Synthesis of heterobinuclear Cu(Ⅱ)-Ni(Ⅱ) complex: Structure, CT-DNA interaction, hydrolytic function and antibacterial studies

Article abstract of DOI:10.1016/j.molstruc.2019.06.081

A new benzyls pendant-armed macrobicyclic heterbinuclear Cu(Ⅱ)-Ni(Ⅱ) complex has been obtained by template-directed synthesis and characterized by elemental analysis, IR spectra, electrospray mass spectra, and single crystal X-ray diffraction. The complex was bridged by two phenolic oxygens and an acetate radical, with the Cu(Ⅱ)-Ni(Ⅱ) distance of 2.9292(8) ?. The hydrolytic function, CT-DNA binding and antibacterial properties were also studied. The initial rate values for the hydrolysis of 4-nitophenylphosphate to 4-nitrophenolate by the Cu(Ⅱ)-Ni(Ⅱ) complex was 1.33 × 10^?5 s^?1, and 10⁴ times faster than that the spontaneous hydrolysis of the phosphate monoester. The complex shows a better binding property to CT-DNA and the intrinsic binding constant is 1.29 × 10⁵ M^?1. The Stern-Volmer constant is 1.25 × 10⁵ M^?1. The viscosity increased obviously with the increase of complex concentration, the results showed that the complex bind to DNA through intercalation mode, which was in accordance with the absorption and emission spectral studies. The antibacterial activities against E.coli was also investigated using the Gentamycinas reference system.

Full text of DOI:10.1016/j.molstruc.2019.06.081

Accepted Manuscript
Synthesis of heterobinuclear Cu(Ⅱ)-Ni(Ⅱ) complex: structure, CT-DNA interaction,
hydrolytic function and antibacterial studies
Peipei Ding, Yang Wang, Huizhi Kou, Jianfen Li, Baoxian Shi
PII:
S0022-2860(19)30804-X
10.1016/j.molstruc.2019.06.081
MOLSTR 26723
DOI:
Reference:
To appear in:
Journal of Molecular Structure
Received Date:
Accepted Date:
11 April 2019
25 June 2019
Please cite this article as: Peipei Ding, Yang Wang, Huizhi Kou, Jianfen Li, Baoxian Shi, Synthesis
of heterobinuclear Cu(Ⅱ)-Ni(Ⅱ) complex: structure, CT-DNA interaction, hydrolytic function and
antibacterial studies, Journal of Molecular Structure (2019), doi: 10.1016/j.molstruc.2019.06.081
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ACCEPTED MANUSCRIPT
Synthesis of heterobinuclear Cu(Ⅱ)-Ni(Ⅱ)
complex: structure, CT-DNA interaction,
hydrolytic function and antibacterial studies
Peipei Ding, Yang Wang, Huizhi Kou, Jianfen Li, Baoxian Shi*ꢀ
School of Chemical and Environmental Engineering, Wuhan Polytechnic University,
Wuhan 430023, China
Abstract
A new benzyls pendant-armed macrobicyclic heterbinuclear Cu(Ⅱ)-Ni(Ⅱ)
complex has been obtained by template-directed synthesis and characterized by
elemental analysis, IR spectra, electrospray mass spectra, and single crystal X-ray
diffraction. The complex was bridged by two phenolic oxygens and an acetate radical,
with the Cu(Ⅱ)-Ni(Ⅱ) distance of 2.9292(8) Å. The hydrolytic function, CT-DNA
binding and antibacterial properties were also studied. The initial rate values for the
hydrolysis of 4-nitophenylphosphate to 4-nitrophenolate by the Cu(Ⅱ )-Ni(Ⅱ)
complex was 1.33×10-5 s-1, and 104 times faster than that the spontaneous hydrolysis
of the phosphate monoester. The complex shows a better binding property to
CT-DNA and the intrinsic binding constant is 1.29×105 M-1. The Stern-Volmer
constant is 1.25×105 M-1. The viscosity increased obviously with the increase of
complex concentration, the results showed that the complex bind to DNA through
intercalation mode, which was in accordance with the absorption and emission
spectral studies. The antibacterial activities against E.coli was also investigated using
the Gentamycinas reference system.
Keywords: heterbinuclear complex, X-ray diffraction, hydrolytic function, CT-DNA
binding, antibacterial activities
Introduction
It’s well known that DNA is the primary target of anticancer drugs due to it plays
* Corresponding author: E-mail address:wyydcc@163.com

