Synthesis, antimicrobial, DNA-binding and photonuclease studies of Cobalt(III) and Nickel(II) Schiff base complexes

Article abstract of DOI:10.1016/j.saa.2012.12.011

New metal complexes of the type [M(nih)(L)](PF₆) _n·xH₂O and [M(nih)₂](PF ₆)·xH₂O (where M = Co(III) or Ni(II), L = 1,10-phenanthroline (phen)/or 2,2′ bipyridine (bpy), nih = 2-hydroxy-1-naphthaldehyde isonicotinoyl hydrazone, n = 2 or 1 and x = 3 or 2) have been synthesized and characterized by elemental analysis, magnetic, IR and ¹H NMR spectral data. The electronic and magnetic moment 2.97-3.07 B.M. data infers octahedral geometry for all the complexes. The IR data reveals that Schiff base (nih) form coordination bond with the metal ion through azomethine-nitrogen, phenolic-oxygen and carbonyl-oxygen in a tridentate fashion. In addition, DNA-binding properties of these six metal complexes were investigated using absorption spectroscopy, viscosity measurements and thermal denaturation methods. The results indicated that the nickel(II) complex strongly bind with calf-thymus DNA with intrinsic DNA binding constant K_b value of 4.9 × 10⁴ M^-1 for (3), 4.2 × 10 ⁴ M^-1 for (4), presumably via an intercalation mechanism compared to cobalt(III) complex with K_b value of 4.6 × 10 ⁴ M^-1 (1) and 4.1 × 10⁴ M^-1 (2). The DNA Photoclevage experiment shows that, the complexes act as effective DNA cleavage agent.

Full text of DOI:10.1016/j.saa.2012.12.011

Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 105 (2013) 229–237
Contents lists available at SciVerse ScienceDirect
Spectrochimica Acta Part A: Molecular and
Biomolecular Spectroscopy
journal homepage: www.elsevier.com/locate/saa
Synthesis, antimicrobial, DNA-binding and photonuclease studies of Cobalt(III)
and Nickel(II) Schiff base complexes
a
a
b
K.R. Sangeetha Gowda a, H.S. Bhojya Naik a, , B. Vinay Kumar , C.N. Sudhamani , H.V. Sudeep ,
⇑
T.R. Ravikumar Naik c, G. Krishnamurthy d
a Department of Studies and Research in Industrial Chemistry, School of Chemical Sciences, Kuvempu University, Shankaraghatta 577 451, India
b Department of Studies and Research in Biotechnology and Bioinformatics, School of Biological Sciences, Kuvempu University, Shankaraghatta 577 451, India
c Department of Organic Chemistry, Indian Institute of Science, Bangalore 560 012, India
d Department of Chemistry, Sahyadri Science College, Shimoga, Karnataka, India
g r a p h i c a l a b s t r a c t
h i g h l i g h t s
"
"
"
Synthesis of Ni(II) and Co(III) mixed
ligand metal complexes.
Characterized by the spectral and
analytical techniques.
DNA binding and photo cleavage
studies of mixed ligand metal
complexes.
"
Screening of newly synthesized
complexes for in-vitro antimicrobial
activity.
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 1 June 2012
Received in revised form 2 December 2012
Accepted 6 December 2012
Available online 14 December 2012
New metal complexes of the type [M(nih)(L)](PF6)nꢀxH2O and [M(nih)2](PF6)ꢀxH2O (where M = Co(III) or
Ni(II), L = 1,10-phenanthroline (phen)/or 2,20 bipyridine (bpy), nih = 2-hydroxy-1-naphthaldehyde isoni-
cotinoyl hydrazone, n = 2 or 1 and x = 3 or 2) have been synthesized and characterized by elemental anal-
ysis, magnetic, IR and 1H NMR spectral data. The electronic and magnetic moment 2.97–3.07 B.M. data
infers octahedral geometry for all the complexes. The IR data reveals that Schiff base (nih) form coordi-
nation bond with the metal ion through azomethine-nitrogen, phenolic-oxygen and carbonyl-oxygen in a
tridentate fashion. In addition, DNA-binding properties of these six metal complexes were investigated
using absorption spectroscopy, viscosity measurements and thermal denaturation methods. The results
indicated that the nickel(II) complex strongly bind with calf-thymus DNA with intrinsic DNA binding
constant Kb value of 4.9 ꢁ 104 Mꢂ1 for (3), 4.2 ꢁ 104 Mꢂ1 for (4), presumably via an intercalation mech-
anism compared to cobalt(III) complex with Kb value of 4.6 ꢁ 104 Mꢂ1 (1) and 4.1 ꢁ 104 Mꢂ1 (2). The
DNA Photoclevage experiment shows that, the complexes act as effective DNA cleavage agent.
Ó 2012 Elsevier B.V. All rights reserved.
