Synthesis and DNA binding studies of Ni(II), Co(II), Cu(II) and Zn(II) metal complexes of N1,N5-bis[pyridine-2-methylene]- thiocarbohydrazone Schiff-base ligand

Article abstract of DOI:10.1016/j.saa.2011.04.018

The thiocarbohydrazone Schiff-base ligand with a nitrogen and sulphur donor was synthesized through condensation of pyridine-2-carbaldehyde and thiocarbohydrazide. Schiff-base ligands have the ability to conjugate with metal salts. A series of metal complexes with a general formula [MCl ₂(H₂L)]·nH₂O (MNi, Co, Cu and Zn) were synthesized by forming complexes of the N¹,N⁵- bis[pyridine-2-methylene]-thiocarbohydrazone (H₂L) Schiff-base ligand. These metal complexes and ligand were characterized by using ultraviolet-visible (UV-Vis), Fourier Transform Infrared (FT-IR), ¹H and ¹³C NMR spectroscopy and mass spectroscopy, physicochemical characterization, CHNS and conductivity. The biological activity of the synthesized ligand was investigated by using Escherichia coli DNA as target. The DNA interaction of the synthesized ligand and complexes on E. coli plasmid DNA was investigated in the aqueous medium by UV-Vis spectroscopy and the binding constant (K_b) was calculated. The DNA binding studies showed that the metal complexes had an improved interaction due to trans-geometrical isomers of the complexes than ligand isomers in cis-positions.

Full text of DOI:10.1016/j.saa.2011.04.018

Spectrochimica Acta Part A 79 (2011) 1050–1056
Contents lists available at ScienceDirect
Spectrochimica Acta Part A: Molecular and
Biomolecular Spectroscopy
journal homepage: www.elsevier.com/locate/saa
Synthesis and DNA binding studies of Ni(II), Co(II), Cu(II) and Zn(II) metal
complexes of N1,N5-bis[pyridine-2-methylene]-thiocarbohydrazone
Schiff-base ligand
A.D. Tiwaria, A.K. Mishrab,∗, S.B. Mishraa, B.B. Mambaa, B. Majic, S. Bhattacharyac
a Department of Chemical Technology, University of Johannesburg, P.O. Box 17011, Doornfontein 2028, South Africa
b UJ Nanomaterials Science Research Group, Department of Chemical Technology, University of Johannesburg, P.O. Box 17011, Doornfontein 2028, South Africa
c Department of Organic Chemistry, Indian Institute of Sciences, Bangalore, India
a r t i c l e i n f o
a b s t r a c t
Article history:
The thiocarbohydrazone Schiff-base ligand with a nitrogen and sulphur donor was synthesized through
condensation of pyridine-2-carbaldehyde and thiocarbohydrazide. Schiff-base ligands have the ability
to conjugate with metal salts. A series of metal complexes with a general formula [MCl2(H2L)]·nH2O
(M Ni, Co, Cu and Zn) were synthesized by forming complexes of the N1,N5-bis[pyridine-2-methylene]-
thiocarbohydrazone (H2L) Schiff-base ligand. These metal complexes and ligand were characterized by
using ultraviolet–visible (UV–Vis), Fourier Transform Infrared (FT-IR), 1H and 13C NMR spectroscopy and
mass spectroscopy, physicochemical characterization, CHNS and conductivity. The biological activity of
the synthesized ligand was investigated by using Escherichia coli DNA as target. The DNA interaction of
the synthesized ligand and complexes on E. coli plasmid DNA was investigated in the aqueous medium by
UV–Vis spectroscopy and the binding constant (Kb) was calculated. The DNA binding studies showed that
the metal complexes had an improved interaction due to trans-geometrical isomers of the complexes
than ligand isomers in cis-positions.
Received 17 January 2011
Received in revised form 16 March 2011
Accepted 13 April 2011
Keywords:
Synthesis
Metal complex
Thiocarbohydrazone
DNA interaction
Schiff-base
© 2011 Elsevier B.V. All rights reserved.
