Synthesis, characterization, DNA binding and cleavage properties of a ternary copper(II) Schiff base complex

Article abstract of DOI:10.1007/s11243-014-9836-7

A ternary coordination complex 2Cu(C₁₄H₈NO ₃Cl)(C₁₂H₈N₂)·3CH ₃OH of a Schiff base (C₁₄H₈NO₃Cl: 4-chloroanthranilic acid-salicylaldehyde) was synt

Full text of DOI:10.1007/s11243-014-9836-7

Transition Met Chem
DOI 10.1007/s11243-014-9836-7
Synthesis, characterization, DNA binding and cleavage properties
of a ternary copper(II) Schiff base complex
•
Xin Li Cai-Feng Bi Yu-Hua Fan
•
•
•
Xia Zhang Xiao-Dan Wei Xiang-Min Meng
•
Received: 23 February 2014 / Accepted: 6 May 2014
Ó Springer International Publishing Switzerland 2014
Abstract A ternary coordination complex 2Cu(C14H8-
NO3Cl)(C12H8N2)ꢀ3CH3OH of a Schiff base (C14H8NO3Cl:
4-chloroanthranilic acid–salicylaldehyde) was synthesized
from 4-chloroanthranilic acid, salicylaldehyde, copper ace-
tate and 1,10-phenanthroline and characterized by physico-
chemical and spectroscopic methods. X-ray crystallography
shows that the copper atom is five coordinated by the imine
nitrogen, two nitrogen atoms from 1,10-phenanthroline, the
hydroxyl oxygen and the carboxylate oxygen atom. The
interaction of the complex with calf-thymus DNA was
investigated by UV absorption spectroscopy, fluorescence
emission spectroscopy and viscosity. In addition, the
cleavage reaction with pBR322 supercoiled plasmid DNA
was investigated. The complex binds to DNA and also
exhibits significant DNA cleavage activity.
nucleases and cancer chemotherapy drugs. Ternary com-
plexes have been at the forefront of these investigations
because of their interesting electronic properties, diverse
chemical reactivity and unusual structures, which can result
in interactions with DNA such as intercalation. In general,
DNA is the primary intracellular target of anticancer drugs
due to the interactions between complexes and DNA,
which cause DNA damage in cancer cells, blocking their
division and resulting in cell death [1]. Bioactivity exper-
iments indicate that complexes with 1,10-phenanthroline as
a secondary ligand have more potent DNA binding capa-
bility and anticancer activity than the analogous complexes
without 1,10-phenanthroline [2, 3]. In recent years, many
reports have shown that Cu(II) complexes play an impor-
tant role in the endogenous oxidative DNA damage asso-
ciated with aging and cancer. Therefore, the synthesis and
biological testing of copper complexes has become an
important area of currant research [4]. In this paper, a
ternary Cu(II) complex of a Schiff base derived from
4-chloroanthranilic acid and salicylaldehyde was synthe-
sized and characterized. The interactions of the complex
with calf-thymus DNA (CT-DNA) were investigated by
UV absorption spectroscopy, fluorescence emission spec-
troscopy and viscometry. In addition, the cleavage reac-
tions of the complex with pBR322 supercoiled plasmid
DNA and the mechanism of cleavage were investigated by
gel electrophoresis experiments.
Keywords Schiff base ꢀ Copper(II) complex ꢀ DNA ꢀ
Intercalative binding ꢀ Cleavage
Introduction
Copper(II) complexes of Schiff base ligands are of con-
siderable interest, since they can show DNA cleavage and
antitumor activity and can therefore be used as chemical
X. Li ꢀ C.-F. Bi (&) ꢀ Y.-H. Fan (&) ꢀ X. Zhang ꢀ X.-D. Wei ꢀ
X.-M. Meng
Key Laboratory of Marine Chemistry Theory and Technology,
Ministry of Education, College of Chemistry and Chemical
Engineering, Ocean University of China,
Qingdao 266100, Shandong, People’s Republic of China
e-mail: bicaifeng301@163.com
Experimental
Measurement and reagents
Infrared spectra were recorded as KBr pellets on a Nicolet
170SX spectrometer in the 4,000–400 cm-1 region.
