Synthesis, X-ray crystal structures, magnetism, and DNA cleavage properties of copper(II) complexes with 1,4-tpbd ligand

Article abstract of DOI:10.1039/b823472h

Four new copper(ii) complexes, [Cu(1,4-tpbd)Br₂] (1), [Cu ₂(1,4-tpbd)(H₂O)₄](ClO₄) ₄ (2), [Cu₂(1,4-tpbd)(1,10-phen)₂(DMF) ₂](ClO₄)₄ (3

Full text of DOI:10.1039/b823472h

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www.rsc.org/dalton | Dalton Transactions
Synthesis, X-ray crystal structures, magnetism, and DNA cleavage properties
of copper(II) complexes with 1,4-tpbd ligand†
Dongdong Li, Jinlei Tian,* Yingying Kou, Fuping Huang, Gongjun Chen, Wen Gu, Xin Liu, Daizheng Liao,
Peng Cheng and Shiping Yan*
Received 5th January 2009, Accepted 18th February 2009
First published as an Advance Article on the web 18th March 2009
DOI: 10.1039/b823472h
Four new copper(II) complexes, [Cu(1,4-tpbd)Br2] (1), [Cu2(1,4-tpbd)(H2O)4](ClO4)4 (2),
[Cu2(1,4-tpbd)(1,10-phen)2(DMF)2](ClO4)4 (3) and [Cu2(1,4-tpbd)(2,2¢-bpy)2(ClO4)2](ClO4)2 (4),
[1,4-tpbd = N,N,N¢,N¢-tetrakis(2-pyridylmethyl)benzene-1,4-diamine], have been synthesized to serve
as artificial nucleases. Single crystal X-ray diffraction reveals that the copper(II) atom has a distorted
intermediate between square pyramidal and trigonal bipyramidal configuration for 1 and a distorted
square pyramidal geometry for 2, while a distorted octahedral environment for 3 and 4.
Variable-temperature magnetic susceptibility studies (2–300 K) indicate the existence of
antiferromagnetic coupling between the copper(II) ions in complex 2. The interactions of the four
complexes with calf thymus DNA (CT-DNA) have been investigated by UV absorption, fluorescent
spectroscopy and cyclic voltammetry, and the modes of CT-DNA binding for the complexes have been
proposed. In the absence of external agents, supercoiled plasmid DNA cleavage by the complexes was
performed under aerobic conditions, the influence on the DNA cleavage process of different complex
concentrations, reaction times was also studied. The DNA cleavage mechanisms were demonstrated
with radical scavengers and anaerobic conditions, indicating all complexes cleaved pBR322 DNA in
50 mM Tris-HCl/18 mM NaCl buffer (pH = 7.2) at 37 ◦C through a hydrolytic process. In the four
copper(II) complexes, complex 2 showed highest cleavage activity with the pseudo-Michaelis–Menten
kinetic parameters kcat = 4.23 h-1 and Km = 2.4 ¥ 10-5 M.
and produce non-natural fragments of DNA which are not suitable
Introduction
further enzymatic manipulation. Furthermore, oxidative cleavage
Phosphodiester linkages of DNA are very stable to hydrolysis, with
is mediated by reactive oxygen species (ROS) that are diffusible
a half-life for spontaneous hydrolysis estimated to be 1011 years
and can cause other severe cytotoxic effects. So these drawbacks
◦
at pH 7 and 25 C.1 In nature, many enzymes that catalyze
hamper their use in vivo. Hydrolytic cleavage in the absence of
phosphate ester hydrolysis are activated by two or more metal
any external agents does not suffer from these disadvantages.
ions.2 Currently there is considerable interest in understanding the
Hydrolytic DNA cleavage involves scission of the phosphoester
reaction mechanism of such enzymes, especially the role of the
bond to generate fragments which could be subsequently re-
metal ions. For a long time, transition metal compounds were
ligated. Hydrolysis requires a metal factor capable of binding
regarded as being synonymous with toxicity. However, in the last
hard oxygen atoms, polarizing bonds and rapid ligand exchange
20 years transition metal complexes have become increasingly
for catalytic turnover. The design and synthesis of the ligands
important as drugs and artificial nucleases. One goal is developing
have been reported, generally featuring tridentate or tetradentate
ligands with free coordination sites of the metal cation.10 On the
other hand, some metal ions have been chosen as active centers,
such as Zn(II),11 Fe(III),10b La(III),12 Cu(II),11 Co(III).13 Many
features of these metal ions, such as their ability in assisting Lewis
activation, nucleophile generation, and leaving group stabilization,
make them suitable for the catalysis of hydrolytic reactions of
DNA.
more reactive chemical systems that can efficiently hydrolyze
phosphate diester bonds under physiological conditions.3–5 Among
them, copper(II) complexes are the most extensively studied
showing potential for the hydrolysis or oxidative cleavage of
DNA.6–9 Most of the oxidative cleavage agents have high efficiency
and versatility, however, they require the addition of an external
agent (oxidative and/or reductive agent, light) to initiate cleavage,
Comparing with mononuclear complexes, dinuclear com-
plexes have higher activity as a result of the cooperative in-
teraction of metal ions in stabilizing the transition state of
phophodiester cleavage. Recently progress has been made in
the study of metal complexes that promote the phosphodi-
ester hydrolysis. There are several reports of copper(II) com-
plexes showing efficient chemical nuclease activity, including
mononuclear,14–16 dinuclear,17–19 trinuclear,20–22 and macromolec-
ular catalytic systems.23–25 For example, a mononuclear divalent
Department of Chemistry, Nankai University, Tianjin, 300071, PR China.
E-mail: yansp@nankai.edu.cn; Fax: +86-022-23502779; Tel: +86-022-
23505063
† Electronic supplementary information (ESI) available: UV-Vis absorp-
tion spectra, emission spectra, cyclic voltammograms, ESR spectra,
agarose gel electrophoresis under aerobic and anaerobic conditions and
corresponding time course of DNA cleavage by the copper(II) complexes.
