A novel bioactive tyramine derived Schiff base and its transition metal complexes as selective DNA binding agents

Article abstract of DOI:10.1016/j.saa.2010.12.056

A novel tyramine derived Schiff base, 3-4-dimethoxybenzylidene-4- aminoantipyrinyl-4-aminoethylphenol(L) and a series of its transition metal complexes of the type, ML₂Cl₂ where, M = Cu(II), Ni(II), Co(II) and Zn(II) have been design

Full text of DOI:10.1016/j.saa.2010.12.056

Spectrochimica Acta Part A 78 (2011) 888–898
Contents lists available at ScienceDirect
Spectrochimica Acta Part A: Molecular and
Biomolecular Spectroscopy
journal homepage: www.elsevier.com/locate/saa
A novel bioactive tyramine derived Schiff base and its transition metal complexes
as selective DNA binding agents
N. Ramana,∗, S. Sobhaa, A. Thamaraichelvanb
a Research Department of Chemistry, VHNSN College, Virudhunagar, Tamil Nadu 626 001, India
b PG and Research Department of Chemistry, Thiagarajar College, Madurai 625 009, India
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 21 July 2010
Received in revised form 6 December 2010
Accepted 14 December 2010
A
novel tyramine derived Schiff base, 3-4-dimethoxybenzylidene-4-aminoantipyrinyl-4-
aminoethylphenol(L) and a series of its transition metal complexes of the type, ML2Cl2 where,
M = Cu(II), Ni(II), Co(II) and Zn(II) have been designed and synthesized. Their structural features and
other properties were deduced from the elemental analysis, magnetic susceptibility and molar conduc-
tivity as well as from mass, IR, UV–vis, 1H NMR and EPR spectral studies. The binding properties of these
complexes with calf thymus DNA (CT-DNA) were investigated using electronic absorption spectroscopy,
viscosity measurement, cyclic voltammetry and molecular docking analysis. The results reveal that the
metal(II) complexes interact with DNA through minor groove binding. The interaction has also been
investigated by gel electrophoresis. Interestingly, it was found that all the complexes could cleave the
circular plasmid pUC19 super coiled (SC) DNA efficiently in the presence of AH2 (ascorbic acid). The
complexes showed enhanced antifungal and antibacterial activities compared to the free ligand.
© 2010 Elsevier B.V. All rights reserved.
Keywords:
Metal complexes
Schiff base
DNA binding
Molecular docking
DNA cleavage
Biological activity
1. Introduction
plasmid DNA efficiently [12,13]. However, to the best of our knowl-
edge, no report has yet appeared on the DNA interaction of Schiff
Medical inorganic chemistry has exploited the unique prop-
erties of metal ions for the design of new drugs [1]. This has,
for instance, demonstrated through numerous biological exper-
iments that DNA is the primary intracellular target for many
anticancer drugs, carcinogens and viruses [2–4]. In this regard,
chemical modification of nucleic acid by transition metal com-
plexes is of paramount importance for designing chemotherapeutic
drugs which regulate gene expression and tools for molecular biol-
ogy [5]. Transition metal complexes have largely been employed for
these purposes not only because of their unique spectral and elec-
trochemical signatures but also due to the fact that the DNA binding
and cleaving ability of metal complexes could be tuned by chang-
ing the ligand environment. These studies are also important for the
development of new probes for nucleic acids and determination of
the mechanism of metal ion toxicity [6,7].
Tyramine is widely used as a pharmacological tool to evaluate
the role of the sympathetic nervous system on human physiol-
ogy and pathology [8]. Schiff base of 4-aminoantipyrine and its
complexes exhibit a great variety of biological activities such as
antitumor, fungicidal, bactericidal, anti-inflammatory and antivi-
ral activities [9–11]. The remarkable biological potencies of these
complexes are due to their ability to bind to cellular DNA and cleave
base metal complexes of 4-aminoantipyrine derivatives contain-
ing the tyramine moiety. Currently, although much attention has
been paid to the interaction of DNA with complexes of Ru(II) [14],
the other metal ion complexes have attracted much less attention
than Ru(II) complexes. This fact prompted us to design a novel
DNA binding and cleaving reagent, 3-4-dimethoxybenzylidene-
4-aminoantipyrinyl-4-aminoethylphenol (L) and its transition
metal(II) complexes.
In this paper, we report the synthesis and characterization of 1:2
type [ML2Cl2] complexes where, M = Cu(II), Ni(II), Co(II) or Zn(II)
and present their DNA binding and cleavage abilities. A molecular
modeling study was performed with special reference to docking to
confirm the minor groove binding mode of these complexes to AT
base pairs in DNA. Biological activities of the said complexes have
also been evaluated. These studies assume significance as they fur-
ther the comprehension of binding of transition metal complexes
to DNA and for developing the next generation of DNA binding and
anticancer drugs.
2. Experimental
2.1. Materials and methods
All chemicals used in the present work viz., 4-aminoantipyrine,
3,4-dimethoxy benzaldehyde, tyramine, and metal(II) chlorides
were of analytical reagent grade (produced by Merck, Germany).
∗
Corresponding author. Tel.: +91 9245 165958 (mobile); fax: +91 4562 281338.
E-mail address: drn raman@yahoo.co.in (N. Raman).
1386-1425/$ – see front matter © 2010 Elsevier B.V. All rights reserved.
doi:10.1016/j.saa.2010.12.056

N. Raman et al. / Spectrochimica Acta Part A 78 (2011) 888–898
889
Commercial solvents were distilled and then used for the prepa-
ration of ligand and its complexes. CT-DNA and pUC19 DNA were
purchased from Bangalore Genei (India). Microanalyses (C, H and
N) were performed in Carlo Erba 1108, Heraeus. Molar conduc-
tivities in DMSO (10−3 M) solution at room temperature were
measured using Deep Vision Model-601 digital direct reading
deluxe conductivity meter. Magnetic susceptibility measurements
of the complexes were carried out by Gouy balance using copper
sulphate pentahydrate as calibrant. IR spectra were recorded as
KBr pellets on Perkin-Elmer FT-IR 783 Spectrophotometer. NMR
spectra were recorded on a Bruker Avance Dry 300 FT NMR Spec-
trometer in CDCl3 with TMS as the internal reference. FAB-MS
Spectra were recorded with a VGZAB-HS Spectrometer at room
temperature in a 3-nitrobenzylalcohol matrix. Electron paramag-
netic resonance spectra of the mixed ligand complexes of copper(II)
were recorded on a Varian E 112 EPR Spectrometer in DMSO
solution both at room temperature (300 K) and at liquid nitro-
gen temperature (77 K) using TCNE (tetracyanoethylene) as the
g-marker. The absorption spectra were recorded using Shimadzu
model UV-1601 Spectrophotometer at room temperature.
