Design, synthesis and DNA binding activities of late first row transition metal(II) complexes of bi- functional tri - and tetratopic imines

Article abstract of DOI:10.1016/j.saa.2012.07.066

A series of novel Co(II), Ni(II), Cu(II) and Zn(II) complexes of tri and tetratopic hydrazones have been prepared. Ligands L¹H₂ and L²H₂ were synthesized by the condensation of 2-formylphenoxyacetic acid with 2-hydrazinobenzothiazole and 2-hydroxy-3-hydrazinebenzopyrazine, respectively. The prepared complexes were characterized by the analytical and spectral techniques. All the complexes were found to be monomeric in nature with octahedral geometry. Both ligands were found to be electrochemically active in the working potential range showing single electron transfer process attributed to the deprotonation of carboxylic group of the 2-formylphenoxyacetic acid. The potency of the ligand and its complexes as antimicrobial agents has been investigated and made to interact with Escherichia coli DNA to investigate the binding/cleaving ability by absorption, hydrodynamic and electrophoresis studies.

Full text of DOI:10.1016/j.saa.2012.07.066

Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 97 (2012) 762–770
Contents lists available at SciVerse ScienceDirect
Spectrochimica Acta Part A: Molecular and
Biomolecular Spectroscopy
journal homepage: www.elsevier.com/locate/saa
Design, synthesis and DNA binding activities of late first row transition
metal(II) complexes of bi- functional tri – and tetratopic imines
⇑
Priya P. Netalkar, Anupama Kamath, Sandeep P. Netalkar, Vidyanand K. Revankar
Department of Studies in Chemistry, Karnatak University, Pavate Nagar, Dharwad 580 003, 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
"
"
"
Series of tri and tetratopic complexes
of bi-functional imines have been
prepared.
Metal complexes exhibit enhanced
antibacterial activity compared to
ligands.
Ligands and their complexes bind to
DNA via intercalative mode of
interaction.
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of novel Co(II), Ni(II), Cu(II) and Zn(II) complexes of tri and tetratopic hydrazones have been pre-
pared. Ligands L1H2 and L2H2 were synthesized by the condensation of 2-formylphenoxyacetic acid with
2-hydrazinobenzothiazole and 2-hydroxy-3-hydrazinebenzopyrazine, respectively. The prepared com-
plexes were characterized by the analytical and spectral techniques. All the complexes were found to
be monomeric in nature with octahedral geometry. Both ligands were found to be electrochemically
active in the working potential range showing single electron transfer process attributed to the deproto-
nation of carboxylic group of the 2-formylphenoxyacetic acid. The potency of the ligand and its com-
plexes as antimicrobial agents has been investigated and made to interact with Escherichia coli DNA to
investigate the binding/cleaving ability by absorption, hydrodynamic and electrophoresis studies.
Ó 2012 Elsevier B.V. All rights reserved.
Received 10 April 2012
Received in revised form 12 July 2012
Accepted 13 July 2012
Available online 24 July 2012
Keywords:
Benzothiazole
2-Hydroxy-3-hydrazinebenzopyrazine
2-Formylphenoxyacetic acid
Antimicrobial activity
DNA binding study
Introduction
in either non-covalent or covalent way. The former way includes
three binding modes, i.e., intercalation, groove binding and exter-
Schiff base metal complexes of multidentate aromatic ligands
have been of current interest in the field of medicinal research
and in development of new therapeutic agents, mainly due to the
fact that they exhibit novel electronic and/or magnetic properties
with fascinating structural features, as well as the ability of these
ligands to be modulated to DNA binding and cleaving abilities
[1–3]. Basically, metal complexes interact with double helix DNA
nal static electronic effects. Among these, intercalation is being
one of the most important. The intercalating ability is known to in-
crease with the planarity of ligands [4]. Additionally, the coordina-
tion geometry and nature of donor atom play key roles in
determining the binding extent of the complexes to DNA. Lastly,
the importance of the central metal ion at the core of the structure
of the DNA binding molecule is noteworthy [5,6]. Many DNA inter-
active heterocyclic compounds, including benzothiazole and ben-
zopyrazine derivatives, have already been evaluated and have
found to be efficient DNA intercalators [7,8].
⇑
Corresponding author. Tel.: +91 9900485652/+91 836 4250821 (Res.).
E-mail address: vkrevankar@rediffmail.com (V.K. Revankar).
