Nucleolytic property promoted by biologically active metal-based complexes formed by neutral quatridentate ligands: Synthesis, characterization, and DNA binding studies

Article abstract of DOI:10.1080/15533174.2012.684262

A series of Ni(II), Cu(II), and Zn(II) complexes have been synthesized and characterized with physicochemical and spectral techniques. Absorption titration study of the interaction of the complexes with calf-thymus (CT) DNA has shown that the complexes can bind to CT DNA. The cyclic voltammograms of all the complexes recorded in the presence of CT DNA-buffer solution have shown that they can bind to CT DNA via intercalation mode, which has also been verified by the viscosity experiments. The antimicrobial and DNA cleavage activities of all the compounds have also been investigated.

Full text of DOI:10.1080/15533174.2012.684262

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Nucleolytic Property Promoted by Biologically
Active Metal-Based Complexes Formed by Neutral
Quatridentate Ligands: Synthesis, Characterization,
and DNA Binding Studies
K. Pothiraj a , T. Baskaran a & N. Raman a
a Research Department of Chemistry , VHNSN College , Virudhunagar , India
Accepted author version posted online: 12 Jul 2012.Published online: 28 Aug 2012.
To cite this article: K. Pothiraj , T. Baskaran & N. Raman (2012) Nucleolytic Property Promoted by Biologically Active Metal-
Based Complexes Formed by Neutral Quatridentate Ligands: Synthesis, Characterization, and DNA Binding Studies, Synthesis
and Reactivity in Inorganic, Metal-Organic, and Nano-Metal Chemistry, 42:8, 1192-1203, DOI: 10.1080/15533174.2012.684262
To link to this article: http://dx.doi.org/10.1080/15533174.2012.684262
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Synthesis and Reactivity in Inorganic, Metal-Organic, and Nano-Metal Chemistry, 42:1192–1203, 2012
Copyright ꢀ Taylor & Francis Group, LLC
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ISSN: 1553-3174 print / 1553-3182 online
DOI: 10.1080/15533174.2012.684262
Nucleolytic Property Promoted by Biologically Active
Metal-Based Complexes Formed by Neutral Quatridentate
Ligands: Synthesis, Characterization, and DNA
Binding Studies
K. Pothiraj, T. Baskaran, and N. Raman
Research Department of Chemistry, VHNSN College, Virudhunagar, India
design of DNA and RNA specific agents capable of controlled
chemical cleavage are of paramount importance due to their po-
tential use as drugs, regulators of gene expression, and tools for
molecular biology.[5] Metal complexes are attractive reagents
for the cleavage of nucleic acids due to their inherently diverse
structure and reactivity. Several studies have been carried out on
the complexes that cleave DNA through an oxidative pathway
which requires a coreactant such as an oxidizing or reducing
agent, light, or redox-active metal center in addition to the prin-
cipal cleavage agent.[6,7]
A series of Ni(II), Cu(II), and Zn(II) complexes have been syn-
thesized and characterized with physicochemical and spectral tech-
niques. Absorption titration study of the interaction of the com-
plexes with calf-thymus (CT) DNA has shown that the complexes
can bind to CT DNA. The cyclic voltammograms of all the com-
plexes recorded in the presence of CT DNA-buffer solution have
shown that they can bind to CT DNA via intercalation mode, which
has also been verified by the viscosity experiments. The antimicro-
bial and DNA cleavage activities of all the compounds have also
been investigated.
In recent years, the interest concerning copper(II) complexes
has been focused on complexes containing Schiff bases de-
rived from ketones and diamines.[8,9] This group of complexes
has been studied due to their antimicrobial and anticancer ac-
tivities. Complexes breaking double-stranded DNA duplex are
thought to be biologically significant sources of cell lethality,
because they appear to be less readily repaired by DNA repair
mechanisms. The potential applications of metal complexes as
non-radioactive probes of nucleic acid structure and possible
DNA cleaving agents have been explored extensively.[10,11] In
these complexes, the metal or ligands can be varied in an easily
controlled way to facilitate the individual applications. Copper
is a bioessential element with relevant oxidation states. Because
of its chemical and biological relevance, a large number of cop-
per(II) complexes have been synthesized and investigated for
their pharmacological properties.[12,13]
Keywords antimicrobial activity, DNA binding, metal complexes,
nucleolytic property
INTRODUCTION
Schiff base is an organic compound in which the nitrogen
atom of an amino group is double bonded to a carbon atom. The
Schiff base acts as an electron sink that greatly stabilizes nega-
tive charge that develops on the adjacent carbon. The chelating
structures, moderate electron donation, and easy tunable elec-
tronic and steric effects proved Schiff bases to be flexible ligands
capable of stabilizing different metals in various oxidation states
with unusual structural features.[1,2] Characteristic Schiff base
provides geometrical cavity control for host–guest interaction
and modulation of its lipophilicity offers remarkable selectivity,
sensitivity, and stability for a specific metal ion.
Very few numbers of Schiff base metal complexes of 2ꢁ-
methylacetoacetanilide and their derivatives have been reported
to possess interesting biological properties such as antitumor,
antioxidant, antimicrobial activities, and laboratory uses and
many industrial applications.[14] A literature search reveals that
the Schiff bases derived from 2ꢁ-methylacetoacetanilide and
ethylenediamine/phenylenediamine have not been reported. Our
interest in the present study deals with the synthesis and spec-
troscopic characterization of nickel(II), copper(II), and zinc(II)
complexes with symmetrical neutral quatridentate (tetradentate)
ligands derived from 2ꢁ-methylacetoacetanilide and diamines.
The investigation of the biological properties of metal com-
plexes has been focused on (a) the binding properties with
Recent works have shown a growing interest in the inter-
action of small molecules/metal complexes with DNA.[3,4] The
Received 13 December 2011; accepted 1 April 2012.
The authors express their heartfelt thanks to the College Managing
Board, Principal and Head of the Department of Chemistry, VHNSN
College, for providing necessary research facilities. Instrumental fa-
cilities provided by Sophisticated Analytical Instrumention Facility
(SAIF), IIT Bombay, for EPR spectra and CDRI, Lucknow, for mass
spectra and CHN analyses are gratefully acknowledged.
Address correspondence to N. Raman, Research Department of
Chemistry, VHNSN College, Virudhunagar 626001, India. E-mail:
drn raman@yahoo.co.in
1192

SYNTHESIS AND DNA BINDING STUDIES OF COMPLEXES
1193
calf-thymus (CT) DNA performed with electronic absorption rer for 3 h and evaporated to room temperature. The product
spectroscopy, cyclic voltammetry, and viscosity measurements; was filtered off and washed with small amount of dilute ethanol
(b) the study of the nucleolytic activity of the complexes; and and petroleum ether. Recrystallization was carried out in hot
(c) the examination of the antimicrobial activity of the ligands ethanol and a yellow-colored amorphous compound was ob-
as well as their complexes.
tained, which was subsequently dried over anhydrous CaCl2
under vacuum.
