Effect of DNA interaction involving antioxidative 4-aminoantipyrine incorporating mixed ligand complexes having alpha-amino acid as co-ligand

Article abstract of DOI:10.1016/j.molstruc.2013.12.018

Few new mixed ligand transition metal complexes of the stoichiometry [ML(A)₂], where M = Co(II), Ni(II), Cu(II) and Zn(II), L = FFAP (furfurylidene-4-aminoantipyrine) and A = amino acid (glycine/alanine/valine), have been designed, synthesized

Full text of DOI:10.1016/j.molstruc.2013.12.018

Journal of Molecular Structure 1060 (2014) 63–74
Contents lists available at ScienceDirect
Journal of Molecular Structure
journal homepage: www.elsevier.com/locate/molstruc
Effect of DNA interaction involving antioxidative 4-aminoantipyrine
incorporating mixed ligand complexes having alpha-amino acid as
co-ligand
Natarajan Raman a, , Arunagiri Sakthivel , Muthusamy Selvaganapathy , Liviu Mitu
⇑
a
a
b
a
Research Department of Chemistry, VHNSN College, Virudhunagar 626 001, India
Department of Chemistry, University of Pitesti, Pitesti 110040, Romania
b
h i g h l i g h t s
g r a p h i c a l a b s t r a c t
Few novel 4-aminoantipyrine incorporating amino acid mixed ligand complexes act as efficient DNA
binding and DNA cleaving agents due to the size of the alkyl group present in the amino acid. They are
good antimicrobial and oxidative activators.
ꢀ
Exploring efficient antioxidative
agents having amino acid as co-
ligand.
ꢀ
ꢀ
ꢀ
b
Excellent K values for valine mixed
ligand complexes.
Effective cleavage of pUC19 DNA via
oxidative pathway.
Better antimicrobial active agents.
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 28 October 2013
Received in revised form 6 December 2013
Accepted 16 December 2013
Available online 21 December 2013
Few new mixed ligand transition metal complexes of the stoichiometry [ML(A) ], where M = Co(II), Ni(II),
Cu(II) and Zn(II), L = FFAP (furfurylidene-4-aminoantipyrine) and A = amino acid (glycine/alanine/valine),
2
have been designed, synthesized and characterized. The molar conductivity of the complexes in DMF at
ꢁ3
10
M concentration shows that they are non-electrolytes. The interaction of these complexes with
CT-DNA indicates that the valine mixed ligand complexes are having higher binding constant than alanine
and glycine mixed ligand complexes. This analysis reveals that binding constant depends on the size of the
alkyl group present in the amino acid. The binding constants of valine mixed ligand complexes are in the
Keywords:
Binding constant
4
5
ꢁ1
order of 10 to 10 M revealing that the complexes interact with DNA through moderate intercalation
mode. The metal complexes exhibit effective cleavage of pUC19 DNA but it is not preceded via radical
cleavage and superoxide anion radical. They are good antimicrobial agents than the free ligand. On com-
4
-Aminoantipyrine
pUC19 DNA
Minimum inhibitory concentration (MIC)
Antioxidant
paring the IC50 values, [Ni(L)(Gly)
2
] is considered as a potential drug to eliminate the hydroxyl radical.
Ó 2013 Elsevier B.V. All rights reserved.
1
. Introduction
transition metal anticancer complexes with better efficiency [1–4].
The interaction of transition metal complexes with DNA has been
a subject of passionate research in the field of bioinorganic chem-
istry, ever since the discovery of cis-platin as an anticancer agent.
As an important intention of anticancer drugs, DNA plays a central
role in replication, transcription, and regulation of genes. DNA has
strong affinity towards many organic compounds and transition
In recent years transition metal complexes have been used
widely by many bioinorganic chemists for developing active
⇑
Corresponding author. Tel.: +91 9245165958; fax: +91 4562281338.
E-mail address: drn_raman@yahoo.co.in (N. Raman).
0
022-2860/$ - see front matter Ó 2013 Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.molstruc.2013.12.018

6
4
N. Raman et al. / Journal of Molecular Structure 1060 (2014) 63–74
metal complexes. In its polyionic form, DNA can attract any cat-
ionic species and also neutral organic compounds. These com-
pounds may react with or bind to DNA with high affinity and
cause the decay of cell at an appropriate time [5].
using copper sulphate pentahydrate as the calibrant. Infrared spec-
ꢁ1
tra (4000–350 cm KBr disc) of the samples were recorded on an
IR Affinity-1 FT-IR Shimadzu spectrophotometer. NMR spectra
were recorded on a Bruker Avance Dry 300 FT-NMR spectrometer
The presence of metal binding sites in DNA structure make dif-
ferent type of interactions possible such as intercalation between
base pairs, minor groove binding, and major groove binding which
play an important role in the efforts of the drug targeted to DNA
6
in DMSO-d , using TMS as the internal reference. Mass spectrome-
try experiments were performed on a JEOL-AccuTOF JMS-T100LC
mass spectrometer equipped with a custom-made electrospray
interface (ESI). EPR spectra were recorded on a Varian E 112 EPR
spectrometer in DMSO solution both at room temperature
(300 K) and liquid nitrogen temperature (77 K) using TCNE (tetra-
cyanoethylene) as the g-marker. The absorption spectra were re-
corded by using Shimadzu model UV-1601 spectrophotometer at
room temperature.
[
6,7]. A large number of transition metal complexes have been
used as cleavage agents for DNA and also for novel potential
DNA-targeted antitumor drugs [8–10]. Additionally, it has been
demonstrated that free radicals can damage proteins, lipids, and
DNA of bio-tissues, leading to increased rates of cancer [11]. Fortu-
nately, antioxidants can prevent this damage, due to their free rad-
ical scavenging activity [12]. Hence, it is very important to develop
compounds with both strong antioxidant and DNA-binding prop-
erties for effective cancer therapy.
This inspires synthetic chemists to search for new metal com-
plexes for bioactive compounds. Transition metals are particularly
suitable for this purpose because they can adopt a wide variety of
coordination numbers, geometries and oxidation state in compar-
ison with carbon and other main group elements. It was reported
that, the treatment with the copper complexes produce remark-
able pharmacological effects, which are not observed when the
parent ligands or inorganic forms of copper are used [13]. From a
coordination chemistry viewpoint, the only atoms available for
coordination are the nitrogen atoms of the pyrazole ring and the
oxygen atom of the carbonyl group. If the nitrogens are blocked
by substitution, such as in antipyrine, coordination can only be
achieved through the oxygen atom. Nowadays, 4-aminoantipyrine
derived transition metal complexes have been extensively used in
various fields like biological, analytical and therapeutic applica-
tions [14]. It has been observed that mixed ligand complexes be-
have as an efficient DNA-targeted material. The incorporation of
2.2. Synthesis of Schiff base (L)
An ethanolic solution of (30 mL) 4-aminoantipyrine (0.01 mol)
was added to an ethanolic solution of furfural (0.01 mol). The
resultant mixture was refluxed for 3 h. The resulting solution
was evaporated under vacuum to remove the solvent. The product
was filtered and the yellow coloured solid was isolated and recrys-
tallized in ethanol before drying over desiccators.
L. Yield: 86%. Anal. Calc. for C16
15 3 2
H N O : C, 68.4; H, 5.3; N, 14.9
ꢁ
1
(%); Found: C, 68.3; H, 5.3; N, 14.9 (%). IR (KBr pellet, cm ):1665
1
m
(C@O); 1645
m
(AHC@N); H NMR (d): (aromatic) 6.6–7.4 (m);
(COOH) 11(s); (CACH
MS m/z (%): 282 [M+1] . kmax (cm ) in DMF, 35,211, 27,778.
3
) 2.3 (s); (NACH ) 3.1(s); (CH@N) 9.3(s).
3
+
ꢁ1
2.3. Synthesis of metal complexes
The metal(II) complexes were prepared by mixing the appropri-
ate molar quantity of ligands and metal salt using the following
procedure. An ethanolic solution of L (10 mM) was heated under
reflux with the ethanolic solution of metal(II) chloride (10 mM)
for 3 h. To the above mixture, amino acid (glycine/alanine/valine)
(20 mM) in alcoholic potash was added and the reflux was contin-
ued for 1 h. The solid product formed was filtered, washed repeat-
an organic compound like
a-amino acid significantly modifies
the structural and biological activities of transition metal com-
plexes [15]. In addition to this, the complexes containing 4-amino-
antipyrine Schiff base derivative along with
to be explored.
This prompted us to synthesize a new series of heterocyclic
compounds containing the antipyrinyl moiety from the Schiff
a
-amino acids are yet
2
edly with de-ionized water and dried over anhydrous CaCl under
vacuum. Schematic diagram for the synthesis of ligand and metal
complexes is given in Scheme 1.
2 23 5 6
[Cu(L)(Gly) ]. Yield: 76%. Anal. Calc. for C20H N O Cu: Cu, 12.9;
bases derived from furfurlyidene-4-aminoantipyrine and a-amino
C, 48.7; H, 4.7; N, 14.2 (%); Found: Cu, 12.5; C, 48.3; H, 4.2; N, 13.8
ꢁ1
acids with Co(II), Cu(II), Ni(II) and Zn(II) metal ions. The amino
acids are known to coordinate with metal ion through N, O-donor
ligand forming five membered chelate ring after dissociation of the
acidic proton [16,17]. They are characterized using analytical and
spectral techniques. Furthermore, their biological studies like
DNA binding, cleavage, antimicrobial, antioxidative responses have
been performed.
(%). IR (KBr pellet, cm ): 1645
m
(C@O); 1597
m
(AHC@N); 3310
ꢁ
ꢁ
ꢁ
m
(NH
2
); 3396, 1406, 1370
(MAO); 450
m
((ACOO ), (ACOO )
m
asy, (ACOO )
+
m
sy); 503
m
m
(MAN). MS m/z (%): 493 [M+1] . kmax
eff (BM): 1.87.
] Yield: 72%. Anal. Calc. for C22
C, 50.7; H, 5.2; N, 13.4 (%); Found: Cu, 11.7; C, 50.2; H, 4.8; N, 13.1
ꢁ1
(cm ) in DMF, 11,001, 35,842.
l
[Cu(L)(Ala)
2
27 5 6
H N O Cu: Cu, 12.2;
ꢁ1
(%). IR (KBr pellet, cm ): 1643
m
(C@O); 1631
m
(AHC@N); 3305
ꢁ
ꢁ
ꢁ
m
(NH
2
); 3427, 1413, 1381
(MAO); 433
m
((ACOO ), (ACOO )
m
asy, (ACOO )
+
m
sy); 503
m
m
(MAN). MS m/z (%): 521 [M+1] . kmax
eff (BM): 1.89.
]. Yield: 68%. Anal. Calc. for C26
C, 54.1; H, 6.1; N, 12.1 (%); Found: Cu, 10.7; C, 53.8; H, 6.0; N, 11.7
ꢁ1
2
. Experimental protocols
(cm ) in DMF, 10,246, 29,240.
l
[
Cu(L)(Val)
2
35 5 6
H N O Cu: Cu, 11.0;
2.1. Reagents and instruments
ꢁ1
(
%). IR (KBr pellet, cm ): 1645
m
(C@O); 1629
m
(AHC@N); 3310
ꢁ
ꢁ
ꢁ
All reagents, 4-aminoantipyrine, furfural, glycine, alanine, va-
m
(NH
2
); 3392, 1406, 1386
(MAO); 428
m
((ACOO ), (ACOO )
m
asy, (ACOO )
+
line and metal(II) chlorides were of Merck products and they were
used as supplied. Commercial solvents were distilled and then
used for the preparation of ligands and their complexes. DNA
was purchased from Bangalore Genei (India). Microanalyses (C, H
and N) were performed in Carlo Erba 1108 analyzer at Sophisti-
cated Analytical Instrument Facility (SAIF), Central Drug Research
Institute (CDRI), Lucknow, India. Molar conductivities in DMF
m
sy); 501
m
m
(MAN). MS m/z (%):578 [M+1] . kmax
eff (BM): 1.93.
]. Yield: 69%. Anal. Calc. for C20
C, 49.2; H, 4.7; N, 14.3 (%); Found: Co, 11.8; C, 48.7; H, 4.4; N, 14.1
ꢁ1
(cm ) in DMF, 11,236, 29,586.
l
[Co(L) (Gly)
2
23 5 6
H N O Co: Co, 12.1;
ꢁ1
(%). IR (KBr pellet, cm ): 1647
m
(C@O); 1595
m
(AHC@N); 3305
ꢁ
ꢁ
ꢁ
2
m(NH ); 3404, 1417, 1390
m((ACOO ), (ACOO )
m
asy, (ACOO )
+
m
sy); 501
m
(MAO); 434
m
(MAN). MS m/z (%): 488 [M+1] . kmax
eff (BM): 4.48.
]. Yield: 53%. Anal. Calc. for C22 Co: Co, 11.4;
C, 51.2; H, 5.3; N, 13.6 (%); Found: Co, 11.1; C, 50.9; H, 5.0; N, 13.2
ꢁ3
ꢁ1
(
10 M) at room temperature were measured by using Systronic
(cm ) in DMF, 12,300, 15,924, 37,313.
l
model-304 digital conductivity meter. Magnetic susceptibility
measurement of the complexes was carried out by Gouy balance
[Co(L) (Ala)
2
27 5 6
H N O

