DNA interaction and efficient antimicrobial activities of 4N chelating metal complexes

Article abstract of DOI:10.1016/j.jphotobiol.2015.04.006

Abstract A new series of metal(II) complexes using a symmetric Schiff base ligand, obtained by the condensation reaction of 4-formyl-N,N-dimethylaniline with benzene-1,2-diamine were synthesized and characterized. The various physico-chemical data indicat

Full text of DOI:10.1016/j.jphotobiol.2015.04.006

Accepted Manuscript
DNA interaction and efficient antimicrobial activities of 4N chelating metal
complexes
S. Packianathan, T. Arun, N. Raman
PII:
S1011-1344(15)00129-3
DOI:
http://dx.doi.org/10.1016/j.jphotobiol.2015.04.006
JPB 10003
Reference:
Journal of Photochemistry and Photobiology B: Bi-
ology
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6 January 2015
30 March 2015
12 April 2015
Please cite this article as: S. Packianathan, T. Arun, N. Raman, DNA interaction and efficient antimicrobial activities
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dx.doi.org/10.1016/j.jphotobiol.2015.04.006
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DNA interaction and efficient antimicrobial activities of 4N chelating metal complexes
S. Packianathan, T. Arun and N. Raman*
Research Department of Chemistry, VHNSN College, Virudhunagar-626 001, India
E-mail: ramchem1964@gmail.com; Tel.: +091-092451-65958; Fax: +091-4562-281338
Abstract
A new series of metal(II) complexes using a symmetric Schiff base ligand, obtained by the
condensation reaction of 4-formyl-N,N-dimethylaniline with benzene-1,2-diamine were
synthesized and characterized. The various physico-chemical data indicate that the complexes
have octahedral geometry. The intrinsic binding constant of the complexes with DNA is
explored. The UV-Vis., circular dichorism, fluorescence emission spectral data and the
viscosity measurements indicate that the complexes bind to calf thymus DNA (CT DNA) by
intercalative mode. The intrinsic binding constants of Cu(II), Ni(II), Co(II) and Zn(II)
complexes are found to be 7.1 × 104, 5.2 × 104, 1.8 × 105 and 6.3 × 104 M-1, respectively.
The cleavage studies of these complexes are investigated by gel electrophoresis method in the
presence of peroxide. The complexes exhibit enhanced biological activities compared to the
free ligand.
Keywords: Octahedral; Fluorescence; Intercalation; Gel electrophoresis; DNA binding
*Corresponding author: Tel.: +91-9245165958; fax: +91-4562281338
Email : ramchem1964@gmail.com (N.Raman)
1

1. Introduction
Schiff bases are one of the most widespread and important class of ligands due to their
simple preparation, selectivity and coordination towards the central metal ions. They are
considered as most ‘privileged ligands’ containing an azomethine group (-CH=N) [1]. These
ligands are able to coordinate with various metal ions to form Schiff base metal complexes
which increase their application in different fields such as catalysis [2] and pharmacology [3].
The efficiency of the Schiff bases as therapeutic agents has often been enhanced upon
coordination to a metal. Over the years there has been a continuous curiosity of the biological
activity in metal complexes [4]. 4-acetylpyrazolone and thiazole Schiff base complexes have
been found a variety of biological applications including antitumor, antihistaminic and anti-
inflammatory activities [5]. The search for new alternative drugs to the eminent cisplatin and
its derivatives, which are still used in more than 50% of the treatment regimes for patients
depression from cancer, is extremely needed [6,7]. Consequently, efforts are being made to
substitute cisplatin with appropriate alternatives and numerous metal-based complexes have
been synthesized and tested for their anticancer activities [8, 9].
Schiff bases imply opportunities for inducing substrate chirality, tuning the metal
centered electronic factor, enhancing solubility and either performing homogeneous or
heterogeneous catalyzes and includes diversified subjects comprising their various aspects in
bio-coordination and bio-inorganic chemistry [10-18].
Transition metal Schiff base complexes are multifaceted tools to probe the structure and
dynamics of DNA. DNA is the primary intracellular target of anticancer drugs due to the
interaction between small molecules and DNA, which cause DNA damage in cancer cells,
blocking the division of cancer cells and resulting in cell death [19-22]. In recent years,
binding studies of these complexes have become very important in the growth of DNA
molecules probes and chemotherapeutics [23-25]. Many transition metal complexes are
known to bind to DNA via both covalent and non-covalent interactions. In covalent binding,
the labile ligand of the complexes is replaced by a nitrogen base of DNA. On the other hand,
the non-covalent DNA interactions include intercalative, electrostatic and groove (surface)
binding of cationic metal complexes along the outside of the DNA helix and the major or
minor groove. 4-formyl-N,N-dimethylaniline, a bioactive ligand, is used as a reagent to detect
urobilinogen in urine. Hence, we are tempted to design and synthesize transition metal Schiff
base complexes using this ligand to study the impact on biological effect. We herein report
the synthesis and characterization of a Schiff base ligand, obtained by the condensation
reaction of the above 4-formyl-N,N-dimethylaniline with benzene-1,2-diamine, and its
2