Y.-H. Fan
e-mail: fanyuhua301@163.com
123

Transition Met Chem
Elemental analyses (C, H and N) were performed on a model
2400 Perkin-Elmer analyzer. 1H NMR spectra were recorded
on an AVANCE III (600 MHz) spectrometer. The UV
spectra were performed on a Unicam UV2 spectrometer. The
fluorescence spectra were measured with a Hitachi F-4500
spectrophotometer. Elemental analyses were carried out on a
Carlo Erba 1106 full-automatic trace organic elemental
analyzer. 4-Chloroanthranilic acid and salicylaldehyde were
purchased from Aladdin, and the other chemicals were
produced in China. CT-DNA (biochemical reagent) and
pBR322 supercoiled plasmid DNA were purchased from
Sigma–Aldrich and used without further purification. A
solution of CT-DNA in Tris–HCl buffer (5 9 10-3 mol L-1
Tris–HCl; 5 9 10-2 mol L-1 NaCl, pH 7.2) gave an
absorbance ratio, A260/A280, between 1.8 and 1.9, indicating
that the CT-DNA was sufficiently free of protein [5].
cm-1): 1,604 (s), 1,584(s), 1,556 (s), 1,519 (s), 1,461 (w),
1,440 (s), 1,397 (m), 1,347 (s), 1,149 (s), 943 (m), 903 (m),
844 (s), 786 (m), 725 (s), 511(m), 457(s). 1H NMR
(DMSO-d6, 600 MHz) d(ppm): 6.796 (d, 1H), 7.068 (d,
1H), 7.187 (d, 1H), 7.325 (d, 1H), 7.499 (d, 1H), 7.528 (d,
1H), 7.795 (s, 1H) 8.064 (s, 1H).
X-ray structure determination
A singlecrystal with dimensions of 0.45 9 0.37 9 0.22 mm
was mounted on an Enraf–Nonius CAD-4 X-ray single-
crystal diffractometer. All data were collected at 293(2) K
with
graphite-monochromatized
MoKa
radiation
˚
(k = 0.71073 A) by using the x - 2h scan mode. A total of
2,969 reflections intensities with I [ 2r(I) were considered
to be observed and used in the subsequent refinements. The
structure was solved by direct methods using SHELXS-97
[6]. The non-hydrogen atoms were defined by the Fourier
synthesis method. Positional and thermal parameters were
refined by the full matrix least-squares method (on F2) to
convergence [7]. Supplementary material has been deposited
with the Cambridge Crystallographic Data Centre (deposi-
tion No. 970004; deposit@ccdc.cam.ac.uk or http://www.
ccdc.cam.ac.uk).
Synthesis of the Schiff base
4-Chloroanthranilic acid (0.171 g, 1.0 mmol) was dis-
solved in methanol (40 mL) with magnetic stirring, and
salicylaldehyde (0.122 g, 1.0 mmol) was added dropwise
to the solution. The reaction mixture was stirred under
reflux at 55 °C for 5 h to give a bright yellow clear solu-
tion. The solution was evaporated to 5 mL, and the yellow
precipitate was filtered off and dried under vacuum. Yield
0.19 g (69 %) based on 4-chloroanthranilic acid. Elemental
Anal Calc (%) for the ligand: C 61.0, H 3.7, N 5.1. Found
(%): C 60.9, H 3.7, N 5.1. IR (KBr, cm-1): 1,617 (s),
1,565(s), 1,487 (m), 1,466 (m), 1,429 (w), 1,364 (s), 1,285
(w), 1,245 (s), 1,224 (w), 1,117 (m), 1,035 (w), 921 (m),
DNA binding experiments
The complex was dissolved in Tris–HCl buffer solution.
Absorption titration experiments were carried out by
gradually increasing the DNA concentration while main-
taining the complex concentration constant (10-5
mol L-1). Absorbance values were recorded after each
successive addition of DNA solution and equilibration.
Ethidium bromide (EB) displacement tests were carried
out by successive additions of the complex to DNA
(2 9 10-6 mol L-1) containing EB (2 9 10-6 mol L-1) in
Tris–HCl buffer. The scanner speed was 240 nm s-1, and the
slit width was 5 nm. These samples were excited at 258 nm.
The fluorescence spectra were recorded between 500 and
750 nm.
