Structure, DNA binding and cleavage of a new Zn(II)Mn(II) macrocyclic complex

Article abstract of DOI:10.1016/j.saa.2012.08.029

A new heterodinuclear complex of an unsymmetrical macrocycle [ZnMnL(CH ₃O)₂]·H₂O has been synthesized by the cyclocondensation between N,N′-bis(3-formyl-5-chlorosalicylidene) ethylenediimine and 2-hydroxyl-1,3-propanediamine in the presence of the metal ions, and characterized by elemental analyses, IR spectra and X-ray determination. The interactions of the complex with DNA have been investigated by UV absorption, fluorescence spectroscopy, viscosity measurements and electrochemical studies. Absorption spectroscopic investigation reveals that the complex has good binding propensity to calf thymus DNA by intercalation with a binding constant of 2.52 × 10⁵ M^-1. Fluorescence spectroscopy shows that the complex can displace ethidium bromide and bind to DNA, with a quenching constant of 4.37 × 10³ M^-1. The agarose gel electrophoresis studies show that pBR322 plasmid DNA can be transformed to nicked form and linear form in air by the complex.

Full text of DOI:10.1016/j.saa.2012.08.029

Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 99 (2012) 329–334
Contents lists available at SciVerse ScienceDirect
Spectrochimica Acta Part A: Molecular and
Biomolecular Spectroscopy
journal homepage: www.elsevier.com/locate/saa
Structure, DNA binding and cleavage of a new Zn(II)Mn(II) macrocyclic complex
⇑
Jing-Jing Zhou, Yu Mei, Zhiquan Pan, Hong Zhou
Key Laboratory for Green Chemical Process of Ministry of Education, Wuhan Institute of Technology, Wuhan 430073, PR China
g r a p h i c a l a b s t r a c t
h i g h l i g h t s
The approximately planar structure of the title complex makes it easier to intercalate into the base pairs
in DNA helix; and has higher binding and cleavage activity.
"
A new heterodinuclear complex has
been synthesized and fully
characterized.
"
"
The complex binds to DNA with
higher binding constant.
The complex can cleavage DNA in
lower concentration.
a r t i c l e i n f o
a b s t r a c t
Article history:
A new heterodinuclear complex of an unsymmetrical macrocycle [ZnMnL(CH3O)2]ꢀH2O has been synthe-
sized by the cyclocondensation between N,N0-bis(3-formyl-5-chlorosalicylidene)ethylenediimine and 2-
hydroxyl-1,3-propanediamine in the presence of the metal ions, and characterized by elemental analyses,
IR spectra and X-ray determination. The interactions of the complex with DNA have been investigated by
UV absorption, fluorescence spectroscopy, viscosity measurements and electrochemical studies. Absorp-
tion spectroscopic investigation reveals that the complex has good binding propensity to calf thymus
DNA by intercalation with a binding constant of 2.52 ꢁ 105 Mꢂ1. Fluorescence spectroscopy shows that
Received 22 February 2012
Received in revised form 10 August 2012
Accepted 11 August 2012
Available online 20 August 2012
Keywords:
Heterodinuclear complex
Unsymmetrical macrocycle
Crystal structure
the complex can displace ethidium bromide and bind to DNA, with
a quenching constant of
4.37 ꢁ 103 Mꢂ1. The agarose gel electrophoresis studies show that pBR322 plasmid DNA can be trans-
formed to nicked form and linear form in air by the complex.
DNA binding and cleavage
Ó 2012 Elsevier B.V. All rights reserved.
Introduction
activities [2,3]. Researches show that this kind of macrocyclic com-
plexes has good DNA binding and cleavage biological properties
Elucidating the effects of affinity and selectivity of small mole-
cules on binding with DNA would be valuable in the application of
chemotherapy and in the development of tools for biotechnology
[1]. Multinitrogen macrocyclic Schiff base ligands containing phen-
oxide groups have received special attention in the past decades
because of their versatile coordination behaviors and biological
[4,5]. The differences of metal ions, ligands and coordination envi-
ronment in the complexes have great effect on the bioactivities [6].
