Exploring the DNA binding mode of transition metal based biologically active compounds

Article abstract of DOI:10.1016/j.saa.2011.09.065

Few novel 4-aminoantipyrine derived Schiff bases and their metal complexes were synthesized and characterized. Their structural features and other properties were deduced from the elemental analysis, magnetic susceptibility and molar conductivity as well

Full text of DOI:10.1016/j.saa.2011.09.065

Spectrochimica Acta Part A 85 (2012) 223–234
Contents lists available at SciVerse ScienceDirect
Spectrochimica Acta Part A: Molecular and
Biomolecular Spectroscopy
journal homepage: www.elsevier.com/locate/saa
Exploring the DNA binding mode of transition metal based biologically
active compounds
N. Raman∗, S. Sobha
Research Department of Chemistry, VHNSN College, Virudhunagar 626 001, India
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 6 July 2011
Received in revised form 6 September 2011
Accepted 30 September 2011
Few novel 4-aminoantipyrine derived Schiff bases and their metal complexes were synthesized and
characterized. Their structural features and other properties were deduced from the elemental analy-
sis, magnetic susceptibility and molar conductivity as well as from mass, IR, UV–vis, 1H NMR and EPR
spectral studies. The binding of the complexes with CT-DNA was analyzed by electronic absorption spec-
troscopy, viscosity measurement, and cyclic voltammetry. The interaction of the metal complexes with
DNA was also studied by molecular modeling with special reference to docking. The experimental and
docking results revealed that the complexes have the ability of interaction with DNA of minor groove
binding mode. The intrinsic binding constants (Kb) of the complexes with CT-DNA were found out which
show that they are minor groove binders. Gel electrophoresis assay demonstrated the ability of the
complexes to cleave the pUC19 DNA in the presence of AH2 (ascorbic acid). Moreover, the oxidative
cleavage studies using distamycin revealed the minor groove binding for the newly synthesized 4-
aminoantipyrine derived Schiff bases and their metal complexes. Evaluation of antibacterial activity of the
complexes against Staphylococcus aureus, Pseudomonas aeruginosa, Escherichia coli, Staphylococcus epider-
midis, and Klebsiella pneumoniae exhibited that the complexes have potent biocidal activity than the free
ligands.
Keywords:
Biologically active complexes
Schiff base
DNA binding
Molecular docking
Chemical nuclease activity
Biocidal activity
© 2011 Elsevier B.V. All rights reserved.
1. Introduction
cis-platin and to develop another class of metallo drugs which are
less toxic and more effective for chemotherapeutic applications,
The study on the metal based biologically active compounds has
attained growing interest largely due to their ability to interact with
DNA [1]. Metal complexes can interact with DNA through three
non-covalent modes viz. intercalation, groove binding and external
static electronic effects. Among these modes, the groove binding
mode plays an important role in the efforts of the drug targeted
to DNA [2]. A large number of transition metal complexes have
been used as cleavage agents for DNA and also for novel poten-
tial DNA-targeted antitumor drugs. This is essential for further
expected applications in many areas like biological and medici-
nal significance as potential artificial gene regulators (or) cancer
chemotherapeutic agents [3].
The most important application is the antitumor action of cer-
tain heavy metals, which work by binding to and distorting DNA,
causing cell death. For example, cis-platin is one of the most potent
and widely used anticancer drugs in use today. Clinical problems of
cis-platin include, acquired cis-platin resistance and limited spec-
trum of cancers that it can cure [4]. To overcome these limitations of
researchers have been continuously investigating the interaction
of other metal complexes with DNA. In addition to this, the coor-
dination chemistry of Schiff base complexes involving oxygen and
nitrogen donor ligands has excited great interest among chemists
in recent years due to their applications in catalysis and their rele-
vance to bioinorganic systems [5].
In recent years, 4-aminoantipyrine transition metal complexes
and their derivatives have been extensively examined due to their
wide applications in various fields like biological, analytical and
therapeutical. Further, they have been investigated due to their
diverse biological properties as antifungal, antibacterial, analgesic,
sedative, antipyretic, anti-inflammatory agents [6] and DNA bind-
ing properties [7].
Having all the above facts in mind, herein we have synthesized
and explored the structural determination of 4-aminoantipyrine
based Schiff bases having oxygen and nitrogen donors, derived
from 4-aminoantipyrine, 2-hydroxy benzaldehyde/2-hydroxy 3-
methoxybenzaldehyde and 2-amino benzoic acid with Cu(II), Ni(II),
Zn(II) metal ions. Binding properties of these metal complexes with
DNA and their antimicrobial evaluation have been researched. The
results would be valuable in understanding the novel agents for
targeting nucleic acids as well as for the synthesis of novel antibac-
terial drugs.
∗
Corresponding author. Tel.: +91 9245165958; 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.2011.09.065

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N. Raman, S. Sobha / Spectrochimica Acta Part A 85 (2012) 223–234
2. Experimental
2.1. Materials and methods
DNA in the buffer (50 mM NaCl, 5 mM Tris–HCl (pH 7.2)) in water
gave a ratio 1.8–1.9, of UV absorbance at 260 and 280 nm, A260/A280
,
indicating that the DNA was sufficiently free from protein [8]. The
concentration of DNA was measured using its extinction coefficient
at 260 nm (6600 M−1 cm−1). Stock solutions were stored at 4 ◦C and
used no more than 4 days. Doubly distilled water was used to pre-
pare solutions. Concentrated stock solutions of the complexes were
prepared by dissolving the complexes in DMSO and diluting suit-
ably with the corresponding buffer to the required concentration
for all of the experiments. The data were then fitted to Eq. (1) to
obtain the intrinsic binding constant (Kb) values for interaction of
the complexes with DNA.
All reagents, 4-aminoantipyrine, 2-hydroxybenzaldehyde/2-
hydroxy-3-methoxybenzaldehyde, 2-aminobenzoic acid and var-
ious metal(II) chlorides, were procured from Merck products.