1. Introduction
Furthermore, the efficiency of these drugs also depends upon the
structure activity relationship (SAR) with DNA that directly influ-
Schiff-base ligands are potential anti-cancer, anti-bacterial and
anti-viral agents and this activity tends to increase in metal(II)
Schiff-base complexes [1,2]. The cleavage of plasmid DNA by square
planar nickel (salen) [bis-(salicylidene) ethylenediamine] under
the influence of magnesium monoperoxyphthalic acid (MPPA)
or iodosylbenzene has been well-reported [3]. The stereospecific
transformation of Cu(II) complexes as groove binder and inter-
calative mode of binding of the complex to the DNA was studied
by electron spin resonance (ESR) [4]. Bhattacharya et al. also
reported the spontaneous cleavage of DNA under ambient aer-
obic conditions by a new water-soluble Co–salen complex [5].
The introduction of the divalent metal cation into the DNA struc-
ture modulates its function [6]. The mechanism of action of metal
complexes as anticancer agents is such that it binds with the
DNA of tumour cells resulting in the inhibition of its growth. An
understanding of DNA binding mode and cleavage is needed to
develop new and efficient antitumor drugs as their effectiveness
depends on the binding mode and affinity towards the DNA [7,8].
ences the drug-action mechanism under physiological conditions
[9–11]. As determined by physical and biochemical methods, the
principle mode of DNA binding is the intercalation of the metal
complex between the base pair within a duplex DNA [12,13]. DNA
offers a variety of binding sites and binding modes for non-covalent
interactions with small molecules. The complexes can bind to
DNA in non-covalent mode such as electrostatic, intercalative and
groove binding [14,15]. The cationic metal complexes possess pla-
nar aromatic ligands in between adjacent base pairs within a duplex
DNA [16–19].
The development of metal complexes as diagnostic or thera-
peutic agents requires techniques that can rapidly and accurately
provide information about the effects of structural alterations on
DNA selectivity and affinity. The non-covalent bonding of metal
complexes with DNA has resulted in various applications such as
synthetic restriction enzymes [20], DNA repair agents [21], devel-
opment of selective probes of DNA structure [22], and artificial
regulators of gene expression [23].
In the study reported in this article we synthesized Schiff-base
N1,N5-bis[pyridine-2-methylene]-thiocarbohydrazone ligand. The
metal(II) Schiff-base complexes using Ni(II), Co(II), Cu(II), and
Zn(II) metals salts were prepared using the Schiff-base lig-
∗
Corresponding author. Tel.: +27 11 559 6180; fax: +27 11 559 6425.
E-mail address: amishra@uj.ac.za (A.K. Mishra).
1386-1425/$ – see front matter © 2011 Elsevier B.V. All rights reserved.
doi:10.1016/j.saa.2011.04.018

A.D. Tiwari et al. / Spectrochimica Acta Part A 79 (2011) 1050–1056
1051
Scheme 1. Synthesis of the metal(II) Schiff-base complex methodology.
and. The ligand and the respective metal complexes were
2.3. Synthesis
characterized by means of physical and spectral analyses. The
complexes were used to study the DNA binding mechanism
and binding constants in an aqueous medium by absorption
spectroscopy.
The Schiff-base ligand was prepared using a previously reported
method and synthesis of the metal(II) Schiff-base complexes is
depicted in Scheme 1 [25,26].
2.3.1. Synthesis of N1,N5
2. Experimental
bis(pyridine-2-methylidene)-thiocarbohydrazone (H2L)
Pyridine-2-aldehyde (10 mmol, 0.94 mL) was added to the thio-
carbohydrazide (5 mmol, 0.536 g) in hot methanolic solution of
Schiff-base with 1 mL HCl (0.1 N). The mixture was heated to reflux
in the water bath for 4 h (Scheme 1). The pale yellow precipitate was
formed by ice cooling. This (precipitate) was filtered off, washed
with methanol and petroleum ether. Re-crystallization was carried
out in methanol and a yellow amorphous compound was obtained,
which was subsequently dried over anhydrous CaCl2 under vacuum
(m.p. 200 ◦C, yield 80%).
2.1. Materials
Plasmid genomic DNA was extracted from Escherichia coli as
per the earlier reported methods [24]. The metal salts ZnCl2,
CuCl2·2H2O, NiCl2·6H2O, and CoCl2·6H2O were purchased from
Merck, South Africa. Thiocarbohydrazide and pyridine-2-aldehyde
were purchased (synthetic grade) from Sigma–Aldrich and Merck,
South Africa, respectively. All the solvents (AR grade) used in the
synthesis and analysis were procured from Merck, South Africa and
used without any further purification.
