DNA Binding and Antimicrobial Activity of cobalt (III) complex containing 2,2’ bipyridyl and 8-hydroxy quinoline as ligands

 

S. Dhakshanamoorthy¹, N. Sri Gayathrai Devi¹, M. Murali Krishnan², M. N. Arumugham^1*

¹Department of Chemistry, Thiruvalluvar University, Vellore – 632115. Tamilnadu, India

²Department of Chemistry, Bannari Amman Institute of Technology, Sathyamangalam - 638401. Tamilnadu, India

 

ABSTRACT:

In this paper cobalt complex of type [Co(Bipy)₂(8HQ)](ClO₄)₂ [Bipy = 2,2’-bipyridyl, HQ = 8-Hydroxy Quinoline) has been synthesized and characterized by elemental analyses, NMR, UV-visible, infrared spectral studies. DNA binding properties of this metal complex was investigated using absorption spectroscopy, fluorescence spectroscopy, viscosity measurements and cyclic voltammetry. The experimental results indicate that the complex bind to DNA through an intercalative mode. The complex tested for biological activity and it has good antibacterial and antifungal activity on comparing with standard drugs.   

 

 

INTRODUCTION:

Transition metal complexes have been used as probes of DNA structure, for electron transfer and specific binding sites of nucleic acids. Recently speaking, ternary metal complexes have considered much attention because of their occurrence and vital role in the transport of metal ions in chemical biology systems [1-4].  DNA is the major target of chemotherapeutic agent due to the interaction between DNA and ligands in medicinal chemistry industry, which influence considerable damage in cancer cells, control the division of cancer cells and resulting in cell death offer new drugs for cancer cells.  This is due to their application as new chemotherapy agents and their photophysical properties that make them potential probes of DNA conformational structure [5-7].  

 

 

 

DNA is cellular host molecule, some of chemicals bring to bear their anticancer effects by binding the molecules to DNA and by this means changes the replication of DNA and inhibits the cancer cell growth, which leads designing new and more efficient chemotherapy agents.  Moreover, their drug activity on cell depends on the way of small molecules binding with DNA strands [8, 9].  Many transition metal complexes have important role as agent for mediation of strand scission of duplex DNA and as chemotherapy drug [10, 11].  The ligands, 1,10-phenanthroline and 2,2’-bipyridyl are strong field bidentate ligands that form more stable chelates with many transition metals. These ligands, as well as some of their derived complexes, do exhibit antimicrobial properties [12-15]. Recently, our group focused on DNA binding activity of important amino acid containing copper (II) complexes are really interested, copper (II) complexes of the type amino acid-Cu-heterocyclic base show efficient DNA binding, cleavage and cytotoxicity [16-18]. In this paper, we discussed effect of thiourea with heterocyclic bases copper (II) complexes on DNA binding, antimicrobial studies.

 

Experimental Section:

Synthesis of [Co(Bipy)₂Cl₂]Cl:

The complex was prepared according to the literature method [19].

 

Synthesis of [Co(Bipy)₂(8HQ)](ClO₄)₂:

2.380 g (5 mmol) of [Co(Bipy)₂Cl₂]Cl complex was dissolved in (25ml) ethanol and stirred well. About 0.725 g (5 mmol) of 8-Hydroxy Quinoline was dissolved in 25 ml of ethanol. Then this solution was added to the above solution the mixture was refluxed for 5 hours in the water bath. On cooling the solution to ambient temperature, an aqueous solution of sodium perchlorate 1.2245 g (10 mmol) was added to the above mixture and then refluxed for 30 minutes. A yellow colored substance was separated out, which was filtered and dried.

 

The DNA binding experiment [2] and computational details [20, 21] were given in the literature.

 

DNA binding studies:

UV-Vis absorption spectra:

Electronic absorption spectra were initially employed to study the binding of cobalt (III) complex with CT-DNA. The absorption spectra in aqueous buffer media of cobalt (III) complex in the absence and in the presence of CT DNA are given in Figure 1.

 

In the UV region, the complex presented bands at 221, 304 for complex which can be attributed to п→п* transition of the coordinated bipyridine ligand.