CCDC reference numbers 639516, 689792, 694617 and 694618 for
complexes 1, 2, 3, and 4, respectively. For ESI and crystallographic data in
CIF or other electronic format see DOI: 10.1039/b823472h
3574 | Dalton Trans., 2009, 3574–3583
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copper complex [Cu(dpq)2(H2O)](ClO4)2 (dpq = dipyrido[3,2-
d:2¢,3¢-f ]quinoxaline) can effectively cleave supercoiled DNA
under hydrolytic conditions with a rate constant of 5.58 h-1
(pH = 7.2).14 Shanta Dhar et al. suggested that a copper-bound
hydroxyl group was the active species in the hydrolysis of the
nucleic acid phosphate backbone. Karlin et al. have discovered a
dicopper complex [Cu2(PD¢O)(H2O)2](ClO4)3·2H2O (PD¢OH = 4-
tert-butyl-2,6-bis[bis(2-pyridylethyl)amino]phenol) that predom-
inantly effects DNA base (rather than ribose) oxidation on G
residues in single-stranded regions of DNA.17 A trinuclear cop-
per complex [Cu3(TDAT)2Cl3]Cl3·2H2O (TDAT = 2,4,6-tris(di-
2-pyridylamine)-1,3,5-triazine) efficiently cleaves the supercoiled
pBR322 DNA into its nicked and linear forms with active oxygen
intermediates which act as active species for the DNA scission.
Synergistic effects of three CuII centers may contribute to the
significant DNA cleavage activity.20
In an attempt to obtain more insight into the selectivity and
efficiency of DNA recognized and cleaved by different copper(II)
complexes, herein we have synthesized and characterized a series
of new mononuclear and dinuclear copper(II) complexes with the
same ligand, [Cu(1,4-tpbd)Br2] (1), [Cu2(1,4-tpbd)(H2O)4](ClO4)4
(2), [Cu2(1,4-tpbd)(1,10-phen)2(DMF)2](ClO4)4 (3) and [Cu2(1,4-
tpbd)(2,2¢-bpy)2(ClO4)2](ClO4)2 (4), and compared the DNA
cleavage activities of mononuclear and dinuclear copper(II) com-
plexes. In this paper, we have studied the magnetism of complex 2,
and variable-temperature magnetic susceptibility studies (2–300
K) showed the existence of antiferromagnetic coupling between
the copper(II) ions in complex 2. The interactions between calf
thymus DNA and the complexes 1, 2, 3, 4 were investigated by
UV absorption, fluorescent spectroscopy and cyclic voltammetry,
and the DNA cleavage experiments induced by complexes were
also demonstrated. All complexes could cleave pBR322 DNA
without addition of external agents and the DNA cleavage was not
inhibited by DMSO, L-histidine or SOD, nor by handling under
anaerobic conditions. The results suggest that the mechanism of
DNA cleavage by these complexes is a hydrolytic cleavage.
Results and discussion
X-Ray structure characterization
Complexes 1, 2, 3 and 4 have been structurally characterized
by X-ray crystallography. The data collection and refinement
parameters are given in Table 1, selected bond lengths and angles
are given in Table 2.
For complex 1, as Fig. 1 shows, it is a neutral mononuclear CuII
complex and the copper atom has a five coordinated geometry with
three N atoms of ligand and two bromine atoms. The trigonality
t value is calculated to be 0.42 according to the Addison–Reedijk
geometric criterion;26 the geometry can be described as a distorted
intermediate between square-pyramidal and trigonal-bipyramidal
configuration. N(1), N(2), N(3), Br(2) atoms form the basal plane
with Cu(1)–N(1) 1.996, Cu(1)–N(2) 1.992, Cu(1)–N(3) 2.132 and
˚
Cu(1)–Br(2) 2.447 A, respectively. The deviations of the four donor
˚
atoms from their mean plane are 0.351, 0.350, 0.432, and 0.271 A,
respectively. The apical position is occupied by Br(1) with a Cu(1)–
˚
˚
Br(1) distance of 2.519 A, and the copper atom is 0.472 A out of
the basal plane. It is close to the analogue [Fe(1,4-tpbd)Cl2],27 in
which the geometry around the Fe atom is best described as being
closer to square pyramidal (with Cl at the apex) than trigonal
Table 1 Crystallographic data and structure refinement parameters for complexes 1, 2, 3 and 4
Complex
1
2
3
4
Empirical formula
C30H28Br2CuN6
695.94
113(2)
0.71070
Monoclinic
P21
8.372(4)
12.813(6)
13.103(7)
90
105.509(5)
90
C30H36Cl4Cu2N6O20
1069.55
294(2)
0.71073
Monoclinic
C30H29Cl2CuN6O9
752.03
294(2)
0.71073
Triclinic
¯
P1
11.848(4)
12.525(4)
13.498(4)
95.499(5)
101.124(6)
115.852(5)
1731.7(10)
2, 1.442
0.844
772
0.24 ¥ 0.20 ¥ 0.14
1.57 to 25.02
-14 to 11, -12 to 14
-16 to 15
8897
C50H52Cl4Cu2N10O20
1381.90
113(2)
0.71073
Triclinic
¯
P1
10.014(2)
12.259(3)
12.887(3)
64.99(3)
78.94(3)
77.72(3)
1391.5(5)
1, 1.649
1.044
Mr
T/K
˚
l/A
Crystal system
Space group
P21/n
˚
a/A
10.428(5)
17.954(8)
10.785(5)
90
107.410(7)
90
1926.7(15)
2, 1.844
1.475
˚
b/A
˚
c/A
a/◦
b/◦
g /◦
3
˚
V/A
1354.4(12)
2, 1.706
3.789
Z, Dc/mg m-3
m/mm-1
F(000)
698
1088
708
Crystal size/mm
0.14 ¥ 0.12 ¥ 0.12
2.26 to 27.88
-11 to 10, -16 to 16
-17 to 17
12782
6328
0.0577
0.22 ¥ 0.20 ¥ 0.16
2.27 to 25.02
-6 to 12, -20 to 21
-12 to 12
9520
3389
0.0963
0.12 ¥ 0.10 ¥ 0.04
1.76 to 25.02
-11 to 11, -14 to 10
-15 to 13
8553
4871
0.0225
q range/◦
Limiting indices h, k, l
Reflections collected
Unique reflections
Rint
6049
0.0242
Absorption correction
Goodness-of-fit on F2
R1, wR2 [I > 2s(I)]
R1, wR2 (all data)
Semi-empirical
1.009
0.0541/0.1007
0.0686/0.1097
0.663/-0.810
Semi-empirical
1.062
0.0427/0.0978
0.0696/0.1130
0.725/-0.616
Semi-empirical
1.058
0.0658/0.1843
0.1086/0.2223
0.678/-0.515
Semi-empirical
1.071
0.0459/0.1318
0.0499/0.1364
0.837/-0.623
-3
˚
Dr (max./min.)/e A
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◦
˚
Table 2 Selected bond lengths (A) and angles ( ) for the complexes 1, 2,
3 and 4
Complex 1
Cu(1)–N(2)
Cu(1)–N(3)
Cu(1)–Br(1)
N(4)–C(16)
1.992(5)
2.132(5)
2.5194(13) N(3)–C(13)
1.376(7)
Cu(1)–N(1)
Cu(1)–Br(2)
1.996(5)
2.4470(14)
1.485(6)
N(2)–Cu(1)–N(1)
N(2)–Cu(1)–Br(2)
N(2)–Cu(1)–Br(1)
N(3)–Cu(1)–Br(1)
N(3)–Cu(1)–Br(2)
C(16)–N(4)–C(25)
C(25)–N(4)–C(19)
C(6)–N(3)–C(13)
162.3(2)
N(1)–Cu(1)–N(3)
N(1)–Cu(1)–Br(2)
N(1)–Cu(1)–Br(1)
80.67(19)
97.54(15)
94.13(13)
95.78(14)
91.46(14)
110.46(13)
137.09(14)
120.6(5)
118.5(5)
112.4(4)
Br(2)–Cu(1)–Br(1) 112.43(4)
N(2)–Cu(1)–N(3)
C(16)–N(4)–C(19)
C(6)–N(3)–C(12)
C(12)–N(3)–C(13)
81.66(19)
119.5(5)
114.2(5)
112.8(5)
Complex 2
Cu(1)–N(3)
Cu(1)–O(2)
1.941(3)
1.995(3)
Cu(1)–N(1)
Cu(1)–N(2)
1.941(3)
2.055(3)
Fig. 1 ORTEP view of the molecular structure and atom labeling scheme
of 1. Thermal ellipsoids are drawn at 30% probability level. Hydrogen
atoms are omitted for clarity.
