versión On-line ISSN 0717-9707
J. Chil. Chem. Soc. v.54 n.4 Concepción dic. 2009
J. Chil. Chem. Soc., 54, N° 4 (2009), págs. 339-344.
INVESTIGATIONS ON NUCLEASE ACTIVITY OF TRANS MIXED LIGAND- COPPER(II) COMPLEXES WITH ORTHO SUBSTITUTED AROMATIC OXIMES AND HETEROCYCLIC BASES
M.S. SURENDRA BABUa, PITCHIKA.G. KRISHNAb, K. HUSSAIN REDDYa* AND G.H. PHILIPb
aDepartment of chemistry, Sri Krishnadevaraya university Anantapur-515003, India. e-mail: firstname.lastname@example.org
bDepartment of zoology, Sri Krishnadevaraya university Anantapur-515003, India.
Mixed ligand complexes of copper(II) with salicyladoxime (SAO), 2-hydroxy acetophenone oxime (HAO) as primary ligands and pyridine( Py), imidazole (Im) as secondary ligands have been synthesized and characterized by molar conductivity, magnetic moments, electronic, IR and ESR spectral data. Cyclic voltammetric studies show quasi-reversible reduction attributable to Cu2+/ Cu+. The binding interactions between metal complexes and calf thymus DNA have been investigated by using UV -Visible titrations and cyclic voltammetry studies. The cleavage activity of complexes was carried out on double-stranded pBR322 circular plasmid DNA by using gel electrophoresis. All complexes show increased nuclease activity in the presence of oxidant (H202). The nuclease activity of mixed ligand complexes are compared with that of the parent copper(II) complexes. Controlled experiments suggest that the complexes cleave DNA predominantly via an oxidative mechanism.
Keywords: Copper(II) complexes; mixed ligand complexes, aromatic oximes, DNA binding, oxidative cleavage.
Investigations on transition metal complexes to probe nucleic acids are becoming more prominent in the research area of bioinorganic chemistry1,7. Copper complexes are well established as chemical nucleases and known to cleave DNA by different mechanisms viz. hydrolytic8 and oxidative910. Oxidative cleavage of DNA could take place by chemical or photochemical means. Chemists are in search of new transition metal complexes as chemical nuclease. Oximes are widely recognized ligands in different fields of chemistry11,14. Survey of literature revealed that metal complexes of such important ligands are not exploited much in DNA studies. Transition metal complexes of ortho substituted aromatic oximes have attracted much attention as they give cis and trans geometrical isomers. Copper complexes are known to assume trans structure while cobalt complexes have cis- structure. Investigations of DNA interactions and cleavage activity of metal complexes with oximes are very limited.
In the light of above and in continuation of our on-going1516 research work on DNA interactions with metal complexes, herein we describe synthesis, characterization, DNA cleavage / binding studies on mixed ligand complexes with salicylaldoxime(SAO), 2- hydroxyacetophenone oxime (HAO) as primary ligands and pyridine (Py) and imidazole (Im) as secondary ligands.
Materials and Methods
All the reagents used in the preparation of ligands and their metal complexes were of reagent grade (Merck). The solvents used in the synthesis of ligands and metal complexes were distilled before use. All other chemicals were of AR grade and used without further purification. pBR322 DNA and Calf thymus DNA were purchased from Bangalore Genie(India). Agarose, Tris. HC1 and ethidium bromide were procured from Sigma-Aldrich. All other chemicals were of Merck make.
