Synthesis, crystal structure, and DNA-binding properties of a new copper (II) complex containing mixed-ligands of 2,2′-bipyridine and p-methylbenzoate

Article abstract of DOI:10.1016/j.ica.2005.12.035

A novel copper complex of [Cu(bpy)(pba)₂·H ₂O]·0.5H₂O (bpy = 2,2′-bipyridine, pba = p-methylbenzoate) was synthesized. The interaction of the complex to native fish sperm DNA was investigated through electrochemistry, electronic absorption spectroscopy and viscosity experiments. In the X-ray crystallography structure, the copper (II) ion is coordinated by two oxygen atoms of two p-methylbenzoate groups, two nitrogen atoms of 2,2′-bipyridine and one water molecule. The observed changes in the physicochemical features of the copper (II) complex on binding to DNA suggested that the complex bind to DNA with intercalation mode via 2,2′-bipyridine ring into DNA base pairs. Electrochemical studies revealed that the complex prefer to bind to DNA in Cu(I) form rather than Cu(II) oxidation state form. Additionally, the nuclease activity of the title complex was assessed by gel electrophoresis assay and the results shown that the copper complex can cleave pBR322 DNA effectively in the presence of ascorbic acid.

Full text of DOI:10.1016/j.ica.2005.12.035

Inorganica Chimica Acta 359 (2006) 1524–1530
www.elsevier.com/locate/ica
Synthesis, crystal structure, and DNA-binding properties of a
new copper (II) complex containing mixed-ligands of 2,20-bipyridine
and p-methylbenzoate
a,
b
b,
c
a
Fa Qian Liu *, Qing Xiang Wang , Kui Jiao *, Fang Fang Jian , Guang Ye Liu ,
a
Rong Xun Li
a
Key Laboratory of Advanced Materials, Qingdao University of Science and Technology, Qingdao 266042, China
College of Chemistry and Molecular Engineering, Qingdao University of Science and Technology, Zhengzhou Road 53#, Qingdao, Shandong 266042, China
b
c
New Materials and Function Coordination Chemistry Laboratory, Qingdao University of Science and Technology, Qingdao 266042, China
Received 26 September 2005; received in revised form 15 December 2005; accepted 20 December 2005
Available online 8 February 2006
Abstract
A novel copper complex of [Cu(bpy)(pba)2 Æ H2O] Æ 0.5H2O (bpy = 2,20-bipyridine, pba = p-methylbenzoate) was synthesized. The
interaction of the complex to native fish sperm DNA was investigated through electrochemistry, electronic absorption spectroscopy
and viscosity experiments. In the X-ray crystallography structure, the copper (II) ion is coordinated by two oxygen atoms of two p-meth-
ylbenzoate groups, two nitrogen atoms of 2,20-bipyridine and one water molecule. The observed changes in the physicochemical features
of the copper (II) complex on binding to DNA suggested that the complex bind to DNA with intercalation mode via 2,20-bipyridine ring
into DNA base pairs. Electrochemical studies revealed that the complex prefer to bind to DNA in Cu(I) form rather than Cu(II) oxi-
dation state form. Additionally, the nuclease activity of the title complex was assessed by gel electrophoresis assay and the results shown
that the copper complex can cleave pBR322 DNA effectively in the presence of ascorbic acid.
ꢀ 2006 Elsevier B.V. All rights reserved.
Keywords: 2,20-Bipyridine ligand; Copper (II) complex; Crystal structure; DNA
1. Introduction
plexes with potential chemical, biological and catalytic
properties.
Copper (Cu) is a physiologically important metal ele-
ment that plays an important role in the endogenous oxida-
tive DNA damage associated with aging and cancer [1].
Some of copper (II) complexes are found to exhibit a vari-
ety of pharmacological activity and superoxide dismutase
activity [2]. Complexes of copper (II) with carboxylate
and imidazole ligands have been studied as model for cop-
per protein that contains both functionalities in the side
chain [3]. These potential and versatile applications are
stimulating researchers to exploit new ligands and com-
Deoxyribonucleic acid (DNA) is the primary target mol-
ecule for most anticancer and antiviral therapies according
to cell biology [4,5]. Investigations of the interactions of
DNA with transition metal complex are basic work to design
new types of the pharmaceutical molecules, to elucidate the
mechanisms involved in the site specific recognition of
DNA and to determine the principles governing the recogni-
tion [6,7]. Since Sigman et al. [8] discovered that copper ion
complexed to 1,10-phenanthroline can cleave DNA in
1979, the copper complexes are widely used as footprint
DNA binding molecules [9,10] and cleaving reagents [11–
19] over the past two decades. Li et al. [19] have synthesized
a Schiff base copper (II) complex, Cu(o-VANAHE)2 (o-
*
Corresponding authors. Fax: +86 532 84023606.
