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Original article
10 (
2_suppl
); S3367-S3374
doi:
10.1016/j.arabjc.2014.01.017

Synthesis, characterization, DNA binding and cleavage studies of mixed-ligand copper (II) complexes

, , ,
 Department of Chemistry, Osmania University, Hyderabad 500 007, Andhra Pradesh, India

⁎Corresponding author. Tel.: +91 9849549376; fax: +91 (40) 27099020. gyanakumari60@gmail.com (C. Gyana Kumari)

Received: , Accepted: ,
© The Author(s) 2014
Disclaimer:
This article was originally published by Elsevier and was migrated to Scientific Scholar after the change of Publisher.

Peer review under responsibility of King Saud University.

Abstract

New two copper complexes of type [Cu(Bzimpy)(L)H2O]SO4 (where L = 2,2′ bipyridine (bpy), and ethylene diamine (en)), Bzimpy = 2,6-bis(benzimidazole-2yl)pyridine have been synthesized and characterized by elemental analyses, molar conductance measurements, magnetic susceptibility measurements, mass, IR, electronic and EPR spectral studies. Based on elemental and spectral studies six coordinated geometries were assigned to the two complexes. DNA-binding properties of these metal complexes were investigated using absorption spectroscopy, fluorescence spectroscopy, viscosity measurements and thermal denaturation methods. Experimental studies suggest that the complexes bind to DNA through intercalation. These complexes also promote the cleavage of plasmid pBR322, in the presence of H2O2.

Keywords

Metal complexes
Fluorescence
Thermal melting
Viscosity
DNA cleavage
1

1 Introduction

Recently, a lot of beautiful complexes of ingenious design based on flexible bis (imidazole) and bis (triazole) ligands have been crystallographically characterized by different groups (Qi et al., 2008; Lan et al., 2008). However, bis(benzimidazole) ligands, which represent a class of aromatic N-donor organic linkers, are still less developed (Li et al., 2010; Chen et al., 2009). Benzimidazole and its derivative are an important class of aromatic heterocyclic compounds with a broad spectrum of biological activities such as antimicrobial (Özkay et al., 2011), anticancer (Kamal et al., 2013), anti-inflammatory (Kulkarni et al., 2013), antivirus (Ujjinamatada et al., 2007), and anticonvulsant (Rostom et al., 2009). DNA is an important cellular receptor, many chemicals exert their antitumor effects by binding to DNA thereby changing the replication of DNA and inhibiting the growth of the tumor cells, which is the basis of designing new and more efficient antitumor drugs and their effectiveness depends on the mode and affinity of the binding (Xu et al., 2012; Sathyaraj et al., 2010). Binding studies of small molecules to DNA are very important in the development of DNA molecular probes and new therapeutic reagents (Mrksich and Dervan, 1993). Transition metal complexes have attracted considerable attention as catalytic systems for use in the oxidation of organic compounds (Jiang et al., 2013), probes in electron transfer reactions involving metalloproteins (Wong-Deyrup et al., 2012), and intercalators with DNA (Liu et al., 2013). Numerous biological experiments have demonstrated that DNA is the primary intracellular target of anticancer drugs; interaction between small molecules and DNA can cause damage in cancer cells, blocking the division and resulting in cell death (Hemmert et al., 2001; Li et al., 1996; Zuber et al., 1998).Since the benzimidazole unit is the key-building block for a variety of compounds which have crucial roles in the functions of biologically important molecules, there is a constant and growing interest over the past few years for the synthesis and biological studies of benzimidazole derivatives (Gellis et al., 2008; Gu¨ven et al., 2007; Kopańska et al., 2004). In this study, we have concentrated on the synthesis, characterization, DNA binding and cleavage studies of mixed-ligand Cu(II) 2,6-bis(benzimidazol-2-yl)pyridine. DNA binding studies were studied by absorption spectra fluorescence spectra, thermal melting and viscosity measurements. The nuclease activity was studied with pBR322 DNA..

