Synthesis, Characterisation, DNA binding/cleavage, anticancer and antimicrobial activity of Ternary Copper(II) Complexes

 

S. Dhakshanamoorthy¹, M. Murali Krishnan², M.N. Arumugham¹*

¹Department of Chemistry, Thiruvalluvar University, Vellore – 632 115, Tamilnadu, India.

²Department of Chemistry, Bannari Amman Institute of Technology, Sathyamangalam – 638 401, Tamilnadu, India

*Corresponding Author E-mail: aru_mugham@yahoo.com

Ternary copper(II) complexes [Cu(L-Ser)(HB)(U)](ClO₄) (1 and 2) where L-Ser = L-Serine, U = Urea, HB is a N,N-donor heterocyclic bases, viz. 1,10-phenanthroline (phen) (1), 2,2’- bipyridine (bpy) (2), are synthesized, characterized, and their DNA binding, cleavage and antimicrobial activities are studied. The one-electron paramagnetic complexes display a d–d band near 600 nm in water.  Binding interactions of the complexes with calf thymus (CT) DNA have been investigated by absorption, emission, viscosity, and cyclic voltammetry studies. The complexes cleave the supercoiled DNA (pBR 322) to its nicked circular form in the presence of gallic acid at appropriate complexes concentration. The phen complex (1) displays significant binding propensity to the CT DNA. The bpy complex (2) does not show any significant binding to the DNA and hence poor cleavage efficiency.

 

 

 

INTRODUCTION:

Transition metal complexes show much interest in cleaving DNA for their vital role in drug design and anti cancer agent [1–8]. The purine base guanine is most susceptible for oxidation among four nucleobases. Spectral properties of transition metal complexes offer a great scope of design for species that are suitable for DNA binding and cleavage activities [9,10]. Also the metal complexes as a drug are more active in presence of heterocyclic donor ligand such as 2,2’-bipyridine (bpy), 1,10-phenanthroline (phen) [11]. These metal complexes are known to interact with DNA via both covalent and non covalent interactions. 

 

In covalent interaction the ligand of the complexes are replaced by a nitrogen base of DNA such as guanine-N7 and in the non-covalent interaction include electrostatic and groove (surface) binding of cationic transition metal complexes along outside of DNA, and along major or minor groove [12]. The transition metal complexes interact with DNA has been demanding investigation with the perspective of development of new anticancer drugs in medicine [13, 14]. Recently our group synthesized [Co(trien)(phen)]³⁺ [15] and phenanthroline copper(II) and Co(III) complexes, and studied their DNA binding/cleavage and anticancer properties. The role of ternary copper(II) complexes in biological systems is well known [16, 17].  In this paper, we reported ternary copper(II) complexes with mixed ligands and study their DNA binding/cleavage properties, antimicrobial and cytotoxicity activities.

 

MATERIALS AND METHODS:

The reagents such as CuCl₂.2H₂O, NaOH, NaClO₄.H₂O, 2,2-bipyridine, L-serine, Urea, 1,10-Phenanthroline, CT DNA, pBR322 DNA, gallic acid, Tris HCl, NaCl and ethidium bromide were purchased from Aldrich.  The spectroscopic titration was carried out in the buffer (50 mM NaCl–5 mM Tris–HCl, pH 7.1) at room temperature. Absorption spectra were recorded on a UV/VIS Shimadzu 2450 Spectrophotometer using cuvettes of 1-cm path length and emission spectra were recorded on a JASCO FP 770 spectrofluorimeter.  FT-IR spectra were recorded on a FT-IR Perkin Elmer spectrophotometer with samples prepared as KBr pellets.  EPR spectra were recorded on Varian E-112 EPR spectrometer at room temperature, the field being calibrated with DPPH = 1, 10-diphenyl-2-picrylhydrazyl (g =2.0037).

 

A solution of calf thymus DNA in the buffer gave a ratio of UV absorbance 1.8 – 1.9:1 at 260 and 280 nm, indicating that the DNA was sufficiently free of protein. Milli-Q water was used to prepare the solutions.  Cyclic voltammetry experiments were recorded on CHI 602D (CH Instruments Co., USA) electrochemical analyzer under oxygen free conditions using a three-electrode cell in DMF solution with TBAP (0.1 M) as the supporting electrolyte.  A pt wire, glassy carbon, and the Ag/AgCl (in saturated KCl solution) electrodes were used as counter, working and reference.