VANAHE = 2-(o-vanillinamino)-1-hydroxyethane)
and
E-mail addresses: fqliu@vip.163.com, qdplastics@163.com (F.Q. Liu),
assessed the interaction between this complex and DNA by
kjiao@qust.edu.cn (K. Jiao).
0020-1693/$ - see front matter ꢀ 2006 Elsevier B.V. All rights reserved.
doi:10.1016/j.ica.2005.12.035

F.Q. Liu et al. / Inorganica Chimica Acta 359 (2006) 1524–1530
1525
viscosity, fluorescence spectroscopy, cyclic voltammetry and
submarine gel electrophoresis. The results suggested that this
copper complex can intercalate into helix DNA and cleave
circular plasmid pBR322 DNA to nicked and linear forms.
A new ligand L, 1-[3-(2-pyridyl)pyrazol-1-ylmethyl]naph-
thalene, and its copper complexe, [Cu(L)3](ClO4)2 have been
synthesized and characterized by Zhang et al. [20]. The inter-
actions of the complex and the ligand L with calf thymus
DNA were then investigated by thermal denaturation, vis-
cosity measurements and spectrophotometric methods.
The experimental results indicated that the copper complex
bind to DNA by intercalative mode via the ligand L. The
intrinsic binding constants are 1.8 · 104 Mꢀ1 and
2.8 · 103 Mꢀ1 for the complex and ligand L with DNA,
respectively. Pasternack and his co-workers [21] have
described the interactions of three cationic water soluble
copper (II) porphyrins, differing in peripheral substituents,
with calf thymus DNA using extinction spectroscopy, circu-
lar dichroism, RLS and resonance Raman spectroscopy. It
was found that tetrakis(N-methylpyridinium-4-yl)porphy-
rin copper (II) behaves as a simple intercalator under the
investigated conditions, whereas tetrakis(4-N,N0,N00-trime-
thylanilinium) porphyrin copper (II), binds externally, with
some limited aggregation under high drug load conditions.
the known molar extinction coefficient value of
6600 Mꢀ1 cmꢀ1 [23]. Supercoiled plasmid pBR322 DNA
was purchased from TaKaRa Biotechnology Co., Ltd.
(China). Ethidium bromide (EB) was obtained from Fluka
Company (Switzerland). The other chemical reagents for
synthesizing copper complex were all purchased commer-
cially and used without further purification. Double dis-
tilled water was used for preparing all the solutions.
2.2. The preparation of the title complex
(pba)2Cu was prepared by mixing aqueous solutions of
copper (II) sulfate and sodium p-methylbenzoate according
to the literature procedures [24]. To a warm solution of
2,20-bipyridine (0.16 g, 1 mmol) in EtOH (50 mL) was
added with stirring (pba)2Cu (0.33 g, 0.1 mmol) and the
mixture was refluxed for 2 h. The resulting solution was fil-
tered and was left to stand overnight. Blue precipitation
appeared. A blue crystal suitable for X-ray analysis was
obtained by recrystallizing this blue solid from ethanol
solution. The C, H and N contents were determined by ele-
mental analysis. Anal. Calc. for C26H25Cu1N2O5.5: C,
60.40; H, 4.87; N, 5.42. Found: C, 60.51; H, 4.95; N, 5.34%.
In
contrast,
trans-bis(N-methylpyridinium-4-yl)diph-
2.3. The physical measurement of the title complex and its
DNA-binding experiments
enylporphyrin copper (II) (t-CuPagg), like the free-base t-
H2Pagg from which it is derived, is capable of forming
extended electronically coupled arrays while bind to the
DNA template.
