DNA binding and nuclease activity of copper(II) complexes of tridentate ligands

Article abstract of DOI:10.1016/j.ica.2011.06.022

Two copper(II) complexes, 1 and 2 with L₁ and L₂ [L₁ = 2-hydroxybenzyl(2-(pyridin-2-yl)ethylamine); L₂ = 2-hydroxybenzyl(2-(pyridin-2-yl)methylamine)] ligands, respectively, have been synthesized and characteriz

Full text of DOI:10.1016/j.ica.2011.06.022

Inorganica Chimica Acta 376 (2011) 264–270
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Inorganica Chimica Acta
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DNA binding and nuclease activity of copper(II) complexes of tridentate ligands
a
a
b
b
a,
⇑
Pankaj Kumar , Basudeb Baidya , Sumit Kumar Chaturvedi , Rizwan Hasan Khan , Debasis Manna ,
a,
⇑
Biplab Mondal
a
Department of Chemistry, Indian Institute of Technology Guwahati, North Guwahati, Assam 781039, India
Interdisciplinary Biotechnology Unit, Aligarh Muslim University, Aligarh 202002, India
b
a r t i c l e i n f o
a b s t r a c t
Article history:
1
Two copper(II) complexes, 1 and 2 with L1 and L2 [L = 2-hydroxybenzyl(2-(pyridin-2-yl)ethylamine);
L = 2-hydroxybenzyl(2-(pyridin-2-yl)methylamine)] ligands, respectively, have been synthesized and
2
Received 22 March 2011
Received in revised form 7 June 2011
Accepted 10 June 2011
characterized. The interaction of both the complexes with DNA has been studied to explore their poten-
tial biological activity. The DNA binding properties of the complexes with calf thymus (CT) DNA were
studied by spectroscopic titration. The complexes show binding affinity to CT DNA with binding constant
Available online 17 June 2011
5
ꢀ1
b
(K ) values in the order of 10 M . Thermal denaturation and circular dichroism studies suggest groove
Keywords:
Copper complexes
DNA
Binding
Cleavage
binding of the complexes to CT DNA. Complexes also exhibit strong DNA cleavage activity in presence of
reducing agents like 3-mercaptopropionic acid and b-mercaptoethanol. Mechanistic studies reveal the
involvement of reactive hydroxyl radicals for their DNA cleavage activity.
Ó 2011 Elsevier B.V. All rights reserved.
1
. Introduction
Irreversible modification of DNA by transition metal complexes
by attractive electrostatic interaction with the phosphate backbone,
but these are rather weak in nature. Hence, strategies to develop
copper(II) complexes which can bind DNA through intercalation
or groove binding has been attracted a great attention.
At the same time, the target specificity is also an important
parameter for the DNA cleavage and nuclease activity studies which
can be attained by right choice of metal–ligand combination.
We have been systematically studying the DNA cleavage and
nuclease activity of various copper(II) complexes of bidentate
and tridentate ligands starting from noncytotoxic to moderately
cytotoxic in nature. In this regard, for the present study two
has drawn considerable interest because of their potential applica-
tions as biological tools or as therapeutic agents. The chemical
nuclease activity of these complexes is attributed to their ability
to mediate oxidative damage to nucleobases and/or to the 2-deoxy-
ribose moiety [1–18]. Knowledge of DNA binding parameters of
these molecules is useful to explore their potential applications,
since their binding mode could be associated with their ability to
cause DNA damage [19–21]. Complexes of essential metals like cop-
per and others are generally less toxic than of nonessential ones and
some of them even have important cellular cytotoxic effects. In
addition, high nucleobase affinity and comparatively strong Lewis
acidity of copper(II) ion induce efficient DNA cleavage activity. As
a result, copper complexes and their interactions with DNA have
1 2 1
ligands, L and L [L = 2-hydroxybenzyl(2-(pyridin-2-yl)ethyla-
mine; L = 2-hydroxybenzyl(2-(pyridin-2-yl)methylamine)] have
2
been chosen considering that the phenolic-OH group may enhance
the affinity of the complexes towards DNA binding through the for-
mation of hydrogen bonding. On the other hand, because of the
geometry of these complexes, they are likely to bind the polyan-
ionic DNA via noncovalent outer-sphere coordination unlike the
square planar complexes.
