Enthused research on DNA-binding and DNA-cleavage aptitude of mixed ligand metal complexes

Article abstract of DOI:10.1016/j.saa.2013.04.054

Five new Mn(II), Co(II), Ni(II), Cu(II) and Zn(II) mixed ligand complexes have been synthesized using a Schiff base precursor (obtained by the condensation of N-(4-aminophenyl)acetamide and 4-chlorobenzaldehyde) as main ligand and 1,10-phenanthroline as c

Full text of DOI:10.1016/j.saa.2013.04.054

Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 112 (2013) 198–205
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Spectrochimica Acta Part A: Molecular and
Biomolecular Spectroscopy
journal homepage: www.elsevier.com/locate/saa
Enthused research on DNA-binding and DNA-cleavage aptitude of mixed
ligand metal complexes
⇑
Rajkumar Mahalakshmi, Natarajan Raman
Research Department of Chemistry, VHNSN College, Virudhunagar 626 001, India
h i g h l i g h t s
g r a p h i c a l a b s t r a c t
The N-(4-aminophenyl)acetamide derived Schiff base mixed ligand complexes act as good DNA binding
and DNA cleaving agents. The research has led to the discovery of new Schiff base mixed ligand com-
pounds for further pharmacological investigation.
ꢀ
ꢀ
Synthesis of good DNA intercalators.
Exploring efficient chemotherapeutic
agents.
ꢀ
ꢀ
ꢀ
Better antimicrobial active agents.
Effective DNA cleavage activators.
Excellent Cu(II) DNA binder.
a r t i c l e i n f o
a b s t r a c t
Article history:
Five new Mn(II), Co(II), Ni(II), Cu(II) and Zn(II) mixed ligand complexes have been synthesized using a
Schiff base precursor (obtained by the condensation of N-(4-aminophenyl)acetamide and 4-chlorobenz-
aldehyde) as main ligand and 1,10-phenanthroline as co-ligand. They have been characterized by micro-
analytical data, IR, UV–Vis, magnetic moment values, conductivity and electrochemical measurements.
The spectral data reveal that all the complexes exhibit octahedral geometry. The high electrical conduc-
tance of the complexes supports their electrolytic nature. The monomeric nature of the complexes has
been assessed from their magnetic susceptibility values. These complexes are better antimicrobial active
agents than the free ligands. DNA (CT) binding properties of these complexes have been explored by UV–
Vis., viscosity measurements, cyclic voltammetry, and differential pulse voltammetry measurements. The
oxidative cleavage activity of the complexes has been studied using supercoiled pUC19 DNA by gel elec-
trophoresis. The experimental results show that the complexes are good intercalators.
Received 27 December 2012
Received in revised form 6 April 2013
Accepted 10 April 2013
Available online 22 April 2013
Keywords:
Biologically active complexes
Schiff base
DNA binding
Chemical nuclease activity
Ó 2013 Elsevier B.V. All rights reserved.
Introduction
increasingly important to explore the possible new drug. Recent
years have a great deal of interest in the synthesis and character-
The interaction of metal complexes with DNA has been the sub-
ject of interest for the development of effective chemotherapeutic
agents. The studies of transition metal complexes are becoming
ization of transition Schiff base metal complexes, containing an
azomethine group (AC@NA). Transition metals are essential for
the normal functioning of living organisms. Therefore, it is not
surprising that transition metal compounds are of great interest
as potential drugs. Schiff bases form an important class of organic
compounds with a wide variety of biological properties [1].
⇑
Corresponding author. Tel.: +91 092451 65958; fax: +91 4562 281338.
E-mail address: ramchem1964@gmail.com (N. Raman).
1386-1425/$ - see front matter Ó 2013 Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.saa.2013.04.054

R. Mahalakshmi, N. Raman / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 112 (2013) 198–205
199
Development of a new chemotherapeutic Schiff base is now
attracting the attention of medicinal chemist [2]. The heterocyclic
rings containing nitrogen and oxygen import special biological
activity to these Schiff bases and their metal complexes. 1,10-phe-
nanthroline (phen) is a chelating bidentate ligand for transition
metal ions that has played a significant role in the development
of coordination chemistry [3–5]. It is a rigid planar, hydrophobic,
electron-poor heteroaromatic system whose nitrogen atoms are
beautifully placed to act cooperatively in cation binding.
Deoxyribonucleic acid (DNA) plays a significant role in the life
process. It is considered as the primary target molecule for most
anticancer and antiviral therapies according to cell biologists. The
structural features determine its coordination ability toward metal
ions. Transition metal complexes bind to DNA in a covalent or non-
covalent fashion. The interaction between DNA and transition me-
tal complexes is an important essential issue in life sciences [6].
Consequently, the design and synthesis of new transition metal
complexes that interact with DNA are of considerable interest in
nowadays [7–9]. Moreover, considerable attention has been fo-
cused on the use of phenanthroline complexes as intercalating
agents of DNA and as artificial nucleases in coordination chemistry
[10].
ratio of 10 mM) in ethanol. After completion of the addition, the
solution was refluxed on a water bath for 3 h and allowed to cool
by standing at room temperature. The formed solid product was
removed by filtration and recrystallized from ethanol, and dried
in vacuum over fused CaCl2.
Synthesis of metal complexes
A solution of Schiff base and 1,10-phenanthroline in ethanol
was added to a solution of MCl2ꢂ2H2O (1:2:1 M ratio of 10 mM)
in ethanol and the mixture was stirred for 1 h and the solution
was refluxed on a water bath for 2 h. The solid product so formed
was separated by filtration and washed thoroughly with ethanol
and dried in vacuo.
