Synthesis, crystal structure, DNA cleavage properties, and protein binding activities of an unsymmetrical dinuclear copper(II) complex

Article abstract of DOI:10.1080/00958972.2014.880784

[Cu₂L(phen)(H₂O) ₃(ClO₄)] (ClO₄)₃ {phen = 1,10-phenanthroline, L = 4-((bis(pyridine2-ylmethyl)amino)methyl)-N,N-bis(pyridin-2-ylmethyl)aniline} was prepared and characterized by physical chemical techniques. The X-ray single-crystal structure analysis shows an unsymmetrical dinuclear species. The interaction of the complex with calfthymus DNA (CT-DNA) was studied by various spectroscopic and viscosity measurements, which indicate that the complex can interact with CT-DNA through intercalation. In the presence of H ₂O₂, the complex can cleave pBR322 DNA more efficiently and hydroxyl radicals (HO^.) may serve as the major cleavage active species. Interactions of the compound with bovine serum albumin (BSA) were also investigated using UV-Vis and fluorescence spectroscopic methods. The results show that the complex quenches the intrinsic fluorescence of BSA by a static quenching mechanism. 2014

Full text of DOI:10.1080/00958972.2014.880784

Journal of Coordination Chemistry, 2014
Vol. 67, No. 2, 300–314, http://dx.doi.org/10.1080/00958972.2014.880784
Synthesis, crystal structure, DNA cleavage properties, and
protein binding activities of an unsymmetrical dinuclear
copper(II) complex
JING LU†‡§, JUN-LING LI†‡§, QIAN SUN†‡§, LIN JIANG†‡§, BI-WEI WANG†,
WEN GU†‡§, XIN LIU*†‡§, JIN-LEI TIAN*†‡§ and SHI-PING YAN†‡§
†Department of Chemistry, Nankai University, Tianjin, PR China
‡Tianjin Key Laboratory of Metal and Molecule Based Material Chemistry, Tianjin, PR China
§Key Laboratory of Advanced Energy Materials Chemistry (MOE), Tianjin, PR China
(Received 28 October 2013; accepted 16 December 2013)
The X-ray single-crystal structure analysis shows that the complex is an unsym-
metrical dinuclear species.
[Cu2L(phen)(H2O)3(ClO4)](ClO4)3 {phen = 1,10-phenanthroline, L = 4-((bis(pyridine2-ylmethyl)
amino)methyl)-N,N-bis(pyridin-2-ylmethyl)aniline} was prepared and characterized by physical
chemical techniques. The X-ray single-crystal structure analysis shows an unsymmetrical dinuclear
species. The interaction of the complex with calfthymus DNA (CT-DNA) was studied by various
spectroscopic and viscosity measurements, which indicate that the complex can interact with
CT-DNA through intercalation. In the presence of H2O2, the complex can cleave pBR322 DNA
more efficiently and hydroxyl radicals (HO.) may serve as the major cleavage active species. Interac-
tions of the compound with bovine serum albumin (BSA) were also investigated using UV−Vis and
fluorescence spectroscopic methods. The results show that the complex quenches the intrinsic
fluorescence of BSA by a static quenching mechanism.
Keywords: Unsymmetrical ligand; DNA Cleavage; Intercalation mode;
A static quenching
mechanism
*Corresponding authors. Email: liuxin64@nankai.edu.cn (X. Liu); tiant@nankai.edu.cn (J.-L. Tian)
© 2014 Taylor & Francis