Scheme 1. Synthesis of 3-4-dimethoxybenzylidene-4-aminoantipyrine.
a magnetic stirrer at room temperature. Then the solution was
reduced to one-third on a water bath. The solid complex precip-
itated was filtered off, washed thoroughly with ethanol and dried
in vacuo.
2.2. Synthesis
2.2.1. Synthesis of 3-4-dimethoxybenzylidene-4-aminoantipyrine
An ethanolic solution of (40 mL) 4-aminoantipyrine
(4.06 g, 0.02 mol) was added to an ethanolic solution of 3,4-
dimethoxybenzaldehyde (3.14 g, 0.02 mol). The resultant mixture
was refluxed for ca. 3 h. The solid product formed was filtered and
recrystallized from ethanol (Scheme 1).
2.3. DNA binding experiments
2.3.1. Absorption spectroscopic studies
The interactions between metal complexes and DNA were
studied 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 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 [15]. The concentration of DNA was measured using its
extinction coefficient at 260 nm (6600 M−1 cm−1). Stock solutions
were stored at 4 ◦C and used no more than 4 days. Doubly dis-
tilled water was used to prepare solutions. Concentrated stock
solutions of the complexes were prepared by dissolving the com-
plexes in DMSO and diluting suitably with the corresponding
buffer to the required concentration for all of the experiments.
The data were then fitted to Eq. (1) to obtain the intrinsic bind-
ing constant (Kb) values for interaction of the complexes with
2.2.2. Synthesis of Schiff base (L)
An ethanolic solution of 3-4-dimethoxybenzylidene-4-
aminoantiyrine (3.51 g, 0.01 mol) was added to an ethanolic
solution of tyramine (1.37 g, 0.01 mol) and the resultant mixture
was refluxed for ca. 6 h after the addition of anhydrous potassium
carbonate. The potassium carbonate was filtered off from the reac-
tion mixture and the solvent was evaporated. The pale orange solid
separated was filtered and recrystallized from ethanol (Scheme 2).
2.2.3. Synthesis of complexes
An ethanolic solution of metal(II) chlorides (1 mmol) was stirred
with an ethanol solution of the Schiff base (2 mmol) for ca. 2 h on
Scheme 2. Synthesis of 3-4-dimethoxybenzylidene-4-aminoantipyrinyl-4-aminoethyl-phenol (L).

890
N. Raman et al. / Spectrochimica Acta Part A 78 (2011) 888–898
DNA.
the entire DNA molecule. The docked poses were generated by the
exhaustive search and optimization step. FRED selects the single
best pose from the set of candidates. This pose is then scored and
the score is used to rank ligands in the output hit list. The consensus
structure step allows multiple scoring functions to vote for the best
docked structure in a rank-by-vote approach.
[DNA]
εa − εf
[DNA]
εb − εf
1
=
+
(1)
K (ε − ε )
[
]
b
b
f
where εa, εf, and εb are the apparent, free and bound metal com-
plex extinction coefficients, respectively. A plot of [DNA]/(εb − εf)
versus [DNA], gave
a
slope of 1/(εb − εf) and
a y-intercept
equal to [Kb/(εb − εf)]−1, Kb is the ratio of the slope to the y-
2.6. Antimicrobial activity studies
intercept.
The in vitro antibacterial and antifungal activities of the ligand
and its complexes were tested against the bacteria Staphylococcus
aureus, Pseudomonas aeruginosa, Escherichia coli, Staphylococcus epi-
dermis and Klebsiella pneumoniae and the fungi Aspergillus niger,
Fusarium solani, Culvularia lunata, Rhizoctonia bataicola and Can-
dida albicans. For the detection of the biocidal activities, the filter
paper disc agar diffusion method was used. Pure Streptomycin
and Nystatin were used separately as standards for antibacterial
and antifungal activity tests respectively. The stock solution was
prepared by dissolving the 1 mg of sample in 10 mL of DMSO to
give the concentration of 100 ␮g/mL and the solution was seri-
ally diluted in order to find the minimum inhibitory concentration
(MIC) value. The suitable medium (nutrient agar for bacteria and
potato dextrose agar medium for fungi) was inoculated with the
test organisms. The standard solutions of Streptomycin and Nys-
tatin were prepared in DMSO to give concentration of 100 ␮g/mL.
The sterilized blank paper discs of 6 mm diameter were impreg-
nated with tested compounds and placed on the surface of the
agar plates previously spread with 0.1 mL of overnight culture of
microorganisms. The incubation was carried out 24 h for bacteria
and 72 h for fungi at 30 ◦C. During the period, the test solution
diffused and the growth of the inoculated microorganisms was
affected. The inhibition zone was developed, at which the concen-
tration was noted.
2.3.2. Electrochemical methods
Cyclic voltammetry were performed on a CHI 620C electro-
chemical 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.
2.3.3. Viscosity measurements
Viscosity experiments 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 sonication in order minimize complex-
ities arising from CT DNA flexibility [16]. Flow time was measured
with a digital stopwatch three times for each sample and an average
flow time was calculated. Data were presented as (Á/Áo)1/3 versus
the ratio of metal(II) complexes to DNA, where Á is the viscosity of
CT DNA solution in the presence of complex, and Áo is the viscos-
ity of CT DNA solution in the absence of complex. Viscosity values
were calculated after correcting the flow time of buffer alone (t0),
Á = (t − t0)/t0 [17].
