Metallation of ethylenediamine based Schiff base with biologically active Cu(II), Ni(II) and Zn(II) ions: Synthesis, spectroscopic characterization, electrochemical behaviour, DNA binding, photonuclease activity and in vitro antimicrobial efficacy

Article abstract of DOI:10.1016/j.saa.2011.03.017

A new ligand [C₂₈H₂₀N₆O₈] (L₂) has been synthesized by the condensation reaction of 3-hydroxy-4-nitrobenzaldehydenephenylhydrazine (L₁) with diethyloxalate. This ligand L₂ is all

Full text of DOI:10.1016/j.saa.2011.03.017

Spectrochimica Acta Part A 79 (2011) 873–883
Contents lists available at ScienceDirect
Spectrochimica Acta Part A: Molecular and
Biomolecular Spectroscopy
journal homepage: www.elsevier.com/locate/saa
Metallation of ethylenediamine based Schiff base with biologically active Cu(II),
Ni(II) and Zn(II) ions: Synthesis, spectroscopic characterization, electrochemical
behaviour, DNA binding, photonuclease activity and in vitro antimicrobial efficacy
N. Raman∗, A. Selvan, S. Sudharsan
Research Department of Chemistry, VHNSN College, Virudhunagar, Tamil Nadu 626 001, India
a r t i c l e i n f o
a b s t r a c t
Article history:
A
new ligand [C28H20N6O8] (L2) has been synthesized by the condensation reaction of
Received 23 December 2010
Received in revised form 17 February 2011
Accepted 9 March 2011
3-hydroxy-4-nitrobenzaldehydenephenylhydrazine (L1) with diethyloxalate. This ligand
L2 is allowed to react with bis(ethylenediamine)Cu(II)/Ni(II)/Zn(II) complexes. It affords
[(L2)Cu(en)2]Cl2(1)/[(L2)Ni(en)2]Cl2(2)/[(L2)Zn(en)2]Cl2(3) complexes, respectively. These complexes
(1–3) have been characterized by the spectral and analytical techniques. The interaction of these
complexes with calf thymus (CT) DNA is characterized by the absorption spectra which exhibit a slight
red shift with hypochromic effect. Electrochemical analyses and viscosity measurements have also
been carried out to determine the mode of binding. The shift in ꢀEp, E1/2 and Ipc values explores the
interaction of CT DNA with the above metal complexes. The slight increase in the viscosity of CT DNA
indicates that these complexes bind to CT DNA through a partial non-classical intercalative mode.
Cleavage experiments using pBR322 DNA in presence of H2O2 indicate that these complexes behave as
efficient artificial chemical nucleases in the order of 1 > 2 > 3. Moreover, the antibacterial and antifungal
studies reveal that complex 1 is highly active against the bacterial and fungal growth.
Keywords:
Metal complexes
DNA interaction
Photonuclease activity
CT DNA
Antimicrobial activity
© 2011 Elsevier B.V. All rights reserved.
1. Introduction
and DNA can often cause DNA damage in cancer cells result-
ing in cell death. Many studies indicate that the transition metal
The design of metal–drug complexes is of particular interest in
the pharmacological research. Metal combinations with pharma-
ceutical agents are known to improve the activity of the drugs
and decrease their toxicity [1]. Schiff bases are potential anti-
cancer drugs and when administered as their metal complexes, the
anticancer activity of these complexes has been enhanced in com-
parison to the free ligand. Schiff base complexes are considered to
be the most important stereochemical models in transition metal
coordination chemistry due to their preparative accessibility and
structural variety. It has been suggested that the azomethine link-
age in Schiff bases is responsible for the biological activities such
as antitumor, antibacterial, antifungal and herbicidal activities [2].
A more precise understanding of the DNA-binding properties
of metal complexes has come about as a result of numerous
motivating factors and developments, including new therapeutic
approaches, exhaustive studies of nucleic acid conformations and
new tools for nanotechnology [3]. Drug researches suggest that
many anticancer, antiviral and antiseptic agents take action by
binding to DNA because the interaction between small molecules
complexes can interact non-covalently with DNA by intercalation,
groove binding or external electrostatic binding [4].
The development of artificial nucleases is an important aspect
of biotechnology, drug designing and molecular biology. Chem-
ical nucleases can cleave DNA by oxidative, photo-induced and
hydrolytic processes. Transition metal complexes have been exten-
sively studied for their nuclease-like activity using the redox
properties of the metal and dioxygen to produce reactive oxygen
species to promote DNA cleavage yielding direct strand scission
or base modification. However, these oxidative cleaving agents
require an external agent (light, oxidative and/or reductive agent)
to initiate cleavage. The hydrolytic cleavage of DNA requires neither
additives nor photo-induced irradiation but follows a mecha-
nistic pathway to target the phosphodiester bonds linking the
nucleosides, and the cleaved products can be amenable to fur-
ther enzymatic religation. Among several types of transition metal
complexes used as synthetic hydrolases, copper(II) and zinc(II)
complexes are better suited for the hydrolysis of DNA due to the
strong Lewis acid properties of these metal ions.
Bearing these facts in our mind, herein we report the synthe-
sis and characterization of a new Schiff base derived from the
mono-condensation of ethylenediamine with carbonyl compound
having a group of NNNN donor site and its different mononuclear
∗
Corresponding author. Tel.: +91 092451 65958; fax: +91 4562 281338.
E-mail address: drn raman@yahoo.co.in (N. Raman).
1386-1425/$ – see front matter © 2011 Elsevier B.V. All rights reserved.
doi:10.1016/j.saa.2011.03.017

874
N. Raman et al. / Spectrochimica Acta Part A 79 (2011) 873–883
complexes. Here, our aim is to establish a comparative electro-
spectrochemical study on the new Schiff-base mononuclear metal
complexes with NNNN donor sites. Additionally, a comparative
study of the interaction of the complexes 1–3 with DNA has been
employed in order to investigate the potential mechanism of their
biological properties. Moreover, the antibacterial and antifungal
activities of these compounds have been carried out against four
bacteria and four fungi using DMSO as negative control.
To the solution of Schiff base (L1) (5.14 g, 0.02 mol) in EtOH was
added diethyloxalate (1.46 g, 0.01 mol) in 2:1 molar ratio. It was
then refluxed for 1 h and cooled to room temperature and then
conc. HCl (6 mL) was added dropwise with constant stirring. The
resulting mixture was refluxed for 6 h and later the volume of the
solution was reduced to half using rotatory evaporator. It was kept
aside for some time to yield brownish red solid product, which
was filtered and dried in vacuo. General formulae of the ligands are
shown in Scheme 1.
The spectral data of the ligands are given below:
L1: red powder, yield 85%, M.F.: C13H11N3O3, M.Wt: 257.24,
M.Pt: 175 ◦C, Anal. Calcd. (%), C(60.67%), H(4.31%), N(16.37%); found
(%) C(60.62%), H(4.29%), N(16.33%); 1H NMR (300 MHz, CDCl3) ı:
10.81 (s, 1H, OH), 6.86–7.52 (m, Ar–H), 8.14 (s, 1H, N CH), 14.08
(s, 1H, NH); IR(KBr) ꢂ (cm−1): 1593(s) (HC N), 1620(s) (C–NO2),
3075(m) (N–H), 3311(s) (O–H), 1327(w) (C–O), 2885(w) (O–H· · ·N),
1421(s) (Ph–C C), 1533(m) (Ph–C–C), 2981(s) (Ph–C–H).
2. Experimental
2.1. Materials and measurements
Phenylhydrazine,
diethyloxalate
and
3-hydroxy-4-
nitrobenzaldehyde were purchased from Aldrich Chemical
Company. Ethidium bromide (EB), CT-DNA and pBR322 plasmid
DNA were obtained from Sigma. All other chemicals used were of
analytical grade. All the experiments involved in the interaction
of metal complexes with CT DNA were carried out in doubly
distilled water buffer containing 5 mM Tris [tris(hydroxymethyl)-
aminomethane] and 50 mM NaCl, adjusted to pH 7.2 with
hydrochloric acid. Solution of CT DNA in Tris–HCl buffer gave ratio
of UV absorbance of about 1.8–1.9:1 at 260 and 280 nm indicating
that the CT DNA was sufficiently free of protein [5]. The CT DNA
concentration per nucleotide was determined spectrophotometri-
cally by employing an extinction coefficient of 6600 M−1 cm−1 at
260 nm [6].
