Synthesis, DNA binding, and antimicrobial studies of novel metal complexes containing a pyrazolone derivative Schiff base

Article abstract of DOI:10.2478/s11696-010-0003-0

A novel series of Co(II), Ni(II), Cu(II), Zn(II), and VO(IV) complexes has been synthesized from the Schiff base derived from 4-[(3,4-dimethoxybenzylidene)amino]-1,5-dimethyl-2-phenyl-1,2-dihydropyrazol-3-one and 1,2-diaminobenzene. Structural features we

Full text of DOI:10.2478/s11696-010-0003-0

Chemical Papers 64 (3) 318–328 (2010)
DOI: 10.2478/s11696-010-0003-0
ORIGINAL PAPER
Synthesis, DNA binding, and antimicrobial studies of novel metal
complexes containing a pyrazolone derivative Schiff base
a
a
a
Natarajan Raman*, Ramaraj Jeyamurugan, Mariyyappan Subbulakshmi,
a
b
Raja Boominathan, Chithu Ramakrishnan Yuvarajan
aResearch Department of Chemistry, VHNSN College, Virudhunagar-626 001, India
bDepartment of Chemistry, Sourashtra College, Madurai-625 004, India
Received 21 June 2009; Revised 31 August 2009; Accepted 4 September 2009
A novel series of Co(II), Ni(II), Cu(II), Zn(II), and VO(IV) complexes has been synthesized
from the Schiff base derived from 4-[(3,4-dimethoxybenzylidene)amino]-1,5-dimethyl-2-phenyl-1,2-
dihydropyrazol-3-one and 1,2-diaminobenzene. Structural features were determined by analytical
and spectral techniques. Binding of synthesized complexes with calf thymus DNA (CT DNA) was
studied by spectroscopic methods and viscosity measurements. Experimental results indicate that
the complexes are able to form adducts with DNA and to distort the double helix by changing the
base stacking. Lower DNA affinity of the VO(IV) complex is caused by the change of coordination
geometry by the vanadyl ion resulting in a somewhat unfavorable configuration for the DNA binding.
Oxidative DNA cleavage activities of the complexes were studied with supercoiled (SC) pUC19 DNA
using gel electrophoresis; the mechanism studies revealed that the hydroxyl radical is likely to be
the reactive species responsible for the cleavage of pUC19 DNA by the synthesized complexes. The
in vitro antimicrobial screening effects of the investigated compounds were monitored by the disc
diffusion method. The synthesized Schiff base complexes exhibit higher antimicrobial activity than
the respective free Schiff base.
ꢀ
c 2009 Institute of Chemistry, Slovak Academy of Sciences
Keywords: pyrazolone, Schiff base, complexes, DNA binding, oxidative cleavage, antimicrobial
Introduction
applications in nucleic acid chemistry as DNA probes
of DNA structure in solutions, reagents for the media-
tion of strand scission of duplex DNA under physico-
chemical conditions, and as chemotherapeutic agents
and in the genomic research (Raman et al., 2008; Lu
et al., 2002).
The interaction between DNA and transition metal
complexes is an important fundamental issue in life
sciences. These complexes can bind to DNA in non-
covalent modes such as electrostatic, intercalative, and
groove binding. The above applications require that
the complex binds to DNA through an intercalative
mode wherein the planar aromatic heterocyclic group
is inserted and stacked between the base pairs of DNA
(Raman & Jeyamurugan, 2009), which is related to
the in vivo replication and transcription of DNA, mu-
Coordination metal complexes have been gaining
increasing importance in recent years; particularly in
the design of repository, slow release long acting drugs
in nutrition and in the study of metabolism (Terenzi
et al., 2009). Recent years have witnessed a great
deal of interest in the synthesis and characterization
of transition metal complexes of pyrazolone deriva-
tives. Among these derivatives, 4-aminoantipyrine is
a remarkable reagent due to its importance in biolog-
ical, pharmacological, clinical, and analytical applica-
tions. Earlier works reported that some drugs show in-
creased activity when administrated as metal chelates
rather than as organic compounds. A number of metal
chelates are of current interest due to their important
*Corresponding author, e-mail: drn raman@yahoo.co.in

N. Raman et al./Chemical Papers 64 (3) 318–328 (2010)
319
tation of genes, variations of species in their charac-
ter, and to the action mechanism of some synthetic
chemical nucleases (Lu et al., 2002). Moreover, ox-
idative cleavage of DNA on irradiation with visible
light has gained considerable interest due to its po-
tential application in photodynamic therapy of cancer
tions of CT DNA in the above buffer gave a ratio of
UV absorbance at 260 nm and 280 nm, A260/A280 of
1.87, indicating that the CT DNA was sufficiently free
from protein (Marmur, 1961). The CT DNA concen-
tration per nucleotide was determined by absorption
spectroscopy at 260 nm using the molar absorption
−
1
−1
(Mitsopoulou et al., 2008). Considering all these facts,
coefficient ε260 (6600 M cm ) (Reichmann et al.,
1954).
