DNA binding mode of novel tetradentate amino acid based 2-hydroxybenzylidene-4-aminoantipyrine complexes

Article abstract of DOI:10.1016/j.saa.2012.07.051

Few transition metal complexes of tetradentate N₂O₂ donor Schiff base ligands containing 2-hydroxybenzylidene-4-aminoantipyrine and amino acids (alanine/valine) abbreviated to KHL¹/KHL² have been synthesized. All the metal complexes have been fully characterized with the help of elemental analyses, molecular weights, molar conductance values, magnetic moments and spectroscopic data. The Schiff bases KHL ¹/KHL² are found to act as tetradentate ligands using N₂O₂ donor set of atoms leading to a square-planar geometry for the complexes around the metal ions. The binding behaviors of the complexes to calf thymus DNA have been investigated by absorption spectra, viscosity measurements and cyclic voltammetry. The DNA binding constants reveal that all these complexes interact with DNA through minor groove binding mode. The studies on mechanism of photocleavage reveal that singlet oxygen ( ¹O₂) and superoxide anion radical (O2-) may play an important role in the photocleavage. The Schiff bases and their metal complexes have been screened for their in vitro antibacterial activities against Staphylococcus aureus, Pseudomonas aeruginosa, Escherichia coli, Staphylococcus epidermidis, Klebsiella pneumoniae and antifungal activities against Aspergillus niger, Fusarium solani, Culvularia lunata, Rhizoctonia bataicola and Candida albicans by MIC method.

Full text of DOI:10.1016/j.saa.2012.07.051

Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 96 (2012) 698–708
Contents lists available at SciVerse ScienceDirect
Spectrochimica Acta Part A: Molecular and
Biomolecular Spectroscopy
journal homepage: www.elsevier.com/locate/saa
DNA binding mode of novel tetradentate amino acid based
2-hydroxybenzylidene-4-aminoantipyrine complexes
⇑
N. Raman , S. Sobha, M. Selvaganapathy, R. Mahalakshmi
Research Department of Chemistry, VHNSN College, Virudhunagar 626 001, 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
Amino acid based 2-hydroxybenzylidene-4-aminoantipyrine complexes are valuable in designing novel
agents for targeting nucleic acids as well as setting the stage for the synthesis of chemical anticarcino-
genic drugs.
"
"
"
"
Synthesis of a new family of amino
acid based complexes.
They were found to act as good
minor groove binders.
These chelates exhibit efficient
cleavage potential of pUC19 DNA.
To develop the novel design of new
metallonucleases.
a r t i c l e i n f o
a b s t r a c t
Article history:
Few transition metal complexes of tetradentate N2O2 donor Schiff base ligands containing 2-hydroxyben-
zylidene-4-aminoantipyrine and amino acids (alanine/valine) abbreviated to KHL1/KHL2 have been syn-
thesized. All the metal complexes have been fully characterized with the help of elemental analyses,
molecular weights, molar conductance values, magnetic moments and spectroscopic data. The Schiff
bases KHL1/KHL2 are found to act as tetradentate ligands using N2O2 donor set of atoms leading to a
square-planar geometry for the complexes around the metal ions. The binding behaviors of the com-
plexes to calf thymus DNA have been investigated by absorption spectra, viscosity measurements and
cyclic voltammetry. The DNA binding constants reveal that all these complexes interact with DNA
through minor groove binding mode. The studies on mechanism of photocleavage reveal that singlet oxy-
Received 29 March 2012
Received in revised form 29 June 2012
Accepted 11 July 2012
Available online 20 July 2012
Keywords:
Alanine complexes
Valine complexes
4-Aminoantipyrine
DNA binding
gen (1O2) and superoxide anion radical (Oꢀꢀ) may play an important role in the photocleavage. The Schiff
2
bases and their metal complexes have been screened for their in vitro antibacterial activities against
Staphylococcus aureus, Pseudomonas aeruginosa, Escherichia coli, Staphylococcus epidermidis, Klebsiella pneu-
moniae and antifungal activities against Aspergillus niger, Fusarium solani, Culvularia lunata, Rhizoctonia
bataicola and Candida albicans by MIC method.
DNA cleavage
Photocleavage
Ó 2012 Elsevier B.V. All rights reserved.
⇑
Corresponding author. Moblie: +91 9245165958; fax: +91 4562 281338.
E-mail address: drn_raman@yahoo.co.in (N. Raman).
1386-1425/$ - see front matter Ó 2012 Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.saa.2012.07.051

N. Raman et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 96 (2012) 698–708
699
various amino acids. The DNA binding behaviors have been studied
with spectroscopic titration, viscosity measurements and photoc-
leavage. In continuation of the study, special attention is given to
the application of electrochemical measurements based on the
interaction of these metal complexes with DNA. The results should
be valuable in designing novel agents for targeting nucleic acids as
well as setting the stage for the synthesis of chemical anticarcino-
genic drugs.
Introduction
Stable, inert and non-toxic metal complexes containing spectro-
scopically active metal centers are exceptionally valuable as probes
of biological systems. Transition metal complexes of pyrazole
derivatives have been the subject of much recent notice because
of their beneficial effects as anticancer agents [1]. Schiff bases are
important class of compounds in medicinal and pharmaceutical
field. In the area of bioinorganic chemistry, interest in Schiff base
complexes has centered on the role of such complexes in providing
synthetic models for the metal-containing sites in metallo-proteins
and metallo-enzymes [2].
Experimental
Materials and methods
They also show biological applications including antibacterial,
antifungal and antitumor activity [3]. Furthermore, tetradentate
and tridentate Schiff base complexes are increasingly important
for designing metal complexes related to synthetic and natural
oxygen carriers. Diamino tetradentate Schiff bases and their com-
plexes have been used as biological models to understand the
structures of biomolecules and biological processes [4]. The inter-
action of metal complexes containing N2O2 Schiff base ligands
has been thoroughly considered. The metal complexes containing
multidentate aromatic ligands with square-planar N4 or N2O2 coor-
dination are among the most studied compounds regarding their
DNA-binding affinity and their cytotoxic properties [5].
All reagents, 4-aminoantipyrine, 2-hydroxybenzaldehyde, ala-
nine, valine and metal(II) chlorides, were procured from Merck
products. Commercial solvents were distilled and then used for
the preparation of ligands and their complexes. pUC19 DNA was
purchased from Bangalore Genei (India). Microanalyses (C, H and
N) were performed in Carlo Erba 1108 analyzer at Sophisticated
Analytical Instrument Facility (SAIF), Central Drug Research
Institute (CDRI), Lucknow, India. Molar conductivities in DMSO
(10ꢀ3 M) at room temperature were measured using Systronic
model-304 digital conductivity meter. Magnetic susceptibility
measurements of the complexes were carried out by Gouy balance
using copper sulfate pentahydrate as the calibrant. IR spectra were
recorded with Perkin-Elmer 783 spectrophotometer in the 4000–
400 cmꢀ1 range using KBr pellets. NMR spectra were recorded on
a Bruker Avance Dry 300 FT-NMR spectrometer in CDCl3 with
TMS as the internal reference. FAB-MS spectra were recorded with
a VGZAB-HS spectrometer at room temperature in a 3-nitrobenzyl-
alcohol matrix. Electron paramagnetic resonance spectra of the
copper complexes were recorded on a Varian E 112 EPR spectrom-
eter in DMSO solution both at room temperature (300 K) and liquid
nitrogen temperature (77 K) using TCNE (tetracyanoethylene) as
the g-marker. The absorption spectra were recorded using Shima-
dzu model UV-1601 spectrophotometer at room temperature.
