Synthesis, crystal structure, dna binding, cleavage

Synthesis and Reactivity in Inorganic, Metal-Organic, and Nano-Metal Chemistry

ISSN: 1553-3174 (Print) 1553-3182 (Online) Journal homepage: http://www.tandfonline.com/loi/lsrt20

Synthesis, crystal structure, dna binding, cleavage and cytotxicity, antimicrobial activity of new copper(ii) complex with l-ornithien and 1,10phenanthroline S. Baskaran, M. Murali Krishnan & M.N. Arumugham To cite this article: S. Baskaran, M. Murali Krishnan & M.N. Arumugham (2016): Synthesis, crystal structure, dna binding, cleavage and cytotxicity, antimicrobial activity of new copper(ii) complex with l-ornithien and 1,10-phenanthroline, Synthesis and Reactivity in Inorganic, MetalOrganic, and Nano-Metal Chemistry, DOI: 10.1080/15533174.2016.1186039 To link to this article: http://dx.doi.org/10.1080/15533174.2016.1186039

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ACCEPTED MANUSCRIPT SYNTHESIS, CRYSTAL STRUCTURE, DNA BINDING, CLEAVAGE AND CYTOTXICITY, ANTIMICROBIAL ACTIVITY OF NEW COPPER(II) COMPLEX WITH L-ORNITHIEN AND 1,10-PHENANTHROLINE S. Baskaran, M. Murali Krishnan, M.N. Arumugham* Department of Chemistry, Thiruvalluvar University, Serkadu, Vellore-632 115, Tamil Nadu, India *

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(M.N.Arumugham) Corresponding author. E-mail: [email protected].

Abstract A new copper(II) complex, [Cu(phen)(L-orn)(NO3)](NO3).2(H2O) (phen = 1,10-phenanthroline, L-orn = L-ornithine) has been synthesized and structurally characterized by elemental analyses, IR, UV-visible and single crystal XRD techniques. The complex crystallized in a monoclinic system with space group P21, a = 10.561(5) Å, b = 6.927(5) Å, c = 15.510(5) Å, α = 90.000(5)°, β = 106.827(5)°, and γ = 90.000(5) °. The complex shows a distorted square-pyramidal (4+1) CuN3O2 coordination sphere. The binding interaction of the present complex with calf thymus DNA (CT–DNA) was investigated and proposed partial intercalation binding mode by UV-Vis, fluorescence, cyclic voltammetry and viscometric techniques. The efficient cleavage activity of the complex on plasmid DNA by complex was observed from gel electrophoresis technique. The complex exhibited potent cytotoxic effects against human cell line (HepG2) and it was found to have good antibacterial and antifungal activities. Keywords Copper(II) complex; L-ornithine; Cytotoxicity; DNA binding; DNA cleavage.

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ACCEPTED MANUSCRIPT 1. Introduction Cisplatin, cis-Pt(NH3)2Cl2 and related Pt(II) complexes are used as anticancer agents in several human cancers particularly testicular and ovarian cancers.[1,2] The action of cisplatin is dependent on the formation of cis-[Pt(NH3)2(OH2)2]2+ by sequential thermal ligand exchange resulting in the covalent binding to GpG DNA sequences forming intrastrand crosslinks disrupting cellular transcription.[3,4] However, it has significant disadvantages including Downloaded by [La Trobe University] at 23:06 02 August 2016

neurotoxicity, ototoxicity, nausea, etc.[5-8] The metal complexes, in this context, with tunable coordination environments and versatile physicochemical properties offer scope for designing and developing highly sensitive diagnostic agents for medicinal applications as exemplified by the chemotherapeutic agent such as cis-platin and bleomycins.[9-11] Many transition metal complexes are known to bind to DNA via both covalent and non-covalent interactions. In covalent binding the labile ligand of the complexes is replaced by a nitrogen base of DNA. On the other hand, the non-covalent DNA interactions include intercalative, electrostatic and groove (surface) binding of cationic metal complexes along outside of DNA helix, major or minor groove. Copper is a biologically relevant element and many enzymes that depend on copper for their activity have been identified. Copper complexes of 1,10-phenanthroline and its derivatives are of great interests since they exhibit numerous biological activities such as antitumor,[12] antiCandida,[13] antimycobacterial,[14] and antimicrobi[15] activity etc. Moreover, considerable attention has been focused on the use of phenanthroline complexes as intercalating agents of DNA[16] and as artificial nucleases.[17–19] It is well known that bis(1,10-phenanthroline)–

