Improving nuclease activity of copper(II)-terpyridine complex through solubilizing and charge effects of glycine

Article abstract of DOI:10.1016/j.jinorgbio.2012.12.018

Copper complexes are potential metallonucleases that may find application in biotechnology and molecular biology. In this study, a ternary copper-terpyridine complex [Cu(ttpy)(Gly)(NO₃)](NO₃) · H₂O (1) (ttpy = 4′-p-tolyl-2,2′:6,2″- terpyridine) is synthesized and characterized by X-ray crystallography and ESI-MS as an artificial nuclease. Glycine (Gly) is introduced into the complex to enhance the water-solubility and electrostatic affinity for the nucleic acid target. The interaction between complex 1 and DNA has been studied by spectroscopy and gel electrophoresis, using a structural analog [Cu(ttpy)(NO₃)₂] (2) as the reference. Complex 1 demonstrates an increased DNA binding ability and oxidative cleavage activity towards supercoiled pBR322 DNA as compared with complex 2. The enhanced water-solubility and positive charge of complex 1 may facilitate its access to DNA and formation of hydrogen bonds with the sugar-phosphate backbone. The results indicate that carefully positioned auxiliary groups in a copper complex can significantly affect the substrate binding or activation ability and consequently the nuclease efficiency of the complex.

Full text of DOI:10.1016/j.jinorgbio.2012.12.018

Journal of Inorganic Biochemistry 121 (2013) 114–120
Contents lists available at SciVerse ScienceDirect
Journal of Inorganic Biochemistry
journal homepage: www.elsevier.com/locate/jinorgbio
Improving nuclease activity of copper(II)–terpyridine complex through solubilizing
and charge effects of glycine
a
b,c,
a
a,
Wen Zhou , Xiaoyong Wang ⁎, Ming Hu , Zijian Guo ⁎⁎
a
State Key Laboratory of Coordination Chemistry, School of Chemistry and Chemical Engineering, Nanjing University, Nanjing 210093, PR China
State Key Laboratory of Pharmaceutical Biotechnology, School of Life Sciences, Nanjing University, Nanjing 210093, PR China
State Key Laboratory of Analytical Chemistry for Life Science, Nanjing University, Nanjing 210093, PR China
b
c
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 23 October 2012
Received in revised form 27 December 2012
Accepted 28 December 2012
Available online 9 January 2013
Copper complexes are potential metallonucleases that may find application in biotechnology and molecular
3 3 2
biology. In this study, a ternary copper–terpyridine complex [Cu(ttpy)(Gly)(NO )](NO )·H O (1) (ttpy=
4′-p-tolyl-2,2′:6,2″-terpyridine) is synthesized and characterized by X-ray crystallography and ESI-MS as an
artificial nuclease. Glycine (Gly) is introduced into the complex to enhance the water-solubility and electrostatic
affinity for the nucleic acid target. The interaction between complex 1 and DNA has been studied by spectroscopy
3 2
and gel electrophoresis, using a structural analog [Cu(ttpy)(NO ) ] (2) as the reference. Complex 1 demonstrates
Keywords:
an increased DNA binding ability and oxidative cleavage activity towards supercoiled pBR322 DNA as compared
with complex 2. The enhanced water-solubility and positive charge of complex 1 may facilitate its access to
DNA and formation of hydrogen bonds with the sugar-phosphate backbone. The results indicate that carefully
positioned auxiliary groups in a copper complex can significantly affect the substrate binding or activation ability
and consequently the nuclease efficiency of the complex.
Artificial nuclease
Copper complex
Crystal structure
DNA cleavage
©
2013 Elsevier Inc. All rights reserved.
1
. Introduction
4′-p-tolyl-2,2′:6,2″-terpyridine) prepared in our laboratory are barely
soluble in aqueous media, which greatly impedes the further study as
nucleases [14]. Therefore, new model systems may need auxiliary
groups capable of increasing water-solubility and substrate binding
to improve their applicability in biological settings.