DNA binding and oxidative cleavage activity of pyra-
zolone derivative transition metal complexes, with in-
teresting physical and spectroscopic properties, have
become our main interest.
Absorption titration experiments were performed
by maintaining constant metal complex concentration
of 25 µM while varying the concentration of CT DNA
within 0 µM to 400 µM. While measuring the absorp-
tion spectra, equal quantity of CT DNA was added to
both the complex solution and the reference solution
to eliminate the absorbance of CT DNA itself. From
the absorption data, the intrinsic binding constant Kb
was determined from a plot of [DNA]/(εa − εf) versus
[DNA] using the equation
Experimental
All reagents and chemicals were procured from
Merck (Darmstadt, Germany). Solvents used for elec-
trochemical and spectroscopic studies were purified
by standard procedures (Perrin et al., 1980). DNA
was purchased from Bangalore Genei (India). Agarose
(molecular biology grade) and ethidium bromide (EB)
[DNA]/(εa−εf) = [DNA]/(εb−εf)+[Kb(εb−εf)]−1(1)
were obtained from Sigma–Aldrich (St. Louis, Mis-
souri, USA). Tris(hydroxymethyl)aminomethane hy-
drochloride (Tris–HCl) buffer solution was prepared
using deionized, sonicated, triply distilled water.
Elemental analyses of the complexes were carried
out on a CHN analyzer Carlo Erba 1108, Heraeus.
where [DNA] is the concentration of CT DNA in base
pairs. The apparent absorption coefficients εa, εf, and
εb correspond to Aobsd/[M] (where M = metal), to the
extinction coefficient for the free M(II) complex and
to the extinction coefficient for the M(II) complex in
the fully bound form, respectively. Kb is given by the
ratio of slope to the intercept.
−
1
Infrared spectra (4000–400 cm , KBr discs) of the
samples were recorded on a Perkin–Elmer 783 se-
ries FTIR spectrophotometer. Electronic absorption
spectra (in DMF at room temperature) in the range
Cyclic voltammetric and differential pulse voltam-
mogram studies were performed on a CHI 620C elec-
trochemical analyzer with a three electrode system of
glassy carbon (GC) as the working electrode, a plat-
inum wire as the auxiliary electrode and Ag/AgCl as
the reference electrode. All voltammetric experiments
were carried out in single-compartment cells of the
volume of 5–15 mL. Solutions were deoxygenated by
purging with N2 prior to the measurements. Increas-
ing amounts of CT DNA were added directly into the
of 200–1100 nm were obtained on a Shimadzu UV-
1
1
601 spectrophotometer. H NMR spectra (300 MHz)
of the ligand and its zinc complex were recorded
on a Bruker Avance DRX 300 FT-NMR spectrome-
ter using CDCl3 and DMSO-d6 as solvents, respec-
tively. Fast atom bombardment mass spectra (FAB-
MS) were obtained in a 3-nitrobenzyl alcohol matrix
using a VGZAB-HS spectrometer. X-band EPR spec-
tra of the complexes were recorded at room temper-
−
3
cell containing the solution (2.5 × 10 M, 5 mM Tris-
HCl/50 mM NaCl buffer, pH = 7.1). The concentra-
tion of CT DNA ranged from 0 µM to 400 µM. The
solution in the cuvette was thoroughly mixed before
each scan. All experiments were carried out at room
temperature.
◦
ature and at −196 C, respectively, using tetracya-
noethylene (TCNE) as the g-marker. Molar conduc-
−
3
tivity of 10 M solutions of the complexes in DMF
were measured at room temperature with a Deepvi-
sion Model-601 digital direct reading deluxe conduc-
tivity meter. Magnetic susceptibility measurements
were carried out by employing the Guoy method at
room temperature on a powdered sample of the com-
plex. CuSO4 · 5H2O was used as the calibrant. Metal
contents of the complexes were determined according
to a literature method (Angelici, 1969). Chloride ion
was determined gravimetrically as silver chloride. Pu-
rity of the ligand and its complexes was evaluated by
column chromatography and thin layer chromatogra-
phy.
Viscosity experiments were carried out on an Ost-
wald viscometer, immersed in a thermostated water-
bath maintained at the constant temperature of (30.0
◦
± 0.1) C. CT DNA samples of approximately 0.5 mM
were prepared by sonication in order to minimize the
complexities arising from CT DNA flexibility (Sathya-
narayana et al., 1993). Flow time was measured with
a digital stopwatch three times for each sample and
the average flow time was calculated. Data were pre-
◦
1/3
sented as (η/η )
versus the concentration of the
All experiments involving the interaction of syn-
thesized complexes with CT DNA were carried out
in a Tris–HCl buffer (50 mM Tris–HCl, pH = 7.2)
containing 5 % of ethanol at room temperature. Solu-
M(II) complexes, where η is the viscosity of the CT
DNA solution in the presence of the complex, and η
is the viscosity of the CT DNA solution in the ab-
sence of the complex. Viscosity values were calculated
◦

3
20
N. Raman et al./Chemical Papers 64 (3) 318–328 (2010)
after correcting the flow time of buffer alone (t0), η =
(t – t0)/t0.
