A Comparative Study of Antimicrobial and DNA Clevage Activity of ...

August 2013 - October 2013, Vol. 3, No. 4; 2482-2491. E- ISSN: 2249 –1929Journal of Chemical, Biological and Physical SciencesAn International Peer Review E-3 Journal of SciencesAvailable online atwww.jcbsc.orgSection A: Chemical ScienceCODEN (USA): JCBPATResearch articleA Comparative Study of Antimicrobial andDNA Clevage Activity of Metal(II)Complexes of Coumarin DerivativesDisha Tilala 1* , Manish Solanki 2 , Denish Karia 31 Kadi Sarva Vishwavidyalaya, Gandhinagar, (Gujarat) India.2 Department of Chemistry, Gujarat Collage, Ahmedabad-382330 (Gujarat) India.3 Department of Chemistry, A. C. Science College, Borsad-388540 (Gujarat) India.Received: 20 August 2013; Revised: 6 September 2013; Accepted: 15 September 2013Abstract: New transition metal complexes of the composition, M[L(H 2 O)] 2 , whereM= Fe(II), Ni(II), Co(II) and Cu(II); L= ligands prepared from 3-acetyl-4-hydroxy-8-methylpyrano[3,2-c]chromene-2,5-dione(L I ) and 4-hydroxy-8-methyl-3-(3-oxobutyanoyl)pyrano [3,2-c]chromen-2,5-dione (L II ), were synthesized and characterized byelemental analyser, conductivity measurement, IR, UV-Vis. and 1H-NMR spectraltechniques. The low conductance data provide evidence for the non-ionic nature of thecomplexes. The antimicrobial activities of the ligands and their complexes have beenstudied by screening the compounds against the bacteria E. coli and B. megaterium andalso the fungi C. albicans and A. niger. The data indicate that most of the metalcomplexes have higher antimicrobial activity than the free ligands. The DNA cleavageexperiments, performed using gel electrophoresis with the corresponding metalcomplexes in the presence of H 2 O 2 showed good results.Keywords: transition metal complexes, antimicrobial, DNA cleavage, gelelectrophoresis2482 J. Chem. Bio. Phy. Sci. Sec. A; Aug. 2013-Oct.2013; Vol.3, No.4; 2482-2491.

A Comparative....DishaTilala et.al.INTRODUCTIONTransition metal complexes with an ability to cleave and bind DNA under physiological conditionsare of current interests for their varied applications in nucleic acid chemistry, viz., as sequencespecified DNA cleaving agents, foot printing agents and diagnostic tools in medical field 1-9 . Metalcomplexes with tunable coordination environments and versatile physicochemical properties offerscope for designing highly sensitive diagnostic agents for medical applications as exemplified by thechemotherapeutic agents like cis-platin and bleomycins 10-12 . DNA cleavage could be achived viaoxidative or hydrolytic pathways 13-15 . Hydrolytic DNA cleavage involves cleavage of phosphodiesterbond to generate fragments which can be subsequenty relegated.Hydrolytic cleavage active species mimic restriction enzymes. Oxidative DNA cleavage involveseither oxidation of the deoxyribose moiety by abstraction of sugar hydrogen or oxidation ofnucleobases 16 . Oxidative cleavage of DNA occurs in the presence of additives or photoinduced DNAcleavage agents 17,18 . Photocleavers require the presence of a photosensitizer that can be activated onirradiation with UV or visible light the redox active ‘chemical nucleases’ are effective cleavers ofDNA in the presence of a reducing agent or H 2 O 2 as an additive 6, 8 . Here in, we present the synthesis,characterization, antimicrobial and oxidative DNA cleavage activity of two novel ligands 3-acetyl-4-hydroxy-8-methylpyrano[3,2-c]chromene-2,5-dione and 4-hydroxy-8-methyl-3-(3-oxobutyanoyl)pyrano[3,2-c]chromen-2, 5-dione and their metal complexes.EXPERIMENTALApparatus and reagents: All chemicals used were of analytical reagent (AR) grade and of thehighest purity available. They include phosphorous oxychloride (Spectrochem), Phenol, malonic acid,zinc chloride, sodium carbonate, hydrochloric acid, ethanol, ammonia, agarose (Merck), and nutrientbroth, glucose, yeast extract (Helini biomolecules). CT DNA was purchased from Genei chemicalcompany, India. Solutions of CT DNA in Tris–HCl/NaCl (pH 7.2) buffer medium gave a ratio ofA 260 /A 280 , of ca. 1.8–1.9, indicating that the DNA was sufficiently free from protein contamination 19 .Stock solutions were kept at 4°C and used within 4 days. Doubly distilled water was used to preparethe buffer. All the melting points were determined in open glass capillaries in a liquid paraffin-bathand are uncorrected. The NMR Spectra were recorded on BRUKER NMR Spectrometer (300 MHz)and IR spectra on Shimadzu, 435-IR in frequency range of 4000-400 cm -1 using KBR pellet technique.The conductivity of metal complexes was determined using Systronic Conductivity Bridge. ElementalAnalyses were performed on a Perkin- Elmer 2400 CHN elemental Analyzer Model 1106.Synthesis of 4-hydroxy-7-methyl-2H-chromen-2-one (1): It was prepared by the reported methodgiven by shah & co-workers 20 . m-cresol (0.1mole) and malonic acid (0.1mole) were added to amixture of phosphorous oxychloride (40ml) and freshly fused zinc chloride (40g). The reactionmixture was heated on water bath at 60-70 o C for 8-10 hrs and poured into ice. The solid residue isfiltered out and treated with hot saturated sodium carbonate. The filtrate was slowly acidified withconcentrated hydrochloric acid. At the point of neutralization, the precipitates were obtained werefiltered and washed with water and further dried and recrystalised with methanol.Synthesis of 4-hydroxy-8-methylenepyrano[3,2-c]chromene-2,5-dione (2): 4-hydroxy-7-methyl-2H-chromene-2-one (0.1mole) and malonic acid (0.1mole) were added to a mixture of phosphorousoxychloride (40mL) and freshly fused zinc chloride (40g). The reaction mixture was heated on waterbath at 60-70 o C for 16-18hrs and poured into ice. The solid residue is filtered out and treated with hotsaturated sodium carbonate. The filtrate was slowly acidified with concentrated hydrochloric acid. At2483 J. Chem. Bio. Phy. Sci. Sec. A; Aug. 2013-Oct.2013; Vol.3, No.4; 2482-2491.

A Comparative....DishaTilala et.al.the point of neutralization, the precipitates are obtained are filtered and washed with water and furtherdried and recrystalised with methanol.Synthesis of 3-acetyl-4-hydroxy-8-methylpyrano [3, 2-c] chromene-2, 5-dione (L I ): Acetylderivatives are prepared 21-26 by acetylation of product-II in presence of acetic acid and phosphorousoxychloride. 4-hydroxy-8-methylenepyrano [3, 2-c] chromene-2, 5-dione (1g) was dissolved inmixture of glacial acetic acid (5.0 mL) and phosphorous oxychloride (4.0 mL). The reaction mixturewas gently refluxed for 4-5hrs., then poured into ice water. 3-acetyl-4-hydroxy-8-methyl-pyrano[3,2-c]chromene-2,5-dione is solid mass, crystallised form glacial acetic acid.Synthesis of 4-hydroxy-8-methyl-3-(3-oxobutanoyl)pyrano [3,2-c]chromene-2,5-dione (L II ):Acetyl derivatives (L I ), on its further reaction with ethyl acetate in presence of sodium gave acetoacetyl derivatives (L II ) 21-26 . 3-acetyl-4-hydroxy-8-methyl-pyrano [3, 2-c] chromene-2, 5-dione (1g)dissolved in ethyl acetate (25.0 mL) was added to pulverized sodium (1g) and refluxed for 8hrs. It wasthen decomposed with ice and extracted with ether. The aqueous layer on acidification gavesubstituted 4-hydroxy-3-(3-oxobutyanoyl) pyrano [3, 2-c] chromene-2, 5-dione, which was thenfiltered out, dried and recrystalised with acetone.Synthesis of metal complexes: The metal solutions (0.1M) were prepared by dissolving metal salt(ferrous ammonium sulphate and chloride of cobalt, nickel & copper) in distilled water andstandardized with 0.1M EDTA solution. Reaction of standardized metal solution (10.0 mL) wascarried out with ligand solution (L I & L II ) (20.0 mL) for two hours in water bath at 100 o C. Few dropsof ammonium hydroxide were added to the reaction mixture to maintain the pH 10.5-11. Precipitatesobtained were filtered, washed with water and alcohol, dried and recrystalised with DMSO.R 4R 3OOOOOHOHOOOH 2OH 3 C-CHOMOC-CH 3OOOOH 2 OR 2R 1R 1R 2R 3R 4R 1R 2R 2R 1R 3R 4OOOH 2OH 3 COCH 2 C-CHOMOC-CH 2COCH 3OOOOH 2 OR 4R 3Structure-IStructure-II2484 J. Chem. Bio. Phy. Sci. Sec. A; Aug. 2013-Oct.2013; Vol.3, No.4; 2482-2491.