2

2 Experimental

2.1

2.1 Materials and chemicals

All other chemicals and solvents were of analytical reagent grade and were used as received unless otherwise noted. 2,6 – pyridine dicarboxylic acid, 2,2′ Bipyridine, ethylene diamine, Tris–HCl, Ethidium bromide (EB) and CT-DNA were purchased from Sigma–Aldrich chemicals. Millipore water was used for preparing buffer.

All the experiments involving with the interaction of the ligand and complexes with CT DNA were carried out in doubly distilled water buffer containing 5 mM Tris [Tris(hydroxymethyl)-aminomethane]. The solution of DNA in the buffer gave a UV absorbance ratio A260/A280 of about 1.8/1.9 indicating that the DNA was sufficiently free from protein (Marmur, 1961). The ligands and complexes were dissolved in a solvent mixture of DMSO and Tris–HCl buffer at the concentration 1.0 × 10−5 M. The stock solution was stored at 4 °C and used within 4 days.

2.2

2.2 Synthesis of ligands and complexes

The ligand Bzimpy (L) was prepared according to the procedure reported earlier (Addison and Burke, 1981). [Cu(Bzimpy)(bipy)H2O]SO4 (Qi et al., 2008).

This complex was prepared by mixing CuSO4.5H2O (0.249 g, 1 mM) in MeOH (50 ml), and 15 ml water and 2,2′ bipyridine (0.156 g, 1 mM) in 15 ml methanol this mixture was refluxed for 2 h. To this Bzimpy (0.312 g, 1 mM) in 15 ml methanol was added and refluxed at refluxing temperature for 3 h. The resulting green solution was slowly evaporated. After 5 days, an amorphous green precipitate was obtained; it was washed thoroughly with acetone, and then evaporated in vacuum over anhydrous CaCl2. The color of the complex is green and yield is 75%.

Analytical data: IR νmax: 3419–3385 cm−1 (νO–H), 3064 cm−1 (νN–H) 1606 cm−1 (νC⚌NIm), 1471 cm−1 (νC⚌

Nphen), 1444 cm−1 1116 cm−1 (νC⚌NImd), 456 cm−1 (νM–N) 619 cm−1 (νM–O); Anal. Calc. for C29H23N7SO5Cu Cal: C 53.99, H 3.59, N 15.21; found: C 54.11, H 3.54, N 15.30; μeff: 1.75 BM; UV–Vis (nm): 307, 402, 637, 927; ESI-MS: 547 [Cu(Bzimpy)(bipy)H2O]+2.[Cu(Bzimpy)(en) H2O]SO4 (Lan et al., 2008).

This complex was prepared by mixing CuSO4.5H2O (0.249 g, 1 mM) in MeOH (50 ml), and 15 ml water and ethylene diamine (0.060 g, 1 mM) in 15 ml methanol this mixture was refluxed for 2 h. To this Bzimpy (0.312 g, 1 mM) in 15 ml methanol was added and refluxed at refluxing temperature for 3 h. The resulting green solution was slowly evaporated. After 5 days, an amorphous green precipitate was obtained; it was washed thoroughly with acetone, and then evaporated in vacuum over anhydrous CaCl2. The color of the complex is green and yield is 72%.

Analytical data: IR νmax: 3308 cm−1 (νO–H), 3219 cm−1 (νN–H), 1575 cm−1 (νC⚌Cphen), 1327 cm−1 (νC⚌Npy) 1456 cm−1 (νC⚌Nphen), 1116 cm−1 (νC⚌NImd), 443 cm−1 (νM–N) 617 cm−1 (νM–O); Anal. Calc. for C21H23N7 SO5Cu Cal: C 45.94, H 4.22, N 17.86; found: C 45.72, H 4.12, N 17.78; μeff: 1.74 BM; UV–Vis (nm): 317, 402, 607; ESI-MS: 452 [Cu(Bzimpy)(en)H2O]+2.