1
843 (w), 783 (w), 747 (w), 547 (w), 469 (w). H NMR
(DMSO-d6, 600 MHz) d(ppm): 6.883 (d, 1H), 7.075 (d,
1H), 7.233 (d, 1H), 7.355 (d, 1H), 7.504 (d, 1H), 7.547 (d,
1H), 7.749 (s, 1H), 7.836 (s, 1H), 8.361 (s, 1H), 11.109 (s,
1H).
Synthesis of the Schiff base complex
The Schiff base (0.275 g, 1.0 mmol) was dissolved in
methanol (20 mL). A solution of Cu(OAc)2ꢀH2O (0.199 g,
1.0 mmol) in methanol (20 mL) was added dropwise to the
above solution with stirring, and the mixture was refluxed
for 5 h to give a green precipitate. A solution of 1,10-
phenanthroline (0.198 g, 1.0 mmol) in methanol (10 mL)
was then added dropwise, and the mixture was stirred for
5 h at 55 °C. The resulting solution was cooled to room
temperature and then filtered. The filtrate was left for slow
evaporation at room temperature. Plate-like crystals were
formed from the solution 2 weeks later, with 58 % yield
based on the 4-chloroanthranilic acid. Elemental Anal Calc
(%) for 2Cu(C14H8NO3Cl)(C12H8N2)ꢀ3CH3OH: C 58.4, H
3.9, N 7.4. Found (%): C 58.4, H 3.9, N 7.5. IR (KBr,
The effect of salt was investigated by successive addi-
tions of NaCl to the mixture while maintaining constant
concentration of DNA and the complex. Fluorescence
intensities were recorded in the absence and presence of
NaCl in mixtures of the complex and DNA.
DNA viscosity measurements were made by adding
different amounts of the complex in Tris–HCl buffer to a
constant concentration of DNA. Viscosity measurements
were made using an Ubbelohde viscometer, immersed in a
thermostated water bath maintained to 298.0 ( 0.1) K.
Data were analyzed as (g/g0)1/3 versus the ratio of the
concentration of the complex and DNA, where g is the
123

Transition Met Chem
Cl
O
OH
O
NH2
OH
N
+
O
OH
HO
Cl
Cl
Cl
N
N
N
Cu(OAc)2
N
+
+
Cu
O
O
O
O
OH HO
N
N
Scheme 1 The synthesis of the Schiff base and the complex
Fig. 1 Crystal structure of 2Cu(C14H8NO3Cl)(C12H8N2). All hydro-
gen atoms and free solvent methanol molecules are omitted for clarity
viscosity of DNA in the presence of the complex, and g0 is
the viscosity of DNA alone.
The crystallographic structural analysis reveals that the
Cu(II) complex crystallizes in the triclinic crystal system,
space group P¯ı. The copper atom is five coordinate, being
coordinated by the imine nitrogen, two nitrogen atoms
from 1,10-phenanthroline, the hydroxyl oxygen and the
carboxylate oxygen atom, forming a ternary Cu(II) com-
plex. There are two complex molecules and three free
solvent methanol molecules in the asymmetric unit. The
crystal structure of the Cu(II) complex is shown in Fig. 1, a
summary of the key crystallographic information is given
in Table 1, and selected bond lengths and angles are listed
in Table 2. The two crystallographically independent
complex molecules are almost identical. The bond lengths
DNA cleavage experiments
The cleavage of pBR322 DNA by the Cu(II) complex was
investigated by agarose gel electrophoresis. The reaction
was carried out by mixing pBR322 DNA and different
concentrations of the complex in 50 mM Tris–HCl buffer,
containing 50 mM NaCl (pH 7.2), to yield a final volume
of 20 lL. The samples were then incubated for 1 h at
37 °C, followed by the addition of loading buffer con-
taining bromophenol blue (0.25 %) and sucrose (40 %).
The reaction products were subjected to electrophoresis on
a 0.8 % agarose gel in TBE buffer (89 mM tris base,
89 mM boracic acid and 2 mM EDTA) at 40 V for 2 h.
The resolved bands were photographed under UV light.