A Comparative....DishaTilala et.al.Reaction scheme:OHC H 3H 2 CC OOHC OOHP OC l 3Zn Cl 2OOH 2 CP O Cl 3ZnCl 2C OOHOOO HCH 3(1 )O HC OOHC H 3( 2 )OOg la. CH 3 CO O HP OCl 3OOOOEth yl ac etateOHNaOHCH 3OCH 3O(L I I )OC OC H 2 CO C H 3( L I )OCO C H 3OOOOO HO HCH 3OCH 3OC-C H 2 CO CH 3C-CH 2 CO C H 3OOOOMMWhere, M = Fe +2 , Co +2 , Ni +2 or Cu +2CONDUCTIVITYThe conductivity of metal complexes was determined using Systronic Conductivity Bridge.Complexes were dissolved in DMF and conductivity was measured. Conductivity of the DMF wasmeasured and solution of the complexes in DMF along with different concentration was measured.The molar conductivity was calculated using the formula.Where, K = Conductivity of the sol. of the complexes in DMF.C = Concentration of the complexes (10 -3 M).The conductivity data are presented in (Table-II) and the data indicates that the complexes are nonelectrolytein nature 27 .2485 J. Chem. Bio. Phy. Sci. Sec. A; Aug. 2013-Oct.2013; Vol.3, No.4; 2482-2491.

A Comparative....DishaTilala et.al.ANTIMICROBIAL ACTIVITYQuantitative antibacterial assay: Different concentrations of compounds were subjected to suitablebroth for quantitative determination of antimicrobial activity. The lowest concentration, whichcompletely inhibits visible microbial growth, was recorded as the minimum inhibitory concentration(MIC, mg/mL).Qualitative antimicrobial assay: The in vitro biological screening effects of the investigatedcompounds were tested against the gram negative bacteria E. coli and gram positive bacteria Bacillusmegaterium by the well diffusion method (agar cup method) using agar nutrient as the medium. Whileantifungal activity was carried out using glucose yeast extract media (GYE) against Aspergillus Nigerand Candida albicans. The stock solutions were prepared by dissolving the compounds in DMSO. Ina typical procedure, a well was made with the help of borer on the nutrient medium plate which waspreviously inoculated with microorganisms. The well was filled with the different concentration oftest solution using a micropipette and incubated at 37 °C for 24 hrs (bacteria) and 48 hrs (fungi).DMSO was used as control. During incubation period, the test solution diffused and the growth of theinoculated microorganisms was affected. Antibacterial activity was indicated by the presence of clearzone of inhibition around the wells. The zone of inhibition was measured in mm.Gel electrophoresis: The DNA cleavage experiment was conducted using CT DNA by gelelectrophoresis with the corresponding metal complex in the presence of H 2 O 2 as an oxidant. Thereaction mixture was incubated before electrophoresis experiment at 35°C for 30minutes as follows:CT DNA 5µl (4×10 -4 µg/µl), 12 µl each compound (1000 ppm), 2 µl H 2 O 2 (35%). The samples wereelectrophoresed for 2 h at 50 V on 0.8% agarose gel using tris-acetic acid-EDTA buffer, pH = 8⋅3.After electrophoresis, the gel was stained using 1 µg/cm 3 ethidiumbromide (EB) and photographedunder UV light using Sony camera.RESULT AND DISCUSSIONAll the compounds are stable at room temperature; ligands are soluble in alcohol, DMF and DMSOwhile all the metal complexes are insoluble in water, methanol, and ethanol, but soluble in DMF andDMSO. The analyses of the compounds are consistent with the stoichiometry proposed and aresummarized in Table I & II. Elemental analyses of metal complexes indicates that the metal: ligand(M:L) ratio is 1:2 for all the divalent metal ions. Because all metal complexes char above temperature270°, their