N. Raman et al. / Journal of Molecular Structure 1060 (2014) 63–74
65
Scheme 1. Synthetic route of Schiff base and its metal complexes.
ꢁ
(
m
m
(
%). IR (KBr pellet, cm 1): 1645
m
(C@O); 1597
m
(AHC@N); 3310
[Ni(L) (Val)
2
]. Yield: 71%. Anal. Calc. for C26
H
35
N
5
O
6
Ni: Ni, 10.3;
ꢁ
ꢁ
ꢁ
(NH
2
); 3429, 1419, 1382
(MAO); 428
m
((ACOO ), (ACOO )
m
asy, (ACOO )
C, 54.6; H, 6.2; N, 12.2 (%); Found: Ni, 9.9; C, 54.1; H, 5.9; N, 11.7
(%). IR (KBr pellet, cm ): 1647
+
ꢁ1
sy); 500
m
m
(MAN). MS m/z (%): 517 [M+1] . kmax
eff (BM): 4.92.
]. Yield: 78%. Anal. Calc. for C26 Co: Co, 10.3;
C, 54.5; H, 6.2; N, 12.2 (%); Found: Co, 10.1; C, 54.1; H, 5.9; N, 11.8
m
(C@O); 1620
m
(AHC@N); 3317
ꢁ
1
ꢁ
ꢁ
ꢁ
cm ) in DMF, 11,099, 12,920, 29,411.
l
m
(NH
503
in DMF, 12,315, 17,211, 29,411.
[Zn(L) (Gly) ]. Yield: 81%. Anal. Calc. for C20
2
); 3427, 1421, 1373
m
((COO ), (ACOO )
m
asy, (ACOO )
m
sy);
)
+
ꢁ1
[
Co(L) (Val)
2
H
35
N
5
O
6
m
(MAO); 428
m(MAN). MS m/z (%): 573 [M+1] . kmax (cm
leff (BM): 3.17.
ꢁ1
(
m
m
(
%). IR (KBr pellet, cm ): 1641
m(C@O); 1628
m
(AHC@N); 3304
2
H
23
N
5
O
6
Zn: Zn, 13.2;
ꢁ
ꢁ
ꢁ
(NH
2
); 3427, 1413, 1381
(MAO); 436
m
((ACOO ), (ACOO )
m
asy, (ACOO )
C, 48.5; H, 4.7; N, 14.2 (%); Found: Zn, 12.7; C, 48.2; H, 4.3; N, 13.9
(%). IR (KBr pellet, cm ): 1646
+
ꢁ1
sy); 501
m
m
(MAN). MS m/z (%): 573 [M+1] . kmax
eff (BM): 4.89.
]. Yield: 73%. Anal. Calc. for C20 Ni: Ni, 12.0;
C, 49.2; H, 4.7; N, 14.3 (%); Found: Ni, 11.7; C, 48.8; H, 4.4; N, 13.9
m
(C@O); 1595
m(AHC@N); 3310
ꢁ
1
ꢁ
ꢁ
ꢁ
cm ) in DMF, 12,422, 16,977, 29,450.
l
m
(NH
503
(CACH
2
); 3442, 1411, 1393
(MAO); 430
) 2.3 (s); (NACH
m
((COO ), (ACOO )
m
asy, (ACOO )
msy);
1
[
Ni(L) (Gly)
2
H
23
N
5
O
6
m
m
(MAN). H NMR (d): (aromatic) 6.5–7.1 (m);
) 3.1(s); (CH@N) 9.6(s). MS m/z (%): 495
in DMF, 29,412, 36,232. eff (BM):
3
3
ꢁ1
+
ꢁ1
(
m
m
(
%). IR (KBr pellet, cm ): 1647
m(C@O); 1595
m
(AHC@N); 3345
[M+1] .
kmax (cm
)
l
ꢁ
ꢁ
ꢁ
(NH
2
); 3442, 1411, 1395
(MAO); 433
m
((ACOO ), (ACOO )
m
asy, (ACOO )
Diamagnetic.
2 27 5 6
[Zn(L) (Ala) ]. Yield: 72%. Anal. Calc. for C22H N O Zn: Zn, 12.5;
C, 50.5; H, 5.2; N, 13.4 (%); Found: Zn, 12.2; C, 50.1; H, 4.8; N, 13.1
+
sy); 503
m
m
(MAN). MS m/z (%):488 [M+1] . kmax
eff (BM): 3.07.
]. Yield: 51%. Anal. Calc. for C22 Ni: Ni, 11.4;
C, 51.2; H, 5.3; N, 13.6 (%); Found: Ni, 11.1; C, 51.8; H, 5.1; N, 13.2
ꢁ
1
cm ) in DMF, 12,453, 13,228, 36,364.
l
ꢁ
1
[
Ni(L) (Ala)
2
H
27
N
5
O
6
(%). IR (KBr pellet, cm ): 1646
m
(C@O); 1625
m(AHC@N); 3302
ꢁ
ꢁ
ꢁ
m
(NH
503
(CACH
2
); 3425, 1413, 1381
(MAO); 435
) 2.3 (s); (NACH
m
((COO ), (ACOO )
m
asy, (ACOO )
msy);
ꢁ1
1
(
m
5
%). IR (KBr pellet, cm ): 1647
m
(C@O); 1629
m(AHC@N); 3315
m
m
(MAN). H NMR (d): (aromatic) 6.5–7.3 (m);
) 3.2(s); (CH@N) 9.7(s). MS m/z (%): 523
in DMF, 29,498, 35,482. eff (BM):
ꢁ
ꢁ
ꢁ
(NH
2
); 3427, 1413, 1381
(MAO); 435
m((COO ), (ACOO )
m
asy, (ACOO )
m
sy);
)
3
3
+
ꢁ1
+
ꢁ1
01
m
m
(MAN). MS m/z (%): 517 [M+1] . kmax (cm
[M+1] .
kmax (cm
)
l
in DMF, 12,360, 13,774, 29,326.
leff (BM): 3.14.
Diamagnetic.

