August 2013 - October 2013, Vol. 3, No. 4; 2482-2491.

E- ISSN: 2249 –1929

Journal of Chemical, Biological and Physical Sciences

An International Peer Review E-3 Journal of Sciences
Available online atwww.jcbsc.org
Section A: Chemical Science

CODEN (USA): JCBPAT Research article

A Comparative Study of Antimicrobial and

DNA Clevage Activity of Metal(II)
Complexes of Coumarin Derivatives
Disha Tilala1*, Manish Solanki2, Denish Karia3
1
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 2013

Abstract: New transition metal complexes of the composition, M[L(H2O)]2, where

M= 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(LI) and 4-hydroxy-8-methyl-3-(3-oxobuty-
anoyl) pyrano [3,2-c]chromen-2,5-dione (LII), were synthesized and characterized by
elemental analyser, conductivity measurement, IR, UV-Vis. and 1H-NMR spectral
techniques. The low conductance data provide evidence for the non-ionic nature of the
complexes. The antimicrobial activities of the ligands and their complexes have been
studied by screening the compounds against the bacteria E. coli and B. megaterium and
also the fungi C. albicans and A. niger. The data indicate that most of the metal
complexes have higher antimicrobial activity than the free ligands. The DNA cleavage
experiments, performed using gel electrophoresis with the corresponding metal
complexes in the presence of H2O2 showed good results.
Keywords: transition metal complexes, antimicrobial, DNA cleavage, gel
electrophoresis

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A Comparative.... DishaTilala et.al.
INTRODUCTION

Transition metal complexes with an ability to cleave and bind DNA under physiological conditions
are of current interests for their varied applications in nucleic acid chemistry, viz., as sequence
specified DNA cleaving agents, foot printing agents and diagnostic tools in medical field1-9. Metal
complexes with tunable coordination environments and versatile physicochemical properties offer
scope for designing highly sensitive diagnostic agents for medical applications as exemplified by the
chemotherapeutic agents like cis-platin and bleomycins10-12. DNA cleavage could be achived via
oxidative or hydrolytic pathways13-15. Hydrolytic DNA cleavage involves cleavage of phosphodiester
bond to generate fragments which can be subsequenty relegated.
Hydrolytic cleavage active species mimic restriction enzymes. Oxidative DNA cleavage involves
either oxidation of the deoxyribose moiety by abstraction of sugar hydrogen or oxidation of
nucleobases16. Oxidative cleavage of DNA occurs in the presence of additives or photoinduced DNA
cleavage agents17,18. Photocleavers require the presence of a photosensitizer that can be activated on
irradiation with UV or visible light the redox active ‘chemical nucleases’ are effective cleavers of
DNA in the presence of a reducing agent or H2O2 as an additive6, 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.

EXPERIMENTAL

Apparatus and reagents: All chemicals used were of analytical reagent (AR) grade and of the
highest purity available. They include phosphorous oxychloride (Spectrochem), Phenol, malonic acid,
zinc chloride, sodium carbonate, hydrochloric acid, ethanol, ammonia, agarose (Merck), and nutrient
broth, glucose, yeast extract (Helini biomolecules). CT DNA was purchased from Genei chemical
company, India. Solutions of CT DNA in Tris–HCl/NaCl (pH 7.2) buffer medium gave a ratio of
A260/A280, of ca. 1.8–1.9, indicating that the DNA was sufficiently free from protein contamination19.
Stock solutions were kept at 4°C and used within 4 days. Doubly distilled water was used to prepare
the buffer. All the melting points were determined in open glass capillaries in a liquid paraffin-bath
and 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. Elemental
Analyses 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 method
given by shah & co-workers20. m-cresol (0.1mole) and malonic acid (0.1mole) were added to a
mixture of phosphorous oxychloride (40ml) and freshly fused zinc chloride (40g). The reaction
mixture was heated on water bath at 60-70oC for 8-10 hrs and poured into ice. The solid residue is
filtered out and treated with hot saturated sodium carbonate. The filtrate was slowly acidified with
concentrated hydrochloric acid. At the point of neutralization, the precipitates were obtained were
filtered 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 phosphorous
oxychloride (40mL) and freshly fused zinc chloride (40g). The reaction mixture was heated on water
bath at 60-70oC for 16-18hrs and poured into ice. The solid residue is filtered out and treated with hot
saturated sodium carbonate. The filtrate was slowly acidified with concentrated hydrochloric acid. At

