Non-enolisable Knoevenagel condensate appended Schiff bases-metal (II) complexes: Spectral characteristics, DNA-binding and nuclease activities

Article abstract of DOI:10.1016/j.saa.2017.03.031

New Schiff base complexes [Cu(L¹)Cl] (1), [Ni(L¹)Cl] (2), [Zn(L¹)Cl] (3), and [Fe(L²)H₂OCl] (4) {L¹?=?(4E)-3-(2-hydroxybenzylidene)-4-(2-hydroxyphenylimino)pentan-2-one, L²?=?2,2

Full text of DOI:10.1016/j.saa.2017.03.031

Accepted Manuscript
Non-enolisable Knoevenagel condensate appended Schiff bases-
metal (II) complexes: Spectral characteristics, DNA-binding and
nuclease activities
Ammavasi Gubendran, Mookkandi Palsamy Kesavan, Srinivasan
Ayyanaar,
Liviu
Mitu,
Periyakaruppan
Athappan,
Jegathalaprathaban Rajesh
PII:
S1386-1425(17)30207-X
doi: 10.1016/j.saa.2017.03.031
SAA 15010
DOI:
Reference:
Spectrochimica Acta Part A: Molecular and Biomolecular
Spectroscopy
To appear in:
Received date:
Revised date:
Accepted date:
22 December 2016
14 March 2017
14 March 2017
Please cite this article as: Ammavasi Gubendran, Mookkandi Palsamy Kesavan,
Srinivasan Ayyanaar, Liviu Mitu, Periyakaruppan Athappan, Jegathalaprathaban Rajesh
,
Non-enolisable Knoevenagel condensate appended Schiff bases-metal (II) complexes:
Spectral characteristics, DNA-binding and nuclease activities. The address for the
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ACCEPTED MANUSCRIPT
Non-enolisable Knoevenagel condensate appended Schiff bases-metal (II)
complexes: Spectral characteristics, DNA-binding and nuclease activities
a,b
c
c
Ammavasi Gubendran , Mookkandi Palsamy Kesavan , Srinivasan Ayyanaar ,
d
a
a,c*
Liviu Mitu , Periyakaruppan Athappan , Jegathalaprathaban Rajesh
a
Department of Inorganic Chemistry, School of Chemistry, Madurai Kamaraj University,
Madurai - 625 021, India.
b
Department of Chemistry, Saraswathi Narayanan College, Madurai-625 022, India.
c
Chemistry Research Centre, Mohamed Sathak Engineering College, Kilakarai–623 806,
Tamilnadu, India.
d
Department of Physics and Chemistry, University of Pitesti, Pitesti-110040, Romania.
*
Corresponding author
Dr. Jegathlaprathaban Rajesh
Associate Professor
Chemistry Research Centre
Mohamed Sathak Engineering College
Killakarai - 623 806
Ramanthapuram (D.T)
Tamilnadu, India.
*
E-mail address: mkuraji@gmail.com

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Abstract
1
1
1
New Schiff base complexes [Cu(L )Cl] (1), [Ni(L )Cl] (2), [Zn(L )Cl] (3), and
2
1
[
Fe(L )H OCl] (4) {L = (4E)-3-(2-hydroxybenzylidene)-4-(2-hydroxyphenylimino)pentan-2-
2
2
one,
L =2,2'-(1E,1'E)-(3-(2-hydroxybenzylidene)-pentane-2,4-diylidene)bis(azan-1-yl-1
idene)diphenol} have been synthesized and characterized by elemental analysis, UV-Vis, IR,
1
2
FAB-mass, EPR, spectral studies and electrochemical studies, the ligands L & L were
1
13
characterized by H and C NMR spectra. Complex 1 show a visible spectral d-d band near 600
nm and display cyclic voltammetric quasireversible response for the Cu(II)/Cu(I) couple vs
Ag/AgCl in DMSO. The EPR spectrum of 1 show g >g suggesting a square planar geometry