1386-1425/$ - see front matter Ó 2012 Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.saa.2012.07.066

P.P. Netalkar et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 97 (2012) 762–770
763
2-Formylphenoxyacetic acid, in combination with benzothia-
zole/benzopyrazine derivatives facilitates the formation of tri-
and tetradentate metal complexes with N and O donor ligand sys-
tem with labile anion in the coordination sphere. The presence of
labile chloride in complexes is an interesting thing in this study
as they allow the binding of the metal centre with N-7 of purine
bases after deprotonation. Furthermore, the coordination model
of the above ligands affords a greater choice and flexibility involv-
ing the N (azomethine nitrogen, ring nitrogen of benzothiazole)
and O (ethereal oxygen, carboxylate oxygen, hydroxyl group of
benzopyrazine). In the present paper, we report the preparation
and characterization of transition metal(II) complexes of tri and
tetratopic Schiff base ligands and their interactions withEscherichia
coli DNA.
for about 15 min, further the reaction mixture was refluxed on
water bath for 4 h. So obtained solids were filtered, washed with
hot ethanol and dried over fused CaCl2. The proposed structures
of the complexes are given in Scheme 3.
Methodology for DNA binding/cleavage analysis
The concentration of E. Coli DNA per nucleotide [C(p)] was mea-
sured by using its known extinction coefficient at 260 nm,
600 Mꢁ1 cmꢁ1. The absorbance of the DNA at 260 nm (A260) and
280 nm (A280) were measured to check its purity. The ratio
A260/A280 was 1.84, indicating that the DNA was satisfactorily
free from protein [12]. Buffer (5 mM tris(hydroxymethyl) amino-
methane, pH 7.2, 50 mM EDTA) was used for the absorption, vis-
cosity and thermal denaturation experiments.
Experimental
Absorption studies
In the spectroscopic absorption studies, the complex was dis-
solved in DMSO to get the desired concentration. The spectroscopic
titrations were carried out by adding increasing amounts (20, 40,
Reagents and apparatus
All chemicals were of reagent grade and solvents were distilled
and dried before use according to the standard procedures. 2-
Formylphenoxyacetic acid [9], 2-hydrazino benzothiazole [10],
and 2-hydroxy-3-hydrazinebenzopyrazine [11] were prepared
according to the standard procedures reported elsewhere. The zinc
chloride used was anhydrous whereas the other metal salts were in
their hydrated form, i.e., CoCl2ꢀ6H2O, NiCl2ꢀ6H2O and CuCl2ꢀ2H2O.
Estimation of the metal(II) ions were done according to the stan-
dard methods. The molar conductivity measurements were made
on ELICO-CM-82 conductivity bridge. The magnetic susceptibility
measurements were made on Faraday balance at room tempera-
ture using Hg[Co(SCN)4] as calibrant. The 1H NMR spectra were re-
corded in DMSO-d6 solvent on Bruker-300 MHz spectrometer at
room temperature using TMS as internal reference. IR spectra were
recorded in a KBr matrix using an Impact-410 Nicolet (USA) FT-IR
spectrometer in 4000–400 cmꢁ1 range. The electronic spectra of
the complexes were recorded on a Hitachi 150–20 spectrophotom-
eter in the range of 1000–200 nm. The cyclic voltametric studies
were performed at room temperature in DMSO under O2 free con-
dition using CH instruments Electrochemical analyzer, CHI-1110A
(USA). The ESR spectra of the copper complexes were scanned on
a Varian E-4X-band EPR spectrometer, using TCNE as the g-marker.
TG and DTA measurements of the complexes were recorded in
nitrogen atmosphere on Universal V2. 4F TA instrument keeping fi-
nal temperature at 800 °C and heating rate was 10 °C/min. The FAB
mass spectra were drawn from JEOL SX 102/DA-6000 mass spec-
trometer using Argon/Xenon (6 kV, 10 mA) as the FAB gas.
60, 80 and 100 ll) of DNA to a solution of the complex (2 ml) at
a fixed concentration contained in a quartz cell, UV–Vis spectra
were recorded after equilibration at 23 °C for 10 min after each
addition. The intrinsic binding constant Kb was determined from
the plot of A0/[A ꢁ A0] versus [DNA]ꢁ1 according to Eq. (1) [13].