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A Comparative.... DishaTilala et.al.
the point of neutralization, the precipitates are obtained are filtered and washed with water and further
dried and recrystalised with methanol.
Synthesis of 3-acetyl-4-hydroxy-8-methylpyrano [3, 2-c] chromene-2, 5-dione (LI): Acetyl
derivatives are prepared21-26 by acetylation of product-II in presence of acetic acid and phosphorous
oxychloride. 4-hydroxy-8-methylenepyrano [3, 2-c] chromene-2, 5-dione (1g) was dissolved in
mixture of glacial acetic acid (5.0 mL) and phosphorous oxychloride (4.0 mL). The reaction mixture
was 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 (LII):
Acetyl derivatives (LI), on its further reaction with ethyl acetate in presence of sodium gave aceto
acetyl derivatives (LII) 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 was
then decomposed with ice and extracted with ether. The aqueous layer on acidification gave
substituted 4-hydroxy-3-(3-oxobutyanoyl) pyrano [3, 2-c] chromene-2, 5-dione, which was then
filtered 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 and
standardized with 0.1M EDTA solution. Reaction of standardized metal solution (10.0 mL) was
carried out with ligand solution (LI & LII) (20.0 mL) for two hours in water bath at 100 oC. Few drops
of ammonium hydroxide were added to the reaction mixture to maintain the pH 10.5-11. Precipitates
obtained were filtered, washed with water and alcohol, dried and recrystalised with DMSO.
R4 R4

R3 O O R3 O O

OH OH
R2 R2

R1
O R1 O
C-CH3 C-CH2COCH3

O O O O

OH2 M H2 O OH2 M H2O

O O O O

H3C-C H3COCH2C-C
O O
R1 R1

R2 R2
HO HO

O O R3 O O R3

R4
R4

Structure-I Structure-II

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A Comparative.... DishaTilala et.al.
Reaction scheme:

OH

C OOH O O POCl3 O O
POCl3 ZnCl2
H 2C
ZnCl2
C OOH
C OOH OH
C H3 H 2C

C OOH
CH 3 OH C H3 O
(2)
(1)
O

gla. CH3COOH
POCl3

O O O O

Ethyl acetate
OH
OH
Na

CH 3 O CH 3 O
C OC H 2CO C H 3
CO C H 3
I
(LII) (L )
O
O

O O O O

OH OH

CH 3 O CH 3 O

-
C C H 2CO CH 3 -
C CH 2CO C H 3

O O O O

M M

Where, M = Fe+2, Co+2, Ni+2 or Cu+2

CONDUCTIVITY

The conductivity of metal complexes was determined using Systronic Conductivity Bridge.
Complexes were dissolved in DMF and conductivity was measured. Conductivity of the DMF was
measured 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 non-
electrolyte in nature27.

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ANTIMICROBIAL ACTIVITY

Quantitative antibacterial assay: Different concentrations of compounds were subjected to suitable

broth for quantitative determination of antimicrobial activity. The lowest concentration, which
completely 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 investigated
compounds were tested against the gram negative bacteria E. coli and gram positive bacteria Bacillus
megaterium by the well diffusion method (agar cup method) using agar nutrient as the medium. While
antifungal activity was carried out using glucose yeast extract media (GYE) against Aspergillus Niger
and Candida albicans. The stock solutions were prepared by dissolving the compounds in DMSO. In
a typical procedure, a well was made with the help of borer on the nutrient medium plate which was
previously inoculated with microorganisms. The well was filled with the different concentration of
test 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 the
inoculated microorganisms was affected. Antibacterial activity was indicated by the presence of clear
zone 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 gel
electrophoresis with the corresponding metal complex in the presence of H2O2 as an oxidant. The
reaction 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 H2O2 (35%). The samples were
electrophoresed 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/cm3 ethidiumbromide (EB) and photographed
under UV light using Sony camera.