2
2
around copper with d
as the ground state. The mass spectral results have confirmed the
x - y
proposed structure for complexes 1-4. DNA binding properties of these complexes 1-4 have been
investigated by absorption titrations, cyclic voltammetric studies and circular dichroism studies.
On titration with DNA, the complexes 1-4 show hypochromism at the MLCT band (13-31%)
with a red shift of 1-8 nm in the electronic spectrum and positive shift of voltammetric E1/2 in the
CV studies are in favour of intercalative binding. CD spectra of 1 showed an increase in molar
ellipticity (θ ) of the positive band with a minor red shift indicating the transition of B-form of
2
78
DNA to A like form. DNA cleavage studies of complexes 1 and 4 with pUC18 DNA were
studied by gel electrophoresis and complex 4 cleaves supercoiled pUC18 DNA in an oxidative
manner in the presence of H O and on photo irradiation at 312 nm.
2
2
Key words: Non-enolisable Knoevenagel condensate; Schiff base complexes; DNA interaction;
CD spectra; DNA cleavage

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1
Introduction
Oxygen and nitrogen donor Schiff base ligands derived transition metal complexes have
been of an intense research interest for many years [1-4], because of the versatility of their steric
and electronic properties. These properties can be brought by choosing the appropriate amine
precursors and ring substituents [5-7]. The value of these tetradentate N O ligands and their
2
2
transition-metal complexes, as catalysts, has attracted significant attention particularly on the
development of agrochemical and pharmaceutical products. Due to the potential catalytic
significance of these metal-salen chelates especially rapid growth in their chemical study has
been developed. Many authors have reported synthesis and characterization of metal complexes
with symmetrical Schiff bases derived from β-diketone and diamine [8, 9]. Diketones, a versatile
ligand system, are long known to form complexes with almost every metal ion. Non-enolisable
Knoevenagel condensate formed by the condensation of the active methylene group of β-
diketone with an aldehydic group of salicylaldehyde can effectively react with amines to form
Schiff bases [10]. Such Schiff bases could be synthesized from salicylidene curcumin and
primary amines, which are expected to form stable complexes with transition metals [11]. The
objective of the present paper is to investigate complexes formed by the reaction of tridentate
1
2
(
L ) and tetradentate (L ) ligands (Scheme 1) derived from non-enolisable Knoevenagel
condensate and o-aminophenol with copper(II), nickel(II), zinc(II) and iron(III). The spectral and
redox behaviour of the complexes 1-4 have been assessed and any change in these behaviours
through titration with DNA attributes to their nucleus properties. The possibility of
photocleavage of DNA by iron(III) complex has also been worked out.

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2
Experimental
2
.1
Materials
Acetylacetone (MERCK), salicylaldehyde (MERCK), 2-aminophenol (MERCK),
Copper(II) chloride, Nickel(II) Chloride, Zinc(II) Chloride, Iron(III) chloride (MERCK), Herring
sperm DNA (SRL), Tris-hydrochloride (SRL) and Sodium chloride (SRL) were purchased and
used. Supercoiled pUC18 DNA was procured from Genei, Bengaluru, India. The other reagents
and solvents were analytical, spectroscopic grade and they were used without further
purification.
2
.2
Physical Measurement
UV-Vis spectral measurements were performed in DMSO solution of the complexes 1-4
using JASCO double beam recording spectrophotometer in the range 200-1100 nm. The infrared
spectra of complexes 1-4 as well as the free ligand were recorded using KBr pellet on a JASCO
-1
FT-IR 410 (double beam infrared spectrophotometer) in the range of 400-4000 cm . Nuclear
magnetic resonance (NMR) spectra were recorded on Bruker 300 MHz spectrometer. Deuterated
chloroform (CDCl ) and Tetramethylsilane (TMS) was used as a solvent and internal standard
3
compound, respectively. An electron paramagnetic resonance spectrum of the complex 1 was
obtained on a Varian-E-112 EPR spectrometer. The spectrum was recorded for solutions of the
complexes 1 in DMSO solvent at room temperature (RT) as well as at liquid nitrogen
temperature (77 K) and the DPPH (2,2-Diphenyl-1-Picrylhydrazyl) was used as the field marker.
Cyclic voltammetric measurements were carried out on an electrochemical analyser of Bio-
Analytical System (BAS) model CV-50W. The electrode cell comprising of a reference Ag/AgCl
2
electrode, working glassy carbon (GC) electrodes with a surface area of 0.07cm and counter
platinum wire electrode. The GC was polished with 0.3 and 0.005 mm alumina before each