2.4. pUC19 DNA cleavage study
The oxidative cleavage experiment was performed using super
coiled pUC19 plasmid DNA Form I (2 ␮L, 10 ␮M) in Tris–HCl buffer
(50 mM) with 50 mM NaCl (pH 7.2) which was treated with the
metal complex (30 ␮M) and ascorbic acid (10 ␮M) followed by
dilution with the Tris–HCl buffer to a total volume of 20 ␮L. The
samples were incubated for 1 h at 37 ◦C. A loading buffer con-
taining 25% bromophenol blue, 0.35% xylene cyanol, 30% glycerol
(3 ␮L) was added and electrophoresis performed at 40 V for each
hour in Tris–acetate–EDTA (TAE) buffer using 1% agarose gel con-
taining 1.0 ␮g/mL ethidium bromide. The cleavage of DNA was
monitored using agarose gel electrophoresis. The gel was visual-
ized by photographing the fluorescence of intercalated ethidium
bromide under a UV illuminator. The cleavage efficiency was mea-
sured by determining the ability of the complex to convert the super
coiled (SC) DNA to nicked circular form (NC) and linear form. Inhibi-
tion reaction was also carried out by prior incubation of the pUC19
DNA with DMSO (4 ␮L) (hydroxyl radical scavenger) and sodium
azide (100 ␮M) (singlet oxygen scavenger).
3. Results and discussion
The Schiff base ligand, L and its Cu(II), Ni(II),Co(II) and Zn(II)
complexes were synthesized and characterized by spectral and ele-
mental analysis data. The complexes were found to be air stable. The
ligand was soluble in common organic solvents but their complexes
were soluble only in CHCl3, DMF and DMSO.
3.1. Elemental analysis and molar conductivity measurements
The results of elemental analysis for the metal complexes were
in good agreement with the calculated values (Table 1) showing
that the complexes have 1:2 metal–ligand stoichiometry of the type
ML2Cl2 wherein L acts as a bidentate ligand.
The formation of these complexes may proceed according to the
equation given below:
MCl2·nH2O + 2L → [ML2Cl2] + nH2O
2.5. Molecular modeling studies
where M = Cu(II), Ni(II), Co(II) and Zn(II).
The metal(II) complexes were dissolved in DMSO and the
molar conductivities of 10−3 M of their solution at room
temperature were measured. The lower conductance values
(18.25–21.47 ꢀ−1 cm−2 mol−1) of the complexes support their
non-electrolytic nature. It also indicates that the chloride anions
bind to the metal ions as ligands and do not ionize.
The interaction of the metal complexes with DNA was also stud-
ied by molecular modeling with special reference to docking. All
calculations were performed in Open Eye with Fast Rigid Exhaus-
tive Docking using the FRED docking software package. The main
function employed for the calculation program was Chemguass
2. Prior to docking, the structure of the metal complexes was
constructed and geometry optimized by MM2 force field. The crys-
tal structure of the complex of netropsin with B-DNA dodecamer
d(CGCGAATTCGCG)2 (NDB code GDLB05), was downloaded from
Protein Data Bank. During the docking analysis, the binding site
of the netropsin drug in the minor groove of the above complex
was assigned as the binding site for our metal complexes across
3.2. Mass spectra
The mass spectrum of Schiff base ligand showed peak at m/z 471
corresponding to [C28H30N4O3] ion. Also the spectrum exhibited
peaks for the fragments at m/z 200, 150, 122 and 77 corresponding

N. Raman et al. / Spectrochimica Acta Part A 78 (2011) 888–898
891
Table 1
Analytical and physical data of Schiff base ligand and its complexes.
a
Compound
Yield (%)
Color
Found (calc)%
M
Formula
(∧m
)
ꢁeff (BM)
C
H
N
[C28H30N4O3]
[CuC56H60N8O6Cl2]
[NiC56H60N8O6Cl2]
[CoC56H60N8O6Cl2]
[ZnC56H60N8O6Cl2]
79
67
73
61
85
Pale orange
Dark green
Pale blue
Pale pink
Yellow
–
71.7 (72.2)
62.5 (62.5)
62.4 (62.8)
62.1 (62.4)
62.3 (62.4)
6.4 (6.5)
5.4 (5.6)
5.6 (5.6)
5.2 (5.6)
5.4 (5.6)
11.9 (12.3)
10.2 (10.4)
10.2 (10.4)
10.1 (10.4)
10.2 (10.4)
471
1075
1071
1071
1077
–
–
5.9 (6.1)
5.2 (5.4)
5.0 (5.0)
5.9 (6.0)
18.25
21.47
19.67
18.76
1.86
3.21
4.88
Diamagnetic
a
The unit of the molar conductance is ꢀ−1 mol−1 cm2.
to [C11H12N4]+, [C9H10O2]+, [C8H10O]+ and [C6H5]+ respectively.
The mass fragmentation pattern of the ligand is shown in Scheme 3.
The spectra of Cu(II), Co(II), Ni(II) and Zn(II) complexes showed a
molecular ion peak [M+] at m/z 1075, 1071, 1071 and 1077 respec-
tively that are equivalent to their molecular weights. The Cu(II)
complex gave a fragment ion peak with loss of 2 chlorine atoms
at m/z 1005. All these fragments leading to the formation of the
species [ML2]+ which further undergoes de-metallation to yield the
species [L+] giving the fragment ion peak at m/z 471.
3.5. Electronic spectra
The geometry of the metal complexes has been deduced from
electronic spectra and magnetic data of the complexes. The elec-
tronic spectra of the complexes were recorded in DMSO solution.
All the complexes show the high energy absorption band in the
region 27,248–33,112 cm−1. This transition may be attributed to
the charge transfer band. The electronic spectrum of copper(II)
complex displays the d–d transition band in the region 13,606 cm−1
[19,20] which is due to 2Eg → T2g transition. This d–d band transi-
2
tion band strongly favors a distorted octahedral geometry around
the metal ion. The absorption spectrum of nickel(II) complex dis-
plays three d–d bands at 14,535, 15,268 and 23,809 cm−1. These
3.3. IR spectra
bands correspond to 3A2g(F) → T2g(F) (ꢂ1), 3A2g(F) → T1g(F) (ꢂ2)
3
3
The coordination mode and sites of the ligand to the metal
ions were investigated by comparing the infrared spectra of the
free ligand with its metal complexes. The characteristic pheno-
lic ꢂ(OH) mode due to the hydroxyl group of tyramine moiety,
present in the Schiff base ligand was observed at 3404 cm−1. The
appearance of this band in all the complexes indicates that phe-
nolic ꢂ(OH) group is free from complexation. In the IR spectrum
of the Schiff base ligand the band observed at 1614 cm−1 was
shifted to lower frequency by 21–35 cm−1 on complexation, sug-
gesting the coordination of the azomethine nitrogen. In addition,
the ligand also revealed a band at 1656 cm−1 due to the ꢂ(C N)
vibration, originating from the condensation of amino group of
tyramine and 3,4-dimethoxybenzalidene-4-aminoantipyrine, was
shifted by 23–31 cm−1 on complexation. This is further substan-
tiated by the presence of new bands, found in the spectra of the
complexes in the regions 503–522 cm−1 and 439–445 cm−1 which
are assigned to ꢂ(M–N) (due to tyramine moiety) and ꢂ(M–N)
(due to 3,4-dimethoxy benzalidene-4-aminoantipyrine moiety)
stretching vibrations. Furthermore, the IR spectra of the all com-
plexes showed another band at 345–368 cm−1, which may be
due to ꢂ(M–Cl) [18] vibrations. Therefore, from the IR spectra, it
is concluded that the Schiff base behaves as a bidentate ligand
coordinated to the metal ions via the (HC N), (C N) and (M–Cl)
groups.