Cu(1)–O(1)
2.213(3)
Cu(1) ◊ ◊ ◊ Cu(1A)
N(1)–Cu(1)–O(2)
N(1)–Cu(1)–N(2)
N(3)–Cu(1)–O(1)
O(2)–Cu(1)–O(1)
N(3)–Cu(1)–N(1)
8.099(3)
N(3)–Cu(1)–O(2)
N(3)–Cu(1)–N(2)
O(2)–Cu(1)–N(2)
N(1)–Cu(1)–O(1)
N(2)–Cu(1)–O(1)
95.04(12)
82.91(12)
157.56(12)
93.72(13)
112.17(12)
95.91(13)
83.87(13)
94.35(13)
90.25(13)
166.36(13)
Complex 3
Cu(1)–N(3)
Cu(1)–N(1)
2.052(5)
2.077(4)
Cu(1)–N(5)
Cu(1)–N(4)
2.057(5)
2.096(4)
Cu(1)–O(1)
2.353(5)
Cu(1)–N(2)
2.511(5)
N(2)–Cu(1)–O(1)
N(2)–Cu(1)–N(3)
N(3)–Cu(1)–N(5)
N(5)–Cu(1)–N(1)
N(5)–Cu(1)–N(4)
N(3)–Cu(1)–O(1)
N(1)–Cu(1)–O(1)
N(2)–Cu(1)–N(5)
165.92(16)
77.89(17)
95.04(12)
93.92(18)
80.80(18)
93.92(18)
93.12(17)
93.73(18)
N(2)–Cu(1)–N(1)
N(2)–Cu(1)–N(4)
N(3)–Cu(1)–N(1)
N(3)–Cu(1)–N(4)
N(1)–Cu(1)–N(4)
N(5)–Cu(1)–O(1)
N(4)–Cu(1)–O(1)
Cu(1) ◊ ◊ ◊ Cu(1A)
75.85(20)
96.65(17)
91.41(18)
92.83(18)
172.16(18)
95.75(18)
93.17(17)
8.458(2)
Complex 4
Cu(1)–N(5)
Cu(1)–N(4)
2.004(3)
2.015(3)
Cu(1)–N(1)
Cu(1)–N(3)
2.007(3)
2.025(3)
Cu(1)–O(1)
2.388(2)
Cu(1)–N(2)
2.427(3)
Fig. 2 ORTEP view of the molecular structure and atom labeling scheme
of 2. Thermal ellipsoids are drawn at 30% probability level. The “A” labeled
atoms are at equivalent positions (-x + 1, -y, -z + 2). Hydrogen atoms
and perchlorate anions are omitted for clarity.
N(5)–Cu(1)–N(1)
N(1)–Cu(1)–N(4)
N(1)–Cu(1)–N(3)
N(5)–Cu(1)–O(1)
N(4)–Cu(1)–O(1)
N(5)–Cu(1)–N(2)
N(4)–Cu(1)–N(2)
O(1)–Cu(1)–N(2)
170.46(11)
93.30(12)
88.83(12)
92.59(10)
96.61(10)
93.93(10)
96.99(10)
165.69(9)
N(5)–Cu(1)–N(4)
N(5)–Cu(1)–N(3)
N(4)–Cu(1)–N(3)
N(1)–Cu(1)–O(1)
N(3)–Cu(1)–O(1)
N(1)–Cu(1)–N(2)
N(3)–Cu(1)–N(2)
Cu(1) ◊ ◊ ◊ Cu(1A)
80.98(11)
96.10(11)
173.74(10)
95.66(11)
89.03(10)
79.12(11)
77.62(10)
8.401(4)
N(2), N(3), O(2), form the basal plane with Cu(1)–N(1) 1.941,
˚
Cu(1)–N(2) 2.055, Cu(1)–N(3) 1.941 and Cu(1)–O(2) 1.995 A, the
apical position is occupied by another O atom of water with Cu(1)–
˚
O(1) distance of 2.213 A. Cu(1) deviates from the basal plane by
bipyramidal, and another analogue [Zn(1,4-tpbd)Cl2],28 in which
the Zn atom is in a tetragonal-pyramidal arrangement with Cl(1)
at the apex.