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scanning and the electrode was sonicated in distilled water for 10 minutes. Dissolved oxygen
was removed by nitrogen gas into the solution for about 15 minutes before each experiment.
2
.3
Spectroscopic Studies on DNA Interaction
Concentrated stock solutions of metal complexes 1-4 were prepared by dissolving them
in 10% DMSO–Tris–HCl/NaCl buffer (5:50 mM) at pH 7.2 and diluting them with the
corresponding buffer to the required concentrations for all experiments.
The interaction of the complexes 1-4 with DNA was investigated by monitoring the
absorption spectra of these complexes in the presence and absence of DNA. HS-DNA in the
buffer gave a ratio of UV absorbance at 260 and 280 nm of about 1.8-1.9:1, which indicating the
DNA was sufficiently free from protein [12]. The DNA concentration was determined by
-1
-1
absorption spectroscopy using the molar extinction coefficient (6600 M cm ) at 260 nm [13].
Differential pulse voltammetry (DPV) of complexes 1-4 in the presence and absence of
DNA were obtained by using Bio-Analytical System (BAS) model CV-50W electrochemical
◦
analyser at 27 C. For a Nernstian electron transfer system, the corresponding equilibrium
constants for each oxidation state binding to DNA, according to the following equation [14],
o´
0´
E ─ E = 0.0591 log (K /K )
b
f
red
ox
o´
o´
where E and E are the formal potentials of M(II)/M(I) or M(III)/M(II) complex, as the case
b
f
may be, couple in the binding and free forms, respectively.
Circular dichroic spectra of DNA alone and with the M(II) complexes 1-4 were recorded
on a JASCO J-810 spectropolarimeter (163-900 nm) using a quartz cuvette with 1 mm optical
path length at increasing complex/DNA ratio (r = 0.1 - 0.5). Each addition was scanned in the
range of 220-320 nm. Each experiment was recurred at least two times to obtain the reliable
values.

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2
.4
DNA Cleavage Studies
Cleavage reactions of supercoiled pUC19 DNA (500 ng) (form I) in 10% DMSO-5 mM
Tris–HCl–50 mM NaCl buffer (pH 7.2) was treated with appropriate complexes. The samples
◦
were incubated at 37 C for 1 h duration. Loading buffer contains 25% bromophenol blue, 0.25%
xylene cyanol and 30% glycerol (3 µL) was added and electrophoresis performed at 60 V for 4 h
in Tris–acetate–EDTA (TAE) buffer (40 mM Tris-base–20 mM acetic acid–1 mM EDTA) using
-1
1
% agarose gel contains 1 g mL ethidium bromide. The cleavage products were irradiated at
room temperature with a UV lamp at 365 nm, 10 W and examined with a BIORAD Model XI
computer controlled electrophoresis power supply.
2
.5
.5.1 Synthesis of L
The non-enolisable Knoevenagel condensate of acetylacetone was prepared by the
Synthesis Protocols
2
reported procedure [15]. Briefly, acetylacetone (10 mmol) was mixed with salicylaldehyde (10
mmol) and 0.5 ml of piperidine was added to it. The solution mixture was stirred thoroughly for
about 3 h with occasional cooling. Then this mixture was kept in the refrigerator for 12 h and
gradually a yellow coloured precipitate was separated in small amounts. To this suspension an
excess of petroleum ether was added, followed by scratching the walls of the beaker with a glass
rod. All the yellow suspension were suddenly solidified and settled down. This was filtered,
washed well with 15 ml of petroleum ether and dried. The crude product was recrystallized from
the mixture of chloroform and petroleum ether (10:1).
1
2
.5.2 Synthesis of L
A mixture of the above product (L) (0.612 g, 3 mmol) and 2-aminophenol (0.327g, 3
mmol) in 50 ml of ethanol was refluxed with magnetic stirrer for 3 h. The resulting yellow