transition metal(II) complexes. The DNA binding and antimicrobial activities have also been
explored.
2. Experimental protocol
The materials and methods, DNA binding, cleavage and antimicrobial procedures are
given in the Supplementary file (S1).
2.1. Preparation of Schiff base ligand (L)
An ethanolic solution of 4-formyl-N,N-dimethylaniline (2.98 g, 0.02 M) was added drop
wise to an ethanolic solution of benzene-1,2-diamine (1.08 g, 0.01 M). Few drops of glacial
acetic acid were added to the reaction mixture and were refluxed for 3 h. It was poured in
crushed ice, wherein orange colored precipitate was obtained. The solid product formed was
filtered, washed, dried and recrystallized from ethanol, dried in vacuum at room temperature.
[L] Yield: 74%. Anal.Calc. (%): C (77.8), H (7.1) and N (15.2); Found (%): C (77.3) H (6.9)
and N (14.9); FT-IR (KBr) (cm-1): 1610 (C=N), 2900-2950 (C-H) and 1400-1600 (C=C); 1H
NMR (DMSO-d6) δ (ppm): 6.7-7.7 (m, aromatic); UV-Vis. in DMSO, (transition) cm-1:
40,485 (π-π*) and 29,293 (n-π*).
2.2. Preparation of metal complexes
To an ethanolic solution of 0.001 M of metal(II) chloride was stirred with twice the
amount of an ethanolic solution of the above ligand (0.74 g, 0.002 M) and the resultant
mixture was stirred for 2 h and refluxed for ca. 3 h. Then the solution was reduced to
one-third on a water bath. The precipitated metal complexes were isolated and washed with
distilled water. They were recrystallized from ethanol and dried in vacuum at room
temperature.
[CuL2Cl2].H2O Yield: 68 %; Anal.Calc. for [C48H52N8CuCl2] .H2O: C 64.51; H 6.09;
N 12.54; Cl 7.94; Cu 7.11; Found (%); C 64.34; H 5.97; N 12.48; Cl 7.85; Cu 7.0; FT-IR
(KBr) (cm-1): 1604 (C=N) and 435 (M-N); ∧m (Ω-1mol-1cm2) 11.13; µeff (BM) 1.78; UV-Vis.
in DMSO, (transition) cm-1: 28,683, 41,666 (LMCT) and 11,778 (d-d).
[NiL2Cl2].H2O Yield: 71%; Anal.Calc. for [C48H52N8NiCl2].H2O: C 64.88; H 6.13; N 12.61;
Cl 7.98; Ni 6.61; Found (%):C 64.6; H 6.02; N 12.52; Cl 7.89; Ni 6.51; FT-IR (KBr) (cm-1):
1604 (C=N) and 433 (M-N); ∧m (Ω-1mol-1cm2) 6.58; µeff (BM) 3.48; UV-Vis. in DMSO,
(transition) cm-1: 28,571, 47,846 (LMCT) and 11,185 (d-d).
3