Elemental analyses (C, H and N) were carried out on a
Perkin–Elmer 1400C analyzer. Voltammetry was per-
formed by using CHI 832 electrochemical analysis system
(China) with three-electrode system consisted of glass car-
bon (GC) electrode (U = 3 mm) as the working electrode,
saturated calomel electrode (SCE) as the reference elec-
trode, and a platinum wire as the auxiliary electrode. All
the electrochemical measurements were carried out in a
10 mL electrolyte cell with 0.01 M pH 6.86 KH2PO4–
Na2HPO4 buffer solution as electrolyte. Electronic
absorption spectrum was measured on a Cary 50 probe
spectrophotometer (Australia) using 0.01 M pH 6.86
KH2PO4–Na2HPO4 as blank solution. Viscosity experi-
ments were carried out using an Ubbelodhe viscometer
maintained at a constant temperature at 30 0.1 ꢁC in a
thermostatic water-bath. Flow time was measured with a
digital stopwatch and each sample was measured three
times, and an average flow time was calculated. Data were
presented as (g/go)1/3 versus binding ratio of [Cu]/[DNA],
where g is the relative viscosity of DNA in the presence
of complex, and go is the relative viscosity of DNA alone.
Viscosity values were calculated from the observed flow
time of DNA-containing solutions corrected for the flow
time of KH2PO4–Na2HPO4 buffer solution alone (t0), g =
t ꢀ t0 [23]. The cleavage of DNA by the title copper
complex was monitored using agarose gel electrophoresis.
Reactions using 10 lM supercoiled (SC) pBR322 plasmid
DNA in 50 mM pH 7.1 Tris–NaCl buffer was treated with
the copper complex and 100 lM ascorbic acid. The samples
wereincubatedfor0.5 hat37 ꢁC. Aloadingbuffercontaining
In this paper, we synthesized and obtained the crystal
structure of a new aqua-2,20-bipyridine copper (II) bis(p-
methylbenzoate) hydrate: [(bpy)Cu(pba)2 Æ H2O] Æ 0.5H2O.
The binding properties of the title complex to fish sperm
DNA were carried out using voltammetry, absorption
spectroscopy and viscosity experiment, and so on. The
binding mode of the copper complex to DNA is accessed
to be intercalation from the experimental results, which
implicated that the copper (II) complex can be a candidate
for DNA-binding reagents, as well as laying the foundation
for the rational design of new useful DNA probes. The gel
electrophoresis experiment shown that the copper complex
can cleave pBR322 DNA effectively in the presence of
ascorbic acid as an effective inorganic nuclease.
2. Experimental
2.1. Materials
Double-strand fish sperm DNA from Beijing Baitai Bio-
chemistry Technology Company (China) was used as
received. The stock solution of DNA was prepared by dis-
solving appropriate amount of DNA in H2O and stored at
4 ꢁC. The ratio of the absorbance at 260 and 280 nm (A260
/
A280) was checked to be ꢁ1.89, indicating that the DNA is
sufficiently free from protein [22]. The concentration of
DNA in nucleotide phosphate [NP] was determined spec-
trophotometrically at 260 nm after 1:100 dilutions using

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F.Q. Liu et al. / Inorganica Chimica Acta 359 (2006) 1524–1530
25% bromophenol blue, 3 lL 30% glycerol was added and
the electrophoresis was performed at 40 V in Tris–acetate–
EDTA (TAE) buffer using 1% agarose gel containing ethi-
dium bromide. Agarose gel electrophoresis of plasmid
DNA was visualized by photographing the fluorescence
of intercalated ethidium bromide under a UV illuminator.
3. Results and discussion
3.1. Crystal structure description
Fig. 1 shows a perspective view of the title complex with
atomic numbering. Selected bond lengths and bond angles
are presented in Table 2.
2.4. X-ray structure determination
In the molecule, each Cu ion is coordinated by two oxy-
gen atoms of two p-methylbenzoate groups, two nitrogen
atoms of 2,20-bipyridine and one water molecule as showed
in Fig. 1. The coordination geometry of the copper center is
essentially square pyramid with Cu(1)N(1)N(2)O(1)O(3)ꢂ ꢂ ꢂ
O(1w) chromospheres. The p-methylbenzoate anion
Single-crystal X-ray diffraction measurement for the title
complex was carried out on a SMART CCD diffractometer
with graphite monochromated Mo Ka (k = 0.071073 nm)
radiation at the temperature of 293(2) K. Intensities were
corrected for Lorentz and polarization effects and empirical
absorption, and the data reduction using SADABS [25] pro-
gram. The structure was solved by direct methods using
SHELXS-97 [26]. All the non-hydrogen atoms were refined
on F2 anisotropically by full-matrix least-squares method.