Ao=½A ꢁ Aoꢂ ¼
eG=½eEꢁH
ꢁ
eGꢂ þ
eG=½eEꢁH
ꢁ
eGꢂ ꢃ 1=K½DNAꢂ
ð1Þ
where A0 and A are the absorbance observed for MLCT absorption
band for the free complex and absorbance observed for MLCT
absorption band at given DNA concentration, respectively. [DNA]
is the concentration of DNA in base pairs, eG and eHꢁG are the appar-
ent absorption coefficients in free and DNA bounded form of com-
plex, respectively. The data were fitted into the above equation to
obtain a graph, with a slope equal to eG/[eEꢁHꢁeG] ꢃ 1/K and inter-
cept equal to eG/[eEꢁHꢁeG] hence Kb was obtained from the ratio of
the intercept to the slope.
Viscosity measurements
DNA binding studies using viscosity measurements were car-
ried out using an Oswald micro-viscometer, maintained at con-
stant temperature (29 °C) with
a
thermostat. The DNA
concentration was kept constant in all samples, but the complex
concentration was increased each time (from 50 to 250 mM). Mix-
ing of the solution was achieved by bubbling nitrogen gas through
the viscometer. The mixture was left for 10 min at 23 °C after addi-
tion of each aliquot of complex. The flow time was measured with
a digital stopwatch. The experiment was repeated in triplicate.
1/3
Data are presented as (
where
g/g0
)
versus the ratio [complex]/[DNA],
Syntheses
g
and g0 are the specific viscosity of DNA in presence and
in absence of the complex, respectively. The values of
were calculated using Eq. (2),
g
and g0
Preparation of the ligands L1H2 and L2H2
The ethanolic solution of 2-hydrazinobenzothiazole (3.3 g;
0.02 mol) and 2-hydroxy-3-hydrazinebenzopyrazine (3.54 g;
0.02 mol) was added slowly to 2-formylphenoxyacetic acid
(3.6 g; 0.02 mol) in ethanol with constant stirring to form L1H2
(Scheme 1) and L2H2 (Scheme 2), respectively. Catalytic amount
of glacial acetic acid was added to the solution and the homoge-
neous reaction mixture was refluxed on water bath for 3 h. The
pale yellow solid that formed upon cooling, was filtered off and
recrystallized from hot ethanol. (L1H2: M.P. 285–287 °C, Yield:
70%; L2H2: M.P. 230–232 °C, Yield: 65%).
s
¼ ðt ꢁ tbÞ=tb
ð2Þ
where tb is the observed flow time of DNA containing solution and t
is the flow time of buffer alone. Relative viscosities for DNA were
calculated from the relation (g/g0) [14].
Electrophoresis
For the gel electrophoresis experiments [15], the solution of
complexes in DMF (1 mg/mL) was prepared and these test samples
(1 lg) were added to the genomic DNA samples of E. Coli and incu-
bated for 2 h at 37 °C. Agarose gel was prepared in TAE buffer
(4.84 g Tris base, PH 8.0, 0.5 M EDTA/lL. PH 7.3), the solidified gel
attained at ꢄ55 °C was placed in electrophoresis chamber flooded
Preparation of complexes
To a stirred ethanolic solution (50 ml) of respective metal chlo-
rides, viz, Co(II), Ni(II), Cu(II), and Zn(II) chloride (0.005 mol), an
ethanolic solution of ligand (0.005 mol) was added and stirred
with TAE buffer. After that 20 lL of each of the incubated complex-
DNA mixtures (mixed with bromophenol blue dye at 1:1 ratio) was

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P.P. Netalkar et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 97 (2012) 762–770
S
H
H
Reflux in ethanol
4hrs
HN
H2N
O
O
O
N
N
O
O
N
HN
2-hydrazinobenzothiazole
OH
OH
S
2-formylphenoxyacetic acid
L1H2
Scheme 1. Schematic representation of synthesis of L1H2.
N
H
N
N
N
H
H
HN
N
NH2
Reflux in ethanol
4hrs
N
O
O
O
OH
HO
3-hydrazine-2-hydroxy
benzopyrazine
O
O
OH
2-formylphenoxyacetic acid
L2H2
OH
Scheme 2. Schematic representation of synthesis of L2H2.
loaded on the gel along with standard DNA marker and electropho-
resis was carried out under TAE buffer system at 50 V for 2 h. At the
end of electrophoresis, the gel was carefully stained with EtBr
in the region 500–470 cmꢁ1 and 400–325 cmꢁ1 are assigned to
v(MꢁN) and v(MꢁO), respectively. The ligand shows a broad med-
ium intensity band at 3202 cmꢁ1 which can be assigned to the
v(NH),which experiences a little shift upon complexation. Non-
involvement of sulfur of the benzothiazole in coordination is as-
sumed in all these complexes, which is in accordance with earlier
reports [18] on the benzothiazole complexes with various transi-
tion metal ions, this is because the sulfur is a poor Lewis base com-
pared to nitrogen in the benzothiazole.