DNA binding experiments
The interaction between metal complexes and DNA was studied
using electronic absorption, viscosity and electrochemical meth-
ods. Disodium salt of calf thymus DNA was stored at 4 °C. All the
experiments involving the interaction of the complexes with calf
thymus (CT) DNA were carried out in Tris–HCl buffer (50 mM
Tris–HCl, pH 7.2) containing 5% DMSO at room temperature. A
solution of CT DNA in the buffer gave a ratio of UV absorbance at
260 and 280 nm of about 1.89:1, indicating the CT DNA sufficiently
free from protein [13]. The concentration of DNA was measured
using its extinction coefficient at 260 nm (6600 Mꢁ1 cmꢁ1) after
1:100 dilution. Stock solutions were stored at 4 °C and used not
more than 4 days. Doubly distilled water was used to prepare solu-
tions. Concentrated stock solutions of the complexes were pre-
pared by dissolving the complexes in DMSO and diluting
properly with the corresponding buffer to the required concentra-
tion for all the experiments.
The use of mixed ligand complexes allows the development of
methods with increased selectivity and has also great importance
in the field of biological and environmental chemistry [11]. Bearing
all the facts in our mind, we herein report the synthesis, structure,
anti-biogram, DNA binding and cleavage studies of mixed ligand
complexes having N-(4-aminophenyl)acetamide Schiff base and
1,10-phenanthroline.
Experimental
Reagents and instruments
Absorption titration experiment was performed by keeping the
concentration of the metal complex as constant at 50
varying the concentration of the CT DNA within 40–400
measuring the absorption spectrum, equal quantity of CT DNA was
added to both the complex solution and the reference solution to
eliminate the absorbance of CT DNA itself. From the absorption
data, the intrinsic binding constant (Kb) was determined from the
l
M while
All reagents N-(4-aminophenyl)acetamide, 4-chlorobenzalde-
hyde, 1,10-phenanthroline and metal(II) chlorides were of Merck
products and used as supplied. Commercial solvents were distilled
and then used for the preparation of ligand and its complexes. DNA
was purchased from Bangalore Genei (India). Microanalyses (C, H
and N) were performed in Carlo Erba 1108 analyzer at Sophisti-
cated Analytical Instrument Facility (SAIF), Central Drug Research
Institute (CDRI), Lucknow, India. Molar conductivities in DMSO
(10ꢁ3 M) at room temperature were measured using Systronic-
model-304 digital conductivity meter. Magnetic susceptibility
measurements of the complexes were carried out by Gouy balance
using copper sulfate pentahydrate as the calibrant. IR spectra were
recorded with Perkin–Elmer 783 spectrophotometer in the 4000–
400 cmꢁ1 range using KBr pellets. NMR spectra were recorded on
a Bruker Avance Dry 300 FT-NMR spectrometer in DMSO-d6 with
TMS as the internal reference. The absorption spectra were re-
corded using Shimadzu model UV-1601 spectrophotometer at
room temperature. Electrochemical measurements were per-
formed on a CHI620C electrochemical analyzer with three elec-
trode system of a glassy electrode as the working electrode, a
platinum wire as auxiliary electrode and Ag/AgCl as the reference
electrode. Solutions were deoxygenated by purging with N2 prior
to measurements.
lM. While
plot of [DNA]/(ea
ꢁef) vs. [DNA] using the following equation:
½DNAꢃ=ðea ef Þ ¼ ½DNAꢃ=ðeb
ꢁ
ꢁ
ef Þ þ ½Kbðeb
ꢁ
ef Þꢃꢁ1
ð1Þ
where [DNA] is the concentration of CT DNA in base pairs. The
apparent absorption coefficients ea ef and eb correspond to Aobs./
,
[M], the extinction coefficient for the free metal(II) complex and
extinction coefficient for the metal(II) complex in the fully bound
form, respectively [14]. Kb is given by the ratio of slope to the
intercept.
Cyclic voltammetry and differential pulse voltammogram stud-
ies were performed on a CHI620C electrochemical analyzer with
three electrode system of glassy carbon as the working electrode,
a platinum wire as auxiliary electrode and Ag/AgCl as the reference
electrode. Solutions were deoxygenated by purging with N2 prior
to measurements.
Viscosity experiments were carried on an Ostwald viscometer,
immersed in a thermostated water-bath maintained at a constant
temperature at 30.0 0.1 °C. CT DNA samples of approximately
0.5 mM were prepared by sonicating in order to minimize com-
plexities arising from CT DNA flexibility [15]. Flow time was mea-
Synthesis of Schiff base ligand and its metal complexes
Synthesis of Schiff base
sured with a digital stopwatch three times for each sample and an
1/3
The preparation of Schiff base ligand was already done by
Raman and Johnson Raja [12]. It was prepared by the dropwise
addition of an ethanolic solution (50 mL) of N-(4-aminophe-
nyl)acetamide into a solution of 4-chlorobenzaldehyde (1:1 M
average flow time was calculated. Data were presented as (
versus the concentration of the metal(II) complexes, where
g/g0)
g
is the
viscosity of CT DNA solution in the presence of complex, and g0 is
the viscosity of CT DNA solution in the absence of complex.