Unsymmetrical dinuclear copper(II) complex
301
1. Introduction
Metal-based pharmaceuticals have received considerable attention in medicinal inorganic
chemistry, of growing significance in both therapeutic and diagnostic medicine [1–4]. The
world’s first anticancer drug, cisplatin (cis-[Pt(NH3)2Cl2]), provided enormous impetus in
establishing the field of medicinal inorganic chemistry in the 1960s [5]. Despite its success,
platinum-based anticancer agents are non-specific, resulting in significant nephrotoxicity,
neurotoxicity, and emetogenesis, limiting its application in clinical applications [6–10].
Designing other transition metal compounds that can bind and cleave double-stranded DNA
under physiological conditions to overcome the toxicity and side effects of platinum
compounds is a major goal.
Copper as an essential element for most organisms plays an important structural and
catalytic role in a number of biological pathways [11]. Copper complexes are regarded as a
potential alternative to the observed cytotoxicity in a variety of cancer cell lines [12–14].
Many copper(II) complexes have been reported to efficiently cleave DNA by oxidative,
hydrolytic, photolytic, or electrolytic mechanisms. In particular, DNA cleavage by copper
complexes containing poly-pyridyl ligands and their derivatives has been extensively
studied [12, 15, 16].
As considerable attention has been paid to interactions of transition metal complexes with
DNA, there is interest in analysis of drug–protein interactions that affect the apparent distri-
bution volume and their elimination rate properties of drugs. Serum albumin is the major
protein constituent of blood plasma with important physiological functions, such as storage
and transport of endogenous and exogenous metabolites of small molecule drugs [17, 18].
Therefore, interactions of metal complexes with bovine serum albumin (BSA) receive more
and more attention in pharmaceutical engineering and pharmacokinetics. Due to its struc-
tural homology with human serum albumin, BSA is the most extensively studied serum
albumin [19–21]. This supports the value of studying the interaction behavior of metal com-
plexes with BSA protein, while evaluating their anticancer properties.
Previously, we synthesized several copper complexes of 1,4-tpbd (N,N,N′,N′-tetrakis-
(2-pyridylmethyl)benzene-1,4-diamine), which exhibited efficient DNA cleavage activity
[22]. As continuation of our previous studies, we have been interested in exploring an
unsymmetrical ligand and obtained a dinuclear copper(II) complex, and investigated its
DNA cleavage activity and protein binding activity.
2. Experimental setup
2.1. Materials
Caution! Perchlorate salts of metal complexes are potentially explosive and therefore should
be prepared in small quantities.
L was synthesized according to a previously reported procedure [23]. Chemicals and sol-
vents were purchased from commercial sources and used as received. Plasmid pBR322
DNA, agarose, ethidium bromide (EB), calfthymus DNA (CT-DNA), BSA, and stock solu-
tions of copper(II) complexes (1.0 × 10−3 M in CH3CN) were stored at 4 °C and prepared at
required concentrations for all experiments. Tris–HCl and phosphate buffer solutions were
prepared using ultrapure water (18 MΩ cm).

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J. Lu et al.
2.2. Measurements
Elemental analyses for C, H, and N were obtained on a Perkin-Elmer analyzer model 240.
Infrared spectroscopy as KBr pellets was performed on a Bruker Vector 22 FT-IR
spectrophotometer from 4000 to 400 cm−1. Electronic spectra were measured on a JASCO
V-570 spectrophotometer. Fluorescence spectral data were obtained on a MPF-4 fluores-
cence spectrophotometer at room temperature. Viscosity measurements were carried out on
an Ubbelodhe viscometer maintained at a constant temperature (37.0 0.1 °C) in a thermo-
stated water bath. The Gel Imaging and documentation DigiDoc-It System were assessed
using Labworks Imaging and Analysis Software (UVI, UK).
2.3. Synthesis of the complex
2.3.1. Preparation of L. A solution of K2CO3 (6.98 g, 50.5 mM) in water (14 mL) was
slowly added to a stirred solution of 2-(chloromethyl)pyridine hydrochloride (11.68 g,
71.2 mM) in water (9 mL) and 4-aminobenzylamine dihydrochloride (2.95 g, 14.8 mM) in
ethanol (21 mL). To the above reaction mixture, 6 mL aqueous solution of sodium hydrox-
ide (0.01 M) was added dropwise under nitrogen. After stirring for seven days in the dark
at room temperature, an oily gum was formed and then extracted with dichloromethane
(3 × 25 mL), and then the organic phases were combined and dried over MgSO4. Most of
the solvent was evaporated under reduced pressure and the remaining crude product was
re-dissolved in absolute ethanol (Yield: 30.8%) (scheme 1).
2.3.2. Synthesis of [Cu2L(phen)(H2O)3(ClO4)](ClO4)3. This complex was prepared by
the addition of an ethanol (10 mL) solution of L (0.2 mM) and 1,10-phenanthroline
(0.2 mM, 0.0396 g) to an aqueous solution of Cu(ClO4)2·6H2O (0.4 mM, 0.1482 g) and then
refluxed for 3–4 h. The solution was cooled to room temperature, filtered, and allowed to
stand for evaporation at room temperature. After several days, blue block crystals suitable
Scheme 1. L.

