Synthesis, structure, magnetic properties and DNA cleavage of binuclear Cu(II) Schiff-base complexes

Article abstract of DOI:10.1039/b819052f

Five binuclear Schiff base copper(ii) complexes [Cu₂(L)(OAc)] ·3DMF (1), [Cu₂(L)(OAc)]₂·3DMF (2), [Cu ₂(L)(BNPP)]·3CH₃CN (3), [Cu₂(L)(Fa)] ·2DMF (4) and [Cu₂(L)(Pa)]·DMF (5) (H

Full text of DOI:10.1039/b819052f

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www.rsc.org/dalton | Dalton Transactions
Synthesis, structure, magnetic properties and DNA cleavage of binuclear
Cu(II) Schiff-base complexes†
Yingying Kou, Jinlei Tian,* Dongdong Li, Wen Gu, Xin Liu, Shiping Yan,* Daizheng Liao and Peng Cheng
Received 28th October 2008, Accepted 16th December 2008
First published as an Advance Article on the web 9th February 2009
DOI: 10.1039/b819052f
Five binuclear Schiff base copper(II) complexes [Cu2(L)(OAc)]·3DMF (1), [Cu2(L)(OAc)]2·3DMF (2),
[Cu2(L)(BNPP)]·3CH3CN (3), [Cu2(L)(Fa)]·2DMF (4) and [Cu2(L)(Pa)]·DMF (5) (H3L =
N,N¢-bis(3,5-tert-butylsalicylidene-2-hydroxy)-1,3-propanediamine, OAc = acetic acid, BNPP =
bis(4-nitrophenyl)phosphate, Fa = 2-tetrahydrofuroic acid, Pa = benzoic acid) have been synthesized
and characterized by X-ray single-crystal structure analysis. Variable-temperature magnetic
susceptibility studies (2–300 K) indicate the existence of ferromagnetic coupling between the copper(II)
ions in complexes 1 and 4, and antiferromagnetic coupling in complexes 3 and 5. The interaction of
these complexes with CT-DNA has been studied by using absorption and emission spectral methods.
The apparent binding constant (Kapp) values for complexes 1, 3, 4 and 5 are 4.67 ¥ 105, 9.48 ¥ 105,
4.30 ¥ 105 and 3.90 ¥ 105 M-1, respectively, which show that the complexes bind to DNA by moderate
intercalative binding modes. Furthermore, all these complexes can cleave plasmid DNA to nicked DNA
in a sequential manner as the concentrations or reaction times are increased in the absence of reducing
agent. Their cleavage activities are promoted in the presence of hydrogen peroxide. The cleavage
mechanisms between the complexes and plasmid DNA are likely to involve singlet oxygen 1O2 and ∑OH
as reactive oxygen species.
efficient intramolecular activation of bound O2 and for binding in
Introduction
a selective manner to particular nucleic acid conformations.11
Nuclease molecules which cleave the DNA duplex have been a
Besides the interest of copper compounds related to nucleases,
significant topic of interest for biochemists. Natural nucleases
polynuclear copper compounds have attracted much attention due
to their coupling interactions of multicopper centers in biological
while chemical nucleases cleave DNA by oxidatively degrading the
systems. As far as we know, most of the bi-bridge binuclear
cleave the phosphate diester backbone of DNA by hydrolysis,1
deoxyribose moiety2 or by hydrolysis of the phosphate ester.3 The
study of artificial nucleases has received attention for their diverse
applications not only as chemical therapeutic agents but also
in genomic research over several decades.4–8 Chemical nucleases
present some advantages over conventional enzymatic nucleases
in that they are smaller in size and thus can reach more sterically
hindered regions of a macromolecule. Many of these utilize the
redox properties of the metal and dioxygen to produce reactive
oxygen species that oxidize DNA, yielding direct strand scission
or base modification.9 In this regard, transition-metal complexes,
especially copper complexes play an important role due to their
structural diversity. Copper complexes are capable of cleaving
DNA by both the hydrolytic and oxidative cleavage modes, the best
studied of these being [Cu(phen)2]2+ which induces direct strand
damage in the presence of H2O2.10 Three equivalents of the Cu(II)
species are required for this process to produce a non-diffusible
intermediate equivalent to a hydroxyl radical. This fact has focused
large attention on copper compounds due to their potential for
copper(II) systems, for example m-hydroxo/alkoxo-m-X (X =
carboxylato) systems, are antiferromagnetically coupled, whereas
ferromagnetic interaction has been observed in only a few m-
hydroxo/alkoxo-m-carboxylato compounds.12–15 In general, most
of the m-alkoxo-X bridged Cu(II) complexes show ferromagnetic
exchange for a Cu–O–Cu angle lower than 116.5◦ and are
antiferromagnetic above this value.16
Recently we have begun to explore the nuclease activity of bin-
uclear Cu(II) complexes.17,18 Moreover, because of our interest in
the binuclear/polynuclear copper(II) complexes, we have reported
the crystal structures and spectroscopic and magnetic properties of
binuclear copper(II) complexes. Here, we present five new binuclear
copper(II) complexes of Schiff-base ligands (H3L) with alkoxo and
carboxylato or phosphato bridges. The single-crystal structures
and magnetic properties and DNA cleavage activities of these
complexes, which efficiently cleave DNA in the presence of H2O2,
have been studied.