For C24H30N4O2 (L1), Yield (%): 68.38, pale yellow, M.pt:
198◦C, M.Wt: 406.52, Anal. Calcd. (%): C, 70.91; H, 7.44;
N, 13.78. Found (%): C, 70.85; H, 7.41; N, 13.74; 1H
NMR (DMSO-d6, δ/ppm): 8.67 (2H, –NH), 7.02–7.71 (8H,
Ar–H), 3.36 (4H,C–CH2C–), 2.86 (12H, –CH3), 1.71–2.32 (4H,
–CH2–CH2); IR (KBr, ν/cm−1): 3211 ν(NH), 3065 ν(Ar–CH),
2984,2945 ν(aliphatic–CH), 1651 ν(–CONH), 1612 ν(C=N),
1548 ν(Ar–C=C); UV–Vis (λmax/ nm) (DMF): 265 and 310.
For C28H30N4O2 (L2):Yield(%):66.95, yellow, M.pt:211◦C,
M.Wt: 454.57, Anal. Calcd. (%): C, 73.98; H, 6.65; N, 12.33.
EXPERIMENTAL
Materials and General Methods
All chemicals and reagents were of reagent grade quality.
Ethylenediamine and phenylenediamine (SD Fine Chemicals,
India) and 2ꢁ-methylacetoacetanilide (Aldrich, USA) were used
as received. The AR grade of NiCl2·6H2O, CuCl2.2H2O and
ZnCl2 were used. Tris-HCl buffer was purchased from Hi-
media (Mumbai, India). The supercoiled (SC) pUC19 DNA
was purchased from Bangalore Genie, India. CT-DNA, 3-
mercaptopropionic acid (MPA), bromophenol blue, xylene
cyanol, agarose (molecular biology grade), and ethidium bro-
mide (EB) were obtained from Sigma Aldrich (USA). Tris–HCl
buffer was prepared using deionized and sonicated triple-
distilled water.
1
Found (%): C, 73.94; H, 6.62; N, 12.28; H NMR (DMSO-
d6, δ/ppm): 8.70 (2H, –NH), 6.96–7.83 (12H, Ar–H), 3.41 (4H,
C–CH2C–), 2.94 (12H, –CH3)); IR (KBr, ν/cm−1): 3232 ν(NH),
3058 ν(Ar–CH), 2991,2939 ν(aliphatic–CH), 1658 ν(–CONH),
1619 ν(C=N), 1552 ν(Ar–C=C); UV–Vis (λmax/ nm) (DMF):
270 and 325.
Carbon, hydrogen, and nitrogen analysis of the ligands and
their complexes were carried out on a CHN analyzer Carlo Erba
1108 (USA). The infrared spectra (4000–400 cm−1 KBr discs)
of the samples were recorded on FT-IR Affinity-I spectropho-
tometer (Shimadzu, Japan). The electronic absorbance spectra
(200–1100 nm) were recorded on a UV-1601 spectrophotometer
(Shimadzu, Japan). Proton nuclear magnetic resonance spectra
were obtained with a 300 MHz Bruker Avance DRX-300 FT-
NMR spectrometer (New York, USA). Chemical shifts (ppm)
were referenced either with an internal standard (Tetramethylsi-
lane) or to the residual solvent peaks. EPR spectra of complexes
in solid state at 300 K and in frozen DMSO at 77 K were recorded
on a Varian E–112 spectrometer (United Kingdom) at X-band,
using a TCNE as marker with 100 kHz modulation frequency
and 9.1 GHz microwave frequency. Mass spectrometry experi-
ments were performed onaJEOL-AccuTOFJMS-T100LCmass
spectrometer (USA) equipped with a custom-made electrospray
interface (ESI). Molar conductance of 10−3 M solution of the
complexes in N,Nꢁ-dimethylformamide (DMF) was measured at
room temperature with an Deep Vision Model-601 digital direct
reading deluxe conductivity meter (India). Magnetic suscepti-
bility measurements were carried out by employing the Gouy
method at room temperature on powder sample of the com-
plex. CuSO4·5H2O was used as calibrant. The metal contents
of the complexes were determined according to the literature
method.[15]
Preparation of Nickel(II), Copper(II), and Zinc(II) Complexes
NiCl2. 6H2O (0.237 g; 1.0 mmol) or CuCl2. 2H2O (0.170 g;
1.0 mmol) or ZnCl2 (0.136 g; 1.0 mmol) dissolved in ethanol
was slowly added to Schiff base ligand L1 (0.406 g; 1.0 mmol)
/ L2 (0.454 g; 1.0 mmol) dissolved in hot ethanol and refluxed
for 2 h. The resulting colored solution was concentrated to ap-
proximately 5 mL, thereby a colored precipitate was formed. It
was filtered, washed with ethanol, and dried in vacuo.
For [NiC24H30N4O2Cl2] ([NiL1Cl2]): Yield (%): 70.73,
green, M.pt: >250◦C, M.Wt: 536.12, Anal. Calcd. (%): Ni,
10.95; C, 53.77; H, 5.64; N, 10.45; Cl, 13.23. Found (%): Ni,
10.91; C, 53.75; H, 5.60; N, 10.42; Cl, 13.21; IR (KBr, ν/cm−1):
3215 ν(–NH), 3064 ν(Ar–CH), 2985, 2944 ν(aliphatic–CH),
1637 ν(C=O),1594 ν(C=N), 1551 ν(Ar–C=C), 578 ν(Cu–O),
486 ν(Cu–N); UV–Vis (λmax/nm) (DMF): 268, 321, 583, 762,
and 960. Molar conductance (DMF): 7.11 ohm−1 cm2 mol−1
;
µeff (BM): 3.06.
For [CuC24H30N4O2Cl2] ([CuL1Cl2]): Yield(%): 73.47, light
green, M.pt: >250◦C, M.Wt: 540.97, Anal. Calcd. (%): Cu,
11.75; C, 53.29; H, 5.59; N, 10.35; Cl, 13.11. Found (%): Cu,
11.69; C, 53.24; H, 5.56; N, 10.32; Cl, 13.09; IR (KBr, ν/cm−1):
3213 ν(–NH), 3067 ν(Ar–CH), 2983, 2945 ν(aliphatic–CH),
1635 ν(C=O),1599 ν(C=N), 1549 ν(Ar–C=C), 585 ν(Cu–O),
493 ν(Cu–N); UV–Vis (λmax/nm) (DMF): 270, 323, 670, and
767; molar conductance (DMF): 8.58 ohm−1 cm2 mol−1; µeff
(BM): 1.79.