Artificial metallonucleases are widely studied because of their
potential use as novel biotechnological tools and therapeutic agents
1,2]. A number of metal complexes with nuclease activity have
[
found application as conformational probes or footprinting reagents
to map the structure of nucleic acids and their adducts with other
molecules, and some may be developed into anti-infective or anti-
tumoral drugs targeting specific types of DNA or RNA in the future
In recent years, we have designed different types of copper com-
plexes in order to find more efficient artificial nucleases [15–17].
Among them, integrating an auxiliary group into copper complexes
proved to be an effective way to promote the nuclease activity [18].
In this study, we try to introduce glycine (Gly) as an auxiliary ligand
to solubilize and ionize the copper(II)–terpyridine complex (Fig. 1).
Since Gly is a highly water-soluble amino acid (25 g/100 mL, 25 °C)
and exists as a zwitterion in neutral solution, we believe that introduc-
tion of Gly into the complex could increase its water-solubility and
affinity for polyanionic nucleic acid targets through solubilization and
electrostatic interaction. To verify our supposition, the DNA binding
and cleavage properties of complex 1 were studied and compared
[3,4]. One of the major working mechanisms of nucleases is oxidative
cleavage, in which hydrogen atoms are abstracted from the sugar and
phosphate components of nucleosides through redox chemistry and
the strand is broken as a result [5]. Copper complexes are able to
promote the generation of reactive oxygen species (ROS) in the pres-
ence of mild reducing agents, which has been exploited to oxidatively
break the DNA strands and further to inhibit the proliferation of
tumor cells [6–8]. A variety of copper complexes have been designed
for these reasons [9,10]; for example, several copper(II)–terpyridine
complexes have shown considerable nuclease property and anti-
tumor activity in physiologically relevant conditions [11–13].
3 2
with that of its analog [Cu(ttpy)(NO ) ] (2) (Fig. 1). As a result, the
water-solubility and DNA cleavage activity of the copper(II)–terpyridine
complex is markedly improved after the optimization.
3 2
However, some of such complexes like [Cu(ttpy)(NO ) ] (ttpy=
2
. Experimental
⁎
Correspondence to: X. Wang, State Key Laboratory of Pharmaceutical Biotechnology,
School of Life Sciences, Nanjing University, Nanjing 210093, PR China. Tel.: +86 25
3594549; fax: +86 25 83314502.
2.1. Materials and methods
8
Reagents such as Cu(NO ) ·3H O, 4-methylbenzaldehyde,
3 2 2
⁎⁎ Corresponding author. Tel.: +86 25 83594549; fax: +86 25 83314502.
E-mail addresses: boxwxy@nju.edu.cn (X. Wang), zguo@nju.edu.cn (Z. Guo).
2-acetylpyridine and Gly were of analytical grade and used without
0162-0134/$ – see front matter © 2013 Elsevier Inc. All rights reserved.
http://dx.doi.org/10.1016/j.jinorgbio.2012.12.018

W. Zhou et al. / Journal of Inorganic Biochemistry 121 (2013) 114–120
115
3 3 2
Fig. 1. Structures of [Cu(ttpy)(Gly)(NO ) ].
)]+ and [Cu(ttpy)(NO
further purification. Supercoiled pBR322 DNA and 6× loading buffer
0.05% bromophenol blue, 0.035% xylene cyanol FF, 36% glycerol and
0 mM EDTA) were purchased from TaKaRa Biotechnology (Dalian,
2.4. DNA binding
(
3
The CT-DNA stock solution was prepared as we described previ-
ously [18]. The UV absorption titration was carried out according
to the method we adopted in previous studies [14]. CD spectra
were recorded at room temperature in the wavelength range of
220–300 nm after CT-DNA (0.1 mM) was incubated without or with
the complex ([complex]/[DNA]=0.4) in the buffer (5 mM Tris–HCl/
50 mM NaCl, pH 7.4) at 37 °C for 12 h. Three scans were performed
for each spectrum. The fluorescence of the EB–DNA system (10 μM EB,
1 mM CT-DNA) was determined in the same Tris–HCl buffer at
room temperature (λex=526 nm, λem=605 nm). Specifically, EB–
DNA (0.3 mL) and aliquot of complex solutions were added to the
buffer, ensuring that the total volume of each system reached 3 mL.