[CoL2Cl2].H2O Yield: 65%; Anal.Calc. for [C48H52N8CoCl2].H2O: C 64.86; H 6.12; N 12.61;
Cl 7.98; Co 6.63; Found (%); C 64.79; H 6.03; N 12.54; Cl 7.86; Co 6.52; FT-IR (KBr)(cm-
1): 1604 (C=N) and 437 (M-N); ∧m (Ω-1mol-1cm2) 9.42; µeff (BM) 4.55; UV-Vis. in DMSO,
(transition) cm-1: 28,901, 45,871 (LMCT) and 11,160 (d-d).
[ZnL2Cl2].H2O Yield: 75%; Anal.Calc. for [C48H52N8ZnCl2].H2O:C 64.39; H 6.08; N 12.52;
Cl 7.92; Zn 7.30; Found (%) ; C 64.26; H 5.91; N 12.39; Cl 7.8; Zn 7.21; FT-IR (KBr)
1
(cm-1): 1604 (C=N) and 438 (M-N); H NMR (DMSO-d6) δ(ppm): 6.60-7.90 (phenyl
(Ω -1mol-1cm2) 5.63; µeff (BM)
−CH
−H);
∧
multiplet), 2.90-3.40 (N 3); 3.9–4.2 (C
m
diamagnetic; UV-Vis. in DMSO, (transition) cm-1: 28,571 and 40,816 (LMCT).
3. Results and discussion
All the compounds are air stable for extended periods and the complexes are remarkably
soluble in DMSO and DMF. The results of elemental analysis for the metal complexes are in
good agreement with the calculated values (Table 1) showing that the stoichiometry of the
complexes is [ML2Cl2] wherein the ligand acts as a bidentate. The formation of these
complexes may proceed according to the equation given below:
MCl2.nH2O + 2L → [ML2Cl2].H2O + nH2O
where, M= Cu(II), Ni(II), Co(II) and Zn(II).
3.1. IR spectra
The IR spectra of the ligand and its corresponding complexes endow with significant
information about the metal ligand bonding. The IR spectra of metal complexes and ligand
were recorded in the range of 300–4000 cm-1. The characteristic bands for aromatic C=C
group are observed in the range of 1400-1600 cm-1 (Fig. S1). Significantly, the expected
characteristic peak of azomethine (C=N) group is also presented at 1610 cm-1 which confirms
the formation of the Schiff base. The C=N band is shifted to lower frequency in the
complexes (1604 cm-1) as expected. This indicates the coordination of -C=N nitrogen to the
metal ion. In the complexes, new peaks formed at 430-440 cm-1 and 356-363 cm-1 region are
assigned to M-N and M-Cl bonds respectively, confirming the complex formation [26].
3.2. Molar conductivity
The molar conductivities (10−3 M) of their solution at room temperature were measured.
The lower conductance values (5-12 ohm−1 cm2 mol−1) of the complexes support their
non-electrolytic nature. The absence of chloride (counter) ion is confirmed from Volhard’s
4

test. It also indicates that the chloride anions bind to the metal ions as ligands and do not
ionize. The formation of the Schiff base ligand and its complexes is given in Scheme 1.
3.3. Electronic spectra and magnetic properties of metal(II) chelates
The electronic spectra of complexes were recorded in DMSO solution and are
consistent with an octahedral metal(II) environment. In electronic spectra of metal complexes
the wide range of bands are due to transition of –CH=N–, charge transfer results from
electrons interaction between the metal and the ligand which involves either a metal to ligand
or ligand to metal electron transfer. The band observed in 40,485 cm-1 is due to π-π*
transition of C=N group. It is shifted to higher range, which is due to nitrogen that involved
in coordination with metal ion. The absorption band observed at 28,571 cm-1 is due to n-π*
transition from azomethine group corresponding to the ligand or metal complexes. The Cu(II)
2
complex shows an absorption peak at 11,778 cm-1, due to the d–d transition (2Eg → T2g) of
Cu(II) ion (Fig. S2), suggesting that the copper ion exhibits an octahedral geometry [27].
Electronic spectrum of the Ni(II) complex shows a band at 11,185 cm-1 which is assigned to
3
3A2g(F) → T1g(F) transition, suggesting an octahedral arrangement for the nickel(II)
complex. The electronic spectrum of Co(II) complex exhibits absorptions at 9,425 and 11,160
-1
4
4
3
3T2g(F) transitions respectively,
→
→
cm which are assigned to T1g(F)
T2g(P) and A2g(F)
corresponding to Co(II) octahedral complex [28]. In difference, Zn(II) complex does not
exhibit any d-d band because of its completely filled d10 transition. However, based on its
elemental analysis and spectral data, an octahedral geometry is proposed for this complex
also.
3.4. 1H NMR spectra
The NMR spectrum is exploiting to determine the identity of prepared ligand and its
diamagnetic metal complex. Ligand and its Zn(II) complex are recorded in DMSO-d6, using
1
tetramethylsilane (TMS) as internal standard (Fig. S3). The proton H NMR spectrum of
ligand shows a singlet peak at 9.65 ppm (1H, CH=N), multiplet in the range 6.60-7.90 ppm
for the aromatic ring protons and a multiplet peak at 2.90-3.40 ppm for (6H, N-CH3) protons.
The proton 1H NMR spectrum of Zn complex shows a singlet peak at 9.55 ppm (1H, CH=N),
multiplet in the range 6.60-7.90 ppm for the aromatic ring protons and a multiplet peak at
2.90-3.40 ppm for (6H, N-CH3) protons. From the above observation, the azomethine group
(CH=N) is involved in metal co-ordination.
5