Results and discussion
Department of Chemistry, Nankai University, Weijin Road 94, Tianjin,
300071, P. R. China. E-mail: yansp@nankai.edu.cn.
† Electronic supplementary information (ESI) available: Additional char-
acterization data (Table S1, Fig. S1–S4). CCDC reference numbers
673944 (1), 702845 (2), 680010 (3), 697012 (4) and 697013 (5). For ESI
and crystallographic data in CIF or other electronic format see DOI:
10.1039/b819052f
X-Ray crystal structure characterization
The crystal structures of complexes 1–5 were determined and are
shown in Fig. 1–5, respectively. Selected bond distances and bond
angles are listed in Table 2.
2374 | Dalton Trans., 2009, 2374–2382
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Table 1 Crystal data and structure refinement for all compounds
Complex
1
2
3
4
5
Empirical formula
C44H73Cu2N5O8
927.15
113(2)
0.71073
Triclinic
¯
P1
9.972(2)
14.743(4)
17.732(5)
108.293(3)
91.948(2)
90.183(3)
2473.5(10)
2
1.245
0.911
988
0.20 ¥ 0.20 ¥ 0.20
2.04–25.01
C79 H121Cu4N7O13
1630.99
113(2)
0.71073
Triclinic
¯
P1
13.670(3)
16.814(3)
19.012(4)
99.93(3)
97.97(3)
98.08(3)
4201.8(15)
2
1.289
1.060
1728
0.20 ¥ 0.18 ¥ 0.12
2.03–25.02
C51H64Cu2N7O11P
1109.14
293(2)
0.71073
Triclinic
¯
P1
10.718(2)
15.912(3)
17.567(4)
102.90(3)
107.26(3)
92.94(3)
2766.5(10)
2
1.331
0.860
1160
0.12 ¥ 0.10 ¥ 0.08
1.58–25.02
C44H68Cu2N4O8
908.10
113(2)
0.71073
Monoclinic
P21/n
19.524(4)
9.7034(19)
24.564(5)
90
97.33(3)
90
4615.5(16)
4
1.307
0.975
C43H58.50Cu2N3O6
840.51
113(2)
0.71073
Triclinic
¯
P1
16.615(3)
16.617(3)
17.076(3)
102.19(3)
99.22(3)
106.83(3)
4285.5(15)
4
1.303
1.040
1774
0.20 ¥ 0.16 ¥ 0.12
1.26–25.02
Mr
T/K
˚
l/A
Crystal system
Space group
˚
a/A
˚
b/A
˚
c/A
a/◦
b/◦
g /◦
3
˚
V/A
Z
Dc/g cm-3
m/mm-1
F(000)
1928
0.12 ¥ 0.10 ¥ 0.08
1.42–27.57
Crystal size/mm
q Range for data
collection/◦
Reflections collected/unique
Rint
Max./min. transmission
Data/restraints/parameters
Goodness of fit on F2
Final R indices [I > 2s(I)]
21731/8694
0.0338
0.8388/0.8388
8694/0/552
1.082
R1 = 0.0485,
wR2 = 0.1273
R1 = 0.0549,
wR2 = 0.1329
1.364/-0.746
24583/14715
0.0273
0.8834/0.8160
14715/162/1004
1.024
R1 = 0.0383,
wR2 = 0.0979
R1 = 0.0481,
wR2 = 0.1040
0.816/-0.720
16379/9614
0.0968
0.9344/0.9039
9614/0/664
1.006
R1 = 0.0843,
wR2 = 0.1887
R1 = 0.1092,
wR2 = 0.2115
1.483/-1.938
32086/10621
0.0461
0.9261/0.8920
10621/357/634
1.039
R1 = 0.0472,
wR2 = 0.1239
R1 = 0.0606,
wR2 = 0.1331
0.934/-0.728
24573/14922
0.0807
0.8853/0.8189
14922/72/1028
1.082
R1 = 0.0809,
wR2 = 0.2170
R1 = 0.1077,
wR2 = 0.2307
1.171/-0.825
R indices (all data)
-3
˚
Dr(max./min.)/e A
◦
˚
Table 2 Selected bond lengths (A) and angles ( ) for all compounds
Complex
1
2
3
4
5
Cu(1)–N(1)
Cu(1)–O(1)
Cu(1)–O(2)
Cu(1)–O(4)
Cu(1)–O(5)
Cu(2)–N(2)
Cu(2)–O(2)
Cu(2)–O(3)
Cu(2)–O(5)
Cu(2)–O(4)
2.000(2)
1.916(2)
1.953(2)
1.983(2)
1.927(2)
1.915(4)
1.920(4)
1.973(4)
1.954(4)
1.929(2)
1.8967(17)
1.9408(17)
1.931(5)
1.881(4)
1.904(4)
1.937(5)
1.9020(18)
1.9570(18)
1.9603(19)
1.9585(18)
1.915(2)
1.9699(17)
1.9072(18)
1.983(2)
1.927(2)
1.887(2)
1.949(2)
1.922(2)
1.934(4)
1.954(4)