0.251 A. This coordination geometry around the copper atom of 2
is very similar to that of compound [Cu2(1,4-tpbd)(H2O)4][S2O6]2,
which is prepared by hydrolysis of Cu2(1,4-tpbd)Cl4.29
˚
The crystal structure of 2 consists of a dinuclear [Cu2(1,4-
The crystal structure of 3 is composed of a dinuclear
-
-
tpbd)(H2O)4]4+ cation and four ClO4 anions (Fig. 2). The 1,4-
[Cu2(1,4-tpbd)(1,10-phen)2(DMF)2]4+ cation and four ClO4 an-
tpbd bridges the two copper atoms which are in a distorted
square-pyramidal arrangement and this is reflected in the index
of trigonality t = 0.15. The complex cation has an exact central
symmetryandtwocopper atoms arelocatedina transarrangement
with respect to the central bridging p-phenylenediamine unit with
the Cu(1) ◊ ◊ ◊ Cu(1A) distance of 8.099 A. Each copper atom is five-
coordinated with N3O2 donor set derived from three N atoms of
the ligand and two O atoms of coordinate water molecules. N(1),
ions (Fig. 3) and 4 is composed of a dinuclear [Cu2(1,4-tpbd)(2,2¢-
bipy)2(ClO4)2]2+ cation and two ClO4 anions (Fig. 4). In both
-
complexes, the copper(II) atom exhibits a distorted octahedral
environment with N5O donor set derived from three ligand N
atoms, two 1,10-phen N atoms and one DMF O atom for 3 and
three ligand N atoms, two 2,2¢-bpy N atoms and one perchlorate O
atom for 4. N(1), N(3), N(4), N(5) atoms consist of the equatorial
˚
˚
˚
plane with Cu–N bond lengths ranging from 2.052 A to 2.096 A
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hyperfine structure were observed for all complexes. The results
exhibit g-tensor parameters with gꢀ > g^ > 2.0023. This indicates
ground state34 characteristic of
2
2
that the copper site has a dx
-y
square planar or octahedral geometry in copper(II) complexes.35
Kivelson and Nieman36 have reported that gꢀ is less than 2.3 for
covalent character and greater than 2.3 for ionic character of
the metal ligand bond in complexes. The gꢀ values for 2 and 4
are less than 2.3, suggesting significant covalent character in the
metal–ligand bonding. For 1 the gꢀ value is slightly higher than 2.3
suggesting a small amount of ionic character of the metal–ligand
bond.
Variable-temperature (2–300 K) magnetic behavior in the form
of cM and cMT versus T plots of 2 is shown in Fig. 5. The cMT
values at 300 K is 0.751 cm3 mol-1 K for 2, which is comparable
to the calculated value (0.750 cm3 mol-1 K, g = 2) of the non-
correlated system of two S = 1/2 spins carriers. The cMT value
decreases slowly upon cooling till about 50 K, then decreases
rapidly and reaches 0.00014 cm3 mol-1 K at 2 K, indicating the
presence of antiferromagnetic exchange interactions between the
adjacent copper ions. Taking into account the dinuclear copper
model, the magnetic susceptibilities of the complex can be fitted
accordingly by the following expression derived from the Bleany–
Bower equation H = -2JS1S2 (S1 = S2 = 1/2):
Fig. 3 ORTEP view of the molecular structure and atom labeling scheme
of 3. Thermal ellipsoids are drawn at 30% probability level. The “A” labeled
atoms are at equivalent positions (-x + 2, -y + 2, -z + 2). Hydrogen atoms
and perchlorate anions are omitted for clarity.
2Ng2 b2
kT
1
cM
=
¥
3 + e-2J / kT
where J is the exchange coupling parameter between S1 and S2,
and other symbols have their normal meanings. The susceptibility
data can be simulated well with the equation with the following
set of converging parameters: g = 2.08, J = -13.3 cm-1 and R =
1.5 ¥ 10-4 for 2. The magnetism of compounds 3 and 4 were not
determined owing to the very similar structure and coordination
environment of copper atoms.
Fig. 4 ORTEP view of the molecular structure and atom labeling scheme
of 4. Thermal ellipsoids are drawn at 30% probability level. The “A” labeled
atoms are at equivalent positions (-x + 2, -y + 1, -z + 1). Hydrogen atoms
and perchlorate anions are omitted for clarity.
˚
˚
for 3 and 2.004 A to 2.025 A for 4, N(2) and O(1) occupy apical
˚
˚
positions with Cu(1)–N(2) = 2.511 A and Cu(1)–O(1) = 2.353 A
˚
˚
for 3 and Cu(1)–N(2) = 2.427 A and Cu(1)–O(1) = 2.388 A for 4,
the angle N(2)–Cu(1)–O(1) is 165.92◦ for 3 and 165.69◦ for 4. The
apical bond lengths are larger than the equatorial bond lengths
due to the Jahn–Teller effects in the d9 copper(II) complexes in 3
and 4. In 3, the distance of Cu(1)–O(1) is comparable to the Cu–O
distances in some of the reported DMF coordinated complexes.30,31
In 4, a perchlorate O atom occupies the apical position; the Cu–
O bond distance observed for 4 is comparable to those observed
previously.32,33 The Cu(1) ◊ ◊ ◊ Cu(1A) distance is 8.458 A for 3 and
˚
˚
8.401 A for 4.
Overall, the co-ordination chemistry of tpbd shows that mono-
coordinated and bridged dinuclear complexes can be prepared, a
trans arrangement is evident in the dicopper complexes, the cause
of the trans arrangement is probably due to crystal-packing effects.
Fig. 5 cM (ꢀ) versus T and cMT (ꢀ) versus T plots for complex 2.
DNA binding properties by UV titration and fluorescence
spectroscopic studies
EPR spectra and magnetic measurements
DNA binding is the critical step for DNA cleavage in most
cases. Therefore, the binding ability of the complexes to CT-
DNA was studied by using UV-Vis absorption and fluorescence
spectroscopy. Firstly, the potential binding ability of the complexes
1, 2, 3, 4 to CT-DNA was studied by UV spectroscopy. The typical
titration curves for complexes are shown in Fig. 2S (ESI†). The
EPR studies of paramagnetic transition metal(II) complexes yield
information about the distribution of the unpaired electrons and
the nature of the bonding between the metal ion and its ligands.
The EPR spectra of complexes 1, 2, and 4 in the solid state
were measured at room temperature (Fig. 1S, ESI†) and without
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absorption peaks of 205 nm and 255 nm for 1, 206 nm and
256 nm for 2, 208 nm and 257 nm for 3, 227 nm and 302 nm
for 4, are attributed to intraligand p–p* transition, as increasing
the concentration of CT-DNA, hypochromism of 41%–61% and
red-shifts of 5–19 nm for 1, 2, 3, 4 are observed at maximal
peaks (Table 1S, ESI†), which suggests intercalation between the
complexes and CT-DNA, because intercalation would lead to
hypochromism and bathochromism in UV absorption spectra due
to the intercalative mode involving a strong stacking interaction
between an aromatic chromophore and the base pairs of DNA.37
The extent of the hypochromism is commonly consistent with the
strength of intercalative interaction.38
HCl/50 mM NaCl buffer (pH = 7.2) at room temperature in
Fig. 4S (ESI†). As can be seen in this figure, the cyclic voltam-
mograms of the four complexes exhibited quasireversible redox
waves which could be assigned to the Cu(II)/Cu(I) couples.47–49 In
the presence of CT-DNA, the cyclic voltammograms of the four
copper(II) complexes exhibited shifts in the anodic and cathodic
peak potentials followed by a decrease in both peak currents,
indicating the interaction existing between the four copper(II)
complexes and CT-DNA. The drop of the voltammetric currents
in the presence of CT-DNA can be attributed to diffusion of
the metal complex bound to the large, slowly diffusing DNA
molecule.50 The E1/2 values for the four copper(II) complexes 1,
2, 3 and 4 in the presence of CT-DNA are -88 mV, -7.5 mV,
-89.5 mV and -188.5 mV, respectively, and exhibit positive shifts
of 6.0 mV, 3.5 mV, 1.0 mV and 1.5 mV, respectively (Table 2S,
ESI†). The results indicate quasi-reversible redox processes (DEp
of 59 mV for a one-electron diffusion and ipa = ipc of about 1
controlled reversible process). Among the three kinds of binding
modes for small molecules to DNA, the interaction modes are
intercalative binding for the four copper(II) complexes according
to the Bard’s report.51 This is consistent with the results obtained
from absorption and fluorescence spectra.