melting points were not recorded. All the complexes have low conductance values, whichindicate that the complexes are nonelectrolytic 28 in nature.IR Spectra: The IR spectra of the ligands a strong broad band observed near ~3440 cm -1 due to theO-H stretching. The broad band suggests the existence of the compounds predominantly in the enolicform 29 . A strong C-O stretching band of alcohol is present at~1080 cm -1 . Attached methyl substituentsgave C-H stretching band near 2932 cm -1 . Multiple bonds of aromatic C=C stretching is observed inbetween 1491-1560 cm -1 while Sharp bands of =C-H bending are observed between 730-100 cm -1 . Asharp band of cyclic ketone between 1726-1732 cm -1 observed while a sharp band appear between1606-1616 cm -1 indicates presence of α,β-unsaturated acetyl ketone. Multiple weak bands C-O-C etherlinkage observed between 1163-1294 cm -1 . Monodentate acetate usually shows two bands at ~1620cm -1 and ~1310 cm -1 due to antisymmetric and symmetric stretching, respectively 30 . Since carbonylabsorption of the compounds also appeared in this region, the band at ~1620 cm -1 could not belocated. However, a medium intensity band observed between 1294-1330 cm -1 suggests themonodentate coordination of the acetate groups.2486 J. Chem. Bio. Phy. Sci. Sec. A; Aug. 2013-Oct.2013; Vol.3, No.4; 2482-2491.

A Comparative....DishaTilala et.al.In case of metal complexes bands of OH remains almost unaffected indicating that the enolic OH isnot replaced during complex formation. In all the spectra of complexes both aromatic and acetylketone frequencies shifted by ~15–20 cm -1 to the lower energy region compared to the free ligand.This phenomenon appears may be due to the coordination of the carbonyl oxygen to the metal ion.That the carbonyl groups are involved in bonding with the metal is further supported by theappearance of a medium intensity bands in the region 476-486 cm -1 assignable to M-O vibrations 30 .Antimicrobial activity: The antimicrobial activity of the ligand and its complexes was evaluated bythe MIC method. The in vitro antimicrobial activity of the investigated compounds was tested againstthe microorganisms, such as E. coli, Bacillus megaterium, Aspergillus, and Candida Albicans by theserial dilution method. On the basis of result of MIC, zone of inhibition was measured (in millimeter)by disc diffusion technique 31-35 are summarized in Table III. A comparative study of the ligands andits complexes indicates that the complexes exhibit higher antimicrobial activity than the free ligand.Such increased activity of the complexes can be explained on the basis of Overtone’s concept andTweedy’s chelation theory.According to Overtone's concept of cell permeability, the lipid membranethat surrounds the cell favors the passage of only the lipid-soluble materials due to whichliposolubility is an important factor, which controls the antimicrobial activity. On chelation, thepolarity of the metal ion will be reduced to a greater extent due to the overlap of the ligand orbital andpartial sharing of the positive charge of the metal ion with donor groups. Further, it increases thedelocalization of π-electrons over the whole chelate ring and enhances the lipophilicity of thecomplexes. This increased lipophilicity enhances the penetration of the complexes into lipidmembranes and blocking of the metal binding sites in the enzymes of microorganisms. Thesecomplexes also disturb the respiration process of the cell and thus block the synthesis of the proteinsthat