The concentration of the complex in the buffer increased by 0.5 μM
after each titration until the fluorescence stopped changing at 5.5 μM.
The apparent binding constant (Kapp) was calculated from the equation
China). Calf thymus DNA (CT-DNA), Tris, and ethidium bromide (EB)
were purchased from Sunshine Bio (Nanjing, China). Distilled water
was used throughout all the experiments. The images of agarose gel
electrophoresis were obtained by using a Bio-Rad Gel-Doc XR imaging
system. The electrospray ionization mass spectrometry (ESI-MS)
spectra were recorded in the positive mode using an LCQ fleet ESI-MS
spectrometer (Thermo Scientific), and the isotopic distribution patterns
of the observed species were simulated using the Isopro 3.0 program.
UV–vis spectra were determined on a Shimadzu UV 3600 (UV–vis-
near-IR) spectrophotometer. Circular dichroism (CD) spectra were
measured by a Jasco J-810 spectropolarimeter (Japan Spectroscopic,
Japan). Fluorescence spectra were recorded on an LS-50B Spectrofluo-
rimeter (Perkin-Elmer, U.S.A.).
2
.2. Synthesis of the ligand and complexes
Kapp½complexꢀ ¼ KEB½EBꢀ
Ligand ttpy was prepared according to the reported procedures [19].
where KEB =1.0×107 M−1, [EB]=1.0 μM, and [complex] is the concen-
tration at which 50% quenching of the fluorescence occurred [23]. The
average value of three independent experimental results was taken as
Complex 1 was prepared as follows. Methanol–water (20 mL, 1:1) solu-
tions of Gly (0.075 g, 1 mmol) and Cu(NO ·3H O (0.241 g, 1 mmol)
3 2
)
2
were mixed and stirred at room temperature for 30 min. Methanol solu-
tion (10 mL) of ttpy (0.323 g, 1 mmol) was slowly added to the mixture
and stirred at 40 °C for 3 h. After removal of the solution, the resultant
solid was washed with acetone and diethyl ether, and dried in vacuo.
Green crystals suitable for single crystal X-ray diffraction were obtained
by vapor diffusion of diethyl ether into the methanol–water solution
the final Kapp
.
2.5. Agarose gel electrophoresis
Supercoiled pBR322 DNA (200 ng) was treated with ascending
concentrations of the complex and 1 μL of ascorbic acid (Vc) at a
100-fold molar excess relative to the complex in the buffer (50 mM
Tris–HCl/50 mM NaCl, pH 7.4). The resulting mixtures (10 μL) were
incubated in the dark at 37 °C for 90 min, and the reactions were
quenched by 2 μL of loading buffer. The solutions were loaded onto
the agarose gel (1%) and subjected to electrophoresis in a TAE buffer
of complex
solution. Yield: 0.35 g, 59.7%. Elementary analysis found (calcd) for
Cu (%): C, 49.21 (49.19); H, 3.62 (3.78); N, 21.57 (21.84).
1 or directly by slow evaporation of the complex
24 22 6 8
C H N O
+
ESI-MS (m/z, positive mode): 542.25, [Cu(ttpy)(Gly)(NO
3
)(H
2
O)]
Cu).
+
(C
24
H
24
N
5
O
6
Cu); 497.25, [Cu(ttpy)(Gly)(OH)(H
2
O)] (C24
H
25
N
4
O
4
Complex 2 was synthesized as reported previously [14].
(
40 mM Tris acetate/1 mM EDTA). DNA bands were stained by EB,
visualized and photographed under UV light. A correction factor of
1.47 was used for the supercoiled DNA (Form I) since the intercalation
of EB into Form I DNA is relatively weak as compared with that into
nicked (Form II) and linear (Form III) DNA [24]. The results of electro-
phoresis were obtained by using a Bio-Rad Gel-Doc XR imaging system.