2
+
attracted great interest. Since the first report of [Cu(phen)
2
]
(
phen = 1,10-phenanthroline) to display highly efficient oxidative
cleavage of DNA, a considerable volume of the literature has been
come out in this direction [22–29]. Recently, some of the mononu-
clear and polynuclear copper complexes with sulfonamides, pyri-
dophenazine, ferrocenyl and others are reported to show strong
chemical nuclease activities [30–42]. However, for an efficient
DNA cleavage activity the metal ion has to be placed into the close
proximity of DNA. It is known that the cations can bind to the DNA
Here we report the syntheses of two copper(II) complexes, 1
and
2
with
L
1
and
2 1
L [L = 2-hydroxybenzyl(2-(pyridin-2-yl)
ethylamine; L
2
= 2-hydroxybenzyl(2-(pyridin-2-yl)methylamine)]
ligands, respectively (Fig. 1); single crystal structure of complex
2 and the interaction of both the complexes with DNA to explore
their potential biological activity. The DNA binding properties of
the complexes with calf thymus DNA (CT DNA) were studied by
spectroscopic titration. The DNA conformational changes and
cleavage activity of the complexes upon binding to the DNA were
also studied in detail.
⇑
Corresponding authors.
E-mail addresses: dmanna@iitg.ernet.in (D. Manna), biplab@iitg.ernet.in (B.
Mondal).
0
020-1693/$ - see front matter Ó 2011 Elsevier B.V. All rights reserved.
doi:10.1016/j.ica.2011.06.022

P. Kumar et al. / Inorganica Chimica Acta 376 (2011) 264–270
265
HO
5% (by volume) for each set of experiment and has no effect on
any experimental results.
N
H
HO
H
N
N
Absorption titration experiments were performed by increasing
the concentration of the CT DNA at constant (20
centration. Samples were initially equilibrated with CT DNA for
min before recording each spectrum. Proper correction was made
lM) complex con-
N
L1
L2
5
to the absorbance of CT DNA. The strength of interactions of com-
Fig. 1. Ligands used for the present study.
plexes to CT DNA was measured by monitoring the absorption
⁄
intensity of the
binding constant (K
p
–
p
transition band (ꢁ262 nm). The intrinsic
2
. Experimental
b
) and the binding site size (s, per base pair)
of the complexes to CT DNA were calculated by nonlinear least
square analysis of the isotherm using the expression of Bard and
co-workers [49] based on McGhee–von Hippel (MvH) model [50]
2
.1. Materials and methods
All reagents and solvents were purchased from commercial
sources and were of reagent grade. The supercoiled (SC) pUC19
DNA (cesium chloride purified) was purchased from Bangalore
Genie (India). Calf thymus (CT) DNA, ethidium bromide (EB),
bovine serum albumin (BSA), 3-mercaptopropionic acid (MPA), b-
mercaptoethanol (BME), bromophenol blue and xylene cyanol
2
2
1=2
ð
e
a
ꢀ
e
f
Þ=ð
e
b
ꢀ
e
f
Þ ¼ ðb ꢀ ðb ꢀ 2K C
t
½DNAꢂ=sÞ Þ2K
b
t
C ;
b
where b = 1 + K
b
C
t
+ K
b
[DNA]/2s,
e
a
is the extinction coefficient
is
observed for the spectral band at a given DNA concentration,
the extinction coefficient of the complex in solution, is the extinc-
tion coefficient of the complex when fully bound to DNA, K is the
equilibrium binding constant, C is the total complex concentration,
[DNA] is the DNA concentration in nucleotides and s is the binding
site size of the complexes in base pairs. The nonlinear least-square
fit analysis was done using OriginLab software.
e
f
e
b
were from Sigma (USA). DMSO, KI, NaN
Merck (Germany). Sodium dodecyl sulfate (SDS), glycerol, agarose
molecular biology grade) were purchased from SRL (India).
3
,
L-histidine were from
b
t
(
Tris(hydroxymethyl)aminomethane–HCl (Tris–HCl) buffer was
prepared using with Milli-Q water. UV–Vis spectra were recorded
on a Perkin-Elmer Lambda 25 UV–Vis spectrophotometer. FT-IR
spectra of the solid samples were recorded on a Perkin-Elmer spec-
trophotometer with samples prepared as KBr pellets. Solution elec-
trical conductivity was checked using a Systronic 305 conductivity
The apparent binding constant (Kapp) values of the copper com-
plexes to CT DNA were determined by ethidium bromide (EB) fluo-
rescence displacement measurements using EB bound CT DNA
solution in 5 mM Tris–HCl buffer (pH 6.8) at room temperature.
The fluorescence intensities at 602 nm (526 nm excitation) of EB
with increasing concentration of copper complexes were recorded.