Keywords:
Mixed ligand metal complexes
DNA binding
DNA photocleavage
Antimicrobial-activity
Introduction
The Schiff base compounds constitute an important class of li-
gands which have been extensively studied in coordination chem-
istry. The nature of the effect of one ligand and its transmission to
another ligand through the central metal ion is very important in
coordination chemistry. These interactions can be observed in
Abbreviations: phen, 1,10-phenanthroline; bpy, 2,20-bipyridine; nih, 2-hydroxy-
1-naphthaldehyde isonicotinoyl hydrazone; CT-DNA, calf thymus DNA.
⇑
Corresponding author. Fax: +91 8282 256255.
E-mail address: hsb_naik@rediffmail.com (H.S. Bhojya Naik).
1386-1425/$ - see front matter Ó 2012 Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.saa.2012.12.011

230
K.R. Sangeetha Gowda et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 105 (2013) 229–237
the thermodynamic and the kinetic aspects of the chemical reac-
tivity [1–4].
their DNA-binding and cleavage properties. In the complexes
1,10-phenanthroline and 2,2-bipyridyl are used as co-complexa-
tion ligands, because they have minimum efficiency at inducing
intercalative binding with DNA as demonstrated previously [13–
15] allowing us to focus on the influence of the side face of asym-
metric ligands on the interaction. Information from this study will
be helpful in the understanding of the mechanism of metal com-
plexes reacting with nucleic acids, as well as laying the foundation
for the rational design of new useful DNA probes and effective
inorganic complex nucleases.
The interaction of transition metal compounds with DNA has
been extensively studied in the past decades. Due to the remark-
able binding properties metal complexes to bind with DNA via
intercalation between base pairs and to cleave the DNA by virtue
of their intrinsic chemical, electrochemical and photochemical
reactivities, these coordination compounds were suitable candi-
dates as DNA footprinting agents, photocleavers and antitumor
drugs [5–7]. Small metal complexes can interact with DNA in
non-covalent fashion and in most important applications, it is by
an intercalative mode. Generally the intercalative complex should
contain an extended aromatic ligand, as its stacking between base
pairs is considered to be a major driving force that leads to binding.
The binding mode and strength are sensitively dependent on the
shape, planar area, size and electron density of the interacting aro-
matic rings. So, a systematic study of the influence of varying
parameters on the interaction of metal complexes with DNA would
be valuable in the rational design of new drugs and therapeutic re-
agents targeted to DNA. And it is possible to systematically vary
parameters of interest by changing the properties of the intercalat-
ing groups [4–6].
On the other hand, cobalt and nickel metal ions showed good
biological activity. Recently, we found that, the large number of co-
balt(III) and nickel(II) complexes exhibited interesting DNA bind-
ing properties and are efficient photonucleases [8–14]. Cobalt
complexes have gained significance as the recent studies have
shown that it can produces more active oxygen species that may
be associated with the induction of chromosomal aberrations and
mutation and because of their application as potential hypoxia-
activated prodrugs [12]. Likewise a rich and varied chemistry of
nickel with DNA has been supported by the existing literature.
Studies have revealed that nickel compounds enter a cell by phago-
cytosis, find their way to the nucleus, and cause oxidative DNA
strand breaks, DNA–DNA cross-links and DNA–protein cross-link.
Ni2+ is known to bind well to guanine via the N7 nitrogen of the
purine’s imidazole ring. Such binding is thought to be responsible
for the fact that sub millimolar concentrations of Ni2+ will induce
conformational change in DNA from the right-handed B helical
form to a left-handed Z helical form [13]. All these collectively sug-
gest that cobalt(III) and nickel(II) coordination compounds would
display interesting binding and cleavage reactivity with nucleic
acids.
Experimental
Chemicals and instrumentations
All reagents and solvents required were of AR grade, purchased
commercially. All the solvents were purified by distillation and
used. 1,10-phenanthroline, 2,20 bipyridine CoCl2ꢀ6H2O, NiCl2ꢀ6H2O,
Tris–HCl and ammonium hexaflurophosphate (NH4PF6) were pur-
chased from Merck (India), calf thymus DNA (CT-DNA) and super
coiled (SC) pUC19DNA were purchased from Bangalore Genie (In-
dia), Agarose (molecular biology grade) and ethidium bromide
were purchased from Himedia. Tris–HCl buffer solution used for
binding and cleavage studies was prepared using deionised double
distilled water. Melting points were determined in open capillaries
and were uncorrected. Microanalyses (C, H and N) were performed
in Carlo-Erba 1106-model 240 Perkin-Elmer analyzer. IR spectra
were recorded with Shimadzu model FT-IR spectrophotometer by
using KBr pellets. UV–visible absorption spectra were recorded
using Shimadzu model UV-1650PC spectrophotometer at room
temperature. 1H-NMRspectra were recorded on a Bruker FT NMR
spectrometer (300 MHz) at 25 °C in DMSO with TMS as the internal
reference. Viscosity measurement swere carried out on semimicro
dilution capillary viscometer (Viscomatic Fica MgW) with a ther-
mostatic bath D40S at room temperature. Thermal denaturation
studies were carried out with a Perkin-Elmer model 554 with a
Shimazdu UV–Vis recording spectrophotometer coupled to a tem-
perature controller (Model TCC-240A) using quartz cuvettes of
10 mm light-path.