1.901(4)
1.956(3)
1.923(5)
1.921(5)
1.895(5)
1.958(4)
1.9430(19)
1.8967(19)
1.938(2)
1.9536(18)
175.00(7)
O(1)–Cu(1)–O(2)
O(1)–Cu(1)–O(4)
O(1)–Cu(1)–N(1)
O(1)–Cu(1)–O(5)
O(2)–Cu(1)–O(4)
O(2)–Cu(1)–N(1)
O(2)–Cu(1)–O(5)
O(5)–Cu(1)–N(1)
O(4)–Cu(1)–N(1)
O(2)–Cu(2)–O(4)
O(2)–Cu(2)–O(5)
O(2)–Cu(2)–N(2)
O(3)–Cu(2)–O(2)
O(3)–Cu(2)–O(4)
O(3)–Cu(2)–O(5)
O(3)–Cu(2)–N(2)
O(4)–Cu(2)–N(2)
O(5)–Cu(2)–N(2)
Cu(1)–O(2)–Cu(2)
Cu(3)–O(8)–Cu(4)
174.22(9)
90.40(8)
93.10(9)
175.52(8)
89.19(8)
93.13(9)
177.62(14)
88.56(15)
92.57(16)
175.6(2)
88.0(2)
92.5(2)
91.42(8)
88.60(7)
90.16(8)
84.90(9)
94.04(8)
84.67(9)
93.23(14)
85.56(16)
94.8(2)
85.1(2)
85.22(8)
93.95(7)
168.54(8)
164.61(9)
163.28(9)
177.03(16)
173.6(2)
92.83(8)
90.72(9)
86.60(9)
174.77(10)
178.78(9)
85.70(9)
168.34(8)
95.69(15)
85.55(16)
173.65(14)
95.25(19)
83.9(2)
174.4(2)
85.06(8)
170.50(7)
89.54(8)
89.29(9)
94.35(10)
87.97(8)
92.59(9)
86.88(15)
93.12(16)
88.50(19)
93.1(2)
92.40(8)
177.70(9)
168.96(10)
107.06(4)
178.78(9)
105.60(9)
105.45(8)
168.14(17)
137.75(9)
170.0(2)
128.25(6)
105.48(4)
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Fig. 1 ORTEP view of the molecular structure and atom-labeling scheme
of 1. Hydrogen atoms and DMF are omitted for clarity.
Fig. 4 ORTEP view of the molecular structure and atom-labeling scheme
of 4. Hydrogen atoms and DMF are omitted for clarity.
Fig. 5 ORTEP view of the molecular structure and atom-labeling scheme
of 5. Hydrogen atoms and DMF are omitted for clarity.
˚
˚
for 1: 0.176 A (Cu(1)) and 0.047 A (Cu(2)), respectively, and for
˚
˚
˚
˚
2: 0.111 A (Cu(1)), 0.087 A (Cu(2)), 0.116 A (Cu(3)) and 0.040 A
(Cu(4)). The dihedral angle between the best coordination planes
in Cu(1)Cu(2) and Cu(3)Cu(4) dinuclear units is 62.6◦ for 1 and
60.8◦ and 63.7◦ for 2, respectively. The twisting is also reflected
in the smaller values of Cu–O(alkoxo)–Cu bridging angles (q)
(Cu(1)–O(2)–Cu(2) = 107.06◦ for 1 and Cu(1)–O(2)–Cu(2) =
105.60◦ and Cu(3)–O(8)–Cu(4) = 105.45◦ for 2), and the shorter
Fig. 2 ORTEP view of the molecular structure and atom-labeling scheme
of 2. Hydrogen atoms and DMF are omitted for clarity.
˚
separations (Cu(1) ◊ ◊ ◊ Cu(2) = 3.120 A for 1 and Cu(1) ◊ ◊ ◊ Cu(2) =
˚
˚
3.107 A, Cu(3) ◊ ◊ ◊ Cu(4) = 3.114 A for 2, respectively) of the
metal centers within the dinuclear units are observed related to
analogues.14,19
The molecular structures of 3–5 are essentially similar to that of
1. The maximum deviations of the copper atoms from the mean
˚
˚
˚
˚
basal planes are 0.036 A (Cu(1)) and 0.050 A (Cu(2)) for 3; 0.131 A
˚
˚
(Cu(1)) and 0.087 A (Cu(2)) for 4 and 0.024 A (Cu(1)) and 0.046 A
(Cu(2)) for 5, respectively. The dihedral angles between the best
coordination planes in Cu(1)Cu(2) dinuclear units are 10.5◦ for 3,
58.3◦ for 4 and 25.4◦ for 5, respectively. The Cu ◊ ◊ ◊ Cu distances
◦
˚
and Cu–O(alkoxo)–Cu◦angles (q) are 3.663 A and 137.75 for
◦
˚
˚
Fig. 3 ORTEP view of the molecular structure and atom-labeling scheme
of 3. Hydrogen atoms and CH3CN are omitted for clarity.
3, 3.112 A and 105.48 for 4 and 3.441 A and 128.25 for 5,
respectively.