The Kapp values were calculated from the equation: KEB ꢃ [EB]
= Kapp ꢃ [complex], where KTD is the binding constant of EB (KEB
1
bridge. H NMR spectra were obtained with a 400 MHz Varian FT
spectrometer. Chemical shifts (ppm) were referenced either with
an internal standard (Me Si) or to the residual solvent peaks. The
4
X-band electron paramagnetic resonance (EPR) spectra were re-
corded on a JES-FA200 ESR spectrometer, at room temperature.
Elemental analyses were obtained from a Perkin-Elmer Series II
Analyzer. The magnetic moment of complexes is measured on a
Cambridge Magnetic Balance.
7
ꢀ1
= 1.0 ꢃ 10 M ), [EB] is the concentration of EB (1.3
lM) and
[complex] is the concentration of the complex at 50% reduction
of the emission intensity [2].
Thermal denaturation is known to cause an easily monitored
hyperchromic effect in the absorption spectra of CT DNA (kmax
,
2
60 nm) [51]. Hence, the thermal denaturation of DNA in absence
2.2. X-ray crystallography
and presence of complexes were carried out by monitoring the
absorbance of the CT DNA at 260 nm within the temperature range
of 40–90 °C in 5 mM Tris–HCl buffer (pH 6.8). The experiments
were carried out with the 3:1 molar ratio of the CT DNA to the
complexes using Perkin-Elmer Lambda 25 spectrophotometer
equipped with a peltier temperature-controller. The values of
Single crystal was grown by slow diffusion followed by slow
evaporation technique. The intensity data were collected using a
Bruker SMART APEX-II CCD diffractometer, equipped with a fine
focus 1.75 kW sealed tube Mo K
a radiation (k = 0.71073 Å) at
2
73(3) K, with increasing (width of 0.3° per frame) at a scan
x
m
DNA melting temperature (T ) were calculated from the derivative
plot (dA260/dT versus T) of the melting profile.
speed of 3 s/frame. The SMART software was used for data acquisi-
tion. Data integration and reduction were undertaken with SAINT
and XPREP software [43]. Multi-scan empirical absorption correc-
tions were applied to the data using the program SADABS [44]. Struc-
tures were solved by direct methods using SHELXS-97 and refined
The circular dichroism (CD) spectroscopy was performed on a
JASCO J-815 CD spectropolarimeter at room temperature. CD spec-
tra of CT DNA in absence and presence of complexes in the 1:1
molar ratio of the CT DNA to the complexes were obtained in the
wavelength range of 220–320 nm in 5 mM Tris–HCl buffer (pH
2
with full-matrix least squares on F using SHELXL-97 [45]. All nonhy-
drogen atoms were refined anisotropically. The hydrogen atoms
were located from the difference Fourier maps and refined. Struc-
tural illustrations have been drawn with ORTEP-3 for Windows [46].
6
.8). Each measurement was the average of three repeated scans,
and the background was subtracted from all of the reagents by
using a corresponding solution without CT DNA as a reference
solution.
2
.3. DNA binding experiments
The interaction of the complexes with calf thymus (CT) DNA
2.4. DNA cleavage experiments
was studied in Tris–HCl buffer (5 mM Tris–HCl, pH 6.8) at room
temperature. CT DNA (ca. 375 M NP) in the buffer medium gave
a ratio of UV absorbance at 260 and 280 nm of 1.86:1, indicating
that the DNA was sufficiently free from protein [47]. The concen-
tration of DNA was measured from its absorption intensity at
l
The chemical nuclease activity of the complexes was monitored
using supercoiled (SC) pUC19 DNA in presence of 3-mercaptopro-
pionic acid (MPA, 500
M) as reducing agents and H
50 mM Tris–HCl buffer containing 10% DMF and 50 mM NaCl (pH
6.8). The extent of cleavage of SC DNA (30 M, 0.2 g) to nicked
circular (NC) and linear form by the complexes (50 M) was mea-
sured by agarose gel electrophoresis. For mechanistic investigation
lM) and b-mercaptoethanol (BME, 200
l
2
O
2
(200 M) as oxidizing agent in
l
2
60 nm using molar absorption coefficient
(
e
)
value of
ꢀ1
ꢀ1
6
600 M cm as reported [48]. The stock solutions of complexes
l
l
were freshly prepared by first dissolving complexes in DMF and
then diluted with buffer. The amount of DMF was kept less than
l

2
66
P. Kumar et al. / Inorganica Chimica Acta 376 (2011) 264–270
of the DNA cleavage reaction was performed in the presence of
different additives like singlet oxygen quencher (NaN , 500 M;
M) and hydroxyl radical scavenger (DMSO, 4 L;
M). The samples after incubation for 1.0 h at 37 °C under
7.61 (d, 1H), 8.53 (d, 1H). 13C NMR (100 MHz, CDCl
): dppm.