 

Figure.1: Electronic spectra of 5.0 х10^-5M complex [Co(Bipy)₂(8HQ)](ClO₄)₃ in the absence (---) and presence (^___) of increasing amount of CT DNA at the ratio r = 0.3, 0.5, 0.7, 1.0. Arrow (↑) shows the absorbance changes upon increasing DNA concentration. Inset: linear plot for the calculation of the intrinsic DNA binding constant. K_b.

 

The absorption intensity of the complex increased (hyperchromism) evidently after the addition of DNA, which indicated the interactions between DNA and the complex. A similarly hyperchromism has been observed for the soret bands of certain porphyrins when interacted with DNA but has not yet been clearly explained. On the other hand, since DNA possesses several hydrogen bonding sites which are accessible both in minor and major grooves, it is likely that the amine groups Schiff bases forms hydrogen bonds with N-3 of adenine or O₂^- of thymine in the DNA, which may contribute to the hyperchromism observed in the absorption spectra. The hyperchromic effect may also be due to the groove binding interaction between positively charged complex and the negatively charged phosphate backbone at the periphery of the double helix CT-DNA. Structurally, intercalation to DNA may be one of the binding patterns, since the cobalt(III) complexes contains bipyridine ligands which should provide aromatic moiety extending from the metal center through which overlapping occurs with base pairs of DNA by an intercalative mode. The intrinsic binding constant (K_b), was determined from the following equation:

 

[DNA]/(ε_a-ε_f) =[DNA]/(ε_b-ε_f)+1/K_b(ε_b-ε_f)

 

The enarappa't’ extinction coefficient (ε_a) was obtained by calculating A _obsd/[Co]. The terms ε_f and ε_b correspond to the extinction coefficients of free (unbound) and fully bound complex, respectively. A plot of [DNA]/(ε_a-ε_f) vs. [DNA] will give a slope 1/(ε_b-ε_f) and an intercept 1/K_b (ε_b-ε_f). K_b is the ratio of the slope and the intercept. The intrinsic binding constant (K_b) for the association of the complex with C-T DNA (inset of Figure.1) can be calculated using the absorption at 272 nm. The K_b value is three order of magnitude lower than that observed for classical intercalators such as ethidum bromide (K_b,1.4 х10⁶ M^-1) in 25mM Tris-HCl/40 mM NaCl buffer (pH 7.9). This indicates the complex have a very low binding affinity to DNA [22,23].

 

Fluorescence spectra.:

Competitive binding studies using DNA with bound ethidium bromide (EtBr) was carried out. The extent of fluorescence quenching of ethidium bromide (EB) by competitive displacement from DNA is a measure of the strength of interaction between the second molecule and DNA. The results in Figure.2 showed that the fluorescence intensity of CT-DNA-EB decreased remarkably with the addition of cobalt complex, which indicated that the complex can bind to DNA and replace EB from the CT-DNA-EB system. The above data was analyzed by means of the Stern-Volmer equation [24]. The quenching plots (inset of Figure.2) illustrates that the fluorescence quenching of EB bound to DNA by the complex is in linear agreement with the Stern-Volmer relationship, which corroborates that the complex bind to DNA. In the plot of I_o/I vs. [complex]/[DNA], K_sq value for the complex are 0.043.

 

 

Figure.2: Emission spectra of EB bound to DNA. [Co(Bipy)₂(8HQ)](ClO₄)₂ [EB]= 40 µM, [DNA] = 40 µM, [Complex] = (0-50 µM). Arrow (↓) shows the intensity changes upon increasing the concentration of the complex. Inset: Stern-Volmer quenching curves.

 

Viscosity measurement.:

Mode of interaction between the metal complex and DNA was clarified by viscosity measurements. Hydrodynamic measurements are sensitive to length change (i.e., viscosity and sedimentation) are regarded as the least ambiguous and the most critical test of binding in solution. A classical intercalation mode demands that the DNA helix lengthens as base pairs are separated to accommodate the bound ligand, leading to the increase of DNA viscosity. In contrast, a partial non-classical intercalation of ligand could bend (or kink) the DNA helix, reduce its effective length and concomitantly its viscosity.