(ethidiumbromide) solution (10 lg/mL) for 10–15 min and
visualized under UV light using a Bio-Rad Trans illuminator. The
illuminated gel was photographed by using a Polaroid camera (a
red filter and Polaroid film were used).
Results and discussion
Ligand L2H2 shows a carboxylic carbonyl group frequency in the
region of 1690 cmꢁ1, which remains almost same in the all the
complexes, suggesting non-involvement of carbonyl oxygen atom
in coordination. Further a weak band around 3429 cmꢁ1 in ligand
is assigned to –OH stretching frequency. It is difficult to account
for the coordination of oxygen of the –OH via deprotonation as
all the complexes shows a strong broad band in the region
3400 cmꢁ1 due to water molecule. The new sharp band at 1682
and 1669 cmꢁ1 in case of Co(II) and Ni(II) complexes, respectively
The analytical and physicochemical data of the complexes are
summarized in Table 1. All complexes are soluble in organic sol-
vents like DMF, DMSO and acetonitrile.
Infrared spectral studies
Infrared frequencies for the ligands and complexes along with
their assignments are presented in Table 2. Ligand L1H2 show a car-
boxylic carbonyl group frequency at 1714 cmꢁ1, which decreases
by 5–10 cmꢁ1 in all the complexes, except in Co(II) complex, indi-
cating the involvement of carbonyl oxygen in coordination. In
Co(II) complex, coordination occurred through carboxylic –OH
after deprotonation. The v(C@N) appears at 1612 cmꢁ1 as an in-
tense sharp peak in the ligand, show negative shift in case of Ni(II)
and Zn(II) whereas positive shift in case of Co(II) and Cu(II) com-
plexes [16], indicating the coordination of azomethine nitrogen
[17]. The v(C@N) of benzothiazole ring observed at 1575 cmꢁ1
show positive shift in case of Co(II) indicating coordination through
ring nitrogen whereas in the case of Ni(II) and Zn(II) complexes, the
band remain unaltered suggesting the nonparticipation of ring
nitrogen in the coordination. On the other hand, in case of Cu(II)
complex, a new band has appeared at 1654 cmꢁ1 attributed to
exo-azomethine group formed at second position of the benzothi-
azole ring due to the tautomerization. In this case coordination has
occurred through ring nitrogen. Asymmetric stretching of C–O–C in
the ligand is assigned in the frequency range of 1292 cmꢁ1, which
shifts to lower energy side in complexes, implying the coordination
of ethereal oxygen. The low frequency bands in all the complexes
assigned to v(C@O) of amide functionality which is absent in the
free ligand suggesting amido-imidol tautomerism [19]. But in the
case of Cu(II) and Zn(II) complexes imidol tautomer is retained as
in case of free ligand and coordination through oxygen via depro-
tonation is seen. Asymmetric stretching of C–O–C in the ligand is
assigned in the frequency range of 1296 cmꢁ1, which shifts to low-
er energy side in complexes, implying the coordination of ethereal
oxygen. The v(C@N) of azomethine group appears at 1653 cmꢁ1 as
an intense sharp peak in the ligand, shifts to a lower frequency side
by about 15–20 cmꢁ1 in complexes, suggesting the coordination of
azomethine nitrogen. The ligand shows a broad medium intensity
band at 3276 cmꢁ1 which can be assigned to the v(NH), which
experiences a little shift upon complexation. The low frequency
bands in all the complexes in the region 500–470 cmꢁ1 and 400–
325 cmꢁ1 are assigned to v(MꢁN) and v(MꢁO), respectively.
1H NMR spectral studies
The Ligand L1H2 is scanned in the range 0–16 ppm for 1H NMR
studies. Within this range it display singlets at 12.6, 8.4, 7.9 ppm,

P.P. Netalkar et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 97 (2012) 762–770
765
2
2
2
2
M:Ni,Zn
2
2
2
2
2
2
2
Scheme 3. Tentative structures of complexes.
which are attributed to carboxylic –OH (1H) (D2O exchangeable)
azomethine proton (1H), and –NH proton, respectively and a mul-
tiplet in the range 6.9–7.8 ppm which are assigned to aromatic
protons. In addition to these prominent peaks, a singlet observed
at 4.8 ppm is assigned to methylene protons of the ethereal link-
age. In contrast, its zinc complex shows a downfield shift of car-
boxylic –OH at 12.79 ppm, azomethine proton at 8.7 ppm,
methylene protons of the ethereal linkage at 4.9 ppm, confirms
the coordination through azomethine nitrogen, carbonyl oxygen
and ethereal oxygen. The peak due to –NH proton does not suffer
any change in complex implying its non-involvement in
coordination.