6
6
N. Raman et al. / Journal of Molecular Structure 1060 (2014) 63–74
[
Zn(L) (Val)
2
]. Yield: 67%. Anal. Calc. for C26
H
35
N
5
6
O Zn: Zn, 11.3;
2.5. Interaction with pUC19 DNA plasmid DNA
C, 53.9; H, 6.1; N, 12.1 (%); Found: Zn, 10.9; C, 53.3; H, 5.8; N, 11.7
ꢁ
1
(
m
5
%). IR (KBr pellet, cm ): 1647
m
(C@O); 1620
m
(AHC@N); 3317
The extent of pUC19 DNA cleavage in presence of activating
ꢁ
ꢁ
ꢁ
(NH
2
); 3427, 1421, 1386
(MAO); 428
) 2.4 (s); (NACH
m
((COO ), (ACOO )
m
asy, (ACOO )
msy);
agent AH
(hydroxyl radical scavenger) and NaN
was monitored using agarose gel electrophoresis. In reactions
using super coiled pUC19 plasmid DNA Form I (10 M) in Tris–
HCl buffer (50 mM) with 50 mM NaCl (pH 7.2) which was treated
with the metal complex and ascorbic acid (10 M) followed by
dilution with the Tris–HCl buffer to a total volume of 20 L. The
2
(ascorbic acid), and two radical scavengers DMSO
1
03
m
m
(MAN). H NMR (d): (aromatic) 6.5–7.2 (m);
) 3.3(s); (CH@N) 9.6(s). MS m/z (%): 579
in DMF, 29,325, 35,335. eff (BM):
3
(singlet oxygen scavenger)
(
[
CACH
M+1] .
3
+
3
ꢁ1
kmax (cm
)
l
l
Diamagnetic.
l
l
samples were incubated for 1 h at 37 °C. A loading buffer contain-
ing 0.25% bromophenol blue was added and electrophoresis was
performed at 40 V for each hour in Tris–Acetate–EDTA (TAE) buffer
2
.4. DNA binding experiments
The interaction between metal complexes and DNA was studied
using 1% agarose gel containing 1.0 lg/mL ethidium bromide. The
by electronic absorption, viscosity and electrochemical methods.
Disodium salt of calf thymus DNA was stored at 4 °C. All the exper-
iments involving the interaction of the complexes with calf thymus
cleavage efficiency was measured by determining the ability of the
complex to convert the super coiled (SC) DNA to nicked circular
form (NC) and linear form (LC). Inhibition reactions were carried
out by prior incubation of the pUC19 DNA (10 mM) with DMSO
(
7
CT) DNA were carried out in Tris–HCl buffer (50 mM Tris–HCl, pH
.2) containing 5% DMF at room temperature. Solution of CT DNA
in the buffer gave a ratio of UV absorbance at 260 and 280 nm,
260/A280 of ca. 1.89:1, indicating that the CT DNA was sufficiently
free from protein [18]. The concentration of DNA was measured by
(4 lL).
A
2
2
.6. Antimicrobial studies
ꢁ1
ꢁ1
using its extinction coefficient at 260 nm (6600 M cm ) after 1:
00 dilution. Stock solutions were stored at 4 °C and used not more
.6.1. Antibacterial activity
1
National Committee for Clinical Laboratory Standard (NCCLS)
than 4 days. Doubly distilled water was used to prepare solutions.
Concentrated stock solutions of the complexes were prepared by
dissolving the complexes in DMF and diluting properly with the
corresponding buffer to the required concentration for all the
experiments.
approved standard nutrient agar was used as medium for testing
the activity of microorganisms as antibacterial agents [21]. For pre-
paring the agar media, 3 g of beef extract, 5 g of peptone, 5 g of
yeast extract and 5 g of sodium chloride were dissolved in
1
000 mL of distilled water in a clean conical flask. The pH of the
The electronic spectra of the complexes were recorded before
and after the addition of DNA in the presence of 5 mM Tris–HCl/
solution was maintained at 7. The solution was boiled to dissolve
the medium completely and sterilized by autoclaving at 15 lbs
pressure (121 °C) for 30 min. After sterilization, 20 mL of media
was poured into the sterilized petri plates. These petri plates were
kept at room temperature for sometime. After few minutes the
medium got solidified in the plates. Then, it was inoculated with
microorganisms using sterile swabs. The stock solutions were pre-
pared by dissolving the compounds in appropriate solvents.
In a typical procedure [18], the antibacterial activities of the
compounds were evaluated by the disc diffusion method against
the bacterial microorganisms. The 5 mm diameter and 1 mm thick-
ness of the disc was filled with the test solutions using a micropi-
pette and the plates were incubated at 37 °C for 24 h. During this
period, the test solution was diffused and affected the growth of
the inoculated bacteria. The zone of inhibition, developed on the
plate was measured.
5
0 mM NaCl buffer (pH 7.2). The intrinsic binding constant for
the interaction of complex with DNA was obtained from absorp-
tion data. A fixed concentration value of complex (10 M) was
titrated with increasing amounts of DNA over the range of
0–150 M. The equilibrium binding constant (K ) values for the
l
2
l
b
interaction of the complexes with DNA were obtained from the
absorption spectral titration data using the following equation
½
DNAꢂ=ð
e
a
ꢁ
e
f
Þ ¼ ½DNA=ð
e
b
ꢁ
e
f
Þꢂ þ 1=K
b
ðe
b
ꢁ
e
f
Þ
ð1Þ
where
absorption at a given DNA concentration,
cient of the complex free in solution, , the extinction coefficient
of the complex when fully bound to DNA, K , the intrinsic binding
constant and [DNA], the concentration in nucleotides. A plot of
DNA]/( ) versus [DNA], gives K as the ratio of the slope to
the intercept.
e
a
the extinction coefficient observed for the charge transfer
e
f, the extinction coeffi-
e
b
b
2.6.2. Antifungal activity
[
e
a
ꢁ
e
f
b
NCCLS approved standard potato dextrose agar was used as
medium for antifungal activity by disc diffusion method [18]. For
preparing the agar media, 200 g of potato extract, 20 g of agar
and 20 g of dextrose were dissolved in 1000 mL of distilled water
in a clean conical flask. The solution was boiled to dissolve the
medium completely and sterilized by autoclaving at 15 lbs pres-
sure (121 °C) for 30 min. After sterilization, 20 mL of media was
poured into the sterilized petri plates. These petri plates were kept
at room temperature for sometime. After few minutes the medium
got solidified in the plates. Then, it was inoculated with microor-
ganisms using sterile swabs. In a typical procedure [18], the anti-
fungal activities of the compounds were evaluated by the disc
diffusion method against the fungal microorganisms. The 5 mm
diameter and 1 mm thickness of the disc was filled with the test
solution using a micropipette and the plates were incubated at
37 °C for 72 h. During this period, the test solution was diffused
and affected the growth of the inoculated fungi. After 36 h of incu-
bation at 37 °C, the diameter of the inhibition was measured. Com-
pounds showing promising antibacterial/antifungal activity were
Cyclic voltammetry studies were performed on a CHI 620C elec-
trochemical 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 deoxygen-
2
ated by purging with N prior to measurements. Viscosity experi-
ments were carried on an Ostwald viscometer, immersed in a
thermostated water-bath maintained at a constant temperature
at 30.0 ± 0.1 °C. CT DNA samples of approximately 0.5 mM were
prepared by sonicating in order to minimize complexities arising
from CT DNA flexibility [19]. Flow time was measured with a dig-
ital stopwatch three times for each sample and an average flow
1/3
time was calculated. Data were presented as (
concentration of the metal(II) complexes, where
of CT DNA solution in the presence of complex, and
ity of CT DNA solution in the absence of complex. Viscosity values
were calculated after correcting the flow time of buffer alone (t ),
= (t ꢁ t )/t [20].
g
/
g
g
0
)
versus the
is the viscosity
is the viscos-
g
0
0
g
0
0

N. Raman et al. / Journal of Molecular Structure 1060 (2014) 63–74
67
selected for minimum inhibitory concentration studies. The mini-
The measured magnetic moment values for [Ni(L)(Gly)
[Ni(L)(Ala) ] and [Ni(L)(Val) ] complexes are 3.07, 3.14 and 3.17
BM respectively, which are in the normal range of nickel(II) com-
plexes having magnetic moments between eff 2.9 and 3.4 BM. This
indicates that the complex of Ni(II) ion is six coordinated and prob-
ably octahedral indicating a small but definite orbital contribution
to the magnetic moment.
2
],
mum inhibitory concentration was determined by assaying at con-
centration of compounds along with standards at the same
concentration. Minimum inhibitory concentration (MIC) is the
lowest concentration of an antimicrobial compound, inhibiting
the visible growth of microorganisms after overnight incubation.
Minimum inhibitory concentrations are important in diagnostic
laboratories to confirm resistance of microorganisms to antimicro-
bial agents and also to monitor the activity of new antimicrobial
agents.
2
2
l
3.2. Electronic spectra
The UV–Vis spectra of L and its mixed ligand complexes were
recorded in DMF solution at room temperature. The bands appear-
2.7. Antioxidant property
*
ing at low energy side are attributed to n–
p
transitions which are
associated with the azomethine chromophore. The bands at higher
Hydroxyl radicals were generated in aqueous media through
*
energy arise from
p–p transitions within the phenyl rings [25].
the Fenton-type reaction [22,23]. The aliquots of reaction mixture
3 mL) contained 1.0 mL of 0.10 mmol aqueous safranin, 1 mL of
.0 mmol aqueous EDTA–Fe(II), 1 mL of 3% aqueous H , and a
The absorption bands of the complexes are shifted to longer wave-
length region compared to those of the ligand [26,27]. All the com-
plexes show the high energy absorption bands in the region
(
1
2 2
O
series of quantitative microaddition of solutions of the test com-
pound. A sample without the tested compound was used as the
control. The reaction mixtures were incubated at 37 °C for
0 min in a water bath. The absorbance was then measured at
20 nm. All the tests were run in triplicate and are expressed as
ꢁ
1
3
5,211–37,313 cm . This transition may be attributed to the
charge transfer band. The electronic spectra of [Cu(L)(Gly) ],
Cu(L)(Ala) ] and [Cu(L)(Val) ] complexes display the d–d transi-
tion bands in the region 11,001, 10,246 and 11,236 cm , respec-
2
[
2
2
3
5
ꢁ1
2
2
tively, which are due to
band strongly favours a distorted octahedral geometry around
the metal ion. Electronic spectra of [Co(L)(Gly) ], [Co(L)(Ala)
and [Co(L)(Val) ] complexes display the d–d transition bands in
the region 12,300, 15,924 and 37,313 cm , 11,099, 12,920 and
g
E ? T2g transition. This d–d transition
the mean and standard deviation (SD) [24]. The scavenging effect
Å
for OH was calculated from the following expression:
2
2
]
Scavenging ratio ð%Þ ¼ ½ðA
where A is the absorbance in the presence of the test compound; A
i
ꢁ A
o
Þ=ðA
c
ꢁ A
o
Þꢂ ꢃ 100%
ð2Þ
2
ꢁ1
ꢁ1
ꢁ1
i
0
29,411 cm , 12,422, 16,977 and 29,450 cm which are assigned
to T1g(F) ? 4T2g(F), T1g(F) ? A2g(F) and T1g(F) ? T2g(P) transi-
tions, respectively. These transitions correspond to the octahedral
geometry, supported by their magnetic susceptibility values
4
4
4
4
4
is the absorbance of the blank in the absence of the test compound;
is the absorbance in the absence of the test compound, EDTA–
Fe(II) and H
A
c
2 2
O .
(
4.84–4.92 BM).
2 2
The absorption spectra of [Ni(L)(Gly) ], [Ni(L)(Ala) ] and
3
. Results and discussion
The Schiff base ligand and the Co(II), Cu(II), Ni(II), and Zn(II)
[
2
Ni(L)(Val) ] complexes display three d–d bands at 12,453, 13,228
ꢁ1
and 36,364 cm , 12,360, 13,774 and 29,326, 14,577, 17,211 and
3
2g(F) ? 3T2g(F), 3
A
2g
2
(
9,411 respectively. These correspond to
A
complexes have been synthesized and characterized by spectral
and elemental analytical data. They are found to be air stable.
The ligand is soluble in common organic solvents and all the com-
plexes are freely soluble in DMF and DMSO.
3
3
3
F) ? T1g(F) and A2g (F) ? T1g(P) transitions, respectively and
being the characteristic of an octahedral geometry. This geometry
is further supported by their magnetic susceptibility values
3.07–3.17 BM). The Zn(II) complexes are diamagnetic and also
(
exhibit INCT bands. According to the empirical formulae, an octahe-
dral geometry is proposed for Zn(II) complexes.
3.1. Physical measurements
1
13
The elemental analytical results for the metal complexes are in
3.2.1. H NMR and C NMR spectra
1
good agreement with the calculated values showing that the
complexes have the stoichiometry of the type [MLA ]. Here L is a
The typical H NMR spectra of L and the zinc(II) complex were
shown in Fig. S1(a and b). H NMR spectrum of the Schiff base
1
2
Schiff-base ligand which is prepared by the condensation of 4-
aminoantipyrine with furfural which acts as a bidentate ligand
and amino acids (A) such as glycine, alanine and valine, used as
co-ligands. The metal(II) complexes were dissolved in DMF and
ligand shows peaks at 6.6–7.4 d which are attributed to phenyl
multiplet of Schiff base ligand (obtained from the condensation
of 4-aminoantipyrine and furfuraldehyde). The ligand also shows
3 3
the following signals: CACH 2.3–2.4 d(s), NACH 3.1–3.3 d(s);
ꢁ3
the molar conductivities of 10 M of their solution at room
temperature were measured. The lower conductance values (15–
CH@N 9.3 d(s) and 6.62, 6.94 and 7.83 d for furfurylidene moiety.
The azomethine proton ACH@N signals in the spectra of the zinc
complexes are shifted to down field (9.6 d) compared to the free li-
gands, suggesting deshielding of azomethine group due to the
coordination with metal ion. There is no appreciable change in
all other signals of the complexes. The peak at 11 d is attributed
to the ACOOH of amino acid (glycine/alanine/valine). The absence
of this peak noted for the zinc complexes confirms the loss of the
COOH proton of amino acid moiety due to complexation. The peak
ꢁ1
2
ꢁ1
3
0
X
cm mol ) of the complexes support their non-electrolytic
nature of the compounds.
2
The measured magnetic moment values for [Cu(L)(Gly) ],
[
2 2
Cu(L)(Ala) ] and [Cu(L)(Val) ] complexes are 1.87, 1.89 and 1.93
BM respectively. These values are also closely agreed with the
magnetic moment values which are calculated from the EPR spec-
tral studies of the above complexes. The observed magnetic mo-
ment values indicate the monomeric nature of the copper
at 5.1 d is attributed to the ANH
valine), but in the zinc complex, it is shifted to down field region
which indicates amino group is involved in complexation. 13
2
of amino acid (glycine/alanine/
2 2 2
complexes. [Co(L)(Gly) ], [Co(L)(Ala) ] and [Co(L)(Val) ], com-
plexes reported herein have magnetic moment values, 4.84, 4.92
and 4.89 BM at room temperature respectively. The magnetic mo-
ment values of all above Co(II) complexes (normal range for octa-
hedral Co(II) complexes is 4.3–5.2 BM) indicate that they adopt an
octahedral geometry.
C
NMR spectrum of L shows aromatic carbons at 116–139 ppm. It
ꢁ
also shows CACH
3
at 14.8 d, NACH
3
at 40.2 d, ACOO at 171 d,
ACH@N at 163 d and 149 d for furfurylidene moiety. From the
1
3
C NMR spectra of zinc complexes, it can be known that AC@O

