Synthesis of new piperazine derived Cu(II)/Zn(II) metal complexes, their DNA binding studies, electrochemistry and anti-microbial activity: Validation for specific recognition of Zn(II) complex to DNA helix by interaction with thymine base

Article abstract of DOI:10.1016/j.saa.2008.12.037

New 3,4:9,10-dibenzo-2,11-dihydroxy-1,12-bispiperazine-5,8-dioxododecane complexes [C₂₄H₃₆N₄O₆Cu] (1), [C₂₄H₃₂N₄O₄Zn] (2) have been synthesized and characterized by el

Full text of DOI:10.1016/j.saa.2008.12.037

Spectrochimica Acta Part A 72 (2009) 1026–1033
Contents lists available at ScienceDirect
Spectrochimica Acta Part A: Molecular and
Biomolecular Spectroscopy
journal homepage: www.elsevier.com/locate/saa
Synthesis of new piperazine derived Cu(II)/Zn(II) metal complexes, their DNA
binding studies, electrochemistry and anti-microbial activity: Validation for
specific recognition of Zn(II) complex to DNA helix by interaction
with thymine base
Irshad-ul-Haq Bhat, Sartaj Tabassum∗
Department of Chemistry Aligarh Muslim University, Aligarh 202002, UP, India
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 17 September 2008
Accepted 25 December 2008
New 3,4:9,10-dibenzo-2,11-dihydroxy-1,12-bispiperazine-5,8-dioxododecane complexes [C24H36
N4O6Cu] (1), [C24H32N4O4Zn] (2) have been synthesized and characterized by elemental analysis, IR,
NMR, Mass, EPR, UV–vis spectroscopy and molar conductance measurements. The complexes are
non-ionic in nature and possess octahedral geometry around Cu2+, Zn2+ central metal ions. The binding
studies of 1 and 2 with calf thymus DNA (CT-DNA) were investigated by UV–vis, fluorescence, cyclic
voltammetery and viscosity measurements. The calculated binding constant Kb for 1 and 2 obtained
from UV–vis absorption studies was 7.6 × 103 M−1, 80.8 × 104 M−1, respectively. The intrinsic binding
constants were also estimated to be 7.0 × 104 M−1 and 7.53 × 105 M−1 for 1 and 2, respectively by using
emission titrations. These experimental results suggest that complexes are groove binders and interact
to CT-DNA with different affinities. Both the complexes in presence and absence of CT-DNA show
quasireversible wave corresponding to CuII/CuI and ZnII/ZnI redox couple. The changes in E1/2, ꢀE, Ipa/Ipc
ascertain the interaction of 1 and 2 with CT-DNA. Further, decrease in viscosity of CT-DNA with increasing
concentration of complexes was observed. In vitro, antimicrobial activity against fungi A. brassicicola, A.
niger and bacteria E. coli, P. aeruginosa of complexes were carried out, which indicate that complex 2 is
more active against both fungal and bacterial strains as shown by % inhibition data.
Keywords:
UV–vis titrations
Fluorescence
Cyclic voltammetry
Antimicrobial activity
Viscosity measurements
© 2008 Elsevier B.V. All rights reserved.
1. Introduction
inal inorganic chemistry [10]. Besides this, metal complexes are
well known to play important roles in various biological processes
Molecular architecture containing heterocyclic piperazine sys-
tem has arisen interest owing to many reasons, notably their
biological properties as antitumour antibiotics [1], PAF (platelet
activity factor) antagonists and HIV-1 replication inhibitors [2], as
anti-inflammatory and antimicrobial activities [3,4], some of these
are highly active against drug resistant strains like staphylococ-
cus [5]. Tethering such biologically active moieties with carboxylic,
hydroxyl or aldehydic molecular framework has rendered multi-
combination agents inherently more potent than either of parent
component [6]. This synthetic strategy is unique and currently in
great demand owing to the use of such multicombination agents
in any therapies viz. cancer, HIV, neurodegenerative diseases, etc.
[7–9].