All hydrogen atoms were placed in calculated positions
assigned fixed isotropic thermal parameters at 1.2 times
the equivalent isotropic U of the atoms to which they were
attached and allowed to ride on their respective parent
atoms. The contributions of these hydrogen atoms were
included in structure-factor calculations. Atomic scattering
factors and anomalous dispersion corrections were taken
from International Table for X-ray Crystallography [27].
A summary of the key crystallographic information is
given in Table 1.
behaves as a monodentate carboxylate ligand. The Cu–O
˚
Cu(1)–O(3) =
distances
[Cu(1)–O(1) = 1.967(5) A;
˚
1.949(5) A] in title complex are normal and are in close
agreement with those found in a similar structure reported
before [28]. The axial Cu(1)–O(1w) distance of 2.359(5) A is
˚
in agreement with that found in a similar reported Cu–
O(1w) bond [32], while, is significantly longer than those
of the equatorial Cu(1)–O(1), O(3) bonds. The 13 atoms
containing one Cu(1) atom and 12 atoms from 2,20-bipyri-
dine molecule are almost coplanar, the deviation of the Cu
atom from the least-squares plane through the 13 atoms
˚
being 0.057(3) A. The dihedral angle between the two p-
methylbenzoate ligands planes is 61.71(2)ꢁ. The p-meth-
ylbenzoate ligand including O(1) made a dihedral of
87.90ꢁ with 2,20-bipyridine ligand. The angles with the axial
atom O(1w) at Cu(1), viz. O(1w)–Cu(1)–O(1), O(3), N(1),
N(2) are 107.6(2)ꢁ, 91.1(2)ꢁ, 88.5(2)ꢁ, 93.7(2)ꢁ, respectively.
In the lattice, there are some intramolecular, intermolec-
ular and potentially weak (C–Hꢂ ꢂ ꢂY hydrogen bonds,
Y = O, N and Cl) interactions. For intramolecular hydro-
Table 1
Summary of crystallographic results for the title complex
Empirical formula
Formula weight
T (K)
C26H25Cu1N2O5.5
517.02
293(2)
0.71073
triclinic
˚
gen bonds, the donor and acceptor distances are 3.0257 A
˚
˚
k (A)
[O(2W)ꢂ ꢂ ꢂO(3)] and 2.6646 A [O(1W)ꢂ ꢂ ꢂO(4)]; for intermo-
Crystal system
Space group
Unit cell dimensions
ꢀ
P1
˚
a (A)
7.0230(14)
˚
b (A)
11.167(2)
16.700(3)
˚
c (A)
a (ꢁ)
b (ꢁ)
c (ꢁ)
103.89(3)
90.77(3)
104.28(3)
1228.4(4)
3
˚
V (A )
Z
1
Dcalc (Mg/m3)
1.398
0.930
536
Absorption coefficient (mmꢀ1
F(000)
)
h Range for data collection (ꢁ)
Limiting indices
1.26–24.50
0 6 h 6 8, ꢀ13 6 k 6 12,
ꢀ19 6 l 6 19
4403/4030 (0.0833)
98.8
full-matrix
least-squares on F2
4030/0/317
1.018
R1 = 0.0705, wR2 = 0.1438
R1 = 0.1920, wR2 = 0.1916
0.590 and ꢀ0.504
Reflections collected/unique (Rint
Completeness to h = 24.50ꢁ (%)
Refinement method
)
Data/restraints/parameters
Goodness-of-fit on F2
Final R indices [I > 2r(I)]
R indices (all data)
Fig. 1. Molecular structure of the title complex with the atomic
numbering scheme.