In general, the crystal structures of the complexes 1 and 2 are
found to be similar, while the complex 2 contains two independent
molecules in the crystallographic asymmetric unit. The structure
1 (Fig. 1) shows that the two copper centers are doubly bridged
by the alkoxo oxygen and the acetate anion. The coordination
geometry around each of the copper atoms is distorted square
planar, and formed by one m-alkoxo oxygen, one-m-acetate oxygen,
one imine nitrogen and one phenoxo oxygen afforded by the
ligand L3-. The displacements of the copper atoms from the
respective least-squares N1O3 coordination planes are only small:
Magnetic properties and ESR spectra
Variable-temperature (2–300 K) magnetic behavior in the form of
cMT vs. T plots of 1, 3, 4 and 5 is shown in Fig. 6. The cMT values
at 300 K are 0.84 cm3 mol-1 K for 1 and 0.81 cm3 mol-1 K for
4, respectively, which are slightly higher than the calculated value
(0.75 cm3 mol-1 K, g = 2) of two uncoupled spins of S = 1/2.
On lowering the temperature from 300 to 24 K for 1 and 100 K
for 4, cMT slowly increases and reaches a maximum value of
0.99 and 0.87 cm3 mol-1 K, respectively, and then decreases
2376 | Dalton Trans., 2009, 2374–2382
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Table 3 Variable-temperature magnetic parameters for the complexes 1,
3, 4 and 5 comparing previous reported
Complex
q/◦
g
J
zJ¢
1
107.06(4) 2.01
105.48(4) 2.00
137.75(9) 2.19
128.25(6) 2.18
56.24 -0.19
50.48 -3.50
4
3
5
-169.23
-71.38
26
0
0
Cu2(L-F)(m-C7H5N2)26
112.0(2)
2.04
2.007
-0.35
-0.82
0
[Cu2(L-H)(m-C7H5N2)]·CH3OH26 114.3(3)
16.7
Cu2(L1)(m-HCO2)28
132.85
131.47
2.075 -156
2.00 -152
Cu2(L1)(m-HCO2)·DMF28
0
Table 4 ESR parameters for complexes 1, 3, 4 and 5 in the solid state at
room temperature
Fig. 6 cMT vs. T plots for complexes 1 (ꢀ), 3 (᭺), 4 (ꢀ) and 5 (ꢁ). Solid
Complex
g1 (gꢀ)
g2
g3 (g^)
G
lines represent the best theoretical fits.
1
3
4
5
2.205
2.275
2.236
2.172
2.070
2.137
2.112
2.070
2.053
2.106
2.096
2.062
3.87
2.59
2.46
2.77
rapidly on further cooling. The rapid decrease of cMT may be
due to intermolecular antiferromagnetic interaction, which is
very common in ferromagnetically coupled systems. The profiles
indicate the existence of intramolecular ferromagnetic exchange
interaction between the copper(II) ions in complexes 1 and 4. In
contrast, the magnetic behavior of the complexes 3 and 5, is typical
of intramolecular antiferromagnetic exchange interaction. In this
and previously reported complexes21,22 in a comparative fashion as
shown in Table 3. The magnetic behaviors of m-alkoxo complexes
are closely related with the Cu–O–Cu bridge angles (q). According
to the linear fitting result between the Cu–O–Cu bridge angles
and the 2J of known complexes, the magnetic exchange is
ferromagnetic below 116.5◦ and antiferromagnetic above this
value.14 For 1, 3, 4 and 5, their magnetic behaviors are all in accord
with this rule.
case, the cMT values: 0.48 cm3 mol-1 K for 3 and 0.73 cm3 mol-1
K
for 5 at 300 K are lower than that (0.75 cm3 mol-1 K) of the
non-correlated system of two S = 1/2 spins carriers. On lowering
temperature, cMT decreases gradually to 0.0085 cm3 mol-1 K for
3 and 0.0126 cm3 mol-1 K for 5 at 2 K.
The ESR spectra of complexes 1, 3, 4 and 5 in the solid state
were measured at room temperature (Fig. S1, ESI†). No hyperfine
structure is observed for any of the complexes. The spectra are
Taking into account the dinuclear copper model, the magnetic
susceptibilities of complex can be fitted accordingly by eqn
(2) derived from the Bleaney–Bowers equation H =-2JS1·S2
(S1 = S2 = 1/2).
quite similar and exhibit g-tensor parameters with gꢀ > g^
>
2
2
2.0023 (Table 4). This indicates that the copper site has a dx
-y
2Ng2 b2
kT
1
Ng2 b2
2kT
ground state23 characteristic for a square-planar geometry.24,25
Also, the observed value, which is <2.3, indicates the covalent
nature between the Cu(II) ion and the ligand.26,27
¢
(1)
cM = (1 -r)
¥
+
r
3 + e-2J / kT
cM
In axial symmetry, G = (gꢀ - 2)/(g^ - 2) where G is the exchange
interaction parameter. The calculated G values of the Cu(II)
complexes are lower than 4 suggesting copper–copper exchange
interactions.24
cM
=
(2)
1 - 7.6853zJ¢cM g2
Here J is the exchange coupling parameter between S1 and S2, r
is the contribution of mononuclear impurity, and other symbols
have their normal meanings.