 

Synthesis of [Cu(L-Ser)(phen)(U)](ClO₄)    (1)

The complex [Cu(L-Ser)(phen)(H₂O)](ClO₄) was synthesized according to a published method [18].  To the aqueous solution of [Cu(L-Ser)(phen)(H₂O)](ClO₄) (1 mmol) was added urea (1 mmol) the colour of the solution change from blue to bluish green.  The resulting solution was stirred for 4 hrs and then solution complex was filtered.  The filtrate was kept for slow evaporation, after two weeks blue green colored complex was separated out.

 

Yield: 68%; Anal. (%) Calc. for C₁₆H₁₈ClCuN₅O₈: C, 37.88; H, 3.58; N, 13.80.  Found: C, 36.82; H, 3.10; N, 13.25.  IR (KBr pellet): 3284, 3120, 3030, 1622, 1598, 1323, 1226, 1168, 1149, 858, 783, 719 cm^-1.  UV-Vis (λ, nm): 272, 294.

 

Synthesis of [Cu(L-Ser)(bpy)(U)](ClO₄)    (2)

Synthesis was described in complex 1, using [Cu(L-Ser)(bpy)(H₂O)](ClO₄) (1 mmol) and urea (1 mmol).  Yield: 66%; Anal. (%) Calc. for C₁₄H₁₈ClCuN₅O₈: C, 34.79; H, 3.75; N, 14.49.  Found: C, 34.54; H, 3.83; N, 14.26.  IR (KBr pellet): 3421, 3230, 2960, 2920, 2331, 1745, 1639, 1597, 1328, 1155, 1035, 1018, 100, 773, 574 cm^-1.  UV-Vis (λ, nm): 239, 300 and 310.

 

The detailed experimental setup of DNA binding, MTT assay and antimicrobial activity described in [15, 17].

 

RESULTS AND DISCUSSION:

Synthesis and general aspects:

The mixed-ligand copper(II) complexes (1 and 2) have been synthesized (Scheme 1) from the complex [Cu(L-Ser)(phen/bpy)(H₂O)](ClO₄) by ligand substitution method.  The copper(II) complexes are soluble in water and most of the organic solvents. The complex is one electron paramagnetic at room temperature, corresponding to d⁹ electronic configuration for the copper(II) center.  The elemental analysis data indicate that the metal-ligand ratio is 1:1:1 in complex, which is consistent with the obtained spectral results.  In the UV–Vis region, the intense absorption bands appeared from 240 to 300 nm is attributed to intraligand transitions.  Another band which appeared around 270 nm is assigned to ligand field transitions, the d-d transitions band centered at 594 and 596 nm for complexes 1 and 2 respectively (Fig. 1), indicating a square-pyramidal geometry in the metal center. In infrared spectra, asymmetric and symmetric COO- stretching vibrations for complex 1 was observed at 1622 and 1309 cm^-1, the differences indicate that carboxylate ion coordinate with metal by monodentate fashion (Fig. 2).   The solid state EPR spectra of the copper (II) complexes were recorded in X-band frequencies.  At room temperature the complexes exhibit well defined single isotropic feature near g = 2.13 and g = 2.11 for complexes 1 and 2 respectively.  Such isotropic lines are usually the results of intermolecular spin exchange, which broaden the lines.

 

 

Scheme 1. Preparation of complexes 1 and 2

 

Fig 1. UV Spectrum of complexes 1 and 2

 

Fig 2. IR spectrum of complexes 1 and 2

 

 

DNA binding properties

Electronic spectral studies

The UV/Vis absorption spectra of the complexes (1 and 2) in the presence and absence of calf-thymus DNA are shown in Fig. 3.  In the presence of CT-DNA, an increase in absorption intensity (hyperchromism) was observed, with slight decrease wavelength (bathochromic shift) for complex 1 and a decrease in absorption intensity (hypochromism) was observed, with slight decrease in wavelength (bathochromic shift) for complex 2.  These shifts are indicated that interaction between the complex and those of the DNA bases.  The changes suggested that complex 1 was consistent with intercalative and complex 2 was electrostatic interaction into the DNA base pairs.   The binding constants (k_b) of the two complexes (inset of Fig. 1) with CT-DNA were 9.49 X 10³ and 5.62 X 10³ M^-1 for complex 1 and 2 respectively.  The higher binding tendency of the phen complex 1 in comparison to its bpy complex 2 analogue could be due to the presence of planar aromatic ring in phen [19].