2
.3. X-ray crystallography
X-ray crystallographic data were collected on a Bruker SMART APEX
CCD area-detector diffractometer operating in the φ-ω scan mode with
graphite-monochromated Mo-Ka radiation (λ=0.71069 Å) at 293 K.
Empirical absorption corrections were carried out using a multiscan
program. The SMART software was used for data acquisition and the
SAINT software for data extraction [20,21]. The structure was solved
by direct methods and refined on F2 by full matrix least-squares
methods using the SHELXTL program [22]. All non-hydrogen atoms
were refined anisotropically. Hydrogen atoms were included in
calculated positions and refined with isotropic thermal parameters.
2.6. Analysis of ROS
Radical scavengers and reaction inhibitors such as dimethyl
sulfoxide (DMSO, 10%), NaN (10 mM), and KI (10 mM) were added
3
respectively to the buffer solutions (50 mM Tris–HCl/50 mM NaCl,
pH 7.4) of pBR322 plasmid DNA (200 ng) and complex 1 or 2. The mix-
ture was incubated in the dark at 37 °C for 15 min prior to the addition

116
W. Zhou et al. / Journal of Inorganic Biochemistry 121 (2013) 114–120
of Vc to initiate the reaction (Vtotal, 10 μL). The subsequent treatment
Table 2
Selected bond lengths (Å) and angles (°) for complex 1.
and analysis were performed as described in Section 2.5.
Bond lengths
3
. Results and discussion
Cu1\N1
Cu1\N2
Cu1\N3
2.033 (2)
1.925 (2)
2.032 (2)
Cu1\O4
Cu1\O7
2.3929 (19)
1.8940 (17)
3
.1. Design and synthesis of the complexes
Bond angles
Complex 1 is an optimized analog of complex 2 that has exhibited
evident binding ability to intracellular DNA and CT-DNA in our previous
study [14]. In the present study, we modified the design by introducing
a solubilizing ligand Gly into the structure, hoping it can improve the
water-solubility of the copper(II)–terpyridine complex. As expected,
complex 1 is not only soluble in water (1.04 g/100 mL), but also soluble
N1\Cu1\N2
N2\Cu1\N3
N3\Cu1\O7
N1\Cu1\O7
N1\Cu1\O4
79.98 (8)
79.94 (8)
95.55 (8)
103.94 (8)
80.80 (7)
N2\Cu1\O4
N3\Cu1\O4
O4\Cu1\O7
N1\Cu1\N3
N2\Cu1\O7
88.50 (7)
106.57 (7)
96.21 (7)
158.39 (8)
174.28 (7)
in common solvents such as methanol and methyl cyanide. In addition,
+
the positive ammonium group (−NH
3
) in Gly could form strong
form the basal plane, and an oxygen atom from nitrate (O4) occupies
the apical position. The dihedral angle between the tolyl and terpyridine
planes is 19.27°; however, the corresponding dihedral angle for complex
2 is 15.8° [14], suggesting that these two planes could rotate along the
C\C bond in solution. The bond length of Cu\O4 is 2.393 Å while that
of Cu\O7 is 1.894 Å, indicating that the Cu\O7 bond is stronger than
Cu\O4 bond and Gly is more steady in the complex. In the crystal,
two molecules can form two N\H···O bonds with each other through
interaction between N\H from Gly and O from nitryl (Fig. S1), and the
bond lengths are both 2.069 Å.
charge-assisted hydrogen bonds with phosphate ester groups, which
can enhance the targeting tendency of the complex toward the
polyanionic phosphate backbone of nucleic acids under physiological
conditions. Although incorporation of an intercalator like ttpy does
not always lead to improved cleavage efficiency, it was found that the
aromatic π cloud is more uniformly distributed in the ttpy–copper
complex, which may lead to better stacking interactions with the DNA
bases and hence a higher binding ability to DNA [12].