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ACCEPTED MANUSCRIPT copper(II) shows an efficient DNA cleavage activity in the presence of thiol and hydrogen peroxide.[20] Amino acids are the basic structural units of proteins that recognize a specific base sequence of DNA. This amino acid with its terminal –C(=O)–NH2 group has the potential to form significant


hydrogen bonding interactions with the double-stranded (ds) DNA and could show good DNAbinding propensity.[21] The continuous of our work,[22, 23,

24]

copper(II)

and

complex

of

1,10-phenanthroline

herein we report the new ternary L-ornithine

[Cu(phen)(L-

orn)(NO3)](NO3).2(H2O), which were synthesized and characterized by various spectroscopic techniques and single X-ray crystallographic method. Studies have been made to explore the role of a DNA binder and amino acid with a terminal amine group along with the mechanistic pathways involved in the chemical nuclease activity and cytotoxicity against (HepG2) cancer cells and antimicrobial activities of L-ornithine copper(II) complex. 2. Experimental 2.1. Materials and instrumentation Common reagent such as ethanol, anhydrous ether, Cu(NO3)2.3H2O, NaOH, and 1,10phenanthroline monohydrate are all analytical grade and used as received. L-ornithine was purchased from Sigma Aldrich. Plasmid pBR322 was purchased from MBI Fermentas. Disodium salt of calf thymus DNA (CT-DNA), Tris(hydroxymethyl) aminomethane (Tris) and ethidium bromide (EB) were purchased from Sigma Aldrich. The infrared spectra were recorded on a Perkin Elmer spectrometer as KBr pellets (4000-400 cm1

), and elemental analysis was performed on a Perkin Elmer 240 C analytical instrument.

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ACCEPTED MANUSCRIPT UV-Vis and Fluorescence spectra of the complex were recorded on Shimadzu UV-2450 spectrophotometer and Jobin Young Fluorolog 3 spectrophotometer respectively. Cyclic voltammograms were obtained on CHI 602D (CH Instruments Co., USA) electrochemical analyzer under oxygen free conditions using a three-electrode cell with a DMF solution of TBAP (0.1M) as the supporting electrolyte. A Pt wire, glassy carbon, and the Ag/AgCl electrode (saturated KCl solution) were used as counter, working and reference electrode respectively. Downloaded by [La Trobe University] at 23:06 02 August 2016

Thermogravimetric analysis (TGA) was performed on a NETZSCH STA 449F3 STA449F3A0927-M instrument. 2.2. Synthesis of [Cu(phen)(L-orn)(NO3)](NO3).2(H2O) To the mixture of L-ornithine (337.0 mg, 2 mmol) and NaOH (80 mg, 2 mmol) in water, an aqueous solution of Cu(NO3)2 . 3H2O (483.0 mg, 2 mmol) was added with stirring. Several minutes later, the ethanolic solution (5 mL) of 1,10-phenanthroline monohydrate (396.4 mg, 2 mmol) was added and then the solution was stirred for about 6 h at 60 °C. On cooling the solution to ambient temperature, the resulting blue color solution was filtered and kept for slow evaporation. Blue blocky single crystals suitable for X-ray quality crystals separated out after a week. It was filtered and washed with ethanol and ether. Yield: 70%. Calculated for C17H24CuN6O10 (%): C 38.06; H 4.47; N 15.67. Found (%): C 38.02; H 4.42; N 15.64. IR (KBr, cm-1): 3493br, 3287w, 3238w, 3125w, 3052w, 1610vs, 1519m, 1473m, 1383s, 1168m, 1085vs, 848m, 720m, 650m cm-1 (br, broad; vs, very strong; s, strong; m, medium; w, weak). 2.3 X-ray crystallography Single crystals of the complex [Cu(phen)(L-orn)(NO3)](NO3).2(H2O) were grown by slow evaporation of the methanol-water mixture. Blue blocky crystal of approximate size 0.30 X 0.30