2.3

2.3 Physical measurements

The percentage composition of C, H, and N of complexes and ligand L were determined using micro analytical methods on Perkin Elmer 240C (USA) elemental analyzer. FT-IR spectra of the ligand and its complexes were recorded by using KBr pellets in the range 4000–400 cm−1 using FT-IR spectrometer. The UV–Visible spectra of the ligand and its metal complexes were carried out in DMSO using Elico SL159 spectrophotometer. The mass spectra of the compounds were recorded by the ESI technique on VG AUTOSPEC mass spectrometer. Magnetic measurements were carried out on a Gouy balance model 7550 using Hg [Co(NCS)4] as standard. Diamagnetic corrections were carried out by Pascal’s constant (Figg’s and Lewis, 1967). ESR spectra of metal complexes were recorded on JEOL JES-FA200ESR spectrometer (X-band microwave unit).

The conductivity measurements were carried out in DMSO (10−3 M) using Digisun Electronic Digital conductivity meter, 0.01 M KCl solution is used for calibration. Melting points of the ligand and decomposition temperature of complexes were determined on Polmon instrument (Model No. MP-96). Emission spectra were recorded on a Hitachi RF-2500 Spectro fluorimeter at room temperature. Thermal melting temperatures were recorded using Hitachi U-2800 double beam UV–Vis spectrophotometer. Viscosity measurements were carried out using an Ostwald viscometer maintained at a constant temperature at 28.0 ± 0.1 °C in a thermostatic bath.

2.4

2.4 DNA binding and cleavage studies

2.4.1

2.4.1 Electronic absorption spectra

Absorption titration experiment was performed with fixed concentrations of the complexes. The complexes were dissolved in a solvent mixture of 1% DMSO and 99% Tris–HCl buffer (5 mM Tris–HCl; 50 mM NaCl, pH 7.1). Absorption titration experiments were performed in the absence and presence of DNA with increasing concentration of CTDNA. 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 CTDNA itself. From the absorption titration data, the binding constant (Kb) was determined using Eq. (1)(Wolfe et al., 1987).

(1)
[ DNA ] / ( ε a - ε f ) = [ DNA ] / ( ε b - ε f ) + 1 / K b [ ( ε a - ε f ) ] where [DNA] is the concentration of DNA in base pairs, the apparent absorption coefficient, εa, εf, and εb correspond to Aobsd/[M], the extinction coefficient of the free compounds and the extinction coefficient of the compound when fully bound to DNA, respectively. In plots of [DNA]/(εa−εf) versus [DNA], Kb is given by the ratio of slope to the intercept.

2.4.2

2.4.2 Fluorescence titration

Ethidium Bromide (EB) emits intense fluorescence in the presence of CT-DNA, due to its strong intercalation between the adjacent CT-DNA base pairs. It was previously reported that the enhanced fluorescence can be quenched by the addition of a second molecule (Song et al., 2006; Baguley and Bret, 1984). The extent of fluorescence quenching of EB bound to CT-DNA can be used to determine the extent of binding between the second molecule and CT-DNA. The competitive binding experiments were carried out in the buffer by keeping [DNA]/[EB] = 1.13 and varying the concentrations of the compounds. The fluorescence spectra of EB were measured using an excitation wavelength of 520 nm and the emission range was set between 550 and 750 nm. The spectra were analyzed according to the classical Stern–Volmer Eq. (2)(Lakowicz and Webber, 1973).

(2)
I 0 / I = 1 + K sv r where I0 and I are the fluorescence intensities at 599 nm in the absence and presence of the quencher, respectively, Ksv is the linear Stern–Volmer quenching constant, r is the concentration of the quencher.

2.4.3

2.4.3 Viscosity measurements

Viscosity experiments were conducted on Ostwald viscometer, immersed in a water bath maintained at 25.0 ± 0.1 °C. Titrations were performed for the compound (3 μM), and each compound was introduced into CT-DNA solution (50 μM) present in the viscometer. Data were presented as (η/η0)1/3 versus the ratio of the concentration of the compound to CT-DNA, where η is the viscosity of CT-DNA in the presence of the compound and η0 is the viscosity of CT-DNA alone. Viscosity values were calculated from the observed flow time of CT-DNA containing solutions corrected from the flow time of buffer alone (t0), η = (t − t0) (Tan et al., 2008).