Synthesis of ligand
Our group has continuously been interested in DNA interactions
of mixed-ligand complexes and has reported the synthesis and
DNA binding and cleavage activity of various Co(III) and Ni(II)
[13–15]. In addition, some hydrazones and their complexes with
metals often have diverse biological and pharmaceutical activities
[15–21]. However, up to now the biological activity and interac-
tions of mixed ligand complexes of 2-hydroxy-1-naphthaldehyde
isonicotinoyl hydrazone with DNA have not been reported. This
aroused our interest in the synthesis of ligand (Scheme 1) and its
Co(III) and Ni(II) mixed ligand complexes in view of evaluating
According to the reported procedures, [22,23] the known 2-hy-
droxy-1-naphthaldehyde isonicotinoyl hydrazone was selected as
the starting material in the present investigation. A solutions of
isonicotinoyl hydrazine (0.05 mol) in anhydrous EtOH (50 cm3)
were added dropwise to the ethanolic solution (50 cm3) of 2-hy-
droxy-1-naphthaldehyde (0.05 mol) with stirring and the mixture
was refluxed for 4 h. The resulting solution was concentrated to
half of its initial volume and then cooled to room temperature.
The obtained yellow precipitate was separated by filtration,
washed with EtOH and dried in a vacuum desiccator.
Scheme 1. Synthesis of ligand.

K.R. Sangeetha Gowda et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 105 (2013) 229–237
231
(NIH) Yellow solid; Yield 72%; mp 181 °C; Anal. Calcd for
17H13N3O2: C, 70.09; H, 4.50; N, 14.42. Found: C, 69.80; H, 4.34;
DNA-binding experiments
C
N, 14.03%. IR (KBr):
m
(cmꢂ1): 3410, 3270, 1665, 1610, 1585,
The DNA-binding and cleavage experiments were performed at
room temperature. The DNA concentration per nucleotide was
determined by absorption spectroscopy using the molar absorp-
tion coefficient (6600 Mꢂ1 cmꢂ1) at 260 nm [27]. The absorption
titration experiments and viscosity measurements experiments
involving the interaction of the complexes with calf thymus DNA
were carried out in Tris–HCl buffer containing (5 mM Tris, pH
7.1, 50 mM NaCl) 5% DMF at room temperature. Phosphate buffer
(1 mM phosphate, pH 7, 2 mM NaCl) was used for thermal dena-
turation. Absorption titration experiments were carried out by
varying the DNA concentration and maintaining the complex con-
centration constant. Due correction was made for the absorbance
of DNA itself. Absorbance values were recorded after each succes-
sive addition of DNA solution and equilibration (ca. 10 min). The
absorption data were analysed for an evaluation of the intrinsic
binding constant Kb using the following equation:
1275. 1H NMR (DMSO-d6) d (ppm): 1H (d, ppm) 7.01 (d, 1H),
7.26 (t, 1H), 7.51 (t, 1H), 7.73 (d, 1H), 8.04 (d, 2H), 8.11 (d, 1H),
8.70 (d, 2H), 9.25 (s, 1H); UV kmax, nm: 364.
Synthesis of metal complexes
Synthesis of [Co(phen)(nih)] (PF6)2ꢀ3H2O (1) and [Co(bpy)(nih)]
(PF6)2ꢀ3H2O (2)
A hot methanolic solution of the 0.001 mol ligand NIH (0.29 g)
was mixed with a methanolic solution 0.001 mol of CoCl2ꢀ6H2O
(0.237 g) 25 cm3 and refluxed for 1 h. Cobalt (III) complexes were
obtained due to air oxidation [24–26]. To the above obtained com-
plex, 1 mmol (0.198 g) of 1,10-phenanthroline/or 2,20 bipyridine
(0.156 g) in 10 ml methanol was added drop wise with constant
stirring and refluxed for 4–5 h under nitrogen. It was then filtered
and the complex was precipitated upon addition of a saturated
ethanolic solution of ammonium hexafluorophosphate to the fil-
trate. It was filtered, washed with cold EtOH and dried under vac-
uum over CaCl2 before being recrystallized (acetone–ether).