200
R. Mahalakshmi, N. Raman / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 112 (2013) 198–205
Viscosity values were calculated after correcting the flow time of
buffer alone (t0),
= (tꢁt0)/t0 [16].
geometry around the metal ion. Its magnetic moment (1.79 BM)
indicates that the complex exits in monomeric nature. The elec-
tronic spectrum of [Co(L)(phen)2]Cl2 complex showed three broad
g
bands in the visible region at 14,705, 15,267 and 30,581 cmꢁ1
,
DNA cleavage study
4
4
which are assigned to T1g(F) ? 4A2g(F), T1g(F) ? 4A2g(F), and
4T1g(F) ? 4T2g(P) transitions respectively [24–26]. The electronic
spectrum of [Ni(L)(phen)2]Cl2 complex exhibited three d–d bands
at 10,695, 11,049 and 25,575 cmꢁ1, attributed to 3A2g(F) ? 3T2g(F),
The extent of cleavage of super coiled (SC) pUC19 DNA
(33.3 M, 0.2 g) to its nicked circular (NC) form was determined
l
l
by agarose gel electrophoresis in 5 mM Tris–HCl buffer (pH 7.2)
containing 50 mM NaCl. The samples after incubation for 2 h at
37 °C in a dark chamber were added to the loading buffer contain-
ing 25% bromophenol blue, 0.25% xylene cyanol, 30% glycerol
3A2g(F) ? 3T1g(F) and A2g(F) ? 3T1g(P) transitions respectively,
3
being characteristic of an octahedral geometry [27]. The magnetic
measurement values of Co(II) complex (4.82 BM) and Ni(II) com-
plex (3.41 BM) suggest steadiness with their octahedral environ-
ment [28,29]. The electronic spectrum of [Mn(L)(phen)2]Cl2
complex revealed four broad, low intensity bands in the visible re-
gion around 15,822, 23,980, 25,510, 28,089 cmꢁ1 respectively
(3
lL) and the solution was finally loaded on 0.8% agarose gel con-
taining 1
lg/mL ethidium bromide [17]. Electrophoresis was car-
ried out in a dark chamber for 3 h at 50 V in Tris–HCl–EDTA
buffer. Bands were visualized by UV light and photographed. To
enhance the DNA cleaving ability by the complexes, hydrogen per-
which are assigned to 6A1g ? 4T1g 6A1g ? 4T2g(G), 6A1g ? 4Eg
,
6A1g ? 4T2g (D) transitions. The electronic spectral data suggest
an octahedral geometry around Mn(II) ion. The magnetic measure-
ment for Mn(II) complex showed magnetic moment value of
4.82 BM. This observed magnetic moment at room temperature
indicates its monomeric nature and octahedral geometry [30].
The complex of Zn(II) is diamagnetic. According to the empirical
formula, an octahedral geometry is proposed for this complex.
oxide (100 l
mol Lꢁ1) was added into each sample.
Antimicrobial studies
Antibacterial activity of the Schiff base ligand and its metal
complexes was tested in vitro against the bacterial species viz.
Staphylococcus aureus, Pseudomonas aeruginosa, Escherichia coli,
Staphylococcus epidermidis, Klebsiella pneumoniae by the paper disk
method using nutrient agar as the medium. Streptomycin was used
as the standard antibacterial agent. The test organisms were grown
on nutrient agar medium in petri plates. Disks were prepared and
applied over the long culture. The compounds were prepared in
DMSO and soaked in filter paper disk of 5 mm diameter and
1 mm thickness. The concentration of ligand and the complexes
1H NMR spectra
The 1H NMR spectra of the Schiff base and its zinc complex were
recorded at room temperature in DMSO-d6. 1H NMR spectrum of
the Schiff base ligand showed peaks at 7.2–7.8 d which are attrib-
uted to phenyl multiplet of Schiff base ligand. The Ph-NH- group
gave singlet at 9.8 d in the free ligand and remained unchanged
in the zinc complex. It showed that the Ph-NH- group was not tak-
ing part in complexation. The azomethine proton (ACH@N) signal
in the spectrum of the ligand showed a signal at 8.5 d. The azome-
thine proton (ACH@N) signal in the spectrum of the zinc complex
showed a downfield shift (8.1 d) compared to the free ligand, sug-
gesting deshielding of azomethine group due to the coordination
with metal ion. The aromatic methyl proton showed (CACH3) sig-
nal at 3.4 d. There was no appreciable change in all other signals of
the complex [31].
used in this study was 0.01 lg/mL. The disks were placed on the
previously seeded plates and incubated at 37 °C and the diameter
of inhibition zone around each disk was measured after 24 h for
antibacterial activity. Growth inhibition was calculated according
to Ref. [18].
Results and discussion
Synthetic routes for the preparation of different compounds are
depicted in Scheme 1. The ligand and its complexes are found to be
stable in air. The ligand (L) is soluble in common organic solvents
but the complexes are soluble only in DMF and DMSO.
IR spectra
The IR spectra provide valuable information regarding the nat-
ure of functional group attached to the metal atom. In order to
study the bonding mode of Schiff base to the metal complexes,
the IR spectrum of the free ligand is compared with the spectra
of the complexes. The main IR bands and their assignments are
listed in Table 3. A sharp band observed at 1625 cmꢁ1 in the IR
spectrum of the Schiff base ligand (>C@N) was shifted downward
by about 1602–1618 cmꢁ1 in all the complexes indicating coordi-
nation through azomethine nitrogen [32]. The band in Schiff base
ligand at 1662 cmꢁ1due to (C@O) vibration, appeared at 1647–
1656 cmꢁ1 in all the complexes. This shows chelation of (C@O)
group with metal ion. The unaltered position of bands due to
Elemental analysis and molar conductivity measurements
The results of elemental analysis for the metal complexes are in
good agreement with the calculated values (Table 1) showing that
the complexes of stoichiometry [M(L)(phen)2]Cl2 [19]. The meta-
l(II) complexes were dissolved in DMSO and the molar conductiv-
ities of 10ꢁ3 M of their solution at room temperature were
measured. The higher conductance values of the complexes sup-
port their electrolytic nature, implying the non-coordination of
chloride anion to the central metal ion. The presence of counter
chloride ion is confirmed from Volhard’s test.