L2: brownish red powder, yield 72%, M.F.: C28H20N6O8,
M.Wt: 568.50, M.Pt: 210 ◦C, Anal. Calcd. (%), C(59.21%), H(3.57%),
N(14.76%); found (%) C(59.14%), H(3.52%), N(14.78%); 1H NMR
(300 MHz, CDCl3) ı: 10.76 (s, 1H, OH), 6.81–7.58 (m, Ar–H), 8.08
(s, 1H, N CH); IR(KBr) ꢂ (cm−1): 1658(m) (C O), 1595(s) (azome-
thine, HC N), 1622(s) (C–NO2), 3309(s) (O–H), 1332(m) (C–O),
2881(w) (O–H· · ·N), 1431(s) (Ph–C C), 1514(m) (Ph–C–C), 2974(w)
(Ph–C–H); UV–Vis (DMF) [nm (frequency, cm−1) (transition)];
424(23,584) (ILCT), 261(38,314) (ILCT), 248(40,322) (ILCT).
2.3. Synthesis of [Cu(en)2]Cl2, [Ni(en)2]Cl2 and [Zn(en)2]Cl2
UV–Vis spectra were recorded on a Shimadzu Model 1601
UV–Visible Spectrophotometer. IR spectra of the ligand and its
metal complexes were recorded on a Perkin-Elmer FTIR-1605 spec-
trophotometer using KBr discs. 1H NMR spectra were measured
on a Bruker Avance DRX 300 FT-NMR spectrometer with tetram-
ethylsilane (TMS) as the internal standard at room temperature.
The complexes were analyzed for their metal contents, follow-
ing standard procedures [7] after decomposition with a mixture
of conc. HNO3 and HCl, followed by conc. H2SO4. Microanalyses
(C, H and N) were carried out on a Perkin-Elmer 240 elemen-
tal analyzer. Mass spectrometry experiments were performed on
a JEOL-AccuTOF JMS-T100LC mass spectrometer equipped with a
custom-made electrospray interface (ESI). The X-band EPR spectra
of the complexes were recorded at RT (300 K) and LNT (77 K) using
DPPH as the g-marker. X-ray diffraction experiments were carried
out on XPERT-PRO diffractometer system. Copper K␣1 line, with
These compounds were synthesized by the method reported
earlier [8,9].
2.4. Synthesis and characterization of 1–3 complexes
To a solution of the ligand (L2) (0.568 g, 0.001 mol) in EtOH
(50 mL) was added [Cu(en)2]Cl2 (0.254 g, 0.001 mol)/[Ni(en)2]Cl2
(0.249 g, 0.001 mol)/[Zn(en)2]Cl2 (0.256 g, 0.001 mol) separately in
1:1 molar ratio in the same solvent. The resulting mixture was mag-
netically stirred and refluxed for 2 h. The colored precipitate formed
was isolated, washed with hexane and dried in vacuo (Scheme 1).
[(L2)Cu(en)2]Cl2 (1): black powder, yield 65%, M.F.:
[CuC32H32N10O6]Cl2, M.Wt: 787.12, M.Pt: >250 ◦C, Anal. Calcd.
(%), C(48.89%), H(4.13%), N(17.81%), Cu(8.11%); found (%)
C(48.81%), H(4.06%), N(17.78%), Cu(8.04%); IR(KBr) ꢂ (cm−1):
1589(s) (azomethine, HC N), 1323(s) (C–N), 671(s) (CH2),
1621(w) (C–NO2), 3311(s) (O–H), 1323(s) (C–O), 3144(s) (–NH2),
2887(s) (O–H· · ·N), 1448(s) (Ph–C C), 1533(w) (Ph–C–C), 2966(s)
(Ph–C–H); UV–Vis (DMF) [nm (frequency, cm−1) (transition)];
˚
wavelength of 1.5406 A generated with a setting of 30 mA and 40 kV
with the electrodes, was used for diffraction. The slit width setting
was 91 mm. The diffracting angle (2ꢁ) was scanned from 10.0251
to 79.9251 continuously with a rate of 2◦/min. The whole process
took place at a temperature of 25 ◦C. Room temperature magnetic
susceptibility measurements were carried out on a modified Gouy-
type magnetic balance, Hertz SG8-5HJ. The molar conductivity of
the complexes in DMSO solution (10−3 M) was measured using a
conductometer model 601/602. Voltammetric experiments were
performed on a CHI 620C electrochemical analyzer in freshly dis-
tilled DMSO solution.
2
604(16,556) (2B1g → A1g) (SP), 420(23,809), 262(38,167) (ILCT).
[(L2)Ni(en)2]Cl2 (2): light yellow powder, yield 69%, M.F.:
[NiC32H32N10O6]Cl2, M.Wt: 782.26, M.Pt: >250 ◦C, Anal. Calcd. (%),
C(49.19%), H(4.17%), N(17.93%), Ni(7.52%); found (%) C(49.11%),
H(4.14%), N(17.87%), Ni(7.46%); IR(KBr)
ꢂ
(cm−1): 1595(s)
(azomethine, HC N), 1309(w) (C–N), 669(s) (CH2), 1622(s)
(C–NO2), 3311(b) (O–H), 1327(w) (C–O), 3161(m) (–NH2),
2881(w) (O–H· · ·N), 1446(s) (Ph–C C), 1531(m) (Ph–C–C), 2930(w)
(Ph–C–H); UV–Vis (DMF) [nm (frequency, cm−1) (transition)];
698(14,326) (1A1g → B1g) (SP), 610(16,393) (1A1g → A2g) (SP),
1
1
2.2. Preparation of ligands
421(23,752), 259(38,610) (ILCT), 244(40,983) (ILCT).
The ligand L2 was prepared by employing a two step synthetic
method (Scheme 1).
[(L2)Zn(en)2]Cl2 (3): brownish yellow powder, yield 61%,
M.F.: [ZnC32H32N10O6]Cl2, M.Wt: 788.95, M.Pt: >250 ◦C, Anal.
Calcd. (%), C(48.82%), H(4.11%), N(17.82%), Zn(8.29%); found (%)
C(48.71%), H(4.06%), N(17.74%), Zn(8.24%); 1H NMR (300 MHz,
CDCl3) ı: 10.78 (s, 1H, OH), 6.97–7.60 (m, Ar–H), 8.10 (s, 1H,
To
a solution of 3-hydroxy-4-nitrobenzaldehyde (1.67 g,
0.01 mol) in EtOH was added phenylhydrazine (1.08 g, 0.01 mol)
dropwise with constant stirring which immediately precipitated to
give a solid red Schiff base (L1). The product was filtered, washed
with hexane and dried in vacuo.
N
CH), 3.74–3.81 (m, CH2), 5.41–5.54 (t, 2H, NH2); IR(KBr) ꢂ
(cm−1): 1600(s) (azomethine, HC N), 1319(s) (C–N), 671(s) (CH2),

N. Raman et al. / Spectrochimica Acta Part A 79 (2011) 873–883
875
Scheme 1. Synthetic steps for metal complexes.
1643(m) (C–NO2), 3315(s) (O–H), 1319(s) (C–O), 3165(m) (–NH2),
2912(b) (O–H· · ·N), 1477(s) (Ph–C C), 1546(s) (Ph–C–C), 2974(w)
(Ph–C–H); UV–Vis (DMF) [nm (frequency, cm−1) (transition)];
426(23,474), 359(27,855) (ILCT).
The absorption titrations of the complex in buffer were per-
formed using a fixed concentration (25 ␮M) for complex to which
increments of the DNA stock solution were added. Metal–DNA solu-
tions were allowed to incubate for 5 min before the absorption
spectra were recorded. The intrinsic binding constants Kb, based on
the absorption titration, were measured by monitoring the changes
of absorption in the MLCT band with increasing concentration of
DNA using the following Eq. (1),
2.5. Procedure for DNA-binding and photo-activated cleavage
experiments
[DNA]
(εa − εf)
[DNA]
(εb − εf)
1
=
+
(1)
The DNA-binding and photo-activated cleavage experi-
ments were performed at room temperature. Buffer A [5 mM
tris(hydroxymethyl)aminomethane (Tris) hydrochloride, 50 mM
NaCl, pH 7.0] was used for absorption titration and viscosity
measurements. Buffer B (50 mM Tris–HCl, 18 mM NaCl, pH 7.2)
was used for DNA photocleavage experiments.
[Kb(εb − εf)]
where [DNA] is the concentration of DNA in base pairs, the apparent
absorption coefficients εa, εf and εb correspond to Aobs/[complex],
extinction coefficient for the free complex and the extinction coef-
ficient of the complex in the totally bound form, respectively. The

876
N. Raman et al. / Spectrochimica Acta Part A 79 (2011) 873–883
data were fitted to Eq. (1), with a slope equal to 1/(εb − ␧f) and y-
intercept equal to 1/[Kb(εb − εf)] and Kb was obtained from the ratio
of the slope to the intercept.
antibacterial and antifungal activities in DMSO solvent against bac-
terial and fungi species.
Suspensions in sterile peptone water from 24 h cultures of
microorganisms were adjusted to 0.5 McFarland. Muller–Hinton
petri discs of 90 mm were inoculated using these suspensions.
Paper discs (6 mm in diameter) containing 10 ␮L of the substance to
be tested were placed in a circular pattern in each inoculated plate.