Synthesis of the Ligands and Their Complexes
Preparation of Schiff Base Ligands
For [ZnC24H30N4O2Cl2] ([ZnL1Cl2]): Yield (%): 71.86, yel-
The ethanolic solution of ethylenediamine (0.60 g, 10 mmol) low, M.pt: >250◦C, M.Wt: 542.82, Anal. Calcd. (%): Zn,
or phenylenediamine (1.08 g, 10 mmol) was added to 2ꢁ- 12.05; C, 53.10; H, 5.57; N, 10.32; Cl, 13.06. Found (%):
methylacetoacetanilide (3.82 g, 20 mmol) hot ethanolic solu- Zn, 12.01; C, 53.08; H, 5.53; N, 10.29; Cl, 13.03; H NMR
1
tion (Scheme 1). The mixture was refluxed with magnetic stir- (DMSO-d6, δ/ppm): 8.69 (2H, –NH), 7.03–7.75 (12H, Ar–H),

1194
K. POTHIRAJ ET AL.
3.37 (4H, C–CH2C–), 2.89 (12H, –CH3), 1.72–2.35 (4H, where [DNA] is the concentration of DNA in base pair, and
–CH2–CH2); IR (KBr, ν/cm−1): 3215 ν(–NH), 3068 ν(Ar–CH), εa, εf, and εb correspond to the apparent absorption coefficient
2984, 2947 ν(aliphatic–CH), 1639 ν(C=O),1597 ν(C=N), 1550 Aobsd/[M], the extinction coefficient for the free complex, and
ν(Ar–C=C), 573 ν(Cu–O), 497 ν(Cu–N); UV–Vis (λmax/nm) the extinction coefficient for the complex in the fully bound
(DMF): 269 and 331; molar conductance (DMF): 9.23 ohm−1 form, respectively.
cm2 mol−1; µeff (B.M.): Diamagnetic.
For [NiC28H30N4O2Cl2] ([NiL2Cl2]): Yield (%): 69.04, red,
Cyclic Voltammetry
Cyclic voltammetry studies were performed on a CH In-
strument Electrochemical analyzer. Cyclic voltammetry exper-
iments were carried out in a 15 mL three-electrode electrolytic
cell. The working electrode was glassy carbon, a platinum wire
was used as the counter electrode and an Ag/AgCl electrode
saturated with KCl was used as the reference electrode. The
M.pt: >250◦C, M.Wt: 584.17, Anal. Calcd. (%): Ni, 10.05; C,
57.57; H, 5.18; N, 9.59; Cl, 12.14. Found (%): Ni, 10.03; C,
57.52; H, 5.14; N, 9.56; Cl, 12.00; IR (KBr, ν/cm−1): 3236
ν(–NH), 3059 ν(Ar–CH), 2988, 2943 ν(aliphatic–CH), 1641
ν(C=O),1601 ν(C=N), 1552 ν(Ar–C=C), 569 ν(Cu–O), 452
ν(Cu–N); UV–Vis (λmax/nm) (DMF): 277, 334, 595, 774, and
987. molar conductance (DMF): 10.85 ohm−1 cm2 mol−1; µeff
cyclic voltammograms of the complexes were recorded in (1:2)
(BM): 3.11.
DMF:buffer solutions at v = 100 mV s−1 where Tris-HCl buffer
For [CuC28H30N4O2Cl2] ([CuL1Cl2]): Yield (%): 71.62,
and 0.1 M Bu4NClO4 solution were used as the supporting elec-
brown, M.pt: >250◦C, M.Wt: 589.02, Anal. Calcd. (%): Cu,
trolytes. Oxygen was removed by purging the solutions with
pure nitrogen that had been previously saturated with solvent
10.79; C, 57.10; H, 5.13; N, 9.51; Cl, 12.04. Found (%): Cu,
10.73; C, 57.06; H, 4.97; N, 9.48; Cl, 12.01; IR (KBr, ν/cm−1):
vapors. All electrochemical measurements were performed at
3235 ν(–NH), 3060 ν(Ar–CH), 2989, 2941 ν(aliphatic–CH),
25◦C.
1639 ν(C=O),1596 ν(C=N), 1553 ν(Ar–C=C), 572 ν(Cu–O),
458 ν(Cu–N); UV–Vis (λmax/nm) (DMF): 274, 339, 694, and
Viscometric Measurements
789; molar conductance (DMF): 12.69 ohm−1 cm2 mol−1; µeff
Viscometric studies were done using a Ubbelodhe viscome-
(BM): 1.84.
ter, thermostated at 37◦C in a constant temperature bath. The
For [ZnC28H30N4O2Cl2] ([ZnL1Cl2]): Yield (%): 70.58, yel-
low, M.pt: >250◦C, M.Wt: 590.87, Anal. Calcd. (%): Zn, 11.07;
C, 56.92; H, 5.12; N, 9.48; Cl, 12.00; Found (%): Zn, 11.03; C,
56.90; H, 5.08; N, 9.46; Cl, 11.97; 1H NMR (DMSO-d6, δ/ppm):
8.71 (2H, –NH), 6.97–7.86 (12H, Ar–H), 3.44 (4H, CH2), 2.96
(12H, –CH3); IR (KBr, ν/cm−1): 3234 ν(–NH), 3059 ν(Ar–CH),
2992, 2940 ν(aliphatic–CH), 1644 ν(C=O),1603 ν(C=N), 1552
ν(Ar–C=C), 580 ν(Cu–O), 455 ν(Cu–N); UV–Vis (λmax/nm)
(DMF): 280 and 346; molar conductance (DMF): 11.77 ohm−1
cm2 mol−1; µeff (B.M.): Diamagnetic.
concentration of DNA was kept constant in all samples, but the
complex concentration was varied and the flow time for each
sample was measured three times. An average flow time was
calculated. The data were presented as (η/η0)1/3 versus binding
ratio (R = [complex]/[DNA]), where η is the viscosity of DNA
in the presence of the complex and η0 is that of DNA alone. Vis-
cosity values were calculated from the observed flowing time
of DNA containing solutions (t) corrected for that of the buffer
alone (t0): η = (t – t0)/t0.
DNA Cleavage and Mechanism Studies
DNA Interaction Studies
Absorption Titration
For the gel electrophoresis analysis, the supercoiled pUC19
DNA (30 µM, 0.2 µg) was treated with complexes in pres-
ence of 3-mercaptopropionic acid in 50 mM Tris–HCl/5 mM
NaCl, buffer (pH 7.2). The solution was then incubated for 1 h
at 37◦C, then a quench buffer solution containing bromophe-
nol blue (0.25%) and sucrose (40%) was added. In the ensuing
step, samples were immediately loaded on 0.8% agarose gel
for electrophoresis in Tris-acetate-EDTA (TAE) buffer (pH 8.0)
containing 1.0 µg/mL EB at 50 V for 2 h. Finally, the gel was
photographed under UV light. Cleavage mechanistic investiga-
tion of pUC19 DNA was carried out in the presence of standard
radical scavengers. These reactions were carried out by adding
standard radical scavengers such as ethanol (EtOH), sodium
azide (NaN3), and superoxide dismutase (SOD) to pUC19 DNA
prior to the addition of complex. Cleavage was initiated by
the addition of complex and quenched with 2 µL of loading
buffer. Further analysis was carried out by the previous standard
The DNA binding experiments were performed in Tris–HCl
buffer (50 mM Tris HCl/5 mM NaCl buffer, pH 7.2). The con-
centration of CT DNA was determined by measuring the ab-
sorption intensity at 260 nm with ε value of 6600 M−1 cm−1 [16]
.
