Inspired research on the DNA binding ability of 4-aminoantiprine derived mixed ligand complexes

Article abstract of DOI:10.1016/j.inoche.2011.12.029

A novel 4-aminoantipyrine derived Schiff base and its four mixed ligand complexes have been synthesized and characterized. The binding properties of metal complexes with DNA have been investigated by electronic absorption spectra and viscosity measurement

Full text of DOI:10.1016/j.inoche.2011.12.029

Inorganic Chemistry Communications 17 (2012) 120–123
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Inorganic Chemistry Communications
journal homepage: www.elsevier.com/locate/inoche
Inspired research on the DNA binding ability of 4-aminoantiprine derived mixed
ligand complexes
⁎
Natarajan Raman , Sivasangu Sobha
Research Department of Chemistry, VHNSN College, Virudhunagar-626 001, India
a r t i c l e i n f o
a b s t r a c t
Article history:
A novel 4-aminoantipyrine derived Schiff base and its four mixed ligand complexes have been synthesized
and characterized. The binding properties of metal complexes with DNA have been investigated by electronic
absorption spectra and viscosity measurements showing that the complexes have the ability of interaction
with DNA by intercalative mode. The effect of the metal complexes on DNA was carried out by pUC19 DNA
agarose gel electrophoresis at 50 V for 2 h. The damage effect of the added ascorbic acid into the medium
is dependent on the free radicals produced from oxidation of ascorbic acid by molecular oxygen and this
damage is considered to be reduced by the metal complexes.
Received 24 October 2011
Accepted 16 December 2011
Available online 27 December 2011
Keywords:
Mixed ligand metal complexes
Schiff base
© 2011 Elsevier B.V. All rights reserved.
DNA binding
DNA damage
Transition metals have an important place within medicinal bio-
chemistry. Research has shown significant progress in utilization of
transition metal complexes as drugs to treat several human diseases
like carcinomas, lymphomas, infection control, anti-inflammatory, di-
abetes, and neurological disorders [1]. Among them, binding of small
molecules to DNA would be invaluable in the rational design of se-
quence specific DNA binding molecules for application in chemother-
apy and in the development of tools for biotechnology [2]. During
several years, tremendous interest has been aroused to explore the
potential applications of metal complexes as non-radioactive probes
of nucleic acid structure and as possible DNA cleaving agents [3].
In recent years, 4-aminoantipyrine transition metal complexes
and their derivatives have been extensively examined due to their
wide applications in various fields like biological, analytical and ther-
apeutical. Further, they have been investigated due to their diverse
biological properties as antifungal, antibacterial, analgesic, sedative,
antipyretic, anti-inflammatory agents [4] and DNA binding properties
[5]. However, to the best of our knowledge no attention was paid on
the interaction of DNA and Schiff base and its mixed ligand metal
complexes derived from 2-hydroxy benzilidene 4-aminoantipyrine
and alanine (Scheme 1). In this communication we describe the syn-
thesis, DNA binding and cleavage abilities of a novel series of 4-
aminoantipyrine based Schiff base and its mixed ligand complexes
with various metal ions.
elemental analyses, magnetic susceptibility, 1H NMR [The 1H NMR
spectra of the ligand and its [ZnL(phen)] complex are given in Fig.
S1 and Fig. S2 (Supplementary data)], IR, Mass, UV spectra and
molar conductivity measurements since no single crystals suitable
for X-ray determination could be isolated. All the instrumental details
are given in Supplementary data.