 

Fig 3. Absorption spectral traces on addition of CT DNA to complexes 1 and 2 (shown by arrow).  Inset plot of [DNA]/(ε_a-ε_f) vs [DNA] for absorption titration of CT DNA with complex at 271 nm.

 

 

Fig 4. Thermal denaturation profiles of CT DNA (140 μM) alone and in the presence of complexes 1 and 2 (40 μM) in 5 mM phosphate buffer (pH 6.85)

 

To investigate the interaction of the complexes with CT-DNA, thermal denaturation study carried out.  On increasing temperature of the CT-DNA solution, the complex 1 exhibits a hyperchromic effect on absorption spectra of DNA bases at 272 nm (λ_max).  Generally, the intercalative mode of binding with DNA increases the DNA melting temperature (T_m).  The T_m has been found to increase in presence of complex 1 suggesting an intercalative mode of binding.  The binding of the complex 2 slightly decrease in melting temperature (T_m) of CT-DNA suggesting electrostatic binding nature of the complex (Fig. 4).

 

Fluorescent spectral studies:

Ethidium bromide (EB) emits fluorescence light in the presence of DNA due to strong intercalation, and its fluorescence behavior quenched by addition of a second, competitive molecule.   In Fig. 5, the emission spectra of EB bound to DNA in the absence and presence of complexes 1 and 2 are shown, the addition of the complexes to DNA with EB produced significant decrease in emission intensity, indicating that the complexes bound with EB in DNA binding [20].  The quenching plots (inset in Fig. 5) demonstrate that the quenching of the EB fluorescence by complexes 1 and 2 is in good agreement with the linear Stern –Volmer relationship, which confirms that the two complexes (1 and 2) bind to DNA.  In the plot of I₀/I vs. [complex]/[DNA], K_sv is given by the ratio of the slope to the intercept.  From these data, K_sv values of 0.456 and 0.277 were determined for 1 and 2, respectively.

 

Fig 5. Emission spectra of EB bound to DNA in the absence (dotted line) and the presence (dashed line) of complexes 1 and 2.  Arrow (↓)  shows the intensity changes upon increasing the concentration of the complex. Inset: Stern–Volmer quenching curves.

Viscosity measurements:

The viscosity of CT-DNA in the presence of the complexes has been measured to investigate further modes of binding of the complexes.  It is well-known that intercalative binding of a compound into DNA produces significant increase in the viscosity of the DNA solution due to an increase in the separation of the base pairs to accommodate the bound species [21,22]. 

 

Fig 6. Effect of increasing amounts of complexes on the relative viscosities of CT DNA at 25⁰C.

 

The plots of relative viscosities with 1/R = [complex]/[DNA] show significant changes in the viscosity for the complexes 1 and 2 (Fig 6).  The increase in viscosity of the complex 1 is observed and there is no significant change in viscosity of complex 2.  The viscosity data suggests intercalative mode of binding of the complex 1 to CT-DNA and electrostatic binding of the complex 2 to CT-DNA.

 

Cyclic voltammetry studies:

The application of CV to study binding of metal complexes to DNA is a useful complement.  The typical cyclic voltammogram of complexes 1 and 2 in the absence and presence of [DNA] is shown in Fig. 7.  The cathode and anode peak currents changed in the presence of DNA.  The change in current may be attributed to molecules bound to DNA.