DMSO impregnated discs were used as negative controls. Toxicity
tests of the solvent, DMSO, showed that the concentration used in
antibacterial activity assays did not interfere with the growth of the
microorganisms.
Cyclic voltammetric experiments were performed in a single
compartmental cell at 25 ◦C with DMSO. A three electrode con-
figuration was used, comprised of a glassy carbon as working
electrode, Pt wire as auxiliary electrode, and an Ag/AgCl electrode
as reference electrode. All the electrochemical measurements were
carried out in a 10 mL electrolytic cell using 5 mM Tris–HCl/50 mM
NaCl buffer (pH = 7.2) as the supporting electrolyte. Solutions were
deoxygenated by purging with N2 prior to measurements.
The difference between forward and backward peak potentials
could provide a rough evaluation of the degree of the reversibility
of one electron transfer reaction. The decreased extents of the peak
currents observed for all metal(II) complexes upon addition of CT
DNA indicated that the complexes interacted with DNA through
binding mode. The Nernst equation was used to estimate the ratio of
equilibrium constants for the binding of the oxidative and reductive
ions to DNA.
2.6.1. Determination of MIC
The in vitro antimicrobial activity was performed against
Gram-positive bacteria: Salmonella aureus, Bacillus subtilis, Gram-
negative bacteria: Escherichia coli, Pseudomonas aeruginosa and
fungal strains: Rhizoctonia bataticola, Rhizopus stolonifer, Candida
albicans, Aspergillus niger. The standard and test samples were
dissolved in DMSO to give a concentration of 100 ␮g/mL. The min-
imum inhibitory concentration (MIC) was determined by broth
microdilution method [11]. Dilutions of test and standard com-
pounds were prepared in nutrient broth (bacteria) or Sabouraud
dextrose broth (fungi) [12]. The samples were incubated at 37 ◦C
for 24 h (bacteria) and at 25 ◦C for 48 h (fungi), respectively, and the
results were recorded in terms of MIC (the lowest concentration of
test substance which inhibited the growth of microorganisms).
ꢀ
ꢁ
Kred
Kox
Eb◦ − Ef◦ = 0.0591 log
(2)
where Eb◦ and Ef◦ are the formal potentials of the bound and free
complex forms, Kred and Kox are the corresponding binding con-
stants for the binding of reduction and oxidation species to calf
thymus (CT-DNA), respectively. The drop of the voltammetry cur-
rents in the presence of DNA was attributed to slow diffusion of the
metal complex bound to CT DNA indicating the extent of binding
affinity of the complex to DNA.
3. Results and discussion
Viscosity measurements were carried out using an Ubbelodhe
viscometer, immersed in a thermostatic water-bath that main-
tained at a constant temperature at 25.0 0.1 ◦C. The compounds
were titrated into the CT-DNA solution (10 ␮M) present in the vis-
cometer. The flow time of each sample was measured by a digital
stopwatch for three times, and an average one was calculated. Data
are presented as (Á/Á0)1/3 vs. binding ratio, where Á and Á0 are the
viscosity of DNA in the presence and absence of complex, respec-
tively.
3.1. Characterization of ligand and its complexes
Several instrumental techniques like IR, UV–vis. spectroscopy,
magnetic measurement, ESI-MS, 1H NMR and EPR were used to
evaluate the structure of the ligand and its complexes. Microana-
lytical data were in good agreement with the proposed structure.
Experimental section comprises physico-chemical parameters of
the ligand and its complexes.
Cleavage products were analyzed by agarose gel electrophoresis
method [10]. The test samples (25 ␮g) were added to the pBR322
DNA (0.2 ␮g). The samples were incubated for 2 h at 37 ◦C and then
20 ␮L of DNA sample (mixed with bromophenol blue dye at 1:1
ratio) was loaded carefully into the electrophoresis chamber wells
along with standard DNA marker containing TAE buffer (4.84 g Tris
base, pH 8.2, 0.5 M EDTA) and finally loaded on agarose gel and
passed the constant 60 V of electricity for around 45 min. Then the
gel was moved and stained with 10.0 ␮g/mL ethidium bromide for
10–15 min and the bands were observed under UV light.
To test the presence of reactive oxygen species (ROS) gener-
ated during strand scission and possible complex–DNA interaction
sites, various reactive oxygen intermediate scavengers and groove
binders were added to the reaction mixtures. Due corrections were
made for the presence of minor quantity of nicked circular (NC)
form in the original SC DNA sample and for the low affinity of
EB binding to SC compared to NC and linear forms of DNA. The
inhibition reactions were carried out at 365 nm by adding differ-
ent reagents [DMSO (4 ␮L), sodium formate (4 ␮L), KI (4 ␮L), NaN3
(200 ␮M), l-His (100 ␮M), 20 U/mL SOD, distamycin (5 mM) and
EDTA (5 mM)] to DNA prior to the addition of the complex (25 ␮M).
3.1.1. Infrared spectra
The IR spectra of the free ligand and its metal complexes were
carried out in 4000–400 cm−1 range. The main IR characteristic
stretching frequencies of the ligand and its metal complexes along
with their proposed assignments are summarized in experimental
part.
The IR spectra of the metal complexes were similar to each other,
except for some slight shifts and intensity changes of few vibration
peaks caused by different metal(II) ions, indicating that the metal
complexes had similar structure. However, there were some sig-
nificant differences between the metal(II) complexes and the free
ligand upon chelation, as expected. The coordination mode and
sites of ligand to the metal ions were investigated by comparing
the IR spectra of the free ligand with its metal complexes.
The ligand L2 showed a medium intensity band at 3309 cm−1
assigned to –OH group, which remained unaltered suggesting the
noninvolvement of the –OH group in coordination with diethylox-
alate [13]. The characteristic bands at 3120 and 1475 cm−1 due to
ꢂ(NH) stretching and bending vibration, were not observed in the
IR spectrum of ligand L2 indicating the ligand formation through
–NH group of phenylhydrazine.
In the complexes, the characteristic band at 1658 cm−1
attributed to the disappearance of ꢂ(C O) [14] whereas the
appearance of a new strong band at 1589–1600 cm−1 region
indicated the condensation of ꢂ(C O) group with NH2 group of
bis-(ethylenediamine)Cu(II)/Ni(II)/Zn(II) complexes. The formation
of ꢂ(C N) band and the disappearance of the ꢂ(C O) band in the
2.6. Evaluation of antimicrobial activity
Qualitative determination of antimicrobial activity was done
using the disc diffusion method. The biological activities of syn-
thesized Schiff base and its metal complexes were studied for their

N. Raman et al. / Spectrochimica Acta Part A 79 (2011) 873–883
877
3.1.4. 1H NMR spectral studies
complexes commensurated with an effective Schiff’s base conden-
sation. A medium intensity band observed at 3144–3165 cm−1 in
the complexes was assigned to ꢂ(NH2) groups of the other side
of bis(ethylenediamine) moiety suggesting 1:1 condensation reac-
tion [15]. In the IR region, the bands appearing at 452, 462 and
447 cm−1 were assigned to ꢂ(Cu–N), ꢂ(Ni–N) and ꢂ(Zn– N), respec-
tively, which further confirmed the formation of the complexes
[16,11].
1H NMR spectrum of the ligand and its complex 3 were scanned
in the range ı: 0–16 ppm. The ligand L1 displayed the signals at
14.08 and 10.81 ppm attributed to the N–H of hydrazine and the
–OH proton of substituted benzaldehyde [21]. In addition to this, a
set of multiplet observed in the range 6.86–7.52 ppm showed the
presence of aromatic protons. The 1H NMR spectrum of the com-
plex 3 displayed signals at 5.41–5.54 and 3.74–3.81 ppm due to
–NH2 and –CH2 protons, respectively [22]. These signals further
supported the formation of complex by 1:1 condensation reaction.
The comparative study of 1H NMR data of ligand and its zinc
3.1.2. Electronic absorption spectra and magnetic moments of
metal complexes
Elemental analysis, IR and molar conductivity data were used
to prove the stoichiometry and formulation of the complexes. A
square-planar geometry was assumed for all the complexes based
on their magnetic data and spectral (UV/Vis) studies.
complex revealed the ligational behaviour of the ligand. In the 1
H
NMR spectrum of ligand L2, the signals due to hydrazine N–H pro-
ton were absent indicating the condensation between Schiff base
ligand L1 and diethyloxalate. There were no significant changes in
the remaining peaks including their intensities.