Complex (1) Brown solid; Yield 63%; Anal. Calcd for C29H22-
CoN5O3: C, 63.62; H, 4.05; N, 12.79; Co, 10.76. Found: C, 63.04;
½DNAꢃ=ðea
ꢂ
ef Þ ¼ ½DNAꢃ=ðeb
ꢂ
ef Þ þ 1=Kbðea
ꢂ
ef
Þ
ð1Þ
where [DNA] is the concentration of DNA in base pairs, ea corre-
sponds to the apparent absorption coefficient Aabs/[M], ef corre-
sponds to the extinction coefficient for the free metal [M]
complex and eb corresponds to the extinction coefficient for the me-
tal [M] complex in the fully bound form. In plots of [DNA]/(ea
ꢂ
ef) vs
m
(cmꢂ1): 3218, 1635,
[DNA], Kb is given by the ratio of slope to the intercept.
H, 4.00; N, 12.09; Co,10.24%. IR (KBr):
Viscosity measurements were carried out using a semimicro
dilution capillary viscometer at room temperature. Flow time
was measured with a digital stopwatch, and each sample was mea-
sured three times, and an average flow time was calculated. Data
1614, 1546, 843, 818, 465, 360; UV kmax, nm: 447.
Complex (2) Brown solid; Yield 60%; Anal. Calcd for C27H22CoN5-
O3: C, 61.95; H, 4.24; N, 13.38; Co, 11.26. Found: C, 61.03; H, 4.14;
N, 12.92; Co, 11.13.%. IR (KBr):
m
(cmꢂ1): 3220, 1642, 1618, 1571,
were presented as (g/g0) versus [complex]/[DNA], where g is the
847, 817, 472, 364; UV kmax, nm: 440.
viscosity of DNA in the presence of the complex and g0 is that of
DNA alone [27–29].
Thermal denaturation experiments were carried out with a
Synthesis of [Ni(phen)(nih)](PF6)ꢀ2H2O (3) and
Shimazdu UV–Vis recording spectrophotometer coupled to
a
[Ni(bpy)(nih)](PF6)ꢀ2H2O (4)
temperature controller by monitoring the absorption at 260 nm
of CT-DNA at various temperatures [30,31].
This complex was prepared in a similar way to that described
for [Co(phen)(nih)](PF6)2ꢀ2H2O.
Complex (3) Brick red solid; Yield 64%; Anal. Calcd for C29H22-
NiN5O3: C, 63.65; H, 4.05; N, 12.80; Ni, 10.73. Found: C, 63.12; H,
DNA cleavage experiments
3.98; N, 12.16; Ni, 10.37%. IR (KBr):
1549, 843, 467, 358; UV kmax, nm: 436;
m
(cmꢂ1): 3217, 1637, 1612,
l
eff: 2.97 0.02 BM.
For the gel electrophoresis experiments, supercoiled pUC19
DNA was treated with Co(III) and Ni(II) complexes in Tris buffer
(50 mM Tris–acetate, 18 mM NaCl, pH 7.2), and the solution was
irradiated at room temperature with a UV lamp (365 nm, 10 W).
After being incubated at 37 °C for 1 h, electrophoresis was carried
out at 50 V for 2 h in Tris–borate EDTA (TBE) buffer. Electrophoresis
was carried out and bands were visualised by UV light and photo-
graphed to determine the extent of DNA cleavage from the inten-
sities of the bands using UVITEC Gel Documentation System
[13,14].
Complex (4) Brick red solid; Yield 60%; Anal. Calcd for C27H22-
NiN5O3: C, 61.98; H, 4.24; N, 13.39; Ni, 11.22. Found: C, 61.12; H,
4.19; N, 13.02; Ni, 11.09%. IR (KBr):
1571, 847, 485, 365; UV kmax, nm: 440;
m
(cmꢂ1): 3221, 1649, 1618,
leff: 3.07 0.02 BM.
Synthesis of [Co(nih)2](PF6)ꢀ3H2O (5) and [Ni(nih)2]ꢀ3H2O (6)
The complexes were synthesised with slight modifications of
the reported procedure [22]. To a refluxing solution of NIH
(0.50 g, 1.72 mmol) in MeOH (125 mL) was added dropwise a solu-
tion of CoCl2ꢀ6H2O and NiCl2ꢀ6H2O, (0.35 g, 0.87 mmol) in MeOH
(30 mL) respectively. An immediate darkening of the solution re-
sulted and reflux was continued for 1 h. The complex was precipi-
tated upon addition of a saturated ethanolic solution of ammonium
hexafluorophosphate. Upon cooling, precipitate was obtained,
which was filtered off, washed with EtOH then diethyl ether and
dried in a vacuum desiccator.