Among the different therapeutic strategies to eradicate cancer
cells, the development of DNA-targeting drugs is a rising topic of
investigations in bioinorganic chemistry [6,7]. The interaction of
such drugs with DNA often gives rise to the formation of coordina-
tion, covalent or non-covalent adducts, thereby disrupting the
transcription and/or replication. After the discovery of the antitu-
mor activities of cisplatin in 1965 [8,9], a great deal of effort has
been accomplished to determine its mechanism of action. Nowa-
days, the main target of cisplatin is generally accepted to be
DNA. The main adduct formed is the very stable intrastrand GpG
cross-link located in the major groove, which is likely to be respon-
sible for the antitumor properties of cisplatin [10]. However, cis-
platin treatment is still accompanied by severe side effects and
by both intrinsic and acquired resistance to the drug [11–13]. To
overcome these problems, there is always a need for developing
new drugs with minimal side effects and maximal curative poten-
tial. Consequently, new DNA targeted drug design alternative strat-
egies are based on different transition metal ions. Amongst the
non-platinum active compounds are the metal based drugs con-
taining essential elements such as copper and zinc. Since any
essential metal which escapes its normal metabolic pathways
can be very toxic to the organism, complexes of such metals may
serve as effective cytotoxic agents. Copper is a bioessential ele-
ment, plays a key role in biological processes, its complexes are
known to exhibit antitumour activity [14]. Besides this, it has also
been demonstrated that copper accumulates in tumors due to
selective permeability of cancer cell membranes to copper com-
pounds [15]. Zinc is the most abundant trace intracellular element
and plays an important role in both genetic stability and function
[16]. Mechanistically, zinc has a significant impact on DNA as a
component of chromatin structure, DNA replication and transcrip-
tion and DNA repair [17].
Synthesis of 2-hydroxy- benzylidene-4-aminoantipyrine
The ligand 2-hydroxy-benzylidene-4-aminoantipyrine was pre-
pared as reported earlier [4,19]. An ethanolic solution of (40 mL) 4-
aminoantipyrine (0.02 mol) was added to an ethanolic solution of
2-hydroxybenzaldehyde (0.02 mol). The resultant mixture was re-
fluxed for ca. 3 h. The solid product formed (2-hydroxy-benzyli-
dene-4-aminoantipyrine) was filtered and recrystallized from
ethanol.
Synthesis of Schiff bases KHL1/KHL2
The potassium salt of amino acid (alanine/valine) was prepared
using the procedure reported by us in the literature [19]. The ami-
no acid (0.01 mol) dissolved in 1:1 water–ethanol (40 mL) was
added to a hot ethanolic solution (30 mL) of KOH (0.01 mol) and
the resulting solution was stirred to obtain a homogeneous solu-
tion. Then, to this solution was added drop-wise an ethanolic solu-
tion of 2-hydroxybenzylidene-4-aminoantipyrine (0.01 mol) and
the resultant mixture was refluxed for ca. 6 h. The dark yellow col-
ored plate like crystalline product (Schiff base, KHL) formed was
filtered and recrystallized from ethanol.
Aim of the present work is to insert NO group in amino acid
Schiff bases. As we know amino acids having Schiff bases readily
form complexes with metal ions which play an important role as
the basic compounds for modeling more complicated amino acid
Schiff bases. They are key intermediates in a variety of metabolic
reactions involving amino acids [18]. Keeping the above knowl-
edge in mind, the aim of this work is to prepare and characterize
new Cu(II), Ni(II) and Zn(II) complexes of Schiff base ligands
derived from 4-aminoantipyrine, 2-hydroxybenzaldehyde and
Synthesis of metal complexes
A solution of metal(II) chloride in ethanol (2 mmol) was stirred
with an ethanolic solution of the Schiff base (2 mmol), for 30 min
on a magnetic stirrer and refluxed for 2 h at room temperature.
The resultant solid product was washed and recrystallized from

700
N. Raman et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 96 (2012) 698–708
Scheme 1. Schematic route for synthesis of Schiff base ligands and their metal complexes.
ethanol. Schematic route for synthesis of Schiff base ligands and
their metal complexes is given in Scheme 1 [19].
to Eq. (1) to obtain the intrinsic binding constant (Kb) values for
interaction of the complexes with DNA [21].
½DNAꢁ=ðea
where ea
ꢀ
ef Þ ¼ ½DNAꢁ=ðeb
ꢀ
ef Þ þ 1=½Kbððeb
ꢀ
ef Þꢁ
ð1Þ
DNA binding experiments
,
e
f, and b are the apparent, free and bound metal complex
e
extinction coefficients, respectively. A plot of [DNA]/(eb
[DNA], gave a slope of 1/(eb f) and a y-intercept equal to [Kb/
f)]ꢀ1, Kb is the ratio of the slope to the y-intercept.
ꢀ
ef) vs
Absorption spectroscopic studies
ꢀ
e
The interactions between metal complexes and DNA were stud-
ied using electrochemical and electronic absorption methods.
Disodium salt of calf thymus DNA was stored at 4 °C. Solution of
DNA in the buffer (50 mM NaCl, 5 mM Tris HCl (pH 7.2)) in water
(eb
ꢀ
e
Electrochemical methods
Cyclic voltammetry was performed on a CHI 620C electrochem-
ical analyzer with three electrode system of glassy carbon as the
working electrode, a platinum wire as auxiliary electrode and Ag/
AgCl as the reference electrode. Solutions were deoxygenated by
purging with N2 prior to measurements.