restricts further growth of the organism.DNA Cleavage Studies: The cleavage efficiency of the complexes compared with that of the controlis due to their efficient DNA-binding ability. The metal complexes were able to degrade DNA. Thegeneral oxidative mechanisms proposed account for DNA cleavage by hydroxyl radicals via.Abstraction of a hydrogen atom from sugar units and predict the release of specific residues arisingfrom transformed sugars, depending on the position from which the hydrogen atom is removed 36 . Thecleavage is inhibited by the free radical scavengers implying that hydroxyl radical or peroxyderivatives mediate the cleavage reaction.The reaction is modulated by a metallocomplexes bound hydroxyl radical or a peroxo speciesgenerated from the co-reactant H 2 O 2 . In the present study, the CT-DNA gel electrophoresisexperiment was conducted at 35 °C using our synthesized complexes in the presence of H 2 O 2 as anoxidant. It was found that, at very low concentrations, few complexes exhibit nuclease activity in thepresence of H 2 O 2 . Control experiment using DNA alone does not show any significant cleavage ofCT-DNA even on longer exposure time. Probably this may be due to the formation of redox couple ofthe metal ions and its behavior. The redox property of the metal complexes mediates oxidation ofnucleic acids. In oxidative DNA cleavage mechanism, metal ions in the complexes react with H 2 O 2 togenerate the hydroxyl radical which attacks at the C4’ position of the sugar moiety and finally cleavesthe DNA 37 . Due to the cleavage, intensity of DNA band decreases which can be observed in figuresIII & IV. A slight increase in the concentration over the optimal value (i.e., the value at which 100%cleavage efficiency was observed) led to extensive degradations, resulting in the disappearance ofbands on agarose gel 38 .2487 J. Chem. Bio. Phy. Sci. Sec. A; Aug. 2013-Oct.2013; Vol.3, No.4; 2482-2491.

A Comparative....DishaTilala et.al.Table- 1: Analytical and physical data of the synthesized compoundsCompoundMolecularFormulaMolecular Melting Elemental Analysis (%) Found (calculated)Weight Point(g) (°C) C H O1 C 10 H 8 O 2 176 210 67.93(68.18) 4.71(4.58) 27.37 (27.25)2 C 13 H 8 O 5 244 110 64.09(63.94) 3.55(3.30) 32.38 (32.76)L I C 15 H 10 O 6 286 88 63.13(62.94) 3.45(3.52) 33.42 (33.54)L II C 17 H 12 O 7 328 104 62.07(62.20) 3.56(3.68) 34.37(34.12)Table -2: Physical characterization, analytical and molar conductance data of metal complexesComplex MolecularFormulaMolecularWeightElemental Analysis (%) Found(calculated)Molarconductance(g) C H O M mho cm 2 mol -1Fe[L I (H 2 O)] 2 C 30 H 26 O 14 Fe 666 54.19 4.12 33.54 8.15 10.14(54.07) (3.93 (33.61) (8.38)Co[L I (H 2 O)] 2 C 30 H 26 O 14 Co 669 53.75 3.83 33.25 9.15 12.07(53.84) (3.91) (33.46) (8.80)Ni[L I (H 2 O)] 2 C 30 H 26 O 14 Ni 668 53.96 3.78 33.61 8.66 11.85(53.84) (3.92) (33.47) (8.77)Cu[L I (H 2 O)] 2 C 30 H 26 O 14 Cu 673 53.32 3.81 33.10 9.76 14.20(53.45) (3.89) (33.23) (9.43)Fe[L II (H 2 O)] 2 C 34 H 30 O 16 Fe 750 54.31 3.89 34.24 7.56 11.49(54.42) (4.03) (34.11) (7.44)Co[L II (H 2 O)] 2 C 34 H 30 O 16 Co 753 54.08 3.94 34.13 7.87 14.17(54.19) (4.01) (3.97) (7.82)Ni[L II (H 2 O)] 2 C 34 H 30 O 16 Ni 753 54.13 4.12 33.90 7.85 15.43(54.21) (4.01) (33.98) (7.79)Cu[L II (H 2 O)] 2 C 34 H 30 O 16 Cu 758 54.02(53.86)4.08(3.99)33.83(33.77)8.05(8.38)13.98Figure 1: Antibacterial data of the Ligands and their Complexes at 200ppm2488 J. Chem. Bio. Phy. Sci. Sec. A; Aug. 2013-Oct.2013; Vol.3, No.4; 2482-2491.