The elemental analyses were performed using Perkin Elmer 2400 CHNS elemental analyzer. Magnetic moments were determined in the polycrystalline state on a PAR model 155 vibrating sample magnetometer operating at field strength of 2-8 kG. High purity Ni metal (Saturation moment 55e.m.u/g) was used as standard. The molar conductance of the complexes in DMF (103 M) solution was measured at 28 ± 2°C with a Systronic model 303 direct-reading conductivity bridge. The electronic spectra were recorded in DMF with a Schimadzu UV-160A spectrophotometer. The FAB-mass spectra were recorded at Indian Institute of chemical Technology, Hyderabad on Joel SX 102/DA-6000 mass spectrometer using m-nitrobenzylalcohol as the matrix. FT-IR spectra were recorded in the range 4000-50 cm with a Bruker IFS 66V in KBr and polyethylene medium. ESR spectra were recorded on Varian E-122 X-band spectrophotometers at liquid nitrogen temperature in DMF. The voltammetric measurements were performed on Bio-Analytical systems, (BAS) CV-27 assembly in conjunction with an X-Y recorder. Measurements were made on the degassed (N bubbling for 5min) solution in DMF (103M) containing 0. lM-tetraethylammonium perchlorate (Et4NC104) as the supporting electrolyte. Three-electrode system consisted of a glassy carbon (working) platinum (auxiliary) and Ag/AgCl (reference) electrode.
Preparation of ligands
Copper oximes complexes
Complexes were prepared by mixing copper(II) chloride (4.3g, 0.025mol) and oxime [SAO (6.9g, 0.05mol) or HAO (7.5 g, 0.05mol)] in 1:2 ratio in 50% aqueous ethanolic medium. The reaction mixture was stirred for 30 min. The green precipitate formed was filtered, washed with hot water and then with cold methanol. The complexes were dried at 110°C. Cu(SAO)2: Yeild = 85 %, M.P. =208-210°C, C= 50.32(50.27), H= 3.38(3.5), N=8.19(8.32). Cu(HAO)2: Yeild = 98 %, M.P.= 234-237 °C, C= 51.67(52.59) H= 4.89(4.92), N=7.62(7.66).
Mixed ligand complexes with pyridine.
Copper(II) complex (0.005 mol) of SAO or HAO was placed in a Schlenk tube and dissolved in pyridine (3ml, 0.981 g/cm3). The solution was stirred magnetically for 30 min and n-hexane (25ml) was added. After standing for 3-4 days, the resulting dark green product formed was washed with water and n-hexane and then dried under reduced pressure over CaCl2. Cu(SAO)2Py2 : Yeild = 60 %, M.P. =180-184°C, C= 57.75(58.32), H= 3.81(4.42), N=11.44(11.39). Cu(HAO)2Py2 : Yeild = 54 %, M.P.= 225-227°C, C= 56.56(56.60) H= 4.91(5.16), N=9.41(9.44).
Mixed ligand complexes with imidazole
The copper(II) complex (1.8 gm, O.Olmol) was placed in a 250-ml round bottom flask. Imidazole (1.5gm, 0.05mol) dissolved in CH2C12 was added to the contents of the flask. The reaction mixture was refluxed on water bath for 2hrs. A dark green precipitate was formed. It was filtered, washed with cold hexane and dried under vacuo over CaCl2. Cu(SAO)2Im2: Yeild = 58 %, M.P. =185-188°C, C= 47.35(47.44), H= 3.78(3.74), N=19.54(19.58). Cu(HAO)2Im2
DNA -binding studies
Assay of DNA Cleavage activity.
The DMF solution containing metal complexes was taken in a clean Eppendroff tube and lul of plasmid DNA was added. The contents were incubated for 2hr at 37 °C and loaded on 0.8% agarose gel after mixing 5 ul of loading buffer (0.25 % bromophenol blue + 25% xylene cyanol + 30% glycerol). Electrophoresis was performed at constant voltage (80 V) till the bromophenol blue reaches to 3á of the gel. Further the gel is stained for 10 min by immersing it in ethidium bromide solution (5 µg/ml of H20). The gel was then de-stained for 10 min by keeping it in sterile distilled water and plasmid band were visualized by viewing the gel under transilluminator and photographed. The efficiency of the DNA cleavage was measured by determining the ability of the complex to form open circular (OC) or nicked circular (NC) DNA from its supercoiled (SC) form by quantitatively estimating the intensities of the bands using the Vilber Lourmat(V 99.01) Gel Documentation System. The reactions were carried out in presence and absence of H202. Control experiments were done in presence of DMSO, glycerol and tert. butyl alcohol as free radical scavenger.