Extent of the cleavage of super coiled (SC) pUC19
DNA (33.3 µM, 0.2 µg) to its nicked circular (NC)
form was determined by agarose gel electrophoresis in
a 50 mM Tris–HCl buffer (pH = 7.2) containing 50
mM NaCl. The gel electrophoresis experiments were
performed by incubation of 30 µM pUC19 DNA, 50
µM of each complex and 50 µM 3-mercaptopropionic
◦
acid (MPA) in the Tris–HCl buffer (pH = 7.2) at 37 C
for 2 h. After the incubation, the samples were elec-
trophorezed for 2 h at 100 V on a 0.8 % agarose gel
using the Tris–acetic acid–EDTA buffer (pH = 7.2).
The gel was then stained using 1 µM ethidium bro-
mide (EB) and photographed under ultraviolet light
at 360 nm.
The synthesized ligand and its complexes were
tested for their in vitro antimicrobial activity against
the Gram-positive bacteria Staphylococcus aureus,
Bacillus subtilis, the Gram-negative bacteria Es-
cherichia coli, Salmonella typhi by the disc diffu-
sion method using agar nutrient as the medium, and
against the fungi Aspergillus niger, Aspergillus flavus,
Candida albicans, and Rhizoctonia bataicola by the
well known diffusion method using potato dextrose
−
2
agar as the medium. The stock solution (10 M) was
prepared by dissolving the compounds in DMSO, the
solutions were then serially diluted in order to find
the minimum inhibitory concentration (MIC) values
(Kannan, 1996). Streptomycin and nystatin were used
as control drugs.
The Schiff base ligand and its Co(II), Ni(II),
Cu(II), Zn(II), and VO(IV) complexes were pre-
pared as follows: an ethanolic solution (20 mL) of 4-
aminoantipyrine (2.03 g, 0.01 mol) was added to an
ethanolic solution (20 mL) of 3,4-dimethoxybenzalde-
hyde (1.06 g, 0.01 mol). On stirring, the solid product
separated. It was filtered, recrystallized from ethanol
and added (2.9 g, 0.01 mol) to an ethanolic solution
Fig. 1. Synthesis of ligand (L). Reaction conditions: i) stirring
at room temperature; ii) 1,2-diaminobenzene, K2CO3,
reflux, 30 h.
solvents but the complexes are soluble only in DMF
and DMSO. The ligand and its complexes were charac-
terized by analytical and spectral techniques. Physical
characterization, microanalytical, and molar conduc-
tivity data of the complexes are given in Table 1. Ana-
lytical data of the complexes correspond well with the
(20 mL) of 1,2-diaminobenzene (0.54 g, 0.005 mol).
After an addition of K2CO3, the mixture was heated
under reflux for about 30 h. Separated solid product
was filtered and recrystallized from ethanol to give the
corresponding Schiff base (ligand L) (Fig. 1). This lig-
and (0.675 g, 0.001 mol), dissolved in hot ethanol (50
mL), was added to a hot ethanolic solution (25 mL)
of metal salts (0.17 g, 0.001 mol) and heated under
reflux for approximately 4 h. The resulting solution
was reduced on a water bath to one third. The pasty
mass obtained was treated with ether and the solid ob-
tained was filtered and washed with ethanol and dried
in vacuo.
general formula [ML]X (where X = Cl2 or SO2 ) ex-
−
4
cept for the cobalt complex whose formula is MLCl2.
Monomeric nature of the complexes was confirmed
from their magnetic susceptibility data. The observed
high molar conductivity of the complexes in DMF at
room temperature is consistent with the electrolytic
nature of the complexes due to counter ions (chlo-
ride/sulfate) in the structure of all the complexes ex-
cept for the cobalt complex which has low molar con-
ductivity indicating its non-electrolytic nature. Ele-
mental analysis results for the metal complexes agree
with the calculated values showing that the complexes
have the metal/ligand ratio of 1 : 1. Presence of the
chloride ion is evident from the Volhard’s test and that
of the sulfate ion from the BaCl2 test.