and 3A2g(F) → T1g(P) (ꢂ3) transitions respectively, being char-
3
acteristic of an octahedral geometry. This geometry is further
supported by its magnetic susceptibility value (3.21 BM). The
electronic spectrum of cobalt(II) complex displays three d–d tran-
sition bands in the region 15,037, 16,529 and 23,752 cm−1 which
4
4
4
4
are assigned to T1g(F) → T2g(F) (ꢂ1), T1g(F) → A2g(F) (ꢂ2), and
4
4T1g(F) → T2g(P) (ꢂ3) transitions respectively. This indicates that
the complex of Co(II) is six coordinate and probably an octahedral
geometry, which is also supported by its magnetic susceptibility
value (4.88 BM). The complex of Zn(II) is diamagnetic. According to
the empirical formula, an octahedral geometry is proposed for this
complex.
3.6. Electron paramagnetic resonance spectra
The EPR spectrum of copper complex provides information
which is important in studying the metal ion environment. The
EPR spectra were recorded in DMSO at LNT and at RT. The spec-
trum of the copper complex at RT shows one intense absorption
band in the high field and is isotropic due to the tumbling motion of
the molecules. However, this complex at LNT shows well resolved
peaks with low field region. The copper complex exhibits the g
||
value of 2.263 and g value of 2.064. These values indicate that the
⊥
Cu(II) lies predominantly in the dx2−y2 orbital, as was evident from
the value of the exchange interaction term G, estimated from the
expression:
3.4. 1H NMR spectra
The 1H NMR spectra of the Schiff base and its zinc complex were
recorded at room temperature in CDCl3. 1H NMR spectrum of the
ligand shows a singlet at 5.3 ı which is attributed to the phenolic
–OH group of tyramine moiety present in the Schiff base ligand.
The presence of this peak noted for the zinc complex confirms the
–OH proton free from complexation. The ligand shows the follow-
ing signals: phenyl multiplet at 6.3–7.4 ı, –CH N at 9.6 ı, –OCH3
at 3.9 ı, –C–CH3 at 3.1 ı, N–CH3 at 2.5 ı, and –CH2 at 1.6–2.1 ı.
The azomethine proton (–CH N) signal in the spectrum of the zinc
complex is shifted down field compared to the free ligand, suggest-
ing deshielding of azomethine group due to the coordination with
metal ion. There is no appreciable change in all other signals of the
complex.
g
|| − 2
G =
g⊥ − 2
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 value
for the exchange interaction parameter for the copper complex
(G = 4.19) suggests that the local tetragonal axes are aligned parallel
or slightly misaligned and the unpaired electron is present in the
d
x2−y2 orbital. This result also indicates that the exchange coupling
effects are not operative in the present complex [21].
Based on the above spectral and analytical data, the proposed
geometry of the metal(II) complexes is given in Fig. 1.

892
N. Raman et al. / Spectrochimica Acta Part A 78 (2011) 888–898
Scheme 3. Mass fragment pattern of Schiff base.
3.7. DNA binding experiments
(bathochromism), due to the strong staking interaction between
aromatic chromophore of the complex and the base pairs of
DNA. The absorption spectra of the Cu(II) and Ni(II) complexes
in the absence and presence of calf thymus DNA are given in
Figs. 2 and 3 respectively. The absorption spectrum of copper
complex showed an intensive absorption band at 318.4 nm and
3.7.1. Absorption titration
Absorption titration can monitor the interaction of metal com-
plexes and DNA. In general, complex bound to DNA through
intercalation usually results in hypochromism and red shift

N. Raman et al. / Spectrochimica Acta Part A 78 (2011) 888–898
893
Fig. 3. Absorption spectra of [NiC56H60N8O6Cl2] in buffer pH = 7.2 at 25 ◦C in the
presence of increasing amount of DNA. Arrows indicate the changes in absorbance
upon increasing the DNA concentration. Inset: plot of [DNA]/(εa − εf) versus [DNA].
269.8 nm and a characteristic absorption band at 397.2 nm in
5 mM Tris–HCl, 50 mM NaCl, at pH 7.2 buffer solution respec-
tively. The latter can be attributed to MLCT band, further the
intense absorption band with maxima of 305, 282.5 nm for
Ni(II), 320.2, 272 nm for Co(II) and 381 nm for Zn(II) complexes
was attributed to ␲–␲* transition. On increasing the concen-
tration of CT DNA resulted in the minor bathochromic shift
in the range ∼1–3 nm and significant hypochromicity lying in
the range ∼18–25% of all the complexes indicating appreciable
groove/surface binding propensity of the complexes to the CT
DNA. The intrinsic binding constants for Cu(II), Ni(II), Co(II) and
Zn(II) complexes are found to be 6.7 × 105, 5.3 × 105, 1.1 × 105
and 5.5 × 105 M−1 respectively. These values are comparable
to the significant DNA groove binding complexes [Fe2(␮-
O)(L-his)2(phen)2](ClO4)2 (Kb = 1.46 × 105 M−1) and [Fe2(␮-O)(L-
his)2(dpq)2](ClO4)2 (Kb = 4.55 × 105 M−1), where his = histidine,
(phen) = phenanthroline and dpq = dipyridoquinoxaline [22]. The
small binding constant value also indicates of more surface
aggregation and/or groove binding than any interaction to the
DNA base pairs. In order to compare the binding strength of
the complexes with CT DNA, the intrinsic binding constants
(Kb) are obtained by monitoring the changes in the absorbance
for the complexes with increasing concentration of DNA. Kb is
obtained from the ratio of slope to the intercept from the plot of
[DNA]/(εa − εf) versus [DNA]. The (Kb) values are shown in Table 2.