Electronic spectra of ligand and its mononuclear copper(II),
nickel(II) and zinc(II) complexes were recorded in the 200–1100 nm
range in DMF solution and their corresponding data are given
in experimental part. In the electronic spectra of the ligand and
its mononuclear metal complexes, the wide range bands were
observed due to either the ␲ → ␲* and n → ␲* of C N chromophore
or charge-transfer transition arising from ␲ electron interactions
between the metal and ligand, which involved either a metal-
to-ligand or ligand-to-metal electron transfer [17]. The electronic
spectra of the free ligand in DMF showed strong absorption bands in
the ultraviolet region (262–424 nm), that could be attributed to the
␲ → ␲* and n → ␲* transitions in the benzene ring or azomethine
(–C N) groups for free ligand. The absorption bands between 262
and 424 nm in free ligand changed a bit in intensity and remained
slightly changed for metal complexes. The absorption shift and
intensity change in the spectra of the metal complexes most likely
originated from the metallation, increased the conjugation and
delocalization of the whole electronic system and resulted in the
energy change of the ␲ → ␲ * and n → ␲* transitions of the conju-
gated chromophore [18].
3.1.5. ESI mass spectral studies
The elemental and analytical data of the copper complex sug-
gested the empirical formula [CuC32H32N10O6]Cl2, which was
further supported by the ESI mass spectrum. The molecular ion
peak at 716 m/z value was observed, corresponding to the mass of
the mononuclear complex. The ESI mass spectrum of L1 Schiff base
showed a molecular ion peak at 257 m/z which is equivalent to its
molecular weight and also exhibited two additional peaks at 258
and 259 m/z corresponding to (M+1) and (M+2) peaks, respectively.
The ESI mass spectrum of L2 exhibited a molecular ion (M+) peak at
568 m/z and an additional peak at 569 m/z corresponding to (M+1)
peak. Apart from these, the spectrum showed few peaks at 166,
255, 256, 402, 403, 236, 333 and 334 m/z which are due to various
fragments of L2. By comparing all the analytical and spectral data of
nickel and zinc complexes, it is evident that these are monomeric
complexes.
3.1.6. EPR studies
The bands observed between 610 and 698 nm can be attributed
to d–d transitions of the metal ions. These values are of particu-
lar importance as they are highly dependent on the geometry of
the molecule. Thus, the smaller values of the wavelength of the
band corresponding to the transitions make a good resemblance
between the geometry of the complex and that of square-planar
complex.
The X-band EPR spectrum of copper(II) complex was recorded in
the solid state at room temperature and also in DMSO at 77 K using
DPPH as the ‘g’ marker (Fig. 1). The solution spectrum of the com-
plex possessed well-resolved g and a broad g region. The four
ꢀ
⊥
peaks in the spectrum were evidently due to the coupling of the
electron spin of the 63Cu nucleus (I = 3/2). The peaks were broad
and had the appearance of ill-resolved triplets. The breadth and
triplet appearance could be attributed to hyperfine splitting by the
nitrogen atom (I = 1) of the ligand. The triplet appearance might
be added as an evidence for nitrogen coordination. Based on the
observation, a distorted square-planar geometry was proposed for
the complex. Various Hamiltonian parameters had been calculated
The electronic spectrum of the complex 1 in DMF revealed a
2
broad band at 604 nm, assigned to 2B1g → A1g transition [19]. It is
characteristic of square-planar environment around the Cu(II) ion.
The complex 2 had diamagnetic behaviour and its electronic
spectrum showed two peaks at 610 and 698 nm ascribed to
1
1
1
1A1g → A2g and A1g → B1g transitions supporting the square-
for this complex (g = 2.22, g = 2.06). Here for the complex 1, g > g
ꢀ
⊥
ꢀ
⊥
planar geometry around Ni(II) ion [19].
suggested a square-planar geometry [23]. For the present copper
complex, gav was calculated using the equation, gav = 1/3(g + 2 g )
The magnetic susceptibility results of transition metal com-
plexes gave an indication of the geometry of the ligands around the
central metal ion. The nickel(II) complex in the present study was
diamagnetic and copper(II) complex was paramagnetic. The mea-
sured magnetic moment value for complex 1 was 1.74 BM which
was very consistent with the expected spin-only magnetic moment
i.e., S = 1/2, a d9 copper(II) system.
ꢀ
⊥
and it was found to be 2.11. The spectrum of the complex had four
hyperfine lines in both parallel and perpendicular regions. A and
ꢀ
A
were calculated to be 200 × 10−4 and 90 × 10−4 cm−1, respec-
⊥
tively. Aav was calculated using the formula Aav = 1/3 (A + 2A ) and
ꢀ
⊥
it was 126.66 × 10−4 cm−1. It has been reported that g value of
ꢀ
copper(II) complex can be used as a measure of the covalent char-
acter of the metal–ligand bond. If the value is more than 2.3, the
metal–ligand bond is essentially an ionic and if the value is less
3.1.3. Molar conductance of metal complexes
With a scope to study the electrolytic nature of the mononu-
clear metal complexes, their molar conductivities were measured
in DMSO at 10−3 M solution. Molar conductance of the complexes
was found in the range 78.32–91.41 ꢃ−1 cm2 mol−1, suggesting the
electrolytic nature of the complexes [20]. The two chloride ions
were found to present in the outside of the coordination sphere
which was confirmed by Volhard’s test.
than 2.3, it is an indicative of covalent character [24]. Here the g
value, 2.22 confirmed the covalent nature of copper complex in
conformity with the presence of copper–nitrogen bonds in these
ꢀ
complexes. Moreover, the trend g > g > ge observed for the com-
ꢀ
⊥
plex indicated the unpaired electron in the dx2–y2 orbital of the
Cu(II) ion, a characteristic of axially symmetric complex [25]. The
empirical ratio g /A was frequently used to evaluate distortions in
ꢀ
ꢀ

878
N. Raman et al. / Spectrochimica Acta Part A 79 (2011) 873–883
Fig. 2. Powder X-ray diffraction pattern of complex 2.
is evaluated by the expression:
ꢀ
ꢁ
ꢀ ꢁ
A
3
7
ꢀ
2
˛
= −
+ (g − 2.0027) +
(g⊥ − 2.0027) + 0.04
ꢀ
0.036
E
ˇ2 = (g − 2.0027)
ꢀ
−8ꢄ˛2
where ꢄ = − 828 cm−1 for the free copper ion and E is the electronic
transition energy. The covalency of the in-plane ␴-bonding, ˛2 = 1
indicates the complete ionic character, whereas ˛2 = 0.5 denotes
100% covalent bonding. The ˛2 value for the copper complex 1 was
0.83, supporting the covalent nature. The ˇ2 parameter gave an
indication of the covalency of the in-plane ␲-bonding. The smaller
the ˇ2 (0.64), the larger is the covalency of the bonding. For the
copper(II) complex, the low value of ˇ2 compared to ˛2 indicated
that the in-plane ␲-bonding was more covalent than the in-plane
␴-bonding. Hathaway [29] pointed out that for pure ␴-bonding,
K ≈ K = 0.77 and for in-plane ␲-bonding K < K , while for out-
ꢀ
⊥
ꢀ
⊥
of-plane ␲-bonding, K < K . The following simplified expressions
⊥
ꢀ
were used to calculate K and K :
ꢀ
⊥
K = (g − 2.0027) d–d transition/8ꢄ
ꢀ
⊥
ꢀ
0
K
= (g⊥ − 2.0027) d–d transition/2ꢄ
0
The observed K (0.5431) < K (0.5728) relation indicated the
ꢀ
⊥
presence of significant in-plane ␲-bonding. Thus, the EPR study of
the copper(II) complex has provided supportive evidence to the
conclusion obtained on the basis of electronic spectrum and mag-
netic moment value.
Fig. 1. EPR spectra of complex 1 at (A) RT (300 K) and (B) LNT (77 K).
tetra coordinated copper(II) complexes [26]. The ratio close to 100,
indicated a roughly square-planar structure around the copper(II)
ion [27] and the values from 170 to 250 indicated a distorted tetra-
3.1.7. X-ray powder diffraction
Single crystal X-ray crystallographic investigation is the most
precise source of information regarding the structure of the com-
plexes, but the difficulty of obtaining crystalline complexes in
proper symmetric form has rendered the powder X-ray diffraction
method for such study. Powder XRD pattern of the Schiff base com-
plexes were recorded over the 2ꢁ = 0–80 range and complex 2 are
shown in Fig. 2. The trend of the curves decreases from maximum to
minimum intensity indicating that these complexes are amorphous
in nature in the present metal–ligand formation.
hedral geometry. Here, the value of the g /A ratio for complex 1
ꢀ
ꢀ
(1 1 1) nearly indicated the square-planar environment around the
Cu(II) ion with small distortions.
In axial symmetry, the g-values were related to the G-factor by
the expression, G = (g − 2)/(g⊥ − 2), which measured the exchange
ꢀ
interaction between copper centers in the solid. According to Hath-
away and Billing [28], if the value of G is greater than 4, the exchange
interaction between copper(II) centers in the solid state is neg-
ligible, whereas when it is less than 4, a considerable exchange
interaction exists in the solid complex. The geometric parameter G
for complex 1 was 3.79 which is less than 4, supporting that a less
exchange interaction is present in the copper complex.