Complex 1 was synthesized by reacting Cu(NO
3 2 2
) ·3H O with Gly
firstly and then with ttpy in methanol–water solution. Complex 2
was obtained directly through reacting Cu(NO ) ·3H O with ttpy in
3 2 2
methanol–water solution.
The crystal structure of complex 2 has been published previously
by our laboratory [14], which is largely similar to the structure of
complex 1.
The stability of complex 1 in aqueous solution was investigated by
ESI-MS. Complex 1 shows two major peaks at m/z 542.25 and 497.25
3
.2. Crystal structure and ESI-MS spectrum
in the ESI-MS spectrum (Fig. 3), which could be assigned to the
+
The structure of complex was characterized by X-ray
1
molecular ion peak [Cu(ttpy)(Gly)(NO
3 2
)(H O)] and hydrolytic
+
crystallography. Crystal parameters and structure refinement details are
summarized in Table 1. Selected bond lengths and angles relevant to
the copper center are listed in Table 2. Detailed crystallographic data of
complex 1 have been deposited at the Cambridge Crystallographic Data
Centre with the deposition number of CCDC 835512. These data are
available for free at http://www.ccdc.cam.ac.uk/deposit, or from CCDC,
2
species [Cu(ttpy)(Gly)(OH)(H O)] , respectively. The result suggests
that the coordinated nitrate is labile in water while Gly is stable,
which is consistent with the above analysis on the bond parameters;
besides, the complex is supposed to exist as a six coordinate species
with one of the coordination sites being occupied by a water molecule
in aqueous solution. Similar hydrolysis was also observed for complex
2 (Fig. S2).
1
2 Union Road, CAMBRIDGE CB2 1EZ, UK; E-mail: deposit@ccdc.cam.ac.
uk.
As the crystal structure shows (Fig. 2), the copper center of
complex 1 adopts a distorted square pyramidal CuN coordination
3.3. DNA binding properties
3 2
O
geometry with ttpy acting as a tridentate equatorial ligand. Three nitro-
gen atoms from ttpy (N1, N2, N3) and one oxygen atom from Gly (O7)
UV, CD, and fluorescence spectroscopies were employed to study
the interaction between CT-DNA and complex 1 or 2. As shown in
Fig. 4, the UV spectrum of complex 1 in aqueous solution shows
intense intraligand bands at 288 and 328 nm, which can be assigned
to the π–π* transition of ttpy [11]. Upon addition of increasing
concentrations CT-DNA to the solution of complex 1, a pronounced
hypochromism was observed at 288 and 328 nm. The hypochromism
Table 1
Crystallographic data and structure refinement parameters for complex 1.
Chemical formula
24 6 9
C H24CuN O
Formula weight
Temperature/K
604.03
296(2)
Crystal size/mm
0.30×0.10×0.10
Crystal system
Triclinic
Space group
P1
a/Å
b/Å
c/Å
α/°
β/°
γ/°
8.706(5)
11.744(5)
12.920(5)
92.599(5)
90.251(5)
109.995(5)
1239.8(10)
3
V/Å
Z
D
2
/g cm−
3
1.618
c
F(000)
622
Limiting indices
θ range/°
Tot., ref. number
Ref. number [I>2σ(I)]
−10≤h≤9, −13≤k≤13, −15≤l≤15
1.85–25.05
6293, 4316
4042
R
R
int
0.0998
0.0415, 0.1247
1.023
1
, wR [I>2σ(I)]
2
Fig. 2. Crystal structure of complex 1. A water molecule and all hydrogen atoms are
omitted for clarity.