ꢀ3
˚
Largest difference in peak and hole (e A
)

F.Q. Liu et al. / Inorganica Chimica Acta 359 (2006) 1524–1530
1527
Table 2
˚
Selected bond lengths (A) and bond angles (ꢁ)
˚
˚
˚
Bond lengths (A)
Bond lengths (A)
Bond lengths (A)
Cu(1)–O(3)
Cu(1)–O(1)
Cu(1)–N(1)
Cu(1)–N(2)
Cu(1)–O(1W)
1.949(5)
1.967(5)
2.001(6)
2.007(6)
2.359(5)
O(1)–C(11)
O(2)–C(11)
O(3)–C(19)
O(4)–C(19)
N(1)–C(1)
1.271(9)
1.267(8)
1.262(9)
1.235(9)
1.313(10)
N(1)–C(5)
N(2)–C(10)
N(2)–C(6)
1.357(9)
1.333(9)
1.350(9)
Bond angles (ꢁ)
Bond angles (ꢁ)
Bond angles (ꢁ)
O(3)–Cu(1)–O(1)
O(3)–Cu(1)–N(1)
O(1)–Cu(1)–N(1)
O(3)–Cu(1)–N(2)
91.2(2)
93.8(3)
163.1(2)
172.5(2)
O(1)–Cu(1)–N(2)
N(1)–Cu(1)–N(2)
O(3)–Cu(1)–O(1W)
O(1)–Cu(1)–O(1W)
92.8(2)
80.5(3)
91.1(2)
107.6(2)
N(1)–Cu(1)–O(1W)
N(2)–Cu(1)–O(1W)
C(11)–O(1)–Cu(1)
C(19)–O(3)–Cu(1)
88.5(2)
93.7(2)
105.5(5)
124.5(5)
lecular hydrogen bonds, the donor and acceptor distances
Addition of DNA results in hypochromism of both the
absorption peaks and moderate bathochromic shift (ca.
2 nm) of the peak at 310 nm. These spectral characteristics
are consistent with a mode of interaction that involves a
stacking interaction between the complex and the base
pairs of DNA, which means that the titled complex can
intercalate into the double helix structure of DNA.
˚
˚
are 2.7641 A [O(1W)ꢂ ꢂ ꢂO(2)] and 2.9672 A [O(2W)ꢂ ꢂ ꢂ
O(1w)], respectively; the O(2w) with C(8) atoms in methyl
group form potentially weak C–Hꢂ ꢂ ꢂO intermolecular
interactions, the donor and acceptor distances are
˚
3.2369(2) A [C(8)ꢂ ꢂ ꢂO(2w)]. All above hydrogen bonds in
this structure form the three-dimensional hydrogen bonds
network that stabilizes the crystal structure.
3.2.2. Viscosity studies
3.2. The studies on the binding of the title complex to DNA
Optical photophysical probes are necessary, but not suf-
ficient clues to support a binding mode of small molecules
to DNA. Further clarification of the interaction mode
between the copper complex and DNA is carried out by vis-
cosity measurements. Hydrodynamic measurements that
are sensitive to length change (i.e., viscosity and sedimenta-
tion) of DNA are regarded as the least ambiguous and the
most critical tests of binding mode in solution in the absence
of crystallographic structural data [32,33]. A classical inter-
calation mode results in lengthening the DNA helix, as base
pairs are separated to accommodate the binding ligand,
leading to the increase of DNA viscosity. The effects of
the complex together with EB on the viscosity of DNA
are shown in Fig. 3. It is found that the viscosity of DNA
3.2.1. Absorption spectroscopic studies
The application of electronic absorption spectroscopy in
DNA-binding studies is one of the most useful techniques
[29–31]. Complex binding with DNA through intercalation
usually results in hypochromism and bathochromism, due
to the intercalative mode involving a strong stacking inter-
action between an aromatic chromophore and the base
pairs of DNA. The absorption spectra of the complex in
the absence and presence of DNA are illustrated in
Fig. 2. In the UV region, the two conjoint peaks of the
complex at 300 and 310 nm, respectively, may attributed
to intraligand p–p* transition of the coordinated groups.
1.08
b
1.07
1.06
a
1.05
o
1.04
1.03
1.02
1.01
1.00
0.99
0.0
0.2 0.4
0.6
r
0.8
1.0 1.2
1.4
Fig. 2. Absorption spectra of 2.00 · 10ꢀ4 M title copper (II) complex in
the absence (a) and presence of 1.02 · 10ꢀ4 M DNA (b) in 0.01 M pH 6.86
KH2PO4–Na2HPO4 buffer solution.