DNA binding properties by UV titration and competitive
fluorescence displacement assay
There are ferromagnetic couplings between the copper(II) ions in
complexes 1 and 4 and the susceptibility data can be simulated well
with the equation with the following set of converging parameters:
J = 56.24 cm-1, g = 2.01 and zJ¢ = -0.19 cm-1 for 1; J =
50.48 cm-1, g = 2.00, r = 2% and zJ¢ = -3.50 cm-1 for 4. These
ferromagnetic interactions between the Cu2+ ions are probably
due to the lower Cu–O(alkoxo)–Cu angles (q) of 107.06◦ for 1
and 105.48◦ for 4, respectively (<116.5◦). In addition, complexes
3 and 5 show antiferromagnetic couplings as a result of the greater
Cu–O(alkoxo)–Cu angles (q) of 137.75◦ for 3 and 128.25◦ for 5
(>116.5◦). The exchange parameters represent typical fits with
J = -169.23 cm-1, g = 2.19 and r = 1.5% for 3, and J = -71.38 cm-1,
g = 2.18 and r = 3% for 5.
DNA binding is the critical step for DNA cleavage in most cases.
Therefore, the binding of complexes 1, 3, 4 and 5 to CT-DNA
were studied by using UV-Vis absorption and fluorescence spec-
troscopy. Electronic absorption spectroscopy is one of the most
useful techniques for DNA-binding studies of metal complexes.
The absorption spectra of copper(II) complexes in the absence
and presence of CT-DNA at different concentrations are given
in Fig. S2 (ESI†). The absorption peaks with maxima of 210 nm
are attributed to intraligand p–p* transitions. The intense ligand
based (p–p*) absorption band is used to monitor the interaction of
the complexes with calf thymus DNA. Complexes bound to DNA
through intercalation, which involves a strong stacking interaction
of the planar aromatic rings of the coordinated ligand with the
base pairs of DNA, usually result in hypochromism and red
A number of m-alkoxo compounds have been structurally
and magnetically studied to reveal the orbital complementarity
effect.14,20 We can list the magnetic properties of 1, 3, 4 and 5
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Table 5 Change in spectral features of the copper(II) complexes on
interaction with CT-DNA in 50 mM Tris-HCl/18 mM NaCl buffer
(pH = 7.2)
Table 6 The Epa, Epc, E1/2 and DE values for the copper(II) complexes in
the absence and presence of CT-DNA
Complex
E
pa/mV
Epc/mV
E
1/2/mV
DEa/mV
Change in
Complex
absorptivity
l
max/nm
Dlmax/nm
De (%)
1
3
4
5
20, -27
19, -36
5, -14
0, -31
-62, -99
-49, -100
-77, -98
-70, -99
-21, -63
-15, -68
-36, -56
-35, -65
42
53
20
30
1
3
4
5
Hypochromism
Hypochromism
Hypochromism
Hypochromism
208
212
218
218
4
6
6
6
33.85
44.26
35.75
37.55
a DE indicates the negative shifts for the four copper(II) complexes in the
absence and presence of CT-DNA.
shift of ligand-band or charge-transfer bands.28 All the present
complexes exhibit significant hypochromism (33.85–44.26%) on
the incremental addition of DNA with varying red shifts (Fig. S2,
ESI;† Table 5). Furthermore, as the extent of hypochromism is
commonly consistent with the strength of intercalative interaction,
this indicates that complex 3 exhibits better DNA binding affinity
compared with complexes 1, 4 and 5.
pBR322 DNA cleavage by the copper(II) complexes
The chemical nuclease activities of the complexes have been
studied using supercoiled pBR322 plasmid DNA in a medium
of 50 mM Tris-HCl/NaCl buffer (pH = 7.2) in the absence
of reducing agent under similar physiological conditions. 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 that migrates
at a rate between that of Form I and Form II. Fig. 7 shows
the results of gel electrophoretic separations of plasmid pBR322
DNA induced by an increasing concentration of complexes in the
absence of reducing agent. As shown in Fig. 7, with the increase
of the concentrations of the complexes, Form I plasmid DNA
is gradually converted into Form II. In addition, the oxidative
cleavage of DNA in the presence of hydrogen peroxide has also
been studied by gel electrophoresis and shown in Fig. 8. The
As a means for further clarifying the binding of complexes,
fluorescence spectral measurements were carried out. The addition
of complex to the DNA bound EB solutions caused obvious reduc-
tion in emission intensities, indicating that complex competitively
bound to DNA with EB. The extent of reduction of the emission
intensity gives a measure of the binding propensity of the complex
to DNA. According to the classical Stern–Volmer equation29
I0/I = 1 + K[Q]; I0 and I are the fluorescence intensities in the
absence and presence of the quencher, respectively. K is a linear
Stern–Volmer quenching constant. [Q] is the concentration of the
quencher. The quenching plots (Fig. S3, ESI†) illustrate that the
quenching of EB bound to DNA by complexes are in agreement
with the linear Stern–Volmer equation, which also indicate the
complexes bind to DNA. In the plot of I0/I vs. the concentrations
of complexes, K is given by the ratio of the slope to intercept.