2.4.4

2.4.4 Thermal denaturation

Thermal studies were carried out with an Hitachi double beam spectrophotometer model U-2800, equipped with temperature-controlling programmer (0.1 °C). The absorbance at 260 nm was continuously monitored for solutions of CT-DNA (100 μM) in the absence and presence of the complexes (10 μM). The temperature of the solution was increased by 1 °C min−1.

2.4.5

2.4.5 DNA cleavage

TE buffer (10 mM Tris–HCl and 1 mM Na2EDTA) was used for dilution of pBR322 DNA. TAE buffer (pH 8.0; 40 mM tris base, 20 mM acetic acid, 1 mM EDTA) was used for gel-electrophoresis. Super coiled pBR322 DNA (0.1 μg/μL) was treated with Cu(II) complexes (15 μM) in a clean Eppendorf tube. The contents were incubated for 1 h at 37 °C and loaded onto a 1% agarose gel after mixing 5 μl of loading buffer (0.25% bromophenol blue). The electrophoresis was performed at a constant voltage 50 V for 2 h until the bromophenol blue had traveled through 75% of the gel. Subsequently, the gel was stained for 10 min by immersion in ethidiumbromide solution. The gel was then destained for 10 min by keeping it in sterile distilled water. The plasmid bands were visualized by viewing the gel under a transilluminator and photographed in a Gel doc system (Alpha InfoTech Corporation).

3

3 Results and discussion

3.1

3.1 Characterization

3.1.1

3.1.1 Characterization by TGA/DTA

The thermo gravimetric analysis (TGA) and differential thermal analysis of complexes 1 and 2 were carried out at temperature ranges from 0 to 600 °C. A small weight loss in the temperature range of 200–250 °C for the complexes indicates the loss of coordinated water molecules from the complexes. The weight loss in the ranges of 400–500 °C is attributed to the decomposition of complexes.

3.1.2

3.1.2 Infrared spectral characterization

In the IR spectrum of Bzimpy the peaks observed in the range of 3384–3225 cm−1 are assigned to v(NH) stretching vibrations (Eriksson et al., 1994), the peak observed in the range of 3060 cm−1 is assigned to v(C–H) stretching frequency, the band at 1573 cm−1 is assigned to v(C⚌N) of the imidazole moiety (Mohani et al., 2002) and a peak at 1492 cm−1 is assigned to v(C⚌C) stretching frequencies. In the IR spectra of complexes the v(NH) is replaced by a new absorption in the region 3385–3432 cm−1, confirming the coordination of the metal ions through N-atom of the imidazole ring (Table 1). In the IR spectra of complexes 1 and 2a new absorption band in the region of 3447–3500 cm−1 is attributed to the v(OH) of the coordinated H2O to the metal ion (complexes 1 and 2). The IR spectra of the complexes also showed, shifted imidazole in plane resonance at 995–1058 cm−1, pointing to further coordination of the imidazole N-atom to the metal. The bipyridine ring stretching frequency i.e., v(C⚌C) and v(C⚌N) typically observed at 1505 and 1421 cm−1 respectively, experienced shifts to higher frequencies indicating the coordination of N-atom of bipyridine to the metal ion (Reddy and Manjula, 2007) (Fig. 1). In the far IR spectra of the complexes reveals ν(M–N) stretching vibrations in the region 420–461 cm−1 and ν(M–O) stretching vibrations are in the region of 580–582 cm−1 confirm the formation of metal complexes (Abdel-Rehman, 1996).

Table 1 FT-IR spectral studies of Bzimpy and Cu(II) complexes.
Compound νO–H νN–H νC–H νC⚌C νC⚌N νM–N νM–O
Bzimpy – 3325–3384 3060 1435 1573 – –
Complex 1 3419 3198 3064 1444 1575 456 582
Complex 2 3417 3198 3218 3055 1456 443 580
Proposed geometries of complexes 1 and 2.
Figure 1
Proposed geometries of complexes 1 and 2.