Fig. 1. Proposed structure of the metal complexes M = Cu(II), Ni(II), Co(II) and Zn(II).
Fig. 2. Absorption spectrum of [CuC56H60N8O6Cl2] in buffer pH = 7.2 at 25 ◦C in the
presence of increasing amount of DNA. Arrows indicate the changes in absorbance
upon increasing the DNA concentration. Inset: plot of [DNA]/(εa − εf) versus [DNA].
Table 2
Electronic absorption spectral properties of Cu(II), Ni(II), Co(II) and Zn(II) complexes.
b
Compound
ꢃmax
ꢄꢃ/nm
H%a
Kb (×105 mol/L)
Free
Bound
397.2
318.8
269.8
399.2
320.8
272
2.0
2.0
2.2
25
15
18
[CuC56H60N8O6Cl2]
[NiC56H60N8O6Cl2]
6.7
320.2
274.2
321.4
275.2
1.2
1.0
24
21
5.36
305
282.5
306.2
284.5
1.2
2.0
19
22
[CoC56H60N8O6Cl2]
[ZnC56H60N8O6Cl2]
1.1
5.5
381.9
384.5
2.6
24
a
H% = [Afree − Abound)/Afree] × 100%.
b
Kb = intrinsic DNA binding constant determined from the UV–vis absorption spectral titration.

894
N. Raman et al. / Spectrochimica Acta Part A 78 (2011) 888–898
Fig. 4. Cyclic voltammogram of [CuC56H60N8O6Cl2] in buffer pH = 7.2 at 25 ◦C in the
presence of increasing amount of DNA. Arrows indicate the changes in voltammetric
currents upon increasing the DNA concentration.
Fig. 5. Cyclic voltammogram of [NiC56H60N8O6Cl2] in buffer pH = 7.2 at 25 ◦C in the
presence of increasing amount of DNA. Arrows indicate the changes in voltammetric
currents upon increasing the DNA concentration.
3.7.2. Electrochemical studies
The application of cyclic voltammetry to the studies of com-
plexes bound to DNA provides a useful complement to the
previously used methods of investigation, such as UV–vis spec-
troscopy and viscosity measurements. The cyclic voltammograms
of [CuC56H60N8O6Cl2] and [NiC56H60N8O6Cl2] complexes in the
absence and in presence of varying amount of DNA are shown
in Figs. 4 and 5 respectively. In the absence of CT DNA, the
first redox cathodic peak appears at 0.456 V for Cu(III) → Cu(II)
[Epa = 0.422 V, Epc = 0.456 V, ꢄEp = 0.034 and E1/2 = 0.44 V] and in
the second redox couple, the cathodic peak appears at −0.122 V
for Cu(II) → Cu(I) [Epa = 0.056 V, Epc = −0.122 V, ꢄEp = 0.178 V and
E1/2 = −0.033 V]. The ipa/ipc ratios of these two redox couples
are 0.78 and 1.0 respectively which indicate that the reac-
tion of the complex on the glassy carbon electrode surface is
quasi-reversible redox process. The incremental addition of CT
DNA to the complexes reveals that the redox couples cause a
less negative shift in E1/2 and decrease of ꢄEp (Table 3). The
ipa/ipc values also decrease in the presence of DNA. Bard has
reported [23] E1/2, shifted to more positive value the inter-
action mode was intercalative binding. In contrast, the slight
decrease in peak potential and voltammetric currents were
occurred and this can be attributed to diffusion of the metal
complexes, bound to the large slowly diffusing DNA molecule.
In nickel complex, in the absence of CT DNA, the first redox
cathodic peak appears at −0.369 V for Ni(III) → Ni(II) [Epa = 0.745 V,
Epc = −0.369 V, ꢄEp = 1.114 V and E1/2 = 0.19 V] and second redox
couple cathodic peak appears at −0.0.955 V for Ni(II) → Ni(I)
[Epa = 0.264 V, Epc = −0.955 V, ꢄEp = 1.219 V and E1/2 = 0.345 V]. In
these two redox couples, the ratio of ipa/ipc is approximately unity
which indicates that the reaction of the complex on the glassy
carbon electrode surface is quasi-reversible redox process. The
incremental addition of CT DNA at the same concentration of the
complex causes considerable decrease in the voltammetric cur-
rents. In addition, the peak potentials, Epc and Epa as well as E1/2
had a shift to less negative potential. The drop of the voltammetric
currents in the presence of CT DNA can be attributed to diffusion
of the metal complexes bound to the large slowly diffusing DNA
molecule [24].
For Co(III) → Co(II) the redox couple cathodic peak appears at
−0.152 V in the absence of CT DNA (Epa = 0.602 V, Epc = −0.152 V,
ꢄEp = 0.754 V and E1/2 = 0.245 V). The ratio of ipa/ipc is approxi-
mately unity. This indicates the quasi-reversible redox process of
the metal complex. The incremental addition of CT DNA to the com-
plexes the redox couples cause a negative shift in E1/2 and a decrease
in ꢄEp. Finally Zn(II) complex exhibits quasi-reversible transfer
process with redox couple [Zn(II) → Zn(0)] cathodic peak appears
at −0.536 V in the absence of DNA (Epa = 0.404 V, Epc = −0.536 V,
ꢄEp = 0.940 V and E1/2 = −0.06 V). The ratio of ipa/ipc is 0.74 V. This
indicates the quasi-reversible redox process of the metal com-
plex. Incremental addition of DNA on Zn(II) complex shows a slight
decrease in the current intensity and negative shift of the oxida-
tion peak potential. The resulting minor changes in the current and
potential demonstrate diffusion of the Zn(II) bound to the large
Table 3
Electrochemical parameters for interaction of DNA with Cu(II), Ni(II), Co(II) and Zn(II) complexes.
a
Compound
Redox couple
E1/2 (V)
ꢄEp (V)b
ipa/ipc
Free
Bound
Free
Bound
Cu(III) → Cu(II)
Cu(II) → Cu(I)
0.440
−0.033
0.450
−0.066
0.034
0.178
0.035
0.136
0.78
1.80
[CuC56H60N8O6Cl2]
Ni(III) → Ni(II)
Ni(II) → Ni(I)
−0.345
−0.373
1.114
1.219
1.245
1.069
1.30
1.10
[NiC56H60N8O6Cl2]
[CoC56H60N8O6Cl2]
0.387
0.376
Co(III) → Co(II)
Zn(II) → Zn(0)
0.245
−0.066
0.224
−0.095
0.754
0.940
0.833
0.958
0.92
0.74
[ZnC56
H60N8O6Cl2]
a
Data from cyclic voltammetric measurements; E1/2 is calculated as average of anodic (Epa) and (Epc) peak potential E1/2 = Epa + Epc/2.