In our previous work, we investigated the DNA binding and
cleavage ability of some homodinuclear multinitrogen macrocyclic
complexes. These complexes cleavage DNA via intercalate mode
with higher binding constants to DNA [7]. To further illustrate
the structure factors on the bioactivity of the complexes, a new
heterodinuclear Zn(II)Mn(II) of an unsymmetrical macrocyclic
ligand (Scheme 1) has been synthesized. And the calf thymus
⇑
Corresponding author. Tel.: +86 8719 4980; fax: +86 8719 4465.
E-mail address: hzhouh@126.com (H. Zhou).
1386-1425/$ - see front matter Ó 2012 Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.saa.2012.08.029

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J.-J. Zhou et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 99 (2012) 329–334
DNA binding activity and plasmid pBR322 DNA cleavage activity of
the complex were investigated via UV absorption, fluorescence
spectroscopy, viscosity, electrochemical studies and agarose gel
electrophoresis measurements.
Physical measurement
IR spectra were measured using KBr disc on a vector 22 FT-IR
spectrophotometer. Elemental analyses were obtained on a Per-
kin-Elmer 240 analyzer. Absorption spectra in the 200–700 nm
range were recorded on a Shimadzu UV-2450 spectrophotometer.
Fluorescence spectra were recorded on a Jasco FP-6500 spectro-
photometer. Cyclic voltammograms were run on a CHI model
750B electrochemical analyzer in DMF solutions containing tet-
ra(n-butyl)ammonium perchlorate (TBAP) as the supporting elec-
trolyte. A three-electrode cell was used, which was equipped
with a glassy carbon-working electrode, a platinum wire as the
counter electrode and an Ag/AgCl electrode as the reference elec-
trode. Scanning rate was 100 mV sꢂ1. The solution was deaerated
for 15 min before measurements.
Experimental
Materials
All solvents and chemicals were of analytical grade and were
used as received, except for methanol and ethanol that were puri-
fied to absolute ones by a general method. 2,6-Diformyl-4-chloro-
phenol was prepared according to the literature method [8].
Ethylenediamine and 2-hydroxyl-1,3-propanediamine were pur-
chased form Alfa Aesar.
Crystal structure determination
Preparation of the complex
The crystals were measured on a Bruker AXS SMART diffractom-
Preparation of N,N0-bis(3-formyl-5-
chlorosalicylidene)ethylenediimine
eter (Mo K
a radiation monochromator). Data reduction and cell
refinement were performed by SMART and SAINT programs [9].
The structures were solved by direct method and refined by full-
matrix least-squares techniques based on F2 [10]. Hydrogen atoms
were located geometrically and refined in riding mode. The non-H
atoms were refined with anisotropic displacement parameters.
Calculations were performed using SHELX-97 crystallographic
software package.
To the solution of 2,6-diformyl-4-chlorophenol (1.845 g, 10
mmol) in absolute ethanol (10 mL), the ethanol solution (15 mL)
of ethylenediamine (0.3 g, 5 mmol) was added dropwise, the
orange deposit appeared. The mixture was stirred at room temper-
ature for 4 h. Then the resulting solution was filtered, the precipi-
tate was washed with ether and absolute ethanol several times and
dried in vacuo. Yield: 1.12 g (57%). Anal. Calc. For C18H14Cl2N2O4
(%): C, 54.98; H, 3.59; N, 7.12. Found: C, 54.64; H, 3.22; N, 7.53.
DNA-binding experiments
IR (KBr,
(C@O), 1221, 1042
m
/cmꢂ1): 3040, 2926
(C–O).
m (C–H), 1645 m (C@N), 1683 m
m
CT-DNA (20 mg) was dissolved in Tris–HCl buffer (100 mL,
50 mM Tris–HCl, 50 mM NaCl, pH = 7.2) and kept at 4 °C for 3 days.
The absorption ratio A260/A280 was within the range of 1.8–2.0. The
DNA concentration was determined via absorption spectroscopy
using the molar absorption coefficient of 6600 Mꢂ1 cmꢂ1
(260 nm) for CT-DNA [11].
The Mn(II)Zn(II) complex was dissolved in DMF at a concentra-
tion of 1.0 ꢁ 10ꢂ4 M. The UV absorption titrations were performed
by keeping the concentration of the complex fixed while varying
the DNA concentration. Complex-DNA solutions were allowed to
incubate for 30 min at room temperature before measurements
were made. Absorption spectra were recorded using cuvettes of
1 cm path length at room temperature.