3.3. Mass spectroscopy
The formation of metal complexes and the speciation of various ionic forms in DMSO
solution were studied with ESI–MS. The mass spectrum of the ligand (L) shows molecular
ion peak at m/z = 238 corresponding to [C15H15N3]+ ion. Also the spectrum exhibits peaks for
the fragments at m/z 371, 222, 194, 134 and 91 corresponding to [C24H26N4]+, [C15H15N2]+,
[C13H9N2]+, [C9H11N]+, and [C7H6]+ respectively (Fig. S4). Besides, the mass spectrum of
Cu(II) complex show peaks at m/z 236 [Base peak], 97, 134, 222, 371, 471 and
863 corresponding to [C15H15N3]+, [CuCl]+, [CuCl2]+, [C15H15N2]+, [C24H26N4]+,
[C24H26N4CuCl]+ and [C48H52N8CuCl2]+ respectively (Fig. S5). The m/z of all the fragments
of Schiff base ligand and its metal complex confirm the stoichiometry of the type [ML2Cl2].
Thus, the mass spectral data reinforce the conclusion drawn from the analytical and
conductance values.
3.5. Electron paramagnetic spectrum of the Cu(II) complex
The EPR spectrum of Cu(II) complex gives an enough information about the structure
of the metal ion environment in the complex. i.e., the geometry, nature of the donating atoms
from the ligand and degree of covalency of the Cu(II)–ligand bonds. The EPR spectrum of
the Cu(II) complex was recorded in DMSO at liquid nitrogen temperature (LNT) which
reveals well resolved peaks in the low filed region (Fig. S6). This is also supported by the
magnetic moment of Cu(II) complex (1.78 BM) which confirms the mononuclear nature of
the complex. The spin Hamiltonian parameters calculated for the Cu(II) complex are precised
in Table 2. The EPR spectrum of the Cu(II) complex exhibits axially symmetric g-tensor
parameters with g|| > g┴> 2.003 indicating that the copper site has a dx2-y2 ground state
characteristic of an octahedral stereochemistry [29]. From the values of g factors, the
geometric parameter G, representing a measure of exchange interaction between Cu(II)
centres in polycrystalline compound can be determined by using the formula :
G = (gll – 2.0027) / (g┴- 2.0027)
If G > 4.0, the local tetragonal axes are aligned parallel or only slightly misaligned. If
G < 4.0, significant exchange coupling is present and the misalignment is appreciable. The
observed value for the exchange interaction parameter for the Cu(II) complex is found to be
4.7 indicating that there is negligible exchange interaction of Cu–Cu in the complex
according to Hathaway [30]. This result also indicates that the exchange coupling effects are
6

not operative in the present complex [31-33]. The isotropic EPR parameters giso = 2.2064 and
Aiso = 94 can be calculated from the position spacing of the resonance lines from room
temperature solution spectrum of the complex.
The bonding parameters of α2 and β2 indicate that the complex has some covalent
character and there is interaction in the out-of-plane π-bonding. The lower value of α2 (0.89)
indicates that the complex has some covalent character. The observed β2 (0.80) [CuL2Cl2])
and γ2 (0.68) [CuL2Cl2] indicate that there is interaction in the out of plane π-bonding whereas
the in-plane π-bonding is completely ionic. This is further confirmed by orbital reduction
factors. For the present case, the observed order is K|| (0.725) > K┴ (0.154) for [CuL2Cl2]
implying a greater contribution from out-of-plane π-bonding than from in-plane π-bonding in
metal-ligand π-bonding. Based on these observations, Cu(II) complex is proposed to have
distorted octahedral geometry. The EPR study has provided supportive evidence to obtain the
conclusion of electronic spectrum and magnetic moment value.
3.6. Thermal Analysis (TGA/DTA)
The complexes were studied for thermal behavior over the temperature range of 25-
300oC under nitrogen atmosphere (Fig S7). The thermograms of Cu(II) and Co(II)
complexes are given in supplementary file. In the present investigation, decomposition of
copper complex takes place in three stages. The first step of decomposition corresponding to
°
a mass loss of 6.63 % in the region 50-100 C, is attributed to the loss of one lattice held
water molecule. In the second step, weight loss of 11.32 % in the region 100–250°C,
corresponds to the loss of azomethine and two counter chloride ions. Further
reduction of mass in the higher temperature range is ascribed to the ligand decomposition and
the final product is found to be copper(II) oxide. Further, the cobalt(II) complex shows
thermal decomposition in three significant steps. The weight loss of 7.23 % in the first step is
attributed to the loss of one coordinated water molecule in the region 70-110°C. In the second
stage, the decomposition corresponds to a mass loss of 21.00% in the range
190–270 °C, showing the combined loss of two chlorides and the ligand. Further reduction of
mass in the higher temperature range is ascribed to the decomposition of remaining part of the
ligand and the final product is analyzed and it is cobalt(II) oxide..
3.7. Powder XRD
The powder X-ray diffraction analysis of Cu(II) and Co(II) Schiff base complexes are
illustrated in Fig. S8. The Schiff base ligand and its metal complexes were recorded in the
7