3
51.85, 53.08, 116.48, 119.16, 122.55, 122.84, 128.74, 128.86,
136.87, 149.44, 157.82, 158.22.
3
l
l
L
-histidine, 500
l
KI, 500
l
dark condition were added with the DNA loading buffer (25%
bromophenol blue, 0.25% xylene cyanol FF and 30% glycerol). The
gel electrophoresis was carried out in a dark room for 2.0 h at
1 4 2
2.6. Synthesis of complex 1, [Cu(L )](ClO )
Copper(II) perchlorate hexahydrate, [Cu(H
5.4 mmol) was dissolved in 10 ml of methanol and to this the
ligand L (2.46 g, 10.8 mmol) was added. The color of the solution
2 6 4 2
O) ](ClO ) (2 g,
4
0 V in a Tris-acetate EDTA (TAE) buffer using 1.0% agarose gel
containing ethidium bromide (1.0 g/ml). The gel images were
l
1
captured by Bio-Rad gel documentation system and the extent of
DNA cleavage was quantitatively calculated by ImageJ software.
Due corrections were made for the low level of the nicked circular
was changed to green. The resulting mixture was stirred for 1 h at
room temperature. The volume of the solution was then reduced to
ꢁ3 ml and kept in freezer for overnight to afford the complex 1 as
green precipitate. It was then filtered off and washed with the
small volume of cold methanol and dried under vacuum. Yield:
(
NC) form present in the original SC DNA sample and for the low
affinity of EB binding to SC compared to NC and linear forms of
DNA.
ꢀ
1
3.2 g (ꢁ82%). UV–Vis (acetonitrile): kmax, 642 nm (
e
= 240 M
ꢀ1
cm ). FT-IR (KBr pellet): 1086, 1120, 764, 2928, 1483, 1258
ꢀ1
ꢀ1
ꢀ1
2
2
.5. Synthesis of the ligands and complexes
cm . Molar conductance: 240 S cm mol . Observed magnetic
moment ꢄ1.66 BM.
.5.1. Synthesis of L
Salicylaldehyde (1.22 g, 10 mmol) in 25 ml methanol was taken
1
2 4 2
2.7. Synthesis of complex 2, [Cu(L )](ClO )
in a 50 ml round bottomed flask fitted with a magnetic stir-bar and
to this pyridine-2-ethylamine (1.22 g, 10 mmol) was added drop-
wise with constant stirring. Immediate formation of yellow precip-
itate of the corresponding Schiff base was observed and the stirring
was continued for 1 h. The Schiff base was then filtered out and
dried under reduced pressure to isolate as yellow solid (Yield:
Complex 2 was prepared from copper(II) perchlorate hexahy-
drate, [Cu(H O) ](ClO (2 g, 5.4 mmol) and L (2.31 g, 10.8 mmol)
following the same procedure like complex 1. Yield: 2.98 g (ꢁ80%).
2
6
4
)
2
2
ꢀ1
ꢀ1
UV–Vis (acetonitrile): kmax, 652 nm (
e = 121 M cm . FT-IR (KBr
ꢀ1
pellet): 1087, 1121, 753, 3254, 1433, 1614 cm . Molar conduc-
ꢀ1
ꢀ1
1
.60 g; ꢁ70%). 1.30 g (5.75 mmol) of the Schiff base was then
tance: 248 S cm mol . The calculated magnetic moment is
found to be 1.62 BM.
added in 20 ml methanol and reduced by the 2.5 equiv. of sodium
borohydride. The colorless solution was dried under reduced pres-
sure to afford a white mass. The mass was dissolved in 25 ml water
and neutralized with dilute acetic acid. The organic product was
3. Results and discussions
then extracted from the aqueous solution using CHCl
portions); removal of the solvent under reduced pressure afforded
3
(3ꢃ 20 ml
The ligands, L1 and L , were prepared by Schiff base reaction
between the appropriate amine and aldehyde in methanol fol-
2
pure
L
1
ligand (Yield: 0.9 g; ꢁ70%). Elemental analyses for
lowed by the reduction of the Schiff base using sodium borohy-
dride (Scheme 1) (see Section 2). The formation of the ligands
was confirmed by various spectroscopic techniques as well as ele-
mental analyses (see Section 2).