Unsymmetrical dinuclear copper(II) complex
303
Table 1. Crystal data and structure refinement for the complex.
Compound
Complex 1
Chemical formula
Formula mass
Crystal system
a/Å
b/Å
c/Å
α/°
β/°
C43H44Cl4Cu2N8O19
1245.74
Triclinic
9.7892(5)
16.1191(6)
16.9868(6)
106.583(3)
93.670(4)
91.180(4)
2561.53(19)
127(2)
γ/°
Unit cell volume/Å3
Temperature/K
Space group
P
Z
2
No. of reflections measured
No. of independent reflections
Rint
17,370
8928
0.0242
Final R1 values (I > 2σ(I))
Final wR(F2) values (I > 2σ(I))
Final R1 values (all data)
Final wR(F2) values (all data)
0.0844
0.2274
0.1048
0.2439
for X-ray crystallography were obtained (Yield ca. 46% (based on the copper salt)).
Elemental analysis (%) : Calcd for C43H44Cl4Cu2N8O19 : C, 41.46; H, 3.56; N, 8.99. Found:
C, 41.38; H, 3.62; N, 8.87. FT-IR (KBr, ν, cm−1): 3238 m (νN−H), 2027 m, 1636 versus
1617 s, 1144 m, 1119 m, 625 s, 484 m.
2.4. X-ray crystallographic studies
Determination of the unit cell and data collection for the complex were performed on
a SuperNova single-crystal diffractometer equipped with graphite-monochromated Mo-
Kα radiation (λ = 0.71073 Å) using the ω-scan technique. The structure was solved by
direct methods (SHELXS-97) and refined with full-matrix least-squares on F2 using
SHELXL-97 [24, 25]. Hydrogens were added theoretically, riding on the concerned
atoms and refined with fixed thermal factors. Details of the crystallographic data and
structure refinement parameters are summarized in table 1. We collected the crystal data
several times at low or room temperature and obtained better quality data with the low
Rint value, but the final R1 value is slightly higher due to structural disorder associated
−
with the pyridine and ClO4 .
2.5. DNA binding studies
The CT-DNA stock solution was prepared by diluting DNA in Tris–HCl/NaCl buffer (pH
7.2, 5 mM Tris–HCl, 50 mM NaCl) and kept at 4 °C for no longer than a week. The
compound was dissolved in ultrapure water (18 MΩ cm) and acetonitrile. The DNA binding
experiments were performed at room temperature in Tris–HCl/NaCl buffer (50 mM Tris–
HCl/1 mM NaCl buffer, pH 7.2) using an acetonitrile (CH3CN) solution of the complex. A
solution of CT-DNA in the buffer (pH 7.2) gave a ratio of UV absorbance of about 1.8–1.9
at 260 and 280 nm, indicating that the DNA was sufficiently free of protein [26]. The DNA