3.1.3

3.1.3 Electronic spectra, magnetic moment and mass spectra

The magnetic momentum (μeff) of the four complexes 1 and 2 is 1.75 BM and 1.78 BM respectively at room temperature, which indicates that the complexes are monomeric.

The electronic absorption spectra are often very helpful in the evaluation of results furnished by other methods of structural investigation. The electronic spectral measurements were used for assigning the stereochemistries of metal ions in the complexes based on the positions and number of d–d transition peaks. The electronic absorption spectra of the Bzimpy and its Cu(II) complexes were recorded at room temperature using DMSO as solvent. Only one broad band is observed at 637 nm (1) and 607 nm (2) in the electronic spectrum of the Cu(II) complex assigned to d–d transitions(2Eg → 2T2g) which is in conformity with octahedral geometry. The visible and ultraviolet spectra of complex compounds in modern coordination chemistry (New York: Interscience), followed by a strong band in the UV region are assigned to metal to ligand charge transfer (LMCT) transitions (Raman et al., 2002).

The ESI-Mass spectra of the complexes 1 and 2 give peaks at m/z: 547, 452 are assigned to [M]+ ion respectively.

3.1.4

3.1.4 EPR spectra

ESR spectral studies of paramagnetic transition metal (II) complexes yield information about the distribution of the unpaired electrons and hence about the nature of the bonding between the metal ion and its ligands. There have been many reports concerning the applications of ESR to square-planar or distorted octahedral complexes of Cu(II) and of the interpretations of the ESR parameters in terms of covalency of the metal-ligand bonding (Raman et al., 2005).

The Cu(II) complexes exhibited well resolved anisotropic signals in the parallel and perpendicular regions as shown in Figs. 1 and 2. The observed data (Table 2) showed that gΠ = 2.3 – 2.49 and g⊥ = 2.04 – 2.07.

Electronic spectra of two metal complexes (10 μM), complex Qi et al., 2008 C, (Lan et al., 2008) D in the absence (—) and presence (colors) of increasing amounts of CT-DNA, [DNA] = 10–100 μM. ([DNA] = 1.727 × 10−4 M). Arrow shows the absorbance changes upon increasing DNA concentration. Inset: Linear plots for the calculation of intrinsic binding constant Kb.
Figure 2
Electronic spectra of two metal complexes (10 μM), complex Qi et al., 2008 C, (Lan et al., 2008) D in the absence (—) and presence (colors) of increasing amounts of CT-DNA, [DNA] = 10–100 μM. ([DNA] = 1.727 × 10−4 M). Arrow shows the absorbance changes upon increasing DNA concentration. Inset: Linear plots for the calculation of intrinsic binding constant Kb.
Table 2 EPR Spectral studies of Cu(II) complexes.
Compound gΠ g⊥ gav G
Complex 1 2.30304 2.0423 2.74 1.25
Complex 2 2.42572 2.0652 3.04 1.344

The gΠ values are greater than g⊥ suggesting major distortion from octahedral symmetry in the Cu(II) complexes (Choi et al., 1977). The gΠ is a moderately sensitive function for indicating covalency. The gΠ > 2.3 is characteristic of anionic environment and gΠ < 2.3 is of covalent environment in M–L bonding. The observed gΠ values for the complexes showed gΠ > 2.3, which is characteristic of anion environment. The trend gΠ > g⊥ > 2.0023 observed for the complexes indicates that unpaired electron is localized in dx2 − y2 orbital of the Cu(II) ion. Thus a tetragonal geometry is proposed for the complexes. Axial symmetry parameter G = (gΠ−2)/(g⊥−2), which measures the exchange interaction between the metal centers in a polycrystalline solid has been calculated, if G > 4 the exchange interaction is negligible and if G < 4 it indicates considerable exchange interaction in the solid (Hathway et al., 1971).