A Comparative....CompoundTable -3: Antimicrobial data of ligands & their metal complexesDishaTilala et.al.(Conc. in ppm & zone of inhibition in millimeter)Escheritia coli Bacillus megaterium Aspergillus niger Candida albicansConc. Zone Conc. Zone Conc. Zone Conc. ZoneL I 100 33 200 30 300 29.5 200 25200 35 300 31.5 400 37 300 26300 36 400 34 500 38 400 29Fe[L I (H 2 O)] 2 100 31 200 32 200 29 200 21200 32.5 300 32.5 300 30 300 22300 34 400 34 400 30.5 400 22.5Co[L I (H 2 O)] 2 50 32 100 33 400 28 200 0100 33 200 34.5 500 32 300 21.5200 34.5 300 35 600 33 400 24Ni[L I (H 2 O)] 2 100 34 200 33.5 300 30 200 23200 35.5 300 35 400 32.5 300 25300 36 400 35 500 35 400 26Cu[L I (H 2 O)] 2 200 35 300 33 400 33 200 0300 36 400 35 500 35 300 18400 36 500 36 600 36 400 18.5L II 50 22 300 20 400 20 200 26100 24 400 21 500 26 300 27.5200 24.5 500 21.5 600 26.5 400 30Fe[L II (H 2 O)] 2 100 22 200 20.5 400 25 200 25200 23 300 22 500 26 300 25.5300 23.5 400 23 600 27 400 26Co[L II (H 2 O)] 2 200 21.5 200 21 300 25.5 200 27300 23 300 22 400 27 300 28400 25 400 24 500 28 400 29Ni[L II (H 2 O)] 2 100 22.5 200 22 400 26 200 28.5200 24 300 23 500 26.5 300 29300 26 400 25.5 600 27 400 29.5Cu[L II (H 2 O)] 2 100 23.5 100 22.5 400 27.5 200 28200 25 200 24 500 28 300 29.5300 26.5 300 25.5 600 30 400 31CONCLUSIONSAll metal complexes are non-electrolytes in DMF (molar conductance < 16 mho cm 2 mol -1 ; 10 -3 Msolution). Elemental analysis (Table 1 & 2), IR and 1H NMR data of the complexes are in agreementwith structure-1&2. Data of antimicrobial activity (Table 3 & 4) shows most of the metal complexesare higher active than their ligands. Figure-I indicates Cu[L I (H 2 O)] 2 & L II are inactive while rest ofthe compounds are active against B. megaterium, whereas all of them showes activity against E. coliat 200 ppm concentration. Comparison of antifungal data at 400 ppm (Figure-II) shows all thecompounds are active against both the fungus. From Figures III it can be concluded thatNi[L I (H 2 O)] 2 & Cu[L I (H 2 O)] 2 complex while from Figure IV it can be concluded that Fe[L II (H 2 O)] 2,Co[L II (H 2 O)] 2, Ni[L II (H 2 O)] 2 and Cu[L II (H 2 O)] 2 complexes cleave DNA in the presence of H 2 O 2 ,whereas Fe[L I (H 2 O)] 2 & Co[L I (H 2 O)] 2 are not effectual. Among all the complexes, Fe[L II (H 2 O)] 2 &Cu[L II (H 2 O)] 2 shows lowest intensity band of DNA, it leads to the conclusion that they have highestDNA cleavage ability.2490 J. Chem. Bio. Phy. Sci. Sec. A; Aug. 2013-Oct.2013; Vol.3, No.4; 2482-2491.

