Synthesis, Characterization, DNA binding, DNA Cleavage and Antibacterial Studies of Ni(II) and Cu(II) Complexes of Pyridoxal Semicarbazone

Introduction

Transition metal complexes of Sciff base ligands based on semicarbazone and thiosemicarbazone have made a major contribution in the development of coordination chemistry [1]. Their study attracted due attention because of their ease of formation, structural diversity, multidentate nature and biological applications [2]. Numerous structural and biological studies have been made on the metal complexes of semicarbazones [3]. Ni(II) complexes of semicarbazone ligand show anticancer activity [4], antibacterial and antifungal activities [5] and Cu(II) complexes containing semicarbazone displayed antifungal [6], antioxidant, radical scavenging and antibacterial activities [7]. Pyridoxal Semicarbazone (PLSC), a Schiff base derived from pyridoxal (a form of vitamin B6) and its transition metal complexes are investigated by many researchers [8,9]. Several reports have been made on the synthesis, structure and the biological activity of transition metal complexes incorporating PLSC ligand. In its metal complexes PLSC can exist as neutral, mono and dianionic forms [10]. It is reported as tridentate ONO ligand, which coordinates through hydrazine nitrogen, phenolic and carbonyl oxygen atoms and an excellent chelating agent [11]. Its metal complexes exhibit enormous potential as biologically active agents. Transition metal complexes of PLSC show antimicrobial activity [12], anticancer activity [13] and cytotoxicity [14]. However, no significant studies were made on the DNA binding and cleavage properties of metal complexes of PLSC.

DNA binding properties of transition metal complexes have been extensively studied during the past two decades as they can be used as anticancer drugs and DNA structural probes [15]. Metal complexes interact with the double helix DNA in either covalent or noncovalent way. The non-covalent way includes three modes of binding, i.e., electrostatic effects, groove binding and intercalation, among which intercalation is the most important binding mode [16]. Small molecules when bind to DNA through intercalation, can damage DNA in cancer cells and hence can be used as anticancer drugs [17]. The ability of transition metal complexes to cleave nucleic acids efficiently with a high level of selectivity for a site or sequence offers many applications for the manipulation of genes and development of novel therapeutics [18].

The aim of the present work is to synthesize the Ni(II) and Cu(II) complexes of pyridoxal semicarbazone, to understand their structure and to evaluate their DNA binding and cleavage properties along with antibacterial activity.

Experimental

Materials and reagents

All the chemicals were procured from Sigma Aldrich and of analytical grade and used without further purification. Ligand was synthesized according to a reported procedure [19].

Methods

Instrumentation

Elemental analyses (% CHN) were obtained using Thermo Finnigan 1112 elemental analyzer. ESI mass spectra of the complexes were recorded on LCMS 2010A, Schimadzu spectrometer. FT-IR spectra of the complexes were recorded using KBr pellets in the range of 4000-250 cm−1 on a Schimadzu IR Prestige-21 FTIR spectrophotometer. UV-Vis spectra in DMSO solution were recorded on Systronics UV-Vis Double beam spectrophotometer 2201 in the range of 200-1000 nm. The molar conductivity was measured with a Digisun digital conductivity bridge using a freshly prepared solution of the complexes in DMSO. Thermogravimetric (TG) analyses were performed using Schimadzu TGA-50H in nitrogen atmosphere in the temperature range of 0°C to 1000°C with a heating rate of 20°C per min. Magnetic susceptibilities were measured at room temperature on Faraday balance model 7550 using Hg[Co(NCS)4] as the internal standard. Diamagnetic corrections were made by using Pascal’s constants [20]. DNA cleavage experiments were performed with the help of Biotech electrophoresis system supported by Genei power supply over a potential range of 50-500 V, visualized and photographed by Biotech Transilluminator system.