2.3.2. Synthesis of metal complexes
All the metal complexes were synthesized by adding the
methanolic solution of metal(II) salts (0.5 mmol) to the hot homo-
geneous solution of the ligand (0.5 mmol) in the methanol. The
mixture of the metal and the ligand was heated to reflux with
constant stirring in a water bath for 4 h (Scheme 1). At room tem-
perature, the product was filtered off. The obtained products were
dried over anhydrous CaCl2 under vacuum.
2.2. Physical and spectral measurements
Melting points were measured using electrothermal digital
melting point apparatus and molar conductivity was measured
on the Crison EC Meter Basic 30+. The entire mass spectrums
were obtained using a Waters Micromass Q-TOF Spectrometer.
Elemental analysis (CHNS/O) was conducted on an elemental Var-
ian analyzer. FT-IR spectra were recorded on the MIDAC FT-IR
(4000 Model) in the range of 400–4000 cm−1 using a KBr disc.
UV–Vis Spectra were recorded on the Varian Cary 50 Spectropho-
tometer (range 200–900 nm) using methanol as a solvent. Metal
complex–DNA titration studies were performed on a Perkin Elmer
Lambda-35 UV-Vis Spectrophotometer at room temperature, and
deionised water was used. The 13C and 1H NMR Spectra were
recorded using Varian Gemini-2000 NMR-300 with d6-DMSO being
the solvent containing tetra-methyl-silane (TMS) as the internal
standard.
2.4. DNA binding studies
Stock solutions (500 ␮M) of the metal(II) Schiff-base complexes
were prepared in the DMSO and then diluted using deionised water.
All the experiments involving the interaction of the complexes
with plasmid DNA were carried out in deionised water contain-
ing Tris–HCl buffer solution (0.6 M HCl) at room temperature and
50 mM NaCl, and adjusted to pH = 7.32. Aliquots of 5 ␮L plasmid
DNA (19.1 ng/␮L and 23.1 ng/␮L) were added continuously while

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A.D. Tiwari et al. / Spectrochimica Acta Part A 79 (2011) 1050–1056
Table 1
Physical properties of the ligand (H2L) and its metal complexes.
Compound
Colour
mp (◦C)
Yield (%)
Conductivity ␮S/cm (10−3
)
(H2L)C13H12N6S
Yellow
200
363
294
320
263
75
79
67
85
75
–
[(H2L)NiCl2]·3H2O C13H14N6OSNiCl2
[(H2L)CoCl2]·3H2O C13H16N6O2SCoCl2
[(H2L)CuCl2]·2H2O C13H14N6OSCuCl2
[(H2L)ZnCl2]·2H2O C13H14N6OSZnCl2
Red brown
Dark black red
Dark green
Lemon yellow
185.1
135.7
149.2
147.6
Table 2
CHNS/O and mass spectral data for ligand and metal complexes.
Compound
Mass
C% obs. (cald)
H% obs. (cald)
N% obs. (cald)
S% obs. (cald)
Cl% obs. (cald)
O% obs. (cald)
–
M% obs. (cald)
–
H2L
307
[M+Na]
341
54.87
(54.91)
32.41
(33.36)
31.83
(33.35)
34.03
(34.48)
33.98
(34.19)
4.34
(4.25)
3.65
(3.88)
3.79
(3.87)
3.51
(3.12)
2.94
(3.53)
27.98
(29.56)
16.87
(17.96)
16.61
(17.95)
17.34
(18.56)
17.67
(18.40)
11.50
(11.28)
5.89
(6.85)
6.85
(7.21)
7.68
(7.08)
7.17
–
C13H12N6S
[(H2L)NiCl2]·3H2O
(15.15)
(15.14)
(15.66)
(15.53)
(10.26)
(10.25)
(7.07)
(12.54)
(12.59)
(14.03)
(14.32)
C
13H12N6SNiCl2·3H2O
[M−2Cl−H]
[(H2L)CoCl2]·3H2O
C13H12N6SCoCl2·3H2O
[(H2L)CuCl2]·2H2O
C13H12N6SCuCl2·2H2O
[(H2L)ZnCl2]·2H2O
C13H12N6SZnCl2·2H2O
413
[M−H]
346
[M−2Cl]
(7.01)
(7.02)
titrating. DNA and compound solutions were allowed to incu-
bate for 10 min at room temperature. Absorption spectrums were
recorded. A fixed amount of the compound, i.e., 500 ␮L (50 ␮M),
was titrated with increasing amounts of DNA, over a DNA concen-
tration range of 0–70 ␮M.
