performed on medium potato dextrose agar contains infusion of 200 g potatoes, 6 g dextrose and 15 g agar (William and Stephen, 1989). Uniform size filter paper disks (three disks per compound) were impregnated by equal volume from the specific concentration of dissolved tested compounds and carefully placed on incubated agar surface. After incubation for 36 h at 27 °C in the case of bacteria and for 48 h at 24 °C in the case of fungus, inhibition of the organism which evidenced by clear zone surround each disk was measured and used to calculate mean of inhibition zones.

2.4 DNA binding studies

The DNA binding experiments were performed in Tris–HCl/NaCl buffer (50 mM Tris–HCl/1 mM NaCl buffer, pH 7.5) using DMF (dimethylformamide) solution (10%) of the metal complexes. The concentration of calf-thymus (CT) DNA was determined from the absorption intensity at 265 nm with a ε value (Li et al., 2010) of 6600 M⁻¹ cm⁻¹. Absorption titration experiments were made using different concentrations of CT-DNA [40, 60, 80 μM], keeping the concentration of the complexes constant, with due correction for the absorbance of the CT-DNA itself. Samples were equilibrated before recording each spectrum.

2.5 DNA cleavage study

The DNA cleavage activity of the Schiff base metal complexes was monitored by agarose gel electrophoresis on CT-DNA. The tests were performed under aerobic conditions with H₂O₂ as an oxidant. Each reaction mixture contained 30 μM of CT-DNA, 40 μM of each complex in DMSO and 50 μM of hydrogen peroxide in 50 mM Tris–HCl buffer (pH 7.2). The reaction was incubated at 37 °C for 2 h. After incubation, 1 μL of loading buffer (bromophenol blue in H₂O) was added to each tube and the mixed samples were loaded on 1% agarose gel. The electrophoresis was carried out for 2 h at 50 V in Tris–acetic acid–EDTA buffer (pH 8.3). After electrophoresis, the gel was stained with 1 μg/cm³ ethidiumbromide (EB) for 30 min prior to being photographed under UV light.

2.6 Synthesis of ligand and its complexes

3,3′-Diamino benzidine (1 mM) in 10 mL of methanol, butanedione monoxime (4 mM) in 20 mL of methanol were mixed and heated at reflux for 2 h at 80 °C. The resulting dark red color solution was allowed to cool. The dark red color product was obtained and dried in desiccator using silica gel as drying agent. The metal complexes were prepared by reacting metal chlorides (2 mM) and ligand (1 mM) in acetonitrile were mixed and refluxed for about 2 h at 90 °C. The resulting product was filtered and dried in desiccator using silica gel (Fig. 1).

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Figure 1

Synthesis of ligand and its complexes.

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3.1 Molar conductivity measurements

The results of the elemental analysis are in good agreement with the calculated values. The metal contents of the complexes were determined according to literature methods (Jeffery et al., 1989). The electrolytic nature of the complexes is measured in DMF at 10⁻³ M. The molar conductivity Λ_m lies between 8 and 13 Ω⁻¹ cm² mol⁻¹. This result shows that the complexes were non-electrolyte in nature, and anions were coordinated inside the coordination sphere (Tabl et al., 2008).

3.2 IR spectral studies

The IR spectra of the complexes are compared with those of the free ligands in order to determine the coordination sites that may involve in chelation. The position and or the intensities of these peaks are expected to be changed upon chelation. The IR spectra of metal complexes and ligand were recorded in the range of 100–4000 cm⁻¹. The azomethine nitrogen (C⚌N) appears around 1644 cm⁻¹ which has been shifted to lower wave number in the range between 1610 and 1640 cm⁻¹. The shift in the frequency due to coordination of two imine and two oxyimino groups with the metal ions. The broad band in the range of 3408–3441 cm⁻¹ is assigned to the stretching frequency of OH/H₂O molecules. The weak band around 460–480 cm⁻¹ could be assigned to the stretching frequency of M–N bands for metal complexes. The bands at 270–280 cm⁻¹ are due to the M–Cl (Murphy et al., 1987).