gave a ratio 1.8–1.9, of UV absorbance at 260 and 280 nm, A260
/
A280, indicating that the DNA was sufficiently free from protein
[20]. The concentration of DNA was measured using its extinction
coefficient at 260 nm (6600 Mꢀ1 cmꢀ1). Stock solutions were
stored at 4 °C and used no more than 4 days. Doubly distilled water
was used to prepare solutions. Concentrated stock solutions of the
complexes were prepared by dissolving the complexes in DMSO
and diluting suitably with the corresponding buffer to the required
concentration for all of the experiments. The data were then fitted
Viscosity measurement
Viscosity experiments were carried out in an Ostwald viscome-
ter, immersed in a thermostated water-bath maintained at a con-

N. Raman et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 96 (2012) 698–708
701
stant temperature at 30.0 0.1 °C. CT DNA samples of approxi-
mately 0.5 mM were prepared by sonication in order to minimize
the complexities arising from CT DNA flexibility. Flow time was
measured with a digital stopwatch three times for each sample
and the average flow time was calculated. Data were presented
ligand (KHL1) shows the molecular ion peak at m/z = 416 corre-
sponding to [C21H21N4O3K] ion. Also the spectrum exhibited peaks
for the fragments at m/z 323, 199, 93 and 77 corresponding to
[C15H15N4O2K] [M+1], [C11H11N4] [M+], [C6H5O [M+] and [C6H5]
[M+] respectively. The mass spectrum of [CuL1] complex shows a
molecular ion peak at m/z = 440, [NiL1] complex shows a peak at
m/z = 435 which corresponds to molecular weight of the respective
compounds while that of [ZnL1] complex shows m/z = 442 which
corresponds to [M+2] peak. This peak supports to the structure of
the complex and confirms the stoichiometry of metal chelates as
ML type. The [CuL1] complex gives a fragment ion peak with loss
of metal m/z = 377. The mass spectrum of Schiff base ligand
(KHL2) shows peak at m/z 460 [M+1] and its [CuL2], [NiL2] and
[ZnL2] complexes show molecular ion peaks at m/z 483 [M+], 478
[M+] and 486 [M+1], respectively. The different molecular ion
peaks appeared in the mass spectra of complexes are attributed
to the fragmentation of the metal complex molecule obtained from
the rupture of different bonds inside the molecule by successive
degradation leading to many more important peaks due to forma-
tion of various radicals. Thus, the mass spectra of complexes show
molecular ion peaks which are in good agreement with the struc-
tures suggested by elemental analysis, spectral and magnetic
studies.
as (
is the viscosity of CT DNA solution in the presence of complex,
g/go)
1/3 vs the concentration of the metal(II) complexes, where
g
and go is the viscosity of CT DNA solution in the absence of com-
plex. Viscosity values were calculated after correcting the flow
time of buffer alone (t0),
g
– (t ꢀ t0)/t0.
Results and discussion
The Schiff base ligands and their Cu(II), Ni(II) and Zn(II) com-
plexes have been synthesized and characterized by spectral and
elemental analytical data. They are found to be air stable. The li-
gand is soluble in common organic solvents and all the complexes
are freely soluble in CHCl3, DMF and DMSO.
Elemental analysis and molar conductivity measurements
The results of elemental analysis for the metal complexes are in
good agreement with the calculated values (Table 1) showing that
the complexes have 1:1 metal–ligand stoichiometry of the type ML
wherein L acts as a tetradentate ligand. The metal(II) complexes
were dissolved in DMF and the molar conductivities of 10ꢀ3 M of
their solution at room temperature were measured. The lower con-
ductance values (11.45–22.10 ohmꢀ1 cm2 molꢀ1) of the complexes
support their non-electrolytic nature.
IR spectra
The IR spectrum of the ligand is compared to know the changes
during complex formation. The characteristic phenolic t(OH) mode
due to the hydroxyl group of 2-hydroxybenzaldehyde moiety pres-
ent in the Schiff base ligands, is observed at 3444/3450 cmꢀ1. The
absence of this band in all the complexes indicates the deprotona-
tion of hydroxyl group. These facts are further supported by the
Magnetic moment study
t
(C–O) bands for all the complexes (1251–1249 cmꢀ1), appearing
The magnetic moment data of the solid-state complexes at
room temperature are reported in Table 1. The magnetic suscepti-
bility measurements show that the Cu(II) complexes are paramag-
netic at ambient temperature with the exception of Zn(II) and
Ni(II) complexes. The copper(II) complexes are paramagnetic with
1.74 and 1.81 BM for [CuL1] and [CuL2], respectively. These values
for Cu(II) complexes are consistent with expected spin-only mag-
netic moment of an S = 1/2 (1.74 and 1.81 BM), Cu(II) d9 system.
at lower frequencies compared to that for free ligands (1276–
1271 cmꢀ1). In the spectra of ligands, the strong band is observed
at 1614/1622 cmꢀ1 that can be assigned to
t(CH@N) frequencies.
These frequencies, which are shifted towards a lower wave number
by 20–25 cmꢀ1 in the spectra of metal complexes, indicate that
coordination of nitrogen of the azomethine group to the central
metal atom in complexes. In addition, the ligands also reveal a
band at 1653/1658 cmꢀ1 due to the
t(C@N) vibration, originating
Mass spectra
from the condensation of amino group of amino acids and carbonyl
group of 2-hydroxybenzylidene-4-aminoantipyrine which are
shifted by 24–15 cmꢀ1 on complexation. Moreover, the coordina-
tion of the ligand to the metal center via the carboxylic group
can also be obvious from the difference of maxima positions as ob-
The FAB-mass spectra of synthesized ligands and their metal
complexes were recorded and the obtained molecular ion peaks
confirm the proposed formulae. The mass spectrum of Schiff base
Table 1
Physical characterization, analytical, molar conductance and magnetic susceptibility data of the ligands and their complexes.
Compound
Yield (%)
Color
Found (calc)%
Formula weight
^
(ohmꢀ1 cm2 molꢀ1
)
leff (BM)
m
M
–
C
H
N
KHL1
71
75
68
56
62
66
52
57
Dark yellow
Dark yellow
Reddish brown
Pale orange
60.2
(60.5)
61.5
(62.7)
56.9
(57.3)
57.1
(57.9)
56.6
(57.0)
59.0
(59.6)
59.6
(60.2)
60.3
5.0
(5.0)
6.0
(6.1)
4.5
(4.5)
4.3
(4.6)
4.4
(4.5)
5.5
(5.6)
5.3
(5.6)
5.6
13.2
(13.4)
12.1
(12.2)
12.5
(12.7)
12.6
(12.8)
12.1
(12.6)
11.0
(11.6)
11.1
(11.7)
(11.0)
(11.5)
416
459
440
435
442
483
478
485
–
–
–
KHL2
–
–
[CuL1]
[NiL1]
[ZnL1]
[CuL2]
[NiL2]
[ZnL2]
14.0
(14.4)
13.0
(13.4)
14.2
(14.8)
12.6
(13.1)
11.5
(12.2)
13.0
14.20
11.45
21.45
22.10
19.30
18.50
1.74
Diamagnetic
Diamagnetic
1.88
Lemon yellow
Dark brown
Pale orange
Diamagnetic
Diamagnetic
Lemon yellow
(13.4)
(59.9)
(5.6)

702
N. Raman et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 96 (2012) 698–708
ꢀ
Table 2
ꢀÞ
served for tsy:ðCOO and tasy:ðCOO Þ. The bands are detected at 1447/
1479 and 1549/1539 cmꢀ1, respectively for free ligands, and
The spin Hamiltonian parameters of the copper complexes in DMSO solution at 300
and 77 K.
