Synthesis, structure, DNA interaction, and hydrolytic function toward bis(p-nitrophenyl) phosphate of a heterobinuclear macrocyclic complex

Article abstract of DOI:10.1007/s11243-012-9600-9

An asymmetrical bis-pyridine pendant-armed macrocyclic heterobinuclear complex, [ZnNiL](ClO₄)₂?CH₃CN (H ₂L was derived from the condensation between 3,3′-((ethane-1,2- diylbis((pyridin-2-ylmethyl)azanediyl))bis(methylene))bis(2-hydroxy-5- methylbenzaldehyde) and 1.3-diaminopropane), has been synthesized and characterized by physico-chemical and spectroscopic methods. The asymmetric unit contains two complete macrocyclic complexes that are nevertheless quite similar to one another. The Zn-Ni separations, bridged by the two phenoxides, are 3.107 and 3.141 A, respectively. The phosphate hydrolysis catalyzed by the complex was investigated using bis(4-nitrophenyl)phosphate (BNPP) as the substrate. The catalytic rate constant (k _cat) is 1.64 × 10^-3 s^-1 at pH 7.4 and 25 °C, which is 10 ⁸-fold higher than that of the corresponding uncatalyzed reaction. The interaction between the complex and calf thymus (CT) DNA was investigated by UV-vis absorption, viscosity experiments, and cyclic voltammetry. The complex shows good binding propensity to calf thymus DNA via intercalation with a binding constant of 5 × 10⁴ M^-1. The agarose gel electrophoresis studies show that the complex has a concentration-dependent DNA cleavage activity.

Full text of DOI:10.1007/s11243-012-9600-9

Transition Met Chem (2012) 37:385–391
DOI 10.1007/s11243-012-9600-9
Synthesis, structure, DNA interaction, and hydrolytic function
toward bis(p-nitrophenyl) phosphate of a heterobinuclear
macrocyclic complex
•
Jia Wei Mao Hong Zhou Yun Feng Chen
•
•
•
Gong Zhen Cheng Zhi Quan Pan
Received: 31 January 2012 / Accepted: 15 March 2012 / Published online: 10 April 2012
Ó Springer Science+Business Media B.V. 2012
Abstract An asymmetrical bis-pyridine pendant-armed
macrocyclic heterobinuclear complex, [ZnNiL]-
The agarose gel electrophoresis studies show that the com-
plex has a concentration-dependent DNA cleavage activity.
(ClO4)2ꢀCH3CN (H2L was derived from the condensation
between 3,30-((ethane-1,2-diylbis((pyridin-2-ylmethyl)aza-
nediyl))bis(methylene))bis(2-hydroxy-5-methylbenzalde-
hyde) and 1.3-diaminopropane), has been synthesized
and characterized by physico-chemical and spectroscopic
methods. The asymmetric unit contains two complete
macrocyclic complexes that are nevertheless quite similar
to one another. The Zn–Ni separations, bridged by the
Introduction
It is known that the metal ions in the active site and the
noncovalent bonding with DNA play major roles in the
highly effective hydrolysis activity of natural phosphatases
[1]. To search for novel restriction enzymes and anticancer
therapeutic agents, much effort has been made in the
synthesis and property investigation of model compounds
for mimicking the hydrolysis activity of natural phospha-
tase. Some macrocyclic binuclear transitional metal com-
plexes are observed to exhibit enhanced DNA-binding and
cleavage activities [2–5]. These complexes interact with
DNA in many modes, such as ionic bonding, hydrogen
bonding, intercalation, hydrolysis, and/or oxidative cleav-
age of phosphate bonds. The key factors dominating the
DNA-binding and cleavage ability of the complexes are the
metal ions and the structures of the ligands [6–8]. The
macrocyclic ligands containing two pyridylmethyl pendant
arms, shown in Scheme 1, are found to be effective mimics
for metalloprotein enzymes [9]. Although the crystal
structures of some mononuclear, homobinuclear, and few
heterobinuclear complexes of such macrocyclic ligands
have been reported, none of their DNA-binding, cleavage,
and phosphatase activity has been reported yet. Herein, a
new asymmetrical macrocyclic heterobinuclear [Zn(II)-
Ni(II)L]2? complex with two pyridylmethyl pendant arms
has been synthesized, and its nuclease activity studied. To
our knowledge, this work provides the first investigation on
the phosphodiesterase activity, DNA-binding and DNA
cleavage ability of this kind of heterobinuclear macrocyclic
complexes.