˚
of N(1)–C(8) and N(4)–C(34) are 1.334 and 1.325 A,
respectively, which are close to the usual –C=N– double
˚
bond at 1.303 and 1.306 A [11]. The bond angles O(1)–
Cu(1)–N(1) (92.7°), O(1)–Cu(1)–N(2) (90.7°), O(1)–
Cu(1)–N(3) (96.1°), O(3)–Cu(1)–N(1) (93.9°), O(3)–
Cu(1)–N(2) (84.6°) and O(3)–Cu(1)–N(3) (110.7°) are all
different to the ideal value of 90°. The Cu(1)–N(3) bond is
significantly longer than other Cu–N bonds, while the
O(3)–Cu(1)–O(1) angle (150.7°) is much closer to 180°
than to 90°, indicating that the central Cu(1) adopts a
distorted square pyramidal geometry [12, 13]. Four atoms
N(1), N(2), O(1) and O(3) define the basal plane, while
N(3) is located in the apical position of the square pyra-
midal structure. Similarly, Cu(2) also has a distorted square
pyramidal geometry, in which N(4), N(6), O(4) and O(6)
define the basal plane, while N(5) is located in the apical
position. The complex molecules are linked into a 1D chain
structure through p–p stacking interactions of the 1,10-
phenanthroline ligands (Fig. 2). Adjacent chains are
bridged by one type of hydrogen bond, involving C–HꢀꢀꢀO
interactions, and also by p–p stacking interactions between
the benzene rings of the ligands. The alternate arrangement
of chains forms a 2D layer structure (Fig. 3). The 2D layer
structure is linked by hydrogen bonds between the Schiff
Results and discussion
The synthesis of the Schiff base and the complex is shown
in Scheme 1. The IR spectrum of the Cu(II) complex dis-
plays a strong absorption at 1,604 cm-1, which is assigned
to the –C=N– stretching mode of the Schiff base ligand [8].
Absorptions at 1,519, 844 and 725 cm-1 are attributed to
1,10-phenanthroline, while bands at 1,556 and 1,347 cm-1
are assigned to mas(COO–) and ms(COO–), respectively.
Bands at 511 and 457 cm-1 are assigned to m(Cu–N) and
1
m(Cu–O), respectively. On comparing the H NMR spectra
of the Cu(II) complex and the free Schiff base, the signal
from the hydrogen atom (11.109 ppm) of –COOH was
absent from the spectrum of the complex, indicating de-
protonation of this group. The hydrogen chemical shift of
–CH=N in the complex was 8.06 ppm, compared to that of
the ligand (8.36 ppm), consistent with coordination to the
copper [9, 10]. Hence, the 1H-NMR study supports the
conclusions drawn from the IR spectra.
123

Transition Met Chem
˚
Table 1 Crystallographic data and structure refinement for complex
Table 2 Selected bond lengths and angles for complex (A, °)
Empirical formula
C55H44Cl2Cu2N6O9
Bond
Dist.
Bond
Dist.
Formula weight
Temperature (K)
1130.94
Cu(1)–N(3)
Cu(1)–O(3)
Cu(1)–N(2)
Cu(1)–N(1)
Cu(1)–O(1)
O(1)–C(1)
O(2)–C(1)
C(10)–O(3)
C(8)–C(9)
C(1)–C(2)
N(1)–C(3)
N(1)–C(8)
C(2)–C(3)
C(9)–C(10)
2.333 (7)
1.934 (6)
2.029 (6)
1.959 (6)
1.966 (6)
1.273 (10)