Commercial solvents were distilled and then used for the prepa-
ration of ligands and their complexes. pUC19 DNA was purchased
from Bangalore Genei (India). Microanalyses (C, H and N) were
performed in Carlo Erba 1108 analyzer at Sophisticated Analyt-
ical Instrument Facility (SAIF), Central Drug Research Institute
(CDRI), Lucknow, India. Molar conductivities in DMSO (10−3 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
sulphate pentahydrate as the calibrant. IR spectra were recorded
with Perkin-Elmer 783 spectrophotometer in the 4000–400 cm−1
range using KBr pellets. NMR spectra were recorded on a Bruker
Avance Dry 300 FT-NMR spectrometer in CDCl3 with TMS as the
internal reference. FAB-MS spectra were recorded with a VGZAB-
HS spectrometer at room temperature in a 3-nitrobenzylalcohol
matrix. Electron paramagnetic resonance spectra of the copper
complexes were recorded on a Varian E 112 EPR spectrome-
ter in DMSO solution both at room temperature (300 K) and
liquid nitrogen temperature (77 K) using TCNE (tetracyanoethy-
lene) as the g-marker. The absorption spectra were recorded
using Shimadzu model UV-1601 spectrophotometer at room
temperature.
[DNA]
(εa − εf)
[DNA]
(εb − εf)
1
=
+
(1)
[Kb(εb − εf)]
where εa, εf, and εb are the apparent, free and bound metal com-
plex extinction coefficients respectively. A plot of [DNA]/(εb − εf)
versus [DNA] gave a slope of 1/(εb − εf) and a y-intercept equal to
[Kb/(εb − εf)]−1, Kb is the ratio of the slope to the y-intercept.
2.3.2. Electrochemical methods
Cyclic voltammetry was performed on a CHI 620C electrochem-
ical 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.
2.3.3. Viscosity measurement
Viscosity experiments were carried out in an Ostwald vis-
cometer immersed in a thermostated water-bath maintained at a
constant temperature at 30.0 0.1 ◦C. CT DNA samples of approxi-
mately 0.5 mM, were prepared by sonication in order to minimize
the complexities arising from CT DNA flexibility [9]. Flow time was
measured with a digital stopwatch three times for each sample
and the average flow time was calculated. Data were presented as
(Á/Áo)1/3 versus the concentration of the metal(II) complexes where
Á is the viscosity of CT DNA solution in the presence of complex and
Áo is the viscosity of CT DNA solution in the absence of complex.
Viscosity values were calculated after correcting the flow time of
buffer alone (t0), Á − (t − t0)/t0 [10].
2.2. Synthesis of Schiff base ligands and their metal complexes
2.2.1. Synthesis of 2-hydroxy-benzylidene-4-aminoantipyrine/2-
hydroxy-3-methoxybenzylidene-4-aminoantipyrine
An ethanolic solution of 4-aminoantipyrine (0.02 mol) was
added to an ethanolic solution of 2-hydroxybenzaldehyde/2-
hydroxy-3-methoxybenzaldehyde (0.02 mol). The resultant mix-
ture was refluxed for ca. 3 h. The solid product formed was filtered
and recrystallized from ethanol.
2.2.2. Synthesis of Schiff base (HL1/HL2)
2.4. Molecular modeling studies
An
ethanolic
solution
of
2-hydroxy-benzylidene-4-
aminoantipyrine/2-hydroxy-3-methoxybenzylidene-4-
2.4.1. DNA–metal complex docking
aminoantipyrine (0.01 mol) was added to an ethanolic solution of
2-aminobenzoic acid (1.37 g, 0.01 mol) and the resultant mixture
was refluxed for ca. 10 h after the addition of anhydrous potassium
carbonate. The potassium carbonate was filtered off from the
reaction mixture and the solvent was evaporated. The orange
color solid separated was filtered and recrystallized from ethanol.
Schematic route for synthesis of Schiff base ligands and their metal
complexes is given in Scheme 1.
The crystal structure of the complex of netropsin with B-DNA
dodecamer d(CGCGAATTCGCG)2 (NDB code GDLB05) was down-
loaded from Protein Data Bank. Crystallographic water molecules
were removed from the DNA. The structures of the metal com-
plexes were constructed using ChemDraw Ultra11.0 and geometry
optimized by MM2 force field geometry and saved as Pdb format.
The interaction of the metal complexes with DNA was also studied
by molecular modeling. All calculations were performed in Open
Eye with Fast Rigid Exhaustive Docking using the FRED docking
software package. The main function employed for the calculation
program was Chemguass 2.
2.2.3. Synthesis of metal complexes
A solution of metal(II) chloride in ethanol (2 mmol) was stirred
with an ethanolic solution of the Schiff base (4 mmol), for ca. 2 h
on a magnetic stirrer at room temperature. Then the solution was
reduced to one-third on a water bath. The solid complex precip-
itated was filtered off and washed thoroughly with ethanol and
dried in vacuo.
2.5. pUC19 DNA cleavage study
2.5.1. DNA cleavage experiments
The oxidative cleavage experiments were performed using
super coiled pUC19 plasmid DNA Form I (2 ␮L, 10 ␮M) in Tris–HCl
buffer (50 mM) with 50 mM NaCl (pH 7.2) which was treated with
the metal complex (30 ␮M) and ascorbic acid (10 ␮M) followed
by dilution with the Tris–HCl buffer to a total volume of 20 ␮L.
The samples were incubated for 1 h at 37 ◦C. A loading buffer con-
taining 25% bromophenol blue, 0.35% xylene cyanol, 30% glycerol
2.3. DNA binding experiments
2.3.1. Absorption spectroscopic studies
The interactions between metal complexes and DNA were
studied using electrochemical and electronic absorption methods.
Disodium salt of calf thymus DNA was stored at 4 ◦C. Solution of

N. Raman, S. Sobha / Spectrochimica Acta Part A 85 (2012) 223–234
225
Scheme 1. Schematic route for the synthesis of Schiff base ligands and their metal complexes.
(3 ␮L) was added and electrophoresis was performed at 40 V for
2.6. Antimicrobial activity studies
each hour in Tris–Acetate–EDTA (TAE) buffer using 1% agarose gel
containing 1.0 ␮g/mL ethidium bromide. The cleavage of DNA was
monitored using agarose gel electrophoresis. The gel was visual-
ized by photographing the fluorescence of intercalated ethidium
bromide under a UV illuminator. The cleavage efficiency was mea-
sured by determining the ability of the complex to convert the super
coiled (SC) DNA to nicked circular form (NC) and linear form. Inhibi-
tion reaction was also carried out by prior incubation of the pUC19
DNA with ethanol (1 ␮M), and sodium azide (100 ␮M). superoxide
dismutase (10 U) and distamycin (100 ␮M).