The mass spectrum of Schiff base ligand showed the molecular ion
peak at m/z 416 [M+1] (91%) abundance corresponding to
[C21H20N4O3K] ion. Also the spectrum exhibited peaks for the frag-
ments at m/z 323, 199, 93 and 77 corresponding to [C15H15N4O2K]
[M+1], [C11H11N4] [M+], [C6H5O] [M+] and [C6H5] [M+] with
0.5%, 0.9%, 0.4% and 0.3% abundances respectively. The spectra of
Cu(II), Ni(II), Co(II), and Zn(II) complexes showed molecular ion
peaks at m/z 619.34 [M+], 615.53 [M+1], 614.33 [M+] and
621.20 [M+] with 96%, 100%, 40% and 69% abundances respectively
that are equivalent to their molecular weights. The Cu(II) complex
gave a fragment ion peak with loss of metal and 1,10 phenanthroline
m/z 376. The m/z of all the fragments of ligand and its complexes con-
firm the stoichiometry of the complexes as [ML(phen)]. It was further
supported by the mass spectra of all the complexes. The observed
peaks were in good agreement with their formulae as expressed
from microanalytical data. The ESI–MS spectra of the ligand and its
[CuL(phen)] complex are given in Fig. S3 and Fig. S4 (Supplementary
data).
The Schiff base ligand and its mixed ligand complexes were pre-
pared by a typical procedure [6–13]. We got the likely composition
of complexes, [ML(phen)] where phen=phenanthroline, through
The binding behavior of the metal complexes to DNA helix is often
investigated using absorption spectral titration, followed by the
changes in absorbance and shift in wavelength. With increasing con-
centration of CT-DNA, the absorption bands of the complexes were af-
fected, resulting in the tendency of hypochromism and a slight red
shift was observed in all the complexes due to the intercalative binding
between DNA and metal complexes. Hyperchromic and hypochromic
⁎
Corresponding author. Tel.: +91 9245165958; fax: +91 4562281338.
E-mail address: drn_raman@yahoo.co.in (N. Raman).
1387-7003/$ – see front matter © 2011 Elsevier B.V. All rights reserved.
doi:10.1016/j.inoche.2011.12.029

N. Raman, S. Sobha / Inorganic Chemistry Communications 17 (2012) 120–123
121
Scheme 1. Schematic route for the synthesis of Schiff base ligand and its metal complexes.
effects are the spectral features of DNA concerning its double helix struc-
ture. This spectral change showed that the change of DNA in its confor-
mation and structure after the complex bound to DNA. Hypochromism
results from the contraction of DNA in the helix axis, as well as from
the change in conformation on DNA, while hyperchromism results
from the damage of the DNA double helix structure [14]. The absorption
spectra of DNA in the absence and presence of copper(II) and nickel(II)
complexes are given in Figs. 1 and 2 respectively.
The absorption spectra of the four metal(II) complexes have been
characterized by metal-to-ligand charge transfer (MLCT) transition in
the visible region. The absorption spectra of complexes exhibit lowest
energy bands at 428.5 nm for copper, 402.3 nm for nickel, 420.6 nm
for cobalt and 404.5 for zinc which are assigned to the metal-to-
ligand charge transfer (MLCT) transition. All the metal complexes
showed decrease in absorption intensity (hypochromism) with a
slight red shift which is due to the intercalative binding between
DNA and metal complexes.
The nature of binding of the complexes to the CT-DNA was further
investigated by viscometric studies. The relative specific viscosity of
DNA was determined by varying the concentration of the added
metal complexes. Measuring the viscosity of DNA is a classical tech-
nique used to analyze the DNA binding mode in solution. In the
Fig. 1. Absorption spectral changes on addition of CT DNA to the solution of [CuL(phen)]
(20 μM) in buffer pH=7.2 at 25 C in the presence of increasing amount of DNA
(0–140 μM). Arrow indicates the changes in absorbance upon increasing the DNA
concentration. Inset: plot of [DNA]/(εa −εf)×10−9 M2 cm versus [DNA]×10−5 M.

122
N. Raman, S. Sobha / Inorganic Chemistry Communications 17 (2012) 120–123
1
2
3
4
5
6
7
Form II
Form III
Form I
Fig. 4. Gel electrophoresis diagram showing the cleavage of pUC19 DNA (10 μM) by
Cu(II), Ni(II) and Zn(II) complexes (30 μM) in a buffer containing 50 mM Tris–HCl
and 50 mM NaCl in the presence of ascorbic acid (AH2, 10 μM) at 37 °C. Lane 1, DNA
control; lane 2, DNA+AH2; lane 3, DNA+[ligand(L)]+AH2; lane 4, DNA+AH2 +
[CuL(phen)]; lane 5, DNA+AH2 +[NiL(phen)]; lane 6, DNA+AH2 +[CoL(phen)];
lane 7, DNA+AH2 +[ZnL(phen)].
