Electrochemical, magnetic, catalytic, DNA binding and cleavage studies of new mono and binuclear copper(II) complexes

Article abstract of DOI:10.1016/j.poly.2010.09.041

A new class of mono [CuL] (1) and binuclear copper(II) complexes [Cu ₂LB](ClO₄)₂ (2 and 3), where L (6,6-piprazine-1,4 diyl-dimethylene bis(4-methyl phenol) is a N ₂O₂ donor ligand and B is a N,N-dono

Full text of DOI:10.1016/j.poly.2010.09.041

Polyhedron 30 (2011) 123–131
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Polyhedron
journal homepage: www.elsevier.com/locate/poly
Electrochemical, magnetic, catalytic, DNA binding and cleavage studies
of new mono and binuclear copper(II) complexes
⇑
S. Anbu, M. Kandaswamy
Department of Inorganic Chemistry, School of Chemical Sciences, University of Madras, Guindy Maraimalai Campus, Chennai 600 025, India
a r t i c l e i n f o
a b s t r a c t
Article history:
A new class of mono [CuL] (1) and binuclear copper(II) complexes [Cu2LB](ClO4)2 (2 and 3), where L (6,6-
piprazine-1,4 diyl-dimethylene bis(4-methyl phenol) is a N2O2 donor ligand and B is a N,N-donor hetero-
cyclic base, viz. 2,2-bipyridyl (bipy) (2) and 1,10-phenanthroline (phen) (3), has been synthesized. These
complexes were characterized by elemental and spectroscopic techniques. The redox, magnetic, catalytic,
DNA binding and cleavage activities of the copper(II) complexes (1–3) were studied. Cyclic voltammetric
investigation of the mononuclear Cu(II) complex (1) shows a quasi-reversible one electron reduction
wave (E1/2 = ꢀ0.87 V) and the binuclear Cu(II) complexes show two quasi-reversible one electron
reduction processes around E11=2 ¼ ꢀ0:28 V, ꢀ0.18 V and E12=2 ¼ ꢀ0:72 V, ꢀ0.66 V versus Ag/AgCl in
DMF, 0.1 M TBAP. ESR spectra of the copper(II) complexes 2 and 3 show a broad signal at g = 2.08 and
2.10, and leff values 1.32 and 1.35 BM respectively, which convey spin–spin interactions between the
two copper(II) ions. Cryomagnetic investigation of the binuclear complexes reveals a weak antiferromag-
netic spin exchange interaction between the Cu(II) ions within the complexes (ꢀ2J = 228.3 (2) and
237.5 cmꢀ1(3)). The initial rate values for the oxidation of 3,5-di-tert-butylcatechol to o-quinone by the
mono and binuclear Cu(II) complexes 1, 2 and 3 are 2.6 ꢁ 10ꢀ7, 6.8 ꢁ 10ꢀ5 and 2.3 ꢁ 10ꢀ5 M sꢀ1 respec-
tively. The complexes 2 and 3 show good binding propensity to calf thymus DNA, giving binding constant
values of 0.37( 0.1) ꢁ 105 (s = 0.1) and 0.44( 0.2) ꢁ 105 Mꢀ1 (s = 0.1) respectively. The complexes dis-
play significant oxidative cleavage of circular plasmid pBR322 DNA in the presence of mercaptoethanol
using singlet oxygen as a reactive species. The phenanthroline containing binuclear Cu(II) complex 3 dis-
plays a better DNA interaction and significant chemical nuclease activity compared to the bypyridyl ana-
log 2 and the mononuclear complex 1.
Received 23 June 2010
Accepted 30 September 2010
Available online 21 October 2010
Keywords:
Binuclear Cu(II) complexes
Magnetic properties
Electrochemical studies
Catecholase activity
DNA binding and cleavage
Ó 2010 Elsevier Ltd. All rights reserved.
1. Introduction
that binuclear copper(II) complexes have greater cleaving effi-
ciency or DNA interaction than the mononuclear complexes [7,9].
The design of small molecules that bind and react at specific
sequences of DNA under physiological conditions via oxidative
and hydrolytic mechanisms has been attracting great interest in
the field of bioinorganic chemistry [1–3]. Intercalators are small
molecules that contain a planar aromatic heterocyclic functionality
which can insert and stack between the base pairs of double helical
DNA [4]. As both spectroscopic tags and functional models for the
active centers of proteins, metal complexes have helped elucidate
the mechanisms by which metalloproteins function. Binucleating
ligands are particularly suited for the synthesis of such complexes
as they are more stable and the two metal ions are fixed in close
proximity, which has important implications for metal–metal
interactions and their reactivity [5]. These properties have turned
chemical nucleases into useful tools as adjuvants in PCR diagnos-
tics [6,7], nucleic acid attacking and cleaving agents [8]. It is known
Transition metal complexes with tunable coordination environ-
ments and versatile spectral and electrochemical properties offer
a great scope of design for species that are suitable for catecholase,
DNA binding and cleavage activities [10]. The present work stems
from our interest in designing new binuclear copper(II) complexes
that are capable of cleaving DNA in the presence of mercap-
toethanol under physiological conditions. Copper(II) complexes
are known to show ‘‘chemical nuclease’’ activity in the presence
of a reducing agent like thiols [11]. Recently, we have reported that
binuclear Cu(II) complexes of an aromatic diamine condensed
Schiff base macrocyclic ligand display significant DNA cleavage
activity (in the presence of mercaptoethanol), greater than the ali-
phatic diamine condensed Cu(II) analogs [12]. A variety of propos-
als have been put forth describing the reactivity of DNA with
copper phenanthroline and bipyridyl complexes [13,14]. The major
pathway for DNA damage by copper phenanthroline complexes is
believed to involve C10-hydrogen atom abstraction, along with
varying amounts of oxidation at the C40- and/or C50-positions in
⇑
Corresponding author. Tel./fax: +91 44 22300488.