A Comparative....DishaTilala et.al.ACKNOWLEDGEMENTAuthors express their gratitude to Xavier Research Foundation and Fr.(Dr.)Vincent Braganza forproviding the necessary research facilities for this research.REFERENCES1. D.S. Sigman, A. Mazumder & D.M. Perrin, Chem Rev, 1993, 93, 2295.2. D.S. Sigman, Acc Chem Res, 1986, 19, 180.3. B. Meunier, Chem Rev, 1992, 92, 1411.4. J. Reedijk, J Inorg Biochem, 2001, 86, 89.5. K.E. Erkkila, D.T. Odom & J.K. Barton, Chem Rev, 1999, 99, 2777.6. B. Armitage, Chem Rev, 1998, 98, 1171.7. D.R. McMillin & K.M. McNett, Chem Rev, 1998, 98, 1201.8. W.K. Pogozelski & T.D. Tullius, Chem Rev, 1998, 98, 1089.9. H.T. Chifotides & K.R. Dunbar, Acc Chem Res, 2005, 38, 146.10. E.R. Jamieson & S.J. Lippard, Chem Rev, 1999, 99, 2467.11. H. Umezawa, Prog Biochem Pharmacol, 1976, 11, 18.12. P.J. Dyson & G. Sava, Dalton Trans, 2006 1929.13. C.J. Burrows & J.G. Muller, Chem Rev, 1998, 98, 1109.14. S.E. Wolkenberg & D.L. Boger, Chem Rev, 2002,102, 2477.15. A. Sreeedhara & J.A. Cowan, J Biol Inorg Chem, 2001, 6, 337.16. R. Rao, A.K. Patra & P.R. Chetana, Polyhedron, 2007, 26, 5331.17. J.A. Cowan, Chem Rev, 1998, 98, 1067.18. E.L. Hegg & J.N. Burstyn, Coord Chem Rev, 1998,173, 133.19. J. Marmur, J. Mol. Biol., 1961, 3, 208–218.20. A. Shah, N. Bhatt, R.V. Raval & V.M. Thakor, Curr. Soc., 1984, 53, 1289-90.21. V.N. Dholakia, M.G. Parekh & K.N. Trivedi, Aust.J.Chem., 1968, 21, 2345-7.22. H.R. Eisenhaur & K.P. Link, J.Am.Chem.Soc., 1953, 75, 2044.23. R. Anschtz, Ber., 1903, 36, 465.24. Pauly & K. Lockmann, ibid., 1915, 48, 28.25. A. Sonn, ibid., 1917, 50, 1292.26. M.A. Stahmenn , I. Wolff & K.P. Link, J.Am.Chem.Soc., 1943, 65, 2285.27. W.J. Geary, Co-ord. Chem. Rec., 1971, 7, 82.28. W.J.Geary, Coord. Chem. Rev., 1970, 7, 81 -122.29. L.J. Bellamy, The Infrared Spectra of Complex Molecules, London, Chapman and Hall, 1980.30. N. Nakamoto, Infrared Spectra and Raman Spectra of Inorganic and Coordination Compounds, NewYork ,John Wiley & Sons, 1997.31. N. Dharmaraj, P. Viswanathamurthi and K. Natarajan, Transition Met. Chem., 2001, 26, 105.32. P.B. Chakrawarti, J. Indian Chem. Soc., 2001, 78, 273.33. M.J. Pelczar, E.C.S. Chan, and N.R. Krieg, In: Microbiology, New York ,Blackwell Science, 1998.34. E.J. Stokes and G.L. Ridgway, Clinical Bacteriology, Edward Arnold, Maryland, USA, 1980.35. C.H. Colins and P.M. Lyne, Microbial Methods, Univ. Park Press, Baltimore, Maryland, USA, , 1970.36. G. Prativel, M. Pitie, J. Bernadou, B. Meunier, Angew. Chem. Int. Ed. Eng., 1991, 30, 702.37. N. Raman, L. Mitu, A. Sakthivel and M.S.S. Pandi, J. Iran. Chem. Soc., 2009, 6(4) 738-748.38. R.R. Joshi and K.N. Ganesh, Proc. Indian Acad. Sci., 1994, 16, 1089.Corresponding author: Disha Tilala; Department Of Chemistry, St. Xavier’s College,Ahmedabad-380009 (Gujarat) India.2491 J. Chem. Bio. Phy. Sci. Sec. A; Aug. 2013-Oct.2013; Vol.3, No.4; 2482-2491.