[11,12] which familiarize their coordination sites with the human
body’s function and thus the development of highly functional
novel metal-based complexes are desired in the pharmaceutical
fields [13]. We have opted for employing designer metal com-
plexes of ligand bearing heterocyclic piperazine system showing
their affinity towards DNA, which is primary target at the molec-
ular site [14]. In view of the fact, that metal ion further tunes and
modulates the organic ligand framework, enhances the efficacy of
the drug components and ensures site specific DNA interaction.
We herein, report the interaction studies of the metal complexes
1 and 2 with CT-DNA, their antimicrobial activity and electrochem-
istry. Complex 2 displays higher binding affinity than complex 1.
Kimura et al. have reported the selective recognition of thymine
base in double-standard DNA by the divalent zinc(II) complexes
with the macrocyclic tetramines or with the derivatives appended
with aromatic groups, it is therefore of great interest that only
Zn(II) effectively functions to recognize thymine base [15–17]. The
Zn(II) complexes in contrast to Cu(II) and Ni(II) break the hydro-
gen bonds of A-T base pairs of DNA helix to selectively bind
Many chemotherapeutics currently in use are metal-based and
this has opened up a new research area in the field of medic-
∗
Corresponding author. Tel.: +91 571 2703893.
E-mail address: tsartaj62@yahoo.com (S. Tabassum).
1386-1425/$ – see front matter © 2008 Elsevier B.V. All rights reserved.
doi:10.1016/j.saa.2008.12.037

I.-u.-H. Bhat, S. Tabassum / Spectrochimica Acta Part A 72 (2009) 1026–1033
1027
thymine, altering the local structure of the AT-regions of DNA
[18–20].
complex added), F and F0 are fluorescence intensities in presence
and absence of CT DNA, respectively. P is the ratio of the observed
fluorescence quantum yield of the bound metal complex to that of
the free metal complex. The value P was obtained as the intercept
by extrapolating from a plot of F/F0 versus 1/[DNA], r denotes ratio
of CB (=CT − CF) to the DNA concentration and c is the free metal
complex concentration and “n” is the binding site number.
2. Experimental
2.1. Reagents
Salicyaldehyde (sd.fine), 1,2-dibromoethane, piperazine hex-
ahydrate, Tris base (Merck), CuCl2·2H2O (Qualigens), ZnCl2
(Rankem) were utilized as received. Calf thymus DNA, thymine and
guanine were purchased from Sigma Chemical Co. All reagent grade
compounds were used without further purification.
2.3. Antifungal screening
Complexes 1 and 2 were tested for their in vitro growth inhibition
activity against pathogenic fungus viz. Alternaria brassicicola and
Aspergillus niger cultured on potato dextrose agar (PDA) medium
and incubated at 25 2 ◦C for 72 h by using poison food technique
[25]. The PDA medium was sterilized in the autoclave in 15 kg/cm2
at 121 ◦C which keeps the medium to retain its normal tempera-
ture and the solution of complexes 1 and 2 is added in the PDA
medium in the different concentration. The concentration is made
in the range of (10–50) × 10−6 M. The inhibition of fungal growth,
expressed in percentage terms was determined on the growth in
test plates compared to respective control plates (untreated), as
given by Vincent equation [26].
2.2. Other physical measurements
Interspec 2020 FTIR spectrometer was used for recording IR
spectra of KBr pellets in the range of 400–4000 cm−1. Electronic
spectra were recorded on USB Ocean Optics 2000 spectrometer.
Data were reported in ꢁmax/nm. Emission spectra were determined
with a Hitachi F-2500 fluorescence spectrophotometer. Microanal-
yses of the complexes were obtained on a Carlo Erba Analyzer
Model 1108. 1H and 13C spectra were recorded on a Bruker DRX-
300 spectrometer. Chemical shifts were reported in ␦ scale. EPR
spectrum of copper(II) complex was recorded on Varian E 112
spectrometer at the X-band frequency (9.1 GHz) at liquid nitrogen
temperature (LNT) using tetracyanoethlene (TCNE) as field marker.
Cyclic voltammetry was carried out at CH instrument electrochem-
ical analyzer. All voltammetric experiments were performed in
single compartmental cell of volume 10–15 ml containing a three-
electrode system comprised of a Pt-disk working electrode, Pt-wire
as auxiliary electrode, and an Ag/AgCl electrode as reference elec-
trode. The supporting electrolyte was 0.4 M KNO3 in milli-Q water.