The in vitro antibacterial activity of the ligands and their com-
plexes were tested against the bacteria Staphylococcus aureus,
Pseudomonas aeruginosa, Escherichia coli, Staphylococcus epider-
midis and Klebsiella pneumoniae by the paper disc method using
nutrient agar as the medium. The stock solution was prepared by
dissolving the 1 mg of sample in 10 mL of DMSO to give the con-
centration of 100 ␮g/mL and the solution was serially diluted in
order to find the minimum inhibitory concentration (MIC) value.
The nutrient agar medium was inoculated with the test organisms.

226
N. Raman, S. Sobha / Spectrochimica Acta Part A 85 (2012) 223–234
present in the Schiff bases was observed at 3443/3452 cm−1
.
The standard solution of streptomycin was prepared in DMSO to
give concentration of 100 ␮g/mL. The sterilized blank paper disks
of 6 mm diameter were impregnated with tested compounds and
placed on the surface of the agar plates previously spread with
0.1 mL of overnight culture of microorganisms. The incubation was
carried out for 24 h at 30 ◦C. During this period, the test solution
diffused and the growth of the inoculated microorganisms were
affected. The inhibition zone developed at which the concentration
was noted.
The absence of this band in all the complexes indicates
the deprotonation of hydroxyl group. The IR spectrum of
the Schiff base ligand showed
a
band at 1624/1622 cm−1
which is assigned to azomethine ꢁ(CH N) linkage, originat-
ing from amino and carbonyl groups of the starting reagents
(4-aminoantipyrine and 2-hydroxybenzaldehyde/2-hydroxy-3-
methoxybenzaldehyde, respectively). These bands were shifted
towards lower frequencies in the spectra of their metal complexes
(1583–1572 cm−1) compared to the above Schiff bases indicating
the involvement of the azomethine nitrogen in chelation with the
metal ion, the coordination of nitrogen to the metal ion could be
expected to reduce the electron density of the azomethine link and
thus caused a shift in the ꢁ(C N) group. In addition, the ligand also
revealed a band at 1656 cm−1 due to the ꢁ(C N) vibration, origi-
nating from the condensation of amino group of 2-aminobenzoic
acid and 2-hydroxy-benzylidene-4-aminoantipyrine/2-hydroxy-
3-methoxybenzylidene-4-aminoantipyrine which was shifted by
30–42 cm−1 on complexation. Moreover, the coordination of the
ligand to the metal centre via the carboxylic group can also be
obvious from the difference of maxima positions as observed
3. Results and discussion
The Schiff base ligands and their Cu(II), Ni(II), and Zn(II)
complexes were synthesized and characterized by spectral and ele-
mental analysis data. The complexes were found to be air stable.
The ligands were soluble in common organic solvents and all the
complexes were freely soluble in CHCl3, DMF and DMSO.
3.1. Elemental analysis and molar conductivity measurements
The results of elemental analysis for the metal complexes were
in good agreement with the calculated values (Table 1) showing
that the complexes have 1:1 metal–ligand stoichiometry of the type
ML wherein L acts as a tetradentate ligand. The metal(II) complexes
were dissolved in DMSO and the molar conductivities of 10−3 M of
their solution at room temperature were measured. The lower con-
ductance values (14.35–24.15 ꢀ−1 cm2 mol−1) of the complexes
support their non-electrolytic nature.
for ꢁsy(COO
−
and ꢁasy(COO− . The bands were detected at 1375
)
)
and 1471 cm−1 respectively for a free ligand and ꢂꢁ = 76 cm−1
For comparison, the ꢁsy(COO and ꢁasy(COO were recorded at
.
−
−
)
)
1315–1319 and 1444–1423 cm−1, respectively, for all the metal
complexes. These results reveal that the organic ligand is involved
in the coordination through the carboxyl group. Conclusive evi-
dence of the bonding was also shown by the observation that new
bands in the spectra of all metal complexes appearing in the low
frequency regions at 503–518 cm−1 and 420–437 cm−1 character-
istic to ꢁ(M–O) and ꢁ(M–N) stretching vibrations respectively that
were not observed in the spectrum of free ligands.
3.2. Mass spectra
The mass spectrum of Schiff base ligand (L1) showed the molec-
ular ion peak at m/z 427 corresponding to [C25H22N4O3] ion. Also
the spectrum exhibited peaks for the fragments at m/z 333, 200, 123
17O2N4]+, [C11H12N4]+, [C5H7ON4
3.4. 1H NMR spectra
+
and 77 corresponding to [C19
H
and [C6H5]+ respectively. The spectra of Cu(II), Ni(II) and Zn(II) com-
plexes showed molecular ion peaks at m/z 488 [M+], 484 [M+1],
and 491 [M+1] respectively that are equivalent to their molecular
weights. The Cu(II) complex gave a fragment ion peak with loss
of metal at m/z 425. The mass spectrum of Schiff base ligand (L2)
showed peak at m/z 458 and its Cu(II), Ni(II) and Zn(II) complexes
showed molecular ion peaks at m/z 519 [M+], 514 [M+] and 523
[M+2] respectively. The mass spectra of ligand (L1) and its zinc
complex are given in Figs. S1 and S2 (supplementary file).
The 1H NMR spectra of the Schiff bases and their zinc complexes
were recorded at room temperature in CDCl3. 1H NMR spectra of
the ligands showed a singlet at 5.4 and 5.6 ␦ respectively, attributed
to the phenolic –OH group of 2-hydroxybenzaldehyde/2-hydroxy-
3-methoxybenzaldehyde, present in the Schiff base ligands. The
absence of these peaks noted for the zinc complexes [ZnL1] and
[ZnL2] confirmed the deprotonation of –OH proton with metal ion
upon complexation. The peaks at 9.3 and 9.6 ␦ are attributable to
the –COOH of 2-aminobenzoic acid moiety present in Schiff bases.
The absence of these peaks noted for the zinc complexes confirmed
the loss of the COOH proton of 2-aminobenzoic acid moiety due to
complexation.