The 1H NMR spectrum of the ligand L2H2 shows the singlets at
12.3 and 11.3 ppm due to carboxylic –OH (1H) and aromatic –OH
(D2O exchangeable) of quinoxaline moiety respectively. Both the
peaks disappeared in the zinc complex, indicating their involve-
ment in coordination to the metal ion via deprotonation. The sing-
lets observed at 8.9 and 7.9 ppm in the spectrum of ligand are

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P.P. Netalkar et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 97 (2012) 762–770
Table 1
Analytical, conductivity data of the ligand L1H2, L2H2 and their complexes.
Compound
Emprical formula
Elemental analysis (%) calculated (found)
Molar cond.
M ohmꢁ1 cm2 molꢁ1
Magnetic moment
in leff. BM
K
C
H
N
M
Cl
L1H2
C16H13N3O3S
58.71
(57.7)
43.60
(42.8)
40.59
(41.1)
41.60
(43.4)
41.26
(41.4)
60.35
(60.7)
43.64
(44.1)
43.64
(43.3)
43.31
(43.2)
46.64
(43.7)
3.97
(3.4)
3.63
(3.5)
3.17
(2.6)
3.50
(3.0)
3.22
(3.1)
4.17
(4.2)
3.68
(3.5)
3.69
(3.4)
4.24
(4.3)
3.68
(3.6)
12.84
(12.2)
9.58
(9.32)
8.87
–
–
–
CoL1H
NiL1H2
CuL1H
ZnL1H2
L2H2
[Co(C16H12N3O3S)ClꢀH2O]
[Ni(C16H13N3O3S)Cl2ꢀH2O]
[Cu(C16H12N3O3S)ClꢀH2O]ꢀH2O
[Zn(C16H13N3O3S)Cl2ꢀH2O]
C17H14O4N4
13.37
(13.2)
13.53
(12.4)
13.77
(14.3)
14.05
(14.1)
–
8.05
(8.15)
14.70
(15.1)
7.68
(7.88)
15.23
(15.1)
–
12.74
13.31
17.12
12.30
4.83
2.87
(8.96)
9.11
1.80
(10.0)
(9.02)
(9.00)
16.56
(15.8)
11.98
(11.5)
11.98
(12.2)
11.88
(11.6)
12.80
(12.1)
Diamagnetic
–
CoL2H
NiL2H
CuL2
[Co(C17H13N4O4)(Cl)(H2O)]ꢀH2O
[Ni(C17H13N4O4)(Cl)(H2O)]ꢀH2O
[Cu(C17H12N4O4)(H2O)2]ꢀ2H2O
[Zn(C17H12N4O4)(H2O)2]
12.60
(12.7)
12.56
(12.3)
13.46
(13.3)
14.94
(14.6)
7.59
(7.45)
7.60
(7.54)
–
7.29
14.69
8.47
4.68
2.92
1.82
ZnL2
–
14.33
Diamagnetic
assigned to the –NH and azomethine proton, respectively and a
multiplet in the range 6.9–7.9 ppm is due to aromatic protons,
shows a small shift in the spectrum of zinc complex. Methylene
protons attached to ethereal linkage shows a singlet at 4.7 ppm,
exhibited a downfield shift at 4.9 ppm in the complex indicating
involvement of ethereal oxygen in coordination. Hence the ligand
coordinates through carboxylic oxygen, enolic oxygen and azome-
thine nitrogen to the metal ion.
Molar conductivity measurements
The molar conductance values of the complexes in DMSO at
10ꢁ3 M concentration are in the range of 7–18 mhoꢁ1 cm2 mole
(Table 1), much less than that expected for 1: 1 electrolytes [20],
hence all complexes are considered as non-electrolytes.
Fig. 1. UV–visible spectra of ligand and its complexes in the range 250–500 nm. (a)
Electronic spectral studies
L1H2, (b) CoL1H, (c) NiL1H2, (d) CuL1H, (e) ZnL1H2.