Results and discussion
The synthesized ligand and its Co(II), Ni(II),
Cu(II), Zn(II), and VO(IV) complexes were found to
be air-stable. The ligand is soluble in common organic

N. Raman et al./Chemical Papers 64 (3) 318–328 (2010)
321
Table 1. Physical characterization, analytical, molar conductance, and magnetic susceptibility data for the ligand and its complexes
wi(calc.) (mass %)
wi(found) (mass %)
Compound
Ligand
Color
Formula
Yield (%)
Molar conductivity
−1
M
C
H
N
(S cm2 mol
)
yellow
black
C46H46N8O4
72
61
48
36
54
49
–
71.3
1.0
5.9
5.7
5.1
5.1
5.1
5.1
5.1
5.1
4.9
5.1
5.1
5.1
14.5
14.1
7.0
–
7
[
[
[
[
[
CuL]Cl2
NiL]Cl2
ZnL]Cl2
VOL]SO4
CoLCl2]
[CuC46H46N8O4]Cl2
[NiC46H46N8O4]Cl2
[ZnC46H46N8O4]Cl2
[VC46H46N8O5]SO4
[CoC46H46N8O4Cl2]
7.8
60.8
60.7
61.1
60.8
60.6
60.3
58.9
58.7
61.1
60.7
129
146
137
128
2.2
7
.6
6.5
.3
7.2
.0
5.4
.1
6.5
.2
6.8
light green
grey
12.4
12.2
12.3
12.1
12.0
11.8
12.4
12.2
6
7
green
5
brown
6
Table 2. Electronic absorption spectral data of the compounds
Absorption (cm 1)
−
Band assignment
µeff (B.M.)
Geometry
Compound
2
B1g → 2A1g
[
[
CuL]Cl2
CoLCl2]
21186
12108
1.69
4.87
Square-planar
Octahedral
4
4
4
T1g (F) → 4T2g (F)
4
1
1
4254
9586
T1g (F) → A2g (F)
4
T1g (F) → T2g (P)
1
1
1
[
[
NiL]Cl2
VO]SO4
22658
7605
19267
3879
A1g → A2g
–
Square-planar
1
1
A1g → B1g
2
2
B2 → A1
1.74
Square-pyramidal
2
2
1
B2 →
E
FAB-MS spectra of the synthesized ligand and its
Schiff base to the metal through the nitrogen atom is
expected to reduce the electron density in the azome-
complexes were recorded and the obtained molecular
ion peaks confirm the proposed formulae. Mass spec-
thine link and to lower the ν(C—N) vibration. All
+
−1
trum of the ligand shows a molecular ion peak (M )
complexes show bands in the 1090–1100 cm
700–750 cm
and
+
−1
at m/z 775 corresponding to [C46H46N8O4] ion. Its
regions which can be assigned to the
+
copper complex shows the M peak at m/z 910 and
phenyl ring vibrations. Assignment of the proposed
coordination sites is further supported by the appear-
another important peak appeared at m/z 838, which
suggests the loss of two chloride ions. These data con-
firm the stoichiometry of the [CuL]Cl2 type. This is
further supported by the mass spectra of all the com-
plexes. The observed peaks are in good agreement
with their empirical formulae as indicated by the mi-
croanalytical data. Thus, the mass spectral data sup-
port the conclusions drawn from the analytical and
high molar conductivity values. The cobalt complex
shows the M+ peak at m/z 905 and the absence of the
peak at m/z 834, which confirms the stoichiometry of
the [CoLCl2] type which is further supported by the
analytical and low molar conductivity values.
−
1
ance of medium bands at 450–500 cm
which can
be attributed to the ν(M—N) vibrations (Firdaus et
al., 2009). The vanadyl complex shows a band at 963
−
1
cm assigned to the ν(V—O) vibration (Raman et
al., 2009).
Electronic spectra serve as a tool to distinguish be-
tween the square-planar, octahedral, and tetrahedral
geometries of the complexes. The absorption regions,
assignment and the proposed geometry of the com-
plexes are given in Table 2. The copper complex is
magnetically normal with a magnetic moment of 1.69
B.M. Electronic absorption of the copper complex in
the DMF solution shows a d–d transition at about
Spectrum of the free Schiff base ligand shows a
−
1
−1
2
2
band of the C—N group in the region of 1635 cm
,
21186 cm which can be assigned as the B1g → A1g
transition, revealing that the Cu(II) complex exists in
the square-planar geometry (Lever, 1968).
which is shifted to lower frequencies in the spectra
−
1
of all complexes (1615–1605 cm ) indicating the in-
volvement of —C—N nitrogen in coordination to the
Electronic spectrum of the cobalt complex exhibits
−
1
metal ion (Priya et al., 2009). Coordination of the
a broad weak band in the regions of 12108 cm
,

3
22
N. Raman et al./Chemical Papers 64 (3) 318–328 (2010)
◦
Fig. 2. EPR spectra of the copper complex at room temperature (a) and at −196 C (b).