DNA binding activity by electronic absorption spectra

The binding of complexes with CT DNA was measured in potassium phosphate buffer solution (pH 7.2). A solution of DNA in the buffer gave a ratio of UV absorbance at 260 nm and 280 nm, A260/A280 of 1.85-1.9, indicating that the DNA was sufficiently free of protein. The concentration of DNA was determined from the UV absorbance at 260 nm using the extinction coefficient ε260=6600 M−1 cm−1. The absorbance titrations were performed at a fixed concentration of complexes and varying the concentration of double stranded CT-DNA (2-20 μM). While measuring the absorption spectra, a proper amount of CT-DNA was added to both compound solution and the reference solution to eliminate the absorbance of CT DNA itself. Concentrated stock solutions of the complexes were prepared by dissolving the complexes in DMSO and diluting suitably with the corresponding buffer to the required concentration (20 μM) for all the experiments. After the addition of DNA to the metal complex, the resulting solution was allowed to equilibrate for 10 min, after which absorption readings were noted. The data were then fit to the following equation to obtain intrinsic binding constant Kb.

[DNA]/[εa –εf]=[DNA]/[εb –εf]+1/Kb[εb–εf] (1)

Where [DNA] is the concentration of DNA in base pairs, εa is the extinction coefficient observed for the absorption band at the given DNA concentration, εf is the extinction coefficient of the complex free in solution, and εb is the extinction coefficient of the complex when fully bound to DNA. A plot of [DNA]/[εa-εf] versus [DNA] gave a slope 1/[εa-εf] and Y intercept equal to (1/Kb) [εb-εf], respectively. The intrinsic binding constant Kb is the ratio of the slope to the intercept [21].

Competitive DNA binding fluorescence experiments

Relative binding of the complexes to CT DNA was studied using fluorescence spectroscopy, by the displacement of ethidium bromide (EB) bound to CT DNA in a potassium phosphate buffer solution (pH 7.2) [22]. In a typical experiment, 480 μl of CT DNA (20 μM) solution was added to 2020 μl of EB in buffer solution {[DNA]/[EB]=1}. The fluorescence intensity was measured upon excitation at λmax=520 nm; maximum emission was observed at λmax 605 nm. The changes in fluorescence intensities at 605 nm of EB bound to DNA were recorded with an increasing amount of the complex concentration (from its 50 μM stock solution). Stern –Volmer quenching constants were calculated using the equation I0/I=1+Ksv.r, where I0 and I are the fluorescence intensities in the absence and presence of the complex respectively, K sv is a linear Stern-Volmer quenching constant and r is the ratio of total concentration of complex to that of DNA. The value of Ksv is given by the ratio of slope to intercept in a plot of I0/I Vs [complex]/ [DNA].

Viscosity measurements

Viscosity measurements were carried out with the Ostwald viscometer, maintained at 25ºC in a thermostatic water bath. Each complex (50 μM) was introduced into CT-DNA solution (300 μM) in phosphate buffer (pH 7.2) present in the viscometer. Flow time of solutions was recorded in triplicate for each sample and an average flow time was calculated. Data were presented as (ƞ'sp/ ƞsp)1/3 versus the ratio of the concentration of the complex to CT-DNA, where ƞ'sp is the viscosity of CT-DNA in the presence of the complex and ƞspis the viscosity of CT-DNA alone [23].

DNA cleavage studies

Agarose gel electrophoresis technique was used to monitor the DNA cleavage ability of the metal complexes on super coiled pBR 322 DNA. Generally, plasmid DNA is converted from super coiled DNA (Form I) to nicked circular (Form II) and linear forms (Form III) [24]. In the experiment, plasmid DNA (300 ng/3 μl) was treated with the complexes in DMSO (20-60 μM) in 5 mM Tris. HCl/50 mM NaCl buffer (pH 7.2). The mixture was incubated for 1 h at 37ºC. A loading buffer containing 1% bromophenol blue and 40% Sucrose (1 μl) was added and loaded onto a 0.8% agarose gel containing EB (1 μg/ml). The gel was run in TAE buffer (40 mM Tris base, 20 mM Acetic acid, 1 mm EDTA, pH 8.3) at a constant voltage 60 V for 2 h until the bromophenol blue had traveled through 75% of the gel. The bands were visualized by viewing the gel on a transilluminator and photographed.