controlled by relaxation of super coiled circular form of pUC19 DNA
into nicked circular form and linear form. When circular plasmid
DNA is conducted by electrophoresis, the fastest migration will be ob-
served for the supercoiled form (Form I). If one strand is cleaved, the
supercoils will relax to produce a slowly moving open circular from
(Form II). If both strands are cleaved, a linear form (Form III) will be
generated that migrates in between [17,18]. The ability of the com-
plexes in affecting DNA cleavage has been investigated by gel electro-
phoresis using super coiled pUC19 DNA in 5 mM Tris–HCl/50 mM
NaCl buffer solution (pH 7.2). All the complexes are found to exhibit
nuclease activity. Fig. 4 shows the result of gel electrophoretic separa-
tions of plasmid pUC19 DNA induced by an addition of metal(II) com-
plexes in the presence of AH2 (ascorbic acid). Under the same
conditions, free AH2 produced no cleavage of pUC19 DNA. When
pUC19 DNA was allowed to interact with mixture of metal complexes
and in presence of ascorbic acid, the mobility of the band was found
to increase slightly as shown in Fig. 4. These phenomena imply that
Cu(II), Co(II), Ni(II) and Zn(II) complexes damage more to plasmid
pUC19 DNA in the presence of AH2.
In summary, a series of the mixed ligand metal complexes with
Schiff base ligand derived from 2-hydroxy-benzylidene-4-aminoanti-
pyrine and alanine was synthesized and characterized. The binding
behavior of metal complexes with DNA was studied by UV spectra,
viscosity and gel retardation assay under physiological conditions.
All the experimental evidences indicate that these four complexes
can strongly bind to CT DNA via an intercalation mechanism. All the
metal complexes show enhanced DNA cleavage activity of pUC19
DNA in the presence of ascorbic acid. The results show that mixed li-
gand metal complexes exhibit more damage to pUC19 DNA in pres-
ence of ascorbic acid. Results obtained from our present work
would be very useful to understand the mechanism of interactions
of the small molecules binding to DNA and helpful in the develop-
ment of their potential applications in biological, pharmaceutical
and physiological fields in future.
Fig. 2. Absorption spectral changes on addition of CT DNA to the solution of [NiL(phen)]
(20 μM) in buffer pH=7.2 at 25 C in the presence of increasing amount of DNA
(0–140 μM). Arrow indicates the changes in absorbance upon increasing the DNA
concentration. Inset: plot of [DNA]/(εa −εf)×10− 9 M2 cm versus [DNA]×10−5 M.
absence of crystallographic structural data, hydrodynamic methods
that are sensitive to DNA length change are regarded as the least am-
biguous and the most critical tests of binding in solution. A classical
intercalation model results in the lengthening 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 and/
or non-classical intercalation of ligand may bend (or kink) DNA
helix, resulting in the decrease of its effective length and concomitantly
its viscosity [15]. The plots of (η/ηo)1/3 vs. [Complex]/[DNA]=R
(where η and ηo are the relative viscosities of DNA in the presence
and absence of complex respectively) give a measure of the viscosity
changes. The effects of all the complexes on the viscosity of CT DNA
are shown in Fig. 3.
A significant increase in the viscosity of DNA on addition of com-
plex results due to the intercalation which leads to the separation
among the DNA bases to the increase in the effective size in DNA
which could be the reason for the increase in the viscosity [16].
Gel retardation assay is a useful tool for identifying metal com-
plexes which interact with DNA and mediate cell functions such as
gene expression, DNA repair and DNA packaging. DNA cleavage is
Acknowledgments
The authors express their sincere thanks to the College Managing
Board, Principal and Head of the Department of Chemistry, VHNSN
College, Virudhunagar, India for providing necessary research facili-
ties. NR thanks University Grants Commission (UGC), New Delhi for
financial support.