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J. Lu et al.
concentration per nucleotide was determined by absorption spectroscopy using the molar
extinction coefficient value of 6600 M−1 cm−1 at 260 nm [27].
Absorption titration experiments were performed by maintaining the concentration of the
complex (25 μM), while gradually increasing the concentration of DNA (0−150 μM). The
absorption was recorded after each addition of CT-DNA. Fluorescence measurements were
made by using a Varian Cary Eclipse spectrofluorometer. For competitive binding experi-
ments, the complex was added to the CT-DNA solution treated with EB (2.4 μM EB and
48 μM CT-DNA) for 30 min. The samples were excited at 510 nm and emission spectra
were recorded at 520–800 nm. The experiments were carried out by adding a certain
amount of a solution of the complex step by step to the EB-DNA solution. The influence of
the addition of the complex to the EB-DNA complex was obtained by recording the varia-
tion of the fluorescence emission spectra with excitation at 510 nm and emission at 602 nm.
Before the emission spectra were recorded, the complex-DNA solutions were incubated at
room temperature for 5 min to complete the reaction.
Viscosity measurements were carried out on CT-DNA by varying the concentration of
the complex in 5 mM Tris–HCl/50 mM NaCl buffer (pH 7.2) using an Ubbelodhe viscome-
ter maintained at 37.0 0.1 °C in a thermostated water bath. Flow time was measured with
a digital stopwatch, each sample was measured thrice and an average flow time was
calculated.
2.6. DNA cleavage and mechanism studies
DNA cleavage experiments were done by agarose gel electrophoresis, which was performed
by incubation at 37 °C as follows: pBR322 DNA (100 ng/μL) in 50 mM Tris–HCl/18 mM
NaCl buffer (pH 7.2) was treated with the complex. The samples were incubated for 3 h
and the loading buffer was added. Then, the samples were electrophoresed for 2 h at 80 V
on 0.9% agarose gel using Tris–boric acid–EDTA buffer. After electrophoresis, bands were
visualized by UV light and photographed. The extent of cleavage of the SC DNA was
determined by measuring the intensities of the bands using the Gel Documentation System.
Cleavage mechanistic investigation of pBR322 DNA was carried out in the presence of
standard radical scavengers and reaction inhibitors. The reactions were carried out by add-
ing standard radical scavengers KI, NaN3, SOD, and catalase to pBR322 DNA prior to the
addition of the complexes. Cleavage was initiated by addition of complex and quenched
with 2 μL of loading buffer. Further analysis was carried out by the above-mentioned
standard method.
2.7. Protein binding studies
The protein binding study was performed by fluorescence quenching experiments using
BSA stock solution (BSA, 1.5 mM) in 10 mM phosphate buffer (pH 7.0). A concen-
trated stock solution of the compound was prepared as used for the DNA binding
experiments, except that phosphate buffer was used instead of a Tris–HCl buffer for all
of the experiments. The fluorescence spectra were recorded at room temperature with
excitation wavelength of BSA at 280 nm and the emission at 342 nm by keeping the
concentration of BSA constant (30 μM). while varying the complex concentration from
0 to 50 μM. In addition, absorption titration experiments were carried out by keeping
the concentration of BSA constant (15 μM), while varying the concentration of the
complex from 0 to 2.5 μM.

Unsymmetrical dinuclear copper(II) complex
305
Figure 1. A view of crystal packing for the complex. Hydrogens and dissociative small molecules are omitted for
clarity.
Table 2. Selected bond lengths (Å) and angles (°) for the complex.
Bond distances (Å)
Cu(1)–N(6)
Cu(1)–N(5)
Cu(1)–O(16)
Cu(2)–N(3)
Cu(2)–O(13)
Cu(2)–O(8)
1.997(5)
2.027(5)
2.335(5)
2.023(6)
2.277(6)
2.645(7)
Cu(1)–N(7)
Cu(1)–N(8)
Cu(1)–N(4)
Cu(2)–N(9)
Cu(2)–N(2)
Cu(2)–O(14)
2.016(5)
2.040(5)
2.508(5)
2.083(16)
1.983(7)
1.957(7)
Bond angles (°)
N(6)–Cu(1)–N(7)
N(7)–Cu(1)–N(5)
N(7)–Cu(1)–N(8)
N(6)–Cu(1)–O(16)
N(5)–Cu(1)–O(16)
N(1)–Cu(2)–O(14)
O(14)–Cu(2)–N(3)
O(14)–Cu(2)–N(9)
N(3)–Cu(2)–N(9)
O(14)–Cu(2)–O(13)
N(3)–Cu(2)–O(13)
171.0(2)
N(6)–Cu(1)–N(5)
N(6)–Cu(1)–N(8)
N(5)–Cu(1)–N(8)
N(7)–Cu(1)–O(16)
N(8)–Cu(1)–O(16)
O(14)–Cu(2)–N(2)
N(2)–Cu(2)–N(3)
N(2)–Cu(2)–N(9)
N(1)–Cu(2)–O(13)
N(2)–Cu(2)–O(13)
N(9)–Cu(2)–O(13)
92.6(2)
91.4(2)
167.4(2)
87.82(19)
95.9(2)
102.4(4)
77.5(3)
147.9(5)
88.9(3)
99.5(3)
96.5(4)
92.8(2)
81.8(2)
98.9(2)
95.3(2)
90.1(4)
172.2(3)
106.9(6)
71.7(5)
83.8(3)
104.0(2)
3. Results and discussion
3.1. The characterization of the complex
The structure of the complex is depicted in figure 1 and selected bond lengths and angles
ꢀ
are listed in table 2. It crystallizes in the P1 triclinic space group. As shown in figure 1, the
complex consists of a dinuclear [Cu2L(1,10-phen)(H2O)3(ClO4)]3+ and three ClO4 anions.
−
L is a linker with Cu1⋯Cu2 distance of 9.694 Å, which is longer than the distances in other
copper(II) complexes (about 8 Å) reported [22]. Compared to 1,4-tpbd reported previously,
we have chosen the p-aminobenzylamine instead of p-phenylenediamine as the raw material