To further clarify the interaction of the complexes with DNA,
the competitive binding experiment was carried out. EB does not
show any appreciable emission in buffer solution due to fluores-
cence quenching of the free EB by the solvent molecules.39 EB
emits intense fluorescence at about 600 nm in the presence of DNA
due to its strong intercalation between the adjacent DNA base
pairs.40 It was previously reported that the enhanced fluorescence
could be quenched by the addition of another molecule.41 Two
mechanisms have been proposed to account for this reduction in
the emission intensity: the replacement of molecular fluorophores
and/or electron transfer.42 No luminescence was observed for the
complexes 1, 2, 3, 4 at room temperature in aqueous solution or in
the present of CT-DNA, so the binding of the complexes cannot
be directly presented in the emission spectra. The relative binding
of complexes 1, 2, 3, 4 to CT-DNA was studied with an EB-
bound CT-DNA solution in 5 mM Tris-HCl/50mM NaCl buffer
(pH = 7.2). Fluorescence intensities at 602 nm (526 nm excitation)
were measured at different complex concentrations. The results
(Fig. 3S, ESI†) showed that the fluorescence intensity of EB-
bound CT-DNA decreased with the addition of the copper(II)
complexes, which indicated that the complexes could bind to CT-
DNA and replace EB from the EB-bound CT-DNA system. Such
a feature is often found in the intercalative DNA interactions.43
According to the classical Stern–Volmer equation,44 the quenching
plot illustrates that the quenching of EB bound to CT-DNA by
complex is in agreement with the linear Stern–Volmer equation,
which also indicates the complexes bind to DNA. In the plot of
I0/I vs. [complex], K is given by the ratio of the slope to intercept.
According to the equation KEB[EB] = Kapp[complex], where the
complex concentration was the value at a 50% reduction of the
fluorescence intensity of EB and KEB = 1.0 ¥ 107 M-1, ([EB] =
50 mM). The apparent binding constants (Kapp) at room tempera-
ture are calculated to be 3.67 ¥ 106 M-1 for 1, 7.51 ¥ 106 M-1 for 2,
4.86 ¥ 106 M-1 for 3 and 1.77 ¥ 106 M-1 for 4, less than the binding
constant of the classical intercalators and metallointercalators
(107 M-1),45 which suggests that the interaction of the copper(II)
complexes with DNA is a moderate intercalative mode.
pBR322 DNA cleavage by the copper(II) complexes
The chemical nuclease activities of complexes 1, 2, 3 and 4
have been studied using supercoiled pBR322 plasmid DNA as
a substrate in a medium of 50 mM Tris-HCl/18mM NaCl buffer
(pH = 7.2) in the absence of external agent. When circular plasmid
DNA is conducted by 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
nicked circular form (Form II). If both strands are cleaved, a
linear form (Form III) will be generated which migrates between
form I and form II.
Effects of complex concentration on plasmid DNA cleavage
The activities of complexes 1, 2, 3 and 4 were assessed by the
conversion of DNA from Form I to Form II and Form III. Firstly,
the concentration-dependent DNA cleavage by complexes was
performed. Fig. 5S (ESI†) shows that the results of gel elec-
trophoretic separations of plasmid pBR322 DNA induced by
increasing the concentration of the four complexes in the absence
of external agent at pH = 7.2 (50 mM Tris-HCl/NaCl buffer) at
37 ◦C. On increasing complex concentrations, the amount of Form
I decreases gradually and Form II increases, all of the examined
complexes are capable of cleaving supercoiled pBR322 plasmid
DNA (Form I) into nicked circular form (Form II), but they
display different cleavage activities. Besides, we also investigated
the DNA cleavage activities of the four complexes under the same
experimental conditions. As Fig. 6 shows, the four complexes all
could convert supercoiled form of pBR322 DNA to nicked circular
form. It seems that complex 2 shows higher cleavage activity than
the other three complexes. These qualitative findings could be
quantified by densitometric analysis of the bands originating from
SC and NC plasmids. The sum of intensity of both bands was
standardized to 100% in all lanes. A plot of relative intensities
is presented in Fig. 7. This analysis shows that complex 2 has
Electrochemical studies
The application of cyclic voltammetry (CV) to study the binding
of metal complexes to DNA can provide a useful complement to
above methods of investigation such as UV-vis and fluorescence
spectra46 and yield information about interactions with both the
reduced and oxidized form of the metal.
Typical cyclic voltammetry (CV) behaviors of complexes in the
absence and presence of CT-DNA were recorded in 10 mM Tris–
3578 | Dalton Trans., 2009, 3574–3583
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Fig. 6 Gel electrophoresis diagrams showing the cleavage of pBR322
DNA (0.1 mg mL-1) at different complex concentrations in Tris-HCl/NaCl
buffer (pH = 7.2) and 37 ◦C. Lane 1: DNA control (3 h); lane 2–4: DNA +
1 (0.05, 0.25, 0.5 mM); lane 5–7: DNA + 2 (0.05, 0.25, 0.5 mM); lane 8–10:
DNA + 3 (0.05, 0.25, 0.5 mM); lane 11–12: DNA + 4 (0.25, 0.5 mM).
the highest cleavage activity of the four complexes. Under the
conditions chosen for our experiments, complex 2 cleaves about
80% of the plasmid to yield the nicked circular form at 0.5 mM,
whereas complex 1, 3 and 4 cause less than 70% of the plasmid to
yield the nicked circular form.
Fig. 7 Quantification of gel electrophoresis bands originating from SC
and NC DNA. The sum of intensities of both bands is standardized to
100% for each individual lane.