3.3 Electronic spectral studies

Electronic spectra of all the complexes were recorded in DMF medium. In electronic spectra of metal complexes the wide range of bands are due to transition of –CH⚌N–, charge transfer results from electrons interaction between the metal and the ligand which involves either a metal to ligand or ligand to metal electron transfer (Tas et al., 2006). The bands observed in 235–265 nm are due to π → π∗ transition of C⚌N group (Serbest et al., 2001). The bands were shifted to higher range, which is due to nitrogen that involved in coordination with metal ion. The absorption bands are observed in the range of 310–370 nm due to n → π∗ transition from imine group corresponding to the ligand or metal complexes. The copper(II) binuclear complex shows a broad absorption peak at 657 nm and arises due to the d–d transition ²Eg → ²T_2g of Cu(II) ion suggest that the copper ion exhibits an octahedral geometry (Lever, 1984; Sathyanarayana, 2001). Electronic spectra of the nickel(II) binuclear complex shows bands at 486, 545 nm which are assigned to ³A_2g(F)→ ³T_1g(P) and ³A_2g(F)→ ³T_1g(P) transitions, respectively, suggesting an octahedral arrangement around the nickel(II) complex (Lever, 1984; Sathyanarayana, 2001). The electronic spectra of binuclear cobalt(II) complexes exhibit absorption at 442, 542, 657 nm are assigned to 4T_1g → 4A_2g, 4T_1g (F) → 4A_2g(F), 4T_1g(F) → 4T_2g transitions, respectively, corresponding to cobalt(II) octahedral complex (Lever, 1984; Sathyanarayana, 2001). The Mn(II) binuclear complex shows bands at 441 and 612 nm, respectively corresponding to ⁶A_1g → ⁴T_2g(G) and ⁶A_1g → ⁴T_1g transitions which are compatible to an octahedral geometry around manganese(II) ion (Lever, 1984; Sathyanarayana, 2001).

3.4 ¹H NMR spectra

The structure of ligand was confirmed by ¹H NMR spectra. The ¹H NMR spectrum of DMSO d₆ solution of oxime ligand showed a well resolved signal as expected. The spectrum of oxime ligand shows singlet at 1.95 ppm and singlet at 2.68 ppm corresponding to CH₃-1 and CH₃-4. The signals between 6.5 and 8.5 ppm were due aromatic protons. The oxime–OH proton appears at 11.25 ppm (Fig. 2).

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Figure 2

¹H NMR spectra of ligand.

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3.5 ESR and magnetic studies

ESR measurement has been made for copper complex using powder sample at room temperature, which could provide only value of g_iso (Fig. 3). The g_iso value of the complex is 2.071. The value of g_iso shows that the copper(II) complex is in octahedral environment. The magnetic moments of Cu(II), Ni(II), Co(II), Mn(II) are 1.95, 2.97, 4.87 and 5.94 B.M., respectively. The values are almost equal spin only value. This indicates that the two metal centers are equivalent and there is no interaction between the two metal centers. The pairing of electron is prevented by greater distance between the two metal centers (Venkatachalam et al., 2008).

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Figure 3

ESR spectrum of [Cu₂(L)Cl₄] complex at room temperature (frequency 9.445 GHz).

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3.6 Thermal studies

The thermal stability of the metal complexes was studied by TGA/DTA. The thermo gravimetric analysis was carried out in temperature range 50 °C to 900 °C. The samples were placed in platinum crucible and Al₂O₃ was used as reference material. Heating was performed under N₂ atmosphere. In copper(II) complex (Fig. 4) one endothermic peak was observed at 120 °C and one exothermic peak was observed at 200 °C which is assigned to the loss of four chloride and four hydroxyl ions 27.14(25.77)%. Another exothermic peak was observed at 490 °C which is attributed to the loss of 8CH₃CN groups 40.12(40.24)%. The decomposition is not completed after 900 °C. In nickel complex (Fig. 5) one endothermic peak was observed at 165 °C which is assigned to the loss of chloride and hydroxyl ions 26(26.09)%. One exothermic was observed at 250 °C and another at 300 °C which is attributed to the loss of 8CH₃CN groups 41.00(40.75)%. After 900 °C the decomposition is not completed.

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Figure 4

TGA/TG studies of [Cu₂(L)Cl₄] complex.

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Figure 5

TGA/TG studies of [Ni₂(L)Cl₄] complex.

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3.7 Anti-microbial studies

Biological activity of the ligand and a series of its metal complexes [Cu(II), Ni(II), Co(II) and Mn(II)] were screened for anti-bacterial activity against S. aureus and S. pyogenes as Gram-positive bacteria and E. coli and K. pneumoniae as Gram-negative and the fungi F. oxysporum and A. fumigatus by using disc-agar diffusion method (Figs. 6–8). The remarkable activity of Schiff base ligands may arise from the presence of four imine and four oxyimino groups which impart in elucidating the mechanism of transformation reaction in biological systems. The results indicate that the complexes show more activity and the ligand have less activity against same microorganisms under identical experimental conditions (Table 2). This would suggest that the chelation could facilitate the ability of a complex to cross a cell membrane and can be explained by Tweedy’s chelation theory (Tweedy, 1964). Chelation considerably reduces the polarity of the metal ion because of partial sharing of its positive charge with donor groups and possible electron delocalization over the whole chelate ring. Such a chelation could enhance the lipophilic character of the central metal atom, which subsequently favors its permeation through the lipid layer of the cell membrane. The copper(II) complex shows higher anti-fungal activity than other complexes and Co(II) complex shows higher anti-microbial activity than other complexes. The variation in the effectiveness of different compounds against different organisms depends either on the impermeability of the cells of the microbes or on differences in ribosome of microbial cells (Mehmet Sonmez et al., 2010; Kurtomg lu et al., 2006).