ꢄEp = Epa − Epc.
b

N. Raman et al. / Spectrochimica Acta Part A 78 (2011) 888–898
895
values. The more negative the relative binding, the more potent the
binding as between DNA and target molecules.
3.7.5. Chemical nuclease activity
The study on the cleavage capacity of transition metal com-
plex to DNA is considerably interesting as it can contribute to
understanding the toxicity mechanism of them and to develop
novel artificial nuclease. DNA cleavage is 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 observed for the
supercoiled form (Form I). If one strand is cleaved, the supercoils
will relax to produce a slowed moving open circular form (Form II).
If both strands are cleaved a linear form (Form III) will be generated
that migrates in between [27,28].
The ability of the complexes in affecting DNA cleavage has been
investigated by gel electrophoresis 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. 8 shows the
result of gel electrophoretic separations of plasmid pUC19 DNA
induced by an addition of metal(II) complexes in the presence
of AH2 (ascorbic acid). Under the same conditions, free AH2 pro-
duced no cleavage of pUC19 DNA. All super coiled (Form I) DNA
was cleaved to form the mixture of Form II and Form III with the
addition of the complex concentration. These phenomena imply
that Cu(II), Co(II), Ni(II) and Zn(II) complexes induce intensively
the cleavage of plasmid pUC19 DNA in the presence of AH2 (Fig. 8).
In order to clarify the cleavage mechanism of pUC19 DNA intro-
duced by metal(II) complexes, the investigation was carried out
further on adding DMSO (hydroxyl radical scavenger). It was found
that no inhibition of DNA cleavage was observed indicating that
hydroxyl radical is not involved in the cleavage process. On the
other hand, addition of sodium azide (singlet oxygen scavenger)
did not show any apparent inhibition in the DNA (Fig. 9) which
reveals that the singlet oxygen lO2 is not responsible for the cleav-
age reaction (Fig. 10). These observations suggested that all the
complexes mediated cleavage reaction did not proceed via radical
cleavage.
Fig. 6. Effect of increasing amounts of [CuC56H60N8O6Cl2] (ꢀ), [NiC56H60N8O6Cl2]
(*) [ZnC56H60N8O6Cl2] (᭹), [CoC56H60N8O6Cl2] (ꢁ) and [EB] (ꢂ) on the viscosity of
DNA. R = [complex]/[DNA] or [EB]/[DNA].
slowly diffusing DNA molecule. The electrochemical parameters of
the Co(II), Ni(II), Cu(II) and Zn(II) complexes are shown in Table 3.
3.7.3. Viscosity measurements
To clarify further the interaction between the metal complexes
and DNA, viscosity measurements was carried out. In the absence of
crystallographic structure data, hydrodynamic methods which are
sensitive to DNA length change are regarded as the least ambiguous
and the most critical tests of binding in solution. A classical inter-
calation model results in lengthening the DNA helix, as base pairs
are separated to accommodate the binding ligand, leading to the
increase of 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
[25]. The plots of (Á/Áo)1/3 versus [complex]/[DNA] = R, gives a mea-
sure of the viscosity changes (Fig. 6). The classical intercalators like
ethidium bromide are known to increase the base pair separation
resulting an increase in the relative viscosity of the DNA. In contrast,
groove (or) surface binding can cause an increase in the effective
length of DNA leading to a minor increase in the effective length of
DNA solution [26]. All the complexes exhibit minor increase in the
relative viscosity of CT DNA suggesting primarily groove binding
nature of the complexes.
3.8. Antimicrobial screening
3.8.1. Antibacterial activity
The Schiff base complexes have provoked wide interest because
they possess a diverse spectrum of biological and pharmaceutical
activities. The synthesized ligand and its complexes were tested
for their in vitro antimicrobial activity. They were tested against
the bacteria S. aureus, P. aeruginosa, E. coli, S. epidermis, K. pneumo-
niae by paper disc method. The antibacterial activity of the newly
synthesized compounds is presented in Table 4. The results indi-
cate that the ligand exhibits moderate antibacterial activity with
respect to the complexes against the same microorganisms under
identical experimental conditions. Further, the antibacterial action
of Schiff base ligand may be significantly enhanced on the pres-
ence of azomethine groups which have chelating properties. These
properties may be used in metal transport across the bacterial
membranes or to attach to the bacterial cells at a specific site from
which it can interfere with their growth. Ligand exhibits MIC in the
range of (14.5–16.9 ␮g/mL) against all the pathogens. The copper
complex shows better antibacterial activity (MIC, 1.9–2.9 ␮g/mL)
against the tested microorganisms than the other complexes which
have MIC values in the range 2.7–4.8 ␮g/mL. It may be attributed to
the atomic radius and the electronegativity of Cu(II) ions. Current
studies reveal thatthe high atomicradius and electronegative metal
ions in their metal complexes exhibit high antimicrobial activ-
ity. Higher electronegativity and large atomic radius decreases the
effective positive charges on the metal complex molecules which
3.7.4. Molecular docking analysis
The results of spectroscopic titration, viscosity and cyclic
voltammetry experiments suggest that Cu(II), Ni(II), Co(II) and
Zn(II) complexes do not interact with DNA via intercalation. The
binding constant values were in the range of 105 per mole for all the
complexes which indicate the possibility of intercalation of these
complexes between the DNA base pairs was little mainly due to
less effective stacking forces between the aromatic nucleus and
DNA bases. Docking experiments were performed in order to find
out the chosen binding site along with preferred orientation of the
ligand inside the DNA minor groove. Docking experiments in the
energy minimized docked structures suggesting the best possible
geometry of the metal complexes located to the AT-rich sequence
of the minor groove as shown in Fig. 7.