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a major role in replication, transcription and regulation of genes [1], so changing the
replication of DNA and inhibiting the growth of tumor cells through the drugs binding
to DNA is the basis of designing and synthesis more effective antitumor agents. For
DNA activity, the most important step is DNA binding affinity with complexes.
Generally speaking, the macrocyclic metal complexes can bind to DNA through
intercalative, outside groove binding and outside stacking [2]. Heterobinuclear metal
complexes are of special interest for its proximity relevant to the active sites of
bimetallic enzymes mimic the catalytic activity. To design more effective complexes,
the choice of the ligand structure and metal ion are the key factors.
Amounts of researches have been done on the coordination chemistry of transition
metal complexes with schiff base ligands due to its numerous applications in the area
of treatment of cancer [3], antibactericide agents [4], antivirus agents [5], fungicide
agents [6], catalysis [7] and other biological properties [8]. The substitutes and
grafting on pendant arms of the ligand have been found to enhance the recognition
and complexation properties. Aromatic rings can enhance the complex planarity,
hydrophobicity and donor property which response for the affinity between the
complex and DNA [9]. Although copper(Ⅱ ) complexes possess biologically
accessible hydrolytically potentials and high nucleobase affinity [10], and nickel
complexes play an important role in bioinorganic chemistry and may provide the basis
for the active sites models of biological systems [11], the copper(Ⅱ) and nickel(Ⅱ)
heterobinulear complex containing two benzyls pendant arms is rarely reported.
In view of all these facts, we herein report the synthesis of a new copper(Ⅱ)-nickel
(Ⅱ) unsymmetrical complex of salicylaldehyde Shiff bases, spectral characterization,
phosphate-hydrolysis, antibacterial properties, DNA binding studies. The synthesis
route was shown in scheme 1.

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CH3
H2N
NH2
NaBH4
N
H
N
N
N
H
O
CHO
CHO
OH
OH
N
N
Et3N
CH3
OH
CH3
CH2Cl2
70.C, reflux
H2SO4
HBr
(HCHO)n
OH
O
Br
O
CH3
H2L
CH3
CH3
CHO
OH
1. Cu(OAc)2
2.
N
O
O
N
N
N
H2N
NH2
Cu
Ni
3. Ni(ClO4)2
N
N
OH
CHO
CH3
CH3
Scheme 1 Synthesis of H2L and the title complex
Experimental
Materials
All chemicals were purchased from commercial sources, CH2Cl2, THF, DMSO,
DMF and CH3OH were purified according to literature [12]. Escherichia coli ATCC
25922 was supplied by Tongji Medical College of HUST. Nickel(II) Perchlorate,
Agarose gel and Calf thymus DNA were purchased from Sigma.
Physical measurements
The FT-IR spectra were performed on a Perkin Elmer FT-IR spectrometer with
samples prepared as KBr disk. Elemental analyses were recorded on a Perkin-Elmer
240 analyzer. Electrospray mass spectra (ES-MS) were done on a Finnigan LCQ
ES-MS mass spectrograph using methanol as the mobile phase with a sample
concentration of about 1.0 mmol·L-1. Fluorescence spectra were recorded on a Jasco
FP-6500 spectrophotometer. UV–Visible spectra were obtained on a Shimadzu
UV-2450 spectrophotometer using cuvette of 1cm path length. Viscosity experiments
were performed on an Ubbelohde viscometer.
The crystallographic data were summarized on a SMART-CCD area-detector
diffractometer at 291 K using graphite-monochromated Mo-Kα radiation (λ=0.71073
Å). Data reduction and cell refinement were performed by SMAET and SAINT
programs. The structures were solved by direct methods (Bruker SHELXTL) and