Electronic absorption spectra of all the compounds were re-
corded in DMSO solution over the range 200–1000 nm. Absorption
spectra of L1H2 and its complexes are shown in Fig. 1. L1H2 and
charge transfer transition and well-resolved, low-intensity band
4
at 600 nm assignable to T1g(F) ? 4A2g(P) transition [22]. CoL2H
L1H2 shows bands at 280 and 276 nm corresponding to p⁄ tran-
p–
exhibits
a charge transfer broad band around 470 nm and
4T1g(F) ? 4T1g(P) transition is obscured into it. NiL1H2 complex
exhibits bands at 680 nm [3A2g ? 3T1g(F)] and 530 nm
[3A2g ? 3T1g(P)] corresponding to d–d transitions and charge trans-
fer transition is observed at 423 nm. The electronic spectrum of
NiL2H shows a charge transfer band at 430 nm. The low intensity
sition whereas the peak at 397 and 308 nm corresponds to nꢁp⁄
transition associated with azomethine linkage respectively [21].
These bands have suffered red shift upon complexation indicating
the coordination of azomethine group. Electronic spectrum of
CoL1H shows a medium-intensity band at 424 nm assignable to
Table 2
IR spectral data (cmꢁ1) of the ligand L1H2, L2H2 and their complexes.
Compound
m
(OH)
m
(NH)
3202
m
(C@O)
m
(C@O) Amidic
m
(C@N)
1612
1618
–O–CH2–
L1H2
3538
3428
3460
3432
3427
3429
3552
3330
3426
3430
1714
1708
1633
1687
1639
1690
1687
1689
1691
1691
–
–
–
–
–
–
1292s
1275
1289
1287
1281
1296(sh)
1283
1290
1280(sh)
1285
CoL1H
NiL1H2
CuL1H
ZnL1H2
L2H2
3150(sh)
3293
3112
3062
3276
3157
3196
3157
2923
1590(sh)
1617
1604
1653
1640
1639
1620
1646
CoL2H
NiL2H
CuL2
1682
1669
–
ZnL2
–

P.P. Netalkar et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 97 (2012) 762–770
767
bands at 850, 560 and 470 nm are assignable to 3A2g ? 3T2g(F),
Table 3
Cyclic voltammetric data of ligand L1H2, L2H2 and their copper complexes.
3A2g ? 3T1g(F) and 3A2g ? 3T1g(P) transitions, respectively (Supple-
mentary Figure-S1), collectively all these offers the octahedral
geometry for the nickel complex [23]. Electronic spectrum of
CuL1H complex exhibits the broad band in the region 380–
500 nm due to charge transfer transition and very low intensity
band at 750 nm assignable to 2Eg ? 2T2g (d–d) transition (Supple-
mentary Figure-S2 (a)). In Case of CuL2 broad band in the region
400–450 and 679 nm are assignable to the charge transfer transi-
tion and 2Eg ? 2T2g (Supplementary Figure-S2 (b)) which is in
agreement with the octahedral geometry of copper complexes. Fi-
nally, both the Zn (II) complexes shows charge transfer bands at
around 350–430 nm.
Complex
Scan rate (V/s)
Epc (V)
Epa (V)
D
Ep (V)
E1/2 (V)
L1H2
CuL1H
L2H2
CuL2
0.1
0.1
0.05
0.05
0.1
ꢁ0.35
ꢁ0.2
0.0
0.05
0.6
0.15
0.55
0.05
0.09
0.15
0.05
0.075
0.125
ꢁ0.025
0.275
⁄DEp = EpaꢁEpc; E1/2 = [Epc + Epa]/2.
Magnetic susceptibility measurements
The magnetic moments measured at room temperature are
listed in Table 1. The electronic spectral data and the observed
magnetic moments for the cobalt, nickel and copper complexes
of both the ligands suggest the existence of octahedral geometry
around metal ions. The magnetic moment values for CoL1H and
CoL2H were found to be 4.83 and 4.68 BM [24], for NiL1H2 and
NiL2H, 2.87 and 2.92 BM [25] and for CuL1H and CuL2 values found
were 1.80 and 1.82 BM, respectively, slightly higher than the spin-
only value which may be due to the spin orbit coupling [26].
Fig. 3a. Graphical representation of antibacterial analysis of L1H2 and its
complexes.
ESR spectral studies
The powder state X-band ESR spectra of copper complexes were
operated in the region of 9000 MHz with corresponding field inten-
sity at 3000 Gauss in powder state at room temperature. Both
CuL1H and CuL2 complexes exhibit isotropic intense broad signal
with giso 2.072 and 2.081, respectively with no hyperfine splitting
[27]. Broad ESR signal may be due to bulkier ligand around the me-
tal ion [28].