306
J. Lu et al.
to synthesize our ligand. The benzyl group which increases the length of the carbon chain
results in the longer distance of Cu⋯Cu than that of [Cu2(1,4-tpbd)(1,10-phen)2(DMF)2]
(ClO4)4. In the cation, each copper is six-coordinate with N5O donor set derived from three
ligand N, two 1,10-phen N, and one coordinated water for Cu1; N3O3 donor set derived
from three ligand N, two coordinated waters, and one perchlorate O for Cu2, forming a dis-
torted octahedral geometry. For Cu1, N(5), N(6), N(7), and N(8) form the equatorial plane
with Cu–N bond lengths from 1.997 to 2.040 Å. N(4) and O(16) occupy apical positions
with Cu(1)–N(4) = 2.508 Å and Cu(1)–O(16) = 2.335 Å. For Cu2, N(2), N(3), N(9), and O
(14) form the equatorial plane with bond lengths from 1.957 to 2.083 Å. O(8) and O(13)
occupy apical positions with Cu(2)–O(8) = 2.645 Å and Cu(1)–O(13) = 2.277 Å. The apical
bond lengths are longer than the equatorial bond lengths due to the Jahn–Teller effects in d9
copper(II) complexes. For Cu2, a perchlorate occupies the apical position; the Cu–O bond
distance observed is comparable to those observed previously [22, 28, 29].
3.2. DNA binding modes and affinity studies
3.2.1. Electronic absorption spectra. Electronic absorption spectroscopy is a useful
method to study the binding of DNA with metal complexes. The potential binding ability of
the complex to CT-DNA was studied by UV spectroscopy. Typical titration curves for the
complex in the absence and presence of CT-DNA at different concentrations are given in
figure 2. The complex shows characteristic charge transfer absorption bands at 200–300 nm,
which are assigned as intraligand electronic spectral π–π* transitions. With increasing
concentration of CT-DNA, significant hypochromicity and a red shift are observed at the
maximal peak for the complex (hypochromism of 37.8% and red shift of 11 nm), which
indicates insert binding nature of the complex with CT-DNA, because intercalation would
Figure 2. (a) Absorption spectra of complex (25 μM) in the absence (black line) and presence of increasing
amounts of CT-DNA at room temperature in 5 mM Tris–HCl/50 mM NaCl buffer (pH 7.2). The arrow shows the
absorbance changes on increasing CT-DNA concentration (b). The inset shows the plot of (εa−εf)/(εb−εf) vs.
[DNA], obtained from absorption spectral titration of the complex.