range of 2θ = 0–80 Å. From the experiment, it is found that Cu(II) and Co(II) metal
complexes show sharp peaks indicating their crystalline nature. The average crystalline size
of the complexes, D was calculated using Scherrer’s formula [9] from equation (1)
D = 0.89λ/(βCosθ) ------------ (1)
where, λ is the wavelength (0.15406 nm), β is the full width at the half- maximum (FWHM)
of the ligand, Cu(II) and Co(II) complexes line and θ is the diffraction angle. The ligand and
the metal complexes Cu(II) and Co(II) have an average grain size of 49.16, 32.23 and 39.32
nm respectively, suggesting that the ligand and complexes are in a nanocrystalline phase. The
crystallite sizes of the Schiff base metal complexes are found to be in the range of 10-100 nm
[34].
3.8. DNA binding studies
3.8.1. Electronic absorption spectroscopy
DNA binding activities of metal complexes have been a clue of vital significance for
the growth of effective metal based chemotherapeutic drugs. One of the most useful
techniques for studying the DNA binding of molecules is electronic absorption spectroscopy
[35]. Any change in the UV–Vis absorption spectra of metal complexes upon the addition of
DNA serves as a proof for the existence of an interaction between them in particular,
hypochromism due to π–π* stacking interaction with a red-shift (bathochromism) may appear
in the case of an intercalative binding leading to stabilization of DNA duplex. The increasing
concentration of CT DNA results in the bathochromic shift in the range 1.5-2.6 nm and
significant hypochromicity lying in the range 17-25 %. The observed hypochromism could be
credited to stacking interaction between the aromatic chromophores of the complexes and
DNA base pairs consistent with the intercalative binding mode, while the red-shift is an
evidence of the stabilization of the CT DNA duplex. UV–Vis spectrum of Cu(II) complex in
the absence and presence of CT DNA is shown representatively in Fig. 1. The UV–Vis
spectra of Ni(II), Co(II) and Zn(II) complexes are shown in Fig. S9 respectively.
The intrinsic binding constant values (Kb) for the complexes Cu(II), Ni(II), Co(II) and
Zn(II) were found to be 7.1×104, 5.2×104, 1.8×105 and 6.3×104 M-1 respectively [36]. The
binding constant (Kb) values of these complexes were compared 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 [37, 38]. 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
8