RESULT AND DISCUSSION

All the compounds are stable at room temperature; ligands are soluble in alcohol, DMF and DMSO
while all the metal complexes are insoluble in water, methanol, and ethanol, but soluble in DMF and
DMSO. The analyses of the compounds are consistent with the stoichiometry proposed and are
summarized 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 temperature
270°, their melting points were not recorded. All the complexes have low conductance values, which
indicate that the complexes are nonelectrolytic28 in nature.
IR Spectra: The IR spectra of the ligands a strong broad band observed near ~3440 cm-1 due to the
O-H stretching. The broad band suggests the existence of the compounds predominantly in the enolic
form29. A strong C-O stretching band of alcohol is present at~1080 cm-1. Attached methyl substituents
gave C-H stretching band near 2932 cm-1. Multiple bonds of aromatic C=C stretching is observed in
between 1491-1560 cm-1 while Sharp bands of =C-H bending are observed between 730-100 cm-1. A
sharp band of cyclic ketone between 1726-1732 cm-1 observed while a sharp band appear between
1606-1616 cm-1 indicates presence of α,β-unsaturated acetyl ketone. Multiple weak bands C-O-C ether
linkage observed between 1163-1294 cm-1. Monodentate acetate usually shows two bands at ~1620
cm-1 and ~1310 cm-1 due to antisymmetric and symmetric stretching, respectively30. Since carbonyl
absorption of the compounds also appeared in this region, the band at ~1620 cm-1 could not be
located. However, a medium intensity band observed between 1294-1330 cm-1 suggests the
monodentate coordination of the acetate groups.

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A Comparative.... DishaTilala et.al.
In case of metal complexes bands of OH remains almost unaffected indicating that the enolic OH is
not replaced during complex formation. In all the spectra of complexes both aromatic and acetyl
ketone 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 the
appearance of a medium intensity bands in the region 476-486 cm-1 assignable to M-O vibrations30.
Antimicrobial activity: The antimicrobial activity of the ligand and its complexes was evaluated by
the MIC method. The in vitro antimicrobial activity of the investigated compounds was tested against
the microorganisms, such as E. coli, Bacillus megaterium, Aspergillus, and Candida Albicans by the
serial dilution method. On the basis of result of MIC, zone of inhibition was measured (in millimeter)
by disc diffusion technique31-35 are summarized in Table III. A comparative study of the ligands and
its 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 and
Tweedy’s chelation theory.According to Overtone's concept of cell permeability, the lipid membrane
that surrounds the cell favors the passage of only the lipid-soluble materials due to which
liposolubility is an important factor, which controls the antimicrobial activity. On chelation, the
polarity of the metal ion will be reduced to a greater extent due to the overlap of the ligand orbital and
partial sharing of the positive charge of the metal ion with donor groups. Further, it increases the
delocalization of π-electrons over the whole chelate ring and enhances the lipophilicity of the
complexes. This increased lipophilicity enhances the penetration of the complexes into lipid
membranes and blocking of the metal binding sites in the enzymes of microorganisms. These
complexes also disturb the respiration process of the cell and thus block the synthesis of the proteins
that restricts further growth of the organism.
DNA Cleavage Studies: The cleavage efficiency of the complexes compared with that of the control
is due to their efficient DNA-binding ability. The metal complexes were able to degrade DNA. The
general 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 arising
from transformed sugars, depending on the position from which the hydrogen atom is removed36. The
cleavage is inhibited by the free radical scavengers implying that hydroxyl radical or peroxy
derivatives mediate the cleavage reaction.
The reaction is modulated by a metallocomplexes bound hydroxyl radical or a peroxo species
generated from the co-reactant H2O2. In the present study, the CT-DNA gel electrophoresis
experiment was conducted at 35 °C using our synthesized complexes in the presence of H2O2 as an
oxidant. It was found that, at very low concentrations, few complexes exhibit nuclease activity in the
presence of H2O2. Control experiment using DNA alone does not show any significant cleavage of
CT-DNA even on longer exposure time. Probably this may be due to the formation of redox couple of
the metal ions and its behavior. The redox property of the metal complexes mediates oxidation of
nucleic acids. In oxidative DNA cleavage mechanism, metal ions in the complexes react with H2O2 to
generate the hydroxyl radical which attacks at the C4’ position of the sugar moiety and finally cleaves
the DNA37. Due to the cleavage, intensity of DNA band decreases which can be observed in figures
III & 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 of
bands on agarose gel38.