Absorption titration experiments were made using different con-
centrations of CT DNA, keeping the concentration of the com-
plexes constant, with due correction for the absorbance of the
CT DNA itself. Samples were equilibrated before recording
each spectrum. To compare quantitatively the affinity of the
complexes bound to DNA, the intrinsic-binding constants Kb
of the complexes were obtained by monitoring the changes in
absorbance with increasing concentration of DNA using the
following equation:[17]
[DNA]/(εa − εf) = [DNA]/(εb − εf) + 1/Kb(εb − εf) [1] method.

SYNTHESIS AND DNA BINDING STUDIES OF COMPLEXES
1195
Determination of Antimicrobial Activity
Paper Disc Method
Sterilized discs were used. Fresh stock solutions (1 mg/mL)
of the synthesized compounds were prepared in redistilled
dimethylsulfoxide (DMSO) according to the required concen-
trations.[18] The discs were impregnated in DMSO. Each disc
was impregnated with 0.1 mL DMSO and so the total amount
of compound contained in each disc was 100 µg. The media
for bacteria and fungi were nutrient agar and dextrose agar re-
spectively. Media was prepared and sterilized by autoclaving
for 15 min at 121◦C, then the medium was cooled to 50◦C in-
oculated with the previously prepared bacteria suspension and
poured in sterilize Petri dishes. The medium was allowed to
solidify after a simple circular movement of the plates. Paper
discs previously impregnated with the solution of compounds
to be tested were placed on the plates. The plates were then
incubated for 24 h at 37◦C in case of bacteria and 48 h at
37◦C in case of fungi. On each plate an appropriate refer-
ence antibiotic disc was applied depending on the test microor-
ganisms. To ensure that the solvent had no effect on bacte-
rial and fungi growth, a control test was performed with test
medium supplemented with DMSO following the same proce-
dure as given in the experiment. DMSO showed no inhibition
zones.
FIG. 1. Absorption spectrum of [CuL2Cl2] in the absence and presence of
increasing amounts of CT DNA at room temperature in 50 mM Tris-HCl/5 mM
NaCl buffer (pH 7.2). Arrow shows the absorbance changing upon increasing
DNA concentration.
Electronic Absorption Spectra and Magnetic Moment
The electronic spectra of the free ligands (L1 and L2) exhibit
two bands in the range of 265–270 and 310–325 nm, assigned to
π → π∗ transition of aromatic rings and azomethine groups, re-
spectively (Figures 1 and 2). The electronic spectra of [CuL1Cl2]
and [CuL2Cl2] complexes (Figures 3 and 4) show a broad band
centered at 670–694 and 767–789 nm, which are attributed to
Dilution Method
To determine the MIC for the all the compounds dilution
method[19] was used. The compounds were dissolved and then
diluted using DMSO, twofold serial concentrations of the com-
pounds were employed to determine the MIC ranging from 200
to 0.78 µg/mL. The MIC value was determined as the low-
est concentration of the compound that completely inhibited
macroscopic growth of microorganism.
2B1g → E1g and 2B1g → A1g transitions characterized by cop-
per(II) ion in octahedral geometry.[20] The octahedral geometry
of copper(II) ion in the above mentioned copper(II) complexes
is confirmed by the measured magnetic moment values in the
range 1.79–1.84 B.M. which is in harmony with the reported
value. The proposed octahedral geometry around the nickel(II)
ion is also confirmed by the position of absorption bands ap-
peared at 960, 762, and 583 nm for [NiL1Cl2] and 987, 774,
and 595 nm for [NiL2Cl2] complexes, attributed to 3A2g(F) →
2
2
RESULTS AND DISCUSSION
The results of the elemental analysis of ligands L1 and L2
and their Ni(II), Cu(II), and Zn(II) complexes were recorded
and are given in Experimental section. All the metal complexes
are colored, non-hygroscopic, and are quite stable at room tem-
perature. They are found to be insoluble in common organic
solvents such as methanol, ethanol, dichloromethane, and ace-
tonitrile, except DMF and DMSO. The molar conductance data
of the complexes in DMF at 10−3 M is in the range 7.11–12.69
ohm−1 cm2 mol−1, which indicates non-electrolytic nature of
the complexes suggesting that the chloride anions are coordi-
nated to metal ion. Different attempts such as crystallization
using mixtures of solvents and low-temperature crystallization
were unsuccessful to obtain a single crystal suitable for X-ray
crystallography. However, the analytical, spectral, and magnetic
data enable us to expect the possible structure of the prepared
complexes.
3
3
3T2g(F), 3A2g(F) → T1g(F) and 3A2g(F) → T1g(P) transitions,
respectively. Themagneticmoment values of 3.06and3.11B.M.
are observed for [NiL1Cl2] and [NiL2Cl2], respectively, corre-
sponding to an octahedral environment around the nickel(II)
ion.
Infrared Spectra of the Ligands and Their
Metal Complexes
The infrared spectra provided valuable information regard-
ing the nature of the functional groups attached to the metal
atom. The intense band in the region 1612–1619 cm−1 in
the IR spectra of free Schiff base ligands is associated with
C N stretching vibration and is shifted to lower frequencies

1196
K. POTHIRAJ ET AL.
FIG. 2. Cyclic voltammagram of [CuL2Cl2] in the absence and presence
of increasing amounts of CT DNA at room temperature in DMF:buffer (1:2)
mixture (pH 7.2). Arrow shows the current changing upon increasing DNA
concentration (color figure available online).
FIG. 3. Effect of increasing amounts of [CuL2Cl2] (ꢀ), [CuL1Cl2] (ꢁ),
[NiL2Cl2] (−), [NiL1Cl2] (ꢂ), [ZnL2Cl2] (•), and [ZnL1Cl2] (ꢀ) on the rela-
tive viscosity of CT DNA versus the [complex]/[DNA] (R) ratio (color figure
available online).
(1594–1603 cm−1) in the spectra of corresponding metal com-
plexes. This indicated the coordination of imine nitrogen to
the metal ion.[21] It was noted that in the infrared spectra of
the ligands as well as all the complexes, a single medium in-
tensity band in the region 3211–3232 cm−1 is present, which
may be assigned to the ν(N–H) stretching vibration of amide
moiety of 2ꢁ-methylacetoacetanilide. The absorption bands at
1651–1658 cm−1 due to ν(C O) stretching frequencies of
amide moiety, in the free ligands shift towards lower values
(1635–1644 cm−1) in the spectra of the complexes of Ni(II),
Cu(II), and Zn(II) ions. This behavior indicates the fact that the
carbonyl oxygen atom of the 2ꢁ-methylacetoacetanilide residue
was coordinated. The infrared spectra of all the metal com-
plexes reveal that (M-O) and (M-N) stretching vibrations are in
the range 452–497 cm−1 and 569–585 cm−1, respectively.