E-mail address: mkands@yahoo.com (M. Kandaswamy).
0277-5387/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.poly.2010.09.041

124
S. Anbu, M. Kandaswamy / Polyhedron 30 (2011) 123–131
CH3
CH3
CH3
OH
2+
OH2
N
O
O
N
N
N
N
O
O
(ii),(iii)
EtOH
-
CHCl3 / CH3OH
(i)
2ClO4
Cu
Cu
Cu
OH
N
N
N
OH2
CH3
CH3
CH3
N
N
N
N
N
(i)
Cu(OAc) .H O
(ii)
(iii)
Cu(ClO4)2 .6H2O
=
2
2
N
Scheme 1. Synthesis of the mono and binuclear Cu(II) complexes.
DNA [15–17]. With in this view, we have prepared side-off mono
and unsymmetrical binuclear copper(II) complexes (each of the
Cu(II) ions is in a different coordination environment, coordinated
by piperazine and bipyridyl or phenanthroline moieties). Similar
types of mono and binuclear complexes have been were reported,
in that one of the copper(II) ions are is coordinated to an azome-
thine or oxamide moiety and the second copper(II) ion is associ-
ated with non-coordinating anions such as perchlorate or
halogens [18–23]. We have already reported the crystal structure
of the ligand (L) [24]. Herein, we report the synthesis of side-off
mono and unsymmetrical binuclear copper(II) complexes (Scheme
1) and their electrochemical, magnetic and catecholase activity.
Complexes 2 and 3 bind to double-stranded DNA in the major
groove and remarkably cleave supercoiled plasmid DNA in the
presence of mercaptoethanol in an oxidative manner.
KBr pellets. UV–Vis spectra were recorded using a Perkin–Elmer
Lambda 35 spectrophotometer operating in the range 200–
1400 nm with quartz cells and
e . A
is given in Mꢀ1 cmꢀ1
CH11008 electrochemical analyzer was used using a three-elec-
trode cell in which a glassy carbon electrode was the working elec-
trode, a saturated Ag/AgCl electrode was the reference electrode
and a platinum wire was used as an auxiliary electrode under oxy-
gen free conditions. The concentration of the complexes was
10ꢀ3 M. TBAP (10ꢀ1 M) was used as the supporting electrolyte.
Electrospray ionization mass spectral measurements were carried
out using a Thermo Finnigan LCQ-6000 Advantage Max-ESI mass
spectrometer. ESR spectra were recorded on powdered samples
of 1–3 using a JEOL TES 100 ESR spectrometer. Low temperature
measurements were made using a liquid nitrogen Dewar.
2.2. Synthesis
2. Experimental
2.2.1. Synthesis of the precursor compound (6,60-piprazine-1,4-diyl-
dimethylene bis (4-methyl phenol) (L)
2.1. Materials and measurements
A formalin solution (10 ml (37%), 0.125 mmol) and piperazine
(5.16 g, 0.05 mmol) were added to a solution of ethanol (50 mL)
and acetic acid (10 mL) with constant stirring. The above mixture
was stirred for 24 h to obtain a clear solution, then 4-methyl phe-
nol (13.05 g, 0.125 mmol) was added and the resulting mixture
was refluxed for about 3 h. A colorless solid was isolated by re-
moval of solvent at reduced pressure. Water (30 ml) and chloro-
form (50 ml) were added to the residue, and the pH of the
aqueous phase was adjusted to 7 with sodium bicarbonate. The
aqueous phase was extracted with chloroform and dried over so-
dium sulfate. Evaporation of the solvent gave the ligand L as a col-
orless solid. Colorless crystals suitable for single crystal X-ray
diffraction studies (Fig. 1) were obtained by recrystallization from
4-Methyl phenol and piperazine were purchased from Loba.
Tetra(n-butyl)ammonium perchlorate (TBAP), used as the support-
ing electrolyte in the electrochemical measurements, was pur-
chased from Fluka and recrystallized from hot methanol. Caution!
TBAP is potentially explosive; hence, care should be taken in han-
dling this compound. All the solvents were purified by reported
procedures [24]. CT-DNA and pBR322 DNA were purchased from
Bangalore Genie (India). All other chemicals and solvents were of
analytical grade and were used as received, without any further
purification. Elemental analysis was carried out on a Carlo Erba
model 1106 elemental analyzer. FT-IR spectra were obtained on a
Perkin–Elmer FT-IR spectrometer with the samples prepared as
Fig. 1. ORTEP diagram of ligand (L).

S. Anbu, M. Kandaswamy / Polyhedron 30 (2011) 123–131
125
chloroform. Yield: 18.5 g (87%). Melting point: 223–225 °C. Anal.
Calc. for C20H26N2O2 (L): C, 73.61; H, 7.97; N, 8.58. Found: C,
73.63; H, 8.06; N, 8.52%. ESI-MS in CH3OH: m/z 327.52 [M+H]+.
1H NMR (CDCl3, 300 MHz): 2.23 (Ar-CH3, s, 6H), 2.4–2.8 (pipera-
zine, bs, 8H), 3.67 (–CH2–, s, 4H), 6.7–6.78 (ArH, m, 16H), 6.96–
6.98 (Ar-H, d, 2H), 5.13 (Ar-OH, s, 2H). 13C NMR (CDCl3, 75 MHz):
20.4 (2CH3), 52.3 (4-CH2), 42.0 (2-CH2), 115.8, 122.7, 129.4,
130.3, 132.9, 155.0 (12-ArC). IR (cmꢀ1, KBr pellet): 2946 (CH
stretching), 1224 (O–C stretching).
wavelength over time intervals of 10 min. The initial reaction rate
values were determined from the slope of the trace at 395 nm dur-
ing the first 30 min of the reaction, until the absorption at 395 nm
increased linearly.