3.3. IR spectra
The coordination mode and sites of the ligands to the
metal ions were investigated by comparing the infrared spectra
of the free ligands with their metal complexes. The char-
acteristic phenolic ꢁ(OH) mode due to the hydroxyl group
of 2-hydroxybenzaldehyde/2-hydroxy-3-methoxybenzaldehyde,
The ligand (L1) showed the following signals: phenyl multiplet
at 6.0–7.8 ␦, –CH N at 8.7 ␦, –C–CH3 at 3.1 ␦, N–CH3 at 2.4 ␦, and
the ligand (L2) exhibited the following signals: phenyl multiplet at
6.0–7.6 ␦, –CH N at 8.2 ␦, –C–CH3 at 3.1 ␦, N–CH3 at 2.4 ␦, OCH3
Table 1
Physical characterization, analytical, molar conductance and magnetic susceptibility data of the ligands and their complexes.
a
Compound
Yield (%)
Color
Found (calc)%
M
Formula weight
(∧m
)
ꢃeff (BM)
C
H
N
L1
74
69
53
51
72
56
42
67
Dark Orange
Dark orange
Dark brown
Pale blue
Yellow
Dark brown
Pale blue
–
–
70.6 (70.4)
68.5 (68.2)
62.3 (61.5)
62.2 (62.1)
61.9 (61.3)
60.8 (60.2)
61.1 (60.7)
60.3 (59.9)
5.2 (5.2)
5.6 (5.5)
4.1 (4.1)
4.2 (4.1)
4.4 (4.1)
4.5 (4.5)
4.8 (4.5)
4.6 (4.4)
13.6 (13.1)
12.5 (12.2)
11.6 (11.4)
12.2 (11.6)
11.6 (11.4)
11.3 (10.8)
11.5 (11.4)
11.2) (10.7)
426
458
488
483
490
519
514
521
–
–
–
–
1.86
–
Diamagnetic
1.88
–
L2
[CuL1]
[NiL1]
[ZnL1]
[CuL2]
[NiL2]
[ZnL2]
13.2 (13.0)
12.3 (12.1)
13.9 (13.3)
12.8 (12.4)
11.7 (11.4)
12.7 (12.5)
20.36
14.35
18.46
17.63
22.26
24.15
Lemon yellow
Diamagnetic
The unit of the molar conductance is ꢀ−1 mol−1 cm2.
a

N. Raman, S. Sobha / Spectrochimica Acta Part A 85 (2012) 223–234
227
at 3.9 ␦. The azomethine proton (–CH N) signals in the spectra
of the zinc complexes were shifted down field compared to the
free ligands, suggesting deshielding of azomethine group due to
the coordination with metal ion. There is no appreciable change in
all other signals of the complexes. The 1H NMR spectra of ligand
(L1) and its [ZnL1] are given in Figs. S3 and S4 (supplementary file).
3.5. Electronic spectra
The electronic spectra of the complexes were recorded in DMSO
solution. All the complexes showed the high energy absorption
bands in the region 28,330–32,786 cm−1. This transition may be
attributed to the charge transfer band. The electronic spectra of
[CuL1]/[CuL2] complexes displayed the d–d transition band in the
region 17,730/18,761 cm−1, which is due to 2B1g → A1g transition.
2
This d–d band strongly favors a square-planar geometry around
the metal ion. It is further supported by the magnetic susceptibility
values (1.84/1.89) BM. The Ni(II) complexes [NiL1]/[NiL2] showed
diamagnetic moment and the electronic spectra of these complexes
showed bands at 15,267/15,174 and 22,675/23,809 cm−1 which are
1
1
1
1
assigned as A1g → B1g and A1g → A2g transitions respectively.
The Zn(II) complexes due to their diamagnetic character and no
d–d transitions and based on stoichiometry of these complexes
and elemental analysis they are four coordinated, which could be
square-planar geometry.
3.6. Molecular modeling studies
Molecular modeling has paid a considerable attention to the
characterization and inferences of geometrical optimization of the
prepared complexes, therefore we could find out the optimized
geometry for all metal complexes by computing the total energy
and the theoretical physical parameters, such as bond length and
bond angles using MM2 CS Chem office version 11.0 molecular
modeling program. The details of some selected bond lengths,
bond angles and total energy of the prepared metal complexes
are given in Table 2. From the table we conclude that the bond-
ing via azomethine nitrogen, phenolic and carboxylic group oxygen
after deprotonation and forms mononuclear complexes with Cu(II),
Ni(II) and Zn(II) metal ion in 1:1 stoichiometric ratio.
3.7. EPR spectra
EPR studies of paramagnetic transition metal(II) complexes
yield information about the distribution of the unpaired electrons
and hence about the nature of the bonding between the metal ion
and its ligands. There have been many reports concerning the appli-
cation of EPR to square-planar or distorted octahedral complexes
of Cu(II) and of the interpretation of the EPR parameters in terms
of covalency of the metal ligand bonding. Despite these numerous
experimentally derived values for the covalency factors, i.e. d orbital
mixing coefficients, there have been only a few molecular orbital
calculations for either four or six coordinate copper(II) system. The
spin Hamiltonian parameters of the copper complexes were calcu-
lated. The g and g values were computed from the spectrum using
||
⊥
TCNE free radical as ‘g’ marker. The EPR spectra of [CuL1] complex
recorded in DMSO solution at 300 and 77 K are shown in Fig. 1. The
g tensor values of this copper(II) complex can be used to derive the
ground state. In square-planar complexes, the unpaired electron
lies in the dx2-y2 orbital [11]. The axial symmetry parameter G is
defined as
g
|| − 2
G =
g⊥ − 2
If G > 4.0 exchange interaction is negligible while G < 4.0 exchange
interaction is considerable and the local tetragonal axes are

228
N. Raman, S. Sobha / Spectrochimica Acta Part A 85 (2012) 223–234
Fig. 2. The proposed model of metal complexes.
of complexes to CT-DNA helix has been followed through absorp-
tion spectral titrations. With increasing concentration of CT-DNA
the absorption bands of the complexes were affected resulting in
the tendency of hypochromism and a minor red shift was observed
in all the complexes.
Hyperchromic effect and hypochromic effect are the spec-
tral features of DNA concerning its double helix structure.