Proton Nuclear Magnetic Resonance Spectra
The Schiff bases and their zinc complexes have been char-
acterized by proton nuclear magnetic resonance spectra to en-
sure the ligands and their zinc complexes purity in solution and
elucidate the differently positioned protons. The NMR spec-
tra of the Schiff base ligands L1 and L2, the singlet signals
ranging at 8.67–8.70, 3.36–3.41, and 2.86–2.94 ppm are as-
cribed to -NH (2H) protons of the amide moiety of the 2ꢁ-
methylacetoacetanilide, C-CH2-C (4H) proton, and aromatic
and aliphatic protons of -CH3 (12H), respectively. In addition
to this the multiplet signals around the ranges at 7.02–7.71 and
6.96–7.83 ppm are due to aromatic protons of L1 and L2, re-
spectively. A characteristic triplet signal at 1.71–2.32 ppm is
assigned to CH2-CH2 (4H) protons of ligand L1. In the case of
diamagnetic zinc complexes, all the signals are slightly shifted
FIG. 4. Gel electrophoresis diagram showing the cleavage of supercoiled pUC19 DNA (0.2 µg) in 50 mM Tris–HCl/5 mM NaCl buffer (pH 7.2) incubated at
37◦C for 2 h with different concentrations of copper complexes in the presence of MPA (100 µM) as an reducing agent: Lane 1, Control DNA; Lane 2, DNA
+ MPA; Lane 3, DNA + [CuL1Cl2L] (30 µM); Lane 4, DNA + [CuL2Cl2] (30 µM); Lane 5, DNA + MPA + [CuL1Cl2] (30 µM); Lane 6, DNA + MPA +
[CuL1Cl2] (60 µM); Lane 7, DNA + MPA + [CuL1Cl2] (100 µM); Lane 8, DNA + MPA + [CuL2Cl2] (30 µM); Lane 9, DNA + MPA + [CuL2Cl2] (60 µM);
Lane 10, DNA + MPA + [CuL2Cl2] (100 µM) (color figure available online).

SYNTHESIS AND DNA BINDING STUDIES OF COMPLEXES
1197
to high value of δ, which reveals that the coordination takes place wide interest in the nature of bonding parameters in the system,
and the data of the complexes are given in the experimental part. we adopted the simplified molecular orbital theory to calculate
the molecular orbital coefficients such as α2 (covalent in-plane
σ bonding) and β2 (covalent in-plane π bonding), which were
Mass Spectra
The mass spectrum has been successfully used to examine calculated by using the following equations:[24]
molecular species in solution. The pattern of the mass spectrum
α2 = (A /0.036) + (g − 2.0027) + (3/7)(g − 2.0027) + 0.04
gives an impression of the successive degradation of the target
compound with the series of peaks corresponding to the various
fragments. In the present investigation the ESI-mass spectra of
L1, L2, and [CuL2Cl2] have been recorded and show a molecular
ion peak at m/z = 406, 455, and 589, respectively, which corre-
sponds to the molecular weight of the respective ligands (L1 and
L2) and the copper complex of L2 ([CuL2Cl2]) and also copper
complex shows 35Cl isotopic peaks observed at 591 (M+2) and
593 (M+4). The fragmentation of [CuL2Cl2] gives the elimi-
nation of two chloride ions followed by the demetallation and
the m/z values are 520 (M-1) and 454 which corresponds to
the fragment ions [CuC28H30N4O2]+. and [C28H30N4O2]+., re-
spectively. Also, some other unstable fragments are observed
in the same copper complex, and the peaks at m/z = 189,
174, and 148 correspond to the fragment ions [C11H13N2O],
[C10H10N2O], and [C9H10NO], respectively. This fragmenta-
tion pattern confirms that the complexes have two chloride ions
inside the coordination sphere and the complex is monomeric
in nature.
||
||
⊥
[2]
β2 = (g − 2.0027)Ed−d/(−8λα2)
[3]
||
If the α2 value is 0.5, it indicates a complete covalent bonding,
while the value of α2 = 1.0 suggests a complete ionic bonding.
The observed value of α2 (0.97) indicates that the complex
has covalent character and β2 = 1.08, which suggests that in
plane π-bonding is also present in the complex. The observed
values indicate that there is interaction in the in plane π-bonding,
whereas in-plane σ-bonding is completely ionic. This is also
confirmed by orbital reduction factors (K and K ), which are
ꢂ
⊥
calculated from the following equations:
K2 = (g − 2.0027)Ed−d/8λ
[4]
||
||
K2⊥ = (g − 2.0027) Ed−d/2λ
[5]
⊥
EPR Spectral Studies
where λ = − 828 cm−1 for the free metal ion. In the case of
EPR studies of copper(II) complexes give information about
the distribution of the unpaired electrons and hence about the
nature of the bonding between the metal ion and its ligand.
The X-band EPR spectra of [CuL2Cl2] complex was recorded
in DMSO solution at liquid nitrogen temperature (LNT) and at
room temperature (RT).
pure σ- bonding K = K , whereas K < K implies consid-
ꢂ
⊥
ꢂ
⊥
erable in-plane π-bonding while for out-plane π-bonding K >
ꢂ
K . For the present complex, the observed order is K (0.75)
⊥
ꢂ
> K (0.56), implying a greater contribution from out-plane
⊥
π-bonding than from in-plane π-bonding in metal ligand π
–bonding. Based on the previous analytical and spectral data,
it is concluded that the copper(II) complex has an octahedral
geometry.
In the present work, there is no information obtained from
the room temperature EPR spectrum of copper complex. The
values of g and g were worked out from the spectra at LNT
ꢂ
⊥
Electrochemical Properties of Metal Complexes
using tetracyanoethylene (TCNE) free radical as a “g” marker.
The ordering of g values [g (2.28) > g (2.05) > ge (2.0027)]
The electrochemical properties of the metal(II) complexes
ꢂ
⊥
observed for the copper(II) complex indicates that the unpaired have been studied by cyclic voltammetry under nitrogen atmo-
electron is localized in dx2-y2 orbital of the copper(II) ion.[22] sphere in DMF solution in the potential range +1.0 to 1.5 V
The value of gav (2.12) was calculated according to the equation versus Ag/AgCl reference and the results are presented in Table
gav = 1/3[g + 2 g ].