Preparation of [ZnMnL](CH3O)2ꢀH2O
A suspension of N,N0-bis(3-formyl-5-chlorosalicylidene)ethyl-
enediimine (0.197 g, 0.5 mmol) in absolute methanol (10 mL)
was added
a
methanol solution (6 mL) of Mn(OAc)2ꢀ4H2O
(0.123 g, 0.5 mmol). The mixture was stirred at ambient tempera-
ture for about 4 h, then triethylamine (5 mL) and a methanol solu-
tion (6 mL) containing Zn(OAc)2ꢀ2H2O (0.110 g, 0.5 mmol) was
added. The mixture was further stirred for about 10 h. A methanol
solution (6 mL) containing 2-hydroxyl-1,3-propanediamine
(0.045 g, 0.5 mmol) was added dropwise, the reaction mixture
was stirred at room temperature overnight. Then the resulting
solution was filtered. The orange single crystals suitable for X-ray
structure analysis were obtained by the slow evaporation of the fil-
trate. Yield: 0.12 g (46%). Anal. Calc. for C23H26Cl2MnZnN4O6 (%): C,
Fluorescence quenching experiments were performed by add-
ing a solution of the complex (1.5
l
L) to EB-bound CT-DNA solu-
M). All
tion (1.5 L) at different concentrations (20–100
l
l
experiments were carried out using cuvettes of 1 cm path length
at room temperature. Samples were excited at 520 nm, and emis-
sion was recorded at 500–750 nm.
42.78; H, 4.06; N, 8.68. Found: C, 42.61; H, 4.50; N, 8.71. IR (KBr, m/
cmꢂ1): 3398
1042 (C–O).
m
(O–H), 3040, 2926
m
(C–H), 1645 m (C@N), 1221,
m
Viscosity measurements were carried out using a capillary vis-
cometer at a constant temperature (23.0 0.1 °C). Flow times were
measured with a digital stopwatch and each sample was measured
three times, and then an average flow time was calculated. Data
are presented as (F/F0)1/3 vs molar ratio of complex to DNA [1],
where F is the viscosity of DNA in the presence of complex and
F0 is the viscosity of DNA in the absence of complex.
Cl
OH
N
N
DNA cleavage
OH
N
N
OH
The cleavage of pBR322 DNA by the complex was examined by
gel electrophoresis experiments. Negative supercoiled pBR322
DNA (0.5
lL, 0.5
l
g/lL) was treated with different concentrations
of complex (1
lL) in Tris–HCl buffer (1
l
L, 50 mM Tris–HCl,
50 mM NaCl, pH = 7.2). After mixing, the mixtures were incubated
at 37 °C for 3 h. The reactions were quenched by the addition of
Cl
Scheme 1. The structure of the macrocyclic ligand (H2L).
sterile solution (1 lL, 0.25% bromophenol blue and 40% w/v

J.-J. Zhou et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 99 (2012) 329–334
331
Table 1
sucrose). The samples were then analyzed by electrophoresis for
Crystal data and structure refinement for the complex.
1 h at 80 V on agarose gel in TAE buffer (40 mM Tris-base,
40 mM acetic acid and 1 mM EDTA, pH = 7.4). The gel was stained
Empirical formula
C23H26Cl2N4O6ZnMn
with EB (1
photographed.
lg/lL) for 0.5 h after electrophoresis and then
Formula weight
Space group
Crystal system
Temperature (K)
a (Å)
645.69
P21/c
Monoclinic
291(2)
10.0293(14)
31.490(4)
9.9344(14)
90
Results and discussions
b (Å)
c (Å)
Synthesis and characterization
a
(deg)
b (deg)
100.353(2)
90
3086.4(7)
4
1.390
1.398
An unsymmetrical macrocyclic heterodinuclear complex was
synthesized by Schiff base condensation of N,N0-bis(3-formyl-5-
chlorosalicylidene)ethylenediimine and 2-hydroxy-1,3-propyldi-
amine in methanol solution containing Zn(OAc)2ꢀ2H2O and
Mn(OAc)2ꢀ4H2O. In the IR spectrum of the complex, the absence
of C@O absorption peak (1680 cmꢂ1) and the presence of C@N
stretching vibration peak (1638 cmꢂ1) indicate that the title com-
plex was formed [12].