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ACCEPTED MANUSCRIPT X 0.25 mm, was used for data collection on Bruker KAPPA APEX-II CCD diffractometer using MoKα radiation. SADABS correction was applied. 11673 reflections were measured. All the data were corrected for Semi-empirical absorption effects. SHELXL[25] was used for structure solution and full matrix least squares refinement on F^2. Hydrogen atoms were included in the refinement as per the riding model. All non-hydrogen atoms were refined anisotropically. The CCDC deposition number of the crystal is 930445. Downloaded by [La Trobe University] at 23:06 02 August 2016

2.4. Spectroscopic Studies on DNA interaction 2.4.1 Electronic spectra The UV absorbance at 260 and 280 nm of the CT-DNA solution in 50 mM NaCl/5 mM Tris– HCl buffer (pH 7.2) give a ratio of~1.9, indicating that the DNA was sufficiently free of protein.[26] The DNA concentration was determined by measuring the UV absorption at 260 nm, taking the molar absorption coefficient (ɛ260) of CT-DNA as 6600 M-1 cm-1.[27] Electronic spectra of [Cu(phen)(L-orn)(NO3)](NO3).2(H2O) (1 X 10-5 M) was recorded before and after addition of CT-DNA (r = 0.0, 0.5, 1.0, 1.5, 2.0, 2.5) where r is the molar ratio of DNA and complex) in the 5 mM Tris–HCl/50 mM NaCl buffer, pH 7.2. The intrinsic binding constant Kb for the interaction of the studied complex with CT-DNA was calculated by absorption spectral titration data using the following equation;[28]

 DNA /  a   f 

  DNA /  b   f   1/ Kb  b   f  …………………..1

where ɛa, ɛf and ɛb correspond to Aobsd/[Cu], the extinction coefficient for the free copper complex, and the extinction coefficient for the copper complex in the fully bound form, respectively. In the plot of [DNA] / (ɛa - ɛf) vs. [DNA], Kb is then given by the ratio of the slope to intercept.

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ACCEPTED MANUSCRIPT 2.4.2 Fluorescence spectra The fluorescence spectra were recorded at room temperature with excitation at 480 nm and emission at 614 nm. The experiment was carried out by titrating [Cu(phen)(Lorn)(NO3)](NO3).2(H2O) (5 mM Tris–HCl/50 mM NaCl buffer) into samples containing 1 X 10-4 M of DNA and 8 X 10-5M ethidium bromide. Stern-Volmer quenching constants were calculated


using the given equation

I0 / I = 1+ Ksv r ……………………….2 Where I0 and I are the fluorescence intensities in the absence and presence of complex, respectively, Ksv is a linear Stern – Volmer and r is the total concentration of complex to that of DNA. In the Plot of I0/ I vs [complex] / [DNA], Ksv is given by the ratio of slop to intercept. 2.4.3 Viscosity Viscosity measurements were carried out using an Ostwald-viscometer maintained at a constant temperature of 28.0 ± 0.1°C in a thermostatic bath. Flow time was measured with a digital stopwatch. Each sample was measured three times and an average flow time was calculated. Data are presented as (ɳ/ɳ0)1/3 versus binding ratio [29] where ɳ is the viscosity of CT-DNA in the presence of complex and ɳ0 is the viscosity of CT-DNA alone. 2.4.4 Cyclic voltammetric Cyclic voltammetry (CV) was performed on a CHI 602D series electrochemical analyzer by a three electrode system. The three electrode system consisted of a glassy carbon (GC) electrode as the working electrode, saturated calomel electrode (SCE) as the reference electrode and Pt wire as the counter electrode. Supporting electrolyte was tetra (N-butyl) ammonium perchlorate.