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refined on F2 by full-matrix least-squares (Bruker SHELXTL) using all unique data
[13]. Hydrogen atoms in the structure were located geometrically and refined in riding
mode. The non-H atoms were refined with anisotropic displacement parameters.
Preparation of 3,3’- (ethane-1,2-diyl bis(benzylazanediyl)) bis(methylene) bis
(2-hydroxy-5-methyl-benzaldehyde)(H2L)
The ligand, H2L, was prepared according to the literature [14]. A solution of
N1,N2-dibenzylethane-1,2-diamine(0.025 mol) in the THF (200 mL) was added to the
mixture of 3-(bromomethyl)-2-hydroxy-5- methylbenzaldehyde (13.0 g, 0.055 mol)
and K2CO3 (20.7 g, 0.15 mol) in the THF(150 mL) by dropwise. The resulting
solution was stirred overnight at room temperature and then filtered. The precipitate
was collected and acidified with an aqueous solution of 32% HCl (15 mL). The
mixture was then extracted with methylene chloride (3×150 mL). By adding a
saturated solution of NaHCO3, the obtained aqueous phase was made alkaline and
then extracted with methylene chloride (3×150 mL) again. After dring over with
anhydrous Na2SO4, filtrated and evaporated, the product (H2L) was obtained by
recrystallization from acetone as white solid (4.85 g, yield: 36.2%). 1H NMR (400Hz,
CDCl3), 10.16 (s, 2H), 7.17-7.35 (m, 14H), 3.59 (s,4H), 3.51 (s, 4H), 2.65 (s,4H).
Synthesis of the complex[CuNiL(μ- OAc)2]ClO4
To the solution of the ligand (0.107 g, 0.20 mmol) resolved in 10mL absolute
methanol, a solution of copper acetate (0.045 g, 0.20 mmol) was added dropwise.
After stirring for 5 h in room temperature, the solution turned grass green suspension,
to which a methanolic solution (10 mL) of 1,3-propanediamino (0.012 g, 0.2 mmol)
was added slowly and stirred for another 5h when the suspension was turned to dark
green. Then the nickel(Ⅱ) perchlorate hexahydrate (0.2 mmol, 0.0677 g) resolved in
10 mL anhydrous methanol was added to above solution, the mixture was stirred for 5
hours resulted in green solution. After filter, the tea green crystals which suitable for
the X-ray measurement were collected by slowly evaporation of the clarify filtrate at
room temperature for one week. (yield: 0.151 g, 56%). Anal. found (%): C, 55.6; H,
5.9; N, 7.2. calcd.(%): C 55.5; H 6.0 ; N 7.3. IR (KBr, ν/cm-1): 1626 ν(C=N), 1107,
621 ν(ClO-4), 1402, 1580, 1313 ν(OAc-).

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Antimicrobial assay
The antimicrobial activities of the synthesized macrocyclic complex against
Gram-negative (E.coli) bacteria were studied by agar well diffusion method [15]. All
the microorganisms were incubated at 37 ℃ for 24 h in Nutrient broth. The
microbial cultures were prepared and adjusted by comparing against 0.5 McFarland
turbidity standard tubes.
The nutrient agar and other experiment materials were sterilized in high
temperature sterilizing oven for 30 min at 120 ℃ and then cooled to room
temperature. For preparation of disks and inoculation, 2 mL of bacterial culture
suspensions were added to 100 mL nutrient agar mentioned above which were heated
to melting and mixed. Then the Agar was poured into the sterilized Petri dish and
allowed to solidify. Samples dissolved in DMSO were filled into the wells which
were cut in agar plates using proper sterile tubes. The plates were plaque covered and
incubated for 24 h at 37 ℃ for the bacteria. At the end of the incubation period,
inhibition zones formed on the medium were evaluated in mm. The inhibition zones
were compared with those of reference discs. All determinations were done in three
replicates.
Cleavage of 4-nitrophenyl phosphate disodium salt hexahydrate (pNPP)
experiments
The hydrolysis of pNPP produces 4-nitrophenolate(4N) which is the chromophoric
leaving group. The kinetic measurements were investigated spectrophotometrically by
monitoring 4N increase at 25℃ in a wavelength of maximum absorbance at 420 nm
in DMF/Tris–HCl buffer (2:3, v:v) solution at pH = 7.2 as a function of regular time
intervals.
DNA binding experiments
Absorption spectral studies
The calf thymus(CT-DNA) was dissolved in Tris-HCl buffer(100 mL 50 mM
Tris-HCl, 50 mM NaCl, pH=7.2) and kept at 4℃ for less than 4 d. The ratio of UV
absorbance at 260 nm and 280 nm was in the range of 1.8-2.0 indicating that the
CT-DNA was free from protein. The DNA concentration per nucleotide was