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 A Comparative.... DishaTilala et.al.
Table- 1: Analytical and physical data of the synthesized compounds

Compound Molecular Molecular Melting Elemental Analysis (%) Found (calculated)

Formula Weight Point
(g) (°C) C H O

1 C10H8O2 176 210 67.93(68.18) 4.71(4.58) 27.37 (27.25)

2 C13H8O5 244 110 64.09(63.94) 3.55(3.30) 32.38 (32.76)
LI C15H10O6 286 88 63.13(62.94) 3.45(3.52) 33.42 (33.54)
LII C17H12O7 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 complexes

Complex Molecular Molecular Elemental Analysis (%) Found Molar

Formula Weight (calculated) conductance
(g) C H O M mho cm2 mol-1
Fe[LI (H2O)]2 C30H26O14Fe 666 54.19 4.12 33.54 8.15 10.14
(54.07) (3.93 (33.61) (8.38)
Co[LI(H2O)]2 C30H26O14Co 669 53.75 3.83 33.25 9.15 12.07
(53.84) (3.91) (33.46) (8.80)
Ni[LI(H2O)]2 C30H26O14Ni 668 53.96 3.78 33.61 8.66 11.85
(53.84) (3.92) (33.47) (8.77)
Cu[LI(H2O)]2 C30H26O14Cu 673 53.32 3.81 33.10 9.76 14.20
(53.45) (3.89) (33.23) (9.43)
Fe[LII(H2O)]2 C34H30O16Fe 750 54.31 3.89 34.24 7.56 11.49
(54.42) (4.03) (34.11) (7.44)
Co[LII(H2O)]2 C34H30O16Co 753 54.08 3.94 34.13 7.87 14.17
(54.19) (4.01) (3.97) (7.82)
Ni[LII(H2O)]2 C34H30O16Ni 753 54.13 4.12 33.90 7.85 15.43
(54.21) (4.01) (33.98) (7.79)
Cu[LII(H2O)]2 C34H30O16Cu 758 54.02 4.08 33.83 8.05 13.98
(53.86) (3.99) (33.77) (8.38)

Figure 1: Antibacterial data of the Ligands and their Complexes at 200ppm

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A Comparative.... DishaTilala et.al.

Figure 2: Antifungal data of the Ligands and their Complexes at 400ppm conc.

Lane: 1 2 3 4 5 6

Figure 3: Agarose gel (0.8 %) showing the changes in the agarose gel
electrophoretic pattern of CT DNA induced by H2O2.

Lane 1: DNA alone, Lane 2: DNA + LI, Lane 3: DNA + Fe[LI(H2O)]2, Lane 4: DNA + Co[LI(H2O)]2, Lane
5: DNA + Ni[LI(H2O)]2, Lane 6: DNA + Cu[LI(H2O)]2- As shown in figure, the intensity of the DNA band
decreased by reaction with metal complexes. This indicated presence of nuclease activity of the complex.

Lane: 1 2 3 4 5 6

Figure 4: Agarose gel (0.8 %) showing the changes in the agarose gel
electrophoretic pattern of CT DNA induced by H2O2.