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ACCEPTED MANUSCRIPT Before each experiment, solution was deaerated by purging pure N 2 for 15 min, and nitrogen atmosphere was kept over the solution during the experiments. 2.5. DNA cleavage DNA cleavage assay was carried out by mixing 1μL of pBR322 DNA, 10μL of sample buffer (Tris- HCl pH 7.4, 0.25% bromphenol bule, 50% glycerol) and 10-4 M solution of complex were added serially (5,7.5,10,12.5,15,17.5,) μL. After 30 min inhibition the complex mixed DNA was Downloaded by [La Trobe University] at 23:06 02 August 2016

subjected to electrophoresis on 1% agarose gel in TAE buffer (40 mM Tris Acetate pH 7.4/1mM EDTA) at 100v and visualized by ethidium bromide staining. Gel Imaging and documentation using progen. 2.6. Cytotoxicity assay Cell cultures were obtained from National Centre for Cell Sciences (NCCS), Pune, India. Tumor cell lines were grown in RPMI-1640 medium supplemented with 10% (v/v) heat-inactivated fetal bovine serum, 2 mM glutamine, 100 U/mL penicillin, and 100 μg/mL streptomycin. Cells were cultured at 37 °C in a highly humidified atmosphere of 5% CO2 in air. The in vitro cytotoxicity of [Cu(phen)(L-orn)(NO3)](NO3).2(H2O) were investigated as follows. The growth inhibitory effect on the hepatocarcinoma cell line HepG2 was measured by [3-(4,5-dimethylthiazol-2-yl)2,5-diphenyltetrazolium bromide, MTT] assay.[30] The growth inhibitory rate of treated cells was calculated by (ODcontrol - ODtest)/ODcontrol X 100%.[31] The morphological examination was performed with a Nikon inverted microscope. 2.7. Antimicrobial activity The in vitro antimicrobial screening of the copper(II) complex was tested for its effect on certain human pathogenic bacteria and fungus by disc diffusion method. The complex was stored dry at

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ACCEPTED MANUSCRIPT room temperature and dissolved in water. Both the Gram positive (Staphylococcus aureus,) and Gram negative (Escherichia coli, Klebsiella pnemoniae) bacteria were grown in nutrient agar medium and incubated at 37 oC for 24 h followed by frequent subculture to fresh medium and were used as test bacteria. The fungi Aspergillus niger grown as sabourad dextrose agar medium were incubated at 27 oC for 48 h followed by periodic subculturing to fresh medium and were used as test fungus. Then the petriplates were inoculated with a loop full of bacterial and fungal Downloaded by [La Trobe University] at 23:06 02 August 2016

culture and spread throughout the petriplates uniformly with a sterile glass spreader. To each disc the test samples (10 ppm) and reference ciprofloxacin (1 μg/disc for bacteria) or Amphotericin-B (10 μg/disc for fungus) was added with a sterile micropipette. The plates were then incubated at 35 ± 2 oC for 24–48 h and 27 ± 1 oC for bacteria and fungus, respectively. Plates with disc containing respective solvents served as control. Inhibition was recorded by measuring the diameter of the inhibitory zone after the period of incubation. All the experiments were repeated thrice and the average values are presented. 3. Results and discussion 3.1. Synthesis and general aspects A ternary copper(II) complex, [Cu(phen)(L-orn)(NO3)](NO3).2(H2O), has been prepared from the reactions of Cu(NO3)2 . 3H2O with 1,10-phenanthroline and L-ornithine. The complex is soluble in water and common organic solvents. The complex is one electron paramagnetic at room temperature, corresponding to d9 electronic configuration for the copper(II) center. The complex display a copper(II) centered d–d band at ~618 nm in addition to the ligand centered bands in the UV region of the electromagnetic spectra (Fig. 1). The electronic spectra of the complex are in good agreement with the previously reported square pyramidal geometry of the

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ACCEPTED MANUSCRIPT complexes.[32,

33]