The structural analysis of docked structures gave signifi-
cant details about the binding pattern of these complexes.
Binding energy of docked metal complexes [CuC56H60N8O6Cl2],
[NiC56H60N8O6Cl2], [CoC56H60N8O6Cl2] and [ZnC56H60N8O6Cl2]
were found to be −120.1, −110.7, −105.6 and −119.1 kJ mol−1
respectively, correlating well with the experimental DNA binding

896
N. Raman et al. / Spectrochimica Acta Part A 78 (2011) 888–898
Fig. 7. Interaction of the complexes [CuC56
H60N8O6Cl2], [NiC56H60N8O6Cl2], [CoC56H60N8O6Cl2], and [ZnC56H60N8O6Cl2] with d(CGCGAATTCGCG) strands of DNA by minor
groove binding approach.
facilitates their interaction with the highly sensitive cellular mem-
branes towards the charged particle [29].
and C. albicans. The minimum inhibitory concentration (MIC) val-
ues of the investigated compounds are summarized in Table 5. A
comparative study of MIC values of ligand (12.9–15.9 ␮g/mL) and
its complexes (2.5–6.8 ␮g/mL) against all the fungi indicates that
the metal complexes exhibit higher antifungal activity than the lig-
and. Such increased activity on metal chelation can be explained on
3.8.2. Antifungal activity
The Schiff base and its metal complexes were screened for their
antifungal activity against A. niger, F. solani, C. lunata, R. bataicola
Table 4
The in vitro antibacterial activity of Schiff base and its metal complexes (MIC in ␮g/mL).
Compound
S. aureus
P. aeruginosa
E. coli
S. epidermis
K. pneumoniae
[C28H30N4O3]
16.9
1.9
3.1
3.7
3.6
1.7
15.8
2.6
3.5
3.4
4.8
1.9
16.4
2.2
3.0
3.9
3.5
1.8
16.2
2.7
2.7
4.1
3.9
1.3
14.5
2.9
3.2
4.2
4.4
2.3
[CuC56H60N8O6Cl2]
[NiC56H60N8O6Cl2]
[CoC56H60N8O6Cl2]
[ZnC56H60N8O6Cl2]
Streptomycin
Streptomycin is used as the standard. MIC (␮g/mL) minimum inhibitory concentration, i.e. the lowest concentration to completely inhibit the bacterial growth.

N. Raman et al. / Spectrochimica Acta Part A 78 (2011) 888–898
897
Table 5
The in vitro antifungal activity of Schiff base and its metal complexes (MIC in ␮g/mL).
Compound
A. niger
F. solani
C. lunata
R. bataicola
C. albicans
[C28H30N4O3]
12.9
2.5
4.2
5.0
5.3
1.1
13.3
2.3
3.4
5.2
6.3
1.6
14.5
3.7
4.5
5.9
6.1
1.2
11.5
4.8
4.7
6.3
6.5
1.0
15.9
5.9
5.3
6.8
6.2
1.5
[CuC56H60N8O6Cl2]
[NiC56H60N8O6Cl2]
[CoC56H60N8O6Cl2]
[ZnC56H60N8O6Cl2]
Nystatin
Nystatin is used as the standard. MIC (␮g/mL) minimum inhibitory concentration, i.e., the lowest concentration to completely inhibit the fungal growth.
and polysaccharides are some important constituents of the cell
wall and membranes which are preferred for metal ion interac-
tion. Apart from this, the cell wall also contains many phosphates,
carbonyl and cystenyl ligands which maintain the integrity of the
membrane by acting as a diffusion barrier and also provide suitable
sites for binding. Furthermore, increased lipophilicity enhances the
penetration of the complexes into lipid membrane and blocking of
the metal binding sites in the enzymes of microorganisms. These
complexes also disturb the respiration process of the cell and thus
block the synthesis of the proteins which restricts further growth
of the organism.
Fig. 8. Gel electrophoresis diagram showing the cleavage of pUC19 DNA (10 ␮M) by
the Cu(II), Ni(II), Co(II) and Zn(II) complexes in a buffer containing 50 mM Tris–HCl
and 50 mM NaCl in the presence of ascorbic acid (AH2, 10 ␮M) at 37 ◦C. Lane 1, DNA
control; lane 2, DNA + ligand+AH2; lane 3, DNA + AH2 + [CuC56H60N8O6Cl2]; lane
4, DNA + AH2 + [NiC56H60N8O6Cl2]; lane 5, DNA + AH2 + [CoC56H60N8O6Cl2]; lane 6,
DNA + AH2 + [ZnC56H60N8O6Cl2].
4. Conclusion
In this paper, the coordination chemistry of a Schiff base
ligand obtained from the reaction of 4-aminoantipyrine, 3,4-
dimethoxybenzaldehyde and tyramine is described. 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 conclusion that the Cu(II) ion assumes a distorted octa-
hedral geometry and the other complexes Ni(II), Co(II) and Zn(II) are
octahedral in nature. In all the complexes, the ligand acts as biden-
tate. The DNA-binding properties of synthetic metal complexes
have been comprehensively studied by different methods includ-
ing electronic absorption spectra, viscosity and cyclic voltammetry
and docking analysis. The experimental and docking results reveal
that all the complexes can interact with DNA through minor groove
approach. The complexes are capable of cleaving pUC19 DNA in the
presence of AH2. The antimicrobial screening data reveal that the
complexes have higher antimicrobial activity than the free ligand.
Fig. 9. Gel electrophoresis diagram showing the cleavage of pUC19 DNA
(10 ␮M) by the Cu(II), Ni(II), Co(II) and Zn(II) complexes in
a buffer con-
taining 50 mM Tris–HCl and 50 mM NaCl in the presence of ascorbic acid
(AH2, 10 ␮M) and DMSO (4 ␮L) at 37 ◦C. Lane 1, DNA control; lane 2,
DNA + ligand + AH2 + DMSO (4 ␮L); lane 3, DNA + AH2 + [CuC56H60N8O6Cl2] + DMSO
(4 ␮L); lane 4, DNA + AH2 + [NiC56H60N8O6Cl2] + DMSO (4 ␮L); lane 5,
DNA + AH2 + [CoC56
H60N8O6Cl2] + DMSO
(4 ␮L);
lane
6,
DNA + AH2 +
[ZnC56H60N8O6Cl2] + DMSO (4 ␮L).