of DNA. Kb was obtained from the ratio of slope to the intercept from the plot
of [DNA]/(εa–εf) versus [DNA]. The Kb values are shown in Table 3.
3.8.2. Cyclic voltammetry
Electrochemistry has been growing helpful in elucidating the basic chemistry of
biological systems. Cyclic voltammetric measurements of Cu(II) complex were carried out.
The cyclic voltammograms of Cu(II) complex in the absence and in presence of varying
amount of DNA are shown in Fig. 2. The incremental addition of CT DNA to the complex
causes shift in the potential of peak in cyclic voltammogram. This result shows that complex
stabilizes the duplex (GC pairs) by intercalating way. The ipc/ipa ratios of these four redox
couples of the complexes are 0.6122, 0.4364, 0.5635 and 0.6571 respectively which indicate
that the reaction of the complex on the glassy carbon electrode surface is quasi-reversible
redox process. The incremental addition of CT DNA to the complex the redox couples causes
a less negative shift in E1/2 and decrease of ∆Ep. The augmentation addition of CT DNA to
the complex causes a positive shift in potential and a decrease in the current intensity. From
these data, it is understood that the entire synthesized complexes interact with DNA through
intercalating way [39]. The cyclic voltammograms of Ni(II) and Zn(II) are shown in Figs.S10
and S11 respectively.
In differential pulse voltammogram of Cu(II) complex in the absence and presence of
varying amount of [DNA] with significant decrease of current intensity (Fig. 3), the shift in
potential is related to the ratio of binding constant by the following equation:
Eb - Ef = 0.0591 log (K[red]/K[oxd]
)
where Eb and Ef are peak potentials of the complex in the bound and free form respectively.
In the present study Schiff base complexes show one electron transfer during the redox
process and its ipc/ipa value is less than unity which indicates the reaction of the complex on
the glassy carbon electrode surface is quasi-reversible. Other complexes [Co(II), Ni(II) and
Zn(II)] show considerable shift in both cathodic and anodic peak potentials in the presence of
incremental addition of CT DNA. Most of the synthesized complexes give both the anodic
and cathodic peak potential shifts which are either positive or negative (Table 4). It indicates
the intercalating mode of DNA binding with the Schiff base complexes.
9

3.8.3. CD spectroscopy
To further investigate the binding mode and interaction affinity of complexes and DNA,
the circular dichorism spectra were carried out. Modifications of the CD signals in the
spectral range of 250–400 nm have shown to be useful to predict and confirm the DNA
conformational changes, damage and/or cleavage upon interaction with metal complexes. CD
spectrum of CT DNA exhibits a positive band at 280 nm due to base stacking and a negative
band at 245 nm due to right-handed helicity of B–DNA. It is generally accepted that classical
intercalation leads to change in intensities of both positive and negative bands, due to
enhancement of base stacking and stabilization of helicity, whereas groove binding and
electrostatic interaction of complexes with DNA shows less or no perturbation on the base
stacking and helicity bands [40, 41]. As shown in Fig. 4, upon increasing the concentration
of complexes to the DNA solution, the intensities of both positive and negative bands
changes in few degrees. In the present complex, the DNA intensity has been found to
decrease in positive band with 2-5 nm and red shifts are observed. Observed changes in those
CD signals of DNA suggest that the stacking mode and the orientation of base pairs in DNA
are disturbed with the binding of the complex. The decrease in intensity clearly suggest that
the binding of the complex induces certain conformational changes, in which the more B- to
more A- conformational changes of DNA could be induced upon the binding of the Cu(II)
complex. In addition, a significant broad induced CD signals with negative ellipticity at
315-320 nm is observed which reveals that the compound firmly bind as intercalation to the
DNA double helix [42].
3.8.4. Emission spectroscopy
In order to get further facts of the aim, illuminating the binding of the metal(II)
complexes to DNA, the fluorescence emission spectrum was studied. In the fluorescence
titrations, the concentration of complex is keeping constant, and varying the CT DNA
concentration. Fig. 5 shows the fluorescence emission intensity curves of Cu(II) and Co(II)
complexes with CT DNA. As seen in both cases, the increasing addition of the CT DNA to
the Cu(II) and Co(II) complexes, the fluorescence emission intensity gradually increases
which clearly delegates that the complexes expose the strong binding with DNA [43]. This
result provides that the binding of complexes with DNA can be intercalation. The
fluorescence intensity fabricates that the complex can insert between base pairs of the
CT DNA. This strengthening of the fluorescence intensity is invented to be one of the
patterns for intercalative binding [44].
10