The IR spectra of the complex shows the asymmetric vas(COO-) and the

symmetric stretching vibration vs(COO-) fall in 1610 and 1383 cm-1, respectively. The difference between vas(COO-) and vs(COO-) stretching frequencies is greater than 200 cm-1, which indicates that the carboxylate groups are coordinated to the metal ion in a monodenate fashion.[34] The bands at 1519 and 1473 cm-1 can be attributed to the ring stretching frequencies [v(C=C) and v(C=N)] of 1,10-phenanthroline. Downloaded by [La Trobe University] at 23:06 02 August 2016

3.2 X-Ray structural characterization The ORTEP diagram of the cation [Cu(phen)(L-orn)(NO3)](NO3).2H2O with the atom numbering is shown in Fig.2. Crystal refinement and data collection is tabulated in Table 1 and selected bond lengths and angles are given in Table 2. It crystallized in the monoclinic P21 space group with two independent molecules in the crystallographic asymmetric unit and a goodness-of-fit on F^2 1.056. The copper(II) complex displays a distorted (4 + 1) squarepyramidal geometry, in which Cu2+ coordinates with two nitrogen atoms of 1,10-phenanthroline, one nitrogen atom of an amino group, one carboxylate oxygen atom of L-ornithine in the equatorial positions and NO3 at the elongated apical position. The configuration at the chiral αcarbon is S shape in the complex. The four equatorial donor atoms are nearly coplanar, The bond lengths of Cu(1)–O(1) (1.9207(14) Å) and Cu(1)–N(3) (1.9821(17) Å) are similar to those found in Cu(L-orn)2.H2O, where the mean Cu–O and Cu–N (O and N atoms of L-ornithine) distances are 1.92(1) and 1.98(1) Å, respectively.[35] The Cu–N(phen) bond lengths of 1.9916(16) and 2.0293(16) Å and the bite angle N(1)–Cu–N(2) of ~82° are close to the corresponding values for some copper – phenanthroline complexes reported.[36–38] The alkyl chain of the cationic amino group –(CH2)3NH3+ remains as a pendant moiety. The X–Cu–Z angles are in the range 175.39(6)

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ACCEPTED MANUSCRIPT – 172.61(6)°, where X and Z are any two atoms which lie trans to each other, and the X–Cu–Y angles range from 82.00(6)° to 102.53(7)°, where X and Y are any two atoms which lie cis to each other. The square pyramidal geometry around the central metal atom is severely distorted. The structure show extensive intermolecular non-covalent interactions. The water molecule is involved in hydrogen bonding interactions with the terminal cationic amine (NH 3+) of L-


ornithine. The terminal amine forms two H bonds with one lattice water and one lattice NO 3 molecules N(4)–O(9)(w1) and N(4)–O(5)(NO3) (distances: 2.868 and 2.845 Å) Table 3. The free oxygen atom of the carboxylate group of L-ornithine forms two H bonds, one with lattice water (O2–O9 (w1)) and other with the lattice water of another asymmetric unit (O2–O10 (w2)) with distances of 2.756 and 2.914 Å respectively. The NO3 axial ligand is H bonded with the lattice Lornithine of another asymmetric unit with a distance of 3.182 Å. The lattice NO 3 is H bonded with lattice water, O9(w1)–O5(NO3)(2) and O10(w2)-O4(NO3)(2), with a distance of 2.862 and 2.909 Å. 3.3. Thermo gravimetric analysis of Copper(II) complex Figure. 3 – shows TGA curves for [Cu(phen)(L-orn)(NO3)](NO3).2(H2O) complex. The complex is stable up to 120 ºC and then gradually loses its weight to decompose. In the weight loss occurs at 120 to 250 ºC corresponding to the loss of lattice water (Calculated: 6.7%; observed 6.4%). The second weight loss the range of 250 to 299.5 ºC is due to the elimination of nitrate ions (Calculated: 12.2%; observed 11.9%). The third weight loss the range of 299.5 to 404.7 ºC is due to the elimination of (L-ornithine) amino acid (Calculated: 24.41%; observed 24.3%) above the 404.7 ºC the complex was decomposed.[39] 3.4. Spectral studies of the interactions with DNA