The above reported complexes showed G values <4 indicating the exchange interaction in complexes. For these complexes, gΠ values between 2.3 and 2.4 further confirm the presence of copper–nitrogen and copper–oxygen bonds in these chelate complexes. From all the spectral data a distorted octahedral geometry has proposed for both the complexes (Fig. 1).

3.2

3.2 DNA binding studies

3.2.1

3.2.1 Electronic absorption titration

Electronic absorption spectroscopy serves as the most common means to study the interactions between metal complexes and DNA (Barton et al., 1984). A complex binds to DNA through intercalation usually results in hypochromism and bathochromism, due to intercalation mode involving a strong stacking interaction between an aromatic chromophore and the base pairs of DNA. Absorption spectra of complexes in presence of DNA are shown in Fig. 2. As the concentration of DNA was increased the MLCT transition bands of complexes exhibited hypochromism and bathochromism, indicates that the complexes bind to CT-DNA through intercalation. The binding strength of the two complexes, the intrinsic binding constants Kb of complexes with DNA were determined from the decay of the absorbance monitored for complexes. The intrinsic binding constant Kb of the complexes with CT-DNA was evaluated from the Eq. (1) (Wolfe et al., 1987). Intrinsic binding constants Kb of complexes 1 and 2 were obtained as 1.41 × 104 and 1.32 × 104 M−1. The stability constants of metal complexes containing Bzimpy are also in the ranges of 106–107 M−1 for example [Cr(Bzimpy)2]+3, [Ru(Bzimpy)2]+2, [Co(Bzimpy)2]+2, [Zn(Bzimpy)2NO3]NO3, [Ru(Bzimpy)(bipy)(H2O)]+3 and [Ru(Bzimpy)(phen)(H2O)]+3 are 1.21 × 104, 1.8 × 104, 1.6 × 105, 3.58 × 10 and 2.87 × 104 M−1 (Vaidyanathan and Nair, 2003a,b, 2002, 2005; Wang et al., 2005). These values are lower than the classical intercalators (Ethidium bromide), whose binding constants are in the range of 106–107 M−1. The binding strength of complex 1 is greater than complex 2, this may be due to the planar structure of the complexes.

3.2.2

3.2.2 Fluorescence studies

In the absence of DNA, both complexes can emit luminescence in Tris buffer at ambient temperature, with a maximum appearance at 420 and 435 nm respectively. Addition of DNA leads to increase in emission intensity of the complexes. Emission intensity of two complexes increases on increasing the concentration of CT DNA is illustrated in Fig. 3. The enhancement of emission intensity is an indication of binding of the complexes to the hydrophobic pockets of DNA, and complexes can be protected efficiently by the hydrophobic environment inside the DNA helix.

Emission spectra of complexes complex, (Qi et al., 2008) C, (Lan et al., 2008) D in the absence (- - -) and presence (—) of the increasing amounts of DNA ([DNA] = 1.727 × 10−4 M).
Figure 3
Emission spectra of complexes complex, (Qi et al., 2008) C, (Lan et al., 2008) D in the absence (- - -) and presence (—) of the increasing amounts of DNA ([DNA] = 1.727 × 10−4 M).

3.2.3

3.2.3 Quenching studies

Ethidium bromide (EB) emits intense fluorescence light in the presence of DNA, due to strong intercalation between the adjacent DNA base pairs. It was previously reported that the enhanced fluorescence can be quenched by the addition of a second molecule (Kelly et al., 1985; Boger et al., 2001). The extent of fluorescence of EB bound to DNA is used to determine the extent of binding between the second molecule and DNA.

The emission spectra of EB bound to DNA in the absence and in the presence of complexes are given in Fig. 6. The addition of the complexes 1 and 2 to DNA pretreated with EB causes obvious reduction in emission intensity, indicating that the complexes compete with EB in binding to DNA.