2.4. Magnetic studies
Variable temperature magnetic measurements were performed
on powdered samples of 2 and 3 using a Quantum Design MPMS
XL-5 SQUID magnetometer in the temperature range 80–298 K
with an applied field of 5000 Oe. The contribution of the sample
holder was determined separately in the same temperature range
and field. Diamagnetic corrections were estimated from Pascal’s
constants.
2.2.2. Preparation of the mononuclear copper(II) complex (1) [CuL]
The mononuclear copper(II) complex 1 was prepared by a gen-
eral procedure in which 1 g (0.5 mmol) quantity of copper(II) ace-
tate monohydrate in 10 mL of methanol was added at 25 °C to the
Mannich base L (1.63 g, 0.5 mmol) taken in 10 mL of a methanol
and chloroform (2:1) mixture. The reaction mixture was stirred
for 3 h, and the product was isolated as a green solid. Yield: 0.8 g
(68%). Anal. Calc. for C20H24CuN2O2: C, 61.93; H, 6.19; N, 7.22;
Cu, 16.38. Found: C, 62.07; H, 6.22; N, 7.29; Cu, 16.30%. ES-MS in
CH3OH: m/z: [CuL+H] (388.27). FT-IR, cmꢀ1 (KBr disc): 3343br,
2923s, 1632s, 1584s, 502w (br, broad; s, strong; m, medium; w,
2.5. DNA binding and cleavage experiments
2.5.1. Absorption spectral studies
The DNA binding experiments were performed in Tris–HCl/NaCl
buffer (50 mM Tris–HCl/1 mM NaCl buffer, pH 7.5) using a di-
methyl formamide (DMF) (10%) solution of complexes 2 and 3.
The concentration of CT DNA was determined from the absorption
weak). KM = 2 S cm2 Mꢀ1 in DMF at 25 °C. kmax, nm (
ꢀ )
, Mꢀ1 cmꢀ1
intensity at 260 nm with a
e
value [25] of 6600 Mꢀ1 cmꢀ1. Absorp-
in DMF: 504 (134), 322 (38 000), 268 (56 000). g = 2.22,
k
g\ = 2.06, A = 176; leff, lB at 298 K: 1.69.
k
tion titration experiments were made using different concentra-
tions of CT DNA, while keeping the complex concentration
constant. Due correction was made for the absorbance of the CT
DNA itself. Samples were equilibrated before recording each spec-
trum. For complexes 2 and 3 the intrinsic binding constant K has
been determined from the spectral titration data using the follow-
ing equations [25]:
2.2.3. Preparation of the binuclear copper(II) complex (2)
[Cu2L(bipy)](ClO4)2
The binuclear Cu(II) complex 2 was prepared from a general
synthetic procedure in which an ethanolic solution of Cu(-
ClO4)2ꢂ6H2O (0.47 g, 1.3 mmol) was added slowly to a vigorously
stirred suspension of the mononuclear complex 1 (0.5 g, 1.3 mmol)
in ethanol (10 ml), and the mixture was stirred for 15 min to obtain
a clear solution. Then an ethanolic solution (5 ml) of 2,2-bipyridyl
(0.2 g, 1.3 mmol) was added dropwise to the above solution and
the resulting solution was stirred for 3 h at 25 °C. A dark green col-
ored solid separated on evaporation of the solvent at ambient tem-
perature and this was recrystallized from acetonitrile. Yield: 0.76 g
(70%). Anal. Calc. for C30H36Cu2Cl2N4O12: C, 42.75; H, 4.04; N, 6.65;
Cu, 15.08. Found: C, 42.62; H, 4.08; N, 6.59; Cu, 15.16%. ES-MS in
CH3OH: m/z: [Cu2L(bpy)–2ClO4–H]+ (641.87), [Cu2L(bpy)–2ClO4]2+
(321.13). KM = 202 S cm2 Mꢀ1 in DMF at 25 °C. FT-IR, cmꢀ1 (KBr
disc): 3364br, 2923s, 1625s, 1089s, 1115s, 626s, 502w. kmax, nm
ð
ea
ꢀ
ef Þ=ðeb
ꢀ
ef Þ ¼ ðb ꢀ ðb2 ꢀ 2K2Ct½DNAꢃ=sÞ1=2=2KCtÞ
ð1:aÞ
ð1:bÞ
b ¼ 1 þ KCt þ K½DNAꢃ=2s
where [DNA] is the concentration of CT DNA in base pairs, the
apparent absorption coefficients ea ef and eb correspond to Aobsd
,
/
[Complex], the absorbance for the free copper(II) complex, and
the absorbance for the copper(II) complex in the fully bound form,
respectively. K is the equilibrium binding constant in Mꢀ1, Ct is the
total metal complex concentration, and s is the binding size. The
non-linear fit analysis was done using Origin Lab, version 6.1. Circu-
lar dichroic spectra of DNA in the presence and absence of the Cu(II)
complexes were obtained by using a J-715 spectropolarimeter at
25 °C with a 0.1 cm path length cuvette.
(ꢀ
, Mꢀ1 cmꢀ1) in DMF: 558 (158), 310 (40 000), 297 (62 000) 273
(113 000). g = 2.1312; leff lB at 298 K: 1.32.