Hypochromism results from the contraction of DNA in the helix
axis as well as from the change in conformation on DNA while
hyperchromism results from the damage of the DNA double helix
structure [14]. The absorption spectrum of [CuL1] complex showed
an intensive absorption band at 370.0 nm and [CuL2] complex at
383.0 nm respectively in 5 mM Tris–HCl and 50 mM NaCl buffer
solution (pH 7.2). Further the intense absorption band with
maxima of 354.2 nm and 383 nm for [NiL1] and [NiL2], 375.8 nm
and 376.3 nm for [ZnL1] and [ZnL2] respectively were obtained.
These are due to intra ligand ␲–␲* transitions. On increasing the
Fig. 1. EPR spectrum of copper complex [CuL1] in DMSO at 300 K (a) and 77 K (b).
misaligned. The observed values of exchange interaction param-
eters for the complexes ranging from 5.32 to 5.23 suggest that the
local tetragonal axes are aligned parallel or slightly misaligned and
the unpaired electron is present in the dx2-y2 orbital. These results
also indicate that the exchange coupling effects are not operating
in the present complexes [12]. The bonding parameters (˛2, ˇ2 and
ꢄ2) of the copper complexes were calculated and summarized in
Table 3. The ˛2, ˇ2 and ꢄ2 values indicate that there is substantial
interaction in the in-plane ␴-bonding whereas ␲-bonding coeffi-
cients are almost ionic. These results are anticipated because there
are no appropriate ligand orbitals to combine with the dxy orbital
of copper(II) ion.
Based on the above spectral and analytical data, the proposed
model of the metal(II) complexes is given in Fig. 2.
3.8. DNA binding studies
3.8.1. Absorption titration
There has been an increasing focus on the interaction between
DNA and other molecules which is hopeful to understand the action
mechanism of some DNA-targeted drugs and toxic agents as well
as the origin of few diseases like gene mutation [13]. The binding
behavior of the metal complexes to DNA helix is often investi-
gated using absorption spectral titration followed by the changes
in the absorbance and shift in the wavelength. The representative
absorption spectra of [CuL1] and [CuL2] complexes in presence and
absence of CT-DNA are shown in Figs. 3 and 4. The binding mode
Table 3
The spin Hamiltonian parameters of the copper complexes in DMSO solution at 300
and 77 K.
2
Fig. 3. Absorption spectral changes on addition of CT DNA to the solution of [CuL1] 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. Inset: plot of
[DNA]/(εa − εf) versus [DNA].
Complex
A
A
Aiso
g
g
⊥
giso
˛
ˇ2
ꢄ2
||
⊥
||
[CuL1]
[CuL2]
156
145
61
67
95.6
83.5
2.21
2.27
2.04
2.05
2.13
2.18
0.70
0.73
0.80
1.03
0.60
0.79

N. Raman, S. Sobha / Spectrochimica Acta Part A 85 (2012) 223–234
229
Fig. 5. Effect of increasing amounts of [CuL1] (ꢀ), [NiL1] (᭹), [ZnL1] (ꢁ), [CuL2] (+),
[NiL2] (×), [ZnL2] (ꢂ), and [EB] (*) on the viscosity of DNA. R = [complex]/[DNA] or
[EB]/[DNA].
Fig. 4. Absorption spectral changes on addition of CT DNA to the solution of [CuL2] 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. Inset: plot of
[DNA]/(εa − ␧f) versus [DNA].
mode in solution. In the absence of crystallographic structural data,
hydrodynamic methods that are sensitive to DNA length change
are regarded as the least ambiguous and the most critical tests
of binding in solution. A classical intercalation model results in
the lengthening of the DNA helix as the base pairs is separated to
accommodate the binding molecule, leading to an increase in the
DNA viscosity. However, a partial and non-classical intercalation of
ligand may bend (or kink) DNA helix, resulting in the decrease of
its effective length and concomitantly its viscosity [17]. The plots
of (Á/Áo)1/3 versus R (where R = [complex]/[DNA]; Á and Áo are the
relative viscosities of DNA in the presence and absence of complex,
respectively) give a measure of the viscosity changes. The effects of
all the complexes on the viscosity of CT DNA are shown in Fig. 5. As
expected, the known DNA-intercalator ethidium bromide increases
the relative viscosity of DNA double helix resulting from intercala-
tion. In contrast, groove (or) surface binding can cause an increase
in the effective length of DNA leading to a minor increase in the
effective length of DNA solution [18]. All the complexes exhibit
minor increase in the relative viscosity of CT DNA compared to
ethidium bromide suggesting primarily groove binding nature of
the complexes.
concentration of CT DNA resulted in the minor bathochromic shift
in the range 1.6–2.5 nm and significant hypochromicity lying in the
range 11–23%. The spectroscopic changes suggested that the com-
plexes showed appreciable groove/surface binding property to the
CT DNA. The (Kb) values are shown in Table 4. The intrinsic binding
constant values (Kb) for the complexes [CuL1], [NiL1], [ZnL1],
[CuL2], [NiL2] and [ZnL2] were found to be 2.7 × 104, 1.7 × 104,
1.0 × 104, 3.7 × 104, 2.3 × 104 and 1.6 × 104 M−1 respectively. The
result suggests that the interaction of metal(II) complexes with
DNA is a significant minor groove binding mode. The binding
constant (Kb) values of these complexes were lower in comparison
to those observed for typical intercalators, ethidium bromide and
[Ru(bpy)2(dppz)]2+ whose binding constants are in the order of
1.4 × 106 and >106 M−1 [15,16]. In order to compare quantitatively
the binding strength of the complexes with CT DNA, the intrinsic
binding constants (Kb) were obtained by monitoring the changes
in the absorbance for the complexes with increasing concentration
of DNA. Kb was obtained from the ratio of slope to the intercept
from the plot of [DNA]/(εa − εf) versus [DNA].