According to the equation KEB[EB] = Kapp[complex], where the
complex concentration is the value for a 50% reduction of the
fluorescence intensity of EB (KEB = 1.0 ¥ 107 M-1, [EB] = 4.0 mM)
the Kapp values are calculated to be 4.67 ¥ 105, 9.48 ¥ 105, 4.30 ¥ 105
and 3.90 ¥ 105 M-1 for complexes 1, 3, 4 and 5, less than the binding
constant of the classical intercalators and metallointercalators
(107 M-1),17 which suggest that the interactions of the complexes
with DNA are all moderate intercalative modes. Complex 3 is the
best binding agent due to its highest planarity.30
Electrochemical studies
Typical cyclic voltammetry (CV) behaviors of complexes in the
absence and presence of CT-DNA are measured. In the presence
of CT-DNA, the cyclic voltammograms of the four copper(II)
complexes 1, 3, 4 and 5 exhibited significant shifts in the anodic
and cathodic peak potentials followed by decrease in both peak
currents, indicating the interaction existing between the four
copper(II) complexes and CT-DNA. The Epa, Epc, E1/2 values and
negative shifts (DE) for the four copper(II) complexes 1, 3, 4 and
5 in the absence and presence of CT-DNA are shown in Table 6.
The results indicated reversible redox processes (DEp of 59 mV for
a one-electron diffusion and ipc = ipa of about 1 for a controlled
reversible process). 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.31
Fig. 7 Agarose gel electrophoresis of pBR322 plasmid DNA treated
with complexes at different concentrations. (a) Lane 0: supercoiled DNA
(control); lanes 1–8: complex 1 (10, 40, 70, 100, 200, 300, 400, 500 mM).
(b) Lane 0: supercoiled DNA (control); lanes 1–8: complex 3 (10, 40,
70, 100, 200, 300, 400, 500 mM). (c) Lane 0: supercoiled DNA (control);
lanes 1–8: complex 4 (10, 40, 70, 100, 200, 300, 400, 500 mM). (d) Lane
0: supercoiled DNA (control); lanes 1–8: complex 5 (10, 40, 70, 100, 200,
300, 400, 500 mM).
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Fig. 8 Agarose gel electrophoresis of pBR322 plasmid DNA treated with
complexes with addition of hydrogen peroxide at different concentrations.
(a) Lane 0: supercoiled DNA (control); lane 1: H2O2 (25 mM); lanes 2–9:
H2O2 (25 mM) + complex 1 (10, 40, 70, 100, 200, 300, 400, 500 mM). (b)
Lane 0: supercoiled DNA (control); lane 1: H2O2 (25 mM); lanes 2–9: H2O2
(25 mM) + complex 3 (10, 40, 70, 100, 200, 300, 400, 500 mM). (c) Lane
0: supercoiled DNA (control); lane 1: H2O2 (25 mM); lanes 2–9: H2O2
(25 mM) + complex 4 (10, 40, 70, 100, 200, 300, 400, 500 mM). (d) Lane
0: supercoiled DNA (control); lane 1: H2O2 (25 mM); lanes 2–9: H2O2
(25 mM) + complex 5 (10, 40, 70, 100, 200, 300, 400, 500 mM).
Fig. 9 Agarose gel electrophoresis of pBR322 plasmid DNA treated with
300 mM complexes for different incubation time. (a) Lane 0: supercoiled
DNA (control) (10 h); lanes 1–8: complex 1 (0.5, 1, 2, 4, 5, 6, 8, 10 h).
(b) Lane 0: supercoiled DNA (control) (10 h); lanes 1–8: complex 3 (0.5, 1,
2, 4, 5, 6, 8, 10 h); (c) Lane 0: supercoiled DNA (control) (10 h); lanes 1–8:
complex 4 (0.5, 1, 2, 4, 5, 6, 8, 10 h). (d) Lane 0: supercoiled DNA (control)
(10 h); lanes 1–8: complex 5 (0.5, 1, 2, 4, 5, 6, 8, 10 h), respectively.
which is indicative of the involvement of the hydroxyl radical in the
cleavage process. In order tofurther clarifythe cleavage mechanism
of pBR322 DNA induced by complexes, it is necessary to perform
the cleavage experiment under anaerobic conditions (Fig. S4, lanes
0–2, ESI†), the results show that the complexes hardly cleave
pBR322 plasmid DNA under anaerobic conditions. According
to the results mentioned above, we propose the hypothesis that the
copper(II) complexes examined here may be capable of promoting
DNA cleavage through an oxidative DNA damage pathway, in
which the active oxygen species involved in the reaction are singlet
oxygen, (1O2) and hydroxyl radical (∑OH).
results indicated that the DNA cleavage activities of the complexes
can be promoted in the presence of hydrogen peroxide compared
with the case of the absence of hydrogen peroxide under the same
experimental conditions (Table S1, ESI†).
A time course of a gel electrophoresis pattern of pBR322 DNA
cleavage during a reaction in the presence of 300 mM complex
at pH = 7.2 and 37 ◦C is shown in Fig. 9. With reaction time
increase, the amount of Form II 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 are dependent on
the concentrations of complexes and reaction times. From Fig. 10,
we can find that all of the processes are typical pseudo-first-order
consecutive reactions, which are consistent with the general model
for enzyme catalyzed reactions.32,33 Fitting the experimental data
with first-order consecutive kinetic equations, rate constants of
2.11 ¥ 10-5 s-1, 2.34 ¥ 10-5 s-1, 1.48 ¥ 10-5 s-1, 2.27 ¥ 10-5 s-1, for the
conversions of supercoiled to nicked DNA are obtained for 1, 3,
4 and 5, respectively. The results indicate that the complex 3 has
better nuclease activity than compounds 1, 4 and 5.