´
˚
two phenoxides, are 3.107 and 3.141 A, respectively. The
phosphate hydrolysis catalyzed by the complex was
investigated using bis(4-nitrophenyl)phosphate (BNPP)
as the substrate. The catalytic rate constant (kcat) is
1.64 9 10-3 s-1 at pH 7.4 and 25 °C, which is 108-fold
higher than that of the corresponding uncatalyzed reac-
tion. The interaction between the complex and calf thymus
(CT) DNA was investigated by UV–vis absorption, vis-
cosity experiments, and cyclic voltammetry. The complex
shows good binding propensity to calf thymus DNA
via intercalation with a binding constant of 5 9 104 M-1
.
Electronic supplementary material The online version of this
article (doi:10.1007/s11243-012-9600-9) contains supplementary
material, which is available to authorized users.
J. W. Mao ꢀ G. Z. Cheng (&)
College of Chemistry and Molecular Sciences,
Wuhan University, Wuhan, People’s Republic of China
e-mail: gzcheng@whu.edu.cn
J. W. Mao ꢀ H. Zhou ꢀ Y. F. Chen ꢀ Z. Q. Pan (&)
Key Laboratory for Green Chemical Process of Ministry
of Education, Wuhan Institute of Technology, Wuhan,
People’s Republic of China
e-mail: zhiqpan@163.com
123

386
Transition Met Chem (2012) 37:385–391
Preparation of 3,30-((ethane-1,2-diylbis((pyridin-2-
ylmethyl)azanediyl))bis(methylene))bis(2-hydroxy-5-
methylbenzaldehyde) (H2L1)
CH3
N
CH3
O
O
N
N
OH
OH
N
Et3N
N
NH HN
CH2Cl2
To a solution of 3-(bromomethyl)-2-hydroxy-5-methylben-
zaldehyde (5.2 g, 22 mmol) in dry CH2Cl2 (50 mL) was
added Et3N (4.2 g, 40 mmol). To the resultant mixture was
Br OH
O
N
added dropwise
a
solution of N1,N2-bis(pyridin-2-
CH3
ylmethyl)ethane-1,2-diamine (2.5 g, 10 mmol)indryCH2Cl2
(40 mL). The red solution was stirred for 18 h at room tem-
perature. The solution was washed with water (3 9 100 mL),
and then, the solvent was removed from the organic phase
under reduced pressure. To the resultant oily residue was
added HCl (5 M, 50 mL). The mixture was stirred for 10 min
and extracted with CH2Cl2 (4 9 40 mL). The aqueous phase
was adjusted to pH & 7 with aqueous NaOH (8 M) and then
to pH & 8.4 with a saturated NaHCO3 solution. The resultant
mixture was extracted with CH2Cl2 (3 9 50 mL). The com-
bined CH2Cl2 fractions were dried over anhydrous Na2SO4
and filtered. Removal of the CH2Cl2 from the filtrate afforded
a brown oil that was purified bycolumn chromatographyupon
silica gel with CH2Cl2–MeOH (95:5) as eluent and the second
band was collected. Removal of the solvent afforded the
desired product as a yellow solid. Yield 3.2 g (59 % based on
starting materials). 1H NMR (300 MHz, CDCl3) d 11.09 (br s,
2H), 10.19 (s, 2H), 8.54 (d, J = 4.6 Hz, 2H), 7.61 (d, J =
1.1 Hz, 2H), 7.37 (s, 2H), 7.33–7.07 (m, 6H), 3.74 (s, 4H), 3.66
(s, 4H), 2.75 (s, 4H), 2.23 (s, 6H). NMR (75 MHz, CDCl3) d
192.25 (s), 158.64 (s), 157.48 (s), 148.54 (s), 137.18 (s), 136.43
(s), 128.44 (s), 127.74 (s), 124.63 (s), 122.84 (s), 121.92 (d,
J = 13.3 Hz), 58.97 (s), 54.57 (s), 50.12 (s), 19.93 (s).
H2L1
CH3
CH3
2+
N
N
N
O
O
N
N
O
O
N
O
H2N
NH2
Ac)
Zn(O
2
Zn
Zn Ni
O
Ni(ClO4)2
N
N
N
N
CH3
CH3
[ZnNiL]2+
Scheme 1 Synthetic route for the preparation of [ZnNiL]2?