Effects of reaction time on plasmid DNA cleavage
The time-dependent cleavage of DNA by complexes 1, 2, 3 and
4 was also studied under similar conditions. With reaction time
increase, the amounts of Form II and Form III increased and Form
I gradually disappeared. The results show that all complexes can
effectively cleave the pBR322 plasmid DNA without addition of
external agents, and cleavages of DNA by the complexes have been
dependent on reaction times, this could be seen in Fig. 6S (ESI†).
Fig. 8 shows the extent on DNA cleavage by Cu(II) complexes of
increasing reaction time. From these experimental results, we can
find that the plots for the appearance of Form II as well as the
disappearance of Form I follow pseudo-first-order kinetic profiles
and fit well to a single-exponential decay curve. This is a typical
Fig. 8 Disappearance of supercoiled (SC, ꢀ) form and formation of nicked circular (NC, ꢀ) and linear (L, ᭹) forms of plasmid DNA in the presence
of complexes 1, 2, 3, and 4 with the incubation time.
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pseudo-first-order consecutive reaction, which is consistent with
the general model for enzyme catalyzed reactions.52 Fitting the
experimental data with first-order consecutive kinetic equations,
the rate constants of 7.24 ¥ 10-5 s-1, 2.97 ¥ 10-4 s-1, 2.00 ¥ 10-4 s-1,
2.66 ¥ 10-5 s-1, for the conversions of supercoiled to nicked DNA
are obtained for 1, 2, 3, and 4, respectively. The results indicate
that complex 2 has a better nuclease activity than compounds 1, 3
and 4.
of external agent. The decrease of Form I was fitted to a single
exponential decay curve (pseudo-first-order kinetics) by use of eqn
(1a) or (1b), where y0 is the initial percentage of a form of DNA,
y is the percentage of a specific form of DNA at time t, a is the
percentage of uncleaved DNA, and kobs is the hydrolysis rate, or
apparent rate constant.
y = (y0 - a)exp(-kobsx) + a
(1a)
(1b)
y = (100 - y0)(1 - exp(-kobsx))
Effects of radical scavengers on plasmid DNA cleavage
In order to determine the hydrolytic cleavage rate, the kinetic
aspect of the hydrolytic DNA cleavage has been investigated.
Reactions were carried out under pseudo-Michaelis–Menten
conditions by using various concentrations of complexes. Based
on the plots of kobs vs. concentration of complex (Fig. 8S, ESI†),
the pseudo-Michaelis–Menten kinetic parameters (kcat and Km)
were calculated to be 2.94 h-1 and 3 ¥ 10-5 M for 1, 4.23 h-1 and
2.4 ¥ 10-5 M for 2, 2.97 h-1 and 2.3 ¥ 10-5 M for 3, 2.95 h-1 and
1.9 ¥ 10-5 M for 4.
The obtained hydrolysis rate constant shows that 2 has a very
high nuclease activity, giving 1.2 ¥ 108 fold rate enhancement
over uncatalyzed rate of cleavage of ds-DNA (3.6 ¥ 10-8 h-1).56
Hydrolytic rate constants of DNA cleavage catalyzed by some
metal complexes have been reported (in Table 3). We can see
from the table that complex 2 exhibits very high nuclease activity
with a rate constant of 4.23 h-1 and Km = 2.4 ¥ 10-5 M at
pH = 7.2. We report here that the dinuclear complex 2 shows
the highest nuclease activity in the four complexes. However, both
dinuclear complex 3 and 4 have similar nuclease activity, due to
the saturated coordination sites around the metal ions and the
To verify if reactive oxygen species (ROS) may be formed in the
DNA cleavage reaction, DNA cleavage experiments were carried
out in the presence of hydroxyl radical scavenger (DMSO),53 super-
oxide scavenger (SOD),54 singlet oxygen quencher (L-histidine)55
and chelating agent (EDTA) under physiological conditions. As
shown in Fig. 9, no obvious inhibitions are observed in the
presence of DMSO, L-histidine and SOD, the results rule out the
possibility of DNA cleavage by hydroxyl radical, singlet oxygen
and superoxide. EDTA can efficiently inhibit DNA cleavage,
indicating copper(II) complexes play a key role in the cleavage.
In order to clarify if O2 in air is responsible for the DNA
cleavage by the copper(II) complexes, it is necessary to perform the
cleavage experiments under anaerobic conditions; the complexes
still cleaved pBR322 plasmid DNA under anaerobic conditions,
this can be seen in Fig. 7S (ESI†). The results indicated that O2 was
not an indispensable cofactor for DNA scission. On the basis of
the above observation, we make the hypothesis that the examined
complexes may be capable of promoting DNA cleavage through a
hydrolytic mechanism. Indeed, direct evidence of DNA hydrolysis
was that the linear DNA fragments (Form III) cleaved by the
complex can be religated by T4 DNA ligase. However, enough
amounts of linear DNA could not be recovered, the ligation
experiments have not been done.
˚
distance of Cu ◊ ◊ ◊ Cu (>8.0 A) which are not favored factor to
cleave DNA. For complex 2, each Cu(II) center is in a distorted
square-pyramidal configuration having an open coordination site,
and two water molecules are coordinated to Cu(II) center, such a
factor facilitates to cleave DNA. It has been widely accepted that
the metal-bound hydroxyl species in enzymes or their models are
the active species in the hydrolysis of the phosphate backbone. In
aqueous solution, the Cu–Br bonds in complex 1 are labile and
The pseudo-Michaelis–Menten kinetics of DNA cleavage
The reaction of supercoiled plasmid DNA cleavage into nicked
and linear DNA by the complexes was carried out in the absence
Fig. 9 Agarose gel showing cleavage of pBR322 DNA (0.1 mg mL-1) incubated with complexes in Tris-HCl/NaCl buffer (pH = 7.2) at 37 ◦C for 3 h. (1)
Complex 1 (0.4 mM); (2) complex 2 (0.04 mM); (3) complex 3 (0.05 mM); (4) complex 4 (0.15 mM). Lane 1: DNA control; lane 2: DNA + complex; lane
3: DNA + complex + DMSO (10 mM); lane 4: DNA + complex + L-histidine (10 mM); lane 5: DNA + complex + SOD (20 U mL-1); lane 6: DNA +
complex + EDTA (5mM).