Deairated solutions were used by purging N2 gas for 15 min prior
to measurements. Molar conductance was measured at room tem-
perature on Digisun electronic conductivity bridge. Interaction of
complexes with calf thymus DNA was performed in 0.01 M buffer
(pH 7.2). Solutions of calf thymus DNA in buffer gave a ratio of
absorbance at 260 nm and 280 nm of ca 1.9:1 indicating that DNA
was free from protein [21]. Solutions of thymine was prepared in
double distilled water (pH 5.2) while the solution of guanine was
prepared in phosphate buffer (pH 2). Viscosity measurements were
carried out using Ostwald’s viscometer at 29 0.01 ◦C. Flow time
was measured with a digital stopwatch. Each sample was measured
three times and an average flow time was calculated. Data are pre-
sented as (ꢂ/ꢂ0) versus binding ratio ([M]/[DNA]) [22]. Where ꢂ is a
viscosity of DNA in the presence of complex and ꢂ0 is the viscosity
of DNA alone. Viscosity values were calculated from the observed
flow time of DNA containing solution (t > 100 s) corrected for the
flow time of buffer alone (t0), ꢂ = t − t0. The intrinsic binding con-
stant, Kb and K of the complexes 1 and 2 to CT-DNA was determined
from Eq. (1) by UV–vis titrations, (2) and (3) (Scatchard’s equations)
by emission titration, respectively [23,24].
100(C − T)
Inhibition % =
(4)
C
where C is the diameter of fungal growth on the control plate and
T is the diameter of fungal on the test plate.
2.4. Antibacterial screening
The solution of complexes ((10–50) × 10−6 M) in DMSO was
applied on a paper disc prepared from sterilized paper (3 mm
diameter) with the help of micropipette. The discs were left in an
incubator for 30 h at 37 ◦C and then applied on the bacteria grown
(Escherichia coli and Pseudomonas aeruginosa) on potato dextrose
agar (PDA) plates. Minimal agar was used for growth of specific bac-
terial species. Agar (50 g) was suspended in freshly distilled water
(1 l), allowed to soak for 15 min and then boiled on water bath until
the agar was completely dissolved. The mixture was autoclaved
for 15 min at 120 ◦C and then poured into previously washed and
sterilized Petri dishes and stored at 40 ◦C for inoculation.
2.5. 3,4:9,10-Dibenzo-1,12-diformyl-5,8-dioxododecane
[C16 H14 O4] L1
The ligand L1 was synthesized using the method reported pre-
viously [27].
2.6. 3,4:9,10-Dibenzo-2,11-dihydroxy-1,12-bispiperazine-5,8-dio-
xododecane [C24H34N4O4] L2
The ligand [C24H34N4O4] L2 was prepared according to the
method reported earlier with a modification [28], and is described
as below.
[DNA]
|εa − εf|
[DNA]
|εb − εf|
ꢁ
1
=
+
(1)
Kb|εb − εf|
ꢀ
To a solution of [C16 H14 O4] L1 (0.27 g, 1 mmol) in 20 ml benzene
was added piperazine hexahydrate (0.388 g, 2.0 mmol) in a Dean-
Stark apparatus. The reaction mixture was refluxed for 1 h. A white
colored solid obtained was filtered, washed with ice-cold methanol
and dried in vacuo. Yield 80%, mp 211 ◦C. IR: (ꢃmax)/cm−1 3412
ꢃ(NH) ꢃ(OH); 2809, 2941 ꢃa(CH2) ꢃs(CH2); 1594, 1506, ꢃ(C C); 1454
ꢃdef(CH2); 1100 ꢃ(C O), ␦(OH) 1286; ꢃa(C N) 1420; ꢃs(C N) 1160;
F
CF = CT
− P (1 − P)
(2)
(3)
F0
r
= K(n − r)
c
where [DNA] represents the concentration of DNA. εa, εf and εb
are the apparent extinction coefficients (Aobs/[M]), the extinction
coefficient for free metal [M] complex and the extinction coefficient
for free metal [M] complex in the fully bound form, respectively. In
plots of |εa − εf| versus, Kb is given by the ratio of slope to intercept.