Experimental
CAUTION
Perchlorate salts of metal complexes are potentially explosive and
therefore should be prepared in small quantities.
Materials and instrumentation
Ligand H3L (N,N¢-bis(3,5-tert-butylsalicylidene-2-hydroxy)-1,3-
propanediamine) was synthesized according to a previously re-
ported procedure.35 Ethidium bromide (EB), calf thymus DNA
(CT-DNA) and pBR322 plasmid DNA was from Sigma. Tris-
HCl buffer solution was prepared using deionized sonicated triple-
distilled water. All other reagents and chemicals were purchased
from commercial sources and used as received. 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. Electronic spectra were measured on a JASCO V-570
spectrophotometer. Fluorescence spectral data were obtained on
The DNA cleavage mechanisms by the complexes were inves-
tigated in the presence of a hydroxyl radical scavenger (DMSO),
a superoxide scavenger (SOD), a singlet oxygen quencher (NaN3)
and a chelating agent (EDTA)34 under our experimental condi-
tions (Fig. 11, lanes 2–5). The SOD enzyme had no effect on the
cleavage reaction (lane 4) suggesting that the superoxide anion
is not involved in the cleavage process. Azide (lane 3) inhibits
1
DNA cleavage by the compound indicating that O2 is involved
in the reaction. The hydroxyl radical scavenger, DMSO (lane 2)
diminishes significantly the nuclease activity of the compound
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Fig. 10 Agarose gel electrophoresis of pBR322 plasmid DNA treated with 300 mM complexes for different incubation times. Densitometric quantitative
results of the gel electrophoresis: (a) complex 1; (b) complex 3; (c) complex 4; (d) complex 5. Scatter: experimental data, line: fitting curves.
a MPF-4 fluorescence spectrophotometer at room temperature.
Cyclic voltammetry measurements were performed on a BAS
Epsilon Electrochemical Workstation. The variable temperature
magnetic susceptibility measurements were carried out with micro-
crystalline samples on a MPMS XL-7 SQUID in the temperature
range 2–300 K. Corrections for the diamagnetism were estimated
from Pascal’s constants. ESR spectra of ground crystals were
carried out at X-band with a Bruker ELESYX instrument.
Preparation of complexes
[Cu2(L)(OAc)]·3DMF (1). To an acetonitrile solution (10 mL)
of Cu(OAc)2·H2O (0.4 mmol, 80 mg) was added a 10 mL
methanol–DMF (1 : 1) mixture of H3L (0.2 mmol) and triethy-
lamine (0.6 mmol). The resulting mixture was stirred for 6 h at
room temperature. After filtration, green block crystals suitable
for X-ray diffraction were obtained by slow evaporation of the
filtrate after several days, which were collected by filtration, washed
with diethyl ether and dried in air (yield: 32 mg, 40%). Elemental
analysis (%): calc. for C44H73Cu2N5O8: C, 57.00; H, 7.94; N, 7.55.
Found: C, 57.02; H, 8.01; N, 7.50.
Fig. 11 Agarose gel electrophoresis of pBR322 plasmid DNA treated
with 300 mM complex in presence of potential inhibitor agents. Incubation
time is 3 h (37 ◦C): (a) complex 1; (b) complex 3; (c) complex 4; (d) complex
5. Lane 0: supercoiled DNA (control); lane 1: complex; lane 2: complex +
DMSO (1 M), lane 3: complex + NaN3 (100 mM), lane 4: complex + SOD
(15 units); lane 5: complex + EDTA (1 mM).
[Cu2(L)(OAc)]2·3DMF (2). To an acetonitrile solution (10 mL)
of Cu(OAc)2·H2O (0.4 mmol, 80 mg) was added 5 mL DMF
of H3L (0.2 mmol) and triethylamine (0.6 mmol). The resulting
mixture was stirred for 4 h at room temperature. After filtration,
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green block crystals suitable for X-ray diffraction were obtained
by slow evaporation of the filtrate after several months, which
were collected by filtration, washed with diethyl ether and dried
in air (yield: 24 mg, 30%). Elemental analysis (%): calc. for
C79H121Cu4N7O13: C, 58.18; H, 7.48; N, 6.01. Found: C, 58.20;
H, 7.44; N, 6.03.
The relative bindings of 1, 3, 4 and 5 to CT-DNA were studied
with an EB–DNA solution in Tris-HCl/NaCl buffer (pH = 7.2).
The fluorescence spectra were recorded at room temperature with
excitation at 510 nm and emission at 602 nm. Such experiments
were carried out by titrating complexes into EB–DNA solution
containing 4.0 ¥ 10-6 M EB and 80 ¥ 10-6 M of DNA.