,
2.5.2. Fluorescence spectral studies
2.2.4. Preparation of the binuclear copper(II) complex (3)
[Cu2L(phen)](ClO4)2
The fluorescence spectral method using ethidium bromide (EB)
as a reference was used to determine the relative DNA binding
properties of complexes 2 and 3 to CT DNA in 50 mM Tris–HCl/
1 mM NaCl buffer, pH 7.5. Fluorescence intensities of EB at
735 nm with an excitation wavelength of 530 nm were measured
at different complex concentrations. Reduction in the emission
intensity was observed with addition of the complexes. The rela-
tive binding tendency of the complexes to CT DNA was determined
from a comparison of the slopes of the lines in the fluorescence
intensity versus complex concentration plot. The apparent binding
The binuclear complex 3 was prepared by following the above
procedure using 1,10-phenanthroline instead of using 2,2-bipyri-
dyl. Green solid, Yield: 0.82 g (73%). Anal. Calc. for C32H36Cu2Cl2-
N4O12: C, 44.34; H, 3.92; N, 6.46; Cu, 14.66. Found: C, 44.41; H,
3.86; N, 6.51, Cu, 14.54%. ES–MS in CH3OH: m/z: [Cu2L(phen)–
2ClO4–H]+ (666.15), [Cu2L(phen)-2ClO4]2+ (333.57). KM = 204 S
cm2 Mꢀ1 in DMF at 25 °C. FT-IR, cmꢀ1 (KBr disc): 3430br, 2925s,
1629s, 1087s, 1113s, 625s, 524w. kmax, nm (
592 (172), 293 (40 000), 268 (138 000), 224, (163 000). g = 2.1221;
lB at 298 K: 1.35.
ꢀ
, Mꢀ1 cmꢀ1) in CH3CN:
constant (Kapp) was calculated using the equation KEB[EB]/
leff
,
Kapp[complex], where the complex concentration equaled the value
at a 50% reduction of the fluorescence intensity of EB and
2.3. Catecholase activity
KEB = 1.0 ꢁ 107 Mꢀ1 ([EB] = 3.3
lM) [26].
The catalytic oxidation of 4,5-di-tert-butyl catechol (3,5-dtbc)
to the corresponding o-quinone (3,5-dtbq) was studied using
10ꢀ3 M of the copper(II) complexes as a catalyst in acetonitrile
solution. The reaction was followed spectrophotometrically by
choosing the strongest absorbance of the product, dtbq, around
395 nm and monitoring the increase in the absorbance at this
2.5.3. DNA cleavage study
The cleavage of plasmid DNA was determined by agarose gel
electrophoresis. The gel electrophoresis experiments were per-
formed on pBR322 DNA (0.020 mg/mLꢀ1) treated with the copper
(II) complexes 1–3 (0.01 mM) in 50 mM Tris–HCl/NaCl buffer (pH

126
S. Anbu, M. Kandaswamy / Polyhedron 30 (2011) 123–131
7.2) in the presence of mercaptoethanol as a reducing agent. The
concentration of the complexes in water or the additives in the
buffer corresponded to the quantity in 2
lL stock solution after
dilution to the 18 L final volume using Tris–HCl buffer. All the
l
samples were incubated for 1 h at 37 °C, followed by addition to
the loading buffer containing 25% bromophenol blue, 0.25% xylene
cyanol, 30% glycerol (3
0.8% agarose gel containing EB (1
l
L). All the samples were finally loaded on
l
g/mLꢀ1). Electrophoresis was
carried out at 50 V for 1 h in TBE buffer (45 mM Tris, 45 mM
H3BO3, 1 mM-EDTA, pH 8.3). The resulting bands were visualized
by UV light and photographed. Quantification of closed circular,
nicked and linear DNA was made by densitometric analysis of ethi-
dium bromide containing agarose gels. Quantification was per-
formed by fluorescence imaging using Gel-Doc 1000 (BioRad) and
data analysis with Multianalysis software (version 1.1), provided
by the manufacturer using the volume quantification method. In
all cases, background fluorescence was subtracted by reference to
a lane containing no DNA. A correction factor of 1.47 was used
for supercoiled DNA since the ability of EB to intercalate into
supercoiled DNA (form I) decreased relative to nicked DNA (form
II). The fraction of each form of DNA was calculated by dividing
the intensity of each band by the total intensities of all the bands
in the lane. All results were obtained from experiments that were
performed at least in triplicate.
Fig. 2. X-band EPR spectrum of the frozen solid (1 and 3) at 77 K.
3.2. Electrochemistry
The electrochemical behavior of the mono and binuclear Cu(II)
complexes 1–3 has been studied by cyclic voltammetry (CV) in
CH3CN containing 0.1 M TBAP. The electrochemical data are sum-
marized in Table 1 and the cyclic voltammograms of the copper(II)
complexes 2 and 3 are depicted in Fig. 3. The mononuclear Cu(II)
3. Results and discussion
complex 1 show one quasi-reversible reduction wave (Ep1c
ꢀ0:95 V and E1pa ¼ ꢀ0:79 V) in the cathodic potential at E1/2
¼
=
3.1. General properties
ꢀ0.87 V versus Ag/AgCl. When compared to the mononuclear
Cu(II) complex reported here, [CuL] reduces at a slightly higher
reduction potential than the mononuclear Cu(II) complex [CuLa]
[29]. This may be due to the aldehyde group present ortho to the
The IR spectra of the binuclear Cu(II) complexes 2 and 3 showed
a strong band in the region around 1000–1100 cmꢀ1 and a sharp
band in the region around 626 cmꢀ1, which could be due to the
antisymmetric stretch and antisymmetric bend of the perchlorate
ions, respectively. No splitting pattern of the perchlorate peak
was observed. This indicates that the perchlorate ions are not coor-
dinated to the Cu(II) ions and are present as counter ions in the
crystal lattice. The important strong band observed at around
3460 cmꢀ1 for both of the binuclear Cu(II) complexes is assignable
to the OH vibration of water molecules.
Table 1
Electrochemical data of the mono and binuclear Cu(II) complexes.
Ep1
E1pa
E11=2
(
D
E1p)
E2pc
E2pa
E21=2 E2p)
(D
Complexes
1
2
3
ꢀ0.95
0.37
0.24
ꢀ0.79
0.18
0.12
ꢀ0.87 (160)
0.28 (190)
0.18 (120)
0.80
0.73
0.63
ꢀ0.59
0.72 (170)
0.66 (140)
The electronic spectra of DMF solutions of complexes 1, 2 and 3
show three intense intraligand bands in the UV region. In the vis-
ible region, complexes 1, 2 and 3 exhibit absorption maxima at
504, 592 and 558 nm, respectively. This strongly suggests that
the coordination geometry around the metal ion in the mononu-
clear complex 1 might be distorted square planar and in the binu-
clear complexes 2 and 3 it might be distorted square pyramidal
[27]. A decrease in kmax (blue shift) of complex 2 may be due to
the larger distortion of the coordination geometry around the
Cu(II) ion than for complex 3.