GOF on F

W. Zhou et al. / Journal of Inorganic Biochemistry 121 (2013) 114–120
117
1
80 mM, the absorption keeps almost steady. We have indicated
that complex 1 exists in solution as a cation. The association between
and DNA would be facilitated by the charge–charge attraction
1
between the cation and the negatively charged DNA phosphate
+
backbone. However, in the presence of NaCl, Na may screen the elec-
trostatic interaction by competing with the complex cation for neutral-
izing the DNA phosphates and make the contribution of electrostatic
interaction to the association less important. At low NaCl concentrations
+
(
b140 mM), the hyperchromism suggests that Na could affect the
affinity of DNA for 1 through non-specific electrostatic interactions
and weaken the intercalative binding between DNA and 1. At high
NaCl concentrations (>140 mM), however, the electrostatic contribu-
tion is saturated and complex 1 becomes “free”, and hence the absorp-
tions return to their starting levels. The UV titration results show that
the binding of complex 1 to DNA is dependent on the salt concentration
of the medium. Therefore, electrostatic forces play a significant role in
the binding process. Complex 2 exhibits no such changes (data not
shown) for its electric neutrality.
Fig. 3. ESI-MS spectrum (positive mode) of complex 1.
The conformational changes of DNA induced by complex 1 or 2
were investigated by CD spectroscopy. The CD spectra of CT-DNA be-
fore and after the interaction with complex 1 or 2 ([complex]/
[DNA]=0.4) are shown in Fig. 6. The positive band at 275 nm is
due to the base stacking and the negative band at 248 nm is due to
the helicity, which is characteristic of B-DNA [26]. Both the positive
and negative bands red-shifted slightly and decreased markedly in
ellipticity in the presence of complex 1 or 2, suggesting that the
copper complexes can unwind the DNA helix and lead to the loss of
helicity [27]. The ellipticity decrease induced by complex 1 seems more
evident than that induced by complex 2 at the same concentration,
which indicates that complex 1 is to some extent more effective than
complex 2 in perturbing the secondary structure of DNA.
existed at two wavelengths is one of the most immediate indications
of possible intercalative binding to DNA [12,25]. The planarity and
extended aromaticity of the terpyridine ligand system may result in
the stacking of the complex between the DNA base pairs. By contrast,
complex 2 only exhibited a moderate hypochromism at 288 nm but
an apparent hyperchromism in the range of 300–350 nm (Fig. S3).
The hyperchromism implies that some interaction other than interca-
lation occurred between 2 and DNA. As the ratio of DNA to complex
increased from 0 to 1.2, the percentage of hypochromism at 288 nm
for 1 is 14.6%, while that for 2 is 3.0%. This suggests that the DNA
binding abilities of complexes 1 and 2 are different from each other,
and the ancillary ligand influences the binding of the complex to
DNA. Since the interaction between complex 1 and DNA could be
stabilized through hydrophilic and electrostatic interactions between
Gly and the phosphate backbone, the DNA binding ability of complex
The DNA binding behavior of complexes 1 and 2 was further
investigated using the EB–DNA system. This system generally gives
a significant increase in the fluorescence emission when intercalator
EB is bound to DNA, and a decrease in the emission when EB is
displaced [28]. As Fig. 7 shows, the fluorescence intensity (I) of the
EB–DNA system at 605 nm (λex =526 nm) decreases remarkably
with the titration of complex 1 or 2, suggesting that the complex
can replace the DNA-bound EB and the binding involves some
intercalative interaction. However, the binding ability to DNA is
different for these two complexes. According to the titration profiles,
the apparent binding constants (Kapp) of complexes 1 and 2 are calculated
1
is higher than that of complex 2.
The presumed electrostatic interaction between complex 1 and
DNA was tested by UV titration in the buffer solution (5 mM Tris–
HCl/NaCl, pH 7.4) with increasing concentration of NaCl from 5 to
2
00 mM. As Fig. 5 shows, in a fixed [DNA]/[complex] ratio (0.4), the
UV spectra of complex 1 change apparently with the variation of
NaCl concentrations. The absorptions at 288 and 328 nm display a
regular and similar changing trend (Inset in Fig. 5). As NaCl increases
from 20 to 140 mM, the absorption also increases gradually; as NaCl
increases from 140 to 180 mM, the absorption decreases; beyond
6
6
to be 4.22×10 and 2.41×10 , respectively [23], which demonstrate that
complex 1 has a higher affinity for DNA than complex 2. The results are
concordant with the UV and CD conclusions.