c
(deg)
Volume (Å3)
Z
Dcalcd (g/cm3)
Mu(Mo K
F(000)
a) [/mm]
1316
Crystal size [mm]
Mo K
radiation(Å3)
range (deg)
0.22 ꢁ 0.24 ꢁ 0.28
a
0.71073
H
1.3–26.0
21157, 6075, 0.059
4090
6075, 345
0.0602, 0.1224, 1.02
0.00, 0.00
ꢂ0.51, 0.40
Tot., uniq. data, R(int)
Observed data [I > 2.0 sigma(I)]
Nref, Npar
Crystal structure of the complex
R, wR2, S
The perspective view of [ZnMnL(CH3O)2]ꢀH2O is given in Fig. 1,
together with the atom numbering scheme. Crystallographic data
and details about the data collection are presented in Table 1 and
selected bond lengths (Å) and angles (°) relevant to the metal coor-
dination spheres of the complex are listed in Table 2.
Max. and Av. shift/error
Min. and Max. Resd. Dens. [e/Åꢂ3
]
Table 2
Selected bond lengths (Å) and bond angles (°) for the complex.
The molecular structure contains one L, two methanol monoa-
nions and one H2O molecule. In the complex, the coordination
polyhedron around each of the metal ions can be better described
approximately as a square pyramid. The two metal ions are
bridged by the phenoxide groups with Zn–Mn separation of
3.249 Å. In each of the coordination sphere, one oxygen atom from
one methoxyl group occupies the apical position with axial Zn–O
and Mn–O distances of 1.956 and 1.968 Å, respectively, which
are much shorter than the counterparts of similar complexes
[13], indicating the stronger coordination interactions between
methanol monoanions and the metal ions in the title complex.
Each of the base plane is composed of two imino nitrogen atoms
and two phenoxide atoms with mean plane deviations of
0.0132 Å for Zn(II) and 0.0595 Å for Mn(II), respectively. In the base
planes, the average Zn–O (2.087 Å) and Zn–N (2.058 Å) distances
are slightly larger than Mn–O (2.036 Å) and Mn–N (2.050 Å),
respectively, due to the larger radius of Zn(II) compared to that
of Mn(II). The atoms except the carbon atoms in the imino groups
in macrocyclic ligand locate in an approximate plane with a mean
plane derivation of 0.0093 Å. Zn(II) and Mn(II) are in the trans po-
sition on the two sides of the macrocycle with the distances of
Bonds
Length (Å)
Bonds
Length (Å)
Zn(1)–O(1)
Zn(1)–O(2)
Zn(1)–O(4)
Zn(1)–N(3)
Zn(1)–N(4)
2.070(3)
2.103(3)
1.956(3)
2.057(4)
2.059(3)
Mn(1)–O(1)
Mn(1)–O(2)
Mn(1)–O(5)
Mn(1)–N(1)
Mn(1)–N(2)
2.025(3)
2.046(3)
1.968(3)
2.043(4)
2.056(3)
Bond angles
Values (°)
Bond angles
Values (°)
O(1)–Zn (1)–O(2)
O(1)–Zn(1)–O(4)
O(1)–Zn(1)–N(3)
O(1)–Zn(1)–N(4)
O(2)–Zn(1)–O(4)
O(2)–Zn(1)–N(3)
O(2)–Zn(1)–N(4)
O(4)–Zn(1)–N(3)
O(4)–Zn(1)–N(4)
N(3)–Zn(1)–N(4)
O(1)–Mn(1)–O(2)
74.45(12)
99.31(12)
85.60(12)
144.06(13)
99.00(13)
145.70(13)
85.71(14)
111.76(15)
113.40(14)
95.26(15)
76.66(12)
O(1)–Mn (1)–O(5)
O(1)–Mn(1)–N(1)
O(1)–Mn(1)–N(2)
O(2)–Mn(1)–O(5)
O(2)–Mn(1)–N(1)
O(2)–Mn(1)–N(2)
O(5)–Mn(1)–N(1)
O(5)–Mn(1)–N(2)
N(1)–Mn(1)–N(2)
Zn(1)–O(1)–Mn(1)
100.61(13)
141.58(14)
86.69(13)
105.32(13)
85.38(15)
134.96(12)
116.92(14)
118.88(13)
82.44(15)
104.97(14)
0.0006 and 0.0005 Å from the corresponding basal plane, respec-
tively (Fig. 1).