t
(NH) moiety of the N-(4-aminophenyl)acetamide in all the metal
complexes indicated that these groups were not involved in coor-
dination. The band at 3061 cmꢁ1
(NH)) did not change for ligand
and complexes. The IR values of
(CAH) observed at 860 cmꢁ1 and
Magnetic susceptibility and ultraviolet spectral measurements
(
m
t
The free Schiff base ligand (L) exhibits two intense bands at
33,783 and 26,737 cmꢁ1 due to
p
–
pꢄ and n–pꢄ transitions respec-
735 cmꢁ1 for phenanthroline were red shifted to 852 cmꢁ1 and
729 cmꢁ1 respectively. These shifts can be explained by the fact
that each of the two nitrogen atoms of phenanthroline ligands do-
nates a pair of electrons to the central metal forming a coordinate
tively. The electronic absorption spectral data for the ligand and
the complexes are given in Table 2. The electronic spectrum of
[Cu(L)(phen)2]Cl2 complex exhibited intraligand charge transfer
bands at ca. 33,989 and 37,037 cmꢁ1 and
a
d–d band at
covalent bond [33]. Ring stretching frequencies (
(C@N)) at 1503 and 1420 cmꢁ1 for free phen were shifted to
higher frequencies upon complexation (5–14 cmꢁ1 and
t(C@C) and
15,243 cmꢁ1 which was due to 2Eg ? 2T2g transition [20–23]. This
(t
d–d band transition band strongly favored a distorted octahedral

R. Mahalakshmi, N. Raman / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 112 (2013) 198–205
201
Scheme 1. Schematic route for synthesis of Schiff base ligand and its metal complexes.
Table 1
Analytical and physical data of Schiff base ligand and its complexes.
a
Compound
Yield (%)
Color
Found (calc)%
Formula weight
(^m
)
leff (BM)
M
C
H
N
[C15H13ON2Cl]
85
74
68
64
62
60
Yellow
65.3
(66.0)
69.8
(67.2)
67.1
(67.7)
67.1
(67.6)
66.7
(67.1)
67.8
4.5
(4.8)
3.9
(4.2)
3.7
(4.2)
3.8
(4.2)
3.9
(4.1)
3.6
10.0
(10.3)
12.2
(12.6)
11.8
(12.1)
11.7
(12.1)
11.8
(12.0)
11.6
–
8.9
(9.1)
8.2
(8.5)
8.3
(8.5)
9.0
(9.3)
7.6
272
696
–
140
–
1.79
[CuC39H29N6OCl]
[NiC39H29N6OCl]
[CoC39H29N6OCl]
[ZnC39H29N6OCl]
[MnC39H29N6OCl]
Dark green
Pale green
Pale brown
Yellow
691
692
698
688
90
3.41
145
138
146
4.82
Diamagnetic
5.75
Dark yellow
(7.9)
(68.0)
(4.2)
(12.2)
a
The unit of the molar conductance is Oꢁ1 molꢁ1 cm2.

202
R. Mahalakshmi, N. Raman / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 112 (2013) 198–205
Table 2
Electronic absorption spectral data of the synthesized compounds at 300 K.
S. no
1
Compound
L
Solvent
DMSO
Absorption (cmꢁ1
)
Band assignment
Geometry
–
33,783
26,737
15,243
37,037
33,898
14,705
15,267
30,581
10,695
11,049
25,575
15,822
23,980
25,510
28,089
INCT
INCT
2
3
4
5
[CuL (phen)2]Cl2
[CoL(phen)2]Cl2
[NiL(phen)2]Cl2
[MnL(phen)2]Cl2
DMSO
DMSO
DMSO
DMSO
2Eg
?
2T2g
Distorted octahedral
Octahedral
INCT
INCT
4T1g(F) ? 4T2g(F),
4T1g(F) ? 4A2g(F),
4T1g(F) ? 4T2g(P)
3A2g (F) ? 3T2g(F),
3A2g(F) ? 3T1g(F),
3A2g(F) ? 3T1g(P)
Octahedral
6A1g
6A1g
6A1g
6A1g
?
?
?
?
4T1g
,
Octahedral
4T2g(G),
4Eg
4T2g(D)
Table 3
IR data of Schiff base ligand and its metal complexes.
Compound
tNAH
tC@O
tHC@N
tMAN
tMAO
[C15H13ON2Cl]
3061
3059
3058
3057
3055
3056
1662
1647
1656
1651
1654
1652
1625
1604
1610
1618
1616
1609
–
–
[CuC39H29N6OCl]
[NiC39H29N6OCl]
[CoC39H29N6OCl]
[ZnC39H29N6OCl]
[MnC39H29N6OCl]
420
424
418
422
416
518
544
520
520
524
3–10 cmꢁ1 for all complexes) indicating coordination of the het-
erocyclic nitrogen. Absorptions at 516–544 and 415–424 cmꢁ1
were ascribed to the formation of MAO and MAN bonds, respec-
tively [34].
DNA binding of the metal complexes
Absorption spectral features of DNA binding
Fig. 1. Absorption spectrum of [CuC39H29N6OCl] complex in buffer pH = 7.2 at 25 °C
in the presence of increasing amount of DNA. Arrow indicates the changes in
absorbance upon increasing the DNA concentration.
Titration with electronic absorption spectroscopy is an effective
method to investigate the binding mode of DNA with metal com-
plexes [35]. Transition metal complexes can bind to DNA via both
covalent and/or non-covalent interactions. In the case of covalent
binding, the labile ligand of the complexes can be replaced by a
nitrogen base of DNA such as guanine N7, while the non-covalent
DNA interactions include intercalative, electrostatic and groove
(surface) binding of metal complexes outside of DNA helix, along
major or minor groove. Generally, the binding of an intercalative
molecule to DNA is accompanied by hypochromism and/or signif-
icant red shift (bathochromism) in the absorption spectra due to
the strong stacking interaction between the aromatic chromophore
of the ligand and DNA base pairs with the extent of hypochromism
and red-shift commonly consistent with the strength of the inter-
calative interaction [36]. Therefore, in order to obtain evidence for
the binding ability of each compound to CT DNA, spectroscopic
titration of compound solutions with CT DNA should be performed
[37]. The electronic absorption spectra of copper complex in the
absence and presence of CT DNA are given in Fig. 1. In the UV re-
gion, the intense absorption bands observed in the metal com-
Table 4
Electronic absorption spectral properties of Cu(II), Ni(II), Co(II) and Mn(II) complexes
with DNA.