The classical Stern–Volmer equation (Kelly et al., 1987) is,

(2)
I 0 / I = 1 + K sv r where I0 and I are the fluorescence intensities in the absence and the presence of complex, respectively, Ksv is a linear Stern–Volmer quenching constant dependent on the ratio of rEB (the ratio of the bound concentration of EB to the concentration of DNA) and r is the ratio of total concentration of complex to that of DNA.The fluorescence quenching curves of EB bound to DNA by 1 and 2 are shown in Fig. 4. The quenching plots illustrate that the quenching of EB bound to DNA by the complexes is in good agreement with the linear Stern–Volmer equation, which proves that the four complexes bind to DNA. In the linear fit plot of I0/I versus [complex]/[DNA], the K values for 1 and 2 are 0.215 and 0.203 respectively. Based on the K values the order of binding strength of metal complexes is 1 > 2.
Emission spectra of EB bound to DNA ([DNA] = 2.33 × 10−5 M) in the absence (- - -) and presence (—) of complexes (10 μM) complex (Qi et al., 2008) C, (Lan et al., 2008) D; Inset: Stern–Volmer quenching curves.
Figure 4
Emission spectra of EB bound to DNA ([DNA] = 2.33 × 10−5 M) in the absence (- - -) and presence (—) of complexes (10 μM) complex (Qi et al., 2008) C, (Lan et al., 2008) D; Inset: Stern–Volmer quenching curves.

3.2.4

3.2.4 Viscosity measurements

For further establishment of the interactions between the complexes and DNA, viscosity measurements were carried out. Optical photo physical probes provide necessary, but not sufficient clues to support a binding model. The hydrodynamic measurements that are sensitive to length change (viscosity and sedimentation) are regarded as the least ambiguous and the most critical test of a binding in solution, in the absence of crystallographic structural data (Kelly et al., 1987). A classical intercalation model demands that the DNA helix must lengthen as base pairs are separated to accommodate the binding ligand, which leads to the increase of DNA viscosity. Ethidium bromide a well known DNA intercalator increases the relative viscosity strongly by lengthening the DNA double helix through intercalation. Upon increasing the concentration of complexes 1 and 2 the relative viscosity of complexes increases steadily similar to the behavior of ethidium bromide. The increased degree of viscosity, which may depend on the binding affinity to DNA, follows the order EB > 1 > 2 (Fig. 5). The increase in viscosity suggests that the complexes could bind to DNA by the intercalation binding mode which are consistent with electronic and fluorescence spectral data.

Effects of increasing amount of Ethidium bromide (E), complex 1 (C) and complex 2 (D) on the relative viscosity of CT-DNA at 29 °C ± 0.1, [DNA] = 15 μM.
Figure 5
Effects of increasing amount of Ethidium bromide (E), complex 1 (C) and complex 2 (D) on the relative viscosity of CT-DNA at 29 °C ± 0.1, [DNA] = 15 μM.

3.2.5

3.2.5 DNA melting studies

Intercalation of the complexes into DNA base pairs causes stabilization of base stacking thereby raising the melting temperature of the double-stranded DNA. The DNA melting experiment is useful in establishing the extent of intercalation. The complexes were incubated with CT DNA and their temperature rose from 25 to 90 °C and the absorbance at 260 nm was monitored. Conductivity and pH measurements were also carried out prior and after heating the complexes to 100 °C for 1 h (Wolfe et al., 1987). The complexes show ΔTm values of 5–8 °C which is characteristic of intercalative binding mode (Figs. 6 and 7).

Plots of absorbance versus temperature (°C) for the melting of CT DNA: D (only DNA), DNA + complex-1 (C).
Figure 6
Plots of absorbance versus temperature (°C) for the melting of CT DNA: D (only DNA), DNA + complex-1 (C).
Plots of absorbance versus temperature (°C) for the melting of CT DNA: D (only DNA), DNA + complex-2 (C).
Figure 7
Plots of absorbance versus temperature (°C) for the melting of CT DNA: D (only DNA), DNA + complex-2 (C).