ꢀÞ
ꢀÞ
D
t
= 102/60 cmꢀ1. For comparison, the tsy:ðCOO and tasy:ðCOO are
Complex
A||
A\
Aiso
g||
g\
giso
a2
b2
c2
observed at 1317–1311 and 1444–1423 cmꢀ1, respectively, for all
the metal complexes. These results reveal that the organic ligand
is involved in the coordination through the carboxyl group. The
[CuL1]
[CuL2]
169
163
67
71
85.2
81.5
2.26
2.23
2.05
2.04
2.25
2.17
0.75
0.75
0.81
0.79
0.76
0.61
presence of new band in the region 518–472 cmꢀ1 due to
t(M–
N) is another indication of the involvement of nitrogen of the azo-
methine. Thus the Schiff bases coordinate through azomethine
nitrogen, phenolic oxygen, carboxylic oxygen of amino acids and
imine group (C@N), which is originated from the condensation of
carbonyl oxygen of 4-aminoantipyrine and amino group of amino
acids.
recorded at 298 and 77 K using TCNE as the ‘g’ marker (Fig. 1).
The solution spectra of the complexes possess well-resolved g||
and a broad g\ region. The four peaks in the spectrum are evidently
due to the coupling of the electron spin of the 63Cu nucleus (I = 3/
2). The peaks are broad and have the appearance of ill-resolved
triplets. The breadth and triplet appearance can be attributed to
hyperfine splitting by the nitrogen atom (I = 1) of the ligand. The
triplet appearance is adduced as an evidence for nitrogen coordina-
tion. Based on this observation, a distorted square-planar geometry
is proposed for the complex. The various Hamiltonian parameters
have been calculated for these complexes (g|| = 2.26 for [CuL1],
2.23 for [CuL2], g\ = 2.03 for [CuL1], 2.07 for [CuL2]. Here, both cop-
UV–Vis spectroscopy
The electronic absorption spectral data for Schiff bases and their
complexes were obtained in 10ꢀ2 M DMF solutions at room tem-
perature. The UV–Vis spectra of the Schiff base ligands and their
metal complexes in DMF show absorption bands between 21,929
and 34,482 cmꢀ1. The spectra of the complexes show intense bands
in the high-energy region at 27,700–34,482 cmꢀ1, which can be as-
signed to charge transfer L–M bands. The Schiff base ligands show
a band at 23,148 and 23,529 cmꢀ1 assigned to n–p⁄ transitions of
the azomethine groups in the KHL1/KHL2 ligands. In the spectra
of complexes, the bands of azomethine n–p⁄ transitions are shifted
to lower frequencies indicating that the imine nitrogen atom is in-
volved in coordination to the metal ion. The spectra of all com-
plexes contain an absorption band in 16,103 and 21,929 cmꢀ1
range which may be assigned to the d–d transition of metal ions.
The electronic spectra of [CuL1]/[CuL2] complexes displayed d–d
transition band in the region 17,921/17,825 cmꢀ1, which is due
to 2B1g ? 2A1g transition. This d–d band strongly favors a square-
planar geometry around the metal ion. The Ni(II) complexes
[NiL1]/[NiL2] show diamagnetic and the electronic spectra of these
complexes show bands at 16,366/16,103 and 21,459/21,929 cmꢀ1
which are assigned as 1A1g ? 1A2g and 1A1g ? 1B1g transitions,
respectively. The zinc(II) complexes show bands at 27,700 and
28,089 cmꢀ1, assigned to charge transfer M–L bands confirming
its tetrahedral geometry [22–24]. They are diamagnetic.
per complexes have g|| > g\ which support the dx
as the ground
2ꢀy2
state, characteristic of square-planar geometry and axially sym-
metric [25]. For the present copper complexes, gav is calculated
using the equation, gav = 1/3 (g|| + 2 g\) and it is 2.105 for [CuL1]
and 2.105 for [CuL2]. Spectra of these complexes have four copper
hyperfine lines in both parallel and perpendicular regions. A|| and
1H NMR
The azomethine proton resonates at 9.3 ppm which is a singlet.
The multiplet observed around 7.0–7.6 ppm is due to aromatic pro-
tons of phenyl ring and phenolic ring. The ligand (KHL1) showed
the following signals: phenyl multiplet at 6.9–7.4 d, –HC@N at
9.8 d, –OH at 5.4 d, –C–CH3 at 2.4 d, –N–CH3 at 2.1 d, alanine–
CH3 at 3.6 d, alanine–CH at 1.24 d. The ligand (KHL2) exhibited
the following signals: phenyl multiplet at 7.0–7.6 d, –HC@N at
9.3 d, OH at 5.2 d, C–CH3 at 3.1 d, N–CH3 at 2.2 d, valine–CH at
1.8 d. The phenolic –OH signal at 5.4/5.2 d observed in the spectra
of the Schiff base ligands is not seen in the spectra of the zinc com-
plexes indicating the participation of the phenolic –OH group in
chelation with proton displacement. The signal due to azomethine
proton 9.8/9.3 d gets shifted upon complexation, which might
probably be due to donation of the lone pairs of electrons by the
nitrogen to the central metal atom, resulting in the formation of
the coordinate linkage (M N). There is no appreciable change
in all other signals of the complexes.
EPR spectra
The EPR spectral parameters of the copper(II) complexes in
DMSO solution at 298 and 77 K are presented in Table 2. The
X-band EPR spectra of copper(II) complexes in DMSO were
Fig. 1. EPR spectra of copper complex [CuL1] in DMSO at 300 K (a) and 77 K (b).

N. Raman et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 96 (2012) 698–708
703
A\ are calculated to be 169 ꢂ 10ꢀ4 for [CuL1], 163 ꢂ 10ꢀ4 for [CuL2]
DNA binding
and 67 ꢂ 10ꢀ4 cmꢀ1 for [CuL1], 71 ꢂ 10ꢀ4 cmꢀ1 for [CuL2], respec-
tively. Aav is calculated using the formula: Aav = 1/3 (A|| + 2A\)
and it is found to be 100.89 ꢂ 10ꢀ4 cmꢀ1 for [CuL1] and
101.56 ꢂ 10ꢀ4 cmꢀ1 for [CuL2]. It has been reported that g|| value
of copper(II) complexes can be used as a measure of the covalent
character of the metal–ligand bond. If the value is more than 2.3,
the metal–ligand bond is essentially ionic and the value less than
2.3 is indicative of covalent character [26]. The present EPR results
show that g|| is lower than 2.3 in this case suggesting that the cop-
per complexes are covalent in nature [27]. It also shows g|| is 2.26/
2.23, which is in conformity with the presence of copper–nitrogen
bonds in these chelates. Also the trend g|| > g\ > ge observed for
these complexes indicate that the unpaired electron is most likely
The design and synthesis of metal complexes that bind to DNA
with specific site in order to develop novel chemotherapeutics,
chemical probes and highly sensitive diagnostic agents for DNA
have received considerable attention [35,36]. The investigation of
the interaction between DNA and other molecules is helpful to
understand the action mechanism of some DNA-targeted drugs
and toxic agents as well as the origins of some diseases like gene
mutation [37]. Basically, a molecule may bind to DNA via covalent
and/or non-covalent interactions (intercalative, electrostatic and
groove binding). In recent years, there is a growing interest in
the application of absorption spectral techniques which can be
used to understand metal–DNA interaction.
in the dx
orbital of the Cu(II) ion and are characteristic for the
2ꢀy2
axial symmetry [28].