275 nm for azomethine (HC N) and (N–N) chromophores, respec-
tively.
The absorption spectra of the Ni(II) complex bands were
observed at 362 nm which is a higher wavelength shifting of the
*
azomethine band of n → ␲ showing the coordination of the Ni(II)
with (HC N). New bands in the complex appeared at ∼418 nm
which is the charge transfer band for the S → Ni(II) and the
absorption band at 485 nm is assigned to the d–d transition band
3. Result and discussion
4
4T1g(F) → A2g(F). The Ni(II) coordination sphere completes its
All the metal complexes were synthesised with an equimo-
lar ratio of ligand and metal salts in methanol. The ligand (H2L)
was synthesized by the condensation of diamine and aldehyde,
and characterized by UV–Vis, IR, mass spectra, 1H and 13C NMR
and physical techniques like CHNS/O. The complexes formed were
[(H2L)NiCl2]·3H2O (1), [(H2L)CoCl2]·3H2O (2), [(H2L)CuCl2]·2H2O
(3) and [(H2L)ZnCl2]·2H2O (4). Due to the amorphous nature of
the complexes, all the efforts were in vain to develop the crystal.
The binding and coordination modes of the ligand and its metal
complexes were determined by spectral data studies.
Table 1 represents the preliminary observations of the synthe-
sized compounds with respect to colour, melting point (mp), yield
and conductivity. The complexes are insoluble in water; however,
they are sparingly soluble in DMSO. The complexes show low molar
conductivity indicating their non-electrolytic behaviour [27]. Ele-
mental analysis of the compounds was conducted and the findings
are given in Table 2.
five-coordination by two nitrogen ions, one sulphur ion and two
chloride ions. From the above spectral data complex the sphere
seems to prefer the distorted tetrahedral geometry [28].
4
4
The Co(II) complex showed the T1g(F) → A2g(F) d–d transi-
tion bands at 529 nm. The 10 nm shift to a higher wavelength
was attributed to the azomethine band observed at 350 nm as a
result of the coordination with C N in the Co(II) complex. The
charge transfer band for the S → Co(II) could be seen at 303 nm
and there was also a 20 nm lower wavelength shift observed
in the N–N chromophore bands of 275 nm of free ligand which
was observed at 255 nm. The Co(II) metal is coordinated to three
donor sites of the ligand and has assumed a square pyramidal
geometry [28].
The UV–Vis spectra of the Cu(II) complex showed peaks at
355 nm and 425 nm. The azomethine band was shifted to higher
wavelength in the Cu(II) complex. The new band was at 425 nm
assigned to the S → Cu(II) showing the coordination. The C N and
C
S groups are coordination sites of the ligand in Cu(II) com-
3.1. Spectral characterization of compounds synthesized
plex. The ligand is coordinated to copper by the two C N and
one C S, and two chloride ions from the metal salts making a
five coordination sphere around the Cu(II). From the above data
and lack of the characteristic peak of Cu(II) at around ∼800 nm the
trigonal bipyramidal geometry is proposed for the Cu(II) complex
[28,29].
3.1.1. UV–Vis spectroscopy
The UV–Vis spectral data of the ligand and its metal complexes
are given in Table 3. Two absorption bands were observed for the
*
ligand corresponding to n → ␲ and n → ␴ bands at 340 nm and
Table 3
UV–Vis Spectral data (nm) for H2L and its metal complexes (methanol 200–900 nm).
*
*
Compound
(HC N) n → ␲ (nm)
N–N n → ␴ (nm)
Intra-ligand charge transfer (nm)
d → d (nm)
H2L
340 (s)
362 (s)
350 (s)
355 (s)
350 (s)
275 (w)
271 (w)
255 (w)
260 (w)
275 (w)
–
418 (m)
303 (m)
–
–
[(H2L)NiCl2]·H2O
[(H2L)CoCl2]·2H2O
[(H2L)CuCl2·2H2O]·H2O
[(H2L)ZnCl2]·2H2O
485 (w)
529 (w)
425 (w)
–
218 (m)
s: small, w: weak, m: medium.