3.2.6

3.2.6 DNA cleavage studies

Gel electrophoresis experiments using pBR322 DNA were performed with complexes in the presence and absence of H2O2 as an oxidant. The nuclease activity was greatly enhanced by the incorporation of metal ion in the respective copolymer; it is evident from Figs. 8 and 9, which shows that the complexes 1 and 2 cleave DNA more efficiently in the presence of oxidant, which may be due to the formation of hydroxyl free radicals. The production of hydroxyl free radical is due to the reaction between the metal complex and oxidant. These hydroxyl radicals participate in the oxidation of the deoxyribose moiety, followed by hydrolytic cleavage of the sugar phosphate backbone (Surendra Babu et al., 2007). The more pronounced nuclease activity in the metal complexes in the presence of oxidant may be due to the increased production of hydroxyl radicals. The cleavage efficiency was measured by determining the ability of the complex to convert the super coiled DNA to nicked (open circular) form or sheared form. As it is evident from Figs. 8 and 9, there is a considerable increase in the intensity of bands for open circular form in the case of complexes 1 and 2. This suggests that samples have nicking activity.

Changes in the Agarose gel electrophoresis pattern of pBR322 plasmid DNA, induced by H2O2 and metal complexes: DNA alone (1); DNA + complex 1 (50 μl) + H2O2 (2); DNA + complex 1 (40 μl) + H2O2 (3); DNA + complex 1 (30 μl) + H2O2 (4); DNA + complex 1 (20 μl) + H2O2 (5); DNA + complex 1 (10 μl) + H2O2 (6).
Figure 8
Changes in the Agarose gel electrophoresis pattern of pBR322 plasmid DNA, induced by H2O2 and metal complexes: DNA alone (1); DNA + complex 1 (50 μl) + H2O2 (2); DNA + complex 1 (40 μl) + H2O2 (3); DNA + complex 1 (30 μl) + H2O2 (4); DNA + complex 1 (20 μl) + H2O2 (5); DNA + complex 1 (10 μl) + H2O2 (6).
Changes in the Agarose gel electrophoresis pattern of pBR322 plasmid DNA, induced by H2O2 and metal complexes: DNA alone (1); DNA + complex 2 (50 μl) + H2O2 (2); DNA + complex 2 (40 μl) + H2O2 (3); DNA + complex 2 (30 μl) + H2O2 (4); DNA + complex 2 (20 μl) + H2O2 (5); DNA + complex 2 (10 μl) + H2O2 (6).
Figure 9
Changes in the Agarose gel electrophoresis pattern of pBR322 plasmid DNA, induced by H2O2 and metal complexes: DNA alone (1); DNA + complex 2 (50 μl) + H2O2 (2); DNA + complex 2 (40 μl) + H2O2 (3); DNA + complex 2 (30 μl) + H2O2 (4); DNA + complex 2 (20 μl) + H2O2 (5); DNA + complex 2 (10 μl) + H2O2 (6).

4

4 Conclusions

Two novel complexes of Cu(II) were synthesized, characterized by elemental analysis, conductance and various spectral techniques. Six coordinated geometries are assigned to complexes 1 and 2. DNA binding studies of the complexes were investigated by UV–Vis, fluorescence, thermal melting and viscosity measurements. These studies confirm that the complex 1 binds to CT-DNA through intercalation. The binding constants of complexes have been determined by the absorption titration spectrofluorimetric method.

The binding constant (Kb) of 1 and 2 was determined as 1.41 × 104 and 1.32 × 104 M−1 respectively. Quenching studies of the four complexes indicated that these complexes strongly bind to DNA, out of two complex 1 binds more strongly. Viscosity measurements indicated the binding mode of complexes with CT DNA by intercalation. Thermal melting studies also support intercalative binding. The nuclease activity of the above metal complexes shows that 1 and 2 complexes cleave DNA through redox chemistry.

Acknowledgments

The author is grateful to Director, Central Facilities Research and Development (Osmania University, Hyderabad). The author is also thankful to Director, Central Instrumentation laboratory, Hyderabad Central University, Hyderabad (India) for providing facility for EPR studies.

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