CF (concentration of metal complex), CT (concentration of metal
ıroc(CH) 755; ı(CNC) 846 cm−1 1H NMR (200 MHz, DMSO-d6):
.
ı = 7.1–7.7 (ArH); 5.70 (NH); 4.58 (OH); 3.2–3.4 (OCH2); 2.37–2.49
(CH2) ppm. 13C NMR (50 MHz, DMSO-d6): ı = 128.64–135.26 (ArC);
46.28–47.21 (CH2); 76.64–77.27 (CHOH); 66.93 (OCH2). ESI-MS

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Scheme 1. (a) Proposed structure of L2, 1 and 2. (b) Cylindrical bonded three-dimensional model of complexes 1 and 2. Color scheme: CuII, dark gray; ZnII, dark gray; N, dark
blue; O, red; C, gray.
m/z 442 [M+]. Anal. Found C, 65.34; H, 7.42; N, 12.52 Calc. for
24H34N4O4: C, 65.12; H, 7.75; N, 12.67.
499 ꢃ(Zn O); 645 ꢃ(Zn N) cm−1 1H NMR (200 MHz, DMSO-d6):
.
ı = 7.1–7.78 (ArH); 5.75 (NH); 3.17–3.4 (OCH2); 2.30–2.60 (CH2).
ESI-MS m/z: 506 [M+]. Anal. Found C, 57.01; H, 6.42; N, 11.02. Calc.
for C24H32N4O4Zn: C, 57.12; H, 6.40; N, 11.11.
C
2.7. 3,4:9,10-Dibenzo-2,11-dihydroxy-1,12-bispiperazine-5,8-
dioxododecane copper(II) [C24H36N4O6Cu] (1)
3. Results and discussion
To a solution of [C24H34N4O4] L2 (0.442 g, 1 mmol) in 10 ml
methanol was added a solution of CuCl2·2H2O (0.170 g, 1 mmol)
in 10 ml methanol dropwise, a green colored precipitate obtained
was stirred for 1 h which was filtered, washed thoroughly with
methanol and dried in vacuo. Yield: 45% mp 160 ◦C. IR: (ꢃmax)/cm−1
3419 ꢃ(NH); 2837, 2958–2997 ꢃa(CH2) ꢃs(CH2);1139 ꢃ(C O);
ꢃa(C N) 1448; ꢃs(C N) 1190; 439 ꢃ(Cu O); 513 ꢃ(Cu N) cm−1. ESI-
MS m/z: 540 [M+]. Anal. Found C, 53.18; H, 6.48; N, 10.12. Calc. for
The spectroscopic studies and analytical data supports proposed
structure (Scheme 1a) and three-dimensional models with cylindri-
cal bonds are depicted in Scheme 1b. Ligand L2 is soluble in many
organic solvents and the complexes 1 and 2 are soluble in solvents
DMSO and DMF. Molar conductive measurements in DMSO show
that complexes are non-ionic in nature.
C24H36N4O6Cu: C, 53.41; H, 6.73; N, 10.39
3.1. IR spectra
2.8. 3,4:9,10-Dibenzo-2,11-dihydroxy-1,12-bispiperazine-5,8-
dioxododecane zinc(II) [C24H32N4O4Zn] (2)
The IR spectra of ligand L1 show a characteristic absorption
band at 1684 cm−1 due to aldehydic ꢃ(C O) stretching vibrations
[29]. Other bands at 1530 cm−1 and 1270–1295 cm−1 are associ-
ated with phenolic ꢃ(C O) stretching and bending vibrations [30].
The absorption bands due to ꢃ(CH2) appear at 2952–2875 cm−1
[31]. In the IR spectrum of ligand L2 the band due to ꢃ(C O) was
not observed. Asymmeteric and symmetric stretching vibration
Complex [C24H32N4O4Zn] was synthesized with ZnCl2
(0.13 g, 1 mmol) by similar procedure as described for complex
[C24H36N4O6Cu]. A white colored product obtained was filtered,
washed thoroughly with methanol and dried in vacuo. Yield: 42%
mp 300 ◦C decomp. IR: (ꢃmax)/cm−1 3417 ꢃ(NH); 2863 ꢃa(CH2);
2932–2979 ꢃs(CH2); 1133 ꢃ(C O); ꢃa(C N) 1441; ꢃs(C N) 1190;
of methylene group was observed at 2809 cm−1 and 2941 cm−1
respectively [32]. The stretching vibration due to ꢃ(NH) and ꢃ(OH)
,

I.-u.-H. Bhat, S. Tabassum / Spectrochimica Acta Part A 72 (2009) 1026–1033
1029
Fig. 1. 1H NMR spectrum of the ligand L2.