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determined using the molar absorption coefficient 6600 M-1 cm-1 at 260 nm for
CT-DNA [16]. The metal complex solution was prepared by using 5% DMF in
Tris-HCl/NaCl buffer and then diluting to the required concentration(50 µM) for all
the experiments. Absorption experiments of the Cu(Ⅱ ) Ni(Ⅱ ) complex in buffer
were carried out by keeping the complex constant (50 µM) while increasing the DNA
concentration (0-100 µM). Before measurement was taken, the complex-DNA
solution was incubated for 30 minutes at room temperature.
Fluorescence spectral studies
To further clarify the binding strength of complex with DNA, fluorescence
quenching experiments were performed by adding the complex of different
concentration (0-60 µM) into EB-bound CT-DNA solution. All samples were excited
at 520 nm, and emission spectra was recorded at 540~750 nm.
Viscosity measurements
A capillary viscometer was used to conduct the viscosity experiment in a
thermostatic water bath maintained at 25 ℃. The desired concentrations of the DNA
and complex were prepared by using Tris-HCl/NaCl buffer (pH=7.2). The flow time
was recorded with a digital stopwatch.
Results and discussion
Synthesis and characterization
An unsymmetrical macrocyclic heterbinuclear Cu(Ⅱ) Ni(Ⅱ) complex was
synthesized by condensation of 3,3′ - (ethane-1,2-diylbis(benzylazanediyl)) bis
(methylene) bis (2-hydroxy-5-methyl-benzaldehyde) with 1,3-propanediamine in the
presence of Cu(OAc)2·4H2O and Ni(ClO4)2·6H2O. The synthetic pathway for the
complex is shown in Scheme 1.
IR spectra
Valuable information about the coordination behavior of the ligand with the metal
ion can be obtained from the IR spectrum. Compared with the free ligand, IR
spectrum of the complex can reveal the changes that might have taken place upon
complexation. In the IR spectrum of the complex, the absence of the stretching
vibrations at 1678 cm-1 υ(C=O) and the presence of a strong band at 1621 cm-1 υ

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(C=N) supported the formation of the Schiff base macrocyclic complex and the
aldehyde groups has been converted into imine groups [17]. The ligand exhibits the
peak 1219 cm-1 due to υ(C-O）of phenolic group is shifted to a higher frequency in
1255 cm-1 upon complexation, indicating the coordination via phenoxide atom [18].
The other peaks at 1090 cm-1 and 623 cm-1 can be ascribed to the perchlorate ion. In
addition, the sharp peak around 1090 cm-1, which is split, clearly explains the
presence of an uncoordinated perchlorate ion [19]. The strong band at 1576 and 1437
cm-1 may reasonably be assigned to the stretching vibration of carboxylate. The Δ υ
value (υas (coo-1) -υa(coo-1))(139 cm-1) separation between the two highest frequency
bands of coordinated carboxyl falls in the range of 130-200 cm-1 , suggested bidentate
bridged coordination of -COO- of the complex, also in agree with the crystal structure
of the complex [20]. Compared to the ligand, some new bands with weak intensity
observed in for the metal complex at 630-500 cm-1 and 480-460 cm-1 can be assigned
to υ(M-O) and υ(M-N) vibration modes respectively [21], which gave conclusive
evidence on the coordinate of phenolic oxygen and azomethine nitrogen of ligand
with metal ions.
ES-MS spectra electrospray mass spectrometry
In order to further characterize the proposed macrocyclic complex, ES-MS was
used. The ES-MS spectra of the complex in methanol solution was given in Figure 1.
The expected dominate molecular ion is m/z 752.17, this chromatographic behaviour
strongly suggests that perchlorate ion dissociate from [CuNi(OAc)L2+]+. No other
fragment peak was observed indicating the stability of the macrocyclic cation in the
methanol solution. The results are consistent with the theoretical and experiment
isotope distributions.