−
1
4254 cm 1, and 19586 cm−1, suggesting an octahe-
B.M. which corresponds to one unpaired electron in-
dicating that the complex is mononuclear. This fact is
also evident from the absence of a half field signal ob-
served in the spectrum at 1600 G due to the ms = ± 2
transitions ruling out any Cu–Cu interaction (Raman
et al., 2009).
dral geometry. The room temperature magnetic mo-
ment (4.87 B.M.) determined for the Co(II) complex
is higher than the spin-only magnetic moment (µeff =
3
.87 B.M.) of three unpaired electrons, which is due to
the spin-orbit coupling contribution and indicates high
spin nature (Sharada & Syamal, 1992). The observed
zero magnetic moment confirms the square-planar en-
vironment of the nickel complex in conformity con-
sidering the fact that all known square-planar com-
plexes of nickel are diamagnetic. The Ni(II) complex
In square-planar complexes, the unpaired electron
2
lies in the d
2
2
orbitals giving B1g as the ground
x −y
ꢀ
state with g > g⊥ > 2, while the unpaired electron
lies in the d orbital giving A1g as the ground state
2
2
z
with g⊥ > g > 2. From the observed values (A =
ꢀ
ꢀ
−
1
1
shows two d–d transitions at about 22658 cm ( A1g
147 > A⊥ = 58; g = 2.29 > g⊥ = 2.06 > 2.0023) it is
ꢀ
1
−1
1
1
→
A2g) and 17605 cm ( A1g → B1g) which reveal
clear that the EPR parameters of the complex coincide
well with the related systems, which suggests that the
complex has square-planar geometry and the system is
axially symmetrical (Ray & Kauffman, 1990). This is
also supported by the fact that the unpaired electron
that the nickel complex exists in the square-planar en-
vironment (Lever, 1968). Electronic absorption spec-
trum of the vanadyl complex exhibits two d–d bands
−
1
2
−1
2
at 19267 cm
and 13879 cm
which are assigned
2
2
to the B2 → A1 and B2 → E transitions, respec-
lies predominantly in the d
2
2
orbital. In the axial
x −y
tively. A square-pyramidal geometry is proposed for
spectra, the g-values are related with the exchange in-
−
1
this system because it shows a band at 13879 cm
teraction coupling constant (G) by the expression: G
2
2
(
B2 → E) (Sharada & Syamal, 1992), which was
= g – 2.0023/g⊥ – 2.0023. According to Dudley and
ꢀ
further confirmed by its EPR spectral data.
Hathaway (1970), if the G value is larger than four,
the exchange interaction is negligible because the local
tetragonal axes are parallel or slightly misaligned. If
the value of G is less than four, the exchange interac-
tion is considerable and the local tetragonal axes are
misaligned. For the presented copper complex, the G
value is 4.9 suggesting that the local tetragonal axes
are parallel or slightly misaligned and consistent with
1
H NMR spectra of the ligand show the following
signals: δ = 7.0–7.5 (m, phenyl), δ = 3.0 (NCH3), δ =
.9 (CH—N), δ = 3.8 (OCH3), δ = 2.2 (C—CH3). The
azomethine proton (CH—N) signal in the spectrum of
the zinc complex is shifted downfield compared to the
free ligand, suggesting the deshielding of the azome-
thine group due to its coordination to the metal ion.
There is no appreciable change in the other signals of
the complex.
7
a d
2
2
ground state.
x −y
Covalent bonding parameters for the Cu(II) ion in
X-band EPR spectrum of the complex at room
temperature (Fig. 2a) shows an intense absorption
band in the high field region, which is caused by the
tumbling motion of the molecules. However, this com-
various ligand field environments are usually deter-
mined. The in-plane σ-bonding covalency parameter
2
α is related to A , g and g⊥ according to the follow-
ꢀ
ꢀ
ing equation
◦
plex in the frozen state (−196 C) shows four well re-
2
solved peaks (Fig. 2b) with low intensities in the low
field region and one intense peak in the high field re-
gion. The value of magnetic susceptibility reveals that
the copper complex has a magnetic moment of 1.69
α = (A /P) + (g − 2.0023) +
ꢀ
ꢀ
+ 3/7(g⊥ − 2.0023) + 0.04
(2)
2
If the α value is 0.5, it indicates complete covalent

N. Raman et al./Chemical Papers 64 (3) 318–328 (2010)
323
the complexes have some covalent character. The out-
2
of-plane π bonding (γ ) and in the plane π-bonding
2
(
β ) parameters are calculated from the following ex-
pressions
2
2
β = (g − 2.0023)E/(−8λα )
(3)
(4)
ꢀ
2
2
γ = (g⊥ − 2.0023)E/(−2λα )
−
1
In these equations λ = −828 cm
for the free
−
1
2
metal ion and E = 17271 cm . The observed β
1.29) and α (0.7) values indicate that there is an in-
2
(
teraction in the out-of-plane π-bonding, whereas the
in-plane π-bonding is completely covalent. This is also
confirmed by the orbital reduction factors (Dodwad et
al., 1989) estimated using the relations: K = (g −
ꢀ
ꢀ
2
.0023)(∆E/−8λ) and K⊥ = (g⊥−2.0023)(∆E/−8λ).