A.D. Tiwari et al. / Spectrochimica Acta Part A 79 (2011) 1050–1056
1053
Table 4
Characteristic IR bands of the H2L and its metal complexes (in KBr disc 400–4000 cm−1).
Compound
ꢀ(N–H)
ꢀ(HC N)
ꢀ(N–N)
ꢀ(H2O)
ꢀ(C S)
ꢀ(S C–NH)
ꢀ(M−N)
H2L
3130
3094
3090
3102
3085
1609
1615
1612
1618
1622
1111
1147
1141
1111
1147
3468
3407
3408
3442
3444
783
764
766
768
777
1529–1111
1519–1147
1521–1091
1563–1010
1541–1147
–
511
503
508
515
[(H2L)NiCl2]·H2O
[(H2L)CoCl2]·2H2O
[(H2L)CuCl2·2H2O]·H2O
[(H2L)ZnCl2]·2H2O
Fig. 1. Tautomeric form and hyper-conjugation behaviour of the ligands.
Table 5
1H NMR (300 MHz) spectral data (ppm) for H2L ligand and its metal complexes (DMSO-d6).
Compound
3CH
4CH
5CH
6CH
7HC
N
9NH
H2L
7.85 (d)
7.84 (d)
–
7.44 (dd)
7.47 (dd)
–
7.90 (dd)
8.05 (dd)
–
8.64 (d)
8.59 (d)
–
8.26 (s)
8.96 (s)
–
8.87 (s)
–
–
–
[(H2L)NiCl2]·H2O
[(H2L)CoCl2]·2H2O
[(H2L)CuCl2·2H2O]·H2O
[(H2L)ZnCl2]·2H2O
–
–
–
–
–
7.83 (d)
7.47 (dd)
8.12 (dd)
8.63 (d)
8.35 (s)
8.84 (s)
d: doublet, and s: singlet.
The UV–Vis spectrum of the Zn(II) complex is dominated by the
free ligand absorption bands. A red shift of 15 nm was observed
in the complex confirming the coordination of the azomethine
group with Zn(II) ion. An inter-ligand charge transfer band was
observed at 218 nm and the metal to ligand charge transfer band
was observed at 470 nm. The zinc metal ion is coordinated by two
In the Co(II), Ni(II), Cu(II) and Zn(II) complexes there were
∼40 cm−1 shifts to a lower frequency, confirming the coordination
to the nitrogen atom with Ni(II) in the complex. The ꢀ(C S) vibra-
tion in the complex (Table 4) shifted to a lower frequency in the
metal complex due to the coordination of the sulphur to the Ni(II)
metal centre. In the Co(II) complex no shift was observed for the
N–N peak data in the complex, suggesting that the bond was not
involved in the coordination (Fig. 1).
The vibration of the thioamide IV bands also shifted with
respect to their participation in the coordination sphere of the
metal(II) ions. Their new band appeared in the complexes due to
the ꢀ(M–N) vibration, suggesting the bonding between the nitrogen
and metal(II) ion.
C
N from the ligand and two chloride ions from the metal salts
making up a four coordination sphere for the tetrahedral coordi-
nated geometry [28].
3.1.2. Infrared (IR) spectroscopy
Spectral analysis data are given in Table 4 for the ligand and
its metal complexes. The infrared spectral data of the Schiff-base
ligand show the characteristic peaks for the functional groups
like –CH N–, C S, N–H, and N–N group at 1609 cm−1, 783 cm−1
,
3.1.3. 1H and 13C NMR spectroscopy
3130 cm−1, and 1111 cm−1, respectively, and thioamide I, II, III, IV
1H NMR spectral data of the ligand in d6-DMSO (Table 5) con-
firm the proposed structure elucidation of the ligand (Scheme 1).
The characteristic peak data of 1H NMR for the ligand and its com-
plexes are given in Table 5. The NMR spectra for the Co(II) and Cu(II)
complexes could not be recorded due to the paramagnetic prop-
erty of metal complexes. There was strong evidence of Schiff-base
ligand synthesis of the N1,N5-bis-(pyridine-2-methylene) thiocar-
bohydrazone (H2L) [30]. The signal at the 8.26 ppm(s) was assigned
to the azomethine (HC N) proton. The NH proton is assigned at
the 8.87 ppm(s). The aromatic region signal was observed from
7.44 ppm to 8.64 ppm (Table 5). In the Ni(II) complexes, the NH pro-
vibrations at 1529 cm−1, 1429 cm−1, 1236 cm−1 and 1111 cm−1
,
respectively, were observed in the ligand [27–30]. The IR spec-
tra of the metal(II) Schiff-base complexes show a broad band at
∼3440 cm−1 for the ꢀ(OH) group of coordinated water. The ꢀ(N–H)
group vibration bands in all the complexes shifted or even disap-
peared or appeared to be very weak due to resonance and hyper
conjugation between the C S and C N with NH. The azomethine
ꢀ(HC N) vibration band in the complex shifted to a higher fre-
quency due to the increase in bond length through coordination
with the metal(II) centre.