 

Effect of the complexes on the viscosity of rod like DNA is shown in the (Figure 3). The viscosity of DNA is increased with the increase of the concentration of the complex in contrast to that of proven DNA intercalator. Based on the viscosity results, it was observed that these complex bind with DNA through intercalation binding [25, 26].

 

Figure.3: Effects of increasing amounts of complex on the relative viscosities of CT DNA at 25 ⁰C [Co(Bipy)₂(8HQ)](ClO₄)_2.

 

Cyclic voltammetry studies:

Cyclic Voltammetric (CV) response for complex in Tris-HCl buffer (pH 7.28) in the presence and absence of CT DNA is shown in (Figure 4). In the forward scan, a single cathodic peak was observed, which corresponds to the reduction of complex. In the reverse scan, no anodic peak was observed, which indicates that the process is irreversible. When CT-DNA is added to a solution of complex, marked decrease in the peak current and potential values were observed. The cyclic voltammetric behavior were not affected by the addition of very large excess of DNA, indicating that the decrease of peak current of complexes after the addition of DNA due to the binding of [Co(Bipy)₂(SB)](ClO₄)₂ complex to the DNA. When the concentration of DNA increased, the changes in peak current and potential become slow. This reveals that the complex were interact with CT-DNA [27].

 

Figure.4: Cyclic voltammogram of 0.5 mM complex [Co(Bipy)₂(8HQ)](ClO₄)₂ in the absence (^__) and presence (---) of 2.5 mM DNA.

Antimicrobial screening:

The cobalt (III) complex were screened in vitro for its antibacterial activity against certain pathogenic bacteria and fungi species using disc diffusion method. These complex were found to reveal considerable activity against Gram +ve, Gram -ve bacteria and fungi. The test solutions were prepared in sterile distilled water and the results of the antimicrobial activities are summarized in Table 1. The complexes were found to reveal considerable activity against Gram +ve, Gram –ve bacteria and fungi. The results of the antimicrobial activities are summarized in Table 1. The complex showed greater extent of inhibition to the growth of bacteria. This may be due to the possible mode of increased toxicity of the cobalt complexes. Chelation considerably reduces the polarity of the metal ion because of partial sharing of its positive charge with donor groups and possible π–electron delocalization over the whole chelate ring. Such a chelation could enhance the lipophilic character of the central metal atom, which subsequently favours it permeation through lipid layers of cell membrane and blocking the metal binding sides on enzymes of microorganism. Either the variation in the effectiveness of different compound against different organisms depends on the impermeability of the cells of the microbes or differences in Ribosome’s of microbial cells [28].

 

Table1:Antimicrobial activity of complex [Co(Bipy)₂(8HQ)](ClO₄)₂

Name of the test organism	Cobalt complex
Gram +ve
Staphylococcus aureus	8
Micrococcus luteus	14
Bacillus cereus	10
Gram -ve
Escherichia coli	10
Klebsiella pneumonia	11
Pseudomonas aeruginosa	-
Fungi
Aspergillus niger	13
Aspergillus flaves	10
Candida albicans	15

 

DFT studies:

The electrostatic potential (ESP) displayed nucleophilic region near oxygen atom of the hydroxyl quinoline, electrophilic region near hyrodgen of heterocyclic base and aromatic ring centre is almost neutral (Fig. 5). The energy gap between HOMO and LUMO is 0.602 ev (Fig. 6). [29-31]

 

Figure.5: Electrostatic potential iso-surface map of complex.

 

Figure.6: HOMO and LUMO energy gap of complex

 

CONCLUSION:

We described here Cobalt (III) complex. Further characterization of the complexes was achieved through physico-chemical and spectroscopic methods. The effectiveness of the binding of the complexes is being confirmed by means of hyperchromism in the electronic spectral studies and change in intensity of emission in the case of emission spectral studies. Besides, the effect of binding is also confirmed by the viscometric and cyclic voltammetric studies. This shows that the complex interact with DNA base pairs effectively. The cobalt (III) complex has exhibit good antimicrobial actives.

 

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Accepted on 18.07.2018        © RJPT All right reserved

Research J. Pharm. and Tech 2019; 12(2):472-476.