1. The cyclic voltammetry of Ni(II) complexes exhibits both
ꢂ
⊥
The axial symmetry parameter G, which is a measure of anodic and cathodic redox potentials. In anodic potential region
the exchange interaction between copper centers in the poly- the reduction wave (Epc, –0.248 for [NiL1Cl2] and –0.395 V
crystalline compound, is calculated using Hathaway expression for [NiL2Cl2]) corresponding to Ni(II)/Ni(I) reaction is ob-
(G = g – 2.0027/ g – 2.0027).[23] According to Hathaway, if tained. During the reverse scan the oxidation of Ni(I)/Ni(II)
ꢂ
⊥
the value of G > 4, the exchange interaction is negligible in solid occurs in the potential range (Epa, –0.078 for [NiL1Cl2] and
complex but G < 4 indicates considerable interaction in solid –0.223 V for [NiL2Cl2]). The ratio of peak current (Ipc/Ipa) is
complex. In the present case, G value (5.86) > 4, indicating no not equal to one and the values of the limiting peak-to-peak
exchange interaction between the copper(II) centers in DMSO separation (ꢀEp = Epa – Epc) are greater than 0.060 V. There-
at 77 K.
fore, the redox couple in voltammetric data can be attributed
Electron paramagnetic resonance and optical spectrum have to a quasireversible one-electron transfer process. The cathodic
been used to determine the covalent bonding parameters for the cyclic voltammetry of Cu(II) complexes displays two cathodic
copper(II) ion in various environments. Since there has been peaks in the potential range (Epc1,–0.249 to –0.584 V and Epc2,

1198
K. POTHIRAJ ET AL.
TABLE 1
Electrochemical behavior of metal(II) complexes in the absence and presence of CT DNA
Epc(V)
Epa(V)
ꢀEp(V)
E1/2(V)
Complex
Redox couple
Free
Bound
Free
Bound
Free
Bound
Free
Bound
[NiL1Cl2]
[CuL1Cl2]
Ni(II)/Ni(I)
Cu(II)/Cu(I)
Cu(I)/Cu(0)
Zn(II)/zn(I)
Ni(II)/Ni(I)
Cu(II)/Cu(I)
Cu(I)/Cu(0)
Zn(II)/Zn(I)
–0.248
–0.249
–0.709
–1.096
–0.395
–0.584
–0.725
–1.112
–0.196
–0.224
–0.663
–1.050
–0.364
–0.571
–0.708
–1.093
–0.078
–0.101
—
–0.029
–0.052
—
0.170
0.148
—
0.167
0.172
—
–0.163
–0.175
—
–0.112
–0.138
—
[ZnL1Cl2]
[NiL2Cl2]
[CuL2Cl2]
—
—
—
—
—
—
–0.223
–0.105
—
–0.201
–0.078
—
0.172
0.481
—
0.163
0.493
—
–0.309
–0.346
—
–0.283
–0.325
—
[ZnL2Cl2]
—
—
—
—
—
—
–0.709 to –0.725 V for [CuL1Cl2] and [CuL1Cl2], respectively), chemical or photophysical properties, thus enhancing the func-
due to the reduction of Cu(II)–Cu(I) and Cu(I)– Cu(0) couples tionality of the binding agent.[25]
respectively. In the reverse anodic scan one anodic peak in the
potential range (Epa1, –0.101 V to –0.105 V for [CuL1Cl2] and
Electronic Absorption Titration
[CuL1Cl2]), due to the oxidation of Cu(I)–Cu(II) couple. The
ratio of peak current (Ipc/Ipa) is less than one and the value
of peak-to-peak separation (ꢀEp) = 0.148 to 0.481 V, which
indicates that the first redox couple for these complexes in-
volved in quasireversible one-electron transfer process, whereas
the observed second redox couple is irreversible in nature. The
cyclic voltammetry measurement upon scanning cathodically,
[ZnL1Cl2] and [ZnL2Cl2] complexes display an irreversible one-
electron peak assigned to the reduction of Zn(II)–Zn(I) at –1.096
and –1.112 V, respectively. The absence of the anodic signal is
indicative of a fast chemical reaction following the charge trans-
fer step and instability of the electrochemically generated Zn(I)
species, respectively.
In general, if a small molecule interacts with DNA, changes
in absorbance (hypochromism) and in the position of the band
(red shift) should occur. This phenomenon indicates that the
small molecule has intercalated into DNA base pairs, and is
involved in a strong interaction in the molecular stack between
the aromatic chromophore and the base pairs. The spectral ef-
fects have been rationalized as[26] the empty π∗-orbital of the
small molecule couples with the π∗-orbital of the DNA base
pairs, which causes an energy decrease, and a decrease of the
π
π∗ transition energy. Therefore, the absorption of the small
molecule should exhibit a red shift. At the same time, the empty
π∗-orbital is partially filled with electrons to reduce the transi-
tion probability, which leads to hypochromism.
To achieve this, the electronic absorption spectra of the metal
complexes of Schiff base ligands (L1 and L2) in the absence and
Binding Characteristics of Metal Complexes With DNA
The interactions of metal complexes with DNA have been the presence of the CT DNA at different concentrations (at a con-
subject of interest for the development of effective chemothera- stant concentration of the complexes) were measured (Table 2)
peutic agents. Transition metal centers are particularly attractive and one representative [CuL2Cl2] complex is given in Figure 5.
moieties for such research since they exhibit well-defined coor- In the absorption spectra of [NiL1Cl2], [CuL1Cl2], [ZnL1Cl2],
dination geometries and also often possess distinctive electro- [NiL2Cl2], [CuL2Cl2], and [ZnL2Cl2], the bands centered at
TABLE 2
Absorption titration of metal(II) complexes with CT DNA
λmax(nm)
Complex
Free
Bound
ꢀλ (nm)
Hypochromicity (%)
Kb × 103 (M–1)
[NiL1Cl2]
[CuL1Cl2]
[ZnL1Cl2]
[NiL2Cl2]
[CuL2Cl2]
[ZnL2Cl2]
321.2
323.0
331.4
334.7
339.1
346.3
321.6
323.8
331.8
335.3
340.5
346.8
0.4
0.8
0.4
0.6
1.4
0.5
12.05
21.30
11.76
15.83
30.88
13.47
2.73
4.19
2.51
3.12
7.82
2.78

SYNTHESIS AND DNA BINDING STUDIES OF COMPLEXES
1199
FIG. 5. Gel electrophoresis diagram showing the cleavage of supercoiled pUC19 DNA (0.2 µg) in 50 mM Tris–HCl/5 mM NaCl buffer (pH 7.2) incubated at
37◦C for 2 h with two different concentration of zinc and nickel complexes in the presence of MPA (100 µM) as an reducing agent: Lane 1, Control DNA; Lane
2, DNA + MPA + [ZnL1Cl2L] (50 µM); Lane 3, DNA + MPA + [ZnL1Cl2] (100 µM); Lane 4, DNA + MPA + [ZnL2Cl2] (50 µM); Lane 5, DNA + MPA +
[ZnL2Cl2] (100 µM); Lane 6, DNA + MPA + [NiL1Cl2] (50 µM); Lane 7, DNA + MPA + [NiL1Cl2] (100 µM); Lane 8, DNA + MPA + [NiL2Cl2] (50 µM);
Lane 9, DNA + MPA + [NiL2Cl2] (100 µM) (color figure available online).