The diffractogram of 2 consists of 10 reflections with maxima
◦
˚
at 2ꢁ = 25.695 corresponding to value of d = 3.4641 A (Table 1). The
main peaks of 2 had been indexed using computer software by trial
and error method, keeping in mind characteristics of various sym-
metry systems until good fit could be obtained between observed
and calculated 2ꢁ and sin2 ꢁ values. The method also yielded hkl
(Miller indices) values. The relative intensities corresponding to
prominent peaks had also been measured. The X-ray data revealed
The parameters g , g , A and A of complex and the energies
ꢀ
⊥
ꢀ
⊥
of the d–d transitions were used to calculate the orbital reduction
parameters (K , K ) and the bonding parameters (˛2 and ˇ2). The
ꢀ
⊥
factors ˛2 and ˇ2 are usually taken as measure of covalency which

N. Raman et al. / Spectrochimica Acta Part A 79 (2011) 873–883
879
Table 1
X-ray diffraction data of complex 2.
2ꢁ (Cal.)
2ꢁ (Obser.)
Sin2 ꢁ (Cal.)
Sin2 ꢁ (Obser.)
d (Å) (Cal.)
d (Å) (Obser.)
h k l
Intensity (%)
15.445
16.079
19.675
25.695
28.805
29.861
32.883
37.366
39.905
50.994
16.797
16.332
19.796
25.853
28.721
29.472
32.978
37.249
39.600
50.444
0.0180
0.0195
0.0291
0.0494
0.0618
0.0663
0.0801
0.1026
0.1164
0.1852
0.0188
0.0201
0.0295
0.0500
0.0615
0.0647
0.0805
0.1019
0.1147
0.1815
5.7321
5.5076
4.5084
3.4641
3.0968
2.9896
2.7237
2.4066
2.2592
1.7894
5.6053
5.4230
4.4813
3.4435
3.1057
3.0283
2.7139
2.4119
2.2740
1.8076
1 1 0
0 0 1
1 1 1
0 2 0
3 1 1
2 0 1
1 1 2
4 0 0
2 2 1
0 0 3
64.76
16.28
20.62
100.00
14.06
14.96
8.08
4.36
4.58
4.30
that the complex 2 had crystallized in the face-centered mono-
clinic system. The azomethine of 2 exhibited the main reflection at
◦
˚
2ꢁ = 25.695 (d spacing 3.4641 A).
3.1.8. Electrochemistry
Electrochemistry of these newly synthesized Schiff base metal
complexes 1–3 was studied by cyclic voltammetry in the scan rate
of 0.080 V s−1 in DMSO solution containing 0.1 M TBAP supporting
electrolyte and redox potentials were expressed with reference to
Ag/AgCl. All the measurements were carried out in the potential
range +1.5 to −1.5 V and are listed in Table 2.
Fig. 3. Cleavage of pBR322 DNA by complex 1 in the presence of peroxide. DNA
(0.2 ␮g) incubated with 1 for 90 min in Tris buffer (pH 7.2). Lane 1: DNA control; lane
2: DNA + 1 (25 ␮M) alone; lane 3: DNA + peroxide (200 ␮M) alone; lane 4: DNA + 1
(25 ␮M) + peroxide (300 ␮M); lane 5: DNA + 1 (25 ␮M) + peroxide (400 ␮M); lane 6:
DNA + 1 (25 ␮M) + peroxide (400 ␮M) + ethanol (15 ␮M).
The electrochemical potentials of these complexes were char-
acterized by well-defined waves in cathodic and anodic regions.
Since the ligands used in this work were not reversibly oxidized or
reduced in the applied potential range, the redox processes were
assigned to the metal centers only.
because the electron-withdrawing anion made the complex more
positively charged and favored the reduction of metal ion. Similarly
the electron-donating groups made the complexes less positively
charged.
The ligand showed an irreversible oxidation at 0.056 V (Epa)
and an irreversible reduction at −1.008 V (Epc) vs. Ag/AgCl in
the scan rate of 0.08 V s−1. Complex 2 exhibited one anodic
wave at Epa = −0.081 V with corresponding cathodic wave at
Epc = −0.808 V. The processes of 2 were quasi-reversible with the
corresponding anodic–cathodic peak separations (ꢀE) for the cou-
ple Ni(II)/Ni(I) and half-wave potentials (E1/2) were calculated as
the average of the anodic and cathodic peak potentials of the pro-
cesses, which are given in Table 2.
Attempting to elucidate correlations between structural and
redox characteristics, the redox properties of the Cu(II)/Cu(I) cou-
ple have been extensively studied and a number of factors which
affect Cu(II)/Cu(I) redox potential have already been identified. The
copper(II) complex 1 showed a quasi-reversible Cu(II)/Cu(I) couple
at formal potential E1/2 = − 0.672 V. This couple can be attributed to
the reduction occurring at the Cu(II) center in the imine nitrogen
site of Schiff base ligand and the strong ␴-donor tendency of the
ethylenediamine moiety [30].
For complex 1, a wave (Epc = − 0.946 V) corresponding to the
reduction of Cu(II) to Cu(I) was obtained. During the reverse
scan, the oxidation of Cu(I) to Cu(II) occurred at the poten-
tial of Epa = − 0.399 V. The values of the limiting peak-to-peak
separation (ꢀEp = −0.547 V) revealed that this process could be
quasi-reversible. Therefore, it was inferred that both the Cu(II) com-
plexes underwent reduction to their respective Cu(I) complexes
which subsequently underwent disproportionation to Cu(0) and
Cu(II). The oxidative responses around 0.596 V might be due to the
Cu(II)/Cu(III) couple.
3.2. Biological evaluation
3.2.1. DNA unwinding studies
3.2.1.1. Hydrolytic cleavage of pBR322 DNA. Copper(II) complexes
are able to cleave DNA through both oxidative and hydrolytic pro-
cesses. The possibility of a hydrolytic mechanism for the DNA
cleavage by the complex 1 must be taken into account.
Fig. 3 shows the electrophoretic pattern of plasmid DNA treated
with complex 1. Control experiments suggested that untreated
DNA and DNA incubated with either complex or peroxide did not
show any significant DNA cleavage (lanes 1–3). However, in the
presence of peroxide, complex 1 was found to exhibit nuclease
activity. In the presence of 25 ␮M of complex 1 and 300 ␮M of per-
oxide, the plasmid DNA was nicked as evident from the formation
of Form II and the disappearance of the supercoiled form in the elec-
trophoretic experiment (lane 4). At the same concentration of the
complex and a slightly higher concentration of peroxide (400 ␮M),
the cleavage was found to be much efficient as seen from the forma-
tion of linear form (Form III) in lane 5. Thus, the nuclease activity of
the complex 1 could be ascribed to the cooperative effect between
Cu(II) ion and L2 ligand. Nuclease activity exhibited by certain cop-
per(II) complex in the presence of hydrogen peroxide indicated the
participation of hydroxyl radical in DNA cleavage. The addition of
ethanol to the reaction mixture before electrophoresis was found
to suppress the DNA cleaving ability of the complexes (lane 6). It
conclusively showed the involvement of the hydroxyl radical in the
observed nuclease activity of complex 1 in the presence of peroxide.
The cyclic voltammogram for 3 recorded at a scan rate of
0.080 V s−1 displayed a reduction peak at Epc = −0.781 V with no
oxidation peak. The reduction wave could be assigned to the reduc-
tion process of Zn(II) to Zn(I).
It was also observed that the ligand moiety had a signifi-
cant effect on E1/2 for all the complexes; electronwithdrawing
groups stabilized the metal(II) in the complexes while the electron-
donating group favored oxidation to metal(III). It was probable,
3.2.1.2. Photo-activated cleavage of pBR322 DNA by complexes. The
cleavage of plasmid DNA could be monitored by agarose-gel
electrophoresis. When circular plasmid DNA was subjected to elec-
trophoresis, relatively fast migration was generally observed for the
intact supercoiled Form I. When scission occurred on one strand

880
N. Raman et al. / Spectrochimica Acta Part A 79 (2011) 873–883
Table 2
Redox couples of the complexes, their potentials and the shift of the potentials in the absence and presence of CT-DNA.
Complex
Redox couple
Ipc (A) × 10−5
Epc (V)
Free
E1/2 (V)
ꢀEp (V)
Kox/Kred
Free
Bound
Bound
Free
Bound
Free
Bound
1
Cu(II)/Cu(I)
Cu(I)/Cu(0)
Ni(II)/Ni(I)
Zn(II)/Zn(I)
2.43
4.05
4.28
1.17
1.88
3.30
4.07
0.99
−0.946
−1.215
−0.808
−0.781
−0.927
−1.193
−0.791
−0.832
−0.672
−0.666
0.547
–
0.727
–
0.522
–
0.645
–
2.09
2.35
1.93
0.13
–
–
2
3
−0.444
−0.468
–
–
scavengers (DMSO, sodium formate, KI), singlet oxygen quenchers
(l-histidine, NaN3), superoxide scavenger (superoxide dismutase
enzyme SOD), minor groove binder (distamycin) and chelating
agent (EDTA) under our experimental conditions.