Antimicrobial study
The antimicrobial activity of the ligand and the complexes were
tested against the standard microbial strains P. aeruginosa ATCC-
20852, K. pneumoniae MTCC-618, S. aureus ATCC-29737, S. typhi
MTCC-3214, E. coli ATCC-25922 and C. albicans ATCC-2091. The pri-
mary screening was performed using agar well diffusion [32] at
fixed concentrations of 10 mg/ml by dissolving in 10% DMSO.
50 mg/ml Ciprofloxacin (fluoroquinolone antibiotic) and Miconaz-
ole as the positive control. The experiments were performed in
triplicates. The MIC of the chemically synthesized compound was
tested against bacterial strains through a macrodilution tube
method [33]. The test concentrations of chemically synthesized
compounds were made from 128 to 0.25 mg/ml in the sterile tubes
no. 1–11.
Complex (5) Brown solid; Yield 74%; Anal. Calcd for C34H24-
CoN6O4: C, 63.88; H, 3.78; N, 13.15; Co, 9.18. Found: C, 63.10; H,
3.45; N, 13.08; Co, 9.11.%. IR (KBr):
m
(cmꢂ1): 3219, 1638, 1616,
1545, 467, 363; UV kmax, nm: 380.
Complex (6) Brick red solid; Yield 71%; Anal. Calcd for C34H24Ni
N6O4: C, 63.85; H, 3.78; N, 13.14; Ni, 9.22. Found: C, 63.17; H, 3.32;
N, 13.00; Ni, 9.07%. IR (KBr): m
(cmꢂ1): 3220, 1640, 1618, 1552, 471,
359; UV kmax, nm: 365.

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K.R. Sangeetha Gowda et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 105 (2013) 229–237
Statistical analysis
complexes, other extra band expected for d–d transitions have
been submerged. Unlike the cobalt(III) complexes, which are dia-
magnetic indicating low spin octahedral, nickel(II) complex were
found to be paramagnetic with leff value of 2.97 0.02 B.M. and
3.09 0.02 B.M. respectively for complex 3 and 4 as expected for
typical d8 octahedral systems. On the basis of above evidence
and analyses, the suggested structure of the complexes are shown
in Fig. 1.
The results of the antibacterial study are expressed as mean
SEM of three replicates in each test. The data were evaluated
by one-way Analysis of Variance (ANOVA) followed by Tukey’s
multiple pair wise comparison tests to assess the statistical
significance. The data were considered at P < 0.05.
Result and discussion
Characterization of complexes
DNA binding studies
The elemental analytical data, IR and magnetic moment data of
the new complexes are summarized in experimental section. These
data were in agreement with the theoretical values within the limit
of experimental error and confirmed the formula of the complexes
were [Co(nih)(L)](PF6)2ꢀ3H2O, [Ni(nih)(L)](PF6)ꢀ2H2O, [Co(nih)2](-
PF6)ꢀ3H2O and [Ni(nih)2]ꢀ3H2O. Complexes are soluble in methanol,
ethanol, DMF, DMSO and in buffer (pH 7.2) solution. The molar
conductance values of the complexes 1–5 in DMF solutions fall in
the region 30.0–65.0 Oꢂ1 cm2 moleꢂ1 shows electrolytic nature.
The Co (III) complexes were obtained due to air oxidation of the
complex formed by Cobalt (II) chloride and ligand [23,24].
Absorption spectroscopic studies
Electronic absorption spectroscopy is usually employed to
determine the binding of complexes with the DNA helix. A com-
plex bound to DNA through intercalation is characterized by the
change in absorbance (hypochromism) and red shift in wave-
length, due to a strong stacking interaction between the aromatic
chromophore and the DNA base pairs. The extent of hypochromism
is commonly consistent with the strength of the intercalative inter-
action [34–36].
The absorption spectra of the complexes in the absence and
presence of calf thymus DNA are shown in Figs. 2–5. In the UV re-
gion, the Co(III) complex exhibit two intense absorption bands: one
at ca. 447 nm which is attributed to the ligand-to-metal charge
transfer transitions (LMCTs), and the other in the region 270 nm
The IR spectra of all complexes indicate that the m(C@N) bands
of the ligands at 1585 cmꢂ1 due to the azomethine linkage were
shifted towards lower frequency 1545–1571 cmꢂ1, indicating that
the ligands coordinate to metal ions via the azomethine nitrogen.
which is assigned to the intraligand (p–
pꢄ) transitions of the aro-
The peak exhibited at 1665 cmꢂ1due to
m(C@O) vibration of the
matic chromophore. With increasing CT DNA, the absorption bands
of the complexes were affected, resulting in hypochromism and
slight shifts to a longer wavelength, which indicates that the aro-
matic N-containing ligand-complexes can interact with CT DNA.