Unsymmetrical dinuclear copper(II) complex
307
lead to hypochromism and bathochromism in UV absorption spectra owing to a strong
stacking interaction between an aromatic chromophore and the base pairs of DNA [30].
The intrinsic equilibrium binding constant (Kb) and the binding site size (s) of the
complex bound to CT-DNA were determined by monitoring the changes in the absorption
intensity of the complex with increasing concentrations of CT-DNA. The values of Kb and
s were calculated according to the equation [31, 32]:
1=2
ðea ꢀ ef Þ=ðeb ꢀ ef Þ ¼ ðb ꢀ ðb2 ꢀ 2Kb2Ct½DNAꢁ=sÞ Þ=2KbCt
(1a)
(1b)
b ¼ 1 þ KbC þ Kb½DNAꢁ=2s
t
where εa is the extinction coefficient observed for the charge transfer absorption band at a
given DNA concentration, εf is the extinction coefficient of the free complex in solution, εb
is the extinction coefficient of the complex when fully bound to DNA, Ct is the total
complex concentration, and [DNA] is the DNA concentration in nucleotides.
The calculated values of Kb and s suggest a relatively strong binding of the complex to
CT-DNA. The Kb value of the complex (3.02 × 105 M−1) is of the same magnitude as that
of the classical intercalator EB (Kb = 1.23 ( 0.07) × 105 M−1). Kb for this complex is higher
than found in previous reports on copper complexes [33–35]. The better binding affinity of
the present dicopper(II) complex may be attributed to more π-electrons provided by the
phenanthroline ring system and aromatic ring for better stacking between the base pairs of
DNA.
3.2.2. Fluorescence spectra. No luminescence is observed for the complex at room
temperature, so the binding of the complex to CT-DNA was studied by a fluorescence
Figure 3. (a) Fluorescence emission spectra (excited at 510 nm) of the CT-DNA-EB system (2.4 μM EB, 48 μM
CT-DNA) in the absence and presence of the complex with increasing the concentration from 0 to 100 μM (from
top to bottom). The dashed line shows the intensity in the absence of complex (b). The fluorescence quenching
curve of EB bound to DNA by the complex.

308
J. Lu et al.
spectral method using the emission intensity of EB as a probe. EB emits intense fluores-
cence in the presence of CT-DNA due to its strong intercalation between the adjacent DNA
base pairs [36]. A competitive binding of the complex to CT-DNA decreases the emission
intensity of EB and the reduction extent of the emission intensity gives a measure to verify
the DNA binding propensity of the complex.
The quenching plots (figure 3) illustrate that quenching of EB bound to CT-DNA by the
complex is in agreement with the linear Stern–Volmer equation I0=I ¼ 1 þ K ½Qꢁ (I and I
sv
0
represent the fluorescence intensities in the absence and presence of quencher and [Q] is the
concentration of quencher) [37]. The values of the apparent binding constant (Kapp) were
calculated on the basis of the equation log KEB½EBꢁ ¼ K ½complexꢁ, where the complex
app
concentration was the value at a 50% reduction of the fluorescence intensity of EB and KEB
= 1.0 × 107 M−1 [38], [(EB) = 2.4 μM]. The calculated Ksv and Kapp values of the complex
are 2.95 × 104 M−1 and 7.1 × 105 M−1, respectively (R = 0.996470 for eleven points), less
than the binding constant of [Cu2(1,4-tpbd)(1,10-phen)2(DMF)2](ClO4)4 (4.86 × 106 M−1)
reported by Li [22]. The Kapp value suggests that the interaction of the copper(II) complex
with DNA is a moderate intercalative mode.
3.2.3. Viscosity measurement. Viscosity measurements were carried out to further
verify the interaction of the complexes with CT-DNA. In classical intercalation, the
complexes result in a lengthening and stiffening of the double helix of DNA, leading to
an increase in viscosity of DNA [39]. In contrast, a partial and/or non-intercalation of
the ligand could result in a less pronounced effect on the viscosity [40]. The plot of
relative specific viscosity (η/η0)1/3 versus [complex]/[DNA] ratio for the complex is given
in figure 4. It shows that the relative viscosity of CT-DNA increased with increasing
concentration of the complexes. The result suggests that the complex binds to CT-DNA
through intercalation.
Figure 4. Effect of increasing amounts of the complex on the relative viscosity of CT-DNA at 37 ( 0.1) °C in
5 mM Tris–HCl/50 mM NaCl buffer (pH 7.2, [DNA] = 0.1 mM).