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coloured solution was allowed to cool at room temperature. The formed pale yellow precipitate
was separated by filtration, washed with 20 ml of ether and recrystallized from ethanol and dried
in a vacuum desiccator.
2
2
.5.3 Synthesis of L
1
This ligand was prepared by a procedure similar to that described above for L except for
1
2
the use of 2 mmol of 2-aminophenol in 60 ml of ethanol. The purity of the ligands L & L were
checked by TLC using a mixture of petroleum ether/ethylacetate (9.5/0.5, v/v) as solvent.
1
2
Synthetic procedure of L & L are shown in scheme 1.
2
.5.4 Synthesis of Complexes 1-3
Complexes 1-3 were prepared by the drop wise addition of the respective hydrated metal
chloride/nitrate (1 mmol) dissolved in 20 ml of ethanol to an ethanolic solution (20 ml) of the
1
ligand L (0.295g, 1 mmol) with stirring and the stirring was continued for 1-1.5 h at room
temperature. The coloured complexes so obtained were filtered, washed with 15 ml of ether and
dried under vacuum over CaCl2.
2
.5.5 Synthesis of Complex 4
A solution of FeCl .5H O (1 mmol) in 20 ml of ethanol was added dropwise to a solution
3
2
2
of the ligand L (0.386 g, 1 mmol) in 20 ml of ethanol with stirring. The complex 4 started to
form immediately upon the addition of the metal solution and during the addition, the solution
turned dark brown and the stirring was continued at room temperature for 30 min. The formed
black precipitate was filtered off, washed with 20 ml of ether and dried under vacuum over
1
2
CaCl . The structure of ligands (L & L ) and complexes (1-4) are shown in Scheme 1 and
2
Scheme 2, respectively.
3
Results and Discussion

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1
13
3
.1
H and C-NMR Spectra
1
1
2
The H NMR spectra of the ligands (L & L ) exhibit singlets in the region of δ, 11.5-
1
1.8 ppm which may be attributed to the phenolic OH group. The single resonance proton
occurring at δ, 5.3 ppm may be due to another phenolic OH group. The methyl protons of
1
3
O=C-CH and N=C-CH appear in the δ, 1.8-2.3 ppm range. The C NMR signals of the ligands
3
3
in the
δ, 162-164 ppm range can be assigned to azomethine carbon atoms. The signals due to
carbon atoms of the benzenoid rings are observed in the δ, 104-158 ppm range. The carbon
atoms of the methyl groups of O=C-CH and N=C-CH appear in the δ, 20-29 and 19-20 ppm
3
3
13
1
ranges respectively. The signal at δ, 196.4 ppm in the C spectrum of L clearly indicates the
13
2
presence of carbonyl carbon and the absence of such a signal in the C spectrum of L indicate
absence of keto carbon (Fig. S1c) thereby confirming the complete condensation of 2-
2
1
13
aminophenol on both sides of β-diketone in L . The H and C NMR spectral data in CDCl for
3
1
13
2
the ligands L (Fig. S1a & S1b) & C NMR spectral data for L (Fig. S1c) exhibit all the
expected signals as per the proposed structure.
3
.2
Mass Spectra
The molecular ion peak for 1 (Fig. S2) is seen at m/z = 391 and the peak at m/z = 357
1
+
1
corresponds to [Cu(L )] . The base peak obtained at m/z = 295 is due to L after the loss of Cl
and Cu atom. The molecular ion peak observed at m/z = 493 in the mass spectrum of 4 (Fig. S2)
+
is in agreement with the calculated mass of M ion, corresponding to the molecular ion formula
+
of the complex 4, C H ClFeN O . The spectrum records the presence of fragments with m/z
2
4
22
2
4
values at 475, 440, 386 and 384 after the successive loss of H O, and HCl from the parent ion.
2
The FAB-mass spectra for other complexes 1 & 4 show peaks in expected molecular weight and
fragmentation pattern in accordance with the molecular formula.