Appendix A. Supplementary data
Supplementary data to this article can be found online at doi:10.
1016/j.inoche.2011.12.029.
References
Fig. 3. Plot of relative viscosity (η/ηo)1/3 vs. [Complex]/[DNA]=R. Effect of increasing
amounts of [CuL(phen)] (▲), [NiL(phen)](■), [ZnL(phen)] (♦), and [ZnL(phen)] (•),
on the viscosity of DNA. [Complex]=0–350 μM, [DNA]=50 μM.
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[10] Anal.Calc. for C33H27N6O3Cu: C, 64.0; H, 4.4; N, 13.5; Cu, 10.2%. Found: C, 63.7; H,
4.3; N, 13.1; Cu, 9.8%. FT-IR (KBr), cm−1: υ(C N): 1604, υ(HC N): 1564, υasy
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was refluxed for ca. 3 h. The solid product formed (2-hydroxy-benzylidene-4-
aminoantipyrine) was filtered and recrystallized from ethanol.
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The alanine (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 solution. Then, to this solution was added
drop-wise an ethanolic solution of 2-hydroxy-benzylidene-4-aminoantipyrine
(0.01 mol) and the resultant mixture was refluxed for ca. 6 h. The dark yellow
colored crystalline product (Schiff base, L) formed was filtered and recrystallized
from ethanol. Yield: 75%.
[12] Anal.Calc. for C33H27N6O3Co: C, 64.4; H, 4.4; N, 13.6; Co, 9.5%. Found: C, 64.2; H,
4.4; N, 13.1; Co, 9.1%. FT-IR (KBr), cm−1: υ(C N): 1636, υ(HC N): 1599, υasy
(COO)−: 1444, υsy (COO)−: 1317, υ(M O): 486, υ(M N): 414, Molar conduc-
tance×10−3, (S cm2 mol−1): 21.83; μeff (BM): 4.72. λmax in (DMSO), cm−1
:
14,070, 16,738 and 21,147 cm−1. ESI MS: m/z 614.33 (40%) [M+].
[13] Anal.Calc. for C33H27N6O3Zn: C, 63.8; H, 4.3; N, 13.5; Zn, 10.5%. Found: C, 63.7; H,
4.3; N, 13.1; Zn, 10.2%. FT-IR (KBr), cm−1: υ(C N):1604, υ(HC N): 1564, υasy
(COO)−: 1442, υsy (COO)−: 1317, υ(M O): 510, υ(M N): 420. (CDCl3, 300 MHz,
δ, ppm): 6.91–7.03 (m, 9H, Ar–H), 7.26–7.58. (m, 8H, (phen) Ar–H), 8.61 (s, 1H,
–HC N), 2.20 (s, 3H, Ar–C–CH3), 1.80 (s, 3H, Ar–N–CH3), 2.62 (d, 3H, alanine–
CH3) 1.64 (q, 1H, alanine–CH). Molar conductance×10−3 (S cm2 mol−1):
,
19.40. μeff (BM): 1.84. λmax in (DMSO), cm−1: 32,679, 35,842 and 37,878 cm−1
.
ESI MS: m/z 621.2 (6 9%) [M+].
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[8] Anal.Calc. for C21H20N4O3K: C, 60.7; H, 4.8; N, 13.4%. Found: C, 60.2; H, 4.7; N,
13.1%. FT-IR (KBr), cm−1
: υ(OH): 3480, υ(C N): 1653, υ(HC N): 1614, υasy
(COO)−: 1549, υsy (COO)−: 1447, 1H NMR (CDCl3, 300 MHz, δ, ppm): 6.91–7.46
(m, 9H, Ar–H), 9.80 (s, 1H, –HC N), 5.42 (s, 1H, –OH), 2.40 (s, 3H, Ar–C–CH3),
2.18 (s, 3H, Ar–N–CH3), 3.69 (d, 3H, alanine–CH3) 1.24 (q, 1H, alanine–CH).
λmax in (DMSO), cm−1
:
37,878, 36,363 and 31,250 cm−1
.
ESI MS: m/z
416(91%) [M+1].
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