Significant information on the nature of bonding in
the Cu(II) complex can be derived from the relative
magnitudes of K and K⊥. In case of pure σ-bonding
ꢀ
K ≈ K⊥ = 0.77, whereas K < K⊥ implies a consid-
ꢀ
ꢀ
erable in-plane π-bonding, while for the out-of-plane
π-bonding K > K⊥ . For the presented complex, the
ꢀ
observed order is K (0.96) > K (0.42) implying a
|
|
ꢀ
greater contribution from the out-of-plane π-bonding
than from the in-plane π-bonding in the metal lig-
and π-bonding. Thus, the EPR study of the copper
complex provided supporting evidence for the optical
results.
EPR spectrum of the vanadyl complex at room
temperature is a typical eight-line pattern, which
shows that a single vanadium atom is present in the
molecule, i.e., it is a monomer. In the frozen solid
state, the spectrum shows two types of resonances: one
set is caused by the parallel features and the other by
the perpendicular features which show an axially sym-
metric anisotropy with a well resolved 16-lines hyper-
fine splitting characteristic of an interaction between
the electron and the vanadium nuclear spin. The ob-
served anisotropic parameter values (g = 1.92, g⊥
ꢀ
=
1.98, A = 174, and A⊥ = 75) indicate that the
ꢀ
unpaired electron is present in the dxy orbital with
the square-pyramidal geometry around the VO(IV)
chelate (Dodwad et al., 1989; Yen, 1969). Based on
the spectral data, structures of the synthesized com-
plexes are given in Figs. 3A–3C.
Absorption spectrum of the copper complex in the
absence and in the presence of CT DNA is shown
in Fig. 4. With increasing CT DNA concentration,
for copper complex, the hypochromism in the band
at 410.5 nm reaches 10.2 % with a blue shift of 4.5
nm. These spectral characteristics obviously suggest
that the copper complex interacts with DNA most
likely through a mode that involves a stacking interac-
tion between the aromatic chromophore and the base
pairs of DNA. After intercalating the base pairs of
DNA, the π* orbital of the intercalated ligand can
couple with the π orbital of the base pairs thus de-
Fig. 3. Structure of complexes: [ML]Cl2 general type (A),
VOL]SO4 (B), [CoLCl2] (C).
[
bonding, while the value of 1.0 suggests complete ionic
2
bonding. The observed value of α (0.7) indicates that

3
24
N. Raman et al./Chemical Papers 64 (3) 318–328 (2010)
Table 3. Absorption spectral properties of synthesized complexes with CT DNA
λmax (nm)
Kb · 10 (dm3 mol
4
−1
)
Complexes
∆λ (nm)
H (%)
Free
Bound
[
[
[
[
[
CuL]Cl2
ZnL]Cl2
CoLCl2]
NiL]Cl2
VOL]Cl2
410.5
402.0
395.0
405.0
404.0
413.0
404.0
392.6
403.3
403.5
2.5
2.0
2.4
2.3
0.5
10.2
8.2
9.8
6.9
0.6
4.4
3.0
3.6
2.9
–
Fig. 4. Changes in the electronic absorption spectrum of
◦
[
CuL]Cl2 in 50 mM Tris-HCl buffer (pH 7.2, 25 C) in
Fig. 5. Cyclic voltammogram of [CuL]Cl2 in the absence
(dashed line) and in the presence (solid lines) of dif-
ferent concentrations of DNA.
the absence (dashed line) and in the presence (solid
lines) of CT DNA.
creasing the π–π* transition energy and resulting in
bathochromism. On the other hand, the coupling π
orbital is partially filled by electrons thus decreasing
the transition probabilities and concomitantly result-
ing in hypochromism. The intrinsic binding constant
Kb can be obtained from the ratio of the slope to the
intercept of the plots of [DNA]/(εa − εf) vs. [DNA].
To compare quantitatively the affinity of the syn-
thesized complexes towards DNA, the intrinsic bind-
ing constants Kb of the synthesized complexes to CT
DNA are shown in Table 3. From these results, very
weak hypochromism and no significant spectral shift
were found after the vanadyl complex was mixed with
DNA which indicates that the vanadyl complex forms
weaker adduct to CT DNA than the other complexes.
The lower DNA affinity of the vanadyl complex might
be explained by the change of the coordination ge-
ometry by vanadyl ion chelation which may result in
a somewhat unfavorable configuration for the DNA
binding.
first redox couple cathodic peak appears at −0.251 V
for Cu(II) → Cu(I) (Epa = −0.893 V, Epc = −0.727
V, ∆Ep = 0.166 V, and E1 = 0.81 V). The ipc/ipa
/2
ratio of this redox couple is less than one. This indi-
cates that reaction of the complex on the glassy car-
bon electrode surface is a quasi-reversible redox pro-
cess. Incremental addition of CT DNA to the com-
plex causes a negative shift in E1/2 and an increase
in ∆Ep. The ipc/ipa values also decrease in the pres-
ence of DNA. The decrease of the anodic and cathodic
peak currents of the complex in the presence of DNA
is due to the decrease in the apparent diffusion coef-
ficient of the Cu(II) complex upon its complexation
with the DNA macromolecule. The second redox cou-
ple, Cu(III) → Cu(II), shows no significant change in
the potential and current intensity in the presence of
CT DNA. These results show that the Cu(II) com-
plex stabilizes the duplex (GC pairs) by intercalation.