Experimental
All chemicals and solvents were of analytical grade and
used as purchased. N1,N2-bis(pyridin-2-ylmethyl)ethane-
1,2-diamine and 3-(bromomethyl)-2-hydroxy-5-methyl-
benzaldehyde were prepared according to the literature
methods [10, 11]. Tris(hydroxymethyl)amino-methane
(Tris), bromophenol blue, ethidium bromide (EB), agarose
gel, and supercoiled pBR322 DNA were purchased from
Toyobo Co.
IR spectra were measured using KBr pellets on a
vector22 FTIR spectrophotometer. NMR spectra were
obtained on a Varian Mercury VX-300 spectrometer at
30 °C in CDCl3 with TMS as the internal reference.
Elemental analyses were obtained on a Perkin-Elmer 240
analyzer. Electrospray mass spectra (ES-MS) were
determined on a Finnigan LCQ ES-MS mass spectro-
graph using methanol as the mobile phase with an
approximate concentration of 1.0 mmol dm-3. UV–vis
spectra in the 190–700 nm range were recorded on a
Shimadzu UV-2450 spectrophotometer. Cyclic voltam-
mograms were run on a CHI model 750 B electro-
chemical analyzer in DMF solutions containing tetra(n-
butyl)ammonium perchlorate (TBAP) as the supporting
electrolyte. A three-electrode cell equipped with a glassy
carbon working electrode, a platinum wire as the counter
electrode, and a Ag/AgCl reference electrode were used.
Scanning rates were in the range of 20–200 mV s-1. The
solution was deaerated with pure argon gas for 15 min
before measurements.
Preparation of [ZnNiL](ClO4)2ꢀCH3CN
To an acetonitrile (20 mL) solution of H2L1 (0.135 g,
0.25 mmol) was added dropwise
a
solution of
Zn(OAc)2ꢀ2H2O (0.055 g, 0.25 mmol) in ethanol (10 mL).
The mixture was stirred for 1 h, and a solution of 1.3-
diaminopropane in ethanol (10 mL) was added dropwise
over a period of 2 h. The resulting bright yellow solution
was stirred for 1 h at ambient temperature, and then, an
ethanol solution (10 mL) containing Ni(ClO4)2ꢀ6H2O
(0.091 g, 0.25 mmol) was added. After stirring for another
5 h, a green precipitate was collected. Dark green single
crystals suitable for X-ray diffraction analysis were
obtained by evaporation of the acetonitrile solution of the
product. Yield: 0.146 g (62 %). Anal. Calc. for
C37H41N7Cl2ZnNiO10: C, 47.3; H, 4.4; N, 10.4. Found: C,
47.6; H, 4.7; N, 10.1. IR (KBr, cm-1): 3,070, 2,918 (C–H),
1,569 (C = Npyridyl), 1,621 (C = Nimine), 1,091, 625
(ClO-4 1). ES-MS (in MeOH): [ZnNiL]?2
[ZnNiL(C2H3N)(OH)]?, 757.25.
,
349.42;
123

Transition Met Chem (2012) 37:385–391
Crystal structure determination
387
buffer(1/3, v/v) solution at pH = 7.4. Stock solutions were
freshly prepared before performing the kinetic measure-
ment, and the ionic strength was maintained at I = 0.10 M
with potassium chloride.
The crystal was measured on a SMART-CCD area-detector
diffractometer at 291 K using graphite monochromatic Mo
˚
Ka radiation (0.71073 A). Data reduction and cell refine-
ment were performed by SMART and SAINT programs
[12]. The structure was obtained by direct methods (Bruker
SHELXTL) and refined on F2 by full-matrix least squares
(Bruker SHELXTL) using all unique data [13]. The non-H
atoms in the structure were treated as anisotropic. Hydro-
gen atoms were located geometrically and refined in a
riding mode. The summary of the crystallographic data is
listed in Table 1.