Fig. 3. Effect of increasing amounts of copper (II) complex (r) and EB
(j) on the relative viscosities of DNA at 30 0.1 ꢁC. r = [Cu]/[DNA] or
[EB]/[DNA], [DNA] = 1.02 · 10ꢀ4 M.

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F.Q. Liu et al. / Inorganica Chimica Acta 359 (2006) 1524–1530
increases steadily with the increase of the concentration of
the complex (Fig. 3a), which is similar to that of a classical
intercalator EB [34] (Fig. 3b). This result demonstrates that
the copper complex and EB bind to DNA through the same
way, i.e., the classical intercalation mode, which also paral-
lels the pronounced hypochromism and spectral red shift of
the complex in the absorption spectrum experiment.
positive shift (10 mV) after forming aggregation with
DNA, suggesting that the copper complex bind to DNA
mainly by intercalation binding mode [36], and this result
also proves the results obtained from viscosity and absorp-
tion spectrum studies again.
In addition to changes in formal potential upon addition
of DNA, the voltammetric current decrease significantly as
shown in Fig. 4. The dependences of cathodic currents of
the copper (II) complex on scan rate (m) are investigated
and the results are shown in the inset of Fig. 4. It is
observed that the cathodic currents of the copper complex
both with and without DNA are linear to the square root
of the scan rate (m1/2), which are expected as the diffu-
sion-controlled processes [37]. While, the slop of the Ipc–
m1/2 plot decreases distinctly after the mixing with DNA,
indicating the reduction in the apparent diffusion coeffi-
cient of complex in the presence of dsDNA [36]. So, we
can interpret the change in current upon DNA addition
in terms of the diffusion of an equilibrium mixture of free
and dsDNA-bound complex to the electrode surface.
3.2.3. Electrochemical studies
Cyclic and differential pulse voltammetric techniques
was employed to study the interaction of the present redox
active metal complex with DNA with a view to further
explore the DNA binding modes assessed from the above
spectral and viscometric studies.
Typical cyclic voltammetry curves for the title complex
in 0.01 M pH 6.86 KH2PO4–Na2HPO4 buffer solution, in
the absence and presence of DNA are shown in Fig. 4. In
the absence of DNA, the complex has a cathodic peak at
ꢀ0.335 V and an anodic peak at ꢀ0.115 V, respectively,
corresponding to the electrochemical process of CuII/CuI
[35]. The separation of the cathodic and anodic peak poten-
tial, DE = 0.240 V, indicates that the electrochemical
behavior of the copper complex on glass carbon electrode
The net shift in E0 can be used to estimate the ratio of
0
equilibrium constants for the binding of the bivalent and
univalent ions to DNA. This is analogous to the treatment
of the association of small molecules with micelles. For a
Nernstian electron transfer system, in which both the oxi-
dized and reduced forms associate with a third species in
solution (DNA), differential pulse voltammetry (DPV)
can be used to calculate the corresponding equilibrium con-
stants for each oxidation state binding to DNA according
to the following equation [37]:
is
a
quasi-reversible process. After addition of
2.72 · 10ꢀ5 M DNA, the value of DE in the presence of
DNA decreases to 0.179 V, showing that the reversibility
of electron-transfer process of the complex is enhanced
after interac0tion with DNA. The formal potential of the
complex, E0 (or voltammetric E1/2), taken as the average
value of cathodic peak and anodic peak potentials, is
ꢀ0.235 V and ꢀ0.225 V, respectively, when DNA is absent
E0b0 ꢀ E0f 0 ¼ 0:059 logðKI=KIIÞ;
and present in the solution. Obviously, E0 undergoes a
0
where E0b0 and E0f 0, the formal potentials of Cu(II)/Cu(I)
complex couple in the binding and0free forms, respectively,
are determined by the formula: E0 = Ep + DEp/2, (Ep, the
DPV peak potential; DEp, the pulse amplitude); KII and KI
are the binding constants of oxidized and reduced forms to
DNA, respectively.
Fig. 5 shows the differential pulse voltammograms of
3.00 · 10ꢀ4 M copper complex in the absence and presence
of DNA. It is observed that the reduction current of Cu(II)
decrease obviously and its potential shifts positively after
reacting with DNA, which is consistent with the results
of the cyclic voltammetry (Fig. 4). Thus, for a limiting
potential shift of +12 mV after adding excess DNA, the
ratio of the binding constants (KI/KII) of the Cu(I) and
Cu(II) complex to DNA was calculated to be 1.6, which
indicates that the intensity of Cu(I) binding to DNA is
stronger than that of Cu(II).