321.2, 323.0, 331.4, 334.7, 339.1, and 346.3 nm exhibit slightly intercalative) with the reduced and oxidized forms of the metal.
red shift in the range of 0.4–1.4 nm with hypochromism of The difference between voltammetric responses of [CuL2Cl2] in
12.05%, 21.30%, 11.76%, 15.83%, 30.88%, and 13.47%, re- the absence and presence of CT-DNA is illustrated in Figure 6.
spectively. The calculated Kb values of [NiL1Cl2], [CuL1Cl2], The significant shift in peak potentials of metal(II) complexes is
[ZnL1Cl2], [NiL2Cl2], [CuL2Cl2], and [ZnL2Cl2] are 2.73 × 103 observed upon addition of CT-DNA. The summary of voltam-
M
−1, 4.19 × 103 M−1, 2.51 × 103 M−1, 3.12 × 103 M−1, 7.82 × metric results for Ni(II), Cu(II) and Zn(II) complexes in the
103 M−1, and 2.78 × 103 M−1, respectively. The observed intrin- absence and presence of CT-DNA is given in Table 1. No new
sic DNA binding constants are similar to the values obtained for redox peaks are appeared after the addition of CT DNA to each
many reported first row transition metal complexes those show complex, but the current intensity decreases significantly, sug-
intercalative binding to DNA,[27,28] but much smaller than the gesting the existence of an interaction between each complex
classical intercalators and metallointercalators where binding and CT DNA, explained in terms of an equilibrium mixture of
constants reported in the order of 107 M−1 [29] From the results, free and DNA-bound complex on the electrode surface.[30] In
.
the [CuL2Cl2] complex shows higher affinity towards CT-DNA. general, the electrochemical potentials of a small molecule will
However from these measurements, it is not clear to conclude shift positively when it intercalates into DNA double helix, and,
that whether DNA binds through an intercalative mode or not. if it is bound to DNA by electrostatic interaction, the potential
So, further studies are needed to elucidate the exact nature of will shift to a negative direction. Additionally, if more poten-
the binding mode.
tials than one present such a shift, a positive shift of Epa and a
negative shift of Epc may imply that the molecule can bind to
DNA by both intercalation and electrostatic interaction.[31]
All the complexes exhibit similar electrochemical behav-
ior upon addition of CT DNA with positive shifts for the
cathodic potentials Epc (ꢀEpc = +0.052 V for [NiL1Cl2],
DNA Interaction of Electrochemical Studies
The electrochemical investigations of metal–DNA interac-
tions could also provide useful supplement to spectroscopic
method and yield information about interactions (electrostatic or
FIG. 6. Gel electrophoresis diagram showing the cleavage of supercoiled pUC19 DNA (0.2 µg) by copper(II) complexes (100 µM) with addition of MPA
(100 µM) and radical scavengers in 50 mM Tris–HCl/5 mM NaCl buffer (pH 7.2) and incubated at 37◦C for 2 h: Lane 1, DNA control; Lane 2, DNA + MPA
+ [CuL1Cl2] + NaN3 (100 µM); Lane 3, DNA + MPA + [CuL1Cl2] + EtOH (4 µL); Lane 4, DNA + MPA + [CuL1Cl2] + SOD (4 Units); Lane 5, DNA +
MPA + [CuL2Cl2] + NaN3 (100 µM); Lane 6, DNA + MPA + [CuL2Cl2] + EtOH (4 µL); Lane 7, DNA + MPA + [CuL2Cl2] + SOD (4 Units) (color figure
available online).

1200
K. POTHIRAJ ET AL.
SCH. 1. Outline of the synthesis of ligands and their complexes.
ꢀEpc = +0.031 V for [NiL2Cl2], ꢀEpc1 = +0.029 V; and for [NiL2Cl2], ꢀEpa1 = +0.051 V for [CuL1Cl2], and
ꢀEpc2 = +0.046 V for [CuL1Cl2], ꢀEpc1 = +0.016 V and ꢀEpa1 = +0.027 V for [CuL2Cl2], respectively). These shifts
ꢀEpc2 = +0.017 V for [CuL2Cl2], ꢀEpc = +0.046 V for show that all the complexes could bind to DNA by intercala-
[ZnL1Cl2], and ꢀEpc = +0.019 V for [ZnL2Cl2], respec- tive interaction.[32] The presence of DNA in the solution at the
tively) and also the anodic potentials Epa shift to positive val- same concentration of six complexes causes a considerable de-
ues (ꢀEpa = +0.049 V for [NiL1Cl2], ꢀEpa = +0.021 V crease in the voltammetric current. The drop of the voltammetric

SYNTHESIS AND DNA BINDING STUDIES OF COMPLEXES
1201
TABLE 3
The in vitro antimicrobial activity of ligands and their metal(II) complexes evaluated by minimum inhibitory concentration
(µg/mL)
Antibacterial activity
Antifungal activity
R. bataicola R. stolonifer
Compound
S. aureus
B. subtilis
E. coli
P. aeruginosa
A. niger
C. albicans
L1
L2
100
100
25
12.5
50
12.5
6.25
25
0.78
—
100
100
12.5
12.5
25
25
12.5
25
200
100
100
25
50
25
12.5
25
0.78
—
200
200
50
50
100
50
25
50
1.56
—
100
100
50
25
25
12.5
6.25
25
—
0.78
200
100
25
25
100
25
12.5
25
—
200
200
100
50
50
50
50
100
—
1.56
200
200
100
25
50
50
25
50
—
1.56
[NiL1Cl2]
[CuL1Cl2]
[ZnL1Cl2]
[NiL2Cl2]
[CuL2Cl2]
[ZnL2Cl2]
Ciprofloxacin
Flucanozole
1.56
—
1.56
currents in the presence of CT DNA could be attributed to dif- plexes in the presence of reducing agent (3-mercaptopropionic
fusion of the metal complex bound to the large, slowly diffusing acid) to pUC19 DNA is investigated by gel electrophoresis in
DNA molecule. Therefore, the results parallel to the previous Tris-HCl buffer (pH 7.2) at 37◦C for 2 h (Figure 8). Control
absorption spectroscopic titration of metal complexes in the experiments suggest that untreated DNA and DNA incubated
presence of DNA.