appears as a single band at 3412 cm−1. The C C bond stretch-
ing absorption of aromatic ring was observed at 1594 cm−1 and
1506 cm−1. The absorption band observed at 1454 cm−1 can be
assigned to mixed band of CH2 deformation and another stretching
absorption of aromatic ring, stretching vibration due to CO bond
of alcohol appears at 1100 cm−1, OH deformation was observed at
1286 cm−1 and the bands observed at 1420 and 1160 cm−1 were
attributed to C N asymmetric and symmetric stretching vibrations,
respectively [33].
plex 2 remain almost unchanged. Therefore, the spectroscopic data
support the proposed structure of the ligand [C24H34N4O4] L2 and
complex 2.
3.3. Electronic spectra
The electronic absorption spectra of 1 and 2 were recorded in
200–1100 nm region at room temperature. The electronic spec-
trum of 1 in DMSO displayed high-energy bands at 230 nm, 306 nm
corresponds to ␲–␲* and ligand metal charge transfer (LMCT)
transitions, respectively. The low energy d–d band at 760 nm
In the IR spectra of complexes 1 and 2 bands due asymmet-
ric and symmetric methylene groups were observed at 2837 cm−1
,
2863 cm−1 and 2958–2958 cm−1, 2932–2979 cm−1, respectively.
The bands observed at 3419 cm−1 and 3417 cm−1 were ascribed
to coordinated ꢃ(NH) stretching vibrations [34]. The band which
appeared at 1100 cm−1 in free ligand [C24H34N4O4] L2 due to
ꢃ(C OH) was shifted to 1139 cm−1 and 1133 cm−1, respectively,
after complexation confirming the coordination of metal ion
through O atom of deprotonated OH group. Similarly the bands
due to C N asymmetric and symmetric vibrations undergo positive
shift of 28 cm−1, 21 cm−1 and 30 cm−1, indicating the involvement
of nitrogen atoms in the formation of complexes. The far IR spectra
of [C24H36N4O6Cu] and [C24H32N4O4Zn] reveal (M O) and (M N)
3
2
was assigned to B1g → E2g transitions suggesting an octahedral
geometery around CuII metal ion [39,40]. The absorption spectrum
of complex 2 revealed bands at 259 nm and 318 nm due to intrali-
gand and LMCT transitions, respectively.
3.4. EPR spectra
The solid state X band EPR spectra of complex 1 acquired at liq-
uid nitrogen temperature (LNT) is anisotropic. The spectrum shows
axial symmetry, with three hyperfine peaks in the parallel region
derived from the coupling of copper nucleus and the unpaired elec-
stretching vibrations in the range 439–499 cm−1 and 513–515 cm−1
respectively.
,
tron. The EPR signals of complex 1 exhibited g⊥ = 2.037, g = 2.36
||
and A = 170 G values. The fact that g > g⊥ confirms an octahedral
||
||
1
geometry with a (dx
)
2
as ground state in complex 1 [30,40].