3.3. Cleavage of pBR322 plasmid DNA by copper (II)
complex
Fig. 4. Cyclic voltammetry of 3.00 · 10ꢀ4 M Copper (II) complex in the
absence (a) and presence 2.72 · 10ꢀ5 M DNA (b) in 0.01 M pH 6.86
KH2PO4–Na2HPO4 buffer solution, Scan rate: 0.04 V/s. Inset: the plots of
the cathodic peak currents of Cu(bpy)(pba)2 in the absence (a) and
presence (b) of DNA vs. the square root of the scan rate (m1/2).
The cleavage ability of the title copper (II) complex to
DNA was investigated by gel electrophoresis using super-
coiled pBR322 DNA in 50 mM Tris–NaCl buffer (pH
7.1). The cleavage efficiency was measured by determining

F.Q. Liu et al. / Inorganica Chimica Acta 359 (2006) 1524–1530
1529
form the Cu(I) species, which then binds to DNA forming
a Cu(I) complex–DNA adduct, then reacts with dioxygen
to form a ‘copper-oxene’ radical, that is the species respon-
sible for the cleavage of DNA.
4. Conclusions
In summary, a new copper complex, [Cu(bpy)(pba)2 Æ
H2O] Æ 0.5H2O, was synthesized and its structure and phys-
icochemical properties were characterized by X-ray crystal-
lography, elemental analysis, electronic absorption
spectrum and electrochemistry. The binding properties of
the copper complex to native DNA were studied by electro-
chemical techniques, electronic absorption spectrophotom-
etry and viscosity experiment, and so on. The experimental
results show that the title copper complex can intercalate
into the double helix structure of the native DNA. The
cleaving ability of the title complex to DNA was assessed
by gel electrophoresis, and the result shows that it has obvi-
ous nuclease activity after mixing with ascorbic acid.
Fig. 5. Differential pulse voltammograms of 3.0 · 10ꢀ4 M copper (II)
complex in the absence (a), and presence of 2.72 · 10ꢀ5 M DNA (b) in
0.01 M pH 6.86 KH2PO4–Na2HPO4 buffer solution. Pulse amplitude,
50 mV; Pulse width, 50 ms; Pulse period, 200 ms; Increasing potential,
4 mV.
Acknowledgements
the ability of the complex to convert the supercoiled DNA
(form I) to nicked circular form (form II) and linear form
(form III). Control experiments using only copper complex
or ascorbic acid (H2A) failed to show any apparent cleav-
age of DNA (Fig. 6, lanes 1 and 2). While, the copper com-
plex can cleave the supercoiled plasmid DNA (form I) to
nicked circular DNA (form II) in the presence of H2A,
and even to linear DNA (form III) when the concentration
of the copper complex increased (Fig. 6, lanes 3–5), sug-
gesting that the copper complex has obvious nuclease activ-
ity after mixing with ascorbic acid. It has been established
that copper (II) complexes are catalytically active in the
oxidation of ascorbic acid by dioxygen involving copper
(I) intermediate species [38], so, according to the DNA
cleavage mechanism proposed by Sigman et al. [39] for
[Cu(phen)2]2+ complex, the cleavage of DNA by the title
copper (II) complex can be expatiated by the following pro-
cedures: the copper (II) is first reduced by ascorbic acid to
The work is supported by the National Nature Science
Foundation of China (Grant Nos. 20375020 and
50372028) and Natural Science Foundation of Shandong
Province (No. Y2002B06).
Appendix A. Supplementary data
CCDC-257520 contains the supplementary crystallo-
graphic data for this paper. These data can be obtained free
of charge at www.ccdc.cam.ac.uk/conts/retrieving.html [or
from the Cambridge Crystallographic Data Centre
(CCDC), 12 Union Road, Cambridge CB2 1EZ, UK; fax:
+44 1222 336 033; e-mail: deposit@ccdc.cam.ac.uk]. Sup-
plementary data associated with this article can be found,
in the online version, at doi:10.1016/j.ica.2005.12.035.
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