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ACCEPTED MANUSCRIPT The interactions of metal complexes with DNA have been the subject of interests for the development of effective chemotherapeutic agents. The binding modes to DNA would give insights into the understanding of the biochemical mechanism of action of the complexes. Recently, studies on some ternary complexes [Cu(phen)(L-orn)] (L-orn = amino acids) indicated that the size, shape, and polarity of the side chains of different amino acids in these ternary


complexes may influence their binding mode to DNA.[40] The absorption spectra of [Cu(phen)(L-orn)(NO3)](NO3).2(H2O), in the absence and presence of CT-DNA were shown in Fig. 4. In the UV region, the complex presented two bands 273 (ɛ = 6.24 X 104 M-1 cm-1) and 294 nm (ɛ = 1.91 X 104 M-1 cm-1), which can be attributed to the π → π* transition of the coordinated phenanthroline ligand. The absorption intensity of the complex increased (hyperchromism) evidently after the addition of DNA, which indicated the interactions between DNA and the complex. A similar hyperchromism was also observed for a copper(II) complex with a ligand bearing –OH group.[41] As DNA double helix possesses many hydrogen bonding sites which are accessible both in the minor and in the major grooves, it is likely that the –OH group of the ternary complex forms hydrogen bonds with DNA, which may contribute to the hyperchromism observed in absorption spectra. The complex bound to DNA through intercalation, which involves a strong stacking interaction of the planar aromatic rings of the coordinated ligand with the base pairs of DNA, usually result in hyperchromism and blue shift.[42] The observed hyperchromism and blue shift indicates that complex involves in partial intercalation to the base pairs of DNA. The binding constant Kb of 1.2144 X 104 M-1 was determined from the plot of [DNA]/(ɛa _

ɛf) versus [DNA] (inset of Fig. 4), and the Kb value is higher than those for related other Cu(II)

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ACCEPTED MANUSCRIPT complexes such as [Cu(phen)(L-Thr)H2O]ClO4 (6.35 × 103 M–1),[43] [Cu(Sal-L-val)phen] (6.48 × 103 M–1)[44] and [Cu(naph-Leu)(phen)]CH3OH.5H2O (4.87 × 103 M–1),[45] [Cu(phen)(pro)(H2O)] ClO4– (3.86 × 103 M–1),[46] [Cu(bipy)(pro)(H2O)]ClO4– (4.6 × 103 M–1).[47] 3.5 Fluorescence spectral studies The emission spectral method is used to study the relative binding of the complex to CT-DNA. The emission intensity of ethidium bromide (EB) is used as a spectral probe. The fluorescence Downloaded by [La Trobe University] at 23:06 02 August 2016

titration experiment, especially the EB fluorescence displacement experiment, has been widely used to characterize the interaction of complex with DNA by following the change in fluorescence intensity of the complex. The intrinsic fluorescence intensities of DNA and that of EB in Tris-HCl buffer is low, while the fluorescence intensity of EB will be enhanced with the addition of DNA as its intercalation into the DNA. Therefore, EB can be used to probe the interaction of complex with DNA. If the complex can intercalate into DNA, the binding sites of DNA available for EB will be decreased, and hence the fluorescence intensity of EB will be quenched.[48] In our experiments, as depicted in Fig. 5, the fluorescence intensity of EB at 614 nm show a remarkable decreasing trend with the increasing concentration of the complex, indicating that some EB molecules are released from EB-DNA after an exchange with the complex which result in the fluorescence quenching of EB. This may be due either to the metal complex competing with EB for the DNA-binding sites thus displacing the EB (whose fluorescence is enhanced upon DNA-binding) or it should be a more direct quenching interaction with the DNA itself. We assume the reduction of the emission intensity of EB on increasing the complex concentration could be caused due to the displacement of the DNA bound EB by the ternary copper(II) complex. Such a quenched fluorescence behavior of EB bound to DNA caused