Antibacterial activity

The complexes are screened for their antibacterial activity using agar well diffusion method against two gram positive bacteria such as Staphylococcus aureus and Bacillus subtilis and two gram negative bacteria such as Escherichia coli and Pseudomonas aeruginosa at a concentration of 1000 μg/mL [25].

Synthesis of Metal Complexes

Synthesis of Ni(II)-PLSC

Ni(II)-PLSC was prepared by the addition of 0.228 g (1 mmol) NiCl2.6H2O(in 10 ml distilled water) to a hot aqueous solution 0.5 g (2 mmol) of the Schiff base ligand (40 ml distilled water) and refluxed for 2 h. A red colored precipitate was formed on adjusting the pH to 7 using a mixture of methanol and ammonia solution. The mixture was continued to reflux for another hr. Then the precipitate was filtered, washed several times with hot distilled water, finally with petroleum ether and air dried.

[Ni(HL-).Cl.2H2O]

(Yield: 0.29 g, 68%).IR: νmax/cm-1: 3437 s, 3331 m, 2873 m,1577 m, 1558 m, 1500 m, 1259 m, 1145 m, 520-550 m(Ni-O), 450-470 m(Ni-N), 310-350 (Ni-Cl). ESI-MS in MeOH: m/Z 355[M++H]. Elemental analysis: Found (calc.)for C9H15NiN4O5Cl: C, 32.03 (32.23); H, 4.73 (4.77); N, 16.70 (16.71) %. μeff=2.8835 B.M. É?M[Ω-1cm2M-1, 10-3, DMSO]: 005

Synthesis of Cu(II)-PLSC

Cu(II)-PLSC was prepared by the by the addition of 0.1636 g (1 mmol)CuCl2.2H2O (in 10 ml distilled water) to a hot aqueous solution 0.5 g (2 mmol) of the Schiff base ligand (in 40 ml distilled water) and refluxed for two hr. A green colored precipitate was formed on adjusting the pH to 7 using a mixture of methanol and ammonia solution. The mixture was continued to reflux for another hr. Then the precipitate was filtered, washed several times with hot distilled water, finally with petroleum ether and air dried.

[Cu(HL-)2].H2O

(Yield: 0.34 g, 71%). IR: νmax/cm-1: 3466 s, 3377 m, 2899 m, 1585 m, 1545 m, 1500 m, 1263 m, 1132 m, 515-570 m(Cu-O), 430- 440 m(Cu-N). ESI-MS in MeOH: m/Z 528[M+-H]. Elemental analysis: Found (calc.) for C18H24CuN8O7: C, 40.92 (40.90); H, 4.51 (4.54); N, 21.18 (21.21) %. μeff=1.6961B.M. É?M[Ω-1cm2M-1, 10-3, DMSO]: 010

Conclusion

Pyridoxal semicarbazone forms complexes with Ni(II) and Cu(II) ions by coordination through ONO atoms. [Ni(HL-).Cl.2H2O] is formed in 1:1(ML) while [Cu(HL-)2].H2O, is formed in 1:2 (ML2) ratios. Both the complexes exhibited octahedral geometry. The complexes were evaluated for their DNA binding activity by absorption titrations, fluorescence spectra and viscometric measurements which suggest that both the complexes are able to bind DNA effectively even at very low concentrations through intercalation. The complexes also exhibited plasmid DNA cleavage ability by hydrolytic pathway. The complexes showed a moderate antibacterial activity against gram positive bacteria.

Acknowledgements

The authors are thankful to University Grants Commission, New Delhi for financial support and also Osmania University, Hyderabad and St. Francis College for Women, Hyderabad for providing necessary facilities to carry out this work.

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