Results and discussions
The present ligands contained two functional groups viz., oxime and phenolic (-OH) groups (Fig. 1). All complexes are stable at room temperature, non hygroscopic, insoluble in water, but partially soluble in methanol, ethanol and completely soluble in dimethylformamide (DMF). The molar conductance and magnetic moment data are summarized in table 1. Analytical data support the formulae of complexes. The molar conductivity data suggest that the complexes are non-electrolytes. The magnetic moment data indicate that the complexes are monomers.
The electronic spectral data of metal complexes recorded in DMF are given in table 2. A single d-d band is observed in the electronic spectra of complexes in 14900 - 17200 cm-1 region. This band is assigned to 2Eg → 2T2g transition, in favor of octahedral structure facilitated by coordination of DMF solvent molecules in axial position. Due to increase in the ligand field strength a blue shift is observed in the d-d band of mixed ligand complexes.19
The FAB mass spectra of parent complexes and mixed complexes are used to compare the stoichiometric composition. The molecular ion peak M+ for parent complexes were observed at m/z =334 and 362, suggesting the stoichiometry of parent complexes as ML2. The molecular ion peak of mixed ligand complexes were observed at m/z =413 and 441 for pyridine adducts and 402 and 430 for imidazole adducts respectively. The stoichiometry of the mixed ligand complexes as ML2L'2.
Elemental analysis values are in close agreement with the values calculate from molecular formula assigned to these complexes, which is further supported by the FAB-mass studies of representative complexes.
The important IR spectral bands of ligands / complexes and their assignments are given in table 3. Strong bands observed at 3300, 3350 cm-1 in the IR spectra of SAO and HAO respectively are assigned to uOH vibration of phenolic group20. The low uOH values are possibly due to intramolecular hydrogen bonding as shown below.
This strong band is absent in copper chelates suggesting the deprotonation of phenolic group and formation of covalent bond between phenolic oxygen and metal. Strong band observed at 1618, 1634cm-1 in the IR spectra of SAO and HAO respectively are assigned to >C=N- stretching vibration. This band is shifted to lower frequency in complexes revealing the participation of azomethine nitrogen in chelation. The non-ligand bands observed in the far IR spectra of metal complexes are assigned (table 3) to uM-N, M-O stretching vibrations. Additional bands observed (1610-1600, 1540-1520, 260-220, 245-230cm-1) in the IR spectra of mixed ligand complexes are presumably due to the binding of bases (pyridine/ imidazole) to copper via nitrogen donor atoms preferably in axial positions21.
In the IR spectra of SAO and HAO bands observed respectively at 3260 and 3210 cm-1 are assigned to oxime OH stretching vibrations. These bands are respectively observed at 3200 to 3150 cm-1 in the chelates, indicating the involvement of oxime OH in strong hydrogen bonding leading to the formation of stable 5-membred ring structure. From above observation it is concluded that oxime -OH is neither deprotonated nor participated in chelation. Observance of three evenly distributed bands in 620-450 cm-1 region is the characteristic of irans-structure forthe complexes22. IR spectral data together with electronic and magnetic moment data suggest the trans square planar structure for cuproxime and trans octahedral structure for mixed ligand copper(II) complexes (Figure 2a-b).
DNA Binding studies
The interaction of copper complexes with DNA was monitored by UV-Vis spectroscopy (Fig. 5). The absorption spectra of copper complexes are compared with and without CT DNA. In presence of increasing amounts of CT DNA the spectra of all complexes showed hypochromicity and bathochromic shift (1-4 nm). The change in absorbance values with increasing amount of CT DNA were used to evaluate the intrinsic binding constants (Kb) for all the complexes which are shown in table 6.
The higher binding constant of IV complex when compared with I is presumably due to the presence of electron donating methyl group present in the former complex. The higher binding constants of mixed ligand complexes with imidazole may be attributed to the presence of additional nitrogen donor atoms of imidazole that can interact more with DNA bases through hydrogen bonding. The higher binding constant of mixed ligands may be attributed to the k stacking or hydrophobic interactions of excess hetero aromatic ligand.