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Figure 1. The ES-MS spectrum of complex
Crystal structure
The crystallographic data and structural refinement data of the complex are listed in
Table 1, selected bond lengths and angles which relevant to the mental coordination
spheres of the complex are showed in Table 2. The crystal structure and hydrogen
bonding stack diagram are presented in Figure 2 and Figure 3 with part of the atom
numbering scheme.
The molecular structure of the complex of just like a winged butterfly shape,
while the four phenyls display the pair of wings. The two metal ions were located in
different coordination environments. One side, the Cu(Ⅱ) center is penta-coordinated,
which is composed of two phenoxo oxgen(O1、 O2), acetate oxygen O3, and the
amine nitrogen(N3、 N4) from the Schiff base macrocyclic, while the Ni(Ⅱ ) was
coordinated with two phenoxo oxgen(O1、 O2), acetate oxygen O4, and the imine
nitrogen (N1、N2) from the Schiff base macrocyclic to give five-coordinate distorted
pyramid configuration on the other side. The nickel ion is placed in a
square-pyramidal environment also with the apical bond length very close to the basal
ones, Ni-O4=2.029(3) Å, Ni-N1=2.032(3) Å, Ni-N2=2.046(4) Å, Ni-O1=1.993(3) Å,
Ni-O2=1.963(3) Å respectively, shorter than that of analogues[14,17]. The copper ion
also can be described as pyramidal but displayed more distorted with the apical bond
length larger than the basal ones, Cu-O3=2.244(3) Å, Cu-O1=1.960(3) Å,
Cu-O2=1.931(3) Å, Cu-N3 =1.978(3) Å, Cu-N4 =1.937(4) Å. The longer Cu-O3 than

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Ni-O4 is due to the weak coordination interactions. The distance of Cu-Ni
endogenous bridge formed by two phenolic oxygens is 2.929(8) Å, which is shorter
than the values foumd in similar dinuclear complexes[22]. The distance is in the range
of appropriate metal-metal distances of the dinuclear complexes for
phosphomonoesterase mimics [23-25]. In the hydrolysis of pNPP, the bimetallic ion
binds to two oxygen atoms of the substrate and promotes the bridged hydroxyl group
to attack the P atom as the nucleophilic reagent [26]. Therefore, this complex with
shorter metal-metal distance than that of analogues [27], are more suitable for
coordination with phosphate monoesters with smaller steric hindrance but greater
alkalinity. The Figure.3 revealed the presence of intermolecular C-H…O interactions
among the two dinuclear complex which are interconnected. In theses interactions, the
perchlorate anions play a key role in the three-dimensional structure of the complex.
The perchlorate anion oxygen O7 as hydrogen-bond acceptors while C16, C25, C35
atoms act as the H bond donor in each macrocyclic unit of the complex. The three
dimensional packing structure are formed through these hydrogen bonds. Details of
the H-bonds are listed in Table 3.
Figure 2. The ORTEP diagram of the complex with 50% ellipsoid probability (all hydrogen
atoms are omitted for clarity)

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Figure 3. The hydrogen bond interactions in intermolecular
Table 1 Crystal data and details of the structure for complex
Empirical
Formula weight
Crystal system
Space group
a,Å
C39H43ClCuN4NiO8
853.47 g/mol
Monoclinic
P 2(1)/n
10.9166(14)
13.2774(2)
27.290(4)
92.53(0)
b, Å
c, Å
β,0
Volume(Å 3)
Z
3951.66(99)
4
D(calc)(g/cm3)
Mu(Moka)[/nm]
F(000)
1.43447
1.138
1772
Crystal size [mm]
0.16x 0.14 x 0.13
Temp.,K
273
Mo Ka radiation(Å3)
Range(deg)
0.71073
2.14,25.5
Nref, Npar
7332,500
Independent reflections(Rint)
Observed data [I＞2.0sigma(I)]
w
7452(0.062)
4109
1/[s2(Fo2)+(0.0745P)2+2.9083P]