Fig. 5. UV spectra of complex 1 (15 μM) in the absence and presence of CT-DNA
([DNA]/[complex]=0.4) in the buffer (5 mM Tris–HCl/NaCl, pH 7.4) with increasing
concentrations of NaCl (5–200 mM). Inset shows the UV absorptions of complex 1 at
288 and 328 nm in buffer solutions with different NaCl concentrations.
Fig. 4. UV spectra of complex 1 (15 μM) in the absence and presence of CT-DNA at
[DNA]/[complex] ratios increasing (the directions for the arrows) from 0.2 to 1.2 in
the buffer (5 mM Tris–HCl/50 mM NaCl, pH 7.4).

118
W. Zhou et al. / Journal of Inorganic Biochemistry 121 (2013) 114–120
Fig. 6. CD spectra of CT-DNA (0.1 mM) in the absence and presence of complex 1 and 2
[complex]/[DNA]=0.4), respectively.
(
3
.4. DNA cleavage activity
The DNA cleavage activity of complexes 1 and 2 was studied using
supercoiled pBR322 plasmid DNA by gel electrophoresis in the buffer
50 mM Tris–HCl/50 mM NaCl, pH 7.4). Fig. 8 shows the electrophoresis
results of the DNA cleavage induced by increasing concentrations of the
complexes in the presence of Vc. Both complexes 1 and 2 can convert
Form I DNA into Form II at 2 μM (Lane 4), and complex 1 is more active
than complex 2 at this concentration. At 6 μM, complexes 1 and 2 cleave
(
Fig. 8. The cleavage patterns of the agarose gel electrophoresis and the corresponding
cleavage extent (%) for pBR322 plasmid DNA (200 ng) by complex 1 (A) or complex 2
(B) and Vc (1 mM) in the buffer solution (50 mM Tris–HCl/50 mM NaCl, pH 7.4) at
37 °C for 90 min. Lane 1, DNA control; Lane 2, DNA+Vc; Lane 3, DNA+complex
(8 μM); Lanes 4–10, DNA+Vc+complex (2, 3, 4, 5, 6, 7, 8 μM, respectively).
100% and 81.14% of Form I DNA (Lane 8), respectively. At 8 μM, Form I
DNA is completely cut up for complex 1, while it is totally transformed
into Forms II and III DNA for complex 2 (Lane 10). Linear DNA (Form
III) appears at 5 μM for both complexes (Lane 7), however, the contents
are quite low (b12%) throughout the tested concentrations. The results
demonstrate that both complexes 1 and 2 have remarkable cleavage
activity, and complex 1 shows higher activity than complex 2. Interest-
ingly, these complexes are about 10 times more active than their coun-
terpart [Cu(ttpy)Cl]Cl in terms of DNA cleavage ability [12].
in Fig. 9, in the whole time course (15–90 min), the content of Form II
DNA in the cleavage products of complex 1 is higher as compared
with that of complex 2 at the same time spot (Lanes 4–9). For both
complexes, Form II DNA displays an ever-increasing trend, in that it
changes from 25.65% to 96.49% for complex 1, and from 21.89% to
6
9
5.34% for complex 2. Form III DNA remains small (b2%) within
0 min. Taken together, both the concentration- and time-dependent
The time-dependent cleavage of pBR322 DNA mediated by the
complexes (5 μM) was followed with agarose gel electrophoresis in
the buffer (50 mM Tris–HCl/50 mM NaCl, pH 7.4) at 37 °C. As shown
data lead to the same conclusion, that is, the DNA cleavage efficiency
of complex 1 is superior to that of complex 2. The result manifestly
indicates that the introduction of Gly can significantly enhance the
nuclease efficiency of the copper–terpyridine complex.
3.5. ROS involved in cleavage
The possible ROS responsible for the DNA cleavage were identified
using common scavengers such as DMSO, KI, and NaN , respectively.