Absorption spectral titrations
The empirical ratio g||/A|| is frequently used to evaluate distor-
tions in tetra coordinated copper(II) complexes [29]. The ratio close
to 100, indicates a roughly square-planar structure around the cop-
per(II) ion [30] and the values from 170 to 250 are indicative of a
distorted tetrahedral geometry. The present value of the g||/A|| ratio
for [CuL1] is 133 and [CuL2] is 136 which indicate nearly square-
planar environments around the Cu(II) ion with small distortions
[31]. In axial symmetry the g-values are related to the G-factor
by the expression
The binding of metal(II) complexes to DNA helix has been char-
acterized through absorption spectral titrations, by following
changes in absorbance and shift in wavelength. The experiments
were performed by maintaining a constant concentration of the
complex while varying the DNA concentration [38]. The absorption
spectrum of complex [CuL1] in the absence and presence of CT-
DNA is given in Fig. 2.
As the DNA concentration is increased, the MLCT transition
bands of [CuL1] complex at 422.2, 399.8 nm, [CuL2] complex at
420.6 nm exhibit hypochromism of about 26.6%, 17.3% and 25.9%,
and bathochromism of 2.8 and 1.8, 2.5 nm, respectively, further
the intense absorption bands with maxima of 376.9, 384.2, 354.8
and 338 nm for [NiL1], [NiL2], [ZnL1] and [ZnL2] exhibit hypochro-
mism of about 21.8%, 20.9%, 15.4% and 17.4%, and bathochromism
of 2.1, 1.7, 1.2 and 1.3 nm, respectively. These spectral characteris-
tics may suggest a mode of binding that involves a stacking inter-
action between the aromatic chromophore and the DNA base pairs.
The hypochromisms observed for the bands of these six com-
pounds are accompanied by a small red shift by less than 4 nm.
The above phenomena imply that these compounds interact with
CT-DNA by appreciable groove/surface binding property. To com-
pare quantitatively the affinity of these three compounds binding
to DNA, the intrinsic binding constant (Kb) has been estimated.
From absorption data, Kb is determined using the following equa-
tion [21]:
G ¼ ðgjj ꢀ 2:0027Þ=ðg? ꢀ 2:0027Þ
which measures the exchange interaction between the copper cen-
ters in polycrystalline solid. The G values lie within the range 4.82–
5.24 for the two copper complexes indicating negligible exchange
interaction of Cu–Cu in the complexes according to Hathaway [32].
The parameters g||, g\, A|| and A\ of complexes and the energies
of the d–d transitions are used to calculate the orbital reduction
parameters (K||, K\) and the bonding parameter (a2 and b2). The
factors a
2 and b2 which are usually taken as measure of covalency,
evaluated by the expression:
a2 ¼ ꢀðA =0:036Þ þ ðg ꢀ 2:0027Þ þ ð3=7Þðg? ꢀ 2:0027Þ þ 0:04
jj
jj
b2 ¼ ðgjj ꢀ 2:0027ÞE= ꢀ 8ka2
c2 ¼ ðg? ꢀ 2:0027ÞE= ꢀ 2ka2
where k = ꢀ828 cmꢀ1 for the free copper ion and E is the electronic
transition energy. The covalency of the in-plane
r-bonding, a
2 = 1
indicates complete ionic character, whereas a
2 = 0.5 denotes 100%
covalent bonding. The a2 values for the present copper complexes
are 0.74 for [CuL1] and 0.75 for [CuL2], supporting that the covalent
nature [33] of their bonding. The b2 parameter gives an indication of
the covalency of the in-plane p
-bonding. The smaller the b2 (0.810)
for [CuL1] and 0.790 for [CuL2] the larger is the covalency of the
bonding. For the copper(II) complex, the high value of b2 compared
to a2 indicates that the in-plane
r
-bonding is more covalent than
-bonding. Hathaway [34] pointed out that for pure
-bonding, K|| ꢃ K\ = 0.77 and for in-plane -bonding K|| < K\,
while for out-of-plane -bonding K\ < K||. The following simplified
expressions are used to calculate K|| and K\:
the in-plane
p
r
p
p
K
K
¼ ðgjj ꢀ 2:0027ÞE=8k0
¼ ðg? ꢀ 2:0027ÞE=2k0
jj
?
The observed K|| (0.611/0.612) < K\ (0.247/0.466) relation indicates
the presence of significant out-of-plane –bonding. The EPR study
p
of the copper(II) complex has provided supportive evidence to the
conclusion obtained on the basis of electronic spectrum and mag-
netic moment value.
Fig. 2. Absorption spectral changes on addition of CT DNA to the solution of [CuL1]
in buffer pH = 7.2 at 25 °C in the presence of increasing amount of DNA. Arrow
indicates the changes in absorbance upon increasing the DNA concentration.

704
N. Raman et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 96 (2012) 698–708
Table 3
Electronic absorption spectral properties of Cu(II), Ni(II) and Zn(II) complexes.
No.
1
Compound
kmax
D
k (nm)
H%
Kb ꢂ 104 (Mꢀ1
)
Free
Bound
[CuL1]
422.2
399.8
420.6
376.9
384.2
354.8
338.0
425.0
398.0
423.1
379.8
385.9
356.0
339.3
2.8
1.4
2.5
2.1
1.7
1.2
1.3
26.6
17.3
25.9
21.8
20.9
15.4
17.4
3.79
2
3
4
5
6
[CuL2]
[NiL1]
[NiL2]
[ZnL1]
[ZnL2]
3.17
2.23
3.18
2.15
1.45
½DNAꢁ=ðea
ꢀ
ef Þ ¼ ½DNAꢁ=ðeb ꢀ ef Þ þ 1=½Kbðeb ꢀ ef Þꢁ
where [DNA] is the concentration of DNA in base pairs, the apparent
absorption coefficients ea f, and eb are the apparent, free and
bound metal complex extinction coefficients, respectively. A plot
of [DNA]/(eb f) vs [DNA], gives a slope of 1/(eb f) and a y-inter-
cept equal to [Kb/(eb
f)]ꢀ1. Kb is the ratio of the slope to the y-
,
e
ꢀ
e
ꢀ
e
ꢀ
e
Fig. 3. Effect of increasing amounts of [CuL1] (j), [NiL1](N), [ZnL1] (ꢄ), [CuL2] (⁄),
[NiL2] (ꢁ), [ZnL2] (X), and [EB] (+) on the viscosity of DNA. R = [complex]/[DNA] or
[EB]/[DNA].
intercept. Intrinsic binding constants of 3.79 ꢂ 104, 3.17 ꢂ 104,
2.23 ꢂ 104, 3.18 ꢂ 104, 2.15 ꢂ 104 and 1.45 ꢂ 104 Mꢀ1 have been
determined for [CuL1], [NiL1], [ZnL2], [CuL2], [NiL2] and [ZnL2],
respectively. The results suggest that the interaction of metal(II)
complexes with DNA is a significant minor groove binding mode.