Compound
k max
D
k/nm
aH%
bKb (Mꢁ1
)
Free
Bound
[CuC39H29N6OCl]
[NiC39H29N6OCl]
[CoC39H29N6OCl]
[MnC39H29N6OCl]
270
269
327
264
265.4
266.2
319.0
261.6
4.6
2.8
8.0
2.4
32
16
21
11
1.4 ꢅ 106
4.2 ꢅ 105
1.9 ꢅ 105
6.4 ꢅ 104
a
H% = [AfreeꢁAbound)/Afree] ꢅ 100%.
b
Kb = Intrinsic DNA binding constant determined from the UV–Vis absorption
spectral titration.
4.2 ꢅ 105 Mꢁ1
,
1.4 ꢅ 106 Mꢁ1 and 6.4 ꢅ 104 Mꢁ1
, respectively
plexes are attributed to the intra ligand
p–
pꢄ transition of the
(shown in Table 4). The results indicate that the binding strength
of the complexes is decreased in the order of Cu(II) > Ni(II) > -
Co(II) > Mn(II). The higher values of Cu(II), Ni(II) and Co(II) com-
plexes and lower value of Mn(II) complex are compared with
that of the reported for classical intercalators (for ethidium bro-
mide in 25 mM Tris–HCl/40 mM NaCl buffer, pH = 7.2 and
[Ru(phen)(dppz)], the binding constants have been found to be in
the order of 106–107 Mꢁ1) [38,39]. Especially, Cu(II), Ni(II) and
Co(II) complexes have strong DNA binding ability as compared
coordinated groups. With increasing concentration of CT DNA, for
the Co(II), Ni(II), Cu(II) and Mn(II) complexes, the absorption bands
at 327, 269, 270 and 264 nm respectively also show hypochromism
of 21%, 16%, 32% and 11%, respectively. The hypochromisms ob-
served for all the metal complexes are accompanied by small red
shifts by about 2, 3, 1.5 and 2 nm respectively. The spectroscopic
changes suggest that the intrinsic binding constants (Kb) of the
Co(II), Ni(II) Cu(II) and Mn(II) complexes are 1.9 ꢅ 105 Mꢁ1
,

R. Mahalakshmi, N. Raman / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 112 (2013) 198–205
203
Fig. 2. Effect of increasing amounts of [CuC39H29N6OCl] ( ), [NiC39H29N6OCl] ( ),
[CoC39H29N6OCl] (ꢅ) [MnC39H29N6OCl] ( ) on the relative viscosity of CT DNA vs
[complex]/[DNA] (1/R) ratio.
Fig. 3. Cyclic voltammogram of [CuC39H29N6OCl] in buffer (pH = 7.2) at 25 °C in the
presence of increasing amount of DNA. Arrow indicates the changes in voltammet-
ric currents upon increasing the DNA concentration.
Table 5
Electrochemical parameters for the interaction of DNA with Cu(II), Ni(II), Co(II) and
Mn(II) complexes.
[46]. As further exploring the binding of the present complexes
with DNA, cyclic and differential pulse voltammetric studies were
carried out both in the presence and absence of DNA. The incre-
mental addition of CT DNA to the complex causes decrease of
anodic and cathodic peak current of the complex. This result shows
that complex stabilizes the duplex (GC pairs) by intercalating way.
The incremental addition of CT DNA to the complex causes shift in
the potential of peak in cyclic voltammogram. Both the cathodic
and anodic peaks show positive shifts which indicate intercalation
of complex to DNA base pairs. Electrochemical parameters for the
interaction of DNA with Cu(II), Ni(II), Co(II) and Mn(II) complexes
are shown in Table 5.
Compound
aE1/2 (V)
Free
bDEp(V)
Ipc/
Ipa
K[red]/
K[oxd]
Bound
Free
Bound
[CuC39H29N6OCl]
[NiC39H29N6OCl]
[CoC39H29N6OCl]
[MnC39H29N6OCl] ꢁ0.677 ꢁ0.686 0.190 0.192
0.309
0.323 0.601 0.646
1.48
1.43
1.42
1.38
1.85
0.85
0.86
0.60
ꢁ0.682 ꢁ0.694 0.282 0.253
ꢁ0.685 ꢁ0.669 0.193 0.237
Data from cyclic voltammetric measurements.
a
E1/2(V) is calculated as average of anodic (EPa) and (Epc) peak potential
E1/2 = EPa + Epc/2.
b
D
Ep(V) = EPaꢁEpc.