6
8
N. Raman et al. / Journal of Molecular Structure 1060 (2014) 63–74
+
Å
and ACH@N groups are involved in complexation through down
field shift. There is no appreciable change in other signals of the
complex.
fragments lead to the formation of the species [M(L)(A)
2
]
which
+Å
further undergoes de-metallation to yield the species [L] giving
the fragment ion peak at m/z 282. The m/z of all the fragments of
ligands and their complexes confirm the stoichiometry of the com-
3.3. IR spectra
2
plexes as [M(L)(A) ].
The coordination mode and sites of the ligand to the metal ions
3.5. EPR spectra
were investigated by comparing the infrared spectra of the free li-
gand with its metal complexes. The IR spectrum shows a band at
Mixed ligand complex is found to be interesting due to the fact
that the arrangement of ligand atoms around Cu(II) is found to
change as one goes from the solid to the solution state. Hence to
obtain further information about the stereochemistry and to inter-
ꢁ1
1
665 cm for the free ligand (L), assigned to
pyrine, shifted towards lower values around 1641–1653 cm
indicating the coordination of the carbonyl oxygen atom of the
-aminoantipyrine derived Schiff base ligand to metal ion. The
tC@O of 4-aminoanti-
ꢁ1
4
pret bonding parameters, the EPR spectra of [Cu(L)(Gly)
2
],
ꢁ1
spectrum of free ligand shows a band in the region 1645 cm , a
characteristic feature of the C@N (azomethine) stretching mode
[Cu(L)(Ala) ] and [Cu(L)(Val) ] complexes have been studied. The
2
2
t
EPR spectra of copper complexes were recorded in DMSO solution
both at room temperature (RT) and at liquid nitrogen temperature
(LNT) and are given in Fig. S2(a and b) and the spectral data are gi-
ven in Table 1. The spectrum at RT shows one intense absorption
band in the high field and is isotropic due to the tumbling motion
indicating the formation of the Schiff base product originating from
amino and carbonyl groups of the starting reagents (4-aminoanti-
pyrine and furfural). This band is shifted towards lower frequencies
ꢁ1
in the spectra of metal complexes (1595–1637 cm ) compared to
the above Schiff base ligand indicating the involvement of the
azomethine nitrogen in coordination with metal ion. Moreover,
the coordination of the co-ligand to the metal centre via the
carboxylic group can also be obvious from the difference of max-
of the molecules. However, [Cu(L)(Gly)
[Cu(L)(Val) ] complexes at LNT show well resolved peaks with
low field region. The anisotropic data (g|| values of 2.3336, 2.3759
and 2.3797 and g values of 2.060, 2.0795 and 2.1665) have been
2 2
], [Cu(L)(Ala) ] and
2
\
ima positions as observed for
are observed at 1390 and 1427 cm respectively for a free amino
acid ligand, For comparison, the and display
t
sy:ðCOO
ꢁ
Þ
and
t
asy:ðCOO
ꢁ
Þ
. The bands
obtained from frozen solution spectra at 77 K which are well re-
solved at low field. g tensor values of Cu(II) complexes can be used
to derive the ground state of the complexes. The observed values,
ꢁ
1
t
sy:ðCOO
ꢁ
Þ
t
asy:ðCOO Þ
ꢁ
ꢁ
1
bands at 1370–1395 and 1406–1421 cm , respectively for all
the metal complexes. These results reveal that the amino acid li-
gand is involved in the coordination through the carboxyl group.
In addition to it, the coordination of the co-ligand to the metal cen-
tre via the amino group is observed. The band is observed at
g
|| > g
\
> g
e \
and A|| > A reveal that the unpaired electron of Cu(II)
2
ion lies predominantly in the dx
2ꢁy2
g
orbital giving E as ground
state. From the values of g factors, the geometric parameter G, rep-
resenting a measure of exchange interaction between Cu(II) cen-
tres in polycrystalline compound can be determined by using the
formula:
ꢁ1
3
340 cm for the amino group of free amino acid ligand but it is
ꢁ
1
shifted to lower frequency region (30 cm ) that indicates amino
group of amino acid is involved in complexation. Conclusive evi-
dence of the bonding is also shown by the observation that new
bands in the spectra of all metal complexes appearing in the low
frequency regions at 501–503 cm and 430–450 cm which are
characteristic to the (MAN) stretching vibrations
respectively that are not observed in the spectrum of free ligand.
G ¼ ðg ꢁ 2Þ=ðg ꢁ 2Þ
ð3Þ
k
?
If G > 4.0, the local tetragonal axes are aligned parallel or only
slightly misaligned. If G < 4.0, significant exchange coupling is
present and the misaligned is appreciable. The observed values
for the exchange interaction parameter for [Cu(L)(Gly)2],
[Cu(L)(Ala) ] and [Cu(L)(Val) ] complexes are 5.8170, 4.8847 and
ꢁ
1
ꢁ1
t
(MAO) and t
2
2
5
.4938 G which suggest that the local tetragonal axes are aligned
3.4. Mass spectra
parallel or slightly misaligned and the unpaired electron is present
in the dx
2ꢁy2
orbital. This result also indicates that the exchange
The FAB mass spectrum of synthesized L is recorded and the ob-
tained molecular ion peaks confirm the proposed formulae. The
coupling effects are not operative in the present complexes [28].
In this study, g|| value is greater than 2.33 suggesting the confor-
mity with the presence of mixed copper–nitrogen and copper-oxy-
gen bonding to the chelates.
mass spectrum of L shows M+1 peak at m/z 282 (91.71%) corre-
+
Å
sponding to (C16
H
15
N
3
O
2
)
ion. Also the spectrum exhibits the
fragments at m/z 214, 202, 188, 172 and 157 corresponding to
The empirical ratio of g |A is frequently used to evaluate distor-
||
||
+Å
+Å
+Å
+Å
[
C
C
12
H
H
12
N
3
O] , [C11
H
11
N
3
O] , [C11
H
11
N
2
O] , [C10
H
8
N
2
O]
and
tion in copper(II) complexes. The ratio is close to 100 indicates a
roughly square-planar structure around the Cu(II) ion and the val-
ues from 170 to 250 are indicative of distorted tetrahedral geome-
try. If the ratio is in between 110 and 170, it indicates nearly
octahedral environment around Cu(II) ion with small distortion.
The observed g |A values for the [Cu(L)(Gly) ], [Cu(L)(Ala) ] and
+Å
+-
[
10
8
N
2
]
respectively (Fig. 4(a)). The spectrum of [Co(L)(Gly) ]
2
Å
complex shows molecular ion peak at m/z 488 [M+1] which is
] complex gives
fragment ion peaks at m/z 471, 428, 384, 326 and 282 correspond-
equivalent to its molecular weight. The [Co(L)(Gly)
2
+Å
+Å
+Å
ing to [C20
[
of [Cu(L)(Ala)
[
[
4
[
[
H
21
N
4
O
6
Co] , [C17
]+Å ions respectively. The spectrum
complex shows molecular ion peak at m/z 521
M+1] which is equivalent to its molecular weight. The
Cu(L)(Ala) ] complex gives fragment ion peaks at m/z 504, 491,
14 4 6 14 4 4
H N O Co] , [C16H N O Co] ,
||
||
2
2
Å
C
16
H
14
N
4
O
4
]+ and [C15
H
14
N
4
O
2
[Cu(L)(Val) ] complexes are 137, 158 and 132 respectively which
2
+Å
2
]
suggest that all the above complexes are having distorted octahe-
dral structure.
2
Electron paramagnetic resonance and optical spectra have been
used to determine the covalent bonding parameters for the Cu(II)
ion in various environments. Since there has been wide interest
in the nature of bonding parameters in the system, the simplified
molecular orbital theory [29] is adopted to calculate the bonding
+
Å
48, 385, 341, 325 and 281 corresponding to [C22
H
25
N
4
O
6
Cu] ,
+
Å
+Å
+Å
+Å
C
21
H
H
24
N
N
4
O
6
Cu] , [C18
H
17
N
4
O
6
Cu] , [C18
H
17
N
4
O
6
]
17 4 4
[C17H N O ]
+Å
+Å
C
17
15
3
O
4
]
and [C16
H
15
N
3
O
2
]
ions respectively. The spectrum
+Å
of [Zn(L)(Val)
2
]
complex shows molecular ion peak at m/z 579
2
2
[
[
5
[
[
M+1] which is equivalent to its molecular weight. Moreover, the
Zn(L)(Val) ] complex gives fragment ion peaks at m/z 562, 549,
coefficients such as
lent in-plane -bonding). The in-plane
related to g and g according to the following equation:
a
(covalent in-plane
r
-bonding) and b (cova-
2
2
p
r
-bonding parameter,
a
is
+
Å
06, 441, 426, 382 and 339 corresponding to [C26
H
33
N
4
O
N
6
Zn] ,
||
\
+Å
+Å
+Å
+Å
25 32 4 6 25 4 6 25 4 6
C H N O Zn] , [C22H N O Zn] , [C22H N O ] , [C21
H
H
22
4 6
O ] ,
2
+
Å
+Å
a
¼ ðA
k
=0:036Þ þ ðg
k
ꢁ 2:0027Þ þ 3=7ðg
?
ꢁ 2:0027Þ þ 0:04
ð4Þ
C
20
22
N
O
4 4
]
and [C17
H
15
N
4
O
4
]
ions respectively. All these