The absorption spectra indicates that, upon addition of DNA to
complex 1, 3 more binding activity observed to that of complexes
2, 4, 5 and 6 as shown in Figs. 2–5.
free ligand were shifted to lower frequencies 1635–1649 cmꢂ1 in
the complexes. This shift confirms that, the group loses its original
characteristics and forms coordinative bonds with metal. The ab-
sence of band due to phenolic OH group at 3410 cmꢂ1 and increase
in frequency of phenolic CAO vibration from 1275 cmꢂ1 of ligand
to 1316–1320 cmꢂ1 in the spectra of all metal complexes suggests
the coordination of ligand to the metal via deprotonation, [22,23]
which infers that azomethine–nitrogen, phenolic-oxygen and car-
bonyl-oxygen as the coordination sites of the monobasic tridentate
ligand. Besides, two non-ligand peaks at 465–485 cmꢂ1 and 345–
The hypochromism observed were 33%, 30%, 34%, 31%, 27% and
25% for complexes (1)–(6) respectively. The [DNA]/(ea ef) ratio
ꢂ
were plotted against [DNA] and intrinsic constant Kb were
obtained from the ratio of the slope to the intercept. The Kb
value obtained for complexes (1)–(6) are 4.6 ꢁ 104 Mꢂ1, 4.1 ꢁ
365 cmꢂ1 of complexes were assigned to
m(MAO) and m(MAN)
stretching vibrations respectively. In addition the IR spectrum of
the PF6 salt of each complex showed a strong band in the region
843–847 cmꢂ1 ascribable to the counter anion and this band was
absent for the corresponding chloride salts.
104 Mꢂ1
,
4.9 ꢁ 104 Mꢂ1
,
4.2 ꢁ 104 Mꢂ1
,
3.6 ꢁ 104 Mꢂ1 and 2.8
ꢁ 104 Mꢂ1respectively. The observed Kb values for complexes 1–6
of Co(III) and Ni(II) complexes are comparable to the values of
classical intercalators Figs. 2–5 [36–38]. This may suggest that
the complexes have intercalative mode of binding that involves a
stacking interaction between the complex and the base pairs of
DNA.
The electronic spectra of the free ligand NIH and complexes
measured in DMSO are dominated by intense ligand-centred tran-
sitions in the near UV. The maxima of complexes and NIH in this
region are almost identical in both wavelength and intensity: com-
plexes kmax 369 nm, 328 nm and 315 nm compared with NIH kmax
368 nm, 328 nm and 315 nm. However, the complex exhibits
prominent transitions in the visible region not seen in the spec-
trum of the faintly coloured free ligand. A broad band were ob-
served at 447 nm, 440 nm, 436 nm, 431 nm, 380 nm and 374 nm
in the spectrum of the complexes (1)–(6) respectively, are assigned
to the LMCT transition and spectral feature of octahedral
Viscosity measurements
In the absence of crystallographic structure data, hydrodynamic
methods which are sensitive to DNA length increases, are regarded
as the least ambiguous and the most critical tests of binding in
solution. A classical intercalative mode demands that the DNA he-
lix must lengthen as base pairs are separated to accommodate the
Fig. 1. Proposed structures of complexes.

K.R. Sangeetha Gowda et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 105 (2013) 229–237
233
Fig. 2. Absorption spectra of mixed ligand complex (1) in Tris–HCl buffer upon addition of DNA. [Co(III)] = 0.5 lM, [DNA] = 0–100 lM. Arrow shows the absorbance changing
upon increase of DNA concentration. The inner plot of [DNA]/(ea
ꢂ
e
f) vs [DNA] for the titration of DNA with complex (1), Kb = 4.6 ꢁ 104 Mꢂ1
.
Fig. 3. UV absorption spectra of complex (3) upon addition of calf thymus (ds) DNA. [Ni(II)] = 0.5 lM, [DNA] = 0–100 lM. Arrow shows the absorbance changing upon the
increase of DNA concentration. The inner plot of [DNA]/(ea
ꢂ
e
f) vs [DNA] for the titration of DNA with complex (3), Kb = 4.9 ꢁ 104 Mꢂ1
.
binding ligand, leading to the increase of DNA viscosity [39,40].
The effects of complexes together with the viscosity of rod-like
DNA are shown in Fig. 6. On increasing the amounts of
complexes 1–6, the relative viscosity of DNA increases steadily.

234
K.R. Sangeetha Gowda et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 105 (2013) 229–237
Fig. 4. Absorption spectra of mixed ligand complex (2) in Tris–HCl buffer upon addition of DNA. [Co(III)] = 0.5 lM, [DNA] = 0–100 lM. Arrow shows the absorbance changing
upon increase of DNA concentration. The inner plot of [DNA]/(ea
ꢂ
e
f) vs [DNA] for the titration of DNA with complex (2), Kb = 4.1 ꢁ 104 Mꢂ1
.