DNA-binding experiments
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 less than 4 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 6,600 M-1 cm-1
(260 nm) for CT-DNA [14]. The complex was dissolved in
DMF at a concentration of 5.0 9 10-5 M. The UV
absorption titrations were performed by keeping the con-
centration of the complex fixed while varying the DNA
concentration. Complex-DNA solutions were allowed to
incubate for 30 min at room temperature before measure-
ments were taken. Absorption spectra were recorded using
cuvettes of 1 cm path length at room temperature. The
intrinsic binding constant Kb was calculated according to
the Eq. 1 [15]:
Cleavage of bis(4-nitrophenyl)phosphate (BNPP)
The hydrolysis of BNPP produces 4-nitrophenolate (NP)
and 4-nitrophenyl phosphate (NPP). The kinetic measure-
ments were performed by monitoring NP spectrophoto-
metrically at 25 °C with a wavelength of maximum
absorbance at 406 nm and an extinction coefficient of
15,150 L mol-1 cm-1 under our experimental conditions.
The catalyzed hydrolysis of bis(4-nitrophenyl)phosphate
(BNPP) by the complex was studied in DMF/Tris–HCl
½DNAꢁ=Eap ¼ ½DNAꢁ=E þ 1=ðKbEÞ
ð1Þ
where Eap ¼ ea ꢂ ef; E ¼ eb ꢂ ef; and ea, ef and eb are the
molar extinction coefficients of solutions containing both
complex and DNA, free complex, and the complex bound
to DNA, respectively.
Table 1 Crystal data and details of the structure determination for
the complex
Empirical formula
Formula weight
Crystal system
Space group
C37H41N7Cl2ZnNiO10
938.75
Viscosity measurements were carried out using a capil-
lary viscometer at a constant temperature (25.0 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 versus
molar ratio of complex to DNA [16], where F is the viscosity
of DNA in the presence of complex, and F0 is the viscosity of
DNA in the absence of complex.
Triclinic
P-1
˚
a (A)
12.7826(16)
17.051(2)
20.653(2)
77.385(2)
74.873(3)
68.404(2)
4,002.7(8)
4
˚
b (A)
˚
c (A)
a (°)
b (°)
c (°)
3
˚
DNA cleavage experiments
Volume (A )
Z
D (calc) (g/cm3)
Mu (Moka) (mm)
F(000)
1.555
The cleavage of pBR322 DNA by the complex was
examined by gel electrophoresis experiments. Negative
supercoiled pBR322 DNA (0.5 lL, 0.5 lg/lL) was treated
with different concentrations of the complex (2 lL) in
Tris–HCl buffer (1 lL, 50 mM Tris–HCl, 50 mM NaCl,
pH = 7.2). The mixture solutions were incubated at 37 °C
for 3 h. The reactions were quenched by the addition of
sterile solution (1 lL, 0.25 % bromophenol blue and 40 %
w/v sucrose). The samples were then analyzed by elec-
trophoresis for 0.8 h at 100 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 with EB (1 lg/lL) for
0.5 h after electrophoresis and then photographed.
1.269
1,936
Crystal size (mm)
Temp., K
0.22 9 0.24 9 0.28
291
˚
Mo Ka radiation (A)
h range (°)
0.71073
2.1, 26.0
Nref, Npar
15,445, 1,051
22,481, 15,445, 0.026
10,106
Tot., uniq. data R(int)
Observed data [I [ 2.0 sigma(I)]
R, wR2, S
0.0557, 0.1246, 1.08
-0.62, 0.64
3
Min. and Max. Resd. Dens. [e/A ]
˚
123

388
Transition Met Chem (2012) 37:385–391
˚
Results and discussion
Synthesis and characterization
Table 2 Selected bond distances (A) and angles (°) for the complex
Bond
Distance
Bond
Distance
Zn1–O1
Zn1–O2
Zn1–N1
Zn1–N4
Zn1–N5
Zn1–N6
Ni1–O1
Ni1–O2
Ni1–N2
Ni1–N3
2.436(3)
2.031(3)
2.154(4)
2.177(4)
2.090(4)
2.137(4)
1.856(3)
1.842(3)
1.869(4)
1.855(4)
Zn2–O4
Zn2–O3
Zn2–N10
Zn2–N9
Zn2–N12
Zn2–N11
Ni2–O3
Ni2–O4
Ni2–N7
Ni2–N8
2.372(3)
2.059(3)
2.165(4)
2.181(4)
2.103(4)
2.181(4)
1.863(3)
1.848(3)
1.884(4)
1.897(4)
In the IR spectrum of the complex, the sharp C=N
stretching vibration corresponding to the imine groups of
the macrocyclic framework is observed at 1,621 cm-1
,
indicating that the macrocyclic complex has been synthe-
sized. Strong bands at 1,091 and 625 cm-1 can be ascribed
to the ClO4- anions [17]. The results are in agreement with
the crystal structure.