Lane 1: DNA alone, Lane 2: DNA + LII, Lane 3: DNA + Fe[LII(H2O)]2, Lane 4: DNA + Co[LII(H2O)]2,
Lane 5: DNA + Ni[LII(H2O)]2, Lane 6: DNA + Cu[LII(H2O)]2– Here also similar observation was noted as
shown in figure 1. This also confirms the nuclease activity of complex.

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A Comparative.... DishaTilala et.al.
Table -3: Antimicrobial data of ligands & their metal complexes

(Conc. in ppm & zone of inhibition in millimeter)

Compound Escheritia coli Bacillus megaterium Aspergillus niger Candida albicans
Conc. Zone Conc. Zone Conc. Zone Conc. Zone
LI 100 33 200 30 300 29.5 200 25
200 35 300 31.5 400 37 300 26
300 36 400 34 500 38 400 29
Fe[LI(H2O)]2 100 31 200 32 200 29 200 21
200 32.5 300 32.5 300 30 300 22
300 34 400 34 400 30.5 400 22.5
Co[LI(H2O)]2 50 32 100 33 400 28 200 0
100 33 200 34.5 500 32 300 21.5
200 34.5 300 35 600 33 400 24
Ni[LI(H2O)]2 100 34 200 33.5 300 30 200 23
200 35.5 300 35 400 32.5 300 25
300 36 400 35 500 35 400 26
Cu[LI(H2O)]2 200 35 300 33 400 33 200 0
300 36 400 35 500 35 300 18
400 36 500 36 600 36 400 18.5
LII 50 22 300 20 400 20 200 26
100 24 400 21 500 26 300 27.5
200 24.5 500 21.5 600 26.5 400 30
Fe[LII(H2O)]2 100 22 200 20.5 400 25 200 25
200 23 300 22 500 26 300 25.5
300 23.5 400 23 600 27 400 26
Co[LII(H2O)]2 200 21.5 200 21 300 25.5 200 27
300 23 300 22 400 27 300 28
400 25 400 24 500 28 400 29
Ni[LII(H2O)]2 100 22.5 200 22 400 26 200 28.5
200 24 300 23 500 26.5 300 29
300 26 400 25.5 600 27 400 29.5
Cu[LII(H2O)]2 100 23.5 100 22.5 400 27.5 200 28
200 25 200 24 500 28 300 29.5
300 26.5 300 25.5 600 30 400 31

CONCLUSIONS

All metal complexes are non-electrolytes in DMF (molar conductance < 16 mho cm2mol-1; 10-3 M
solution). Elemental analysis (Table 1 & 2), IR and 1H NMR data of the complexes are in agreement
with structure-1&2. Data of antimicrobial activity (Table 3 & 4) shows most of the metal complexes
are higher active than their ligands. Figure-I indicates Cu[LI(H2O)]2 & LII are inactive while rest of
the compounds are active against B. megaterium, whereas all of them showes activity against E. coli
at 200 ppm concentration. Comparison of antifungal data at 400 ppm (Figure-II) shows all the
compounds are active against both the fungus. From Figures III it can be concluded that
Ni[LI(H2O)]2 & Cu[LI(H2O)]2 complex while from Figure IV it can be concluded that Fe[LII(H2O)]2,
Co[LII(H2O)]2, Ni[LII(H2O)]2 and Cu[LII(H2O)]2 complexes cleave DNA in the presence of H2O2,
whereas Fe[LI(H2O)]2 & Co[LI(H2O)]2 are not effectual. Among all the complexes, Fe[LII(H2O)]2 &
Cu[LII(H2O)]2 shows lowest intensity band of DNA, it leads to the conclusion that they have highest
DNA 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.
ACKNOWLEDGEMENT

Authors express their gratitude to Xavier Research Foundation and Fr.(Dr.)Vincent Braganza for
providing the necessary research facilities for this research.

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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.