The Kb value (Table 3) obtained here is lower than that reported
for a few intercalating complexes [Ru(bpy)2(dppz)]2+ (4.90 ꢂ 106
Mꢀ1) [39] and [Ru(bpy)2(HBT)]2+ (5.71 ꢂ 107 Mꢀ1) [40].
Cyclic voltammetry
Cyclic voltammogram of [CuL1] in the absence of DNA features
reduction of +3 to +2 form at a cathodic peak potential, Epc of
0.393 V and reduction of +2 to +1 form at a cathodic peak potential,
Epc of ꢀ0.137 V vs Ag/AgCl. Reoxidation of the +2 form occurs at
0.456 V and +1 occurs at 0.034 V upon scan reversal. In the absence
of DNA, the separation of the anodic and cathodic peak potentials,
Viscosity measurement
To further make clear the interaction between the complex and
DNA, viscosity measurements are studied. Optical photophysical
studies are not enough to explain a binding between DNA and
the complex. The viscosity measurements of CT-DNA are regarded
as the least ambiguous and the most critical tests of a binding
model in solution in the absence of crystallographic structural
data. A classical intercalation model demands that the DNA helix
lengthens as base pairs are separated to accommodate the bound
ligand, leading to the increase of DNA viscosity. In contrast, a par-
tial, non-classical intercalation of ligand could bend (or kink) the
DNA helix, reducing its length and, concomitantly, its viscosity.
In addition, complexes that binds exclusively in the DNA grooves
by partial and/or non-classical intercalation, under the same condi-
tions, typically cause less pronounced (positive or negative) or no
change in DNA solution viscosity. The values of the relative specific
D
Ep = ꢀ0.063 V, the formal potential E1/2 taken as the average of
Epa and Epc is 0.424 V for Cu(III) ? Cu(II) redeox couple, Ep =
D
0.171 V and E1/2 = ꢀ0.051 V for Cu(II) ? Cu(I) redeox couple. The
ipa/ipc ratios of these two redox couples are 1.35 and 1.46 which
indicate that the reaction of the complex on the glassy carbon elec-
trode surface is quasi-reversible redox process. The presence of
DNA in the solution at the same concentration of the complex
causes a considerable decrease in the voltammetric current. In
addition, the peak potentials, both Epa and Epc as well as E1/2 have
a shift to less negative potential which is shown in Fig. 4.
viscosities for DNA and the complexes are plotted against of (
g/
go)
1/3 vs [Complex]/[DNA] = R, (Fig. 3). With the increase in amount
of metal complexes, the relative viscosity of DNA increases stea-
dily, which is consistent with DNA groove binding suggested
above, which is also known to enhance DNA viscosity [41]. Inter-
estingly, for metal(II) complex there is an increase in specific vis-
cosity of DNA, which is not as pronounced as those observed for
the classical intercalator ethidium bromide (EB) [42] and the
metallointercalators like [Co(phen)2(PIP)]3+ [43], [Co(phen)2(H-
PIP)]3+ [44] and [Ru(phen)2(dppz)]2+ [44]. As intercalation causes
a significant increase in viscosity of DNA solutions due to lengthen-
ing of the DNA helix as base pairs are separated to accommodate
the aromatic chromophore of the bound molecule, it is tempting
to ascribe the observed increase in viscosity to intercalative inter-
action of the complex. However, the effective intercalations of the
phenyl rings of Schiff base in metal complexes are discouraged by
several steric factors. Therefore, it is obvious that the complex pre-
fers to engage in DNA surface binding with its large size affecting
an increase in DNA viscosity, rather than in intercalative DNA
interaction. The extent of increase in viscosity may depend on
the DNA binding mode and affinity of metal complexes.
Fig. 4. Cyclic voltammogram of [CuL1] in the presence of increasing amount of DNA
in buffer pH = 7.2 at 25 °C. Arrow indicates the changes in voltammetric currents
upon increasing the DNA concentration.

N. Raman et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 96 (2012) 698–708
705
The cyclic voltammogram of [CuL2] complex in buffer pH = 7.2
at 25 °C features two anodic peaks (Epa1 = 0.421 V and Epa2
ꢀ0.050 V vs Ag/AgCl) and two cathodic peaks (Epc1 = 0.399 V and
Epc2 = ꢀ0.098 V vs Ag/AgCl). The oxidation of peak Epa1 belongs to
Cu(III)/Cu(II) and reduction of Cu(II) occurred, upon scan reversal
at 0.399 V. The separation of the anodic and cathodic peak poten-
DNA photo cleavage
=
Redox active chemical nucleases that are able to irreversibly
damage DNA have received a lot of interest because of their poten-
tial application as biological tools or as drugs. As shown in Figs. 5
and 6, all the complexes cause extensive damage to DNA when
irradiated with UV light at 360 nm. Plasmid DNA serves as a conve-
nient probe for DNA photocleavage, whereby gel electrophoretic
analysis with ethidium-bromide staining is used to distinguish
three distinct topological forms of DNA: Form I (supercoiled), Form
II (relaxed), and Form III (linear). The topological state of plasmid
DNA, in turn, reflects the nature of DNA damage mediated by a
photoactivatable agent: single or double-strand breaks in the back-
bone as well as unwinding of the double helix.
tials,
DEp = 0.022 V and the ratio of anodic to cathodic peak poten-
tial is ipa/ipc = 1.11, indicating a quasi-reversible redox process.
The oxidation of peak Epa2 belongs to Cu(II)/Cu(I), and reduction
of Cu(II) occurred, upon scan reversal at ꢀ0.098 V. The separation
of the anodic and cathodic peak potentials,
DEp = 0.048 V and the
ratio of anodic to cathodic peak potential is ipa/ipc = 1.38, indicat-
ing a quasi-reversible redox process. The formal potential E1/2 of
these two redox couple has been observed at 0.41 and ꢀ0.074 V,
respectively. The presence of DNA in solution at the same concen-
tration of this complex caused a considerable decrease in the vol-
tammetric current. In addition, the peak potentials, Epa and Epc,
as well as E1/2 had a shift to less negative potential. The decrease
extents of the peak currents observed for metal complex upon
addition of CT-DNA may indicate that the binding affinity of metal
complex.