3.8.2. Viscosity measurements
3.8.3. Electrochemical studies
The viscometric measurement is also an important tool to find
the nature of binding of metal complexes to the DNA. The relative
specific viscosity of DNA is determined by varying the concen-
tration of the added metal complexes. Measuring the viscosity
of DNA is a classical technique used to analyze the DNA binding
Cyclic voltammetric technique provides a useful complement
to the previously used methods of investigation like absorption
spectral titration and viscosity studies. The cyclic voltammograms
of [CuL2] and [NiL1] complexes in the absence and in presence
of varying amount of DNA are shown in Figs. 6 and 7 respec-
tively. Summary of voltammetric results of metal complexes is
given in Table 5. The cyclic voltammetric data of [CuL1] complex
in the absence of DNA featured the reduction of +3 to the +2 and
+2 to the +1 form at cathodic peak potential. In the absence of
CT DNA, the first redox cathodic peak appeared at −0.143 V for
Cu(III) → Cu(II) [Epa = 0.037 V, Epc = −0.143 V, ꢂEp = 0.180 V and
E1/2 = −0.053 V] and in the second redox couple, the cathodic peak
appeared at 0.398 V for Cu(II) → Cu(I) [Epa = 0.462 V, Epc = 0.398 V,
ꢂEp = 0.064 V and E1/2 = 0.43 V]. The ipa/ipc ratios of anodic to
cathodic peak currents were 1.28 and 1.02 respectively indi-
cating that the reaction of the complex on the glassy carbon
electrode surface is a quasi-reversible redox process. In [CuL2]
complex, in the absence of CT DNA, the first redox cathodic
peak appeared at −0.048 V for Cu(III) → Cu(II) [Epa = 0.647 V,
Table 4
Electronic absorption spectral properties of Cu(II), Ni(II) and Zn(II) complexes.
No
Compound
ꢂ
(nm)
aH%
bKb × 104 (M−1
)
max
Free
Bound
1
2
3
4
5
6
[CuL1]
[NiL1]
[ZnL1]
[CuL2]
[NiL2]
[ZnL2]
370.0
354.2
375.8
383.0
344
371.7
355.4
376.9
385.5
345.6
377.6
1.7
1.2
1.1
2.5
1.6
1.3
21
18
11
23
16
13
2.7
1.7
1.0
3.7
2.3
1.6
376.3
a
H% = [Afree − Abound)/Afree] × 100%.
b
Kb = Intrinsic DNA binding constant determined from the UV–vis absorption
spectral titration.

230
N. Raman, S. Sobha / Spectrochimica Acta Part A 85 (2012) 223–234
Fig. 8. Interaction of the complexes [CuL1] and [NiL1] with d(CGCGAATTCGCG)
strands of DNA by minor groove binding approach.
Fig. 6. Cyclic voltammogram of [CuL2] in the presence of increasing amount of DNA
in buffer pH 7.2 at 25 ◦C. Arrow indicates the changes in voltammetric currents upon
increasing the DNA concentration.
Reoxidation of +1 form occurred at −0.076 V and −0.149 V for
[NiL1] and [NiL2] respectively upon scan reversal. In the absence of
DNA, the separation of the anodic and cathodic peak potential was
ꢂEp = 0.001 V for [NiL1] and 0.354 V for [NiL2]. The formal potential,
E1/2 was taken as the average of Epc and Epa (−0.082 V and −0.326 V
respectively). Finally the zinc complexes showed redox cathodic
peak at −0.123 V for [ZnL1] and −0.147 V for [ZnL2] and anodic
peak appeared at −0.594 V and −0.591 V respectively, the sepa-
ration of the anodic and cathodic peak potential, ꢂEp = −0.471 V
and −0.444 V for [ZnL1] and [ZnL2]. The formal potentials were
E1/2 = −0.3585 V and −0.369 V. The incremental addition of CT DNA
to the complexes revealed that the redox couples caused a less
negative shift in E1/2 and decrease of ꢂEp. The ipa/ipc values also
decreased in the presence of DNA. Bard has reported [19] that if E1/2
is shifted to more positive value, the interaction mode is intercala-
tive binding. In contrast, the slight decrease in peak potential and
voltammetric currents were occurred and this can be attributed
to diffusion of the metal complexes, bound to the large, slowly
diffusing DNA molecule.
Epc = −0.048 V, ꢂEp = 0.0.695 V and E1/2 = 0.299 V] and in the
second redox couple, the cathodic peak appeared at −0.458 V
for Cu(II) → Cu(I) [Epa = 0.003 V, Epc = −0.458 V, ꢂEp = 0.461 V and
E1/2 = −0.227 V]. The ratios of anodic to cathodic peak currents were
1.22 and 0.99 respectively, indicating a quasi-reversible redox pro-
cess. The cyclic voltammogram of nickel complexes in the absence
of DNA showed a reduction of +2 to the +1 form at a cathodic
peak potential, Epc of −0.083 V for [NiL1] and −0.503 V for [NiL2].
3.8.4. Docking of metal complexes with DNA
We have further tried to explore the minor groove binding
profile of metal(II) complexes with the help of docking methods.
The DNA binding constant values, Kb obtained from the absorp-
tion titration (Table 4) were in the range of 104 M−1 for all the
metal complexes indicating that the possibility of intercalation of
these metal(II) complexes between the DNA base pairs was little
and mainly due to less-effective stacking forces between the metal
complexes and the DNA bases because H-bond donor/acceptor
atoms present in the metal complexes to stay in the minor groove
with the help of van der Waals’ forces and H-bonds. Fig. 8 shows
the docked conformation of the [CuL1] and [NiL1] complexes with
Fig. 7. Cyclic voltammogram of [NiL1] in the presence of increasing amount of DNA
in buffer pH 7.2 at 25 ◦C. Arrow indicates the changes in voltammetric currents upon
increasing the DNA concentration.
Table 5
Electrochemical parameters for the interaction of DNA with Cu(II), Ni(II) and Zn(II) complexes.
No
Compound
Redox couple
aE1/2 (V)
bꢂEp (V)
ipa/ipc
Free
Bound
Free
Bound
1
2
3
4
5
6
[CuL1]
[NiL1]
[ZnL1]
[CuL2]
[NiL2]
[ZnL2]
Cu(III) → Cu(II)Cu(II) → Cu(I)
Ni(II) → Ni(I)
−0.0530.430
−0.079
−0.0740.411
−0.107
0.1800.064
−0.007
0.471
0.6950.461
0.354
0.187 0.171
−0.214
1.281.02
0.89
1.18
1.220.99
0.86
1.09
Zn(II) → Zn(0)
−0.358
−0.371
0.464
Cu(III) → Cu(II)Cu(II) → Cu(I)
Ni(II) → Ni(I)
0.299−0.227
−0.326
0.266−0.272
−0.340
0.678−0.544
0.377
Zn(II) → Zn(0)
−0.369
−0.395
0.444
0.452
a
Data from cyclic voltammetric measurements; E1/2 is calculated as average of anodic (Epa) and (Epc) peak potential E1/2 = Epa + Epc/2.