2
−y
3.2. NMR spectra
4. DNA interaction studies
1H and 13C NMR spectra of ligand L2 and complex 2 were
recorded in DMSO-d6. The 1H NMR of ligand [C24H34N4O4] L2
revealed characteristic signals at 7.10–7.70, 3.2–3.4 and 4.58
assigned to aromatic, OCH2 and OH protons [35,8]. The resonance
at 2.37–2.49 ppm and 5.7 ppm can be ascribed to CH2 CH2 and NH
protons of piperazine ring [8,36,37]. In the complex 2, resonances
due to the protons of aromatic ring remain unaltered. A singlet at
5.75 ppm can be assigned to NH protons [36,37]. Resonating sig-
nals due to OCH2 and CH2CH2 were observed at 3.17–3.40 ppm
and 2.3–2.6 ppm, respectively [35,8,3]. The slight shift in NH and
absence of OH protons, respectively suggests that complexation of
metal ion takes place through NH and deprotonation of OH groups
respectively. The 13C NMR spectrum of ligand L2 was character-
ized by various resonances due to aromatic carbon (C C), CH2,
CHOH, OCH2 at 128.64–135.26, 46.28–47.21, 76.64–77.27 and 66.93,
respectively [38,37,8]. The 1H NMR spectrum of L2 is depicted in
Fig. 1. On the other hand resonances due to carbon skeleton of com-
The binding of complexes with CT-DNA was ascertained by elec-
tronic absorption titrations. Any interaction between the complexes
and DNA is expected to perturb the ligand centered transi-
tions of complexes. The changed in the ␲–␲* transitions of 1
and 2 were determined as function of added concentration of
((0.067–0.33) × 10−4 M) and the typical absorption titration curves
for 1 and 2 are given in Fig. 2a and b. A hyperchromism of 23% and
29% for 1 and 2 with a red shift of 7–8 nm was observed. The intrin-
sic binding constant Kb has been calculated from Eq. (1), to compare
quantitatively the affinities of 1 and 2 to CT-DNA were 7.6 × 103 M−1
and 80.8 × 104 M−1, respectively. The complex 2 shows a multifold
increase in Kb value in comparison to the Kb values of complex 1,
which has been attributed to the stronger affinity of zinc complexes
for selective recognition of nucleobase of double strand CT-DNA.
Literature survey reveals that Zn(II) complexes in contrast to Cu(II)
and Ni(II) break the hydrogen bonds of A-T base pairs of DNA

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Fig. 2. (a) Absorption spectral traces of 1 in Tris–HCl buffer upon addition of CT-DNA. Inset: plots of [DNA]/εa − εf versus [DNA] for the titration of CT-DNA with complexes
(ꢀ), experimental data points; full lines, linear fitting data. (b) Absorption spectral traces of 2 in Tris–HCl buffer upon addition of CT-DNA. Inset: plots of [DNA]/εa − εf versus
[DNA] for the titration of CT-DNA with complexes (ꢁ), experimental data points; full lines, linear fitting data.
helix, bind selectively to thymine, altering the local structure of
the AT-regions of DNA [20]. These hyperchromic spectral changes
observed are typical of complex bound to DNA grooves, leading to
small perturbations and has been observed in case of complexes
upon their interaction with DNA [41]. The “hyperchromic effect”
was due to the dissociation of ligand aggregates [42] or due to its
external contact (surface binding) with the duplex by engaging in
hydrogen-bonding interactions between coordinated NH and OH
with functional groups positioned on the edge of DNA bases.
In order to gain support for selective binding of the zinc com-
plexes to thymine base of DNA helix involving possibly N3 thymine
coordination, the interaction of 2 with DNA bases thymine and
guanine was investigated using electronic absorption spectral tech-
nique. Addition of thymine to complex 2 results in an increase
in an absorbance (88% hyperchromism) in the intraligand bands
with no blue or red shift (Fig. 3a). The hyperchromicity implies
that the interaction between complex 2 and the thymine is com-
bination coordination of Zn metal ion after ligand dissociation or
reorganization [42] and hydrogen bonding with O2 and O4 oxy-
gen atoms of thymine [20]. However, on addition of guanine to
the complex 2 exhibited relatively small hyperchromic changes
(70%) in absorbance with no shift absorbance intensity (Fig. 3b).
These spectral changes reveal that complex 2 has stronger binding
affinity towards thymine in comparison to guanine. The complexes
show preference for thymine due to two effects. When thymine
binds, hydrogen bonds are formed between the NH groups of lig-
and secondly, a significant stronger molecular orbital interaction
is identified in thymine in comparison to guanine. The presence of
electron withdrawing two oxo group at the C2 and C3 position of
the thymine ring lowers the energy of the lone-pair orbital at N3
Fig. 3. (a) Absorption spectral traces of 2 in H2O upon addition of thymine. (b) Absorption spectral traces of 2 in phosphate buffer upon addition of guanine.