The DNA cleavage experiments were done by agarose gel
electrophoresis, which was performed by incubation at 37 ◦C
for 3 h as follows: pBR322DNA (0.1 mg mL-1) in 50 mM Tris–
HCl/18 mM NaCl buffer (pH = 7.2) was treated with complex
in the absence of additives. The samples were incubated for
3 h, and then loading buffer was added. Then the samples were
electrophoresed for 4 h at 80 V on 0.8% agarose gel using Tris–boric
acid–EDTA buffer. After electrophoresis, bands were visualized by
UV light and photographed. Quantification of cleavage products
was performed by UVIpro software, Version 10.03.40 Supercoiled
plasmid DNA values were corrected by a factor of 1.3, based on
average literature estimate of lowered binding of ethidium.41
Cleavage mechanistic investigations of pBR322 DNA were done
using different reagents such as DMSO, NaN3, SOD and EDTA
added to pBR322 DNA prior to the addition of complex. The
anaerobic conditions were achieved using an MBRAUN LAB-
Star glove box. Deoxygenated solutions were prepared by four
freeze–pump–thaw cycles. Before each cycle the solutions were
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.
[Cu2(L)(BNPP)]·3CH3CN (3). To an acetonitrile solution
(5 mL) of Cu(ClO4)2·6H2O (0.4 mmol, 144.8 mg) was added
10 mL CH3CN–DMF (1 : 1) of H3L (0.2 mmol) and triethylamine
(0.6 mmol). Then 0.2 mmol BNPP and 0.2 mmol NEt3 dissolved
in 3 mL CH3CN was added to the above mixture after stirring
for 1 h at room temperature. The green solution was stirred for
another 3 h. After filtration, green block crystals suitable for X-ray
diffraction were obtained by slow evaporation of the filtrate after
several months, which were collected by filtration, washed with
diethyl ether and dried in air (yield: 65.2 mg, 45%). Elemental
analysis (%): calc. for C51H64Cu2N7O11P: C, 55.23; H, 5.82; N,
8.84. Found: C, 55.20; H, 5.86; N, 8.81.
[Cu2(L)(Fa)]·2DMF (4). Complex 4 was prepared similarly to
3, but adding 0.2 mmol 2-tetrahydrofuroic acid instead of BNPP to
the reaction mixture. After filtration, green block crystals suitable
for X-ray diffraction were obtained by slow evaporation of the
filtrate after several months, which were collected by filtration,
washed with diethyl ether and dried in air (yield: 72.4 mg, 50%).
Elemental analysis (%): calc. for C44H68Cu2N4O8: C, 58.19; H,
7.55; N, 6.17. Found: C, 58.22; H, 7.53; N, 6.19.
[Cu2(L)(Pa)]·DMF (5). Complex 5 was prepared similarly to 3,
but adding 0.2 mmol PhCOONa instead of BNPP and NEt3 to the
reaction mixture. After filtration, green block crystals suitable for
X-ray diffraction were obtained by slow evaporation of the filtrate
after several days, which were collected by filtration, washed with
diethyl ether and dried in air (yield: 60.8 mg, 42%). Elemental
analysis (%): calc. for C43H58.50Cu2N3O6: C, 61.44; H, 7.02; N,
5.00. Found: C, 61.50; H, 6.99; N, 4.98.
Conclusions
In summary, we have synthesized and characterized five binuclear
copper(II) complexes with Schiff base ligand H3L. Variable-
temperature magnetic susceptibility studies (2–300 K) indicate the
existence of ferromagnetic coupling between the copper(II) ions in
complexes 1 and 4, and antiferromagnetic coupling in complexes 3
and 5, and have a clear dependence on Cu–O(m-alkoxo)–Cu angles.
The complexes can all effectively promote cleavage of plasmid
DNA without addition of external agents and in the presence
of hydrogen peroxide at pH = 7.2 and 37 ◦C. DNA cleavage
mechanism studies show that complexes examined here may be
capable of promoting DNA cleavage through an oxidative DNA
damage pathway.
X-Ray crystallography
Diffraction data for 1, 2, 4 and 5 were collected at 113 (2) K
and 3 at 293 (2) K, with a Rigaku Saturn CCD diffractometer
˚
using Mo-Ka radiation (l = 0.71073 A) with the w–2q scan
technique. An empirical absorption correction was applied to the
raw intensities.36 The structures were solved by direct methods
(SHELX-97) and refined with full-matrix least-squares technique
on F2 using SHELX-97.37,38 The hydrogen atoms were added
theoretically, and riding on the concerned atoms and refined with
fixed thermal factors. The details of crystallographic data and
structure refinement parameters are summarized in Table 1.
Abbreviations
EB
ethidium bromide
Tris
EDTA
DMSO
tris(hydroxymethyl)aminomethane
ethylenediaminetetraacetic acid
dimethyl sulfoxide
DNA-binding and cleavage experiments
The UV absorbance at 260 and 280 nm of the CT-DNA solution
in 18 mM NaCl–50 mM Tris–HCl buffer (pH = 7.2) give a
ratio of 1.8–1.9, indicating that the DNA was sufficiently free of
protein.33 The concentration of CT-DNA was determined from its
absorption intensity at 260 nm with a molar extinction coefficient
of 6600 M-1 cm-1.39 The absorption spectra of 1, 3, 4 and 5 binding
to DNA were performed by increasing amount of CT-DNA to the
complexes in Tris-HCl buffer (pH = 7.2).
CT-DNA calf thymus DNA
TBE Tris-boracic-EDTA
Acknowledgements
This work was supported by the National Natural Science
Foundation of China (No. 20771063).
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