1.256 (10)
1.309 (9)
1.421 (11)
1.528 (12)
1.427 (10)
1.334 (9)
1.426 (11)
1.407 (11)
Cu(2)–N(5)
Cu(2)–O(6)
Cu(2)–N(6)
Cu(2)–N(4)
Cu(2)–O(4)
O(4)–C(27)
O(5)–C(27)
C(36)–O(6)
C(34)–C(35)
C(27)–C(28)
N(4)–C(29)
N(4)–C(34)
C(28)–C(29)
C(35)–C(36)
2.358 (7)
1.935 (6)
2.014 (8)
1.956 (7)
1.986 (7)
1.235 (11)
1.273 (11)
1.335 (9)
1.436 (12)
1.572 (15)
1.420 (11)
1.325 (10)
1.412 (12)
1.430 (12)
298 (2)
˚
Wavelength (A)
0.71073
Crystal system
Space group
Triclinic
P¯ı
˚
a (A)
8.1253 (5)
˚
b (A)
11.5385 (11)
27.964 (3)
˚
c (A)
a (°)
b (°)
79.2250 (10)
86.591 (2)
c (°)
Volume (A )
88.422(2)
3
˚
2,570.6 (4)
2
Z
Calculated density (g/cm3)
Absorption coefficient (mm-1
F(000)
1.461
)
0.995
Bond
Angle
Bond
Angle
1,160
O(3)–Cu(1)–N(1)
O(3)–Cu(1)–O(1)
N(1)–Cu(1)–O(1)
O(3)–Cu(1)–N(2)
N(1)–Cu(1)–N(2)
O(1)–Cu(1)–N(2)
O(3)–Cu(1)–N(3)
N(1)–Cu(1)–N(3)
O(1)–Cu(1)–N(3)
N(2)–Cu(1)–N(3)
C(8)–N(1)–C(3)
C(8)–N(1)–Cu(1)
C(3)–N(1)–Cu(1)
C(1)–O(1)–Cu(1)
C(10)–O(3)–Cu(1)
O(2)–C(1)–O(1)
O(2)–C(1)–C(2)
O(1)–C(1)–C(2)
N(1)–C(8)–C(9)
O(3)–C(10)–C(9)
93.9 (2)
150.7 (3)
92.7 (2)
O(6)–Cu(2)–N(4)
O(6)–Cu(2)–O(4)
N(4)–Cu(2)–O(4)
O(6)–Cu(2)–N(6)
N(4)–Cu(2)–N(6)
O(4)–Cu(2)–N(6)
O(6)–Cu(2)–N(5)
N(4)–Cu(2)–N(5)
O(4)–Cu(2)–N(5)
N(6)–Cu(2)–N(5)
C(34)–N(4)–C(29)
C(34)–N(4)–Cu(2)
C(29)–N(4)–Cu(2)
C(27)–O(4)–Cu(2)
C(36)–O(6)–Cu(2)
O(5)–C(27)–O(4)
O(5)–C(27)–C(28)
O(4)–C(27)–C(28)
N(4)–C(34)–C(35)
O(6)–C(36)–C(35)
93.5 (3)
144.2 (3)
90.3 (3)
Crystal size (mm)
h range for data collection (°)
Limiting indices
0.45 9 0.37 9 0.22
2.85–25.02
-9 B h B 9
-13 B k B 12
-30 B l B 33
12,384/8,734 [Rint = 0.0453]
96.2
84.6 (2)
86.2 (3)
175.5 (3)
90.7 (2)
175.3 (3)
92.6 (3)
Reflections collected/unique
110.7 (2)
100.1 (3)
96.1 (2)
117.9 (2)
99.4 (3)
Completeness to h = 25.02 (%)
Max. and min. transmission
Data/restraints/parameters
Goodness of fit on F2
RA1 , wRB2 [I [ 2r(I)]
RA1 , wRB2 (all data)
0.8108 and 0.6631
8,734/0/670
1.082
96.5 (2)
76.5 (3)
76.7 (3)
117.6 (6)
123.1 (6)
119.3 (5)
129.1 (5)
126.0 (5)
122.1 (8)
119.0 (8)
118.8 (8)
127.0 (7)
125.4 (8)
118.8 (8)
123.7 (6)
117.5 (5)
128.0 (7)
126.7 (6)
125.1 (11)
115.3 (10)
119.5 (9)
127.9 (8)
124.4 (8)
R1 = 0.0909, wR2 = 0.2407
R1 = 0.1600, wR2 = 0.2757
0.575 and -1.058
-3
)
˚
Largest diff. peak and hole (e. A
w = 1/[r2 (F2o) ? (0.0975P)2 ? 5.1937P], with P = (Fo2 ? 2 F2c)/3
base ligands and methanol molecules to give a 3D structure
(Fig. 4).
DNA binding studies
UV absorption spectroscopy provides a simple and con-
venient way to investigate the interactions between mole-
cules and DNA [14, 15]. The UV spectra of the Cu(II)
complex in the absence and presence of CT-DNA are given
in Fig. 5. The absorption peaks of the complex showed a
slight red shift with increasing concentrations of DNA. A
significant hypochromic effect was observed at 289 nm.