The cyclic voltammogram of [CuC39H29N6OCl] complex in the
absence and in presence of varying amount of DNA is shown in
Fig. 3. In the absence of CT DNA, the redox cathodic peak appeared
with the other complex Mn(II). The electronic absorption spectra of
cobalt and nickel complexes in the absence and presence of CT
DNA are given in supplementary files (Fig. S1, Fig. S2). These results
suggest an intimate association of the compounds with CT-DNA
and it is also likely that compounds bind to the helix via
intercalative mode. Hypochromism has been suggested for the
interaction between the electronic state of the intercalating chro-
mophore and that of the DNA bases [40–44].
at 0.0077 V for Cu-complex [DEp = 0.601 V and E1/2 = 0.309 V]. The
ratio of ipc/ipa is approximately 1.48 which indicates that the reac-
tion of the complex on the glassy carbon electrode surface is quasi-
reversible redox process. The incremental addition of CT-DNA to
the complex causes a positive shift in potential and a decrease in
the current intensity. Among the three kinds of binding modes for
small molecules to DNA, Bard has reported [47] that if E1/2 is shifted
to more negative value when small molecules interact with DNA,
then the interaction mode is electrostatic binding. On the contrary,
if E1/2 is shifted to more positive value, the interaction mode is
Viscosity measurements
To know the nature of DNA binding of the mixed ligand com-
plexes, viscosity measurements are carried out on CT DNA by vary-
ing the concentration of the added complexes. The values of
changes in the relative specific viscosities of CT DNA in presence
and absence of the complex respectively are plotted against 1/R,
[complex]/[DNA]. A classical intercalation mode causes a signifi-
cant increase in viscosity of DNA due to an increase in separation
of base pairs at intercalation sites and hence an increase in overall
DNA length. The results indicate that the presence of the metal
complex increases the viscosity of the DNA solution, as illustrated
in Fig. 2. As general rule metal complexes can increase the viscosity
of DNA when they intercalate into the double-stranded DNA (or)
bind to the phosphate group of DNA backbone [45]. The plots show
that the relative viscosity increases with increase in concentration
of complexes. The observed order of DNA-binding in viscosity mea-
surements suggests that metal complexes bind DNA with a moder-
ate intercalative mode.
DNA binding of electrochemical behavior
The application of cyclic voltammetry (CV) to the study of bind-
ing of metal complexes to DNA provides a useful complement to
the method for the investigation of electronic absorption spectra
Fig. 4. Cyclic voltammogram of [NiC39H29N6OCl] in buffer (pH = 7.2) at 25 °C in the
presence of increasing amount of DNA. Arrow indicates the changes in voltammet-
ric currents upon increasing the DNA concentration.

204
R. Mahalakshmi, N. Raman / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 112 (2013) 198–205
In differential pulse voltammogram of the complex, [MnC39H29-
N6OCl] in the absence and presence of varying amount of [DNA]
with significant decrease of current intensity (Fig. 5), the shift in
potential is related to the ratio of binding constant by the following
equation:
Eb ꢁ Ef ¼ 0:0591 logðK =K½oxdꢃÞ
½redꢃ
where Eb and Ef are peak potentials of the complex in the bound and
free form respectively. All other complexes show considerable shift
in both cathodic and anodic peak potentials in the presence of
incremental addition of CT DNA. It is found that our synthesized
complexes give both the anodic and cathodic peak potential shifts
towards positive direction, i.e. positive (in copper complex) or from
more negative region to less negative region (in other complexes). It
indicates the intercalating mode of DNA binding with phen mixed
ligand Schiff base complexes.
DNA cleavage study
Fig. 5. Differential pulse voltammograms of [MnC39H29N6OCl] in buffer (pH = 7.2)
at 25 °C in the presence of increasing amount of DNA. Arrow indicates the changes
in voltammetric currents upon increasing the DNA concentration.
DNA cleavage can be achieved by targeting its basic compo-
nents like phosphodiester linkages, deoxyribose sugar or nucleo-
bases. Transition metal complexes of polypyridyl ligands are
known to cleave DNA under irradiation by UV or visible light. Gel
electrophoresis experiments using pUC19 plasmid DNA were per-
formed with free ligand and its complexes in the presence and ab-
sence of H2O2 as an oxidant. When super coiled DNA is conducted
by electrophoresis, faster migration will be observed for DNA of
closed circular confirmation (Form I). If one strand is cleaved, the
supercoiled DNA will relax to produce a slower moving nicked cir-
cular form (Form II). If both strands are cleaved, a linear confirma-
tion (Form III) that migrates between Form I and Form II will be
generated [48]. This method using supercoiled pUC19 DNA in the
presence of the ligand and its metal complexes was carried out
intercalative binding. Hence, it is concluded that the [CuC39H29N6-
OCl] complex could bind to DNA by intercalative mode.
For the CV behavior of [NiC39H29N6OCl] in the absence of DNA,
the redox couple cathodic peak appeared at ꢁ0.824 V for Ni-com-
plex (
D
Ep = 0.282 V and E1/2 = ꢁ0.682 V). Ipc/Ipa ratio of redox cou-
ple is approximately 1.43. This indicates that the reaction of the
complex on the working electrode surface is quasi-reversible redox
process. The results are similar to the above spectroscopic and vis-
cosity data of the complexes in the presence of DNA. The cyclic vol-
tammogram of [NiC39H29N6OCl] complex in the absence and in
presence of varying amount of DNA is shown in Fig. 4.
For the CV behavior of [CoC39H29N6OCl] in the absence of DNA,
the redox couple cathodic peak appeared at ꢁ0.781 V for Co-com-
in a medium of 50 lM Tris–HCl/NaCl buffer (pH 7.2). From figure
(Fig. 6) it is evident that the complexes cleave DNA more efficiently
in the presence of an oxidant (H2O2). This may be attributed to the
formation of hydroxyl free radicals. All the complexes showed pro-
nounced nuclease activity in the presence of oxidant H2O2 which
may be due to the increased production of hydroxyl radicals. Con-
trol experiments using DNA alone did not show any significant
cleavage of pUC19 DNA even on longer exposure time. From the
observed results, it is concluded that all the complexes effectively
cleave the DNA as compared to control DNA.
plex (
D
Ep = 0.193 V and E1/2 = ꢁ0.685 V). The ratio of ipc/ipa is
approximately 1.42. It indicates that the reaction of the complex
on the glassy carbon electrode surface is quasi-reversible redox
process. The cyclic voltammogram of [CoC39H29N6OCl] complex
in the absence and in presence of varying amount of DNA is shown
in Supplementary file (Fig. S3).