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Figure 6

Anti-bacterial studies (Gram-negative) of Schiff base ligand and its metal complexes. 1. E. coli. 2. K. pneumoniae. Standard = chloramphenicol inhibition zone in mm, concentration 100 μg/mL.

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Figure 7

Anti-bacterial studies (Gram-positive) of Schiff base ligand and its metal complexes. 1. S. aureus. 2. S. pyogones. Standard = tetracycline inhibition zone in mm, concentration 100 μg/mL.

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Figure 8

Anti-fungal studies of Schiff base ligand and its metal complexes. 1. F.oxysporum. 2. A. fumigatus. Standard = clotrimazole inhibition zone in mm, concentration 100 μg/mL.

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3.8 DNA binding studies

Electronic absorption spectroscopy is universally employed to determine the binding characteristics of metal complex with DNA. The absorption spectra of copper(II) complex in absence and presence of CT-DNA are shown in Fig. 9. In UV region two intense bands absorbed at 370 nm attributed to the ligand to metal charge transfer absorption and another at 265 nm which is assigned to the π→π∗ transition of aromatic chromophore. It has been reported that the intercalating ability of the complex depends on the planarity of ligands, the coordination geometry, ligand donor atom type and the metal ion type (Xu et al., 2003). Intercalative mode of binding usually results in hypochromism and red shift due to the strong stacking interaction between an aromatic chromophore and the base pairs of DNA. The extent of red shift and hypochromism are commonly found to correlate with the intercalative binding strength. But, metal complexes which bind non-intercalatively or electrostatically with DNA may result in hyperchromism or hypochromism. In general, the absorption spectra of metal complexes bound to DNA through intercalation exhibit significant hypochromism and red shift due to the strong π→π∗ stacking interaction between the aromatic chromophore ligand of metal complex and the base pairs of DNA (Terenzi et al., 2009). In copper(II) complex the decrease in absorption intensity (hypochromism) with a slight red shift is due to the intercalative binding between DNA and metal complexes. The absorption bands of complex at 265 nm shifted to red nearly 6, 8, 10 nm, respectively, with increasing concentrations (40, 60, 80 μM) (Colak et al., 2010).

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Figure 9

Absorption spectra of copper(II) complex in the absence and in the presence of CT-DNA. (a) Metal complex only, (b) complex with CT-DNA(40 μM), (c) complex with CT-DNA(60 μM), and (d) complex with CT-DNA(80 μM).

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3.9 DNA cleavage studies

Gel electrophoresis experiments were performed using CT-DNA with ligand, complexes in presence and absence of H₂O₂. Complexes exhibit cleavage ability at low concentration (40 μM). The ligand exhibits no significant activity in the presence of oxidant. The activity was much higher for the complexes in presence of H₂O_2. When calf-thymus DNA is subjected to electrophoresis, relatively fast migration will be observed for the intact super coil form (Form I). If scission occurs on one strand (nicking), the super coil will relax to generate a slower moving open circular form (Form II). If both strands are cleaved, a linear form (Form III) that migrates between Forms I and II will be generated (Zhang et al., 2002). From Fig. 10 the complexes show more activity in the presence of oxidant which may be due to the reaction of hydroxyl radical with DNA. These hydroxyl free radicals participate in the oxidation of the dexoyribose moiety followed by hydroxyl cleavage of sugar phosphate backbone. The results of DNA cleavage studies have been shown in Fig. 10. All metal complexes were able to convert DNA (Form I) into open circular (Form II). The binuclear Cu(II) complex was found to be highly active in cleaving DNA in the presence of hydrogen peroxide. The H₂O₂ is coordinated to the copper ions of the complex, affording a peroxo-dicopper species. This coordinated peroxide ion attacks the DNA phosphate bond via a nucleophilic mechanism and hydrolyzes the P–O bond (Gao et al., 2000).

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Figure 10

Cleavage of CT-DNA (30 μM) by the metal complexes (40 μM) in the presence of reducing agent H₂O₂ 50 μM, in 50 mM of Tris–HCl buffer (pH 7.2). From left to right Lane 1. Control; Lane 2. DNA + Ligand + H₂O₂; Lane 3. DNA + copper(II) complex; Lane 4. DNA + copper(II) complex + H₂O₂; Lane 5. DNA + nickel(II) complex + H₂O₂; Lane 6. DNA + cobalt(II) complex + H₂O₂; Lane 7. DNA + manganese(II) complex + H₂O₂.

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