The hydroxyl radical scavengers DMSO, sodium formate and KI
(lanes 2–4) diminished the DNA breakdown of complex 1, which
indicated that the hydroxyl radical was participated in the oxidative
cleavage. Sodium azide and l-histidine (lanes 5 and 6) had no signif-
icant effect on the DNA cleavage. This fact ruled out the involvement
of the participation of 1O2 or singlet oxygen-like entities in the reac-
tion. The significant reduction in the ability of complex 1 to cleave
DNA in the presence−of the superoxide- dismutase enzyme (lane
7) suggested that O2 was one of the reactive species that actu-
ally broke the DNA. No apparent inhibition of DNA damage was
observed in the presence of the minor groove binder distamycin
(lane 8), suggesting the lack of interaction of complex 1 through the
DNA minor groove. The EDTA, a copper(II)-specific chelating agent
that strongly bound to copper(II) forming a stable complex, could
efficiently inhibit DNA cleavage, indicating copper(II) complexes
played the key role in the cleavage.
Fig. 4. Gel electrophoresis diagram showing the cleavage of pBR322 DNA (0.2 ␮g)
at different complex concentrations in Tris–HCl/NaCl buffer (pH = 7.2) at 37 ◦C on
irradiation with UV light for 1.5 h. Photograph showing the effects of transition metal
complexes on DNA, lane 1: DNA control; lanes 2–5: DNA + 1 (10, 20, 30, 40 ␮M),
respectively; lanes 6–9: DNA + 2 (10, 20, 30, 40 ␮M), respectively.
(nicking), the supercoil relaxed to generate a slower-moving, open
circular Form II. When both strands were cleaved, a linear Form III
was generated, migrating between Form I and Form II DNA [31].
In Fig. 4, the gel electrophoresis pattern of pBR322 DNA is shown
after incubation with complex 1 or 2 and irradiation at 365 nm.
No DNA cleavage was observed for negative control (lane 1). With
increasing concentration of the copper(II) complex 1 (lanes 2–5)
and nickel(II) complex 2 (lanes 6–9), the amount of Form I of
pBR322 DNA diminished gradually, whereas Form II increased and
Form III was also produced. Whereas for complex 1, at the con-
centration of 40 ␮M, significant amounts of linear DNA (Form III)
were visible, which indicated that, under comparable experimen-
tal conditions, complex 1 exhibited more effective DNA cleavage
activity than complex 2. These different cleavage efficiencies par-
alleled the observed DNA-binding affinities of the two complexes,
as was reported in other cases [32].
3.2.1.3. Viscosity studies. To further clarify the binding mode
between the complexes and DNA, viscosity measurements were
carried out on CT-DNA by varying the concentration of the com-
plexes. Intercalation of the complex to DNA is known to cause
a significant increase in the viscosity of DNA solution due to an
increase in the separation of the base pairs at the intercalation site
and, hence, an increase in the overall DNA molecular length. In
contrast, a partial and/or non-classical intercalation could bind (or
kink) the DNA helix and concomitantly reduce its effective length
and viscosity.
The involvement of reactive oxygen species (ROS) such as
hydroxyl, superoxide, singlet oxygen-like species and hydrogen
peroxide in the nuclease mechanism was determined by mon-
itoring the quenching of the DNA cleavage in the presence of
ROS scavengers which are shown in Fig. 5. Control experiments
suggested that untreated DNA did not show any cleavage in the
dark and even upon irradiation by light. In order to observe the
reactive species that were responsible for the DNA damage, we
investigated the DNA cleavage in the presence of hydroxyl radical
The effect of each investigated complex on the viscosity of CT-
DNA solution was studied in order to assess the binding mode
and strength of these complexes with DNA. Representative plots
of Á/Á0 vs. [complex]/[DNA] are shown in Fig. 6 for the investigated
complexes. With an increasing amount of complex 3, the relative
viscosity of DNA decreased and then increased gradually while the
increasing amount of complex 1 and 2 further reduced the effec-
tive length of DNA supporting that both the complex 1 and 2 bound
through partial non classical intercalation mode but with different
affinity. However, complex 1 showed strong binding in comparison
to complex 3 presumably due to electrostatic interaction with DNA
[33]. The effect on the CT-DNA shown in Fig. 6 revealed that the
relative viscosity of DNA increased steadily in the following order,
1 > 2 > 3 with an increasing amount of the above compounds.
Several factors which include the shape and hydrophobicity of
the complex as well as extension of planarity and the presence of
additional donor functionalities on the ligand have been cited to be
an importance in rendering these complexes to be strong intercala-
tive, electrostatic binding agents. It should be noted here that each
ethylenediamine-based complex investigated in the present study
also exhibited spectral, electrochemical and viscosity changes in
the presence of DNA that were analogous to those exhibited by
the complexes mentioned above. The increased degree of viscosity
may depend on the affinity of the compounds to DNA, which is also
consistent with our foregoing hypothesis.
Fig. 5. Gel electrophoresis diagram showing the cleavage of SC pBR322 DNA (0.2 ␮g)
by the complex 1 (25 ␮M) in Tris–HCl/NaCl buffer (50 mM, pH 7.2) in the presence of
various reagents on photo-irradiation with UV light for 1 h: lane 1: DNA control; lane
2: DNA + DMSO (4 ␮L) + 1; lane 3: DNA + sodium formate (4 ␮L) + 1; lane 4: DNA + KI
(4 ␮L) + 1; lane 5: DNA + NaN3 (200 ␮M) + 1; lane 6: DNA + l-His (100 ␮M) + 1; lane
7: DNA + 20 U/mL SOD + 1; lane 8: DNA + distamycin (5 mM) + 1; lane 9: DNA + EDTA
(5 mM) + 1.

N. Raman et al. / Spectrochimica Acta Part A 79 (2011) 873–883
881
Fig. 6. Effect of increasing amount of EB (*), [Ru(bpy)3]2+ (᭹) and in presence of
increasing concentration of complexes of 1 (ꢀ), 2 (ꢀ) and 3 (ꢁ) on the relative
viscosity of CT-DNA at 30 ◦C. [DNA] = 10 ␮M, R = [complex]/[DNA] or [EB]/[DNA].
Fig. 7. Absorption spectra of complex 1 in presence of DNA in Tris–HCl buffer upon
addition of CT DNA. [complex] = 25 ␮M. Arrow shows the absorbance changing upon
the increase of DNA concentration.
3.2.2. Interactions with DNA
The interactions of metal complexes with DNA have been the
subject of interest for the development of effective chemothera-
peutic agents. Transition metal centers are particularly attractive
moieties for such research since they exhibit well-defined coordi-
nation geometries and also possess distinctive electrochemical or
photophysical properties, thus enhancing the functionality of the
binding agent. The interaction of the complexes with CT DNA has
been studied with UV spectroscopy and CV in order to investigate
the possible binding mode to CT DNA and to calculate the binding
constants (Kb) [34].
sition (LMCT), and the other in the region 240–262 nm which was
assigned to the intraligand (␲–␲*) transition of the aromatic chro-
mophore. With increasing CT DNA, the absorption bands of the
complexes were affected, resulting in the obvious tendency of
hypochromism and slight shifts to longer wavelengths, which indi-
cated that the four N-containing 1–3 complexes could interact with
CT DNA. The results showed that upon addition of DNA to complex
1, more binding activity was observed than that of 2 and 3. The
hypochromism observed were 13.60% for 1, 12.31% for 2 and 14.67%
for 3, respectively. The [DNA]/(εa − εf) ratio was plotted against
[DNA] and intrinsic constants Kb were obtained from the ratio of
the slope to the intercept. The Kb values obtained for complexes 1,
3.2.2.1. Absorption spectral features in DNA binding. The absorption
spectra are the most common means to examine the interaction
between metal complex and DNA. In general, the absorption spec-
tra of metal complexes bound to DNA through intercalation exhibit
significant hypochromism and red shift due to the strong ␲–␲
stacking interaction between the aromatic chromophore ligand of
metal complex and the base pairs of DNA [35].
2 and 3 were 9.37 × 105 M−1, 3.16 × 105 M−1 and 4.59 × 105 M−1
,
respectively. The significant difference in DNA-binding affinity of
the three metal(II) complexes could be understood as a result of
the fact that the complex with higher numbers of metal(II) chelates
showed stronger binding affinity with DNA. Our results are consis-
tent with earlier reports on preferential binding to CT DNA in the
metal complexes [38].