with either copper complexes or 3-mercaptopropionic acid do
not show any significant DNA cleavage (Lanes 1–4). At low to
medium concentration (30–60 µM), both the [CuL1Cl2] and
[CuL2Cl2] complexes show efficient DNA cleavage activity
while the [CuL2Cl2] complex shows higher cleavage activity
than [CuL1Cl2] complex in the presence of reducing agent. At
the higher concentration (100 µM), the DNA cleavage reac-
tion of [CuL1Cl2] complex exhibits the supercoiled form (Form
I), which is converted into nicked form (Form II) only but the
[CuL2Cl2] complex shows the supercoiled form (Form I) is
completely converted into nicked form (Form II) and linear
form (Form III) due to the complex having higher DNA binding
properties. The concentration dependent DNA cleavage prop-
erty of Ni(II) and Zn(II) complexes (50 µM and 100 µM) in
the presence of MPA to pUC19 DNA is studied by gel elec-
trophoresis in Tris-HCl buffer (pH 7.2) at 37◦C for 2 h (Figure
9). In different concentrations, the above two Ni(II) and Zn(II)
complexes display lower DNA cleavage and therefore the DNA
cleavage mechanistic study focuses only on the Cu(II) com-
plexes. In general, copper complexes can cleave DNA through
hydrolytic and oxidative processes. For the oxidative process,
the complexes have been shown to react with molecular oxygen
or hydrogen peroxide to produce a variety of active oxidative
intermediates (reactive oxygen species or nondiffusible copper-
oxene species). In order to obtain information about the active
chemical species that is responsible for the DNA cleavage that
occurs in the presence of hydroxyl radical scavengers (EtOH),
singlet oxygen quenchers (NaN3), and superoxide scavenger
(SOD) under our experimental conditions. Figure 10 shows that
NaN3 (Lanes 2 and 5) and EtOH (Lanes 3 and 6) significantly
DNA-Binding Study with Viscosity Measurements
DNA viscosity is sensitive to DNA length change; therefore,
its measurement upon addition of a compound is often con-
cerned as the least ambiguous method to clarify the interaction
mode of a compound with DNA and provides reliable evidence
for the intercalative binding mode.[33] In the case of classic inter-
calation, DNA base pairs are separated in order to host the bound
compound resulting in the lengthening of the DNA helix and
subsequently increase the DNA viscosity. On the other hand, the
binding of a compound exclusively in DNA grooves by means
of partial and nonclassic intercalation causes a bend or kink in
the DNA helix reducing its effective length and, as a result, DNA
solution viscosity is decreased or remains unchanged. Viscosity
measurements have been carried out on CT DNA solutions upon
addition of increasing amounts of metal complexes (Figure 7).
The addition of the complexes results in an increase in the rela-
tive viscosity of DNA, which may be explained by the insertion
of the compounds in between the DNA base pairs, leading to an
increase in the separation of base pairs at intercalation sites and,
thus, an increase in overall DNA length.[34,35] Thus, the results
obtained from the viscosity experiments validate those obtained
from the spectroscopic and electrochemical studies.
Nucleolytic Property of Complexes
The study on the cleavage capacity of transition metal com-
plexes to DNA is considerably interesting, as it can contribute
to understanding the toxicity mechanism of them and to de-
velop novel artificial nuclease. The cleavage ability of com-

1202
K. POTHIRAJ ET AL.
reduce the nuclease activity of the complexes, which is indica-
tive of the involvement of the singlet oxygen and hydroxyl radi-
cal in the cleavage process. Further, the addition of SOD (Lanes
4 and 7) has no significant effect on the DNA cleavage. This
fact rules out the involvement of the participation of superoxide
anion in the DNA cleavage reaction. This fact implies that DNA
cleavage reaction by the complexes should be realized by an
oxidative cleavage.
of 4-(2-pyridylmethyl)-1,7-dimethyl-1,4,7-triazonane-2,6-dione and 4-(2-
pyridylethyl)-1,7-dimethyl-1,4,7-triazonane-2,6-dione. Eur. J. Med. Chem.
2009, 44, 1607–1614.
13. Berners-Price, S.J.; Johnson, R.K.; Giovenella, A.J.; Faucette, L.F.;
Mirabelli, C.K.; Sadler, P.J. Antimicrobial and anticancer activity of tetra-
hedral, chelated, diphosphine silver(I) complexes: comparison with copper
and gold. J. Inorg. Biochem. 1988, 33, 285–295.
14. Raman, N.; Pothiraj, K.; Baskaran, T. DNA interaction, antimicrobial, elec-
trochemical and spectroscopic studies of metal(II) complexes with triden-
tate heterocyclic Schiff base derived from 2ꢁ-methylacetoacetanilide. J. Mol.
Struct. 2011, 1000, 135–144.
Antimicrobial Evaluation of Ligands and Their Complexes
The synthesized neutral quatridentate ligands and their metal
complexes have been screened for their antibacterial and anti-
fungal activities. From the antimicrobial screening observation
(Table 3), the nickel and zinc complexes show moderate activity
against all microorganisms, which is appreciable as compared
to the ligands, whereas the copper complexes show higher an-
timicrobial activity against S. aureus and A. niger under identi-
cal experimental conditions. This enhancement in antimicrobial
property is brought about upon complexation that can be re-
lated to the increased lipophilicity that powers the rate of entry
of molecules into the cell and inertness of certain metal-ligand
linkages, which protects the molecule against enzymatic degra-
dation.[36]
15. Angellici, R.J. Synthesis and Techniques in Inorganic Chemistry; Philadel-
phia: W. B. Saunders, 1969.
16. Reichmann, M.E.; Rice, S.A.; Thomas, C.A.; Doty, P. A further examination
of the molecular weight and size of desoxypentose nucleic acid. J. Am.
Chem. Soc. 1954, 76, 3047–3053.
17. Wolfe, A.; Shimer, G.H. Jr.; Meehan, T. Polycyclic aromatic hydrocarbons
physically intercalate into duplex regions of denatured DNA. Biochem.
1987, 26, 6392–6396.
18. Collins, C.H.; Lyre, P.M.; Grange, J.M. Microbiological Methods, 6th edn.;
London: Butterworths, 1989.
19. Jones, R.N.; Barry, A.L.; Gaven, T.L.; Washington, J. A. In Manual of
Clinical Microbiology, 4th edn., Lennette, E.H.; Balows, A.; Shadomy W.J.
(Eds.); Washington, DC: American Society for Microbiology, 1984.
20. Lever, A.B. P. Inorganic Electronic Spectroscopy, 2nd edn.; Amsterdam:
Elsevier, 1984.
21. Meghdadi, S.; Mereiter, K.; Amirnasr, M.; Fadaee, F.; Amiri, A. Synthesis,
crystal structure, and electrochemistry of [Co{(Me-sal)2dien}(N3)] and
[Co{(Me-sal)2dpt}(N3)]. J. Coord. Chem. 2009, 62, 734–744.
22. Speir, G.; Csihony, J.; Whalen, A.M.; Pierpont, C.G. Studies on aerobic
reactions of ammonia/3,5-Di-tert-butylcatechol Schiff-Base condensation
products with copper, copper(I), and copper(II). Strong copper(II)−radical
ferromagnetic exchange and observations on a unique N−N coupling reac-
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23. Hathaway, B.J. A new look at the stereochemistry and electronic properties
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