Acknowledgements
The authors express their sincere thanks to the College Man-
aging Board, Principal and Head of the Department of Chemistry,
VHNSN College, Virudhunagar, India for providing necessary
research facilities and financial support. They also wish to acknowl-
edge the help rendered by Thiagarajar College with regard to
computational facilities.
References
[1] P.C.A. Bruijnincx, P.J. Sadler, Curr. Opin. Chem. Biol. 12 (2008) 197–206.
[2] V.S. Li, D. Choi, Z. Wang, L.S. Jimenez, M.S. Tang, H. Kohn, J. Am. Chem. Soc. 118
(1996) 2326–2331.
[3] R. Bera, B.K. Sahoo, K.S. Ghosh, S. Dasgupta, Int. J. Biol. Macromol. 42 (2008)
14–21.
[4] S.M. Hecht, J. Nat. Prod. 63 (2000) 158–168.
[5] M.S. Surendra Babu, P.G. Krishna, K.H. Reddy, G.H. Philip, J. Serb. Chem. Soc. 75
(2010) 61–74.
Fig. 10. Gel electrophoresis diagram showing the cleavage of pUC19 DNA (10 ␮M)
by the Cu(II), Ni(II), Co(II) and Zn(II) complexes in a buffer containing 50 mM Tris–HCl
and 50 mM NaCl in the presence of ascorbic acid (AH2, 10 ␮M) and NaN3 at 37 ◦C.
Lane1, DNA; lane 2, DNA + AH2 + NaN3; lane 3, DNA + AH2[CuC56H60N8O6Cl2] + NaN3;
lane
4,
DNA + AH2 + [NiC56H60N8O6Cl2] + NaN3;
lane
5,
DNA +
[6] R. Vijayalakshmi, M. Kanthimathi, V. Subramanian, B.U. Nair, Biochim. Biophys.
Acta 1475 (2000) 157–162.
AH2 + [CoC56H60N8O6Cl2] + NaN3; lane 6, DNA + AH2 + [ZnC56H60N8O6Cl2] +NaN3.
[7] K.S. Ksaprzak, Chem. Res. Toxicol. 4 (1991) 604–615.
[8] G. Jacob, A. Gamboa, A. Diedrich, C. Shibao, D. Robertson, I. Biaggioni, J. Hyper-
tens. 46 (2005) 355–359.
[9] (a) A.M. Farghaly, A. Hozza, Pharmazie 35 (1980) 596–601;
(b) T. Hitoshi, N. Tamao, A. Hideyuki, F. Manabu, Takayuki, Polyhedron 16 (1997)
3787–3794.
the basis of Tweedy’s chelation theory [30]. Chelation reduces the
polarity of the metal ion considerably because of the partial shar-
ing of its positive charge with the donor groups and also due to
␲-electron delocalization on the whole chelating ring. The lipids

898
N. Raman et al. / Spectrochimica Acta Part A 78 (2011) 888–898
[10] M.E. Hossain, M.N. Alam, J. Begum, A.M. Akbar, M. Nazimuddin, F.E. Smith, R.C.
Hynes, Inorg. Chim. Acta 249 (1996) 207–213.
[21] N. Raman, S. Thalamuthu, J. Dhaveethuraja, M.A. Neelakandan, S. Banerjee, J.
Chil. Chem. Soc. 53 (2008) 1439–1443.
[11] D. Chiaramonte, J.M. Steiner, J.D. Broussard, K. Baer, S. Gumminger, E.M.
Moeller, D.A. Williams, R. Shumway, Can. J. Vet. Res. 67 (2003) 182–188.
[12] N. Raman, R. Jeyamurugan, M. Subbulakshmi, R. Boominathan, C. Yuvarajan,
Chem. Pap. 64 (2010) 318–328.
[22] M. Roy, T. Bhowmick, R. Santhanagopal, S. Ramakumar, A.R. Chakravarty, Dalton
Trans. 24 (2009) 4671–4682.
[23] M.T. Carter, M. Rodriguez, A.J. Bard, J. Am. Chem. Soc. 111 (1989)
8901–8911.
[13] N. Raman, A. Selvan, P. Manisankar, Spectrochim. Acta Part A 76 (2010)
161–173.
[24] L.-Z. Li, C. Zhao, T. Xu, H.-W. Ji, Y.-H. Yu, G.-Q. Guo, H. Chao, J. Inorg. Biochem.
99 (2005) 1076–1082.
[14] G. Sathyaraj, T. Weyhermuller, B.U. Nair, Eur. J. Inorg. Chem. 45 (2010) 284–291.
[15] J. Marmur, J. Mol. Biol. 3 (1961) 208–218.
[25] S. Sathyanarayana, J.C. Dabroniak, J.B. Chaires, Biochemistry 31 (1992)
9319–9324.
[16] J.B. Charies, N. Dattagupta, D.M. Crothers, Biochemistry 21 (1982) 3933–3940.
[17] S. Satyanarayana, J.C. Daborusak, J.B. Charies, Biochemistry 32 (1993)
2573–2584.
[18] M.J. Kharodawala, M.N. Patel, Synth. React. Inorg. Met.-Org. Nano-Met. Chem.
32 (2002) 1483–1495.
[26] C. Rajput, R. Rutkaite, L. Swanson, I. Haq, J.A. Thomas, Chem. Eur. J. 12 (2006)
4611–4619.
[27] D.S. Sigman, Acc. Chem. Res. 19 (1986) 180–186.
[28] A.S. Sitlani, E.C. Long, A.M. Pyle, J.K. Barton, J. Am. Chem. Soc. 114 (1992)
2303–2312.
[19] L. Casella, M. Gullotti, J. Am. Chem. Soc. 103 (1981) 6338–6347.
[20] Y. Prashanthi, K. Kiranmai, N.J.P. Subhashini, Shivaraj, Spectrochim. Acta Part A
70 (2008) 30–35.
[29] N.A. Negma, M.F. Zakia, M.A.I. Salemb, Colloids Surf. B: Biointerfaces 77 (2010)
96–103.
[30] R. Ramesh, S. Maheswaran, J. Inorg. Biochem. 96 (2003) 457–462.