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3
.3
EPR Spectra
The EPR spectra of copper(II) complex 1 (Fig. S3) gave g and g values in the regions


2
2
2
.29 and 2.09 respectively, which indicate the unpaired electron lie in a d
orbital of the
x -y
copper(II) ion. It has been reported that the magnitude of the ratio G = (g -2) / (g -2) indicates


the possibility of exchange interaction in the copper(II) complexes [16]; In the present case, the
values appeared in 3.22, which indicate (G < 4) considerable exchange interaction in the solid
complexes [17]. Often the quotient g A is empirically treated as an index of tetrahedral
/


distortion. The g A values are expected to be 105-135 and 150-250 range, respectively for
/


squar planar and tetrahedrally distorted copper(II) complexes. In the present case the g A are
/


in between 178-185. It is assumed that the geometry of the present complex 1 is distorted from
planarity to tetrahedral.
3
.4
Infrared Spectra
1
2
The IR spectral data of free Schiff base ligands (L & L ) and their complexes (1-4)
1
recorded using KBr discs are summarized in Table 1. The IR spectra of the ligand L and
-1
complex 1 are shown in Fig. S4. Bands at 1620 and 1668 cm are assigned respectively to
1
υ(C=N) and υ(C=O) of one of the uncondensed β-diketones thereby confirming formation of L .
-1
2
Absence of any band in between 1580 and 1620 cm in the IR spectrum of ligand L confirms
complete condensation of both keto groups of β-diketone with two moles of 2-aminophenol.
–
1
Bands at 1620-1615 cm in the free ligands are assigned to υ(C=N). These bands are shifted to
lower wave numbers in the complexes 4 due to the coordination of the nitrogen atom of the
-1
azomethine group to the metal ion. The band at 1668 cm , corresponding to υ(C=O) of the free
1
ligand L is shifted to lower frequency on complex formation due to the coordination of oxygen
atom of the keto form 1-3. New bands observed after complex formation between 560–524 and

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–
1
4
30–410 cm were metal sensitive and are assigned to υ (M–O) and υ (M–N) respectively.
1
2
Thus the IR spectral data confirm the mode of coordination of ligands (L & L ) to the metal in
complexes 1-4.
3
.5
Electronic Absorption Spectra
The electronic absorption spectral data are given in Table 2. All these complexes 1-4 in
DMSO show bands at 232-246 and 409-425 nm that are attributed respectively to the intraligand
transition and the charge transfer transition from the metal ions to phenoxide of the ligands
(MLCT). The electronic spectrum of copper(II) complexes 1 exhibit bands at ca 600 nm which
2
2
2
2
can be assigned to the combination of B1g
A and B
Eg transitions for a square planar
1g
1g
environment in coordinating solvents [18]. The nickel(II) complex 2 exhibits, band at 495 nm
1
1
1
1
which may be assigned to the overlap of A → B and A → A indicating the square
1
g
1g
1g
2g
planar geometry around the nickel (II) ion. The d–d bands are observed only for 1 & 2 not for 3
4 in the visible region. Since for six coordinate Fe(III) complexes and four coordinate Zn(II)
complexes, all d-d transitions are forbidden, absorption bands are generally not observed for 3 &
&
4
3
[19].
.6
Electrochemical Behaviour
The cyclic voltammogram of complex 4 dissolved in DMSO is shown in Fig. S5 and the
various peak potentials for the complexes 1, 2, 4 except 3 are given in Table 3. With the ratio of
anodic to cathodic peak current less than unity and high separation between cathodic and anodic
peak potential, ΔEp = (E -E ) > 200 mV, the redox process for the complex 1 may be
pc pa
considered quasireversible with one electron redox process. The cyclic voltammogram of the
complex 4 shows cathodic peak at 0.143 V and anodic peak at 0.225 V the peak separation ΔEp
=
82 mV and i i = 0.67 indicate the quasireversible character with one electron redox process.
pa/ pc