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ACCEPTED MANUSCRIPT by the interaction between the copper(II) complex and DNA is also found in other ternary copper complex.[49, 50] The Kq values of ternary copper(II) complex with copper chelates are 2.51 X 104 M-1. 3.6 Viscosity measurements To further explore the binding mode of the present copper(II) complex, viscosity measurements


of CT–DNA solution with complex were carried out. Since the relative specific viscosity (ɳ/ɳ0), (ɳ and ɳ0 are the specific viscosities of DNA in the presence and absence of the complex, respectively) of DNA reflects the increase in contour length associated with separation of DNA base pairs caused by intercalation, a classical intercalator such as ethidium bromide could cause a significant increase in viscosity of DNA solutions. In contrast, a partial and/or non-classical intercalation of the ligand could bend or kink DNA, resulting in a decrease in its effective length with a concomitant increase in its viscosity,[51,

52]

while the electrostatic and groove binding

cause little or no effect on the relative viscosity of DNA solution.[53] Therefore viscosity measurement, which is sensitive to the changes in the contour length of DNA, is regarded as the least ambiguous and most critical means of studying the interaction of metal complex with DNA in solution and provides stronger arguments for an intercalation binding mode. The plots of relative specific viscosities versus 1/R = ([Complex] / [DNA]) are shown in (Fig. 6). The complex–DNA solution increases steadily in relative specific viscosity with increasing the concentration of the complex. However, the increase in the viscosity was much less compared to that of classical intercalators like ethidium bromide in the same DNA concentration range.[54] This observation leads us to support the above spectral studies which suggest that the complex interacts with DNA via partial intercalation between DNA base pairs. A partial intercalation of

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ACCEPTED MANUSCRIPT complex leads to lengthening of DNA helix as base pairs are separated to accommodate the binding ligand leading to an increase in viscosity of DNA solution. 3.7 Cyclic voltammetry The application of cyclic voltammetry to study of the interaction between complex and DNA provides a useful compliment to the previously utilized methods of investigation such as UV-Vis


and viscosity experiments. The typical cyclic voltammogram of a 0.01 mM solution of [Cu(phen)(L-orn)(NO3)](NO3).2(H2O) complex without and with DNA at glassy carbon (GC) electrode in DMF were carried out (Fig. 7). 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 height and shifts of peak potential to more -ve values were observed. The cyclic voltammetric behavior was not affected by the addition of very large excess of DNA, indicating that the decrease of peak current of complex after the addition of DNA due to the binding of [Cu(phen)(L-orn)(NO3)](NO3).2(H2O) complex to the DNA. When the concentration of the DNA increased with changes the in peak current and potential become slowly. This reveals that the complex were interacting with CT-DNA.[55] 3.8. Nuclease activity It is reported that many antitumor agents interacted with DNA and caused DNA strand scission,[56] so we continued to study the capacity of [Cu(phen)(L-orn)(NO3)](NO3).2(H2O), to cleave DNA by gel electrophoresis using plasmid pBR322 DNA. The gel electrophoresis result reveals that the first lane consists of pBR322 DNA alone, and in this lane there is no DNA cleavage occurred. The addition of [Cu(phen)(L-orn)(NO3)](NO3).2(H2O), to supercoiled

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ACCEPTED MANUSCRIPT plasmid (Form I) pBR322 DNA converted into both nicked (Form II) and linear (Form III) products. Fig. 8 shows the cleavage of DNA at different concentration of the complex for 30 min reaction time (pH 7.4, 37 °C). With the increase of complex concentration, the supercoiled DNA converted completely into nicked and linear form. The cleavage efficiency of [Cu(phen)(Lorn)(NO3)](NO3).2(H2O), reaches about 75% at a concentration of 5μL (Fig.7, lane 2) in converting Form I to Form II. When the concentration of the complex was 10μL or above, Form Downloaded by [La Trobe University] at 23:06 02 August 2016