I.-u.-H. Bhat, S. Tabassum / Spectrochimica Acta Part A 72 (2009) 1026–1033
1031
Fig. 4. Cyclic voltammogram of 1 at a scan rate 0.3 V s−1 in DMSO/H2O (a) in absence
CT-DNA and (b) in presence of CT-DNA.
of thymine base. Our results are consistent with earlier reports on
preferential binding to N3 position of thymine in the zinc complexes
[17,18,20].
5. Electrochemical studies
The redox behavior of complexes 1 and 2 in the absence and
presence of CT-DNA were studied in DMSO/H2O 5:95 by means of
cyclic voltammetery at room temperature within potential range
of 2.0 V to −1.2 V. Cyclic voltammetry is one of the most impor-
tant electroanalytical techniques employed for interaction of metal
complexes with biomolecules due to the similarity between various
redox chemical and biological processes [43]. Cyclic voltammogram
of complex 1 exhibits a quasireversible redox wave corresponding
to CuII/CuI with Epc and Epa values −0.590 V and −0.195 V, respec-
tively at the scan rate of 0.3 V s−1. The ratio of anodic to cathodic
Fig. 6. (a) Emission spectra of 1 in the presence of DNA in Tris–HCl buffer. Inset: plots
of F/F0 versus [DNA]. (b) Emission spectra of 2 in the presence of DNA in Tris–HCl
buffer. Inset: plots of F/F0 versus [DNA].
peak currents Ipa/Ipc was 0.407. The formal electrode potentials E1/2
,
ꢀEp (difference in cathodic Epc and anodic Epa peak potentials) were
−0.393 V and 0.394 V, respectively. The ꢀEp value is larger than
Neristian value observed for a one electron transfer redox couple
[44]. At constant parameters, the addition of CT-DNA results the
shift in E1/2 = −0.363 V and ꢀEp = 0.537 V (Fig. 4), respectively. The
ratio of Ipa/Ipc is 0.303 for CT-DNA bound metal complex 1. The
shift in potentials and decrease in current ratio suggest the binding
of complex 1 to CT-DNA [45].
The cyclic voltammogram of complex 2 (DMSO/H2O; 5:95, 25 ◦C)
at scan rate 0.3 V s−1 features a quasireversble redox wave ZnII/ZnI
[46] with E1/2, ꢀEp and Ipa/Ipc values −0.384 V, 0.495 V and 0.383,
respectively. Upon addition of CT-DNA under the same recording
conditions complex 2 experience a shift in E1/2 (−0.355 V), ꢀEp
(0.419 V) and ratio of anodic to cathodic peak currents Ipa/Ipc is 0.247
(Fig. 5). At different scan rates both the complexes do not show
any major change. The larger decrease in potentials and current
ratios of complex 2 suggest that 2 binds to CT-DNA more strongly
in comparison to complex 1.
6. Fluorescence studies
Complexes 1 and 2 exhibit luminescence either in DMSO or in
presence of CT-DNA. Hence, binding studies of both the complexes
to CT-DNA have been carried out by fluorescence spectral titrations
[24]. Fixed amount of complexes (0.067 × 10−4 M) was treated with
increasing amount of CT-DNA ((0.067–0.33) × 10−4 M). The results
of emission titration of complexes 1 and 2 with DNA are illustrated
in Fig. 6a and b. Upon addition of DNA the emission intensity was
decreased steadily. The binding constants estimated for complexes
1 and 2 by Scatchard’s Eqs. (2) and (3) were 7.0 × 104 M−1 and
7.53 × 105 M−1, respectively. The binding constant K values suggest
that the interaction of 2 with the CT-DNA is stronger in comparison
Fig. 5. Cyclic voltammogram of 2 at a scan rate 0.3 V s−1 in DMSO/H2O (a) in absence
of CT-DNA and (b) complex 2 in presence of CT-DNA.

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I.-u.-H. Bhat, S. Tabassum / Spectrochimica Acta Part A 72 (2009) 1026–1033
Fig. 7. Effects of increasing amount of complexes 1 and 2 on relative viscosity of
CT-DNA at 29 0.1 ◦C. [DNA] = 1 × 10−3, R = [complex]/[DNA].
to 1. These results are consistent with the findings obtained from
UV–vis spectral studies as well.