Form I DNA (Fig. 5, lane 1), often referred to as closed, circular
DNA, represents native plasmid DNA that is negatively supercoiled
in the absence of DNA damaging agents. When single-strand
breaks occur, superhelical tension is removed and the DNA relaxes
1
2
3
4
5
6
The Cu(II)/Cu(I) and Cu(II)/Cu(III) redox processes are influ-
enced by the coordination number, stereochemistry and the
hard/soft character of the ligands, donor atoms However due to
inherent difficulties in relating coordination number and stereo-
chemistry of the species present in solution, redox processes are
generally described in terms of the nature of the ligands present
[45]. Patterson and Holm [46] have shown that softer ligands tend
to produce more positive E values, while hard acids give rise to
negative E values. The observed E values for the copper complexes
indicate considerable ‘‘hard acid’’ character, as reported earlier
[47]. This is likely to be due to azomethine nitrogen donor and
nitrogen atoms of the heterocyclic bases involved in coordination.
The cyclic voltammogram of both nickel complexes in the ab-
sence of DNA feature reduction of +2 to +1 form at a cathodic peak
potential, Epc of ꢀ0.083 V for [NiL1] and ꢀ0.094 V for [NiL2] vs Ag/
AgCl. Reoxidation of the +1 form occurs at ꢀ0.112 and ꢀ0.251 V,
respectively, upon scan reversal. In the absence of DNA, the sepa-
Form II
Form III
Form I
Fig. 5. Gel electrophoresis diagram showing the photocleavage of pUC19 DNA by
the synthesized complexes in DMF–Tris buffer medium on irradiation with UV light
of 360 nm: lane 1, DNA control (60 min); lane 2, DNA + ligand (60 min); lane 3,
DNA + [CuL1] (dark, 90 min); lane 4, DNA + [CuL1] (60 min); lane 5, DNA + [NiL1]
(60 min); lane 6, DNA + [ZnL1] (60 min).
1
2
3
4
5
6
ration of the anodic and cathodic peak potentials,
DEp = 0.029 V
for [NiL1] and 0.157 V [NiL2], the formal potential E1/2 taken as
the average of Epa and Epc is ꢀ0.097 V for [NiL1] and ꢀ0.172 for
[NiL2].
Form II
Form III
Form I
Finally the zinc complexes showed redox cathodic peak at
ꢀ0.159 V for [ZnL1] and ꢀ0.201 V for [ZnL2] and anodic peak
appeared at ꢀ0.565 V and ꢀ0.673 V, respectively, the separation
of the anodic and cathodic peak potential,
D
Ep = ꢀ0.406 and
ꢀ0.472 V for [ZnL1] and [ZnL2]. The formal potential of these two
complexes are found to be E1/2 = ꢀ0.362 and ꢀ0.437 V. The incre-
mental addition of CT DNA to the complexes revealed that the re-
Fig. 6. Gel electrophoresis diagram showing the photocleavage of pUC19 DNA by
the synthesized complexes in DMF–Tris buffer medium on irradiation with UV light
of 360 nm: lane 1, DNA control (60 min); lane 2, DNA + ligand (60 min); lane 3,
DNA + [CuL2] (dark, 60 min); lane 4, DNA + [CuL2] (60 min); lane 5, DNA + [NiL2]
(60 min); lane 6, DNA + [ZnL2] (60 min).
dox couples caused a less negative shift in E1/2 and decrease of
DEp.
The ipa/ipc values also decreased in the presence of DNA. The elec-
trochemical parameters of the Cu(II), Co(II), Ni(II) and Zn(II) com-
plexes are shown in Table 4.
Table 4
Electrochemical parameters for the interaction of DNA with Cu(II), Ni(II) and Zn(II) complexes.
No.
1
Compound
Redox couple
E1/2 (V)
D
Ep (V)
ipa/ipc
Free
Bound
Free
Bound
[CuL1]
Cu(III) ? Cu(II)
Cu(II) ? Cu(I)
Ni(II) ? Ni(I)
Zn(II) ? Zn(0)
Cu(III) ? Cu(II)
Cu(II) ? Cu(I)
Ni(II) ? Ni(I)
Zn(II) ? Zn(0)
0.424
ꢀ0.051
ꢀ0.097
ꢀ0.362
0.410
ꢀ0.074
ꢀ0.172
ꢀ0.437
0.374
ꢀ0.061
ꢀ0.189
ꢀ0.381
0.373
ꢀ0.085
ꢀ0.189
ꢀ0.465
ꢀ0.063
0.171
0.029
0.374
0.152
ꢀ0.183
ꢀ0.353
0.039
1.35
1.46
1.59
1.14
1.11
1.38
1.26
1.01
2
3
4
[NiL1]
[ZnL1]
[CuL2]
ꢀ0.406
0.022
0.048
0.157
ꢀ0.472
0.140
5
6
[NiL2]
[ZnL2]
ꢀ0.182
ꢀ0.500

706
N. Raman et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 96 (2012) 698–708
to produce Form II, which migrates more slowly through the aga-
rose gel as nicked, circular DNA. If two single-strand lesions occur
within approximately 15 base pairs [48] or if frank double-strand
cleavage takes place, the result is linear, Form III DNA(Fig. 5, lanes
4 and 5), which travels between Forms I and II on the gel. DNA
photocleavage assays with pUC19 plasmid and [CuL1] and [CuL2]
complexes show no DNA damage in the dark (Fig. 5, lane 3 and
Fig. 6, lane 3). Similarly, irradiation of the plasmids alone for
60 min with 360 nm light results in no discernible DNA damage.
However, irradiation of all the metal complexes in the presence
of pUC19 plasmid produces double-strand breaks. The result indi-
cates the importance of the metal complexes for observing the
photo induced-DNA cleavage activity. To establish the reactive spe-
cies responsible for the photoactivated cleavage of pUC19 DNA, the
following experiments were carried out. Photoactivated cleavage is
pUC19 DNA in the presence of metal complexes and different
inhibitors are shown in Figs. 7–9.
1
2
3
4
5
6
Form II
Form III
Form I
Fig. 9. Photoactivated cleavage of pUC19 DNA in the presence of DNA + [ZnL1]
(60 min) and different inhibitors after irradiation at 360 nm for 60 min: lane 1 DNA
control (60 min); lane 2, DNA + [ZnL1] (50 M); lane 3, DNA + [ZnL1] + sodium azide
l
(100
l
M); lane 4, DNA + [ZnL1] + SOD (1 U); lane 5, DNA + [ZnL1] + DMSO (4
M).
lL);
lane 6, DNA + [ZnL1] + distamycin (100
l
The cleavage of the plasmid DNA was not inhibited in the pres-
ence of hydroxyl radical scavengers such as DMSO, indicating that
hydroxyl radical (OH) was not likely to be the cleavaging agent.