FAB mass spectral studies
The FAB mass spectrum of copper complexes was recorded by
using m-NO2 benzyl alcohol as matrix. FAB mass spectra of CuL1H
and CuL2 complexes show molecular ion peak corresponding to m/
z at 443 and 435, respectively, consistent with monomeric nature
of complexes. Apart from this, spectra show some other peaks,
which are due to molecular ions of various fragments of the com-
plexes. By comparing all the analytical and spectral data of cobalt,
nickel and zinc complexes, it is evident that all complexes are
monomeric.
Fig. 3b. Graphical representation of antibacterial analysis of L2H2 and its
complexes.
Thermal study
The thermogravimetric analyses of all the complexes have been
carried out in nitrogen environment over the temperature range of
30–800 °C. The TG/DTA analysis of CuL1H is used as representative
example (Fig. 2). TG–DTA curves of CuL1H show weight losses in
three considerable stages. In the first stage around 8.0%, in the tem-
perature range 35–250 °C is attributed to the weight loss of one
coordinated water molecule along with one lattice held water
molecule, which is further supported by the endothermic signal
in DTA. Further weight loss in the region 250–325 °C is attributed
to the loss of one chloride along with the ligand and in the final
part degradation of remaining ligand has been observed. Finally
the graph became plateau because of the formation of stable metal
oxide.
Similarly, TG–DTA curves of CuL2 show weight losses in three
considerable stages. In the first stage 7.9%, in the temperature
range 55–160 °C is attributed to the weight loss of two lattice held
water molecules, which is further supported by the DTA peak at
Fig. 2. TG–DTA spectrum of CuL1H.

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P.P. Netalkar et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 97 (2012) 762–770
108 °C. In the next step the weight loss around 8% is attributed to
two coordinated water molecules. Further weight loss in the tem-
perature range 350–600 °C is attributed to the loss of ligand to
form stable metal oxide. TG–DTA graphs of cobalt, nickel and zinc
complexes follow the same pattern with slight differences.
deprotonation of carboxylic group of the 2-formylphenoxyacetic
acid [29,30] (Supplementary Figure-S4). The separation between
anodic and cathodic peak potentials (DEp) is 50 and 150 mV,
respectively indicating a reversible and quasireversible redox pro-
cess for both electroactive ligands supported by Ipc/Ipa ꢅ1 values.
The cyclic voltammograms of both Cu(II) complexes show a single
electron transfer process, an anodic peak was observed at 0.6 and
0.55 V, respectively. In the reverse scan, the cathodic peak was ob-
served at ꢁ0.35 and 0.0 V, respectively. The separation between
Cyclic voltammetric studies
The cyclic voltammograms were scanned in the potential range
of ꢁ1.0 to +1.0 V with scan rates 0.05, 0.1, and 0.15 Vsꢁ1 in DMSO
under oxygen free conditions and data are summarized in Table 3.
The cyclic voltammograms of L1H2 and CuL1H complex are used as
representative examples in Supplementary material (Figure-S3(a)
and Figure-S3(b)). Both the ligands were found to be electrochem-
ically active in the working potential range showing single electron
transfer process, an anodic peak was observed at 0.05 and 0.15 V,
respectively. In the reverse scan, the cathodic peak was observed
at 0.1 and ꢁ0.2 V, respectively. This spectacular behavior of both
the ligands in the working potential range is attributed to the
anodic and cathodic peak potentials (DEp) is 95 and 55 mV, respec-
tively indicating a quasireversible and reversible redox process for
both electroactive copper complexes supported by Ipc/Ipa values
which are nearly equal to 1.
Fig. 5. Absorption spectra of NiL2H in absence (very first line from pinnacle) and in
presence of increasing amounts of E. coli DNA. Arrow mark shows the absorption
changes upon changing the E. coli DNA concentration.
Table 4
Intrinsic binding constants of the complexes.
Sl No.
Complex
Kb
1
2
3
4
5
6
7
8
CoL1H
NiL1H2
CuL1H
ZnL1H2
CoL2H
NiL2H
CuL2
1.43 ꢃ 104
2.2 ꢃ 104
4.3 ꢃ 104
1.3 ꢃ 104
1.5 ꢃ 104
1.9 ꢃ 104
1.03 ꢃ 105
1.9 ꢃ 104
Fig. 4a. Gel electrophoresis pictures of ligand L1H2 and their complexes. Photo-
graph showing the effects of transition metal complexes on DNA of E. coli. Lane M:
DNA marker, Lane C: Untreated DNA, Lane 1: L1H2, Lane 2: CoL1H, Lane 3: NiL1H2,
Lane 4: CuL1H, Lane 5: ZnL1H2.