The positive ion ESI mass spectrum shows a signal for 1 pre-
dominantly at m/z = 388.27, corresponding to the [CuL+H] species
[28]. The binuclear Cu(II) complexes 2 and 3, [Cu2L(B)(H2O)2](2-
ClO4) (B = bpy or phen), show a major peak at m/z = 321.13 and
333.57 respectively, corresponding to the [Cu2L(B)-2ClO4]2+ ion.
The ESI mass spectral data of the copper(II) complexes confirm
the proposed formula of the complexes. The solid-state ESR spec-
trum of the mononuclear copper(II) complex 1 shows four lines
with nuclear hyperfine spin 3/2 due to hyperfine splitting. For the
mononuclear complex 1, the observed g , g\ and A values are
k
k
2.22, 2.06 and 176 respectively. The observed ESR spectral values
are similar to a piperazine based Mannich base mononuclear Cu(II)
complex [29]. Broad spectra centered at g = 2.10–2.11 are observed
for the binuclear copper(II) complexes due to an antiferromagnetic
interaction between the two copper ions. Fig. 2 shows the ESR spec-
tra of the mono and binuclear copper(II) complexes 1 and 3.
Fig. 3. Cyclic voltammograms of the Cu(II) complexes 2 and 3.

S. Anbu, M. Kandaswamy / Polyhedron 30 (2011) 123–131
127
237.5 cmꢀ1 respectively. The singlet–triplet energy separation
(ꢀ2J) values are comparable to the ꢀ2J values of a similar type of
binuclear copper(II) complexes reported in the literature [20–22].
A plot of vM and leff versus T for complex 3 is depicted in Fig. 4.
Magnetostructural correlation in the phenoxo-bridged dicopper(II)
complexes reveals that the major factor that controls the exchange
interactions is the Cu–O–Cu bridge angle; however other factors,
such as the degree of distortion from planarity and the reduction
in electron density on the copper atoms, reduce the effective over-
lap of the metal orbitals with those of the bridging ligand, and
hence reduce the antiferromagnetic spin exchange between the me-
tal centers [31]. Thus the antiferromagnetic exchange interaction
between the copper centers is likely to be influenced by the degree
of planarity of the oxygen bridges, phenoxide bridge angle [32] and
phenolate ions of the reported [CuLa], which reduces the electron
density of the phenolate ion and makes the reduction easier. The
binuclear Cu(II) complexes 2 and 3 are associated with two qua-
si-reversible reduction waves. The first and second reduction
potentials for the binuclear Cu(II) complexes 2 and 3 are observed
at E1pc ¼ ꢀ0:37 and ꢀ0.24 V and Ep2c ¼ ꢀ0:80 and ꢀ0.73 V respec-
tively. Coulometric titrations by potentiostatic exhaustive electrol-
ysis performed at 100 mV more negative to the first (n ꢄ 0.96) and
second (n ꢄ 0.94) reduction waves consumed approximately one
electron for each step. This indicates that each process corresponds
to a single electron transfer process, and this can be assigned to the
redox couples CuIICuII/CuIICuI and CuIICuI/CuICuI.
3.3. Magnetochemistry
the extent of out-of-plane displacement (s) of the phenyl ring from
the oxygen atom within the Cu2O2 core [33]. It is noted that the
exchange interaction value of the binuclear Cu(II) complex 3 is
higher than the binuclear Cu(II) complex 2. This indicates that the
exchange interaction value increases with the increasing coplana-
rity of the heterocyclic bases as well as the deviation from coplan-
arity of the phenyl ring [34] from the oxygen atom within the Cu2O2
core.
The observed magnetic moment value of the mononuclear
Cu(II) complex at room temperature is 1.68 lB, which is somewhat
lower than the spin only value. This is due to the monomeric nat-
ure of the complex and there is no possibility of an exchange inter-
action. The magnetic susceptibility of the binuclear Cu(II)
complexes 2 and 3 were measured at 5000 Oe in the temperature
range 80–298 K. The observed vM values of 2 and 3 at 298 K were
found to be 2.9 ꢁ 10ꢀ3 and 3.1 ꢁ 10ꢀ3 cm3 molꢀ1 respectively, and
upon lowering the temperature, the vM value increased (Fig. 4).
3.4. Catecholase activity
The magnetic moment values 1.32 and 1.35 lB/Cu are observed
at room temperature for complexes 2 and 3 respectively. This indi-
cates the presence of antiferromagnetic coupling by super ex-
change through the phenolic oxygen atoms [27]. To evaluate the
singlet–triplet energy separation (ꢀ2J), variable temperature mag-
netic studies for complexes 2 and 3 were performed in the temper-
ature range 80–298 K and the extent of the exchange interaction
has been calculated using the modified Bleany–Bowers equation
[30],
The mono and binuclear copper(II) complexes 1, 2 and 3 were
tested for catecholase activity. The time dependent formation of
DTBQ in the presence of 1, 2 and 3 is shown in Fig. 5. The initial rate
values for the mono and binuclear copper(II) complexes 1, 2 and 3
are 2.6 ꢁ 10ꢀ7, 6.8 ꢁ 10ꢀ5 and 2.3 ꢁ 10ꢀ5 M sꢀ1 respectively. The
oxidation of catechol to o-quinone requires the presence of two
metal ions in close proximity; hence the initial rate values of the
binuclear complexes are comparatively higher than the mononu-
clear complex [35–37]. The kinetic data reveal that the binuclear
Cu(II) complexes 2 and 3 show a nearly 100-fold rate enhancement
of catechol oxidation compared to the mononuclear Cu(II) complex
1. Catechol oxidation studies show that the bipyridyl based com-
plex 2 has a higher catalytic activity than the phenanthroline con-
taining Cu(II) analog 3. This is because the intrinsic flexibility of the
bipyridyl containing ligand is higher than the phenanthroline con-
taining ligand system, which leads to a distortion in the geometry
of the complex.