N. Raman et al. / Journal of Molecular Structure 1060 (2014) 63–74
69
Table 1
The spin Hamiltonian parameters of copper complexes in DMSO solution at 300 and 77 K.
2
b2
2
2
k
2
?
Complex
A
||
A
\
A
iso
g
||
g
\
g
iso
G
g
||/A||
a
c
K
K
[
[
[
Cu(L)(Gly)
2
]
]
]
170
150
171
120
110
94
136
123
120
2.3336
2.3759
2.3797
2.0600
2.0795
2.1665
2.1512
2.1783
2.2375
5.8170
4.8847
5.4938
137
158
132
0.8669
0.8620
0.7860
0.6857
0.6688
0.6184
0.4715
0.5476
0.0929
0.5945
0.5765
0.7017
0.1021
0.1180
0.3043
Cu(L)(Ala)
Cu(L)(Val)
2
2
2
2
If
a
= 1.0, it indicates complete ionic character whereas
a
= 0.5
concentration of CT-DNA, the absorption bands of the complexes
are affected, resulting in the tendency of hypochromism and a min-
or red shift is observed in all the complexes. With increasing DNA
concentration, the absorption bands at 336.4, 281.0 and 260.9 nm
for the complexes [Cu(L)(Gly) ], [Cu(L)(Ala)] and [Cu(L)(Val) ] are
2 2
appeared with hypochromism of 11.3%, 11.61% and 18.24% respec-
tively. The absorption bands at 339.8, 342.3 and 341.8 nm for co-
balt complexes [Co(L)(Gly) ], [Co(L)(Ala)] and [Co(L)(Val) ] are
2 2
appeared with hypochromism of 3.08,%, 3.55% and 11.50% respec-
tively. The absorption bands at 339.8, 340.2 and 330.2 nm for
denotes 100% covalent bonding, with assumption of negligible
small values of the overlap integral. Here, in the present study
2
the values of
Cu(L)(Val)
indicate that metal–ligand bonds have some covalent character
in-plane bonding. The out-plane bonding is not strong in the
present study. This is due to demand of crystal packing forces on
a
for the complexes [Cu(L)(Gly)
2
], [Cu(L)(Ala)
2
] and
[
2
] are 0.8669, 0.8620 and 0.7860 respectively which
r
r
2
the interstitial site. The out-of-plane
-bonding (b ) parameters have been calculated from the follow-
p
bonding (
c
) and in-plane
2
p
ing expressions:
2 2
[Ni(L)(Gly) ], [Ni(L)(Ala)] and [Ni(L)(Val) ] complexes are appeared
with hypochromism of 7.34%, 13.10% and 22.0% respectively. The
absorption bands at 339.6, 339.6 and 339.0 nm for [Zn(L)(Gly)2],
2
2
b ¼ ðg ꢁ 2:0027ÞE= ꢁ 8k
a
ð5Þ
ð6Þ
k
[
2
Zn(L)(Ala)] and [Zn(L)(Val) ] complexes are appeared with hypo-
2
2
c
¼ ðg? ꢁ 2:0023ÞE=ðꢁ2k
a
Þ
chromism of 1.10%, 9.11% and 8.15% respectively. The electronic
absorption spectra of most of the complexes in 5 mM Tris–HCl buf-
fer (pH 7.2) are similar in shape and exhibit broad absorption
bands in the range of 321.5–306.5 nm which are typical for transi-
ꢁ1
where k = ꢁ828 cm for free Cu(II) ion and E is the electronic tran-
2
? T2g. The observed values of b2 for the com-
], [Cu(L)(Ala) ] and [Cu(L)(Val) ] are 0.6857,
.6688 and 0.6184 and the values of2c are 0.475, 0.5476 and
2
sition energy of
plexes [Cu(L)(Gly)
E
g
2
2
2
2
tions between the p-electronic energy levels (Figs. 1 and 2). It is
0
0
2
also reported that the intense absorption bands (in the UV region)
.0929 respectively. In this study, b >
-bonding. Hathaway [30] pointed
-bonding, K|| ꢄ K and for in-plane -bonding
, while for out-of-plane -bonding K < K|| the following
simplified expressions were used to calculate K|| and K
c which indicates that there
ꢅ
observed for the complexes are attributed to intraligand
p–
p
tran-
is an interaction in out-of-plane
out that for pure
p
sition of the coordinated groups. On increasing the concentration
of CT-DNA results in the minor bathochromic shift in the range
ꢆ0.2–2.6 nm and significant hypochromicity lying in the range
ꢆ3.08–22.0% which indicates that all the complexes are weak
intercalator to the CT-DNA.
r
\
p
K|| < K
\
p
\
\
:
K
k
¼ ðg ꢁ 2:0027ÞE=8k
0
ð7Þ
k
The intrinsic binding constants (K
CT-DNA have been obtained by monitoring the changes in the intral-
igand band with increasing concentration of DNA. The K values are
shown in Table 2, which shows the intrinsic binding constants for
the complexes [Cu(L)(Gly) ], [Cu(L)(Ala)] and [Cu(L)(Val) ] are found
b
) of the metal complexes with
K
?
¼ ðg? ꢁ 2:0027ÞE=2k
The observed K|| for the complexes [Cu(L)(Gly)
0
ð8Þ
b
2
], [Cu(L)(Ala)
2
]
and [Cu(L)(Val) ] are 0.5945, 0.5765 and 0.7017 and K
.1021, 0.1180 and 0.3043 respectively which imply a greater con-
tribution from out-of-plane -bonding than from in-plane -bond-
ing in metal ligand -bonding. Thus, the EPR study of the copper(II)
2
\
are
2
2
0
3
3
4
ꢁ1
to be 5.559 ꢃ 10 , 8.613 ꢃ 10 and 3.8271 ꢃ 10 M respectively.
p
p
p
complexes has provided supportive evidence to the conclusion ob-
tained on the basis of electronic spectra and magnetic moment
values.
Increasing DNA
3.6. DNA binding studies of metal complexes by optical method
One of the primary methods of observing and quantifying DNA
is through the use of optics. Electronic absorption spectroscopy is
universally employed to determine the binding characteristics of
metal complex with DNA. Binding of the macromolecule leads to
changes in the electronic spectrum of the metal complex. Base
binding is expected to perturb the ligand field transition of the me-
tal complex. A complex bound to DNA through intercalation is
characterized by the change in absorbance (hypochromism) and
red shift in wavelength, due to the intercalative mode involving a
stacking interaction between the aromatic chromophore and the
DNA base pairs. The extent of hypochromism is commonly consis-
tent with the strength of the intercalative interaction [31]. On the
other hand, metal complexes, which bind non-intercalatively or
electrostatically with DNA may result in hyperchromism or hypo-
chromism [32,33].
Fig. 1. Electronic absorption spectra of [Ni(L)(Ala)
2
] in the absence and presence of
M from top to
increasing amounts of DNA. {[Complex] = 10 M, [DNA] = 20–180
l
l
The binding mode of complexes to CT-DNA helix has been
followed through absorption spectral titrations. With increasing
bottom}. Arrow indicates the change in the absorbance upon increasing the DNA
concentration.

7
0
N. Raman et al. / Journal of Molecular Structure 1060 (2014) 63–74
binding constant depends on the size of the alkyl group present in
the mixed ligand of amino acid. As the size of the alkyl group in-
creases, it makes more damage on the double helical structure of
the DNA. Moreover, the enhanced binding constant for the valine
mixed ligand complexes may be due to higher magnetic moments,
electronic spectral vibrations, increased in hypochromism value,
higher DNA cleavage ability and lower current density.
Increasing DNA
3.7. Viscosity measurements
The nature of binding of the metal complexes to the CT-DNA is
further investigated by viscometric studies. The relative specific
viscosity of DNA has been determined by varying the concentration
of the added metal complexes. Measuring the viscosity of DNA is a
classical technique used to analyze the DNA binding mode in solu-
tion. In the absence of crystallographic structural data, hydrody-
namic methods that are sensitive to DNA length change are
regarded as the least ambiguous and the most critical tests of bind-
ing in solution. A classical intercalation model results in the length-
ening of the DNA helix as the base pairs are separated to
accommodate the binding molecule, leading to an increase in the
DNA viscosity. However, a partial or weak or non-classical interca-
lation of ligand may bend (or kink) DNA helix, resulting in the de-
crease of its effective length and concomitantly its viscosity [36].
Fig. 2. Electronic absorption spectra of [Ni(L)(Val)
increasing amounts of DNA. {[Complex] = 10
bottom}. Arrow indicates the change in the absorbance upon increasing the DNA
2
] in the absence and presence of
M, [DNA] = 20–180 lM from top to
l
concentration.
1
/3
2 2
The binding constants for [Co(L)(Gly) ], [Co(L)(Ala)] and [Co(L)(Val) ]
The plot of (
g/
0
g )
0
vs [complex]/[DNA] = R, g and g are the rela-
3
3
4
ꢁ1
complexes are 1.034 ꢃ 10 , 2.766 ꢃ 10 and 3.1055 ꢃ 10 M
tive viscosities of DNA in the presence and absence of compound,
respectively, gives a measure of the viscosity changes. The effects
of all the complexes on the viscosity of CT-DNA are shown in
Fig. 3. As expected, the known DNA intercalator ethidium bromide
increases the relative viscosity of DNA double helix resulting from
intercalation. In contrast, weak intercalation can cause an increase
in the effective length of DNA leading to a minor increase in the
effective length of DNA solution [37]. All the complexes exhibit
minor increase in the relative viscosity of CT-DNA compared to
ethidium bromide suggesting primarily weak interaction nature
of the complexes.
respectively. For the nickel complexes the binding constant values
3
4
5
ꢁ1
are 3.6955 ꢃ 10 , 4.4614 ꢃ 10 and 3.8991 ꢃ 10 M respectively.
3
Finally for the zinc complexes the binding values are 1.573 ꢃ 10 ,
4
4
ꢁ1
9
.3590 ꢃ 10 and 2.3355 ꢃ 10 M respectively. The result suggests
that the interaction of metal(II) complexes with DNA is significantly
through weak intercalation mode. The binding constant (K ) values
of these complexes are lower in comparison to those observed for
b
2
+
typical intercalators, ethidium bromide and [Ru(bpy)
2
(HBT)]
7
whose binding constants are in the order of 7 ꢃ 10 and
7
ꢁ1
5
.71 ꢃ 10 M [34,35]. Based on the observed results, it is con-
cluded that valine mixed ligand complexes are having higher bind-
ing constants than alanine and glycine mixed ligand complexes.
Among the four complexes all nickel mixed ligand complexes are
having higher binding constants than other complexes. Next higher
binding constants have been observed in all copper mixed ligand
complexes and the least binding values are found in the all zinc
mixed ligand complexes. It can also be explained with the help of
hypochromism which is directly related to the binding constant of
the complexes. From this analysis (Table 2), it can be known that
3
.8. Electrochemical detection binding of metal complexes with DNA
Cyclic and differential pulse voltammetric techniques are
extremely useful in probing the nature and mode of DNA binding
of metal complexes. Typical cyclic voltammogram of complex,
[
2
Cu(L)(Val) ] in the absence and in the presence of varying amount
of [DNA] is shown in Fig. 4. The incremental addition of CT-DNA to
the complex causes decrease of anodic and cathodic peak current
of the complex. This result shows that complex stabilizes the du-
plex (GC pairs) by intercalating way. The incremental addition of
CT-DNA to the complex causes shift in the potential of peak in
Table 2
Electronic absorption spectral properties of Cu(II), Co(II), Ni(II) and Zn(II) amino acid
mixed ligand complexes.
aH%
bK
(M
ꢁ1
)
No
Compound
k
max
D
k (nm)
b
Free
Bound
3
3
3
3
3
3
4
3
4
4
5
4
1
2
3
4
5
6
7
8
9
[Cu(L)(Gly)
2
]
]
]
]
]
336.4
339.8
339.8
339.6
281.0
342.3
340.2
339.6
260.9
341.8
340.2
339.0
337.8
339.6
339.4
340.2
280.6
341.0
338.8
339.8
261.7
339.2
341.4
339.8
1.4
0.2
0.4
0.6
0.4
1.3
1.4
0.2
0.8
2.6
1.2
0.8
11.33
3.08
7.34
1.10
11.61
3.55
13.10
9.11
18.24
11.50
22.00
8.15
5.559 ꢃ 10
1.034 ꢃ 10
3.695 ꢃ 10
1.573 ꢃ 10
8.613 ꢃ 10
2.766 ꢃ 10
4.4614 ꢃ 10
9.3590 ꢃ 10
3.8271 ꢃ 10
3.1055 ꢃ 10
3.8991 ꢃ 10
2.3355 ꢃ 10
[Co(L)(Gly)
2
[Ni(L)(Gly)
[Zn(L)(Gly)
2
2
[Cu(L)(Ala)
[CoL)(Ala)
[Ni(L)(Ala)
[Zn(L)(Ala)
[Cu(L)(Val)
[Co(L)(Val)
2
2
]
2
]
]
]
]
2
2
10
11
12
2
[Ni(L)(Val)
2
]
[Zn(L)(Val)
2
]
a
H% = [Afree ꢁ Abound)/Afree] ꢃ 100%.
b
b
K = Intrinsic DNA binding constant determined from the UV–Vis absorption
Fig. 3. Effect of increasing amounts of [Ni(L)(Val)
2 2
] ( ), [Cu(L)(Val) ] ( ),
spectral titration.
[
Co(L)(Val) ] ( ), [Zn(L)(Val)
2
2
] (Ç) and [EB] (ꢅ) on the relative viscosity of CT-DNA.