Unsymmetrical dinuclear copper(II) complex
3.3. The cleavage of pBR322 DNA
309
The DNA cleavage activities of the complex have been studied using supercoiled pBR322
plasmid DNA as a substrate in a medium of 50 mM Tris–HCl/NaCl buffer (pH 7.2) in the
absence of external agents under physiological conditions for 3 h. When the original super-
coiled form (Form I) of plasmid DNA is nicked, an open circular relaxed form (Form II)
will exist in the system and the linear form (Form III) can be found upon further cleavage.
Using electrophoresis, the compact Form I migrates relatively faster, while the nicked Form
II migrates slowly, and the linearized form (Form III) migrates between Forms I and II.
Concentration-dependent DNA cleavage by the complex without any external additives
in natural light was performed. The result of gel electrophoretic separations of plasmid
pBR322 DNA induced by increasing concentration is shown in figure 5. No obvious DNA
cleavage was observed for controls in which the compound was absent (lane 6). With
increasing concentration of the complex, Form I plasmid DNA is gradually converted into
Form II, the complex shows poor DNA cleavage activity. At 65 μM, the complex cleaves
about 83% of the plasmid to yield the nicked circular form.
Figure 5. Agarose (1%) gel electrophoresis showing concentration-dependent cleavage of pBR322 DNA (200 ng)
by complex in different condition for an incubation time of 3.0 h at 37 °C. Lane 6: DNA control; lanes 1–5: DNA +
5, 20, 35, 50, 65 μM of the complex.
Figure 6. Agarose (1%) gel electrophoresis showing time-dependent cleavage of pBR322 DNA (200 ng) by
complex in same condition at 37 °C. Lane 0: DNA control; lanes 1–5: DNA + 50 μM of complex for 0, 15, 45, 75,
135, 180 min.

310
J. Lu et al.
Figure 7. Agarose (1%) gel electrophoresis showing concentration-dependent cleavage of pBR322 DNA (200 ng)
by complex in different condition for an incubation time of 3.0 h at 37 °C. Lane 0: DNA control; lanes 1–5: DNA +
5, 20, 35, 50, 65 μM of the complex + 0.25 mM H2O2.
Scheme 2. Proposed mechanism for DNA cleavage by copper(II) complexes in the presence of H2O2.
Time-dependent cleavage of DNA by the complex was also studied under similar
conditions. With increasing reaction time, the amounts of Form II increased and Form I
decreased, showing that cleavage of DNA by the complex depends on reaction time
(figure 6).
In the presence of H2O2, the DNA cleavage efficiencies of the complex exhibit remark-
able increases. As shown in figure 7, in the control experiments with DNA alone and DNA
with H2O2 alone, no DNA cleavage is observed (lane 0 and lane 1). Obviously, Form I dis-
appeared and the linear form (Form III) can be found at 5 μM, which implies that H2O2 is a
reductant (proposed mechanism for DNA cleavage is shown in scheme 2) [41].
3.4. DNA cleavage mechanism
In order to obtain further knowledge of the active chemical species responsible for the
DNA damage activities by the complex, we investigated the influence of different poten-
tially inhibiting agents including hydroxyl radical scavenger (KI), singlet oxygen quenchers
(NaN3), superoxide scavenger (SOD), hydrogen peroxide scavenger (catalase), and chelat-
ing agent (EDTA) under our experimental conditions. No obvious inhibitions were observed