3580 | Dalton Trans., 2009, 3574–3583
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Table 3 Pseudo-Michaelis–Menten kinetic analysis for hydrolytic
cleavage of DNA
Preparation of complexes
[Cu(1,4-tpbd)Br2] (1). To a methanolic solution (10 mL) of
1,4-tpbd (0.1 mmol, 47.3 mg) was added dropwise an aqueous
solution (5 mL) of CuBr2 (0.2 mmol, 44.7 mg), and the resulting
solution was stirred for 12 h at room temperature, filtered and
brown block crystals of 1 suitable for X-ray diffraction were
obtained by slow evaporation the filtrate, collected by filtration,
washed with diethyl ether and dried in air. Yield 37% (based
on the copper salts). Elemental analysis data: calculated. (%) for
C30H28Br2CuN6 (659.94): C, 51.78; H, 4.06; N, 12.08. Found (%):
C, 51.55; H, 4.18; N, 12.01. IR (KBr, n/cm-1): 1607, 1518, 1384,
764.
Complex
Km/M
k
cat/h-1
Reference
This work
[Cu2(1,4-tpbd)(H2O)4]-
(ClO4)4
Cu-neamine
2.4 ¥ 10-5
3.9 ¥ 10-6
4.23
1.86
56
57
14
15
58
58
58
59
59
59
60
61
Cu(9aneN3)
Not determined ~0.04
[Cu(dpq)2(H2O)]2+
Cu-tach
Not determined
9.55 ¥ 10-5
6.5 ¥ 10-5
4.7 0.2
4.34 0.77
4.15
0.43
0.61
4.42
4.21
0.50
0.79
[Cu(L1)2(Br)](ClO4)5
[Cu(L2)2(Br)](ClO4)5
[Cu(bipy)(NO3)2]
7.5 ¥ 10-5
3.3 ¥ 10-4
[Cu(L1)Cl2](ClO4)2·H2O 7.46 ¥ 10-5
[Cu(L2)Cl2](ClO4)2
[Cu(bipy)Cl2]
1.07 ¥ 10-4
3.5 ¥ 10-4
9.8 ¥ 10-4
3.80 ¥ 10-6
Co3+-cyclen
[Cu2(1,4-tpbd)(H2O)4](ClO4)4 (2). To a methanolic solution
(10 mL) of 1,4-tpbd (0.1 mmol, 47.3 mg) was added dropwise an
aqueous solution (5 mL) of Cu(ClO4)2·6H2O (0.2 mmol, 74.2 mg),
the mixture solution was refluxed for 3 h in the presence of air,
cooled and filtered, green block crystals of 2 suitable for X-ray
structure analysis were obtained by slow evaporation the filtrate,
collected by filtration, washed with diethyl ether and dried in air.
Yield 39% (based on the copper salts). Elemental analysis data:
calculated. (%) for C30H36Cl4Cu2N6O20 (1069.55): C, 33.69; H,
3.39; N, 7.86. Found (%): C, 33.55; H, 4.15; N, 7.75. IR (KBr,
n/cm-1): 1612, 1506, 1284, 1121, 1068, 778, 760, 622.
[Ru(bpy)2BPG]2+
0.06 0.003
the Br- anions exchange easily with water molecules and then lead
to form the active species via deprotonation of the bound water
molecule. In complex 2, the bound water molecules are directly
deprotonated to form the active species, then direct interaction
between the phosphodiester groups and active species facilitates
the formation of an intermediate, which allows the DNA to be
cleaved readily.
[Cu2(1,4-tpbd)(1,10-phen)2(DMF)2](ClO4)4 (3). To a methano-
lic solution (10 mL) of 1,4-tpbd (0.1 mmol, 47.3 mg) was
added dropwise an aqueous solution (5 mL) of Cu(ClO4)2·6H2O
(0.2 mmol, 74.2 mg), the mixture solution was refluxed for 3 h in the
presence of air, then 1,10-phen (0.2 mmol, 39.6 mg) was added and
stirred for further 5 h at room temperature, filtered, green block
microcrystals were obtained and dissolved in DMF. After two
weeks later, green block crystals of 3 suitable for X-ray structure
analysis were obtained by slow evaporation the filtrate, collected
by filtration, washed with diethyl ether and dried in air. Yield 39%
(based on the copper salts). Elemental analysis data: calculated.
(%) for C30H29Cl2CuN6O9 (752.03): C, 47.91; H, 3.89; N, 11.17.
Found (%): C, 47.75; H, 4.04; N, 11.09. IR (KBr, n/cm-1): 1607,
1506, 1443, 1283, 1145, 1120, 1086, 767, 626.
Experimental
Caution
Although no problems were encountered in this work, transition-
metal perchlorate salts are potentially explosive and should thus
be prepared in small quantities and handled with care.
Materials and instrumentation
The 1,4-tpbd ligand was synthesized according to the procedure.62
Ethidium bromide (EB), calf thymus DNA (CT-DNA) and
pBR322 plasmid DNA were from Sigma. Tris-HCl buffer solution
was prepared using deionized sonicated triple-distilled water. All
other chemicals used were of analytical grade. Solution of the
copper(II) complex and other reagents used for strand scission
were prepared freshly in triple-distilled water before use. Solvents
used in this research were purified by standard procedures.
Elemental analyses for C, H and N were obtained on a Perkin-
Elmer analyzer model 240. Infrared spectroscopy on KBr pellets
was performed on a Bruker Vector 22 FT-IR spectrophotometer in
the 4000–400 cm-1 regions. The electronic spectra were measured
on a JASCO V-570 spectrophotometer. The fluorescence spectral
data were obtained on a MPF-4 fluorescence spectrophotometer
at room temperature. Cyclic voltammetry measurements were
performed on a BAS Epsilon Electrochemical Workstation. The
Gel Imaging and documentation DigiDoc-It System were assessed
using Labworks Imaging and Analysis Software (UVI, UK).
The variable temperature magnetic susceptibility measurements
were carried out with a microcrystalline sample on MPMS XL-
7 SQUID in the temperature range 2–300 K. Corrections for
the diamagnetism were estimated from Pascal’s constants. EPR
spectra of ground crystals were carried out at X-band with a
Bruker ELESYX instrument.
[Cu2(1,4-tpbd)(2,2¢-bpy)2(ClO4)2](ClO4)2 (4). To a methanolic
solution (10 mL) of 1,4-tpbd (0.1 mmol, 47.3 mg) was added drop-
wise an aqueous solution (5 mL) of Cu(ClO4)2·6H2O (0.2 mmol,
74.2 mg), the mixture solution was refluxed for 4 h in the presence
of air, then 2,2¢-bpy (0.2 mmol, 31.2 mg) was added and refluxed
for further 1 h, cooled and filtered, green block crystals of 4 suitable
for X-ray structure analysis were obtained by slow evaporation the
filtrate, collected by filtration, washed with diethyl ether and dried
in air. Yield 30% (based on the copper salts). Elemental analysis
data: calculated. (%) for C50H52Cl4Cu2N10O20 (1381.90): C, 43.46;
H, 3.79; N, 10.14. Found (%): C, 43.35; H, 3.90; N, 10.07. IR (KBr,
n/cm-1): 1606, 1515, 1474, 1444, 1089, 773, 626.