ꢂEp = Epa − Epc.
b

N. Raman, S. Sobha / Spectrochimica Acta Part A 85 (2012) 223–234
231
Fig. 9. Gel electrophoresis diagram showing the cleavage of pUC19 DNA (10 ␮M) by Cu(II), Ni(II) and Zn(II) complexes in a buffer containing 50 mM Tris–HCl and 50 mM
NaCl in the presence of ascorbic acid (AH2, 10 ␮M) at 37 ◦C. Lane 1, DNA control; lane 2, DNA + [ligand(L1)] + AH2; lane 3, DNA + AH2 + [CuL1]; lane 4, DNA + AH2 + [NiL1]; lane
5, DNA + AH2 + [CoL1]; and lane 6, DNA + AH2 + [ZnL1].
DNA. The docked structure revealed that the complexes perfec-
tively fit within the minor groove with a binding site of base
pairs long that more desirably involves AT residues. A similar
type of interaction has been reported with [Cu(L-arg)2]2+ bind-
ing to d(CGCGAATTCGCG)2 [20]. In general, minor groove-binding
molecules can form hydrogen bonds to bases, typically to N-3 of
adenine and O-2 of thymine. Most minor groove binding drugs bind
to AT rich sequences. This preference in addition to the designed
property for the electronegative pockets of AT sequences is prob-
ably due to better van der Waals’ contacts between the complex
and groove walls in this region. Thus, the narrower AT regions com-
pared to G-C regions produce a better fit of these types of molecules
into the minor groove and lead to van der Waals’ interactions
with the DNA functional groups that define the groove. Hydrogen
bonding from the C-2 carbonyl oxygen of T or the N-3 nitrogen
of A to the minor groove binders is important [21]. The binding
energy for the docking of CuL1, NiL1, ZnL1, CuL2, NiL2 and ZnL2
with DNA were found to be −78.52, −69.56, 67.63, −80.40, −72.40
and −68.19 kJ mol−1 respectively which correlated well with the
experimental DNA binding values. The larger negative value of
the binding energy establishes the greater binding potential of the
metal complexes with DNA.
by metal complexes (lanes 3–6) in the presence of AH2 (ascorbic
acid). Under the same conditions, free AH2 produced no cleavage
of pUC19 DNA (lane 2). All the metal complexes are able to
convert supercoiled (Form I, SC) to nicked circular (Form II, NC)
and the open circular form (Form III, LC) thus revealed that metal
complexes behave as efficient chemical nuclease for double strand
cleavage of DNA. These phenomena imply that metal complexes
induced intensively the cleavage of pUC19 DNA in the presence of
ascorbic acid. To establish the reactive species responsible for the
cleavage efficiency of pUC19 DNA induced by metal complexes, the
following experiments were carried out. The cleavage of pUC19
DNA did not inhibit in the presence of hydroxyl radical scavenger
like ethanol indicating that hydroxyl radical (OH•) was not likely
to be the cleaving agents (Fig. 11, lanes 3–8). On the other hand,
addition of singlet oxygen quencher like sodium azide decreased
the cleavage efficiencies but slightly (Fig. 12, lanes 3–8) which
reveals that 1O2 is not the activated oxygen intermediate respon-
sible for the cleavage. The slight suppression of cleavage may be
ascribed to the affinity of sodium azide for transition metals.
Superoxide dismutase (SOD) is one of the most important
antioxidant enzymes that catalyzes superoxide neutralization by
converting it to hydrogen peroxide and oxygen [22]. The redox
reaction is shown below:
2O2− + 2H+ ⇒ O2− + H2O2
3.8.5. Gel electrophoresis studies
3.8.5.1. Chemical nuclease activity. The results of spectroscopic
titration, viscosity and cyclic voltammetry experiments suggest
that metal complexes bind to DNA. Binding of these complexes
with DNA was also studied by gel electrophoresis using pUC19
DNA. This moves on agarose gel under the influence of electric
field. This movement is retarded in agarose gel electrophoresis
when they are bound to metal complexes. Figs. 9 and 10 show the
results of gel electrophoretic separations of pUC19 DNA induced
Superoxide ion is known to be involved in oxidative stress and
this reactive species is responsible for several diseases like reper-
fusion injury, arthritis, neuronal apoptosis, cancer and acquired
immunodeficiency syndrome [23]. These prompted us to inves-
tigate the SOD activity of the metal complexes with pUC19
DNA. While in presence of SOD, a facile superoxide anion radical
−
O2 quencher, the cleavage was improved which indicated that
Fig. 10. Gel electrophoresis diagram showing the cleavage of pUC19 DNA (10 ␮M) by Cu(II), Ni(II) and Zn(II) complexes in a buffer containing 50 mM Tris–HCl and 50 mM
NaCl in the presence of ascorbic acid (AH2, 10 ␮M) at 37 ◦C. Lane 1, DNA control; lane 2, DNA + [ligand(L2)] + AH2; lane 3, DNA + AH2 + [CuL2]; lane 4, DNA + AH2 + [NiL2]; lane
5, DNA + AH2 + [CoL2]; and lane 6, DNA + AH2 + [ZnL2].

232
N. Raman, S. Sobha / Spectrochimica Acta Part A 85 (2012) 223–234
Fig. 11. Gel electrophoresis diagram showing the cleavage of pUC19 DNA (10 ␮M) by Cu(II), Ni(II) and Zn(II) complexes in a buffer containing 50 mM Tris–HCl
and 50 mM NaCl in the presence of ascorbic acid (AH2, 10 ␮M) at 37 ◦C. Lane 1, DNA control; lane 2, DNA + AH2 + ethanol; lane 3, DNA + AH2 + [CuL1] + ethanol;
lane 4, DNA + AH2 + [NiL1] + ethanol; lane 5, DNA + AH2 + [ZnL1] + ethanol; lane 6, DNA + AH2 + [CuL2] + ethanol; lane 7, DNA + AH2 + [NiL2] + ethanol; and lane 8,
DNA + AH2 + [ZnL2] + ethanol.