N. Raman et al. / Journal of Molecular Structure 1060 (2014) 63–74
71
cyclic voltammogram. Both the cathodic and anodic peak show po-
sitive or negative shift which indicates intercalation of complex to
DNA of base pairs. If one of the shifts is positive and another one is
negative, it reveals the intercalation and electrostatic binding of
the complex to CT-DNA or it may break the secondary structure
of DNA.
2
In differential pulse voltammogram of the complex, [Cu(L)(Val) ]
in the absence and presence of varying amount of [DNA] with signif-
icant decrease of current intensity (Fig. 5), the shift in potential is
related to the ratio of binding constant by the following equation:
E
b
ꢁ E
f
¼ 0:0591 logðK½redꢂ=K½oxdꢂ
Þ
ð9Þ
where E
b
f
and E are peak potentials of the complex in the bound and
free form respectively. In the present study, gly, ala and val mixed
ligand complexes show one electron transfer during the redox
process and its Ipc/Ipa value is less than unity which indicates the
reaction of the complex on the glassy carbon electrode surface is
quasi-reversible. Other complexes [Co(II), Ni(II) and Zn(II)] show
considerable shift in both cathodic and anodic peak potentials in
the presence of incremental addition of CT-DNA.
Fig. 4. Cyclic voltammogram of [Cu(L)(Val)
presence ( ) of increasing amount of DNA concentration 10
M, 100 M, 150 M, 200 M, 250
Supporting electrolyte, 5 mM Tris–HCl + 50 mM NaCl in water (pH 7.2).
2
]
in the absence
(
)
and
M,
l
M, 20
l
4
0
l
l
l
l
lM, respectively with scan rate of 10 mV/s.
Most of the synthesized complexes give both the anodic and
cathodic peak potential shifts which are either positive or negative.
(Table 3). It indicates the intercalating mode of DNA binding with
gly/ala/val mixed ligand Schiff base complexes. It is interesting to
note that val mixed ligand complexes give greater decrease in cur-
rent intensity than ala/gly mixed ligand complexes (Figs. 4 and 5).
From this observation it is concluded that intercalative mode of
DNA binding to val mixed ligand complexes is stronger than
ala/gly mixed ligand complexes.
3
3
.9. DNA cleavage studies
.9.1. DNA cleavage without adding reluctant
The ability of complex to cleave supercoiled DNA is determined
by agarose gel electrophoresis. When circular plasmid DNA in the
presence of an inorganic molecule is subjected to electrophoresis,
relatively fast migration will be observed for the intact supercoiled
form (Form I). If scission occurs on one strand, the super coil will
relax to generate a slower moving open circular form (Form II). If
both the strands are cleaved, a linear form (Form III) that migrates
in between Form I and II will be generated [38]. The DNA cleavage
ability of gly, ala and val mixed ligand complexes in DMF in the
presence of Tris buffer medium is found out by Agarose gel
electrophoresis pattern for the cleavage of pUC19 plasmid DNA.
Fig. 5. Differential pulse voltammogram of 100
absence and presence of DNA concentration 10
50 M, 200 M, 250 M, respectively with scan rate of 10 mV/s. Supporting
l
M [Co(L)(Val)
2
] complex in the
l
M, 20 M, 40
l
l
M, 100 M,
l
1
l
l
l
electrolyte, 5 mM Tris–HCl + 50 mM NaCl in water (pH 7.2).
Table 3
Electrochemical parameters for the interaction of DNA with metal(II) complexes of amino acid co-ligands.
S.No
Complexes
aE1/2 (V)
bD
E
p(V)
K
R
/K
O
cIpc/Ipa
Free
Bound
Free
Bound
1
2
3
4
5
6
7
8
9
[Cu(L)(Gly)
2
]
]
]
]
]
]
ꢁ0.1235
ꢁ0.1790
ꢁ0.6663
+0.1724
ꢁ0.139
ꢁ0.0985
ꢁ0.1615
ꢁ0.6128
+0.1987
ꢁ0.136
+0.037
+0.036
+0.065
+0.031
0.8590
1.1640
1.1294
0.7421
0.398
0.0341
0.8590
0.7357
1.2415
2.1543
0.8264
0.6813
0.6782
0.6900
0.5789
0.7660
0.7312
0.6260
0.8512
1.0236
0.8419
0.8240
0.9265
0.8740
[Co(L)(Gly)
2
[Ni(L)(Gly)
[Zn(L)(Gly)
2
ꢁ0.0335
+0.0513
ꢁ0.0600
+0.0750
ꢁ0.0640
ꢁ0.0650
ꢁ0.1298
+0.0630
+0.0330
+0.0728
ꢁ0.0164
+0.0806
ꢁ0.0880
+0.0650
ꢁ0.0620
ꢁ0.0200
ꢁ0.2185
+0.0220
+0.0620
+0.0242
2
[Cu(L)(Ala)
[Co(L)(Ala)
2
2
ꢁ0.2015
ꢁ0.1340
ꢁ0.4903
ꢁ0.5267
ꢁ0.5045
ꢁ0.1045
ꢁ0.1657
ꢁ0.2215
ꢁ0.1160
ꢁ0.4300
ꢁ0.3882
ꢁ0.4800
ꢁ0.0800
ꢁ0.1637
[Ni(L)(Ala)
2
]
[Zn(L)(Ala)
2
]
]
]
[Cu(L)(Val)
2
1
1
1
0
1
2
[Co(L)(Val)
2
[Ni(L)(Val)
2
]
[Zn(L)(Val)
2
]
Data from cyclic voltammetric measurements:
a
E
D
1/2 is calculated as the average of anodic (EPa) and cathodic (Epc) peak potentials. E1/2 = Epa + Epc/2.
= Epa ꢁ Epc
Error limit: ±5%.
b
c
E
p
.