The intrinsic binding constant Kb of the complex with CT-
DNA was determined according to the following equation
[16]:
where [DNA] is the concentration of DNA in the base
pairs, Kb is the intrinsic binding constant, ea corresponds to
the apparent extinction coefficient, and eb and ef correspond
to the extinction coefficients of the bound and free forms of
the complex, respectively. Through this formula, the
binding constant Kb was calculated as 1.93 9 104 L mol-1
.
Interestingly, this Kb value is higher than those for similar
complexes such as [Cu(naph-leu)phen]CH3OHꢀ0.5H2O
(Kb, 4.87 9 103 L mol-1) [17] and [Cu(sal-L-val)phen]
(Kb, 6.48 9 103 L mol-1) [18].
½DNAꢁ=ðeaꢂefÞ ¼ ½DNAꢁ=ðebꢂefÞ þ 1=KbðebꢂefÞ;
123

Transition Met Chem
Fig. 2 1D chain structure of the Cu(II) complex. All hydrogen atoms
and free solvent methanol molecules are omitted for clarity
Fig. 4 3D structure of the Cu(II) complex. All hydrogen atoms and
free solvent methanol molecules are omitted for clarity, except for
those engaged in hydrogen bonding
Fig. 3 2D layer structure of the Cu(II) complex. All hydrogen atoms
and free solvent methanol molecules are omitted for clarity, except
for those engaged in hydrogen bonding
Ethidium bromide (EB) is a common reagent used to
probe the interactions between complexes and DNA. When
intercalated into DNA, EB shows strong fluorescence at
600 nm, but this is diminished if the EB is displaced from
the DNA helix [19–21]. Therefore, in order to further probe
the interactions of our complex with DNA, a fluorescence
titration experiment was carried out. The emission spectra
of EB bound to DNA in the absence and presence of
complex are given in Fig. 6. With increasing concentra-
tions of the complex, the fluorescence intensity of the EB-
DNA system decreased significantly at 594 nm. This
indicates that the complex can displace EB from the DNA
helix, resulting in quenching of the fluorescence intensity.
The quenching constant Ksq was determined according to
the classical Stern–Volmer equation [22]:
Fig. 5 UV spectra of the Cu(II) complex with increasing concen-
tration
of
DNA.
[Complex] = 10-5 mol L-1
,
[DNA] =
0–5 9 10-5 mol L-1. From a to f: r = 0, 1.0, 2.0, 3.0, 4.0, 5.0
(r = [DNA]/[complex]). Inset Fitted equation of between [DNA]/
(ea - ef) and [DNA]
where I0 is the emission intensity in the absence of
quencher, I is the emission intensity in the presence of
quencher, and r corresponds the ratio of the total concen-
tration of complex to that of DNA. The quenching constant
Ksq was calculated as 0.76, which is higher than that of
[Cu(L-Phe)(TATP)(H2O)]? (Ksq, 0.41) [23], suggesting
that our Cu(II) complex binds quite strongly with DNA.
The effect of salt concentration was investigated by
adding increasing amounts of NaCl to the mixture of DNA
I0=I ¼ 1 þ Ksq ꢀ r;
123

Transition Met Chem
Fig. 8 Effects of different concentration of the Cu(II) complex
(b) and EB (a) on the relative viscosities of DNA. [DNA] =
Fig. 6 Emission spectra of EB–DNA system on fluorescence
quenching with different concentration of the Cu(II) complex.
2 9 10-5 mol L-1
, . r = 0,
[complex] = 0–0.14 9 10-5 mol L-1
[EB] = 2 9 10-6 mol L-1
,
[DNA] = 2 9 10-6 mol L-1
,
[com-
0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07 (r = [complex]/[DNA])
plex] = 0–2.4 9 10-6 mol L-1, kex = 258 nm. From a to f: r = 0,
0.15, 0.30, 0.60, 0.90, 1.20 (r = [complex]/[EB-DNA]). Inset Fitted
equation of between I0/I and [Complex]/[DNA]
Hydrodynamic measurements that are sensitive to length
change (i.e., viscosity and sedimentation) are regarded as
the least ambiguous and the most critical tests of a binding
model in solution in the absence of crystallographic
structural data [26]. The classical intercalation model pre-
dicts that the DNA helix must lengthen as base pairs are
separated to accommodate the binding ligand, leading to an
increase in DNA viscosity. On the other hand, a partial
intercalation mode can bend (or kink) the DNA helix,
resulting in a decrease in the effective length and viscosity,
while non-intercalative groove binding causes no obvious
increase in DNA viscosity [27]. The effects of the Cu(II)
complex and EB on the viscosity of DNA are shown in
Fig. 8. With increasing concentrations of both the complex
and EB, the viscosity gradually increased, although the
increment for the complex was less than that for EB. These
results indicate that the complex binds to DNA via an in-
tercalative mode.