C
14
H
16
N
2
O: Calc. (%): C, 73.66; H, 7.06; N, 12.27. Found: C, 73.61;
1
H, 7.07; N, 12.19%. H NMR (400 MHz, CDCl
3
): dppm 2.98 (t, 2H),
3
7
4
1
.06 (t, 2H), 3.97 (s, 2H), 6.77 (m, 2H), 6.93 (d, 1H), 7.12 (m, 3H),
.57 (t, 1H), 8.49 (d, 1H). 13C NMR (100 MHz, CDCl
3
): dppm, 37.30,
The complex 1 was prepared by stirring a methanol solution of
[Cu(H O) ](ClO ) with 2 equiv. of L at room temperature for 1 h.
7.76, 52.52, 116.40, 118.97, 121.61, 122.68, 123.48, 128.43,
28.68, 136.65, 149.43, 158.42, 159.62.
2
6
4 2
1
After reducing the volume of the resulting deep blue solution, the
solution was kept in freezer for overnight. The blue crystals of the
complex were obtained from the mixture (Scheme 2).
The complex 2 was reported earlier [52]. The complex 2 was
2 6 4 2 2
synthesized by the reaction of [Cu(H O) ](ClO ) and L following
the same procedure. The formulation of the complexes has been
supported by various spectroscopic analyses (Supporting informa-
tion). The single crystal X-ray structure of complex 2 was deter-
mined. The ORTEP diagram is shown in Fig. 2. The crystallographic
2
.5.2. Synthesis of L
The ligand L
1.08 g, 10 mmol) and salicylaldehyde (1.22 g, 10 mmol) following
the same procedure like L
. Yield: (ꢁ75%). Elemental analyses
O: Calc. (%): C, 72.87; H, 6.59; N, 13.07. Found: C, 72.95;
2
2
was prepared from pyridine-2-methylamine
(
1
13 14 2
C H N
1
H, 6.61; N, 13.11%. H NMR (400 MHz, CDCl
3
): dppm, 3.87 (s, 2H),
3
.94 (s, 2H), 6.72 (t, 1H), 6.81 (d, 1H), 6.92 (d, 1H), 7.13 (m, 3H),
HO
N
NH2
CHO
OH
I) MeOH
II) stirring for 2 h
+
N
N
2.5 eqv. NaBH4
HO
H
N
N
L1
1
Scheme 1. Synthesis of ligand L .

P. Kumar et al. / Inorganica Chimica Acta 376 (2011) 264–270
267
H
O
H
N
MeOH
N
HN
NH
N
+
[
Cu(H O) (ClO ) ]
2
6
4 2
Cu
2
(ClO4)2
N
OH
L1
O
H
1
Scheme 2. Synthesis of complexes 1.
Table 1
O2
Crystallographic data for complex 2.
Formula
26 26 4 10
C H Cl2CuN O
O5
Cl1
O3
Molecular weight
Crystal system
Space group
Temperature (K)
Wavelength (Å)
a (Å)
688.96
monoclinic
P 2 /n
O4
1
296(2)
0.71073
7.1126(5)
21.6213(15)
9.6947(7)
90.00
107.685(3)
90.00
1420.43(17)
2
1.611
1.021
multi-scan
706.0
3416
C3
C2
b (Å)
c (Å)
C1
Cu1
C4
C5
a
(°)
b (°)
(°)
C6
N1
c
3
N2
C7
V (Å )
Z
Density (mg mꢀ3)
Absolute coefficient (mm
Absolute correction
F(0 0 0)
O1
C9
ꢀ1
)
C8
C13
Total number of reflections
Reflections, I > 2 (I)
C10
r
2524
C11
Maximum 2h (°)
Index ranges
28.13
ꢀ9 6 h 6 9
C12
ꢀ
ꢀ
28 6 k 6 27
12 6 l 6 12
Fig. 2. ORTEP diagram for complex 2 (50% thermal ellipsoid plot). Hydrogen atoms
Complete to 2h (%)
98.2%
Full-matrix least-squares on F2
are omitted for clarity.