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Complex 2 exhibits irreversible cyclic voltammogram showing only cathodic peak at 0.725 V
1
0
II
and no anodic counterpart. Due to stable electronic structure (d ), Zn could not undergo
further oxidation as well as reduction process. Because of this, complex 3 failed to show redox
behavior.
3
.7
DNA Binding Studies
3
.7.1 Absorption Spectroscopic Titration
The interaction of the complexes 1-4 with DNA was investigated by monitoring the
absorption spectra of these complexes in the presence and absence of DNA. The absorption
titrations of the complexes 1-4 in buffer were performed using a fixed metal complex
concentration (20 µM) to which increments of the herring sperm DNA stock solution was added
(0-160 µM). The absorption spectra of complexes 1 and 4 in the presence of increasing amounts
of DNA are shown in Fig. 1. Percentage of hypochromism, a shift in wavelength and binding
constants are given in Table 4. Both intra-ligand and charge transfer bands are monitored for 4
and MLCT bands alone were monitored for complexes 1-3. With the increasing concentration
of DNA, the absorption bands are affected and hypochromism accompanied by red shift in
wavelength are observed for the complexes 1-4. The red shift obtained for 1-3 is very small in
contrast to 4 where an appreciable red shift (8 nm) is observed in addition to hypochromism.
These spectroscopic changes suggest interactions between the metal complexes and DNA and
such hypochromicity has been attributed to intercalative binding [20]. Notably, the
hypochromicities of the peaks are lower than those of classical intercalators such as ethidium
2
+
bromide and [Ru(phen) (dppz)] whose percentage hypochromicities are > 25% [21].
2
As the strength of intercalation is related to red shift along with the hypochromism, the
intercalative ability of these complexes is different. The large shift observed with π-π* transition

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reveals intercalation for 4 compared to other complexes. Intrinsic binding constants (Kb)
3
-1
determined from the decay of the absorbance for complexes 1-3 and 4 are around 2.5 x 10 M
4
-1
and 2.0 × 10 M respectively. The obtained binding constant of these complexes 1-4 with DNA
7
-1
are lower than those reported for classical intercalators (ethidium–DNA, 7.0 ×10 M in 40 mM
6
-1
tris–HCl buffer, pH 7.9 and 1.4 × 10 M in 40 nM NaCl-25 nM tris–HCl, proflavin with
5
-1
Escherichia coli DNA, 50% GC content, 4.1 × 10 M in 0.1 M Tris-HCl) with a established
DNA-binding mode involving the complete intercalation of the planar molecules between the
base pairs [22]. This is indicative of binding of the complexes 1-4 with DNA host with an
affinity less than the classical intercalators.
The important difference in DNA binding affinity of the complex 4 as against rest of the
complexes 1-3, that may not fully intercalate, can be understood as a result of the fact that
symmetric planar aromatic moiety as well as the structural change from octahedral to square
planar in pH 7.1 may permit the complex to stack into DNA base pairs. Therefore, the
conclusion can be made preliminary that while complexes 1-3 are able to bind poorly to DNA by
weak intercalation, complex 4 is found to bind to DNA by strong intercalation.
3
.7.2 Circular Dichroism Study
The observed circular dichroism spectrum of herring sperm DNA in Tris-HCl buffer
consisting of a positive band at 278 nm due to base stacking and a negative band at 248 nm due
to helicity with zero-cross over at 260 nm is characteristic of right-handed B-form DNA [23].
Fig. 2 show the CD spectra of DNA treated with different concentrations of complex 1. The
increasing concentration of complexes 1-3, peaks at 278 nm, 248 nm and zero-cross over at 260
nm are perturbed. As for complex 1-3 negative band is slightly increased but there is a
significant increase in intensity of positive band at 278 nm with small red shift (5 nm) suggesting