Fig. 12. Gel electrophoresis diagram showing the cleavage of pUC19 DNA (10 ␮M) by Cu(II), Ni(II) and Zn(II) complexes in a buffer containing 50 mM Tris–HCl and 50 mM NaCl
in the presence of ascorbic acid (AH2, 10 ␮M) at 37 ◦C. Lane 1, DNA control; lane 2, DNA + AH2 + NaN3; lane 3, DNA + AH2 + [CuL1] + NaN3; lane 4, DNA + AH2 + [NiL1] + NaN3;
lane 5, DNA + AH2 + [ZnL1] + NaN3; lane 6, DNA + AH2 + [CuL2] + NaN3; lane 7, DNA + AH2 + [NiL2] NaN3; and lane 8, DNA + AH2 + [ZnL2] + NaN3.
O2− might be an inhibitor in the cleavage of the plasmid and reduc-
ing the amount of O2 could improve the cleavage effect (Fig. 13).
data (done by paper disc method) of the investigated compounds
are summarized in Table 6. From the table, it is inferred that all the
metal complexes have higher inhibitory effect than free ligands.
This higher antimicrobial activity of the metal complexes compared
to Schiff bases may be due to the change in structure due to coordi-
nation and chelating effect to make metal complexes to act as more
powerful antibacterial agents, thus killing the microbe or by inhibit-
ing multiplication of the microbe by blocking their active sites [25].
Moreover, a capable of existing mode for the toxicity increase may
be considered in light of Tweedy’s chelation theory [26]. Chelation
reduces the polarity of the metal ion considerably because of the
partial sharing of its positive charge with the donor groups and also
due to ␲-electron delocalization on the whole chelating ring. The
lipids and polysaccharides are some important constituents of the
cell wall and membranes which are preferred for metal ion interac-
tion. Apart from this, the cell wall also contains many phosphates,
carbonyl and cystenyl ligands which maintain the integrity of the
membrane by acting as a diffusion barrier and also provide suitable
sites for binding. Furthermore, increased lipophilicity enhances the
penetration of the complexes into lipid membrane and blocking of
−
To determine the groove selectivity of the complexes, control
experiments were performed using minor groove binder. When
pUC19 DNA was treated with distamycin, a minor groove binder,
the cleavage reaction mediated by metal complexes was quenched.
This clearly suggests that the complexes prefer to bind to DNA
minor groove (Fig. 14, lanes 3–8). The result of agarose gel elec-
trophoresis indicates that the metal complexes are able to perform
an efficient cleavage of pUC19 DNA.
3.9. Antimicrobial screening
The synthesized ligands and their complexes were tested for
their in vitro antimicrobial activity. They were tested against the
bacteria because bacteria can achieve resistance to antibiotics
through biochemical and morphological modifications [24]. The
organisms used in the present investigation were S. epidermidis, K.
pneumoniae, S. aureus, P. aeruginosa and E. coli. Streptomycin was
used as the standard for comparing the results. The biocidal activity
Fig. 13. Gel electrophoresis diagram showing the cleavage of pUC19 DNA (10 ␮M) by Cu(II), Ni(II) and Zn(II) complexes in a buffer containing 50 mM Tris–HCl and 50 mM
NaCl in the presence of ascorbic acid (AH2, 10 ␮M) at 37 ◦C. Lane 1, DNA control; lane 2, DNA + AH2 + SOD; lane 3, DNA + AH2 + [CuL1] + SOD; lane 4, + AH2 + [NiL1] + SOD; lane
5, DNA + AH2 + [ZnL1] + SOD; lane 6, DNA + AH2 + [CuL2] + SOD; lane 7, DNA + AH2 + [NiL2] + SOD; and lane 8, DNA + AH2 + [ZnL2] + SOD.

N. Raman, S. Sobha / Spectrochimica Acta Part A 85 (2012) 223–234
233
Fig. 14. Gel electrophoresis diagram showing the cleavage of pUC19 DNA (10 ␮M) by the Cu(II), Ni(II) and Zn(II) complexes in a buffer containing 50 mM Tris–HCl and
50 mM NaCl in the presence of ascorbic acid (AH2, 10 ␮M) at 37 ◦C. Lane 1, DNA control; lane 2, DNA + AH2 + distamycin; lane 3, DNA + AH2 + [CuL1] + distamycin; lane
4, DNA + AH2 + [NiL1] + distamycin; lane 5, DNA + AH2 + [ZnL1] + distamycin; lane 6, DNA + AH2 + [CuL2] + distamycin; lane 7, DNA + AH2 + [NiL2] + distamycin; and lane 8,
DNA + AH2 + [ZnL2] + distamycin.
Table 6
Minimum inhibitory concentration values of the synthesized compounds against the growth of five bacteria (mg/mL).
Compound
S. aureus
P. aeruginosa
E. coli
S. epidermidis
K. pneumoniae
L1
45
48
14
16
25
17
19
28
12
44
43
15
21
28
16
23
23
16
46
47
13
29
23
15
26
31
14
48
51
11
26
28
19
25
34
10
44
48
10
24
27
22
25
29
12
L2
[CuL1]
[NiL1]
[ZnL1]
[CuL2]
[NiL2]
[ZnL2]
Streptomycina
a
Streptomycin is used as the standard.
the metal binding sites in the enzymes of microorganisms. These
complexes also disturb the respiration process of the cell and thus
block the synthesis of the proteins which restricts further growth
of the organisms.
research facilities. They also wish to acknowledge the help ren-
dered by Thigarajar College with regard to computational facilities.
NR thanks University Grants Commission, New Delhi for financial
assistance.
4. Conclusion
Appendix A. Supplementary data
Six novel transition metal(II) complexes have been synthesized
and characterized. The Schiff base ligands are behaving as N2O2
donor tetradentate ligands. The structure of the complexes has been
determined on the basis of elemental analyses, molar conductivi-
ties, IR spectra, 1H NMR, mass spectra and UV–vis spectra. All these
studies give good evidence for the proposed structure. The absorp-
tion spectroscopy, viscosity measurement, cyclic voltammetry and
molecular docking studies have been carried out on the interac-
tion of the complexes with DNA. The experimental and docking
results reveal that all the complexes can interact with DNA through
minor groove approach. The complexes are shown to act as active
DNA binding agents. Gel electrophoresis experiment shows that
presence of ascorbic acid enhances the DNA cleavage to a signif-
icant extent. The studies on mechanism of pUC19 DNA cleavage
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.saa.2011.09.065.
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