Refinement method
Goodness-of-fit (GOF) on F2
1.095
R indices [I > 2
R indices (all data)
r
(I)]
0.0553
0.0698
data and a list of important bond angles and distances were given
in Tables 1 and 2, respectively. The room temperature magnetic
moment measurement showed one electron paramagnetism for
both the complexes (1.66 and 1.62 BM for complex 1 and 2, respec-
tively). The complexes 1 and 2 displayed axial spectra in X-band
EPR at room temperature in acetonitrile solvent which are charac-
the increase in molar ratio (r) of DNA to complexes 1 and 2
(r = 0–3.6), the absorption intensity at ꢁ262 nm increases by ꢁ8-
and 6-fold, respectively. This exceptionally strong hyperchromism,
along with minor blue shift for complexes 1 and 2, indicate strong
interaction of the complexes with CT DNA mainly through groove
binding [57]. It is known that the hyperchromicity of the UV absor-
bance band is caused by the unwinding of the double helix as well
as its unstacking and the concomitant exposure of the bases;
whereas, red- or blue-shift indicates that the complex may have
some effect on DNA [11,57,58]. To compare the binding parameters
2
2
teristics of square planar Cu(II) complexes with d ꢀ d ground
x
y
state (gav = 2.07 and 2.14 for complexes 1 and 2, respectively)
[
53,54]. Both the complexes were found to behave as 1:2 electro-
ꢀ1
2
ꢀ1
lyte in acetonitrile solution [
K
M
(
cm mol ), 247 and 259 for
complexes 1 and 2, respectively] [55].
3.1. DNA binding studies
quantitatively, the intrinsic equilibrium binding constant (K
b
) for
the complexes 1 and 2 (shown in Table 3) have been determined
4
ꢀ1
4
ꢀ1
The UV–Vis spectroscopy is used widely to study the binding of
to be 8.56 ꢃ 10 M and 7.15 ꢃ 10 M , and the binding site size
(s) to be 0.79 and 0.77 (b.p.), respectively (Table 3). The higher
binding constant values could be due to the presence of aromatic
rings, which might facilitate the interaction of the complexes with
the metal complexes with DNA. It has been found that intercala-
tion or electrostatic interaction between the metal complex and
DNA leads to hypochromism; whereas hyperchromism indicates
the break down of the secondary structure of DNA [56]. The UV–
Vis absorption titrations have been employed to ascertain the
DNA binding strength of complexes 1 and 2 by monitoring the
changes in the absorption intensity of the ligand-centered bands
around 262 nm. Fig. 3 illustrates the representative UV–Vis spectra
for the binding of complexes in absence and presence of CT DNA (at
the DNA bases through noncovalent p–p interaction. The lower va-
lue of s (s < 1) suggests groove binding and/or surface aggregation
of the complexes on DNA, which also correlates with the hyper-
chromicity of the UV-absorptions in presence of higher DNA con-
centrations (Fig. 3) [59].
The binding strength of the complexes to CT DNA has been fur-
a constant complex concentration, 20
lM) and the inset shows
ther verified by measuring apparent DNA binding constants (Kapp
)
binding isotherm generated from the UV–Vis spectral data. With
from ethidium bromide (EB) fluorescence displacement assay

2
68
P. Kumar et al. / Inorganica Chimica Acta 376 (2011) 264–270
Table 2
Selected bond lengths (Å) and bond angles (°) for complex 2.
Angle (°)
Bond length (Å)
N1–Cu1–N2
N1–Cu1–O1
N1–Cu1–N1
N1–Cu1–N2
N1–Cu1–O1
N2–Cu1–O1
N2–Cu1–N1
N2–Cu1–N2
N2–Cu1–O1
O1–Cu1–N1
O1–Cu1–N2
O1–Cu1–O1
N1–Cu1–N2
N1–Cu1–O1
N2–Cu1–O1
81.6(1)
97.43(8)
180.0(1)
98.4(1)
82.57(8)
86.52(9)
98.4(1)
180.0(1)
93.48(9)
82.57(8)
93.48(9)
180.00(7)
81.6(1)
Cu1–N1
Cu1–N2
Cu1–O1
Cu1–N1
Cu1–N2
Cu1–O1
N1–O5
N1–C1
2.022(2)
2.037(2)
2.635(3)
2.022(2)
2.037(2)
2.635(3)
1.346(3)
1.338(4)
1.493(4)
1.491(5)
N2–C6
N2–C7
97.43(8)
86.52(9)
Fig. 4. Representative fluorescence emission spectra of DNA-EB in Tris–HCl buffer
pH 6.8) with the increase in molar ratio of complex 1 to DNA (0–3.4) at room
temperature. The inset shows the plot of I/I vs. [complex] for fluorescence
quenching curves of DNA-EB by complexes 1 and 2.
(
0
ence in apparent binding constant values also in consistent with
the intrinsic binding constants. Thus, both intrinsic and apparent
binding parameters clearly show that complexes 1 and 2 strongly
interact with CT DNA.