For the CV behavior of [MnC39H29N6OCl] in the absence of DNA,
the redox couple cathodic peak appeared at ꢁ0.783 V for Mn-com-
plex (D
Ep = 0.190 V and E1/2 = ꢁ0.677 V). The ratio of ipc/ipa is
approximately 1.38. It indicates that the reaction of the complex
on the glassy carbon electrode surface is quasi-reversible redox
process. The cyclic voltammogram of [MnC39H29N6OCl] complex
in the absence and in presence of varying amount of DNA is shown
in Supplementary file (Fig. S4). From these data it is understood
that all the synthesized complexes interact with DNA through
intercalating way.
Antimicrobial studies
The antibacterial activity of the Schiff base ligand, its metal
complexes and Streptomycin (as a standard compound) were
tested against bacteria. The organisms used in the present
investigations included S. aureus, P. aeruginosa, E. coli, S. epidermi-
dis, K. pneumoniae. The diffusion agar method was used to evaluate
Fig. 6. The gel electrophoretic separation of plasmid pUC19 DNA treated with [M(L)(phen)2]Cl2 complexes. Lane 1; DNA control ; Lane 2: DNA + ligand + H2O2; Lane 3:
DNA + [Co(L)(phen)2]Cl2; Lane 4: DNA + [Cu(L)(phen)2]Cl2 + H2O2; Lane 5: DNA + [Zn(L)(phen)2]Cl2 + H2O2; Lane 6: DNA + [Ni(L)(phen)2]Cl2 + H2O2; Lane 7:
DNA + [Mn(L)(phen)2]Cl2 + H2O2.

R. Mahalakshmi, N. Raman / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 112 (2013) 198–205
205
References
Table 6
The in vitro antibacterial activity of Schiff base and its metal complexes (zone of
inhibition in mm).
[1] B. Witkop, L.K. Ramachandran, Metabolism 13 (1964) 1016–1025.
[2] B. Katia, L. Simon, R. Anne, C. Gerard, D. Francoise, M. Bernard, Inorg. Chem. 35
(1996) 387–396.
Compound
S.
P.
E. coli S.
K.
aureus aeruginosa
epidermidis pneumoniae
[3] L.A. Summers, Adv. Heterocycl. Chem. 22 (1978) 1–69.
[4] P.G. Sammes, G. Yahioglu, Chem. Soc. Rev. 23 (1994) 327–334.
[5] C.R. Luman, F.N. Castellano, in: J.A. McCleverty, T.J. Meyer, A.B.P. Lever (Eds.),
Comprehensive Coordination Chemistry, vol. 1, Elsevier, Oxford, UK, 2004, p.
25.
[6] Q. Zhang, J. Liu, H. Chao, G. Xue, L. Ji, J. Inorg. Biochem. 83 (2001) 49–55.
[7] X.B. Yang, Y. Huang, J. Zhang, S.K. Yuan, R.Q. Zeng, Inorg. Chem. Commun. 13
(2010) 1421–1427.
[C15H13ON2Cl]
++
+
++
+
++
[CuC39H29N6OCl]
[NiC39H29N6OCl]
[CoC39H29N6OCl]
[MnC39H29N6OCl] +++
Streptomycin
DMSO
++++
++++
+++
+++
+++
++
++
+++
ꢁ
+++
+++
++++
+++
+++
ꢁ
+++
++
+++
++
++
ꢁ
++++
+++
++
++++
++
++
ꢁ
ꢁ
[8] S. Anbu, M. Kandaswamy, Polyhedron 30 (2011) 123–131.
[9] J. Sun, S. Wu, H.Y. Chen, F. Gao, J. Liu, L.N. Ji, Z. Mao, Polyhedron 30 (2011)
1953–1959.
Streptomycin is used as the standard.
DMSO was used as antimicrobial inert solvent.
+++++ = Excellent activity (100% inhibition).
+++ = Good activity (60–70% inhibition).
++ = Moderate activity (30–50% inhibition).
+ = Less activity (10–20% inhibition).
[10] W.K. Pogozelski, T.D. Tullius, Chem. Rev. 98 (1998) 1089–1107.
[11] E. Casassas, A. Izquierdo-Ridora, R. Tauler, J. Chem. Soc., Dalton Trans. (1990)
2341–2345.
[12] N. Raman, S. Johnson Raja, J. Serb. Chem. Soc. 72 (2007) 983–992.
[13] J. Marmur, J. Mol. Biol. 3 (1961) 208–218.
[14] L.F. Tan, X.H. Liu, H. Chao, L.N. Ji, J. Inorg. Biochem. 101 (2007) 56–63.
[15] J.B. Charies, N. Dattagupta, D.M. Cromers, Biochemistry 21 (1982) 3933–3937.
[16] S. Satyanarayanan, J.C. Davorusak, J.B. Charies, Biochemistry 32 (1983) 2573–
2584.
[17] N. Raman, S. Sobha, Spectrochim. Acta A 93 (2012) 250–259.
[18] A. Rahman, M.I. Choudhary, W.J. Thomsen, Bioassay Techniques for Drug
Development, Harwood Academic Publishers, The Netherlands, 2001. pp. 16.
[19] J.D. Ranford, P.J. Sadler, D.A. Tocher, J.Chem. Soc., Dalton Trans. (1993) 3393–
3399.
the antibacterial activity of the synthesized metal complexes. The
results of the bactericidal study of the synthesized compounds
are displayed in Table 6. All the metal complexes were found to
have higher antibacterial activity than Schiff base ligand. The
DMSO control showed no activity against any bacterial strain. Such
increased activity of the complexes can be explained on the basis of
the Overtone’s concept [49] and Tweedy’s chelation theory [50].