The ES-MS spectrum of the complex in methanol
solution is dominated by two peaks at m/z 394.42 and
757.25, which are assigned to [ZnNiL]2? (calc. 394.39)
and [ZnNiL(C2H3N)(OH)]? (calc. 756.86), respectively,
indicating that [ZnNiL]2? is very stable in methanol
solution.
Bond
Angles
Bond
Angles
O2–Ni1–O1
O2–Ni1–N3
O1–Ni1–N3
O1–Ni1–N2
O2–Ni1–N2
N3–Ni1–N2
O2–Zn1–N5
O2–Zn1–N6
N5–Zn1–N6
O2–Zn1–N1
N5–Zn1–N1
N6–Zn1–N1
O2–Zn1–N4
N5–Zn1–N4
N6–Zn1–N4
N1–Zn1–N4
O2–Zn1–O1
N5–Zn1–O1
N6–Zn1–O1
N1–Zn1–O1
N4–Zn1–O1
78.70(14)
91.76(16)
169.50(16)
94.15(16)
172.78(16)
95.45(17)
113.32(14)
93.78(13)
95.65(14)
137.29(13)
81.19(15)
125.81(14)
90.59(14)
155.84(14)
78.60(15)
83.34(15)
62.55(12)
78.89(13)
149.83(12)
83.05(12)
117.50(13)
O4–Ni2–O3
O3–Ni2–N7
O4–Ni2–N7
O4–Ni2–N8
O3–Ni2–N8
N7–Ni2–N8
O3–Zn2–N12
O3–Zn2–N11
N12–Zn2–N11
O3–Zn2–N9
N12–Zn2–N9
N11–Zn2–N9
O3–Zn2–N10
N12–Zn2–N10
N11–Zn2–N10
N9–Zn2–N10
O3–Zn2–O4
N12–Zn2–O4
N11–Zn2–O4
N9–Zn2–O4
N10–Zn2–O4
77.22(13)
91.60(15)
168.47(16)
93.65(16)
170.81(15)
97.57(17)
116.05(13)
90.21(13)
96.17(14)
137.48(12)
80.20(14)
128.73(13)
90.41(13)
153.25(14)
78.90(14)
82.61(15)
62.53(12)
79.19(13)
145.33(12)
84.74(12)
119.48(12)
Crystal structure of [ZnNiL](ClO4)2ꢀCH3CN
A perspective view of the complex is illustrated in Fig. 1
with the atom numbering scheme. Selected bond lengths
and angles are listed in Table 2. There are two crystallo-
graphically different molecules in the unit cell (A and
B) with slightly different geometric parameters. The two
molecules have the same macrocyclic unit with little dif-
ferences in relative bond length and angle. Each unit of the
complex shows that the Ni atom resides at the N(imine)2O2
site and the Zn atom at the N(pyridyl)2N(amine)2O2 site
with both pyridyl ligands found on the same side of the
mean molecular plane. Ni-donor bond distances are in the
˚
range of 1.842–1.897 A [18, 19]. The Zn–N distances are
˚
in the range of 2.090–2.181 A and two Zn–O distances are
˚
2.031–2.059 A. These distances are comparable with those
reported in other similar complexes [18–20]. The longer
˚
Zn1–O1 2.436 A and Zn2–O4 2.372 A are due to the weak
˚
coordination interactions [19, 20]. The Zn atom is out of
˚
the approximate N(amine)2O2 plane by 0.936 A (A) and
˚
0.908 A (B) and Ni atom lies almost on the N(imine)2O2
plane in both A and B. The distances between the Zn and
˚
Ni centers are 3.107 and 3.141 A, which are in the range of
˚
appropriate metal–metal distances 3.0–5.4 A of the dinu-
clear complexes for phosphoesterase mimics [21–23].
Therefore, the complex is predicted to have phosphatase
activity.