7. Viscosity measurements
Hydrodynamic measurements, which are sensitive to length
change, are regarded as the least ambiguous and most critical test
of binding in solution [47]. A classical intercalative mode causes a
significant increase in viscosity of DNA solution due to its increase
in separation of base pairs at intercalation sites and increases in
overall DNA length. In contrast, complexes those bind exclusively
in the DNA grooves by partial and/or non-classical intercalation,
under same conditions, typically cause less positive or negative or
no change in DNA solution viscosity. However partial non-classical
intercalator causes a kink in the DNA helix and reduces the length
and its viscosity. As a means for further clarifying the binding of
the metal complexes, viscosity measurements were carried out
on CT DNA by varying the concentration of the added complex.
The increase in relative viscosity of CT-DNA is due to formation
of bridged structures leading to extension of DNA duplexes [48],
therefore binding may involve inner sphere complex formation via
phosphate oxygen and indicates that the binding of 1 and 2 is sur-
face binding or by forming bridged adducts. Thus, these adducts
formed by bridging the duplexes are stabilized by hydrophobic
interaction or columbic interaction as reported earlier in some cop-
per complexes [49,50]. The plot of relative specific viscosities ꢂ/ꢂ0
versus [1/R] is shown in Fig. 7.
Fig. 8. Antimicrobial activity of 1 and 2 ((10–50) × 10−6 M) in terms of percent inhi-
bition against (a) A. brassicicola, A. niger and (b) E. coli, P. aeruginosa.
which subsequently favours its permeation through lipid layers of
cell membrane resulting in the interference with normal cell pro-
cess [52,53].
9. Conclusion
The synthesis and characterization of new piperazine derived
metal complexes 1 and 2 have been described. DNA binding stud-
ies by spectroscopic titrations viz. UV–vis and fluorescence reveal
that complex 2 binds more strongly to CT-DNA as compared to com-
plex 1. This enhanced binding capacity of complex 2 is attributed
to the selective recognition of DNA bases. The results from cyclic
voltammetry and viscosity measurements also support the above
observations. In addition complexes selectively inhibited the fungal
(A. brassicicola and A. niger) and bacterial (E. coli and P. aeruginosa)
growth, which has been explained on the basis of chelate theory.
These studies reveal that complexes 1 and 2 can be used as poten-
tial chemotherapeutic agents and hold much promise in the field
of drug discovery.
8. Biological activities
The susceptibility of certain strains of fungi and bacteria towards
complexes 1 and 2 were screened by measuring the size of the
inhibition diameter. % inhibition data was calculated by using Vin-
cent Eq. (4) and depicted in (Fig. 8a and b). Antimicrobial activity
against fungus (A. brassicicola and A. niger) and bacteria (E. coli and P.
aeruginosa) at (10–50) × 10−6 M was tested in vitro, respectively. The
results revealed that the complex 2 is more effective in comparison
to complex 1 for both fungal and bacterial strains. The increase in
biological activity of complexes may be due to the effect of the metal
ion on the cell metabolism. A possible mode of toxicity increase may
be considered in the light of chelation theory [51]. Chelation con-
siderably reduces the polarity of the metal ion because of partial
sharing of its positive charge with the donor groups and possible
␲-electron delocalization over whole chelate ring. Such chelation
could enhance the lipophilic character of central metal ion atom
Acknowledgements
We are grateful to the Third World Academy of Science, Italy
(Grant No. 01-268RG/CHE/AS) for generous financial support to
purchase electrochemical analyzer and Ocean Optics USB-2000
spectrometer and University Grants Commission, New Delhi for
financial support through research grant No. 31-100/2005. Thanks
to Regional Sophisticated Instrumentation Center, Central Drug

I.-u.-H. Bhat, S. Tabassum / Spectrochimica Acta Part A 72 (2009) 1026–1033
1033
Research Institute, Lucknow, for providing CHN-analysis data, Mass
and NMR spectra and Regional Sophisticated Instrumentation Cen-
ter, Indian Institute of Technology, Bombay, for EPR measurements.
The authors gratefully acknowledge Mahmud Khan Department
of Plant Protection and Javaid Ahmad Bhat Department of English
Aligarh Muslim University, Aligarh, India for providing antimicro-
bial activity and improving the English language of the manuscript,
respectively.
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