As shown in Fig. 2, hydrogen bonding interactions among the
molecular units play an important role in maintaining the three-
dimensional network of the complex. Each molecular unit is con-
nected with adjacent five molecular units through abundant
hydrogen bonding interactions, involving coordination methanol
monoanions, chlorine atoms and hydroxyl in the macrocyclic li-
gand as well as water molecules. The selected hydrogen bonding
parameters are as follows: dO3ꢀ ꢀ ꢀH8 = 2.51 Å, dO4ꢀ ꢀ ꢀH19 = 2.36 Å,
dO2Wꢀ ꢀ ꢀH23C = 2.44 Å, dCl1ꢀ ꢀ ꢀH2Y = 2.16 Å, dCl2ꢀ ꢀ ꢀH23B = 2.73 Å, \C8–
H8ꢀ ꢀ ꢀO3 = 133,
\C19–H19ꢀ ꢀ ꢀO4 = 171,
\C23–H23Cꢀ ꢀ ꢀO2W = 150,
\O2W–H2Yꢀ ꢀ ꢀCl1 = 133 and \C23–H23Bꢀ ꢀ ꢀCl2 = 135. Besides, there is
also a kind of hydrogen bonding interaction inside the molecular,
that is N–Hꢀ ꢀ ꢀO, its parameters are as follows: dN3ꢀ ꢀ ꢀH3D = 2.49 Å,
\O3–H3Dꢀ ꢀ ꢀN3 = 106.
Absorption spectroscopic studies
Electronic absorption spectroscopy is one of the most useful
methods for DNA-binding studies of metal complexes. The
Fig. 1. Perspective view of the complex, water molecules and hydrogen atoms are
omitted for clarity.

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J.-J. Zhou et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 99 (2012) 329–334
Fig. 4. Plot of [DNA]/(ea
Zn(II)Mn(II) complex.
ꢂ ef) vs [DNA] for absorption titration of CT-DNA with the
the ratio of slope to the intercept from the plot of [DNA]/Eap vs
[DNA] [17]. The Kb value is 2.52 ꢁ 105 Mꢂ1, which is higher than
those of the similar phenoxide bridged homodinuclear complexes
[6]. Considering the similarities in their structures, the higher bind-
ing activity of the title complex with DNA may be contributed to
the three factors: Zn(II)Mn(II) coordinative effects on the binding
with DNA, the hydrogen bonding interactions derived from the hy-
droxyl groups in the macrocyclic ligand with DNA and intercala-
tion interactions between the base pairs in DNA and aromatic
groups in the complex.
Fig. 2. View of the hydrogen bonding interactions in the complex, hydrogen atoms
except those involving in the hydrogen bonding are omitted for clarity.
absorption spectra of the complex in the absence and presence of
calf thymus DNA at different concentrations (8–256
shown in Fig. 3.
lM) are
The spectrum of the complex shows a very strong absorption at
373 nm, which is attributed to a metal-to-ligand charge transfer
(MLCT) [14,15]. Hypochromism in the band reaches as high as
62.5% after adding DNA, indicating that the interaction mode be-
tween CT-DNA and the complex is intercalative [16]. In order to
quantitatively investigate the binding strength of the complex with
CT-DNA, the intrinsic binding constant Kb was obtained by moni-
toring the changes in absorbance at 373 nm for the complex as
increasing concentration of CT-DNA using the equation, [DNA]/
Fluorescence spectroscopic studies
It is well known that DNA solutions are not fluorescent and EB
(ethidium bromide) has weak fluorescent, while the solutions of
DNA with EB show very strong fluorescence emission [18]. In the
experiment, when the Zn(II)Mn(II) complex was added to the EB-
DNA system, the emission intensity was reduced. The emission
spectra of EB bound to DNA in the absence and presence of the
complex are given in Fig. 5. It can be noted that the fluorescence
intensity of the EB-DNA solutions decreases with the addition of
the complex obviously. The results suggest that the complex can
replace the EB and bind to the DNA molecule.