Electrochemical parameters of the synthesized Co(II),
Ni(II), Cu(II), and VO(IV) complexes are shown in
Table 4. These results indicate that the synthesized
complexes may stabilize the duplex DNA. In the ab-
sence of CT DNA, the redox couple anodic peak ap-
pears at −0.286 V. Incremental addition of DNA to
the Zn(II) complexes shows a decrease in the current
intensity and a negative shift of the oxidation peak
Cyclic and differential pulse voltammetric tech-
niques are extremely useful in probing the nature and
mode of DNA binding of metal complexes. Typical
cyclic voltammogram of copper complex in the ab-
sence and in the presence of varying amount of [DNA]
is shown in Fig. 5. In the absence of CT DNA, the

N. Raman et al./Chemical Papers 64 (3) 318–328 (2010)
325
Table 4. Electrochemical parameters of the interaction of DNA with Co(II), Ni(II), and Cu(II) complexes
E1/2 (V)
Bound
∆Ep (V)
Bound
Complexes
Redox couple
K[red]/K[oxd]
Ipc/Ipa
Free
Free
[
[
[
CoLCl2]
NiL]Cl2
CuL]Cl2
Co(III)/Co(II)
Ni(II)/Ni(I)
Cu(II)/Cu(I)
0.438
−0.325
0.810
0.407
−0.312
0.795
0.348
0.315
0.166
0.382
0.332
0.194
1.34
0.75
0.84
0.82
0.76
0.91
indicates that the zinc ion stabilizes the duplex (GC
pairs) by intercalation. Hence, for the complex of the
electroactive species (Zn(II)) with DNA, the electro-
chemical reduction reaction can be divided into two
steps
[
Zn(II)-DNA] ↔ Zn(II) + DNA
(6)
(7)
Zn(II) + 2e ↔ Zn◦
−
The dissociation constant (Kd) of the Zn(II)-DNA
complex was obtained using the following equation
Kd
2
2
p
2
2
p
i =
(i
o
− i ) + i
o
− [DNA]
(8)
p
p
[
DNA]
where Kd is the dissociation constant of the com-
plex Zn(II)-DNA, i2
and i represent the reduction
2
o
p p
Fig. 6. Differential pulse voltammogram of [CuL]Cl2 in the ab-
sence (dashed line) and in the presence (solid lines) of
different concentrations of DNA.
current of Zn(II) in the absence and in the presence
of DNA, respectively. Using the above equation, the
dissociation constant was determined. The low value
−
10
of the Zn(II) ion dissociation constant (5.4 × 10
−
1
potential. The resulting changes in the current and
potential demonstrate that there is an interaction be-
tween Zn(II) and DNA.
mol L ) was indispensable for the replication, degra-
dation and translation of the genetic material of all
species.
Differential pulse voltammogram of the copper
complex in the absence and in the presence of varying
amount of [DNA] is shown in Fig. 6. An increase in
the concentration of DNA causes a negative potential
shift along with a significant decrease of the current
intensity. The shift in the potential is related to the
ratio of the binding constants
In addition to the peak potential shift in cyclic
voltammetry, the spectral shift in UV absorption titra-
tion and the dependence of the ionic strength on the
binding constant, viscosity measurements were carried
out to provide further information on the nature of the
interaction between the complex and DNA. A classi-
cal intercalation model demands that the DNA helix
lengthens as base pairs are separated to accommodate
the bound ligand leading to an increase of the DNA
viscosity. In contrast, a partial non-classical intercala-
tion of the ligand could bend (or kink) the DNA he-
lix, reduce its effective length and concomitantly also
its viscosity in order to further elucidate the binding
mode of the present complex; viscosity measurements
were carried out by varying the concentration of the
added complex. The effect of the complexes on the
viscosity of DNA is shown in Fig. 7.
Relative viscosity of DNA increases with the in-
crease in the concentration of the metal complexes,
which is similar to that of the classical intercalators
(Wang et al., 2007). The viscosity results unambigu-
ously show that complexes bind with DNA in the
intercalation mode. The vanadyl complex predomi-
nantly shows surface binding characteristic. This is
oꢁ
− E fo ꢁ = 0.0591 log(K+/K2+)
E
(5)
b
oꢁ
oꢁ
where Eb and Ef are formal potentials of the
Co(III)/Co(II) or Ni(II)/Ni(I) or Cu(II)/Cu(I) or
Zn(II)/Zn(0) complex couples in the bound and in
the free form, respectively. The ratio of the binding
constants (K[red]/K[oxd]) for DNA binding of the syn-
thesized complexes were calculated and found to be
less than one. The above electrochemical experimen-
tal results indicate preferential stabilization of Co(II),
Ni(II), Cu(II), and Zn(II) forms on binding to DNA
over other forms.