I was degraded completely to form (Form II) and (Form III) (Fig. 7, lines 4, 5, 6 and 7). The amount of Form III reached 35% at a complex concentration of 17.5μL, which indicates that [Cu(phen)(L-orn)(NO3)](NO3).2(H2O), is a potent DNA cleavage under the present experimental conditions. 3.9. Cytotoxic activity The in vitro cytotoxic effects was carried out [Cu(phen)(L-orn)(NO3)](NO3).2(H2O), against tumor cell lines (HepG2). The results shown in (Fig. 9), after treatment for 48 h, lower dose of copper(II) complex (5μL) has no cytotoxic effect on HepG2 cells. The complex solution was prepared 10-4 M concentration. However, at concentrations of (10, 15, 20, 25μL), copper(II) complex treatment resulted in a significant decrease in cell viability by 70%, 50%, 40%, and 30% respectively. The IC50 (inhibitory concentration 50%) of copper(II) complex-induced cell death was about at 15μL after 48 h. The cell morphology changed to spheroid shape, which was accompanied by chromatin condensation (Fig. 10). These results suggest that copper(II) complex-induced cell death is mainly due to apoptosis. 4. Antimicrobial activity

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ACCEPTED MANUSCRIPT The copper(II) complex was screened in vitro for its microbial activity against certain pathogenic bacterial and fungal species using disc diffusion method. The complex was found to exhibit considerable activity against Gram positive and Gram negative bacteria and the fungus Aspergillu niger. The test solutions were prepared in water and the results of the antimicrobial activities are summarized in Table 4. Zoroddu et al.[57] Have reported that copper(II) complex shows any significant activity against the Gram positive and Gram negative bacteria. Recently Downloaded by [La Trobe University] at 23:06 02 August 2016

Patel et al. have indicated that the copper(II) complex with L-ornithine has exhibited considerable activity against some human pathogens.[58] In our biological experiments, using copper(II) complex, we have observed antibacterial activity against Gram positive bacteria Staphylococcus aureus and Gram negative bacteria E. coli and Klebsiella pnemoniae. The copper (II) complex has shown high activity against Gram positive than Gram negative bacteria. The copper(II) complex is also very active against the fungus Aspergillus niger, Rhizopus sps than the standard antifungal drug, Amphotericin-B. It may be concluded that our copper(II) complex inhibits the growth of bacteria and fungi to a greater extent. 5. Conclusion A new copper(II) complex having L-ornithine and heterocyclic bases in a CuN3O2 coordination geometry are prepared and structurally characterized by X-ray crystallography. The crystal structure of the complex shows a square pyramid (4+1) coordination geometry in which the amino acid and heterocyclic bases bind at the basal positions. The DNA interaction of the complex was investigated by electronic absorption, fluorescence, viscosity and cyclic voltammetry techniques. This result indicates that a partial intercalative binding mode of complex with DNA. The DNA cleaves of the complex was investigated by gel electrophoresis

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ACCEPTED MANUSCRIPT techniques. This result indicates that the complex cleaves plasmid DNA efficiently. The introduction of L-ornithine to the copper complex give rise to potential selective interactions with DNA, which may result in the variation of nuclease activity of Cu–phen system as well as its cytotoxicity towards tumor cell lines. The complex exhibited highly in vitro cytotoxic effects toward HepG2 tumor cell lines. As hypothesized, we find that copper(II) complex modulates


apoptosis induction in HepG2 cells at IC50 (15μL) and the complex [Cu(phen)(Lorn)(NO3)](NO3).2(H2O), exhibit good antimicrobial activity. Acknowledgements We are grateful to IIT Madras, Chennai, for providing instrumental facilities such as IR spectroscopy, X-ray crystallography, Emission spectroscopy and TGA.

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Table. 1. Crystal data and structure refinement for [Cu(Phen)(L-orn)(NO3)](NO3).2H2O Identification code

shelxl

Empirical formula

C17 H24 Cu N6 O10

Formula weight

535.96

Temperature

295(2) K

Wavelength

0.71073 A

Crystal system, space group

Monoclinic, P21

Unit cell dimensions

a = 10.561(5) A alpha = 90.000(5) deg. b = 6.927(5) 106.827(5) deg.

A beta =

c = 15.510(5) A gamma = 90.000(5) deg. Volume

1086.1(10) A^3

Z, Calculated density

2, 1.639 Mg/m^3

Absorption coefficient

1.074 mm^-1

F (000)

554

Crystal size

0.30 x 0.30 x 0.25 mm

Theta range for data collection

2.08 to 27.50 deg.

Limiting indices

-13