Binding nature of these complexes is further confirmed by redox investigation studies. Figure 6 show profile diagram of Cu (HAO)2Py2 in presence and absence of CT-DNA. On addition of CT-DNA, the complexes experience a shift in E values as well as AEp values at the scan rate of 50mVs-1. The ratio of anodic to cathodic peak currents Ip /Ip in free copper complexes is decreased on addition of CT-DNA (table-5), suggesting that CT-DNA moiety is bound to the complexes28,29. The change in formal potential of free copper(II) complex in the presence of DNA reveal the binding of complex with DNA .The binding constant Kb and redox potential values suggest these complexes are week binders.
DNA Cleavage Activity
The nuclease activity of present copper complexes has been investigated on pBR322 supercoiled plasmid DNA by agarose gel electrophoresis in the presence and in the absence of oxidant (H202) at 120 min incubation period. The gel electrophoresis diagrams are shown in the Figure 7. The nuclease activity of all copper(II) complexes has fairly increased in presence of oxidant (H202) (Figure 7a; all even no. lanes). Cu(SAO)2 complex cleaved super coiled form(SC) to nicked circular (NC) both in presence and absence of H202 which is evident from lanes 4 & 3 respectively. The higher cleavage activity in presence of oxidant (lane 4) is evident from Table 7. Cu(HAO)2 complex show complete degrading of SC form to NC and OC(open circular or linear form) in presence of oxidant (lane 10). In lanes 6 & 12, Cu(SAO)2Py2 and Cu(HAO)Py2 respectively converted SC form to NC in presence of H202. No such cleavage is observed in the absence of oxidant (lane 5 & 11). SC form has been degraded into NC and OC forms in lanes 8 & 14 containing imidazole complexes. In the absence of oxidant no such conversion was observed (lanes 7 & 13). From table 7 the cleavage efficiency of mixed ligand complexes are arranged in order based on cleavage efficiency (Table 7). The order is as follows:
Control DNA cleavage experiment reveal the involvement of OH free radical as cleavage active species. In lanes 6, 7 & 8 containing DMSO, glycerol and tert. butyl alcohol are added to reaction mixture as hydroxyl radical scavenger agents. In the presence of hydroxyl radical scavengers, especially with DMSO and ferf-butyl alcohol (lanes 5 and 6), cleavage activity is diminished significantly indicating the involvement of the hydroxyl radical as active species in the cleavage process.
While reports on DNA interactions of copper complexes of polypyridyl and phenanthroline ligands are numerous in the literature, those of interactions of metal oximates and, more so of mixed ligand copper complexes with oximes and aromatic bases are not investigated so far. In this study, we have attempted to unravel the DNA interactions and nuclease activity of mixed ligand copper complexes. The reduction in peak intensity (absorption spectra) is in analogy
with similar observation made earlier for other mixed ligand complexes30,31. Since the binding constants values are appreciable and less than 106 M-1 and since the complexes contain aromatic hydrophobic groups these complexes may be better regarded as partial intercalators of DNA. Nuclease activity of imidazole complexes is more than the corresponding pyridine complexes. The mixed ligand complexes of imidazole are found to be better DNA binding agents and efficient nucleases than the corresponding pyridine complexes possibly due to the additional nitrogen atom that may facilitate to strengthen DNA interaction via hydrogen bonding with DNA bases.
The present studies revealed that the copper complexes show insignificant nuclease activity in the absence of oxidant. However the activity is greatly enhanced in the presence of oxidant facilitated by the production of hydroxyl free radicals which can damage DNA via oxidative path. Insignificant nuclease activity of complexes in the absence of oxidant is presumably due to the more stability of complexes attributed to their trans structure.
The authors thank University Grant Commission [F12-118/2001], New Delhi, India for financial support.
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(Received: September 26, 2008 - Accepted: August 25, 2009).