Eap = [DNA]/E + 1/(KbE), where Eap
=
ea
ꢂ
e
f, E = eb
ꢂ
ef, ea, ef and eb
correspond to Aobs/[complex], the extinction coefficients for the
free complex and the complex in the fully bound form, respec-
tively. As is shown in Fig. 4, the value of Kb was obtained from
According to the classical Stern–Volmer equation I0/I = 1 + K[Q],
in which I0 and I are the fluorescence intensities in the absence and
presence of complex, respectively; K is the linear Stern–Volmer
quenching constant and [Q] is the concentration of the complex,
the relative binding propensity of the complex to CT-DNA can
be determined [19]. The fluorescence intensity at 607 nm (kex
=
520 nm) of EB in the bound form was plotted against the com-
pound concentration. The quenching constant K of the complex is
obtained by the slopes of the plot of I0/I vs [Q] in Fig. 6. The calcu-
lated quenching constant K is 4.37 ꢁ 103 Mꢂ1, which is lower than
the binding constant calculated from the absorption experiment.
The reason for this difference may be ascribed to the different spec-
troscopy and different calculation method. The absorption spec-
troscopy study is relative to the metal-to-ligand charge transfer.
Fluorescence spectroscopic study is based on the replacement of
EB by the complex. The changes related to the coordination envi-
ronment of the metal ions in the complex will have some effect
on the results of absorption spectroscopy, which includes coordi-
nation interaction as well as intercalation interaction. So, in our
experimental condition, the binding constant should be larger than
the quenching constant. The quenching constant of the Zn(II)Mn(II)
Fig. 3. UV–visible spectra of the Zn(II)Mn(II) complex in the presence of increasing
amounts of CT-DNA in 50 mM Tris–50 mM NaCl aqueous buffer solution (pH = 7.2).
[M] = 100 lM, [DNA] = 0 (1), 8 (2), 16 (3), 32 (4), 64 (5), 128 (6), 256 (7) lM.

J.-J. Zhou et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 99 (2012) 329–334
333
Fig. 7. Effects of increasing amounts of the Zn(II)Mn(II) complex on the relative
viscosities of CT-DNA at 23 ( 0.1)°C; [DNA] = 100 M, r = [M]/[DNA].
Fig. 5. Emission spectra of EB bound to DNA in the absence and presence of the
Zn(II)Mn(II) complex. [EB] = 10 M, [DNA] = 25 M, [complex] 1–6 = 0, 10, 20, 30,
40, 60 M, kex = 520 nm. Arrow shows the intensity changing upon increasing the
l
l
l
l
complex concentration.
complex is little smaller than those of DNA-intercalative com-
plexes [20], indicating that the Zn(II)Mn(II) complex binds to
DNA via both intercalative mode and coordination interactions.
Viscosity study
Viscosity, sensitive to length increase, is regarded as the one of
the least ambiguous and the most critical tests of binding model
with DNA in solution in the absence of crystallographic structural
data [21]. To clarify the binding mode of the complex with DNA, a
viscosity study of DNA solutions containing varying amount of
added complex was carried out (Fig. 7). The specific viscosity of
the DNA increases obviously with the addition of the increasing
amounts of the complex. The results suggest that the main binding
mode between the complex and DNA may be intercalation [22–
23].
Fig. 8. Cyclic voltammograms of the Zn(II)Mn(II) complex (3 ꢁ 10ꢂ4 M) in the
absence (1) and presence (2) of CT-DNA (3 ꢁ 10ꢂ4 M) in 50 mM Tris–HCl/50 mM
Electrochemical studies
NaCl buffer solution (pH = 7.2), scan rate: 0.1 V sꢂ1
.
The electrochemical properties of the complex were studied by
cyclic voltammetry in 50 mM Tris–HCl/50 mM NaCl buffer solution
Fig. 9. Agarose gel electrophoresis of pBR322 plasmid DNA in the presence of
different concentrations of the complex. DNA (0.5 g/ L) with complex was
incubated for 3 h in 50 mM Tris–HCl/NaCl buffer (pH = 7.2) at 37 °C. Lanes 1–5,
l
l
DNA + complex (12.5
lM, 25 lM, 50 lM, 100 lM, 200 lM), respectively; Lane 6,
DNA control.