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that there exists interaction between aromatic rings of the complex and base pairs of the DNA.
These spectral features suggest that some conformational changes have taken place and the DNA
becomes more form A like upon its interaction with these complexes. Similar phenomenon was
observed in La(III) complexes [24,25]. B→A transition was reported on the interaction with
certain multinuclear Pt(II) complexes [26]. Patra et al. reported that if the positive band at 278
nm showed increase in molar ellipticity (θ ) with a minor red shift of the band maximum with
2
78
an increase in band intensity upon addition of the copper complexes to DNA, the phenomenon
could be due to groove binding that stabilizes the B form (right-handed) of DNA [27].
As for complex 4 is concerned the intensity of both positive and negative bands are
increased and in addition obvious changes in intensity with new bands are also observed making
the CD curve rather wavy upon successive addition of the complex (Fig. 3). This spectral change
could be due to intercalation leading to cleavage of DNA. The CD spectral studies of the present
complexes 1-4 clearly reveals that the complex 1, 2 and 3 are involved in weak intercalation
binding different from 4, which cleaves the DNA through intercalation.
3
.7.3 Electrochemical Studies
Cyclic voltammograms of 1, 2 and DPV of 4 in the absence and presence of DNA are
shown in Fig. S6 & Fig. 4 respectively, and the voltammetric data are reported in Table 5. The
separation of the cathodic and anodic peak potentials is large ( ΔE > 600 mV) and, the ratio of
p
cathodic to anodic peak current i /i is less than unity for 1 indicating irreversible redox process
pa pc
(
ΔEp = 59 mV for one electron diffusion and i /i of about 1 controlled reversible process).
pa pc
Complex 2 exhibits irreversible electrochemical wave both in DMSO and in Tris-HCl buffer
solution. Such an irreversible electrochemical behaviour is due to constraints in the ligand
geometry of the complex [28] and complex 4 shows, no anodic peak in Tris-HCl buffer solution.

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Owing to the absence of anodic peaks in 2 and 4, E from DPV measurements were used to
pc
calculate the binding constants.
0
The formal potential E (or voltammetric E ), taken as the average of E and E , in the
1
/2
pc
pa
absence of DNA for the complex 1 is -0.384 V which upon addition of DNA, undergoes a
positive shift. However, while cathodic peak potential E moves towards more negative potential
pc
and E moves towards less negative side coupled with the decrease of its voltammetric current
pa
in the case of 1. The noticeable shift in the peak potentials indicates the strong interaction of the
complex and DNA [29]. It is clear that (Table 5) peak potential shift occurs in the complexes 1,
2
, 4 and the interaction of the reduced form of the complexes with DNA is not the same as that
of their oxidized form. Though the half-wave potentials E1/2 of all the complexes undergo a
positive shift, the shift made by 1 is least and by 4 is highest. The highest shift observed for the
complex 4 upon addition of CT DNA indicates that it possesses high-binding affinity than other
complexes 1 and 2. For 1 and 2 K is higher than K (ratio K /K > 1) which suggest that the B-
+
2+
+
2+
I
II
DNA tends to stabilize the M over M state of the complexes by partial intercalation mode. A
ratio of K /K = 2.02 at R = 4 was estimated for 4 indicates that the reduced form, interacts
2
+
3+
with the DNA on the DNA/Fe more strongly than the oxidized form, which is characteristic of
intercalative interactions. The conclusions could be drawn in the light of Bard’s report that the
complex 4 would intercalate into DNA and other complexes may bind to DNA by weak
intercalation. The results parallel to the above spectroscopic and CD data of complexes in the
presence of DNA.
3
.8
DNA Cleavage Studies by Gel electrophoresis

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Transition metal-mediated radical production may result in an efficient DNA cleavage
and which was controlled by relaxation of the supercoiled circular form of pUC18 into the
nicked circular and linear forms [30].
All supercoiled (Form I) DNA are cleaved to open circular form (Form II) in presence of
H O (lanes 3-6) and complex 1. In the absence of H O the complex 1 (Fig. 5) could not
2
2
2
2
promote complete conversion of supercoiled form into Form II and Form III (lane 2). It can be
seen that neither the complex 1 alone nor the H O without complex (not shown) is cleavage
2
2
active. Even in presence of higher concentrations of H O the complex could cleave supercoiled
2
2
form (Form I) into slower-moving open circular form (Form II) only and complete conversion
into Form II and Form III was not possible. The cleavage mechanism may involve hydroxyl
radical oxidative mechanisms. It is evident from the Fig. 5 that both the complex 1 and a
co-oxidant such as H O are required to cleave plasmid DNA.
2
2
Complex 4 exhibit concentration independent single strand cleavage of supercoiled form
to nicked form (lanes 3 and 4). The photonicking of DNA could be due to the guanine base of
DNA by the photo activated metal complex. Control experiment (lane 1, Fig. 6) suggests that
untreated DNA doesn’t show any cleavage upon irradiation. Untreated DNA + H O alone (lane
2
2
2
) also does not show any cleavage. The DNA cleavage activity is enhanced in the presence of
H O all supercoiled (Form I) DNA is cleaved and to form a mixture of Form II and Form III in
2
2,
the presence of H O (lanes 5 and 6). In the absence of H O the complex could not promote
2
2
2
2
complete conversion supercoiled form of DNA into Form II and Form III even at high
concentrations (lane 4). It is seen that the complex 4 is not fully cleavage active (lanes 3 and 4).
The cleavage mechanism may involve hydroxyl radical oxidative mechanisms. It is evident from