Fig. 2. Proposed structure for the metal complexes.

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A.D. Tiwari et al. / Spectrochimica Acta Part A 79 (2011) 1050–1056
Fig. 3. (H2L), ligand–DNA titration graph of 50 ␮M of ligand in 500 ␮L of Tris–HCl
buffer, 50 mM NaCl, pH 7.32 with E. coli plasmid DNA (19.1 ng/␮L). Arrows show the
absorption change upon increasing DNA concentration.
Fig. 5. (a) Co(II)L1–DNA titration graph of 50 ␮M of complex in 500 ␮L of Tris–HCl
buffer, 50 mM NaCl, pH 7.32 with E. coli plasmid DNA (19.1 ng/␮L); (saturation curve
is given in insert). (b): Co(II)L1–DNA: half-reciprocal plot of D/εap vs. D. The binding
constant, Kb = (slope/intercept) × 106 = 2.1 × 105 M−1
.
ton disappeared in deuterium exchange showing the involvement
of the NH in the bonding. However, in the case of the Zn(II) com-
plexes, the NH proton does not disappear in deuterium exchange
due to the bonding with –C N.
Since the aromatic group did not participate directly in the
coordination there was a very small shift in that region but the
azomethine proton shifted down the field due to the positive
centre of the metal coordination with the ligand HC N group
(Scheme 1 and Fig. 2). From Fig. 2 the tautomeric effect was
explained by the 13C NMR spectra. Two types of carbon peaks were
observed for the C S and C–SH at 175.48 cm−1 and 176.73 cm−1
,
respectively. Other peaks are also given in Table
Scheme 1 [31,32].
6 and in
3.2. Electronic absorption titration of DNA-binding studies
UV–Vis absorption spectroscopy is one of the most commonly
used techniques for investigation of the binding mode of metal
complexes to DNA [34]. Absorption titrations were carried out by
keeping the concentration of the probe constant while adding a
concentrated solution of E. coli genomic DNA. The saturation in
hypochromism was observed. The saturation in hypochromism is
quantitative in most of the cases which is shown by plotting the
A0/A vs. [DNA] where A0 and A are the absorption intensities of the
Fig. 4. (a) (1), Ni(II)L1–DNA titration graph of 50 ␮M of complex in
500 ␮L of Tris–HCl buffer, 50 mM NaCl, pH 7.32 with E. coli plasmid DNA
(23.1 ng/␮L);(complex-DNA saturation curve is given in insert). (b) Ni(II)L1–DNA:
half-reciprocal plot of D/εap vs. D. The binding constant, Kb = (slope/intercept) ×
106 = 2.3 × 105 M−1. Arrows show the absorption change upon increasing DNA
concentration.

A.D. Tiwari et al. / Spectrochimica Acta Part A 79 (2011) 1050–1056
1055
Table 6
13C NMR (75 MHz) spectral data (ppm) for the ligand (DMSO-d6).
Ligand/gp
H2L
C
S
C–SH
HC
N
HS–C
N
Py2C
N
Py3C
Py5C
Py6C
N
Py4C
137.02
175.48
176.73
151.32
152.72
149.71
124.37
126.59
139.32
individual metal-complexes in the absence and presence of vari-
ous concentrations of DNA. The binding constants were calculated
from the following equation:
it cannot bind in any possible way to the DNA. The absorption
reduces every time after addition of the DNA due to the dilution
factor.
The titration absorption curve for the DNA binding activity with
the Ni(II)L1 complex is shown in Fig. 4 showing the best interac-
tion with DNA among all the metal complexes. They interact more
strongly with DNA and they have a higher binding constant than
the other complexes, due to the possible geometry of the Ni(II)
complex of the distorted tetrahedral. The best result for the Ni(II)
complex was shown towards plasmid DNA by UV–Vis spectroscopy.