Fig. 5. Absorption spectra of mixed ligand complex (4) in Tris–HCl buffer upon addition of DNA. [Ni(II)] = 0.5 lM, [DNA] = 0–100 lM. Arrow shows the absorbance changing
upon increase of DNA concentration. The inner plot of [DNA]/(ea
ꢂ
e
f) vs [DNA] for the titration of DNA with complex (4), Kb = 4.2 ꢁ 104 Mꢂ1
.
The experimental results suggest that complexes bind to DNA
through a classical intercalation mode. This helps complexes inter-
calate into the DNA base pairs deeply [37,38].
any added complex. The Tm of DNA was increased by 4–7 1 °C in
presence of complexes (1)–(6) at [DNA nucleotide phosphate]/
[complex] = 25 Fig. 7. The rT values of DNA were also increased
by 4 1 °C for these complexes. The increase Tm and rT of DNA
could be interpreted in terms of the stabilization that results from
the intercalation of these metal complexes with DNA. The observa-
tions made during the absorption titration, viscosity measure-
ments and thermal denaturation experiments are reminiscent of
those reported earlier for various metallointercalators, thus
suggesting that the complexes bound to DNA by intercalations
[41–43].
Thermal denaturation studies
The thermal behaviour of DNA is a measure of the stability of
DNA double helix temperature. An interaction between DNA and
complexes were indicated by the increase in the thermal melting
temperature (Tm). Thermal denaturation experiments also revealed
the intercalation of these complexes with DNA. The CT-DNA alone
melts at 61 1 °C (10 mmol NaCl, 1 mmol phosphate) in absence of

K.R. Sangeetha Gowda et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 105 (2013) 229–237
235
Fig. 6. Plot of relative viscosity vs [Complex]/[DNA]. Effect of Co(III) complexes and Ni (II) complexes on the viscosity of CT-DNA at room temperature. Complex = 0–160 lM,
[DNA] = 150 lM.
Fig. 7. Thermal denaturation of CT-DNA in the absence and presence of Co(III) and Ni(II)complexes. [DNA] = 50 lM, [complex] = 10 lM, buffer: phosphate.
DNA cleavage activity
DNA cleavage was analysed by monitoring the conversion of
supercoiled DNA (Form I) to nicked DNA (Form II) and linear
DNA (Form III) under anaerobic conditions. No DNA cleavage was
observed for the control in which metal complex was absent (lane
1). Complex 1–6 can induce the obvious cleavage of the plasmid
DNA at the concentration of 20 lM, 40 lM and 60 lM. It was
shown that, at lower concentrations, DNA cleavage was not effec-
tive (lanes 2–4) Figs. 8–10 respectively. At the concentration of
60 lM, complexes can almost promote 75% conversion of super-
coiled DNA to nicked and linear DNA (Form I–III). With increasing
concentration of these complexes (lanes 2–7), the amount of Form
I of pUC19 DNA diminished gradually, whereas Form III increased.
However, nickel complexes 3 and 4 exhibited much higher cleav-
ing efficiency for pUC19 DNA. Even at the concentration of
Fig. 8. Photo-induced DNA cleavage by the mixed ligand complexes (1) and (3).
Supercoiled DNA runs at position I (SC), nicked DNA at position II (NC) and Linear
DNA at position III (LC). The complexes were irradiated with UV light at 365 nm,
Lane; 1: control DNA (without complexes), Lane; 2: 20
Co(III), Lane; 4: 60 M; Co(III), Lane; 5: 20 M; Ni(II), Lane; 6: 40
7: 60 M; Ni(II).
lM; Co(III), Lane; 3: 40
lM;
M; Ni(II), Lane;
40
lM, it can promote almost 65% conversion of DNA from form
l
l
l
I–III. The nucleolytic efficiencies of nickel complexes at the
l

236
K.R. Sangeetha Gowda et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 105 (2013) 229–237
60 lM concentration (lanes 5–7) Figs. 8–10 respectively, the super-
coiled pUC19 DNA was completely converted to form II and form
III. Higher concentrations were not examined because of the pre-
cipitation of complex in the reaction mixture. It was found that
nickel complexes were the most potent nuclease mimic in terms
of molecular structure. Chemical environment and their geometric
structures may also affect the nucleolytic efficiency of nickel com-
plexes. The different DNA-cleavage efficiency of the complexes
may be considered due to the different binding affinity of the com-
plexes to DNA [44,45].
Antimicrobial studies
Fig. 9. Photo-induced DNA cleavage by the mixed ligand complexes (2) and (4).
Supercoiled DNA runs at position I (SC), nicked DNA at position II (NC) and Linear
DNA at position III (LC). The complexes were irradiated with UV light at 365 nm,
The synthesized ligand and complexes were tested for their
in vitro antimicrobial activity. Significant inhibitory spectrum was
observed in the primary screening Table 1. The minimum inhibi-
tory concentrations of the investigated compounds are summa-
rized in Table 2.