ꢀ
ꢁ
vM
¼
N2b2=3Kt ½3 þ expðꢀ2J=kTÞꢀ1ð1 ꢀ
q
Þ þ 0:45 =T þ Naꢃ
q
g
ð2Þ
In this equation, the values of N and g have been fixed as
a
60 ꢁ 10ꢀ6 cm3 Mꢀ1 and 2.20, respectively. vM is the paramagnetic
susceptibility per metal atom after correction for diamagnetism, g
is the average gyromagnetic ratio and
q is the percentage of mono-
meric impurities; the other symbols have their usual meaning. The
ꢀ2J values were evaluated by a non-linear regression analysis, in
which ꢀ2J,
q
and g are the variables. The ꢀ2J values of the unsym-
metrical binuclear Cu(II) complexes
2 and 3 are 228.3 and
Fig. 4. Experimental variation of the molar susceptibility (per Cu) and effective
magnetic moment ( eff) with temperature for the Cu(II) complex 3.
Fig. 5. A comparison of time dependent oxidation of 3,5-DTBC by different
complexes (a) 1, (b) 2, and (c) 3.
l

128
S. Anbu, M. Kandaswamy / Polyhedron 30 (2011) 123–131
3.5. DNA binding and cleavage properties
and 312 nm (for complex 3) with increasing concentration of
DNA. The extent of hypochromism gives a measure of the strength
of the intercalative binding. The observed trend in hypochromism
among the present complexes follows the order phenanthroline
based binuclear Cu(II) complex 3 > bipyridyl based binuclear Cu(II)
complex 2. The binding constants (K) of complexes 2 and 3 are cal-
culated as 0.37( 0.1) ꢁ 105 (s = 0.1) and 0.44( 0.2) ꢁ 105 Mꢀ1
(s = 0.1) respectively. The better binding of complex 3 than com-
plex 2 may be due to the extended aromaticity and coplanarity
of the phenanthroline ring system in complex 3, which is expected
to be stacked between the base pairs upon interaction of the com-
plex with DNA [40]. These spectral characteristics obviously sug-
gest that the two complexes in our paper interact with DNA,
most likely through a mode that involves a stacking interaction be-
tween the aromatic chromophore and the base pairs of DNA. Com-
plex 3 shows a minor bathochromic shift of the spectral band of
ꢅ3 nm, suggesting a groove-binding preference of the complex.
Structurally, the ligand in 3 should provide a more aromatic moiety
to overlap with the stacking base pairs of the DNA helix by interca-
lation which results in hypochromism and bathochromism. The in-
creased aromatic moiety in the phenanthroline based ligand of the
Cu(II) complex facilitates its potential intercalative and/or DNA
major groove binding, while complex 2, with a bipyridyl moiety,
may prefer minor groove binding.
3.5.1. Absorption spectral studies
The binding ability of complexes 2 and 3 with calf thymus (CT)
DNA is characterized by measuring their effects on the UV spectra.
Complex binding with DNA through intercalation usually result in
hypochromism and bathochromism due to the intercalative mode
involving a strong stacking interaction between an aromatic chro-
mophore and the base pairs of DNA [38]. In the present investiga-
tion the interaction of the bipyridyl and phenanthroline based
binuclear Cu(II) complexes 2 and 3 in DMF solutions (10%) with
CT DNA have been investigated (Fig. 6). Absorption titration exper-
iments of the Cu(II) complexes in buffer were performed by using a
fixed copper concentration to which increments of the DNA stock
solution were added. The binding of the Cu(II) complexes to duplex
DNA led to a decrease in the absorption intensities with a small
amount of red shift in the UV–Vis absorption spectra. After interca-
lating the base pairs of DNA, the
*
p orbital of the intercalated li-
gand can couple with the
decreasing the p–p transition energy and resulting in the batho-
p orbital of the base pairs, thus
*
chromism [39]. To compare quantitatively the affinity of the two
complexes toward DNA, the binding constant K of the two com-
plexes to CT DNA was determined by monitoring the changes of
absorbance (Fig. 6) at 268 and 293 nm (for complex 2) and 298
Fig. 6. Absorption spectra of complexes 2 and 3 (20
l
M) in the absence (line a) and presence of increasing amounts of CT DNA (0–250
lM) at room temperature in 50 mM
Tris–HCl/NaCl buffer (pH 7. 5). The arrow shows the absorbance changing upon increasing the DNA concentration. Inset shows the plot of (ea
titration of DNA to the Cu(II) complexes.
ꢀ
e
f)/(eb f) vs [DNA] for the
ꢀ e

S. Anbu, M. Kandaswamy / Polyhedron 30 (2011) 123–131
129
Fig. 7. Emission spectrum of EtBr bound to DNA in the presence of 3. ([EtBr] = 3.3
lM, [DNA] = 40
lM, [complex] = 0–25
lM, kex = 510 nm). The arrow shows the intensity
change upon increasing Cu(II) complex (3) concentrations. Inset shows the plots of emission intensity Io/I vs [DNA]/[complex].