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the Fig. 6 that both the generation of hydroxyl radicals and/or activated oxygen mediated by the
complex 4 may result in DNA cleavage.
4
Conclusion
1
2
The non-enolisable Knoevenagel condensate derived Schiff base ligands L & L and its
metal complexes 1-4 have been successfully synthesized and characterized. The DNA binding
studies have been successfully demonstrated using absorption spectra, CD spectra and
electrochemical studies. On absorption spectral titration with DNA, the complex 4 shows
uppermost red shift of 8 nm and shows a positive shift of voltammetric E1/2 in the CV studies.
These reveal that the complex 4 has the highest binding capability than the other complexes 1-3.
Consequently, complex 4 strongly cleaves supercoiled pUC18 DNA in an oxidative manner in
the presence of H O and on photo irradiation at 312 nm. From these preliminary investigations
2
2
we concluded that complex 4 can act as a potent chemotherapeutic agent against human cancer
cells.
Acknowledgment
This work was supported by the DST-SERB (Grant. No: SB/FT/CS-130/2012). The
authors honestly express their heartfelt thanks to Madurai Kamaraj University and Mohamed
Sathak Engineering College, Kilakarai for their lab facilities and their instrumentation facilities.

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Schemes
1
2
Scheme 1 Synthetic procedure of L & L
Scheme 2 Synthesis of complexes 1-4.

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Scheme 1

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Scheme 2

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Figures
Fig. 1 Absorption spectra of 1 (a) and 4 (b) (20 µM) in the absence and presence of
increasing amounts of DNA (0-160 µM) in Tris-HCl buffer upon addition of DNA.
Arrow mark indicates the absorbance changes upon increasing the DNA concentration.
Fig. 2 CD spectra of herring sperm DNA in the absence (a) and presence (b) of complex 1,
[
DNA] = 200 µM, [complex] = 100 µM, (1/R = 0.5)
Fig. 3 CD spectra of herring sperm DNA in the absence (a) and presence (b-d) of complex 4,
DNA] = 200 µM, [complex] = 0-100 µM, (1/R = 0.1- 0.5)
[
-4
Fig. 4 DPV of 10 M of 4 in DMSO: buffer (1:10) mixture with increasing amounts of DNA
-
1
[
R = 0, 2, 4, 6, and 8] at 50 mVs
Fig. 5 Agarose gel electrophoresis diagram showing the cleavage of SC pUC18 DNA
500 ng) by complex 1 in Tris-HCl/NaCl Buffer (50 mM, pH = 7.2); Lane1, DNA
(
control; Lane 2, DNA + 100 µM complex; Lane 3, DNA + 50 µM complex + 50 µM
H O ; Lane 4, DNA + 100 µM complex + 50 µM H O Lane 5, DNA+ 100 µM
2
2
2
2;
complex + 100 µM H O ; Lane 6, DNA + 100 µM complex + 200 µM H O .
2
2
2
2
Fig. 6 Agarose gel electrophoresis diagram showing the cleavage of SC pUC18 DNA (500 ng)
by complex 4 in Tris-HCl/NaCl Buffer (50 mM, pH = 7.2); Lane1, DNA contro l; Lane
2
, DNA +100 µM H O ; Lane 3, DNA + 50 µM complex; Lane 4, DNA + 100 µM
2 2
complex Lane 5, DNA + 100 µM complex + 100 µM H O ; Lane 6, DNA + 100 µM
;
2
2
complex + 200 µM H O
2
2

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Fig. 1

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Fig. 2

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Fig. 3

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Fig. 4