When the L2 ligand of the ethylenediamine based metal(II) com-
plexes intercalated into the base pairs of DNA, its ␲* orbital was
coupled with the ␲ orbital of DNA base-pairs to give rise to the
decrease in the ␲–␲* transition energies. As a result, the ꢄmax of
the intra ligand transition of the L2 ligand was shifted to the longer
wavelength (red shift). On the other hand, the coupled ␲* orbital
of the L2 ligand of the ethylenediamine based metal(II) complexes
were also partially occupied by electrons due to overlapping with
the ␲ orbital of the DNA base pairs (effect of charge transfer) to
decrease the transition probabilities. Consequently, the transition
intensity was weakened, resulting in hypochromic effect [36]. The
hypochromic effect, characteristic of intercalation has been usu-
ally attributed to the interaction between the electronic states of
the compound chromophores and those of the DNA bases [37].
The binding of complexes 1–3 to DNA helix have been character-
ized through absorption spectral titrations, by following changes in
absorbance and shift in wavelength. In Fig. 7, the absorption spec-
tra of the metal(II) complex 1 (at a constant concentration) were
shown in the absence and presence of CT-DNA. The absorption
spectra of the metal(II) complex 2 (at a constant concentration)
in the absence and presence of CT-DNA were included in the
supplementary material (Fig. S1). In the UV region, the 1–3 com-
plexes exhibited two intense absorption bands: one at 400–425 nm
which was attributed to the ligand-to-metal charge transfer tran-
3.2.2.2. Effect of electrode potential on metal complex and CT-DNA
interactions. The electrochemical investigations of metal–DNA
interactions could also provide a useful supplement to spec-
troscopic methods and yield information about interactions
(electrostatic or intercalative) with both the reduced and oxidized
form of the metal. The difference between voltammetric responses
of 1 in the absence and presence of CT-DNA are depicted in Fig. 8
whereas the difference between voltammetric responses of 3 in the
absence and presence of CT-DNA is included in the supplementary
file (Fig. S2). The significant shift in peak potentials and current
ratios of 1–3 were observed upon addition of CT-DNA. The sum-
mary of voltammetric results for 1–3 in the absence and presence of
CT-DNA is given in Table 2. No new redox peaks had appeared after
the addition of CT DNA to each complex, but the current intensity
decreased significantly, suggesting the existence of an interaction
between each complex and CT DNA, explained in terms of an equi-
librium mixture of free and DNA-bound complex on the electrode
surface [39].
The electrochemical investigations of metal–DNA interactions
are a useful complement to spectroscopic methods and can provide
information about interactions with both the reduced and oxidized

882
N. Raman et al. / Spectrochimica Acta Part A 79 (2011) 873–883
Table 3
Antimicrobial activity, expressed in MIC (␮g/mL), of complexes and ligands on the positive and negative bacteria and fungal strains.
Complex
Antibacterial activity
Antifungal activity
S. aureus
B. subtilis
E. coli
P. aeruginosa
A. niger
R. stolonifer
C. albicans
R. bataicola
L1
L2
1
2
19.27
18.96
5.37
7.64
6.38
1.98
–
19.34
17.34
6.48
8.71
5.72
2.32
–
17.08
15.24
4.41
7.82
6.37
1.04
–
18.21
16.57
5.58
6.18
5.27
1.16
–
21.06
16.27
7.19
7.28
7.11
–
18.32
18.17
5.26
5.37
6.31
–
17.95
16.22
4.97
6.37
5.14
–
18.28
18.31
6.23
8.46
7.56
–
3
Streptomycin
Nystatin
1.82
2.35
2.18
1.96
form of the metal. The electrochemical potential of a small molecule
will shift positively when it intercalates into DNA double helix,
and, if it is bound to DNA by electrostatic interaction, the potential
will shift to a negative direction. Additionally, if more potentials
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 [40].
3.2.3. Antimicrobial studies
From the in vitro antimicrobial screening results (Table 3 and
supplementary Fig. S3), it was observed that the ligand was mod-
erately active against the bacteria and fungi used. All the complexes
exhibited higher activity in comparison with the ligand against
both the bacteria and fungi. The remarkable activity of the Schiff
base metal complexes may be arise from the hydroxyl groups,
which may play an important role in the antibacterial activity, as
well as the presence of imine groups which import in elucidating
the mechanism of transformation reaction in biological systems.
Among the complexes, the complex 1 exhibited better activity than
2 and 3 against all the microorganisms used. On the other hand,
the electron-withdrawing substituent such as nitro group on the
benzene ring exhibited a comparable growth-inhibitory activity
against microorganisms. Our results showed that the tested com-
pounds exhibited a specific antimicrobial activity, both concerning
the microbial spectrum and the MIC value.
The comparative screening of the antimicrobial properties of
complexes 1–3 exhibited an improved microbicidal effect as com-
pared with the ligands. In case of E. coli compound 1 emerged as
effective antibacterial agent having MIC value 4.41 ␮g/mL. In case
of P. aeruginosa, compounds 1 and 3 had shown good antibacterial
capability at MIC values 5.58 ␮g/mL and 5.27 ␮g/mL, respectively,
and in case of B. subtilis compound 3 had shown good antibacterial
capability at a MIC value 5.72 ␮g/mL. For antifungal activity against
C. albicans, compounds 1 and 3 showed marked antifungal poten-
tial having MIC values 4.97 ␮g/mL and 5.14 ␮g/mL, respectively, as
compared to other synthesized complexes. In case of R. stolonifer,
compounds 1 and 2 were found to be most active with MIC val-
ues 5.26 ␮g/mL and 5.37 ␮g/mL, respectively. However, it was to
be mentioned that zinc(II) complex exhibited good antimicrobial
activity, followed by copper species. The reason for this higher
antimicrobial efficacy could be related to the inhibition by several
structural enzymes, which play a key role in vital metabolic path-
ways of the microorganisms. It was demonstrated that the above
metal complexes showed a higher effect on E. coli and P. aeruginosa
than on S. aureus and this difference could be due to the Gram-
status. It is known that the membrane of Gram-negative bacteria is
surrounded by an outer membrane containing lipopolysaccharides.
The newly synthesized Schiff bases seem to be able to combine with
the lipophilic layer in order to enhance the membrane permeabil-
ity of the Gram-negative bacteria. The lipid membrane surrounding
the cell favors the passage of only lipid soluble materials; thus the
lipophilicity is an important factor that controls the antimicrobial
activity. Moreover, the increase in lipophilicity enhances the pen-
etration of Schiff bases into the lipid membranes and thus restricts
further growth of the organism. This could be explained by the
charge transfer interaction between the Schiff base molecules and
the lipopolysaccharide molecules which lead to the loss of perme-
ability barrier activity of the membrane.
Complexes
1 and 2 exhibited the same electrochemical
behaviour upon addition of CT DNA with a positive shift for the
cathodic potential Epc (ꢀEpc = +0.019 V and +0.017 V for 1 and
2, respectively) (Table 2) and the anodic potential Epa shifting
to negative values (ꢀEpa = (−0.006 V and −0.065 V for 1 and 2,
respectively). These shifts showed that 1 and 2 could bind to
DNA by both intercalative and electrostatic interaction [40,41]. On
the other hand, a negative shift of the cathodic potentials for 3
(ꢀEpc = −0.051 V) and no anodic potential was observed. Thus, the
existence of electrostatic interaction between 3 and CT DNA bases
may be suggested [40]. The presence of DNA in the solution at
the same concentration of three complexes caused a considerable
decrease in the voltammetric current. The drop of the voltammetric
currents in the presence of CT DNA could be attributed to dif-
fusion of the metal complex bound to the large, slowly diffusing
DNA molecule. The decrease extents of the peak currents observed
for three complexes upon addition of CT DNA indicated that the
DNA binding affinity increased in the order: 3 < 2 < 1. The results
paralleled to the above spectroscopic and viscosity data of three
complexes in the presence of DNA.
Fig. 8. Cyclic voltammograms in DMSO:buffer [mixture 50 mM Tris–HCl/NaCl buffer
(pH, 7.0)] (1:2) solution of complex 1 carried out at scan rate = 0.08 V s−1 with incre-
mental addition of CT DNA. 0.1 M n-Bu4NClO4 as supporting electrolyte and the
arrow mark indicates the current changes upon increasing DNA concentration.
Such an enhanced activity of the complexes can be explained
on the basis of Overtone’s concept and Tweedy’s chelation theory
[42]. According to Overtone’s concept of cell permeability, the lipid

N. Raman et al. / Spectrochimica Acta Part A 79 (2011) 873–883
883
membrane that surrounds the cell favors the passage of only lipid
Acknowledgments
soluble materials due to which liposolubility is an important fac-
tor that controls antimicrobial activity. On chelation, the polarity
of the metal ion is 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 delocaliza-
tion of ␲-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 block-
ing of metal binding sites on the enzymes of the 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. The variation in the effectiveness of the
different compounds against different organisms depends on the
impermeability of the cells of microbes or difference in ribosome
of the microbial cells.