Thermal behavior of DNA in the presence of the metal com-
plexes has also been studied. The dsDNA at its melting temperature
unwinds to give a single strand DNA, resulting in the increase of
absorbance at 260 nm. In general, intercalation of small molecules
to dsDNA is associated with high melting temperatures. A minor
shift in the CT DNA T
respectively, confirm predominant groove binding mode of the
complexes (Fig. 5a and Table 3). The higher value for complex
m
value in presence of complexes 1 and 2,
DT
m
1
(+1.37 °C) than complex 2 (ꢀ2.7 °C) could be due to the extended
flexibility of the ligand in complexes 1 compare to 2, which has
been reflected in binding parameters.
Fig. 3. Representative absorption spectra of complex 1 (20
l
M) in Tris–HCl buffer
(
pH 6.8) with the increase in molar ratio of DNA to complex (0–3.6) at room
temperature. The inset shows the nonlinear least square fit of bf vs. [DNA]
using the expression of Bard and co-workers based on McGhee–von Hippel model.
D
e
af
/
D
e
For further understanding of the DNA structural changes in
presence of complexes and to reconfirm the mode of interactions
the CD studies have been performed. The positive absorption band
at 274 nm and the negative one at 245 nm in the CD spectrum are
due to the base stacking and the right-handed helicity of B-DNA,
respectively [56,61]. The CD spectrum of CT DNA in presence of
complex 1 at 1:1 molar ratio shows an increase in positive band
(98%) and decreases in negative band (37%) with a blue shift of
3 nm and 1 nm, respectively, compared to the only CT DNA. Under
similar condition complex 2 shows an increase in positive band
Table 3
DNA binding parameters for complexes.
1
2
a
ꢀ1
4
4
K
K
D
b
(M ) [s]
8.56 (±0.3) ꢃ 10 , [0.79]
7.15 (±0.3) ꢃ 10 , [0.77]
b
ꢀ1
5
4
app (M
)
1.76 (±0.3) ꢃ 10
8.45 (±0.2) ꢃ 10
c
T
m
(°C)
+1.37
ꢀ2.7
d
ꢀ1
4
4
K
BSA (M
)
3.3 (±0.3) ꢃ 10
1.3 (±0.1) ꢃ 10
(
81%) and decrease in negative band (18%) with a blue shift of
a
b
c
Intrinsic equilibrium binding constant from UV–Vis absorption titration.
Apparent DNA binding constant from fluorescence displacement assay.
Change in DNA melting temperature of CT DNA.
1 nm for positive band only. These changes in CD spectrum of CT
DNA indicate strong conformational changes by the complexes.
The major change at 274 nm absorption band clearly indicates that
both the complexes interact with dsDNA through groove and/or
surface and decreases the helicity of the dsDNA. The larger CD
spectral changes by 1 than 2 are in correlation with their DNA
binding parameters.
d
Bovine serum albumin (BSA) binding constant from fluorescence spectral
measurements.
(Fig. 4). EB is known to show reduced emission intensity in buffer
solution due to solvent quenching, and an enhancement of the
emission intensity when EB intercalatively binds to dsDNA [60].
The competitive binding of the complexes to DNA has been mea-
sured from the extent of reduction of the EB emission intensity
at 602 nm. The inset shows the binding isotherm generated from
the fluorescence spectra of EB. The Kapp values indicate that com-
plex 1 binds better than the complex 2 (Table 3). The small differ-
3.2. DNA cleavage study
To investigate whether the DNA binding and DNA conforma-
tional changing properties of the complex 1 and 2 are associated
with further pharmacological activities, chemical nuclease activity
assay have been performed. The cleavage activity of complexes to-

P. Kumar et al. / Inorganica Chimica Acta 376 (2011) 264–270
269
0
0
0
0
0
.05
.04
.03
.02
.01
0
a
b
2
DNA
1
-
0.01
5
0
60
70
80
90
0
T / C
Fig. 5. (a) Derivative plot of thermal denaturation of CT DNA (150
l
M) only and in presence of complexes (50
l
M) in Tris–HCl buffer (pH 6.8). (b) Circular dichroism spectra
of (A) CT DNA only, (B) CT DNA in presence of complex 2 and (C) CT DNA in presence of complex 1 in Tris–HCl buffer (pH 6.8) at room temperature. [complex] = 150
DNA] = 150 M.
lM,
[
l
3
1
3
2
4
3
98 91 34 15 % NC
Table 4
Chemical nuclease activity of SC pUC19 DNA by the complexes in presence of various
additives.
(a)
NC
SC
Complex % NC DNA
DNA
only
Complex + MPA +DMSOa +NaN b
3
+KIc
+L-
histidine
4
5
6
7
Lane
d
5
93
41 90 83
91 % NC
1
2
5
6
93
44
41
11
90
48
83
23
91
46
(
b)
c)
NC
SC
[
complex] = 50
l
l
M.