Fig. 7 The relative fluorescent intensity curve of DNA bound to the
Cu(II) complex with increasing concentration of NaCl.
[DNA] = 10-6 mol L-1
0–1.4 9 10-6 mol L-1
,
[complex] = 10-6 mol L-1
,
[NaCl] =
DNA cleavage studies
and Cu(II) complex. If the complex binds to DNA by an
electrostatic interactive mode, DNA will be increasingly
surrounded by sodium ions with increasing ionic strength,
making it more difficult for the complex to interact with
DNA. As a result, fluorescence quenching should occur
with increasing concentrations of NaCl [24]. As shown in
Fig. 7, however, increasing concentrations of NaCl had
little effect on the fluorescence intensity. This result con-
firms that the complex binding to CT-DNA is not primarily
via electrostatic interaction [25].
To further investigate the interactions of the complex with
DNA, cleavage experiments were carried out, in which the
cleavage reaction of supercoiled plasmid DNA was moni-
tored by agarose gel electrophoresis. DNA cleavage is
controlled by relaxation of the supercoiled circular form of
pBR322 DNA into nicked circular and linear forms. When
circular plasmid DNA is subjected to electrophoresis, the
fastest migration will be observed for the supercoiled form
(form I). If one strand is cleaved, the supercoils will relax
to produce a slower moving open circular form (form II). If
both strands are cleaved, a linear form (form III) will be
generated that migrates in between forms I and II [28]. The
To further clarify the interaction between the complex
and DNA, viscosity measurements were taken.
123

Transition Met Chem
NaN3 (lanes 5 and 6), which are known to be scavengers of
superoxide and singlet oxygen [30]. Hence, these results
suggest that the DNA cleavage mechanism involves
hydroxyl radical species [31].
Fig. 9 Photoactivated cleavage of pBR322 DNA (0.2 lg) of the
Cu(II) complex. Lane 1 DNA control; Lane 2–7 DNA ? the Cu(II)
complex, [complex] = 20, 30, 40, 50, 60 and 70 lM, respectively
Conclusion
In this paper, we have described a ternary Cu(II) complex
of a Schiff base ligand derived from 4-chloroanthranilic
acid and salicylaldehyde. The complex binds to CT-DNA
via an intercalative mode, and also shows significant DNA
cleavage activity. The result of this study may be helpful in
better understanding the interactions between DNA and
copper complexes, which may facilitate the design of new
anticancer drugs.
Acknowledgments This research was supported by the National
Natural Science Foundation of China to C. F. Bi (Nos. 21371161 and
21071134), the Specialized Research Fund for the Doctoral Program
of Higher Education of China to Y. H. Fan (No. 20120132110015),
the Natural Science Foundation of Shandong Province to X. Zhang
(No. ZR2012BQ026), the Special Foundation for Young Teachers of
Ocean University of China to X. Zhang (No. 201113025).
Fig. 10 Photoactivated cleavage of pBR322 DNA (0.2 lg) in the
presence of ROS scavengers of the Cu(II) complex (70 lM). Lane 1
DNA control; Lane 2 DNA ? the Cu(II) complex; Lane 3–6
DNA ? the Cu(II) complex ? ROS scavengers: (3) DMSO
(5 mM), (4) t-BuOH (5 mM), (5) KI (5 mM), (6) NaN3 (5 mM)
gel electrophoresis pattern of pBR322 DNA after incuba-
tion with the Cu(II) complex and irradiation at 245 nm is
shown in Fig. 9. No DNA cleavage was observed for the
control for which the metal complex was absent (lane 1).
With increasing concentrations of the Cu(II) complex
(lanes 2–5), the amount of form I DNA diminished grad-
ually, whereas form II DNA increased, showing the
potential chemical nuclease activity of the complex.
Additionally, on further increasing the concentration of the
complex (lanes 6 and 7), the linear DNA (form III)
appeared, indicating that this complex is a potent DNA
cleavage agent.
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