3.5.2. Fluorescence spectral studies
The fluorescence spectroscopy technique is an effective method
to study metal interaction with DNA. Ethidium bromide (EB) is one
of the most sensitive fluorescence probes that can bind with DNA
[41]. The fluorescence of EB increases after intercalating into
DNA. If the metal intercalates into DNA, it leads to a decrease in
the binding sites of DNA available for EB, resulting in a decrease
in the fluorescence intensity of the EB-DNA system [42]. The emis-
sion spectra of EB bound to DNA in the absence and presence of
complex 3 are shown in Fig. 7. The addition of the complex to
DNA pretreated with EB causes an appreciable reduction in the
fluorescence intensity, indicating that complex 3 competes with
EB to bind with DNA. The reduction of the emission intensity gives
a measure of the DNA binding propensity of the complexes and
stacking interaction (intercalation) between adjacent DNA base
pairs [43]. The fluorescence quenching curve of DNA-bound EB
by complexes 2 and 3 illustrates that the quenching of EB bound
to DNA by complexes 2 and 3 is in good agreement with the linear
Stern–Volmer equation. In the linear fit plot of I0/I versus [com-
plex]/[DNA], K is given by the ratio of the slope to the intercept.
The K values for complexes 2 and 3 are 2.13 (R = 0.991) and 3.9
(R = 0.992) respectively (I0 is the emission intensity of EB-DNA in
the absence of complex; I is the emission intensity of EB-DNA in
the presence of complex). The concentrations of the complexes
were taken for observing a 50% reduction of the emission intensity
of EB [44]. From the data in Fig. 8, we know that 50% of EB mole-
cules were replaced from DNA-bound EB at a concentration ratio
of [Cu2 complex]/[EB] of 4.84 for 2 and 3.63 for 3. By taking a
DNA binding constant of 1.0 ꢁ 107 Mꢀ1 for EB [45], apparent DNA
binding constants of 1.83 ꢁ 106 Mꢀ1 and 2.75 ꢁ 106 Mꢀ1 were de-
rived {Kb(EB)/([Cu2 complex]/[EB])} for complexes 2 and 3 respec-
tively. The Kapp values imply that both complexes can strongly
interact with DNA and are protected by DNA efficiently, since the
hydrophobic environment inside the DNA helix reduces the acces-
sibility of solvent water molecules to the complex and the com-
plex’s mobility is restricted at the binding site [46]. The greater
Kapp value for complex 3 may be due to the presence of the aro-
matic moiety, which enhances the binding propensity of the mol-
ecule to DNA. The presence of a 4-methylphenol group and also
the hydrophobic property of the rigid ligand also facilitate the
DNA binding [47].
Fig. 8. The effect of addition of complex 2 (
D) and 3 (j) on the emission intensity of
40 M CT DNA-bound EtBr in Tris–HCl/NaCl buffer (50 mM, pH 7.5) at 25 °C; (h) on
l
the emission intensity of the EtBr in absence of CT DNA but at different
concentrations of 2 and 3.
DNA exhibits band at 272 nm (UV: kmax 260 nm) due to base stack-
ing and a negative band at 239 nm due to the helicity of B-DNA
[50]. Incubation of the DNA with complexes 2 and 3 induced con-
siderable changes in the CD spectrum. Fig. 9 illustrates the effect of
bipyridyl and phenanthroline based binuclear ternary Cu(II) com-
plexes on the CD spectrum of CT DNA. At a [Cu2L3 and 4]:CT DNA ra-
tio of 2:1, all the copper(II) complexes produced shifts to a higher
energy for the positive CD signal, as well as an enhancement of the
CD elipticity at 272 nm. Examination of Fig. 9 shows that the mag-
nitude of the increases in elipticity at 272 nm increase in the fol-
lowing order: 2 < 3. The results reveal that the interaction is
through preferential groove binding with CT DNA and induce con-
formational changes from B-DNA to Z-DNA. However, the changes
induced by 3 are more significant than those by 2, which suggest
that 3 has a higher affinity for CT DNA than complex 2. It is reason-
able to suggest that the higher affinity of complex 3 with CT DNA
due to the presence of phenanthroline in the compartment of the
complex, which enhances the interaction. The bipyridyl based
Cu(II) complex 2 induced only slight conformational changes in
DNA, such as the conversion from a more B-like to C-like structure
within the DNA molecule. Interaction with cations effectively
screens the negative charge on N(7) base sites as well as phosphate
3.5.3. CD spectral studies
Circular dichroism (CD) is a powerful method for distinguishing
the three main DNA binding modes, namely intercalative (negative
CD), outside groove binding (positive CD), and outside stacking
(bisignate CD) [48,49]. The UV circular dichroic spectrum of CT

130
S. Anbu, M. Kandaswamy / Polyhedron 30 (2011) 123–131
strands are cleaved, a linear form (Form III) that migrates between
Form I and Form II will be generated [51]. The cleavage mechanism
of pBR322 DNA induced by complexes 2 and 3 was investigated
(Fig. 11A and B) and clarified in the presence of singlet oxygen
quenchers L-histidine [52] (lanes 2 and 3), sodium azide (Fig. 12
(lane 2)) and superoxide dismutase (SOD) (lane 3), and hydroxyl
radical scavengers 0.4 M DMSO and methanol (lanes 4 and 5)
[53]. It is remarkable that DMSO, methanol and SOD are com-
pletely ineffective, and these results rule out the possibility of
DNA cleavage by hydroxyl radicals and superoxide anions. Fig. 12
shows that there is no DNA damage under an argon atmosphere
(lanes 3–6). This indicates the necessity of oxygen in the cleavage
reactions. Complexes 2 and 3 do not exhibit DNA damage activity
in the presence of singlet oxygen quenchers like L-histidine and the
azide anion. These observations suggest that 2 and 3 mediated
cleavage reactions proceed via an oxidative pathway mechanism
and imply that singlet oxygen plays a role in the cleavage chemis-
try. In the presence of mercaptoethanol, the Cu(II) complexes 2 and
3 remarkably degrade pBR322 DNA by an oxidative (O2-dependent
pathway) cleavage mechanism using singlet oxygen as the reactive
species [54]. The phenanthroline based binuclear Cu(II) complex 3,
at a low concentration (0.50 mM), shows better chemical nuclease
activity (above 89%) than the bipyridyl based binuclear Cu(II) com-
plex 2 (84%) and the mononuclear Cu(II) complex 1 (68%). The
structure of the ligand plays an important role in the cleavage
[48]. A rigid structure of the ligand shows a better DNA cleavage
ability after combining to the metal ions, because it may involve
a synergistic mechanism with the rigid complex. The disappear-
ance of the SC versus time followed pseudo first-order kinetics.