ZnL2
Fig. 4b. Gel electrophoresis pictures of ligand L2H and their complexes. Photograph
showing the effects of transition metal complexes on DNA of E. coli. Lane M: DNA
marker, Lane C: Untreated DNA, Lane 6: L2H, Lane 7: CoL2H, Lane 8: NiL2H, Lane 9:
CuL2, Lane 10: ZnL2.
Fig. 6. Effects of increasing amounts of CoL2H, NiL2H, CuL2 and ZnL2 on the relative
viscosities of E. coli DNA at 29 °C.

P.P. Netalkar et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 97 (2012) 762–770
769
Biochemistry
DNA binding analysis using viscosity measurement
Hydrodynamic (viscosity) measurements are sensitive to
changes in the length of DNA base pairs and are regarded as the
least ambiguous and the most critical tests of a binding model in
solution in the absence of crystallographic structural data. Interca-
lative binding is confirmed by the lengthening of the DNA helix as
base pairs are separated to accommodate the binding compound,
leading to an increase in viscosity of DNA solution [14,32]. The
present complexes show such an increase, suggesting an intercala-
tive binding to DNA as shown in Fig. 6.
Anti-biogram analysis
In order to study the comparative biological activities, the com-
pounds were screened for antibacterial (against E. coli) in the range
25–800 mg concentrations. From the antibacterial studies it is in-
ferred, that both the ligands were found to be inactive against
E. coli. The results obtained are graphically represented in Figs.
3a and b. From the graph it is clear that there is enhancement in
the inhibitory activity of the complexes, which is represented by
the high degree of activity in case of copper and cobalt complexes
derived from L1H2, whereas, in the case of L2H2, moderate activity
has been showed by both these complexes. All the other complexes
were found to be less active against the bacterium tested. Inhibi-
tory activities of metal complexes against bacterial species have
enhanced as compared to the ligand indicating that complexation
is able to modulate or alter antibacterial property.
Conclusion
It is concluded from the analytical and spectral data that these
ligands act as tri and tetradentate chelates with N2O2, NO2 and
NO3 donating sites. The Ligand L2H2 and its complexes show ami-
do-imidol tautomerism. All the complexes are found to be octahe-
dral and monomeric in nature. Cyclic voltammograms of ligands
and its copper complexes show single electron transfer process.
The metal complexes have higher antimicrobial activity than the
ligand, especially Co(II) and Cu(II) complexes. Spectroscopic stud-
ies together with viscosity experiments support that, both the li-
gands and their complexes bind to DNA via intercalative mode of
interaction whereas no significant cleavage of DNA have been
observed.
DNA cleavage studies by gel-electrophoresis method
The interactions between the free ligand and complexes with
DNA are shown in Figs. 4a and b. Gel-electrophoresis technique
works on the migration of DNA under the influence of electric po-
tential. The photograph shows the bands with different bandwidth,
compared to the control, is the differentiating criteria for binding
ability of complexes with E. coli in this study. Control experiment
using DNA alone does not show any significant cleavage of DNA
even after a longer exposure time. In the photograph Fig. 4a, un-
treated DNA (lane c) is more intense and has slightly bigger width
compared to the bands of free ligand L1H2 and the complexes ex-
cept lane 3 NiL1H2. This result could be attributed to the interac-
tion of DNA to a small extent for L1H2 and its complexes except
NiL1H2. On the other hand, in Fig. 4b, lane 9, corresponding to
CuL2 shows decrease in intensity and bandwidth indicating the
interaction of complex with DNA whereas L2H2 and its complexes
shows no significant changes in the intensity and bandwidth com-
pared to the control. We conclude from the gel electrophoresis
studies that there is no significant cleavage of DNA by the action
of either free ligands or its complexes.
Acknowledgments
The authors thank the USIC and Department of chemistry, Kar-
natak University, Dharwad for providing spectral facility. Record-
ing of FAB mass spectra (CDRI Lucknow) and ESR spectra (IIT
Bombay) are gratefully acknowledged. The authors thank UGC for
providing RFSMS and DST for INSPIRE fellowship.
Appendix A. Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.saa.2012.07.066.
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