L.
] = 500
[KI] = 500 M.
-Histidine] = 500 lM.
a
DMSO = 6
b
c
[
NaN
3
lM.
1
6
2
3
4
5
6
Lane
l
d
[
L
44
11 48 23
46 % NC
(
NC
SC
similar cleavage activity (Supporting information). Control experi-
ments using only reducing agents like MPA and BME, oxidizing
1
2
3
4
5
6
Lane
2 2
agents like H O or the complexes alone (Supporting information)
have not shown appreciable DNA cleavage under similar experi-
mental conditions.
To understand the basis of chemical nuclease activity of the
complexes in presence of reducing agents we have systematically
investigated the mechanistic aspects using various additives. The
addition of hydroxyl radical scavengers like DMSO, KI and the
singlet oxygen quenchers like NaN , L-histidine in absence of any
3
reducing agent have almost modest or no effect on DNA cleavage
activity, respectively (Table 4).
This indicates their noninvolvement in DNA cleavage activity.
Whereas, in presence of reducing agents only the hydroxyl radical
scavengers showed significant inhibitory effect in the chemical
nuclease activity (Fig. 6b and c). This mechanistic data suggest that
the DNA cleavage reaction of the complexes in presence of MPA,
presumably, proceeds via hydroxyl radical pathway.
Fig. 6. (a) Representative gel electrophoresis diagram showing the chemical
nuclease activity of the complexes (50 M) using SC pUC19 DNA (30 M, 0.2 g)
in presence of 3-mercaptopropionic acid (MPA, 500 M) and b-mercaptoethanol
BME, 200 M): lane 1, DNA control; lane 2, DNA + MPA; lane 3, DNA + BME; lane 4,
DNA + 1 + MPA; lane 5, DNA + 1 + BME, lane 6, DNA + 2 + MPA; lane 7,
DNA + 2 + BME. (b) and (c) Representative gel electrophoresis diagram showing
l
l
l
l
(
l
the mechanistic aspects of chemical nuclease activity of SC pUC19 DNA (30
.2 g) by the complexes 1 and 2, respectively, in presence of singlet oxygen
quencher like, NaN (500 M), -histidine (500 M) and hydroxyl radical scaveng-
ers like DMSO (6 L), KI (500 M): lane 1, DNA control; lane 2, DNA + 1/2 + MPA;
lane 3, DNA + 1/2 + MPA + DMSO; lane 4, DNA + 1/2 + MPA + NaN ; lane 5, DNA + 1/
+ MPA + KI; lane 6, DNA + 1/2 + MPA + -histidine.
lM,
0
l
3
l
L
l
l
l
3
2
L
wards SC pUC19 DNA has been investigated by measuring the
extent of formation of nicked circular (NC) DNA (Fig. 6a) in the
presence of reducing agents. Complex 1 found to be highly active
forming ꢁ98% and ꢁ91% NC in presence of reducing agents like
3
-mercaptopropionic acid (MPA) or b-mercaptoethanol (BME),
4. Conclusions
respectively. Complex 2 also exhibits good chemical nuclease
activity showing formation of ꢁ34% and ꢁ15% NC in presence of
reducing agents like MPA and BME, respectively. The extent of
DNA cleavage by the complexes as observed from the agarose gel
electrophoresis also follows their DNA binding propensity. The
The synthesis and structural characterization of Cu(II) com-
plexes 1 and 2 were carried out with an aim to study their DNA
binding and nuclease activity. Both the complexes effectively inter-
act with CT DNA through groove binding mode resulting in strong
conformational changes of the CT DNA. The chemical nuclease
2 2
complexes in presence of oxidizing agent like H O also exhibit

2
70
P. Kumar et al. / Inorganica Chimica Acta 376 (2011) 264–270
[20] A. Sancar, G.B. Sancar, Annu. Rev. Biochem. 57 (1998) 29.
activity study indicates that 1 has stronger cleavage activity than 2.
The mechanistic studies indicate that the hydroxyl radicals are the
main driving force and the extent of DNA cleavage in presence of
hydroxyl radicals, is governed to a large extent by their DNA bind-
ing mode and propensity. This work presents a good overall corre-
lation between DNA binding and DNA cleavage activity of the
complexes. Further biological studies are beyond the scope of this
investigation. The results are of importance towards further
designing and developing Cu(II) based complexes and systematic
assessment of DNA binding, cleavage activity for their potential
applications as therapeutic agents.
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