The authors express their sincere thanks to the College Man-
aging Board, Principal and Head of the Department of Chemistry,
VHNSN College for providing necessary research facilities and
financial support. Instrumental facilities provided by Sophisticated
Analytical Instrument Facility (SAIF), IIT Bombay and CDRI, Luc-
know are gratefully acknowledged.
Appendix A. Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.saa.2011.03.017.
References
[1] B. Lippert, Coord. Chem. Rev. 200–202 (2000) 487–516.
[2] S. Rekha, K.R. Nagasundara, Indian J. Chem. A45 (2006) 2421–2425.
[3] A.R. Banerjee, J.A. Jaeger, D.H. Turner, Biochemistry 32 (1993) 153–163.
[4] J.G. Liu, B.H. Ye, H. Li, Q.X. Zhen, L.N. Ji, Y.H. Fu, J. Inorg. Biochem. 76 (1999)
265–271.
4. Conclusion
[5] W.Y. Zhong, J.S. Yu, W.L. Huang, K.Y. Ni, Y.Q. Liang, Biopolymers 62 (2001)
315–323.
The synthesis and characterization of three new metal(II)
complexes of Cu(II), Ni(II) and Zn(II) have been realized with
physicochemical and spectroscopic methods. Each metal is
four-coordinate and hence, the geometry can be described as
square-planar. In the cyclic voltammograms of the complexes
recorded in DMSO, the irreversible or, in most cases, quasi-
reversible waves attributed to redox couples, characteristic for each
metal complex, have been recorded at potentials expected for the
central metal. The study of the interaction of complexes with CT
DNA has been performed with UV spectroscopy and cyclic voltam-
metry. It reveals the ability of synthesized metal complexes 1–3
to bind to DNA. The binding strength of the complexes with CT
DNA calculated with UV spectroscopic titrations have shown that
complex 1 exhibits the highest Kb value among the compounds
examined. Cyclic voltammetric study proposes both intercalative
and electrostatic interaction as the most possible binding mode
to DNA. The better binding properties of the complexes should
be attributed to the good coplanarity of the ligand after coordina-
tion with metal ions. Meanwhile, nature of the central metal ions
also affects the intercalative ability. These results indicate that DNA
might also serve as the primary target of these compounds; in addi-
tion, they should have many potential practical applications, just
like the promising therapeutic drug candidates. All the complexes
can effectively cleave plasmid DNA without addition of external
agents. DNA cleavage mechanism studies show that the complexes
examined here may be capable of promoting DNA cleavage through
both oxidative and hydrolytic DNA damage pathways. They display
higher nuclease activity of the Cu(II) complex than other complexes
and hence, the cooperative interaction of metal ions is a favored
factor to cleave DNA.
[6] A.K. Patra, S. Roy, A.R. Chakravarty, Inorg. Chim. Acta 362 (2009) 1591–1599.
[7] A. Vogel, Text Book of Quantitative Inorganic Analysis, third ed., ELBS, Longman,
London, 1969.
[8] G. Wilkinson, R.D. Gillard, J.A. McCleverty, Comprehensive Coordination Chem-
istry, Pergamon Press, Oxford, 1987.
[9] F. Arjmand, B. Mohani, S. Ahmad, Eur. J. Med. Chem. 40 (2005) 1103–1110.
[10] V. Uma, M. Kanthimathi, T. Weyhermuller, B. Unni Nair, J. Inorg. Biochem. 99
(2005) 2299–2307.
[11] M. Sonmez, M. Celebi, I. Berber, Eur. J. Med. Chem. 45 (2010) 1935–1940.
[12] R. Senthil Kumar, S. Arunachalam, Polyhedron 26 (2007) 3255–3262.
[13] B.S. Hammes, C.J. Carrano, J. Chem. Soc., Dalton Trans. 19 (2000) 3304–3309.
[14] A.A. Tak, F. Arjmand, S. Tabassum, Transit. Met. Chem. 27 (2002) 741–747.
[15] A.S. El-Tabl, T.I. Kashar, R.M. El-Bahnasawy, A.E. Ibrahim, Pol. J. Chem. 73 (1999)
245–254.
[16] Q. Yun Chen, H. Jian Fu, J. Huang, R. Xian Zhang, Spectrochim. Acta A 75 (2010)
355–360.
[17] M. Odabasoglu, F. Arslan, H. Olmez, O. Buyukgungor, Dyes Pigments 75 (2007)
507–515.
[18] Z. Chen, Y. Wu, D. Gu, F. Gan, Spectrochim. Acta A 68 (2007) 918–926.
[19] H. Ünver, Z. Hayvali, Spectrochim. Acta A 75 (2010) 782–788.
[20] M. Patil, R. Hoonur, K. Gudasi, Eur. J. Med. Chem. 45 (2010) 2981–2986.
[21] S. Budagumpi, N.V. Kulkarni, G.S. Kurdekar, M.P. Sathisha, V.K. Revankar, Eur.
J. Med. Chem. 45 (2010) 455–462.
[22] P.M. Angus, R.J. Geue, N.K.B. Jensen, F.K. Larsen, C.J. Qin, A.M. Sargeson, J. Chem.
Soc., Dalton Trans. 22 (2002) 4260–4263.
[23] T.A. Reena, M.R. Prathapachandra Kurup, Spectrochim. Acta
322–327.
A 76 (2010)
[24] P. Kamalakannan, D. Venkappayya, Russ. J. Coord. Chem. 28 (2002) 423–433.
[25] F.A. Cotton, G. Wilkinson, Advanced Inorganic Chemistry. A Comprehensive
Text, fourth ed., John Wiley and Sons, New York, 1986.
[26] J. Muller, K. Felix, C. Maichle, E. Lengfelder, J. Strahle, U. Weser, Inorg. Chim.
Acta 233 (1995) 11–19.
[27] V.P. Daniel, B. Murukan, B. Sindhu Kumari, K. Mohanan, Spectrochim. Acta A 70
(2008) 403–410.
[28] B.J. Hathaway, D.E. Billing, Coord. Chem. Rev. 5 (1970) 143–207.
[29] B.J. Hathaway, Struct. Bond. 14 (1973) 49–67.
[30] V.A. Sawant, B.A. Yamgar, S.K. Sawant, S.S. Chavan, Spectrochim. Acta A 74
(2009) 1100–1106.
[31] J.E. Coury, J.R. Anderson, L. McFail-Isom, L.D. Williams, L.A. Bottomley, J. Am.
Chem. Soc. 119 (1997) 3792–3796.
We have evaluated in vitro antibacterial and antifungal activ-
ities for our newly synthesized Schiff base and its mononuclear
copper(II), nickel(II) and zinc(II) complexes. The results obtained
from this research demonstrate that newly synthesized compounds
have good to moderate antibacterial and antifungal activity against
the bacterial and fungal strains. Besides, the ligands and complexes
1 and 3 exhibit selective and effective activity against Candida
species. Multi-drug resistant microorganisms pose a serious chal-
lenge to the medical community and there is therefore an urgent
need to develop new agents. In this sense, we think that the ligands
and three metal complexes 1–3 might be effective as antibacterial
and antifungal agents. Thus, the remarkable DNA binding affinity,
antibacterial and antifungal activities suggest that the above com-
pounds would have potential application for developing new drugs
for cancer.
[32] L. Tan, Y. Xiao, X. Liu, S. Zhang, Spectrochim. Acta A 73 (2009) 858–864.
[33] S. Mathur, S. Tabassum, Cent. Eur. J. Chem. 4 (2006) 502–522.
[34] E.K. Efthimiadou, A. Karaliota, G. Psomas, J. Inorg. Biochem. 104 (2010)
455–466.
[35] Y. Jun Liu, C. Hui Zeng, H. Liang Huang, L. Xin He, F. HaiWu, Eur. J. Med. Chem.
45 (2010) 564–571.
[36] A.M. Pyle, J.P. Rehmann, R. Meshoyrer, C.V. Kumar, N.J. Turro, J.K. Barton, J. Am.
Chem. Soc. 111 (1989) 3051–3058.
[37] J. Jiang, X. Tang, W. Dou, H. Zhang, W. Liu, C. Wang, J. Zheng, J. Inorg. Biochem.
104 (2010) 583–591.
[38] D. Dong Li, J. Lei Tian, W. Gu, X. Liu, S. Ping Yan, J. Inorg. Biochem. 104 (2010)
171–179.
[39] T. Hirohama, Y. Kuranuki, E. Ebina, T. Sugizaki, H. Arii, M. Chikira, P.T. Selvi, M.
Palaniandavar, J. Inorg. Biochem. 99 (2005) 1205–1219.
[40] G. Psomas, J. Inorg. Biochem. 102 (2008) 1798–1811.
[41] K. Jiao, Q.X. Wang, W. Sun, F.F. Jian, J. Inorg. Biochem. 99 (2005) 1369–1375.
[42] N. Raman, A. Kulandaisamy, C. Thangaraja, P. Manisankar, S. Viswanathan, C.
Vedhi, Transit. Met. Chem. 29 (2004) 129–135.