7
2
N. Raman et al. / Journal of Molecular Structure 1060 (2014) 63–74
It has been observed that valine mixed ligand complexes have
more DNA cleavage ability than alanine and glycine mixed ligand
complexes. Among the valine mixed ligand complexes, nickel and
copper complexes are having higher cleavage ability. These results
are closely agreed with results of the binding studies. It is shown in
Fig. 6. The DNA cleavage ability of [Ni(L)(Val)
investigated using pUC19 DNA in Tris buffer medium at 37 °C for
5 min. Control experiments suggest that untreated DNA does
not show any cleavage upon irradiation, however with increasing
concentration of [Ni(L)(Val) ] complex, the amount of Form I of
2
] complex has been
Fig. 8. Agarose gel electrophoresis pattern for the cleavage pattern of pUC 19
plasmid DNA (20 M) by [Ni(L)(Val) ] (30 M) in DMF–Tris buffer medium in
presence of different activating agents at 37 °C after incubation for 30 min. Lane 1,
DNA control; Lane 2, DNA + complex + H (20 M); Lane 3, DNA + complex + Asc
M); Lane 4, DNA + complex + GSH (20 M); Lane 5, DNA + complex + MPA
M).
4
l
2
l
2
O
2
l
2
(
(
20
20
l
l
l
pUC19 DNA gradually diminishes, whereas that of Form II increases
suggesting single strand DNA cleavage (Fig. 7).
3.9.2. In presence of activators
In order to probe the cleavage mechanism of pUC19 DNA in-
2
duced by [Ni(L)(Val) ] complex, the DNA cleavage activity has been
evaluated in presence of different activators, viz. ascorbic acid
(
(
(
Asc), H
GSH). As shown in Fig. 8 the nuclease activity of [Ni(L)(Val)
30 M) in DMF–Tris buffer medium in presence of activators fol-
2 2
O , 3-mercaptopropionic acid (MPA) and glutathione
2
]
l
2 2
lows the order MPA > Asc > GSH > H O .
Fig. 9. Gel electrophoresis diagram showing the cleavage of supercoiled pUC19
DNA (20 M) by [Ni(L)(Val) ] (50 M) with addition of MPA (100 M) and radical
scavengers in 5 Tris buffer medium and incubated at 37 °C for 1 h: Lane 1, DNA
control; Lane 2, DNA + [[Ni(L)(Val) ] + DMSO (4 L); Lane 3, DNA + [Ni(L)(Val) ] +
ethanol (4 L); Lane 4, DNA + [Ni(L)(Val) ] + t-BuOH (100 M); Lane 5, DNA +
Ni(L)(Val) ] + -histidine (100 M); Lane 6, DNA + [Cu(L)(Val) ] + NaN (100 M);
Lane 7, DNA + [Ni(L)(Val) ] + NaN (100 M); Lane 8, DNA + [Cu(L)(Val) ] + SOD
4 Units) Lane 9, DNA + [Ni(L)(Val) ] + SOD (4 Units) respectively.
3
.9.3. In presence of radical scavengers
In order to clarify the cleavage mechanism of pUC19 DNA intro-
l
2
l
l
duced by metal(II) complexes, the investigation is carried out fur-
ther on adding DMSO, ethanol and t-BuOH (hydroxyl radical
scavengers). It is found that there is no inhibition of DNA cleavage
which indicates that hydroxyl radical is not involved in the cleav-
2
l
2
l
2
l
[
2
L
l
2
3
l
2
3
l
2
(
2
age process (Fig. 9, lanes 2–4). On the other hand, addition of L-his-
tidine and sodium azide (singlet oxygen scavenger) does not show
any apparent inhibition in the DNA which reveals that the singlet
l
oxygen O
2
is not responsible for the cleavage reaction (Fig. 9, lanes
3.10. Antimicrobial screening
5
–7). These observations suggest that all the complexes mediated
cleavage reaction have not proceeded via radical cleavage. SOD
addition (Fig. 9, lanes 8 and 9) does not have any apparent effect
on the cleavage activity indicating the non-involvement of super
oxide radical in the cleavage reaction. Therefore, from these obser-
vations one can understand that the DNA cleavage proceeds via
oxidative pathway.
The main aim of the production and synthesis of any antimicro-
bial compound is to inhibit the causal microbe without any side ef-
fects on the patients. In addition, it is worthy to stress here on the
basic idea of applying any chemotherapeutic agent which depends
essentially on the specific control of only one biological function
and not multiple ones. The chemotherapeutic agent affecting only
one function has a highly sounding application in the field of treat-
ment by anticancer, since most of the anticancer used in the pres-
ent time affect both cancerous diseased cells and healthy ones
which in turns affect the general health of the patients. Therefore,
there is a real need for having a chemotherapeutic agent which
controls only one function. Introducing metal ions into a biological
system may be carried out for therapeutic and diagnostic purpose.
To support in the field of bioinorganic chemistry, subsequently,
the compounds synthesized have been evaluated for their antibac-
terial and antifungal actions. The antibacterial and antifungal tests
have been carried out using the disc diffusion method. The organ-
isms used in present investigation include the bacteria Staphylo-
coccus aureus, Pseudomonas aeruginosa, Escherichia coli,
Staphylococcus epidermidis, Klebsiella pneumoniae and the fungal
species Aspergillus niger, Aspergillus flavus, Culvularia lunata, Rhizoc-
tonia bataicola and Candida albicans. The effectiveness of the inves-
tigated ligand and its metal complexes as good antimicrobial
agents has been screened in addition to evaluation of few known
antibiotics using streptomycin as standard antibacterial agent
and nystatin as antifungal agent. The results of the antimicrobial
studies of the synthesized compounds are displayed in Table 4
and Table 5. All the complexes are more potent than the ligand to-
wards the specified bacterial and fungal species. Moreover, all the
complexes are having antimicrobial activities that are very close to
the standard streptomycin and nystatin. Most of the complexes are
having better activities towards P. aeruginosa, E. coli, S. epidermidis
and fungal species.
Fig. 6. Agarose gel electrophoresis pattern for the cleavage of pUC19 plasmid DNA
(
20
Ligand 30
Cu(L)(Val)
Lane 6: [Ni(L)(Ala)
plex + DNA; Lane 8: [Co(L)(Ala)
M complex + DNA; Lane 10: [Ni(L)(Gly)
l
mL) with complex in DMF–Tris buffer medium; Lane 1: DNA control; Lane 2:
M complex + DNA; Lane 3: [Ni(L)(Val) ] 30 M complex + DNA; Lane 4:
] 30 M complex + DNA; Lane 5: [Co(L)(Val) ] 30 M complex + DNA;
] 30 M complex + DNA; Lane 7: [Cu(L)(Ala) ] 30 M com-
] 30 M complex + DNA; Lane 9: [Cu(L)(Gly)
] 30 M complex + DNA.
l
2
2
l
[
l
2
l
2
l
2
l
2
l
2
]
3
0
l
2
l
Fig. 7. Agarose gel electrophoresis pattern for the cleavage of pUC19 plasmid DNA
20 mL) with [Ni(L)(Val) ] in DMF–Tris buffer medium; Lane 1: DNA control; Lane
: 10 M complex + DNA; Lane 3: 15 M complex + DNA; Lane 4: 20 M com-
plex + DNA; Lane 5: 25 M complex + DNA; Lane 6: 30 M complex + DNA.
(
2
l
2
l
l
l
l
l

N. Raman et al. / Journal of Molecular Structure 1060 (2014) 63–74
73
Table 4
The in vitro antibacterial activity of ligand and its amino acid mixed ligand complexes evaluated by MIC (Minimum Inhibitory Concentration,
l
g/mL).
Compound
FFAP (L)
S. aureus
P. aeruginosa
E. coli
S. epidermidis
K. pneumoniae
23.8 ± 0.12
6.0 ± 0.82
7.2 ± 1.02
5.7 ± 0.23
4.8 ± 1.12
7.2 ± 0.23
5.4 ± 0.38
5.4 ± 0.51
7.2 ± 1.18
12.3 ± 0.16
13.4 ± 1.02
12.3 ± 0.25
13.5 ± 0.96
–
8.9 ± 0.61
4.5 ± 0.24
6.3 ± 0.86
1.6 ± 0.19
1.8 ± 0.88
2.4 ± 0.42
2.1 ± 0.75
2.3 ± 0.28
3.0 ± 1.06
1.8 ± 0.24
2.6 ± 1.15
2.1 ± 0.17
1.6 ± 0.92
–
20.3 ± 0.65
3.6 ± 0.18
2.1 ± 0.78
1.8 ± 0.42
1.9 ± 1.16
1.6 ± 0.25
1.9 ± 0.14
3.0 ± 0.49
1.8 ± 0.40
1.5 ± 0.18
2.4 ± 0.28
1.9 ± 0.13
1.5 ± 0.48
–
21.7 ± 0.23
6.0 ± 0.46
1.4 ± 1.03
6.3 ± 0.39
4.2 ± 0.68
1.8 ± 0.44
1.3 ± 0.52
3.0 ± 0.58
3.0 ± 1.08
1.8 ± 0.11
1.2 ± 0.64
1.2 ± 0.25
2.4 ± 0.68
–
24.4 ± 0.45
4.8 ± 0.15
13.5 ± 0.69
6.0 ± 0.47
2.9 ± 1.03
6.36 ± 0.36
4.8 ± 0.73
6.6 ± 0.14
7.8 ± 0.87
11.2 ± 0.14
12.3 ± 0.18
10.5 ± 0.44
13.4 ± 0.28
–
[
[
[
[
[
[
[
[
[
[
[
[
Cu(L)(Gly)
Co(L)(Gly)
2
]
]
]
2
Ni(L)(Gly)
Zn(L)(Gly)
2
2
]
]
Cu(L)(Ala)
Co(L)(Ala)
2
2
]
Ni(L)(Ala)
2
]
Zn(L)(Ala)
2
]
Cu(L)(Val
2
]
Co(L)(Val)
2
]
Ni(L)(Val)
2
]
Zn(L)(Val)
2
]
DMSO
Streptomycina
1.7 ± 0.08
1.9 ± 0.11
1.8 ± 0.05
1.3 ± 0.14
2.3 ± 0.18
a
Streptomycin is used as a standard drug; Error limit: ±2%.
therefore also conducted an investigation to explore whether the
synthesized complex has the hydroxyl radical scavenging property.
The ability of one of the present compounds is compared to scav-
enge hydroxyl radicals with those of the well-known natural anti-
oxidants mannitol and vitamin C, using the same method as
reported by Wu et al. [43]. The 50% inhibitory concentration
Table 5
The in vitro antifungal activity of ligand and its amino acid mixed ligand complexes
evaluated by MIC (Minimum Inhibitory Concentration,
lg/mL).
Compound
FFAP (L)
A. niger
A. flavus
C. lunata
R. bataicola C. albicans
25.3 ± 1.02 8.4 ± 0.98 18.3 ± 0.86 21.7 ± 1.13 26.4 ± 1.26
[
[
[
[
[
[
[
[
[
[
[
[
Cu(L)(Gly)
2
]
]
]
]
]
]
7.0 ± 0.23 6.0 ± 0.26
9.2 ± 0.28 7.8 ± 0.36
6.7 ± 0.85 3.1 ± 0.49
5.8 ± 1.12 3.3 ± 0.92
9.2 ± 0.11 3.9 ± 0.15
7.4 ± 0.13 3.6 ± 0.22
7.1 ± 0.68 3.8 ± 0.95
10.2 ± 0.28 4.5 ± 0.85
15.3 ± 0.06 4.2 ± 0.10
17.4 ± 0.08 3.6 ± 0.13
14.3 ± 0.86 3.6 ± 0.58
15.5 ± 1.06 3.1 ± 0.69
4.1 ± 0.32
2.6 ± 0.43
2.3 ± 0.58
2.4 ± 0.86
2.2 ± 0.23
2.6 ± 0.30
3.7 ± 0.15
2.5 ± 0.57
3.1 ± 0.15
3.0 ± 0.21
1.8 ± 0.17
2.1 ± 0.87
8.0 ± 0.41
3.4 ± 0.51 14.5 ± 0.68
5.8 ± 0.52
ꢁ3
(
IC50) values of mannitol and vitamin C are about 9.6 ꢃ 10 and
Co(L)(Gly)
2
ꢁ3 ꢁ1
Ni(L)(Gly)
Zn(L)(Gly)
2
8.3 ± 0.62
6.2 ± 0.78
3.8 ± 0.28
3.3 ± 0.36
5.0 ± 0.46
5.2 ± 0.63
3.0 ± 0.09 12.2 ± 0.12
3.2 ± 0.15 13.3 ± 0.18
3.2 ± 0.59 11.5 ± 0.65
5.4 ± 0.53 14.3 ± 0.41
– –
7.0 ± 0.87
3.9 ± 0.82
7.3 ± 0.31
5.8 ± 0.38
7.6 ± 0.52
8.8 ± 0.88
8.7 ꢃ 10
M , respectively. According to the antioxidant experi-
2
ments, the IC50 values of [Ni(L)(Gly)
] and [Cu(L)(Gly)
] complexes
2
2
Cu(L)(Ala)
Co(L)(Ala)
2
ꢁ8
ꢁ1
ꢁ6
ꢁ1
are 2.12 ꢃ 10
that [Ni(L)(Gly)
droxyl radical. In view of the observed IC50 values, the [Ni(L)(Gly)
M
and 1 ꢃ 10
M
respectively which imply
2
2
] complex has preferable ability to scavenge hy-
Ni(L)(Ala)
2
]
]
Zn(L)(Ala)
2
]
]
]
2
Cu(L)(Val)
Co(L)(Val)
2
complex can be considered as a potential drug to eliminate the hy-
droxyl radical. The antioxidant behaviour is also observed in all
2
Ni(L)(Val)
2
]
alanine mixed ligand complexes. The IC50 values of [Cu(L)(Ala)
2
],
complexes are
respectively. These
Zn(L)(Val)
2
]
[
Co(L)(Ala)
2
], [Ni(L)(Ala)
2
]
2
and [Zn(L)(Ala) ]
DMSO
–
–
–
Nystatina
1.1 ± 0.12 1.7 ± 0.09
1.2 ± 0.25
1.0 ± 0.04
1.5 ± 0.16
ꢁ6
ꢁ7
ꢁ5
ꢁ7
ꢁ1
1 ꢃ 10 , 1 ꢃ 10 , 1 ꢃ 10 and 1 ꢃ 10
M
values show that Co(II), Zn(II), Cu(II) and Ni(II) alanine mixed li-
gand complexes have preferable ability to scavenge hydroxyl rad-
ical. On comparing those IC50 values, it can be shown that
a
Nystatin is used as a standard drug; Error limit: ±2%.
[
[
Ni(L)(Gly)
Ni(L)(Ala)
2
], [Co(L)(Ala)
] complexes can be considered as potential drugs to
2
], [Zn(L)(Ala)
2
], [Cu(L)(Ala)
2
]
and
The results obtained show that the mixed ligand complexes are
2
more toxic than the ligand against the same microorganisms. This
enhancement in the activity is rationalized on the basis of the
structure of the metal complexes by possessing an additional azo-
methine (HC@N) linkage which imports in elucidating the mecha-
nism of transamination and resamination reactions in biological
system. It has been suggested that the ligands with nitrogen and
oxygen donor systems might restrain enzyme production, since
the enzymes which require these group for their activity appear
to be especially more susceptible to deactivation by the metal ions
upon chelation. All the complexes are more active against more
organisms comparable to the Schiff base ligand. The rise in the
antimicrobial activity of the mixed ligand complexes may be owing
to the effect of the metal ion on the normal cell processes. This is
probable due to the greater lipophilic nature of the complexes.
Such increased activity of the metal chelates can be explained on
the basis of chelation theory. While chelation is not the only crite-
rion for antibacterial activity, it is an intricate blend of several fac-
tors such as the nature of the metal ion and the ligand, the
geometry of the metal complexes, the lipophilicity and the pres-
ence of co-ligands, the steric and pharmacokinetic factors.
eliminate the hydroxyl radical.
4
. Conclusion
Novel mononuclear 4-aminoantipyrine derived Schiff base li-
gand and amino acid mixed ligand Cu(II), Ni(II), Co(II), and Zn(II)
complexes have been characterized by spectral and analytical data.
The IR, electronic transition and g tensor data lead to the conclu-
sion that the Cu(II) ion assumes a distorted octahedral geometry
and the other complexes Ni(II), Co(II) and Zn(II) are octahedral in
nature. In all the complexes, the Schiff base ligand acts as a biden-
tate. DNA-binding properties of synthetic metal complexes have
been comprehensively studied by different methods including
electronic absorption spectra, viscosity measurements, cyclicvol-
tammetry analysis. All results suggest that the complexes interact
with DNA through a weak intercalation binding mode. In addition,
the DNA cleavage ability of glycine, alanine and valine mixed li-
gand complexes in DMF in presence of Tris buffer medium is found
out by Agarose gel electrophoresis pattern for the cleavage of
pUC19 plasmid DNA. It has been observed that valine mixed ligand
complexes have more DNA cleavage ability than alanine and gly-
cine mixed ligand complexes. Among the valine mixed ligand com-
plexes, nickel and copper complexes are having higher cleavage
ability. These results are closely agreed with the results of the
3.11. Antioxidant potency
According to relevant reports in the literature [39–42], few
transition metal complexes may exhibit antioxidant activity. It is