While in the presence of superoxide dismutase (SOD), a facile
superoxide anion radical (Oꢀ2ꢀ) quencher, the cleavage was im-
proved, which indicated that (Oꢀ2ꢀ) might be an inhibitor in the
photoactivated cleavage of the plasmid and reducing the amount
of (Oꢀ2ꢀ) can improve the cleavage effect. Moreover, the DNA cleav-
ages of the plasmid by metal complexes were inhibited in the pres-
ence of the singlet oxygen (1O2) scavenger histidine suggested that
(1O2) is likely to be the reactive species responsible for the cleavage
reaction. Control experiments data suggest the formation of singlet
oxygen (1O2) as the reactive species in a type-II process in the me-
To determine the groove selectivity of the complexes [49], con-
trol experiments are performed using minor groove binder dista-
mycin. The addition of distamycin inhibits the DNA cleavage by
the metal complexes. The results suggest minor groove preference
for the metal complexes.
Antimicrobial activity
Antibacterial activity
The Schiff base complexes have provoked wide interest because
they possess a diverse spectrum of biological and pharmaceutical
activities. The antibacterial activity of the Schiff base ligands and
their metal complexes and streptomycin (as a standard compound)
was tested against bacteria because bacteria can achieve resistance
to antibiotics through biochemical and morphological modifica-
tions [50]. The organisms used in the present investigations in-
cluded Staphylococcus aureus, Pseudomonas aeruginosa, Escherichia
coli, Staphylococcus epidermidis and Klebsiella pneumoniae. The dif-
fusion agar method was used to evaluate the antibacterial activity
of the synthesized metal complexes.
tal assisted photo-excitation process involving ligand n–p⁄ and
p⁄ transitions.
p–
1
2
3
4
5
6
The results of the bactericidal study of the synthesized com-
pounds are displayed in Table 5. The MIC values obtained reflect
that the Schiff base ligands have moderate activity against S. aureus
and E. coli and less active against P. aeruginosa and S. epidermidis.
Ligands also show a moderate activity towards K. pneumoniae.
The remarkable activity of the Schiff base ligand may arise from
the two imine groups which import in elucidating the mechanism
of trans formation reaction in biological system. All the metal com-
plexes are found to have higher antibacterial activity against Schiff
base ligands. The antibacterial results evidently show that the
activity of the Schiff base compounds becomes more pronounced
when coordinated to the metal ions. The studies reveal that a sig-
nificant antibacterial activity can be induced for complexes by
Form II
Form I
Fig. 7. Photoactivated cleavage of pUC19 DNA in the presence of DNA + [CuL1]
(60 min) and different inhibitors after irradiation at 360 nm for 60 min: lane 1 DNA
control (60 min); lane 2, DNA + [CuL1] (50 M); lane 3, DNA + [CuL1] + sodium azide
l
(100
l
M); lane 4, DNA + [CuL1] + SOD (1 U); lane 5, DNA + [CuL1] + DMSO (4
M).
lL);
lane 6, DNA + [CuL1] + distamycin (100
l
1
2
3
4
5
6
Table 5
Minimum inhibitory concentration values of the synthesized compounds against the
growth of five bacteria (MIC in lg/mL).
Form II
Form I
Compound
S.
P.
E. coli S.
epidermidis
K.
aureus
aeruginosa
neumoniae
L1
17.7
18.3
5.4
6.6
7.5
6.7
9.9
10.8
21.5
22.5
5.5
7.1
8.8
8.6
8.7
8.3
1.9
17.8
17.2
5.3
7.9
8.3
5.1
7.6
5.9
1.8
21.3
25.1
7.1
9.6
10.8
8.9
11.5
10.4
1.3
18.9
18.5
7.0
8.4
7.5
6.2
8.5
10.9
2.3
L2
[CuL1]
[NiL1]
[ZnL1]
[CuL2]
[NiL2]
[ZnL2]
Fig. 8. Photoactivated cleavage of pUC19 DNA in the presence of DNA + [NiL1]
(60 min) and different inhibitors after irradiation at 360 nm for 60 min: lane 1 DNA
control (60 min); lane 2, DNA + [NiL1] (50 M); lane 3, DNA + [NiL1] + sodium azide
l
(100
l
M); lane 4, DNA + [NiL1] + SOD (1 U); lane 5, DNA + [NiL1] + DMSO (4
M).
lL);
Streptomycin 1.7
lane 6, DNA + [NiL1] + distamycin (100
l

N. Raman et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 96 (2012) 698–708
707
Table 6
through minor groove binding. When irradiated at 360 nm, metal
complexes can efficiently cleave the plasmid pUC19 DNA. The
mechanism studies of photoactivated cleavage reveal that singlet
Minimum inhibitory concentration values of the synthesized compounds against the
growth of five fungi (MIC in lg/mL).
oxygen (1O2) and superoxide anion radical (Oꢀꢀ) may play an
Compound
A. niger
F. solani
C. lunata
R. bataicola
C. albicans
2
L1
22.6
24.8
8.0
9.1
8.9
9.2
11.5
9.3
25.5
27.6
7.3
10.3
8.2
10.5
10.1
11.7
1.6
23.3
25.7
8.4
7.4
10.4
7.9
10.5
10.3
1.2
21.5
22.3
8.1
25.9
24.1
10.2
8.1
important role in the photocleavage. However, these observations
and a more extensive study would be necessary in order to assert
that the complexes act as cleavage agents. These results should be
considered as promising lead compounds for the development of
targeted drugs for cancer treatment. The Schiff base ligands and
their complexes show antimicrobial activity against selected kinds
of bacteria and fungi, results of this work can show that the ap-
proach of coordinating 4-aminoantipyrine derivatives with phar-
macologically interesting metals such as copper, nickel and zinc
could be a suitable strategy to develop novel therapeutic tools
for the medical treatment.
L2
[CuL1]
[NiL1]
[ZnL1]
[CuL2]
[NiL2]
[ZnL2]
Nystatin
7.5
10.2
11.5
10.5
12.2
1.0
9.5
10.1
11.3
11.0
1.5
1.1
using either a biocation or a metallic ion exhibiting inhibitory
properties as well as a ligand that displays such activity. On the
other hand, important characteristics that can be correlated with
a good antimicrobial activities are as follows: (i) the presence of
uncoordinated groups that permits the recognition by living organ-
ism and enhances the solubility (hydrophilic or hydrophobic) (ii)
the ability to form hydrogen bonding with a counter anion or sol-
vent molecules (iii) the dimension and nature of the substituted
groups attached to the donor atoms that influence the ligand lipo-
philicity and membrane permeability, controlling thus the cell
penetration (iv) the small coordination number or the presence
of some ligands easy to be removed in interaction with biomole-
cules (v) a stereochemistry that allows a favorable three-dimen-
sional interaction with biomolecules and (vi) a high kinetic and
thermodynamic stability in order to control the dissociation in
the acidic medium from the stomach, the metabolization in san-
Acknowledgments
The authors express their sincere thanks to the College Manag-
ing Board, Principal and Head of the Department of Chemistry,
VHNSN College, Virudhunagar, India for providing necessary re-
search facilities. N.R. thanks the University Grants Commission
(UGC), New Delhi for financial assistance.
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