 

In the absence of DNA, the cathodic peak appears at 0.370V and anodic peak appears at -0.163V, the separation of the anodic and cathodic peak potential (ΔE_p) is -0.207V for complex 1, for complex 2 the cathodic and anodic peak appears at 0.110V and -0.220V respectively, and the ΔE_p is -0.330V.  The redox couples ratio of Ipc/Ipa is approximately unity, indicating that the reaction of the complexes on the working electrode surface is quasireversible.  In the addition of DNA to the complex 1, the considerable changes with redox couple causes a negative shift in E_1/2 (-0.522V) and complex 2 redox couple shift in E_1/2 (-0.341V). The electrochemical potential shift after reacting with DNA is suggested that complexes interact with DNA.

 

DNA cleavage:

DNA cleavage is also a useful method to probe DNA-complex interactions.  Supercoiled plasmid DNA (Form I) cleavage by the Cu(II) complexes into nicked DNA (Form II) and linear DNA (Form III) was studied in the presence of gallic acid (Fig. 8).  We found that the supercoiled DNA was completely cleaved by complex 1 in the presence of gallic acid when increase ratio of complex/gallic acid, but complex 2 didn’t cleave significantly compared to complex 1.  This result revealed that complex 1 was a potent DNA cleavage agent in the presence of gallic acid as a reducing agent.

 

 

Fig 7. Cyclic voltammogram of complexes (1 and 2) in the absence (dashed line) and presence (dotted line) CT DNA.

 

 

Fig 8. Cleavage of supercoiled pBR322 DNA by complexes 1 and 2 at different concentrations in the presence of gallic acid. Lane 1, DNA + complex (10μM) + Gallic acid (1mM); Lane 2, DNA + complex (20μM) + Gallic acid (1mM); Lane 3, DNA + complex (30μM) + Gallic acid (1mM); Lane 4, DNA + complex (40μM) + Gallic acid (1mM); Lane 5, DNA + complex (50μM) + Gallic acid (1mM); Lane 6, DNA + complex (60μM) + Gallic acid (1mM); Lane 7, DNA + complex (10μM) + Gallic acid (1mM).

 

MTT assay:

The cytotoxicity of the complexes to be used as chemotherapeutic agents was studied using MTT assay.  The ability of the complexes on HepG2 cells was tested with or without various concentrations (10–50 μg/ml) of complex 1.  Cells incubated with different concentration (Fig. S4) of Doxorubicin served as positive control.  After incubation period, MTT assay was carried out to calculate the cell death percentage [23].  For each concentration, of the complexes cells were incubated in triplicate. The (Fig. 9) clearly illustrates that there is a clear decrease in the live cells number in the cells incubated with complex in a concentration dependent manner.  Viability of cells incubated without any compound was considered as 100% and the percentage of live cells incubated with compound is given as relative to the control.  The IC₅₀ value of the complex 1 is 40.08 μg/ml.

 

Fig 9. Cell viability of HepG2 cells after treatment with complex 1 at different concentrations.

 

Antibacterial and antifungal activity:

The copper (II) complexes were screened in vitro for its microbial activity against certain pathogenic bacterial and fungal species using disc diffusion method.  The complexes were found to exhibit considerable activity against bacteria and the fungus.  Zoroddu et al [24] reported that copper complexes show considerable activity against the bacteria.  Recently Patel et al [25] have indicated that the copper(II) complexes with L-phenylalanine has exhibited considerable activity against some human pathogens.  In our biological experiments, using copper (II) complexes, we have observed antibacterial activity antifungal activity.  The complex 2 has shown high antibacterial activity (Table 1) against Enterococcus faecalis and Staphylocuccus aureus and complex 1 has shown high antifungal activity against Mucor sps.  It may be concluded that our complexes 1 and 2 inhibit the growth of bacteria and fungi to a greater extent.

 

Table 1. Antimicrobial activity of copper(II) complexes

CONCLUSION:

Ternary copper(II) complexes having heterocyclic bases and L-serine are prepared and characterized.  The copper(II) complexes with heterocyclic base in CuN₄O coordination geometry shows DNA binding ability.  The complex 1 and 2 show intercalative and electrostatic binding respectively.  The DNA cleavage activity of complex 1 is more significant than complex 2.  The MTT assay of complex 1 (higher IC₅₀) and antimicrobial activity of complexes (1 and 2) suggested that complexes have potential role in medicinal line.

 

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Received on 15.04.2017         Modified on 03.05.2017

Accepted on 23.05.2017         © AJRC All right reserved