Fig. 1 The perspective view of the complex. Hydrogen atoms,
perchlorate anions, and acetonitrile molecules have been omitted for
clarity (ellipsoids are drawn at 15 % probability level)
123

Transition Met Chem (2012) 37:385–391
Kinetic study
389
1.0
0.8
0.6
0.4
0.2
0.0
0.12
0.10
0.08
0.06
0.04
0.02
0.00
1
6
BNPP is usually used as a DNA model compound in the
investigation of phosphatase activity. The study of phospha-
tase hydrolysis ability of the complex was carried out in
pH = 7.4 buffer solution in order to mimic corresponding
biological conditions. The initial hydrolysis rate of the com-
plexhasbeendeterminedbyfollowingtheabsorptionincrease
at 406 nm due to the formation of 4-nitrophenolate ion with
time. A plot of initial rate versus substrate concentration is
presentedinFig. 2. Theprofilerevealssaturationkineticswith
Michaelis–Menten-like behavior in the system. The initial
ratesobtainedfor therange ofBNPP concentrationsusedwere
linearized by means of the Lineweaver–Burk method (Fig. 2
inset), giving the kinetic parameters as Vmax = 1.64 9
10-7 mol L-1 s-1, Michaelis constant Km = 2.4 9 10-2
mol L-1, catalytic rate constant kcat (=Vmax/[Complex]) =
1.64 9 10-3 s-1, second-order rate constant kBNPP (=kcat/
KM) = 6.8 9 10-2 mol-1 L s-1. The kcat of the title com-
plex is 350 times higher than that observed for the corre-
sponding binuclear zinc complex at the same pH [24] and
1.5 9 108 times faster than the BNPP uncatalyzed hydrolysis
[25]. It suggests that the title complex has a good phosphate
diester hydrolysis activity. Compared with the similar binu-
clear complexes [8], the larger macrocyclic ring size and the
distorted structure of the title complex are likely to be
responsible for its higher hydrolysis activity.
E
0
20 40 60 80 100 120 140 160 180
[DNA]/10-6
M
300
350
400
450
500
550
600
Wavelength/nm
Fig. 3 Absorption spectral traces of the complex in 50 mM Tris–
HCl-NaCl buffer (pH = 7.2) on increasing the quantity of CT-DNA.
The arrow shows the absorbance changing upon increasing DNA
concentrations. Inset shows the plot of [DNA]/Eap versus [DNA] for
the absorption titration
[26]. The absorption spectra of the Zn(II)Ni(II) complex in
the absence and presence of CT-DNA at different con-
centrations (0–150 lM) are given in Fig. 3. The 12.7 %
hypochromism and a slight red shift at 350 nm indicate that
the interaction mode of the complex with DNA is inter-
calative binding. By monitoring the changes of absorbance
at 360 nm for the complex with increasing concentrations
of CT-DNA, the intrinsic binding constant Kb was obtained
Absorption spectroscopic studies
from the Eq. 1. The calculated Kb value is 5 9 104 M-1
.
Comparing the intrinsic binding constant of this complex
with those of DNA-intercalative macrocyclic metal com-
plexes, the complex interaction with CT-DNA can be
predicted as by moderate intercalation [3, 27].
Electronic absorption spectroscopy is employed to deter-
mine the binding characteristics of metal complexes with
DNA. Intercalative binding of a complex to DNA generally
results in hypochromism along with a red shift of the band
Viscosity study
9
Viscosity, sensitive to volume increases, is regarded as the
one of the least ambiguous and the most critical tests of
binding interactions with DNA in solution in the absence of
crystallographic structural data. To further confirm the
interaction mode between the Zn(II)Ni(II) complex and
DNA, a viscosity study was carried out. As shown in
Fig. 4, the specific viscosity of the DNA increases obvi-
ously with increased concentration of the complex. This
again suggests that the binding mode between the complex
and DNA should be a classical intercalation mode [28, 29].