Differential pulse voltammogram of the presented
Zn(II) complex shows a negative potential shift along
with a significant decrease of current intensity dur-
ing the addition of increasing amounts of DNA. This

3
26
N. Raman et al./Chemical Papers 64 (3) 318–328 (2010)
Table 6. Minimum inhibitory concentration of the synthesized
compounds against the growth of selected fungi
MIC (mg mL 1)
−
Compound
L
A. niger A. flavus C. albicans R. bataicola
77
42
32
35
22
31
10
68
31
27
39
24
36
8
82
44
39
32
18
42
12
79
51
41
39
27
47
14
[
CuL]Cl2
[
NiL]Cl2
[
ZnL]Cl2
[
[
VOL]Cl2
CoLCl2]
Nystatin
Fig. 7. Effect of [VOL]SO4 (∗), [ZnL]Cl2 (×), [CoLCl2] (ꢀ),
NiL]Cl2 ( ), [CuL]Cl2 (ꢁ) on the viscosity of DNA
relative specific viscosity vs. R = [Complex]/[DNA]).
DNA cleavage of the ligand alone is inactive
in the presence of a reducing agent like the 3-
mercaptopropionic acid (MPA). The results indicate
the importance of the metal in the complex for the ob-
servation of the chemical nuclease activity. Oxidative
cleavage of pUC19 DNA in the presence of an external
reducing agent like MPA (5 mM) was studied by gel
electrophoresis using supercoiled (SC) pUC19 DNA
[
(
8
7
6
5
4
3
2
1
(0.2 µg, 33.3 µM) in 50 mM Tris–HCl/50 mM NaCl
buffer (14 µL, pH = 7.2) and the complexes (50 µM).
From the obtained results, synthesized complexes ex-
hibit significant DNA cleavage activity. Further, the
presence of a smear in the gel diagram indicates the
presence of radical cleavage (Fig. 8).
Fig. 8. Gel electrophoresis diagram showing the cleavage of SC
pUC19 DNA (0.2 µg) by the synthesized complexes (50
µM) in the presence of MPA (5 mM): DNA control
With regard to redox chemistry of the synthe-
sized complexes, a plausible mechanism is proposed
here. The reaction of M(II)-L (M = Cu(II), Co(II),
Ni(II), and Zn(II)) with MPA forms an L-M(I) com-
(lane 1), DNA + MPA (lane 2), DNA + ligand + MPA
(lane 3), DNA + [CuL]Cl2 + MPA (lane 4), DNA +
[
VOL]SO4 (lane 5), DNA + [CoLCl2] + MPA (lane 6),
DNA + [ZnL]Cl2 + MPA (lane 7), DNA + [NiL]Cl2 +
MPA (lane 8).
ꢁ
plex species which abstract C4 -hydrogen atom of the
ꢁ
nucleotide adjacent to the 3 -side of guanine. Elec-
trophoretic behavior of the DNA product treated with
the L-VO(IV) complex in the presence of MPA sug-
Table 5. Minimum inhibitory concentration of the synthesized
ꢁ
ꢁ
compounds against the growth of selected bacteria
gests a 3 ,4 carbon-carbon scission of the deoxyribose
ꢁ
ring initiated by the abstraction of the C4 -hydrogen
MIC (mg mL 1)
−
atom as a trigger reaction. On the other hand, the re-
action between the L-VO(IV) complex and MPA may
form an active L-V(III) complex species.
Compound
L
S. typhi S. aureus B. subtilis E. coli
66
33
35
39
22
34
18
58
37
38
42
19
48
12
61
33
29
30
24
33
10
69
27
22
31
18
28
14
For in vitro antimicrobial activity, the investigated
compounds were tested against some bacteria and
fungi. MIC values of the investigated compounds are
summarized in Tables 5 and 6. A comparative study
of the ligand and its complexes (MIC values) indi-
cates that the complexes exhibit higher antimicrobial
activity than the free ligand. From the MIC values re-
sults that compound [VOL]SO4 is more potent than
the other investigated complexes and the Schiff base.
Such increased activity of the complexes can be ex-
plained on the basis of the Overtone’s concept (An-
janeyulu & Rao, 1986) and the Tweedy’s chelation
theory (Dharmaraj et al., 2001). According to the
Overtone’s concept of cell permeability, the lipid mem-
brane surrounding the cell favors the passage of only
[
CuL]Cl2
[
NiL]Cl2
[
ZnL]Cl2
[
VOL]Cl2
CoLCl2]
Streptomycin
[
not surprising since the VO(IV) complex has a square-
pyramidal geometry and hence, stacking of the com-
plex between the base pairs is difficult. The other com-
plexes being four coordinate with a planar tetraden-
tate ligand are capable of stacking between the base
pairs.

N. Raman et al./Chemical Papers 64 (3) 318–328 (2010)
327
lipid-soluble materials; therefore, liposolubility is an
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