There is a hypochromism effect as well as a red-shift at ꢃ = 368 nm
(ꢂꢃ = 4.9 nm) with a binding constant of 2.3 × 105 M−1 and having
isosbestic point at ꢃ = 498.2. The presence of isosbestic point in this
titration suggests that there exist chemical equilibrium between
the bound and free metal complexes or the ligand with no spec-
troscopic detectable intermediate states in the presence of DNA.
Strong hypochromism and spectral broadening in absorption inten-
sities indicates intense interaction between the electronic states of
D
D
1
=
+
(1)
ꢁεap
ꢂε
ꢂε × K
where D is the concentration of DNA in the base molarity,
ꢂεap = |εa − εf|, and ꢂε = |εb − εf|, where εb and εf are respective
extinction coefficients of the ligand in the presence and absence of
DNA.
The apparent extinction coefficient εap was obtained by calculat-
ing Aobs/[ligand] where Aobs was the observed absorbance. The data
were fitted to Eq. (1) to obtain a straight line with a slope = 1/ꢂε and
y-intercept = 1/(ꢂε × K). The binding constants K were determined
from the ratio of the slope to the y-intercept [33,35].
The DNA–ligand titration absorption curve in Fig. 3 shows that
there is no interaction between the DNA and the ligand because
there is no charge on the ligand and even the ligand is planar so
Fig. 6. (a) (AD4), Cu(II)L1–DNA half-reciprocal plot of D/εap vs. D. The binding con-
stant, Kb = (slope/intercept) × 106 = 4.67 × 104 M−1. (b) Cu(II)L1–DNA titration graph
of 50 ␮M of complex in 500 ␮L of Tris–HCl buffer, 50 mM NaCl, pH 7.32 with E. coli
plasmid DNA (19.1 ng/␮L). DNA titration saturation curve is given in insert.
Fig. 7. (a) Zn(II)–L1–DNA half-reciprocal plot of D/εap vs. D. The binding constant,
Kb = (slope/intercept) × 106 = 1.52 × 105 M−1. (b): Zn(II)–L1–DNA titration graph of
50 ␮M of complex in 500 ␮L of Tris–HCl buffer, 50 mM NaCl, pH 7.32 with E. coli
plasmid DNA (19.1 ng/␮L). DNA titration saturation graph is given in insert.

1056
A.D. Tiwari et al. / Spectrochimica Acta Part A 79 (2011) 1050–1056
the complex chromophore with that of DNA bases. That means that
Ni(II) complex acts as groove binder with the DNA.
Acknowledgements
The titration absorption curve for the DNA binding activity with
the Co(II)L1 complex is shown in Fig. 5. There is a hypochromism
effect as well as a red-shift at ꢃ = 360 nm (ꢂꢃ = 6.9 nm) with a
binding constant of 2.1 × 105 M−1 and having isosbestic point at
ꢃ = 494.2. The Co(II) complexes show less affinity towards DNA
than the Ni(II) complexes. Complexes of Cu(II) and Zn(II) have
much less binding affinity than Ni(II) and Co(II) complexes; they
even have different binding modes of interaction or bonding to
the DNA. From Fig. 6 for Cu(II) and Fig. 7 for Zn(II) there is no
isosbestic point but the absorption bands of chromophores are
reduced after addition of DNA which shows the interaction of the
complexes’ chromophores with DNA bases and their binding with
plasmid DNA.
The Ni(II) and Co(II) complexes show better DNA binding activity
and interaction than Cu(II) and Zn(II) complexes due to availabil-
ity of the d-orbital in the case of the Ni(II) and Co(II) complexes.
DNA is also acting as a ligand and making binary complexes along
with H2L as different coordinating species and metals are behaving
as the central core. In the case of Cu(II) and Zn(II) complexes the
ligand chromophores have free coordinating sites even after the
coordination with the metal so that their chromophores interact
with the DNA.
Financial support from DST/Mintek Nanotechnology Innovation
Centre, the National Research Foundation (NRF), Pretoria, South
Africa, the University of Johannesburg, and the UJ Commonwealth
Fellowship for author Anand D. Tiwari is gratefully acknowledged.
Special thanks to the MRDG Department, Indian Institute of Sci-
ences, Bangalore, India, for making available the plasmid genomic
E. coli DNA.
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