3.8.5. Viscosity measurements
To get further evidence and clarifying the binding of the metal(II) complexes to DNA,
viscosity measurements were carried out on CT DNA by varying the concentration of the
complexes added. The viscosity of DNA is significantly improved by complete or partial
intercalation of complexes into the DNA base-pair stack, but it is slightly disturbed by the
electrostatic or covalent binding of molecules. A classical intercalation model results in the
lengthening of the DNA helix as the base pairs are separated to accommodate the binding
molecule, leading to an increase in the DNA viscosity. The changes in the relative specific
viscosity of CT DNA in the presence and absence of the complex were plotted against
1/R = [complex]/[DNA]. A classical intercalation mode causes a significant increase in the
viscosity of DNA due to an increase in base pair separation at intercalation sites and hence an
increase in the overall DNA length [44]. The effects of all the complexes on the viscosity of
CT DNA are shown in Fig. 6. A significant increase in the viscosity of DNA on addition of
complex results due to the intercalation which leads to the separation among the DNA bases
to the increase in the effective size in DNA which could be the reason for the increase in the
viscosity [46].
3.9. DNA cleavage efficacy
Gel electrophoresis experiments were performed using ligand and complexes in
presence of H2O2. Complexes exhibit cleavage ability at low concentration. The ligand
exhibits no major activity in the presence of oxidant. The activity was much higher for the
complexes in presence of H2O2. When CT DNA is subjected to electrophoresis, relatively fast
migration will be observed for the intact super coil form (Form I). If scission occurs on one
strand (nicking), the super coil will relax to generate a slower moving open circular form
(Form II). If both strands are cleaved, a linear form (Form III) that migrates between Forms I
and II will be generated [47, 48]. The complexes show more activity in the presence of
oxidant which may be due to the reaction of hydroxyl radical with DNA. The results of DNA
cleavage studies have been shown in Fig. 7. Further, [CuL2Cl2] and [NiL2Cl2] complexes
have shown greater cleavage potential compared to ligand and other complexes studied.
3.10. Antimicrobial activity
Biological activity of the ligand and a series of its metal complexes [Cu(II), Ni(II),
Co(II) and Zn(II)] were screened for anti-bacterial activity against P. putida (Gram-negative
bacteria), N.asteroides and L. lactis (Gram-positive bacteria) and anti-fungal activity against
the fungi A.flavus, C.ablicans and A.niger. The minimum inhibitory concentration (MIC)
11

was determined by assaying at serial dilution of concentrations along with standards at the
same concentrations. The antimicrobial results of Schiff base and its metal complexes are
systematized in Tables 5 and 6. The remarkable activity of Schiff base ligand may arise from
the presence of azomethine group which may be one of the reasons for impart in elucidating
the mechanism of transformation reaction in biological systems [49]. The results indicate that
the complexes show more activity and the ligand has less activity against the same
microorganisms under identical experimental conditions.
As previously mentioned, the metal complexes show improved antimicrobial activity
than their corresponding ligand. According to the Overtone′s concept of cell permeability, the
lipid membrane surrounding the cell favors the passage of only lipid-soluble materials and
therefore, liposolubility is an important factor which controls the antimicrobial activity. On
chelation, polarity of the metal ion is reduced to a greater extent due the overlapping of the
ligand orbital and partial sharing of the positive charge of the metal ion with donor groups.
The increased lipophilicity enhances the penetration of the complexes into the lipid
membranes and blocks 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
proteins, which restricts further growth of the organism [50].
4. Conclusion
A novel biological important Schiff base ligand and its metal complexes of Cu(II),
Co(II), Ni(II) and Zn(II) have been synthesized and characterized. The complexes have been
employed to bind CT DNA and such binding ability has been explored using diverse
techniques. The above data have proved the CT DNA binding of the complexes through the
intercalation mode. Among the synthesized metal complexes Co(II) complex has the
strongest DNA binding affinity. The biological screening data reveal that the complexes have
higher antimicrobial activity than the free ligand. The collective biological activities of the
complexes may be helpful for the designing of metal based drugs.
Acknowledgments
The authors express their heartfelt thanks to the College Managing Board, Principal and
Head of the Department of Chemistry, VHNSN College for providing necessary research
facilities. IIT Bombay and CDRI, Lucknow, are gratefully acknowledged for providing
instrumental facilities.
12

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17

Figure Captions
Scheme 1. Schematic route for synthesis of metal complexes.
0
Fig. 1. Absorption spectrum of Cu(II) complex in buffer pH =7.2 at 25 C in presence of
increasing amount of DNA. Arrow indicates the changes in absorbance upon increasing the
DNA concentration.
Fig. 2. Cyclic voltammograms of Cu(II) complex in buffer (pH = 7.2) at 25 0C in presence of
increasing amount of DNA.
Fig. 3. Differential pulse voltammograms of Cu(II) complex in buffer (pH = 7.2) at 25 0C in
the presence of increasing amount of DNA.
Fig. 4. CD spectra of DNA in buffer pH =7.2 at 25 0C in presence of increasing amount of
Cu(II) complex. Arrow indicates the changes in ellipticity upon increasing the DNA
concentration.
0
Fig. 5. Emission spectrum of Cu(II) complex in buffer pH =7.2 at 25 C in presence of
increasing amount of DNA.
Fig. 6. Effect of increasing amounts of [CuL2Cl2] ( ), [NiL2Cl2] ( ), [CoL2Cl2] ( ),
[ZnL2Cl2] (X), and [EB] (X) on the relative viscosity of CT DNA vs [complex]/[DNA] (1/R)
ratio.
Fig. 7. The gel electrophoretic separation of plasmid pUC19 DNA treated with
[ML2Cl2].H2O complexes. Lane 1; DNA control; Lane 2: DNA + ligand + H2O2; Lane 3:
DNA + Cu + H2O2; Lane 4: DNA + Co + H2O2; Lane 5: DNA + Ni + H2O2; Lane 6: DNA +
Zn + H2O2.
18