7
4
N. Raman et al. / Journal of Molecular Structure 1060 (2014) 63–74
binding studies. The cleavage studies suggest that all the com-
plexes mediated cleavage reaction have not proceed via radical
cleavage. Antimicrobial activity studies show that the complexes
exhibit good biological activity against different organisms as com-
pared to ligand and standards. These studies assume significance
as they provide the comprehension of binding of transition metal
complexes to DNA and for developing the next generation of
DNA binding and anticancer drugs.
[12] W.J. Chen, P. Guo, J. Song, W. Cao, J. Bian, Bioorg. Med. Chem. Lett. 16 (2006)
582–3585.
3
[
[
13] J.R.J. Sorenson, Chem. Br. 20 (1984) 1110–1113.
14] N. Raman, S. Sobha, Spectrochim. Acta 85A (2012) 223–229.
[15] P. Kumar, A.K. Singh, J.K. Saxena, D.S. Pandey, J. Organomet. Chem. 694 (2009)
570.
16] C.M. Metcalfe, S. Spey, H. Adams, J.A.J. Thomas, Chem. Soc. Dalton Trans. (2002)
732–4739.
[17] A. Amboise, B.J. Maiya, Inorg. Chem. 39 (2000) 4256–4263.
3
[
4
[
[
[
18] J. Marmur, J. Mol. Biol. 3 (1961) 208–218.
19] J.B. Charies, N. Dattagupta, D.M. Crothers, Biochemistry 21 (1982) 3933–3940.
20] S. Satyanarayanan, J.C. Davorusak, J.B. Charies, Biochemistry 32 (1993) 2573–
2
584.
Acknowledgements
[
21] J.G. Cappuccino, N. Sherman, Microbiology Lab Manual, fourth ed., Benjamin/
Cummings Publishing Company, Inc, 1996..
The authors express their sincere thanks to the College Manag-
ing Board, Principal and Head of the Department of Chemistry,
VHNSN College for providing necessary research facilities. They
also thank the Sophisticated Analytical and Instrumentation Facil-
ity, Central Drug Research Institute, Lucknow, for providing CHN-
analysis data and FAB-Mass spectra and Sophisticated Analytical
and Instrumentation Facility, Indian Institute of Technology,
Bombay, for EPR measurements.
[22] C.C. Winterbourn, Biochem. J. 198 (1981) 125–131.
[
[
23] C.C. Winterbourn, Biochem. J. 182 (1979) 625–628.
24] Z. Guo, R. Xing, S. Liu, H. Yu, P. Wang, C. Li, P. Li, Bioorg. Med. Chem. Lett. 15
(
2005) 4600–4603.
[25] T.R. Holman, M.P. Hendrich, L. Que, Inorg. Chem. 31 (1992) 937–939.
[26] J. Manonmani, M. Kandaswamy, V. Narayanan, R. Thirumurugan, S. Shanmuga
Sundura Raj, G. Shanmugam, M.N. Ponnuswamy, H.K. Fun, Polyhedron 20
(
2001) 3039–3048.
[
[
27] K. Karaoglu, T. Baran, K. Serbest, M. Er, I. Degirmencioglu, J. Mol. Struct. 922
(2009) 39–45.
28] N. Raman, S. Thalamuthu, J. Dhaveethuraja, M.A. Neelakandan, S.J. Banerjee, J.
Chil. Chem. Soc. 53 (2008) 1439–1443.
[
[
29] R.K. Ray, G.B. Kauffman, Inorg. Chim. Acta 173 (1990) 207–214.
30] B.J. Hathaway, Structure and Bonding, vol. 14, Springer Verlag, Heidelberg,
Appendix A. Supplementary material
1
973. p. 60.
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.molstruc.2013.12.
[31] C.N. Sudhamani, H.S. Bhojya Naik, T.R. Ravikumar Naik, M.C. Prabhakara,
Spectrochim. Acta 72A (2009) 643–647.
[
32] D. Lawrence, V.G. Vaidyanathan, B. Unni Nair, J. Inorg. Biochem. 100 (2006)
244–1251.
0
18.
1
[
[
[
33] Q.S. Li, P. Yang, H.F. Wang, M.L. Guo, J. Inorg. Biochem. 64 (1996) 181–195.
34] M.J. Waring, J. Mol. Biol. 13 (1965) 269–282.
35] D.L. Arockiasamy, S. Radhika, R. Parthasarathi, B.U. Nair, Eur. J. Med. Chem. 44
References
(
2009) 2044–2051.
36] S. Sathyanarayana, J.C. Dabrowiak, J.B. Chaires, Biochemistry 32 (1993) 2573–
584.
37] C. Rajput, R. Rutkaite, L. Swanson, I. Haq, J.A. Thomas, Chemistry 12 (2006)
611–4619.
38] D.D. Li, J.L. Tian, W. Gu, X. Liu, H.H. Zeng, S.P. Yan, J. Inorg. Biochem. 105 (2011)
94–901.
39] S.B. Bukhari, S. Memon, M. Mahroof-Tahir, M.I. Bhanger, Spectrochim. Acta
1A (2009) 1901–1906.
[
[
1] H.K. Liu, P.J. Sadler, Acc. Chem. Res. 44 (2011) 349–359.
2] U. Jungwirth, C.R. Kowol, B.K. Keppler, C.G. Hartinger, W. Berger, P. Heffeter,
Antioxid. Redox Signal. 15 (2011) 1085–1127.
[
[
[
[
[
2
[
3] S.L.H. Higgins, T.A. White, B.S.J. Winkel, K.J. Brewer, Inorg. Chem. 50 (2011)
4
4
63–470.
[
[
[
[
[
4] M. Cusumano, M.L. Di Pietro, A. Giannetto, Inorg. Chem. 45 (2006) 230–235.
5] K. Abdi, H. Hadadzadeh, M. Weil, M. Salimi, Polyhedron 31 (2012) 638–648.
6] C. Li, S.L. Liu, L.H. Guo, D.P. Chen, Electrochem. Commun. 7 (2005) 23–28.
7] L.H. Hurley, Nat. Rev. 2 (2002) 188–200.
8
7
40] F.V. Botelho, J.I. Alvarez-Leite, V.S. Lemos, A.M.C. Pimenta, H.D.R. Calado, T.
Matencio, C.T. Miranda, E.C. Pereira-Maia, J. Inorg. Biochem. 101 (2007) 935–
8] S. Tabassum, M. Zaki, M. Afzal, F. Arjmand, Dalton Trans. 42 (2013) 10029–
1
0041.
9
43.
[
9] Z.D. Matovic, E. Mrkalic, G. Bogdanovic, V. Kojic, A. Meetsma, R. Jelic, J. Inorg.
Biochem. 121 (2013) 134–144.
[
[
[
41] Q. Wang, Z.Y. Yang, G.F. Qi, D.D. Qin, Eur. J. Med. Chem. 44 (2009) 2425–2433.
42] T.R. Li, Z.Y. Yang, B.D. Wang, D.D. Qin, Eur. J. Med. Chem. 43 (2008) 1688–1695.
43] H. Wu, J. Yuan, Y. Bai, G. Pan, H.W.X. Shu, J. Photochem. Photobio. B: Biol. 107
[
[
10] S. Ambika, S. Arunachalam, R. Arun, K. Premkumar, RSC Adv. 3 (2013) 16456–
6468.
11] D. Dreher, A.F. Junod, J. Cancer 32 (1996) 30–38.
1
(
2012) 65–72.