(pH = 7.2) using tetrabutyl ammonium perchlorate (TBAP) as sup-
porting electrolyte in the scan range of ꢂ1.2 to ꢂ0.4 V. The cyclic
voltammograms of the complex in the absence and presence of
CT-DNA are shown in Fig. 8. In the absence of DNA, the CV curve
of the complex has a cathodic peak at ꢂ0.752 V and an anodic peak
Fig. 6. Stern–Volmer quenching plots of EB bound to DNA by the Zn(II)Mn(II)
complex. I0 is emission intensity of EB-DNA in the absence of complex; I is emission
intensity of EB-DNA in the presence of complex; [Q] = [complex].
at ꢂ0.869 V ( E = 0.117 V) with E1/2 = ꢂ0.811 V. Upon addition of
D
DNA, both the anodic and cathodic peak potentials shifted to more

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J.-J. Zhou et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 99 (2012) 329–334
Comparing with similar dinuclear nickel complexes, the Zn(II)M-
n(II) complex has higher cleavage activity [27]. We think that the
reason can be classified as follows: the approximately planar struc-
ture of the title complex makes it easier to intercalate into the base
pairs in DNA helix; and the synergetic effect of Zn(II) and Mn(II)
promotes the hydrolysis of the phosphate bonds in DNA.
Fig. 10. Time dependence of the cleavage of pBR322 DNA (0.5 lg/lL) with the
Conclusion
complex after incubation in 50 mM Tris–HCl/NaCl buffer (pH = 7.2) at 37 °C: Lanes
1–5, DNA + complex (100 M) for 1 h, 2 h, 3 h, 4 h, 5 h, Lane 6, DNA control.
l
In conclusion, an unsymmetrical macrocyclic heterodinuclear
complex has been synthesized and structurally characterized.
Hydrogen bonding interactions are the main factors to maintain
the three-dimensional networks of the complex. The title complex
binds to DNA via both intercalative mode and coordination interac-
tions. The approximately planar structure and the synergetic effect
of Zn(II) and Mn(II) of the title complex are contributed to the
higher binding and cleavage activity.
Acknowledgments
Fig. 11. Gel electrophoresis diagram showing the cleavage of pBR322 DNA (0.5
lg/
l
L) by the complex at different scavenging agents in 50 mM Tris–HCl/NaCl buffer
We are grateful to the support from the foundation of National
Nature Science Foundation of China (No. 21171135, 20871097 and
20971102).
(pH = 7.2) and 37 °C for 3 h: Lanes 1–4, DNA + complex (100 lM) + 5 mM EtOH;
DMSO; KI; NaN3. Lane 5, DNA + complex (100 lM); Lane 6, DNA control.
positive values vs a solution without DNA, Epc = ꢂ0.670 V, Epa
=
References
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DNA cleavage
In order to evaluate the function of the complex to incise
pBR322 DNA, the cleavage reaction of supercoiled pBR322 DNA
was monitored by agarose gel electrophoresis. The cleavage prod-
ucts were subjected to gel electrophoretic separation and the gels
analysed after ethidium bromide staining. As shown in Fig. 9, with
increasing concentrations of the complex, the amount of Form I
(supercoiled form) of DNA diminished obviously, whereas Form II
(nicked form) and Form III (linear form) increased. This suggests
that the complex can cleave the DNA effectively. Fig. 10 shows
the time dependence of the cleavage reactions of DNA with the
complex after incubation (pH = 7.2, 37 °C). With the increase of
the incubation time, the cleavage of DNA increased obviously.
In order to further clarify the DNA cleavage mechanism, some
scavenging agents, hydroxyl radical scavengers (5 mM DMSO and
5 mM EtOH), superoxide scavenger (5 mM KI) and singlet oxygen
scavenger (5 mM NaN3), were added in the incubation solution.
No obvious change of DNA cleavage activity of the complex is ob-
served in the presence or absence of the scavenging agents
(Fig. 11). Therefore, DNA cleavage promoted by this complex might
not occur by an oxidative pathway but rather by a hydrolytic
pathway and/or intercalation. This result is consistence with the
deduction from UV–vis and fluorescence spectroscopy studies.