According to the Overtone’s concept of cell permeability, the lipid
membrane that surrounds the cell favors the passage of only the li-
pid-soluble materials due to which liposolubility is an important
factor that controls the antimicrobial activity.
[20] A.B.P. Lever, Inorganic Electronic Spectroscopy, 2nd ed., Elsevier, New York,
1968.
[21] L. Sacconi, M. Chiampolini, J. Chem. Soc. A 276 (1964) 1442–1454.
[22] L.N. Sharada, M.C. Ganorkar, Ind. J. Chem. 27A (1988) 617–621.
[23] L. Sacconi, Trans. Metal. Chem. 4 (1968) 199–298.
[24] F.A. Cotton, G. Wilkinson, C.A. Murillo, M. Bochman, Advanced Inorganic
Chemistry, Sixth ed., Wiley, New York, 1999.
[25] G.G. Mohamed, Z.H. Abd, El. Wahwb, J. Therm. Anal. 73 (2003) 347–359.
[26] M.M. Omar, G.G. Mohamed, Spectrochim. Acta A 61 (2005) 929–936.
[27] G.G. Mohamed, N.A. Ibrahim, H.A.E. Attia, Spectrochim. Acta A 72 (2009) 610–
615.
Conclusion
[28] P.P. Hankare, S.R. Naravane, V.M. Bhuse, S.D. Delekar, A.H. Jagtap, Ind. J. Chem.
43A (2004) 1464–1468.
[29] T.R. Rao, P. Archan, Synth. React. Inorg. Metal.-Org. Chem. 35 (2005) 299–304.
[30] S. Roy, T.N. Mandal, A.K. Barik, S. Gupta, R.J. Butcher, M. Nethaji, S.K. Kar,
Polyhedron 27 (2008) 593–601.
[31] N. Raman, S. Sobha, Spectrochim. Acta A 85 (2012) 223–234.
[32] G. Mukherjee, S.N. Poddar, Ind. J. Chem. 25 (A) (1986) 275.
[33] R. Senthil Kumar, S. Arunachalam, Polyhedron 26 (2007) 3255–3262.
[34] B.S. Creaven, M. Devereux, A. Foltyn, S. McClean, G. Rosair, V.R. Thangella, M.
Walsh, Polyhedron 29 (2010) 813–822.
[35] J.M. Kelly, A.B. Tossi, D.J. McConnell, C.O. Uigin, Nucl. Acids Res. 13 (1985)
6017–6034.
[36] H. Chao, W.J. Mei, Q.W. Huang, L.N. Ji, J. Inorg. Biochem. 92 (2002) 165–170.
[37] Y.M. Song, Q. Wu, P.J. Yang, N.N. Luan, L.F. Wang, Y.N. Liu, J. Inorg. Biochem.
100 (2006) 1685–1691.
In this paper, a novel N-(4-aminophenyl)acetamide derived
Schiff base and its Cu(II), Ni(II), Co(II), Mn(II) and Zn(II) complexes
have been synthesized and characterized by spectral and analytical
data. Binding of these complexes to CT-DNA has been investigated
in detail by electronic absorbance titrations, viscosity, cyclic
voltammetry and differential pulse voltammogram analysis. The
binding order is found to be in the following order Cu(II) > Ni(II)
> Co(II) > Mn(II). In addition, all of these metal complexes are
found to promote the photocleavage of pUC19 DNA. All the metal
complexes are found to have higher antibacterial activity than
Schiff base ligand.
[38] M. Cory, D.D. McKee, J. Kagan, D.W. Henry, J.A. Miller, J. Am. Chem. Soc. 107
(1985) 2528–2536.
[39] M.J. Waring, J. Mol. Biol. 13 (1965) 269–282.
Acknowledgements
[40] E.C. Long, J.K. Barton, Acc. Chem. Res. 23 (1990) 271–273.
[41] G. Dougherty, W.J. Pigram, Crit. Rev. Biochem. 12 (1982) 103–132.
[42] H.M. Berman, P.R. Young, Annu. Rev. Biophys. Bioeng. 10 (1981) 87–114.
[43] C. Cantor, P.R. Schimmel, W.H. Freeman, Biophys. Chem. San Francisco 2
(1980) 398–404.
[44] T.A. Tysco, R.J. Morgan, A.D. Baker, T.C. Strekas, J. Phys. Chem. 97 (1993) 1707–
1711.
[45] F. Gao, H. Chao, F. Zhou, Y.X. Yuan, B. Peng, L.N. Ji, J. Inorg. Biochem. 100 (2006)
1487–1494.
[46] S. Mahadevan, M. Palaniandavar, Bioconjugate Chem. 7 (1996) 138–143.
[47] Q.-L. Zhang, J.-G. Liu, H. Xu, H. Li, J.-Z. Liu, H. Zhou, L.-H. Qu, L.-N. Ji, Polyhedron
20 (2001) 3049–3055.
[48] J.K. Barton, A.T. Danishefsky, J.M. Goldberg, J. Am. Chem. Soc. 106 (1984) 2172–
2176.
The authors express their heartfelt thanks to the College Man-
aging Board, Principal, and Head of the Department of Chemistry,
VHNSN College, for providing necessary research facilities. SAIF,
IIT Bombay and CDRI, Lucknow, are gratefully acknowledged for
providing instrumental facilities. NR thanks UGC, New Delhi for
financial assistance.
Appendix A. Supplementary material
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.saa.2013.04.054.
[49] Y. Anjaneyalu, R.P. Rao, Synth. React. Inorg. Org. Chem. 16 (1986) 257–272.
[50] N. Raman, A. Sakthivel, K. Rajasekaran, J. Coord. Chem. 62 (2009) 1661.