The reaction profile for the complex 3 mediated reaction displayed
approximately pseudo-first-order kinetic behavior (Fig. 13), with
kobs ꢅ 0.07 minꢀ1 and R2 = 0.984. On comparison of the cleavage
property with some reported results [55,56] of Cu(II) complexes,
we note that a lesser amount of the reducing agent (here mercap-
toethanol) and complex are required in our case for maximum
cleavage of DNA. This is well reflected from the cleavage data
which show that more than 85% of cleavage occurs with a very
Fig. 9. CD spectra recorded over the wavelength range 230–320 nm for solutions
Containing a 2:1 ratio of CT DNA (200
(100 M).
lM) and binuclear Cu(II) complexes 2 and 3
l
oxygens simultaneously, both along the deoxyribophosphate back-
bone and in the groove of the helix, to promote such a transconfor-
mational change.
3.5.4. DNA cleavage activity
The cleavage of supercoiled pBR322 DNA was studied in a med-
ium of 50 mM Tris–HCl/NaCl buffer (pH 7.2) in the presence of
mercaptoethanol as a reducing agent. All the mono and binuclear
complexes 1–3 showed remarkable cleavage. The results of the
gel electrophoresis separations of plasmid pBR322 DNA by the
complexes 1–3 in the presence of mercaptoethanol are depicted
in Fig. 10. Under similar conditions, no cleavage of pBR322 DNA oc-
curred for free mercaptoethanol or the complexes (lanes 1–5). No
cleavage was found at low concentration (0.30 mM) of the mono-
nuclear Cu(II) complex (lane 6). All the supercoiled (Form I) DNA
was cleaved to Form II with a concentration of 0.50 mM of mono-
nuclear complex (lane 7). At a same concentration of the binuclear
complexes (0.50 mM), the cleavage is found to be much more effi-
cient, as is seen from the formation of the linear form (Form III) in
lanes 8 and 9. In the presence of the binuclear complex, the prob-
ability of double strand scission is enhanced once the DNA has
undergone a single strand break. It is clear that the cleavage of
pBR322 DNA is highly dependent on the number of metal ions as
well as the presence of an aromatic moiety in the complexes. At
the concentration of 0.50 mM, complex 1 can almost promote the
complete conversion of DNA from Form I to Form II (68%). When
circular plasmid DNA is subjected to electrophoresis, relatively fast
migration will be observed for the intact supercoil form (Form I). If
scission occurs on one strand (nicking), the supercoil will relax to
generate a slower moving open circular form (Form II). If both
low complex concentration (50 lM). Meanwhile, due to the rigid
Fig. 11. Cleavage of SC pBR322 DNA (0.2 lg, 33.3 lM) by Cu(II) complexes 1 and 2
(0.50 mM) in the presence of the reducing agent mercaptoethanol (0.71 mM) in
50 mM Tris–HCl/NaCl buffer (pH 7.2). (A) Lane 1, DNA control; lane 2,
DNA + mercaptoethanol +
toethanol + SOD (4 units) + 2; lane 4, DNA + mercaptoethanol + DMSO (2
lane 5, DNA + mercaptoethanol + methanol (2 L) + 2. (B) lane 1, DNA control; lane
2, DNA + mercaptoethanol + -histidine (0.4 mM) + 3; lane 3, DNA + mercap-
toethanol + SOD (4 units) + 3; lane 4, DNA + mercaptoethanol + DMSO (2 L) + 3;
lane 5, DNA + mercaptoethanol + methanol (2 L) + 3.
L-histidine (0.4 mM) + 2; lanes 3, DNA + mercap-
lL) + 2.
l
L
l
l
Fig. 10. Cleavage of SC pBR322 DNA (0.2 lg, 33.3 lM) by Cu(II) complexes 1–3
(0.50 mM) in the presence of the reducing agent mercaptoethanol (0.71 mM) in
50 mM Tris–HCl/NaCl buffer (pH 7.2). Lane 1, DNA control; lane 2, DNA + mercap-
toethanol; lanes 3–5, DNA + 1, 2 and 3 (0.30 mM) respectively; lanes 6 and 7,
Fig. 12. Anaerobic cleavage of supercoiled plasmid DNA (0.2
complexes 1–3 (0.50 mM). Lane 1: DNA control; lane 2: DNA + 0.71 mM mercap-
toethanol + NaN3 (90 M) b + 3 (aerobic condition); lanes 3–5, DNA + mercap-
toethanol + 1, 2 and 3 respectively.
lg, 33.3 lM) by
DNA + mercaptoethanol + 1 (0.30
lM and 0.50
l
M) respectively; lanes 8 and 9,
l
DNA + mercaptoethanol + 2 and 3 (0.50
l
M) respectively.

S. Anbu, M. Kandaswamy / Polyhedron 30 (2011) 123–131
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L-histidine and azide ions were obviously inhibiting the cleavage
activity. In conclusion, we have performed a comparative study
of the influence of aliphatic and aromatic moieties in acyclic li-
gands on DNA cleavage activity. Cu(II) complexes with phenan-
throline based ligands display a better DNA interaction and
significantly greater chemical nuclease activity than the bipyridyl
based Cu(II) analogs.
Acknowledgements
S.A. is grateful to CSIR (SRF), New Delhi, Government of India,
for a fellowship. We thank the Department of Science and Technol-
ogy (DST-FIST), New Delhi, Government of India, for financial
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