M= Cu(II), Ni(II), Co(II) and Zn(II)
Scheme 1. Schematic route for the synthesis of metal complexes.
19

0
Fig. 1. Absorption spectrum of Cu(II) complex in buffer pH=7.2 at 25 C in presence of
increasing amount of DNA. Arrow indicates the changes in absorbance upon increasing the
DNA concentration.
20

Fig. 2. Cyclic voltammograms of Cu(II) complex in buffer (pH = 7.2) at 25 0C in presence of
increasing amount of DNA.
21

Fig. 3. Differential pulse voltammograms of Cu(II) complex in buffer (pH = 7.2) at 25 0C in
the presence of increasing amount of DNA.
22

Fig. 4. CD spectra of DNA in buffer pH =7.2 at 25 0C in presence of increasing amount of
Cu(II) complex. Arrow indicates the changes in ellipticity upon increasing the DNA
concentration.
23

0
Fig. 5. Emission spectrum of Cu(II) complex in buffer pH =7.2 at 25 C in presence of
increasing amount of DNA.
24

Fig. 6. Effect of increasing amounts of [CuL2Cl2] ( ), [NiL2Cl2] ( ), [CoL2Cl2] ( ),
[ZnL2Cl2] (X), and [EB] (X) on the relative viscosity of CT DNA vs [complex]/[DNA] (1/R)
ratio.
25

1
2
3
4
5
6
From II
Form III
Form I
Fig. 7. The gel electrophoretic separation of plasmid pUC19 DNA treated with
[ML2Cl2].H2O complexes. Lane 1; DNA control; Lane 2: DNA + ligand + H2O2; Lane 3:
DNA + Cu + H2O2; Lane 4: DNA + Co + H2O2; Lane 5: DNA + Ni + H2O2; Lane 6: DNA +
Zn + H2O2.
26

Table Captions
Table 1.
Table 2.
Table 3.
Elemental and physical data of Schiff base ligand (HL) and its
metal(II) complexes
The spin Hamiltonian parameters of Cu(II) complex in DMSO
solution at 77 K
Electronic absorption spectral properties of Cu(II), Co(II), Ni(II) and
Zn(II) complexes with DNA
Table 4.
Table 5.
Table 6.
Electrochemical parameters for interaction of DNA with Cu(II), Ni(II),
Co(II) and Zn(II) complexes
Minimum inhibitory concentration of the synthesized compounds
against the growth of bacteria (µg/mL)
Minimum inhibitory concentration of the synthesized compounds
against the growth of fungi (µg/mL)

Table 1. Elemental and physical data of Schiff base ligand (HL) and its metal(II)
complexes
Calculated(Found) %
m
(ohm-1
cm2mol-1)
Compound
µeff (BM)
M
C
H
N
77.8
(77.3)
64.51
7.1
15.2
(14.9)
12.54
L
-
-
-
1.78
(6.9)
6.09
7.11
Cu(II)
Ni(II)
Co(II)
Zn(II)
11.13
6.58
9.42
5.63
(7.01) (64.34) (5.97)
6.61 64.88 6.13
(6.51) (64.66) (6.02)
6.63 64.86 6.12
(6.52) (64.79) (6.03)
7.30 64.39 6.08
(7.21) (64.26) (5.91)
(12.48)
12.61
3.48
(12.52)
12.61
4.55
(12.54)
12.52
Diamagnetic
(12.39)

Table 2. The spin Hamiltonian parameters of Cu(II) complex in DMSO solution at
77 K
Hyperfine constant
× 10–4 cm–1
Complex
g
g
giso
2
2
2
G
A
[CuL2Cl2] 90
A
Aiso
94
104
2.4319 2.0937 2.064
0.89
0.80
0.68
4.7