3
As Fig. 10 presents, the cleavage activity of complexes 1 and 2 is
reduced dramatically in the presence of hydroxyl radical scavenger
DMSO (Lane 5), indicating that diffusible ∙OH plays a key role in the
cleavage process. Hydrogen peroxide scavenger KI enhances the
cleavage activity of 1 by 7.5% but inhibits that of 2 by 10.6% (Lane
6
3
); singlet oxygen scavenger NaN increases the cleavage activity of 1
and 2 by 5.5% and 7.3%, respectively (Lane 7). These results seem to
1
suggest that H
to some extent. The cleavage activity of the complexes in D
the lifetime of 1
is significantly longer than that in water, further
demonstrates the involvement of
2
O
2
and O
2
are also involved in the cleavage mechanism
2
O, where
O
2
1
2
O in the cleavage, especially for
complex 2 (Lane 8). In summary, hydroxyl radicals are the crucial ROS
responsible for the cleavage activity of these complexes; hydrogen
peroxide and singlet oxygen are less influential in the cleavage. The
Fig. 7. Emission profiles (λex=526 nm, λem=605 nm) of the EB–DNA system in the
buffer (5 mM Tris–HCl/50 mM NaCl, pH 7.4) upon addition of complex 1 (▼) and
complex 2 (▲) at 25 °C.
2
distinct impact of D O on the cleavage activity of 1 and 2 suggests that

W. Zhou et al. / Journal of Inorganic Biochemistry 121 (2013) 114–120
119
4
. Conclusion
The development of artificial nucleases is of great significance for
improving our knowledge of enzyme action and developing new
biotechnological tools as well as therapeutic agents. Copper complexes
have been studied as potential probes of nucleic acid structure and
antitumor drugs through mediating DNA oxidation in the presence of
reducing agents. These oxidation reactions may induce some damage
within the sugar or base moieties of DNA, leading to strand scission or
base modification. Artificial nucleases must be highly efficient in order
to compete with the innate cellular DNA repair machinery. In this
study, we introduced an auxiliary ligand glycine into the copper–
terpyridine complex to raise its nuclease efficiency by increasing the
accessibility of the complex to the nucleic acid target. This try is proved
to be a very successful method to improve the suboptimal cleavage
activity of a copper complex. The enhanced water-solubility and cationic
form of the complex in solution could facilitate its approach to DNA and
formation of hydrogen bonds with the sugar-phosphate backbone. The
auxiliary group may not be directly involved in the cleavage reaction,
but it could be helpful to achieve the synergy between the copper ion
and ligand, to stabilize the transition states, and as a result, to increase
the DNA cleavage activity of the complex.
Abbreviations
Fig. 9. The cleavage patterns of the agarose gel electrophoresis and the corresponding
cleavage extent (%) for pBR322 plasmid DNA (200 ng) by complex 1 (A) or complex 2
CD
circular dichroism
(
B) (5 μM) and Vc (1 mM) in the buffer solution (50 mM Tris–HCl/50 mM NaCl,
CT-DNA Calf thymus DNA
pH 7.4) at 37 °C. Lanes 1–3, DNA, DNA+Vc, DNA+complex at 90 min, respectively;
Lanes 4–9, DNA+Vc+complex at 15, 30, 45, 60, 75, and 90 min, respectively.
EB
ethidium bromide
ttpy
ROS
Vc
4′-p-tolyl-2,2′:6,2″-terpyridine
reactive oxygen species
ascorbic acid
these complexes may follow different pathways in the cleavage process.
Obviously, auxiliary ligand Gly in complex 1 is the primal factor
causative of the difference; however, the exact mechanism is unknown.
Acknowledgments
We are grateful to the financial support from the National Basic
Research Program of China (Grant 2011CB935800) and the National
Natural Science Foundation of China (Grants 21271101, 21131003,
2
1021062, 91213305).
Appendix A. Supplementary data
Supplementary data to this article can be found online at http://
dx.doi.org/10.1016/j.jinorgbio.2012.12.018.
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