Lane; 1: control DNA (without complexes), Lane; 2: 20
Co(III), Lane; 4: 60 M; Co(III), Lane; 5: 20 M; Ni(II), Lane; 6: 40
7: 60 M; Ni(II).
lM; Co(III), Lane; 3: 40
lM;
M; Ni(II), Lane;
l
l
l
l
A comparative study of the ligand and the complexes indicates
that the metal complexes exhibit higher antimicrobial activity than
the free ligand. The potent antimicrobial activity of metal com-
plexes could be attributed to the fact that metal complexes with la-
bile ligands have long been known to undergo ligand-substitution
reactions with biomolecular targets. This pronounced activity can
be explained on the basis of Tweedy’s chelation theory [46]. There
is a decrease in the polarity of the metal ion significantly after che-
lation, because of the partial sharing of its positive charge with the
donor group and also due to p-electron delocalization on the whole
chelate ring. The metal ion interaction is preferred with the lipids
and polysaccharides which are the important constituents of the
cell wall and membranes. Other components of cell wall viz., many
phosphates, carbonyl and cysteinyl ligands which maintain the
integrity of the membrane by acting as a diffusion barrier also pro-
vide suitable sites for binding. Furthermore, the reduction in polar-
ity increases the lipophilic character of the chelates and an
interaction between the metal ion and the lipid is favoured which
may lead to the subsequent breakdown of the permeability barrier
of the cell resulting in obstruction with the normal cell processes.
Fig. 10. Photo-induced DNA cleavage by the mixed ligand complexes (5) and (6).
Supercoiled DNA runs at position I (SC), nicked DNA at position II (NC) and Linear
DNA at position III (LC). The complexes were irradiated with UV light at 365 nm,
Lane; 1: control DNA (without complexes), Lane; 2: 20
Co(III), Lane; 4: 60 M; Co(III), Lane; 5: 20 M; Ni(II), Lane; 6: 40
7: 60 M; Ni(II).
lM; Co(III), Lane; 3: 40
lM;
M; Ni(II), Lane;
l
l
l
l
Table 1
Antimicrobial activity of synthesized compounds against standard microbial strains (inhibition zone in mm).
Microbial strains
Complexes
1
Nih
Ciprofloxacin
Miconazole
2
3
4
5
6
Pa
Kp
Sa
St
Ec
Ca
17.97 0.62
15.40 0.49
16.80 0.17*
17.23 0.75*
18.67 0.24*
16.63 0.60*
17.67 1.01
14.57 0.70
17.57 0.23
15.10 0.26*
19.20 0.17*
19.57 0.35*
16.97 0.79*
14.77 0.35*
16.23 0.43*
15.73 0.93*
19.53 0.29*
18.23 0.65
18.17 1.01
16.03 0.09*
15.63 0.44*
17.37 0.35*
20.03 0.29*
19.07 0.48
15.00 0.29*
14.00 1.04*
14.83 0.44*
15.17 0.44*
15.00 1.03*
14.50 0.32*
13.17 0.93*
13.17 0.44*
13.67 0.88*
13.33 0.88*
14.30 0.35*
13.53 0.43*
11.93 0.22*
12.93 0.43*
13.50 0.29*
12.63 0.22*
13.47 0.24*
13.07 0.58*
20.00 0.58
21.00 0.87
20.17 1.01
20.77 0.54
21.83 0.38
-
–
–
–
–
–
20.15 0.64
The values are the mean of three experiments SE.
Abbreviations: Pa, Pseudomonas aeruginosa (Pa ATCC-20852); Kp, Klebsiella pneumoniae (Kp MTCC-618); Sa, Staphylococcus aureus (Sa ATCC-29737); St, Salmonella typhi (St
MTCC-3214); Ec, Escherichia coli (Ec ATCC-25922); Ca, Candida albicans (Ca ATCC 2091).
*
P < 005 vs. standard.
Table 2
Minimum inhibitory concentrations (in lg/ml) of compounds using macrodilution method.
Bacterial strains
Complexes
1
Nih
Ciprofloxacin
Miconazole
2
3
4
5
6
Pa ATCC-20852
Kp MTCC-618
Sa ATCC-29737
St MTCC-3214
Ec ATCC-25922
Ca ATCC-2091
4
4
4
2
2
8
2
4
8
4
4
2
4
4
4
2
2
4
4
4
8
4
4
4
16
8
8
4
8
8
8
16
4
8
32
4
8
8
16
16
4
2
4
2
2
-
–
–
–
–
–
4
16
8
Abbreviations: Pa, Pseudomonas aeruginosa; Kp, Klebsiella pneumoniae; Sa, Staphylococcus aureus; St, Salmonella typhi; Ec, Escherichia coli; Ca, Candida albicans.

K.R. Sangeetha Gowda et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 105 (2013) 229–237
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