8
2.2
7
2.0
1.8
1.6
6
1.4
1.2
5
1.0
30 40 50 60 70 80 90 100 110
1/[BNPP] / mol-1
L
4
1.0
1.5
2.0
2.5 3.0
Electrochemical study
[BNPP]/ 10-2M
The electrochemical properties of the complex with
CT-DNA were studied by cyclic voltammetry in 50 mM
Tris–HCl/50 mM NaCl buffer solution (pH = 7.2) using
Fig. 2 Kinetic experiments for the cleavage of BNPP by the complex
in DMF/Tris–HCl buffer (1/3, v/v) solution at pH = 7.4 and 25 °C,
I = 0.10 M (KCl), [Complex] = 10-4
M
123

390
Transition Met Chem (2012) 37:385–391
tetrabutyl ammonium perchlorate (TBAP) as supporting
electrolyte in a sweep range of -1.0 to -0.4 V. The cyclic
voltammograms of the complex in the absence and in the
presence of CT-DNA are shown in Fig. 5. In the absence of
DNA, the cathodic peak potential (Epc) and the anodic peak
potential (Epa) are -0.833 and -0.689 V. Addition of CT-
DNA to the Zn(II)Ni(II) complex results in a decrease in
the cathodic and anodic peak currents, and both the anodic
and cathodic peak potentials shift to more positive value
with Epc = -0.813 V and Epa = -0.601 V. These results
could be explained by a reduction in the apparent diffusion
coefficient of the complex on binding to DNA and again
indicate that an intercalative interaction occurred between
the complex and DNA [30, 31]. The larger negative
potential (Epc = -0.813 V at 100 mV/s) of the reduction
peak reveals that the metal ions in the complex are not
easily reduced in the Tris–HCl buffer solution (pH = 7.2),
which is a supplementary evidence that the DNA cleavage
by the complex may not be an oxidation mode.
1.05
1.04
0
F
F
1.03
1.02
DNA cleavage
0.2
0.4
0.6
0.8
1.0
1.2
[C]/[DNA]
In order to evaluate the function of the complex to incise
DNA, the cleavage reaction of plasmid pBR 322 DNA by
the complex was monitored by agarose gel electrophoresis.
The DNA-cleaving ability was investigated with various
concentrations and incubation time intervals (Fig. 6). The
results show that this complex can transform the Form I
(supercoiled form) to Form II (nicked form) obviously. The
transformation from Form I to Form II at the concentration
of 200 lM is about 36 %. Furthermore, 7 % transforma-
tion was observed at the lower concentration of 25 lM.
The cleavage activity of the complex increased on both
increasing the concentration of the complex and the incu-
bation time.
Fig. 4 Effects of increasing amounts of the complex on the relative
viscosities of CT-DNA at 25.0 ( 0.1) °C; [DNA] = 30 lM
a
3
b
2
1
0
-1
-2
Conclusions
In summary, we prepared and structurally characterized a
new asymmetrical bis-pyridine pendant-armed macrocyclic
heterobinuclear Zn(II)Ni(II) complex. The complex is very
stable in acetonitrile solution and showed high phosphate
diester hydrolysis activity. The investigations of UV
absorption, viscosity, electrochemical studies, and agarose
-1.4
-1.2
-1.0
-0.8
-0.6
-0.4
-0.2
0.0
0.2
E/V
Fig. 5 Cyclic voltammograms of the complex (1 9 10-4 M) in the
absence (a) and presence (b) of CT-DNA (1.6 9 10-5 M) in 50 mM
Tris–HCl/50 mM NaCl buffer solution (pH = 7.2), scan rate: 0.1 V/s
B
A
Form I
Form II
1
2
3
4
5
1
2
3
4
5
Fig. 6 Agarose gel electrophoresis of pBR322 plasmid DNA in the
presence of different concentrations of the complex. DNA (0.5 lL) in
Tris–HCl buffer (1 lL, 50 mM Tris-50 mM NaCl, pH = 7.2) at
37 °C a in different complex concentration (incubation time 3 h).
Lane 1 DNA control; Lane 2–5 DNA ? complex(25, 50, 100, and
200 lM); b in different incubation time: Lane 1 DNA control; Lane
2–5 DNA ? complex (200 lM) for 0.5, 1, 1.5, 2 h
123

Transition Met Chem (2012) 37:385–391
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Crystallographic data have been deposited with the Cam-
bridge Crystallographic Data Center, under deposition
number CCDC 857556 for the complex [ZnNiL](ClO4)2ꢀ
CH3CN. Copy of the data can be obtained free of
charge on application to The Director, CCDC, 12 Union
Road, Cambridge CB2, 1EZ, UK (fax: ?44-1223-336033);
E-mail:deposit@ccdc.cam.ac.uk or http://www.ccdc.cam.ac.uk).
Acknowledgments We are thankful for the financial support from
the National Nature Science Foundation of China (20971102 and
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