X-Ray crystallography
Single crystals of complexes 1, 2, 3, and 4 were used for X-ray
diffraction analyses by mounting on the tip of a glass fiber in air.
Crystal data were collected at 113(2) K (for 1 and 4) or 294(2) K
(for 2 and 3), using a Bruker SMART CCD 1000 diffractometer.
Diffraction intensities for the four complexes were collected by
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using the w-scan technique. The structures were solved by direct
methods using the program SHELXS-9763 and subsequent Fourier
difference techniques, and refined anisotropically by full-matrix
least-squares on F2 using SHELXL-97.64 Restrained refinements
(ISOR, SIMU, DFIX) are applied for the big thermal vibration
of C(13) atom (for 1) and the disordered structure of perchlorate
anions (for 3 and 4). All the non-hydrogen atoms were refined
anisotropically and all the hydrogen atoms were located in
the Fourier difference maps. Further crystallographic data and
experimental details for structural analyses of complexes 1, 2,
3 and 4 are summarized in Table 1. Selected bond angles and
distances are listed in Table 2.
equilibrated with nitrogen to aid the deoxygenation process. The
deoxygenated solutions were stored under a nitrogen atmosphere
prior to use. Reaction mixtures were prepared in a glove box by
addition of the appropriate volumes of stock solutions to the
reaction tubes, incubated at room temperature for 3 h and then
quenched by the addition to the loading buffer in the glove box.
All other conditions were the same as those listed for the aerobic
cleavage reactions.
Conclusions
CCDC reference numbers for complexes 1, 2, 3, and 4 are
639516, 689792, 694617 and 694618, respectively. For crystal-
lographic data in CIF or other electronic format see DOI:
10.1039/b823472h
Mononuclear and dinuclear copper(II) complexes of the tpbd
ligand with diimine coligands have been synthesized and char-
acterized by single crystal X-ray diffraction. Variable-temperature
magnetic susceptibility studies (2–300 K) indicate the existence of
antiferromagnetic coupling between the copper(II) ions in complex
2. The interaction of complexes with calf thymus DNA (CT-DNA)
has been investigated by UV absorption, fluorescent spectroscopy
and cyclic voltammetry, and results show that the interaction
between the complexes and CT-DNA is an intercalation mode.
DNA cleavage mechanism studies show that all complexes may
hydrolytically cleave pBR322 DNA in 50 mM Tris-HCl/18mM
NaCl buffer (pH = 7.2) in the absence of external agents. Complex
2 exhibits significantly highest nuclease activity in the four
complexes, with the pseudo-Michaelis–Menten kinetic parameters
kcat = 4.23 h-1 and Km = 2.4 ¥ 10-5 M. The results of the experiments
showed that although all the complexes could cleavage DNA
hydrolytically, complex 2 displayed the highest cleavage efficiency
due to presence of coordinated water molecules and unsaturated
coordination sites of the Cu atoms in the complex 2. This work is
a good example of rational design of efficient hydrolytic cleavage
nucleases.
DNA binding and cleavage experiments
The DNA-binding experiments were performed at room tempera-
ture. Using the electronic absorption spectral method, the relative
bindings of the four complexes to CT-DNA were studied in 5 mM
Tris-HCl/50 mM NaCl buffer (pH = 7.2). The solution of CT-
DNA gave a ratio of UV absorbance at 260 nm and 280 nm,
A260/A280, of 1.8–1.9, indicating that the DNA was sufficiently free
of protein.65 The CT-DNA stock solutions were prepared in 5 mM
Tris-HCl/50mM NaCl buffer, pH = 7.2 (stored at 4 ◦C and used
within 4 days after their preparation). The concentration of CT-
DNA was determined from its absorption intensity at 260 nm with
a molar extinction coefficient of 6600 M-1cm-1.66
By the fluorescence spectral method, the relative bindings of
complexes to CT-DNA were studied with an EB-bound CT-DNA
solution in 5 mM Tris-HCl/50mM NaCl buffer (pH = 7.2). The
fluorescence spectra were recorded at room temperature with
excitation at 526 nm and emission at 602 nm. The experiment
was carried out by titrating complexes into EB-DNA solution
containing 5 ¥ 10-5 M EB and 5 ¥ 10-5 M CT-DNA.
Abbreviations
Cyclic voltammetry were performed on a BAS Epsilon Electro-
chemical Workstation with three-electrode system consisted of a
platinum glassy carbon as the working electrode, a platinum wire
as the auxiliary electrode, and a saturated calomel electrode (SCE)
as the reference electrode. All the electrochemical measurements
were carried out in a 10 mL electrolytic cell by using 10 mM
Tris–HCl/50 mM NaCl buffer (pH = 7.2) as the supporting
electrolyte. Solutions were deoxygenated by purging with N2 prior
to measurements.
1,4-tpbd
N,N,N¢,N¢-Tetrakis(2-pyridylmethyl)benzene-
1,4-diamine
1,10-Phenanthroline
2,2¢-Bipyridine
N,N¢-Dimethylformamide
Ethidium bromide
Calf thymus DNA
Tris(hydroxymethyl)aminomethane
Tris-boracic-EDTA
1,10-phen
2,2¢-bpy
DMF
EB
CT-DNA
Tris
TBE
The DNA cleavage experiment was done by agarose gel
electrophoresis. The reaction was carried out following a literature
method.67 Bands were visualized by UV light and photographed.
The resolved bands were visualized by ethidium bromide staining
and were then quantified. A correction factor of 1.22 was utilized
to account for the decreased ability of ethidium bromide to
intercalate into Form I DNA compared with Form II.68 Cleavage
mechanistic investigation of pBR322 DNA was done using
different reagents such as DMSO, L-histidine, SOD, and EDTA
added to PBR322 DNA prior to the addition of complexes.
The anaerobic conditions were achieved using an mbraun LAB-
Star glove box. Deoxygenated solutions were prepared by four
freeze–pump–thaw cycles. Before each cycles the solutions were
EDTA
DMSO
SOD
SC
NC
L
Ethylenediaminetetraacetic acid
Dimethyl sulfoxide
Superoxide dismutase
Supercoiled circular
Nicked circular
Linear
Infrared spectroscopy
IR
Acknowledgements
This work was supported by the National Natural Science
Foundation of China (No. 20771063).
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Soc., Dalton. Trans., 2002, 8, 1757.
34 B. Singh, B. P. Yadav and R. C. Agarwal, Indian J. Chem., 1984, 23A,
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