Unsymmetrical dinuclear copper(II) complex
311
Figure 8. Agarose (1%) gel electrophoresis showing cleavage of pBR322 DNA (200 ng) by complex in different
condition for an incubation time of 3.0 h at 37 °C. Lane 0, DNA control; lane 1, DNA + 0.25 mM H2O2; lane 2,
DNA + Complex (5 μM) + 0.25 mM H2O2; lane 3, DNA + Complex (5 μM) + 0.25 mM H2O2 + KI (20 mM); lane 4,
DNA + Complex (5 μM) + 0.25 mM H2O2 + NaN3 (20 mM); lane 5, DNA + Complex (5 μM) + 0.25 mM H2O2 +
SOD (20 U/mL); lane 6, DNA + Complex (5 μM) + 0.25 mM H2O2 + Catalase (20 U/mL); lane 7, DNA + Complex
(5 μM) + 0.25 mM H2O2 + EDTA (10 mM); lane 8, DNA + Complex (5 μM) + 0.25 mM H2O2 + Methyl Green; lane
9, DNA + Complex (5 μM) + 0.25 mM H2O2 + SYBR.
for the complex in the presence of NaN3 (lane 4), SOD (lane 5), and catalase (lane 6) in
figure 8. These results rule out the possibility of DNA cleavage by singlet oxygen, superox-
ide, or hydrogen peroxide. The addition of KI (lane 3) diminishes the nuclease activity of
the compounds, which indicates that the hydroxyl radical is involved in the cleavage
process. The chelating agent EDTA can efficiently inhibit DNA cleavage (lane 6),
indicating that the Cu(II) complex plays a key role in DNA breakage.
3.5. Protein binding studies
Qualitative analysis of the binding of chemical compounds to BSA is usually detected by
inspecting the fluorescence spectra. Generally, the fluorescence of BSA is caused by
tryptophan and tyrosine. Changes in the emission spectra of tryptophan are common in
response to protein conformational transitions, subunit associations, substrate binding, or
denaturation [42]. Therefore, the intrinsic fluorescence of BSA can provide considerable
information on their structure and dynamics and is often utilized in the study of protein
Figure 9. The emission spectrum of BSA (30 μM; λexi = 280 nm; λemi = 345 nm) in the presence of increasing
amounts of complex. The dashed line shows the intensity in the absence of complex. The arrow shows the fluores-
cence quenching upon increasing the concentrations of the complex (a). The inset shows the Stern−Volmer plots
(b) and Scatchard plots (c) of the complex with BSA.

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J. Lu et al.
Figure 10. Absorption spectra of BSA (15 μM) and BSA with the complex (2.5 μM).
folding and association reactions. The interaction of BSA with our complex was studied by
fluorescence measurement at room temperature (figure 9).
The fluorescence quenching is described by the Stern–Volmer equation and the quench-
ing rate constant Kq was calculated with the plot of I0/I versus [Q]. Quenching can occur by
different mechanisms, which are usually classified as dynamic quenching and static quench-
ing; dynamic quenching refers to a process in which the fluorophore and the quencher come
into contact during the transient existence of the excited state. Static quenching refers to
fluorophore-quencher complex formation in the ground state. A simple method to explore
the type of quenching is UV−Vis absorption spectroscopy. UV−Vis spectra of BSA in the
absence and presence of the compound (figure 10) show that the absorption intensity of
BSA was enhanced as the compound was added, and there was a little blue shift, revealing
a static interaction between BSA and the added compound due to the formation of ground
state BSA-compound [43].
If it is assumed that the binding of compound with BSA occurs at equilibrium, the equi-
librium binding constant can be analyzed according to the Scatchard equation [44]:
log½ðI0 ꢀ IÞ=Iꢁ ¼ log Kbin þ n log½Qꢁ, where Kbin is the binding constant of the compound
with DNA and n is the number of binding sites [figure 9(b) and (c)]. The Kbin and n values
of the complex obtained are 1.53 × 104 M−1 and 0.93 (R = 0.98983 for ten points). The cal-
culated value of n is 1 for the complex, indicating the existence of just a single binding site
in BSA for the complex [44].
4. Conclusion
A new unsymmetrical dinuclear Cu complex, [Cu2L(phen)(H2O)3(ClO4)](ClO4)3, has been
synthesized and characterized by single-crystal X-ray diffraction. The interaction of the
complex with CT-DNA has been investigated by UV absorption, fluorescence spectroscopy,
and viscosity, and the results indicate that the complex binds to CT-DNA through intercala-
tion. DNA cleavage studies showed that the complex exhibits DNA cleavage in the pres-
ence of H2O2 and hydroxyl radicals generated in the presence of the complex may act as
active species for DNA scission. The ability to bind to proteins has also been explored,

Unsymmetrical dinuclear copper(II) complex
313
which is the requisite for a drug to act as an anticancer agent for probing and targeting
nucleic acids and proteins.
Supplementary material
Crystallographic data (excluding structure factors) for the structures in this article have been
deposited with the Cambridge Crystallographic Data Center as supplementary publication
CCDC 967951. Copies of the data can be obtained, free of charge, on application to the
CCDC, 12 Union Road, Cambridge CB2 1EZ, UK.
Funding
This work was supported by the National Natural Science Foundation of China [21171101 21071083
and 21001066]; NFFTBS [No. J1103306]; Tianjin Science Foundation [No. 12JCYBJC13600].
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