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P=(Fo2+2Fc2)/3
R,wR2,S
Min. and Max. Resd. Dens.[e/ Å 3]
0.053,0.1556,1.061
-0.71,0.578
Table 2 Selected bond distances (Å) and angles (º) for complex
Bonds
Distance
1.931(3)
Bonds
Distance
1.963(3)
Cu1-O2
Ni1-O2
Cu1-N4
1.937(4)
Ni1-O1
1.993(3)
Cu1-O1
1.960(3)
Ni1-O4
2.029(3)
Cu1-O3
2.244(3)
Ni1-N1
2.032(3)
Cu1-N3
1.978(3)
Ni1-N2
2.046(4)
Bond
Angle
Bond
Angle
O2-Cu1-N4
O2-Cu1-O1
N4-Cu1-O1
O2-Cu1-N3
N4-Cu1-N3
O1 -Cu1-N3
O2-Cu1-O3
N4-Cu1-O3
O1-Cu1-O3
N3-Cu1-O3
O2-Cu1-Ni1
N4-Cu1-Ni1
O1-Cu1-Ni1
N3-Cu1-Ni1
O3-Cu1-Ni1
169.21(14)
77.95(12)
92.00(14)
89.63(14)
99.25(15)
162.19(14)
94.46(13)
89.58(15)
90.93(13)
102.84(14)
41.61(8)
O2-Ni1-O1
O2-Ni1-O4
O1-Ni1-O4
O2-Ni1-N1
O1-Ni1-N1
O4-Ni1-N1
O2-Ni1-N2
O1-Ni1-N2
O4-Ni1-N2
N1-Ni1-N2
O2-Ni1-Cu1
O1-Ni1-Cu1
O4-Ni1-Cu1
N1-Ni1-Cu1
N2-Ni1-Cu1
76.44(12)
98.42(13)
100.54(13)
160.46(14)
94.95(13)
100.41(13)
91.33(13)
156.22(13)
101.40(14)
90.03(14)
40.79(9)
130.69(11)
42.63(8)
41.76(8)
85.14(9)
129.76(11)
76.09(9)
136.16(11)
131.86(10)
Table 3 Selected hydrogen-bonding distances (Å) and angles (º)
D-H…A
d(D-H)
0.9700
0.9700
0.9700
d(H…A)
2.42
d(D…A)
3.389(7)
3.507(8)
3.202(7)
<(DHA)
173.700
168.300
127.700
C(16)-H(16A)…O(7)#1
C(25)-H(25A)…O(7)#1
C(35)-H(35A)…O(7)#2
2.55
2.52
Symmetry transformations used to generate equivalent atoms
#1= 1/2-x,-1/2+y,1/2-z, #2= 1/2+x,1/2-y,-1/2+z
Antibacterial activity

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For in vitro, the zone of growth inhibition against E. coli with zone reader was
determined the antibacterial activities of the compound. An inhibition zone diameter
over 7 mm indicates that the tested compound is active against the bacteria under
investigation [28]. The inhibitory activity of media with only solvent DMSO and
media with only Gentamycin (standard antibiotic drug) as controls were also tested
[29]. The screening results of the negative control which medium with only solvent,
the positive control whereas media with Gentamycin, and the metal complex for
antibacterial were shown in Table 4. It shows that the activity of the complex against
E. coli was increased with the growing concentration of the complex, which is higher
than the ligand, but lower than the standard drug that of Gentamycin. The chelation
theory and the permeability of the cell of the microbes can explain the difference of
the antibacterial activity. According to Overston’s concept of cell permeability [30],
the lipid membrane surrounding the cell favors the passage of only lipid soluble
materials. Therefore the liposolubility is an important factor that influence the
antibacterial activity. On chelation [31], due to the partial sharing of its positive
charge with the donor groups in the macrocyclic chelate ring system, the polarity of
the metal ion was reduced. Further, the chelation increases the delocalization of π
-electrons over the whole chelating ring which increase the lipophilic nature, and
enhance the penetration of the complex into the lipid layer of the microorganism.
Table 4 The diameters of inhibition against E.coli (mm)
concentration
compounds
2mM
1mM
10
complex
ligand
12
11
10
Gentamycin
DMSO
14
13
8
8
The catalyzed hydrolysis of pNPP
This assay mainly discusses the influence of medium concentration in the range of
5×10-3 to 1×10-3 M at pH 7.2 on the hydrolysis rate of the pNPP. From the equation(1),
the rate of hydrolysis can be expressed as the concentration of reactants decreases or
production increases with the time increasing as follow:

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d[S] d[NP]
V  

(1)
dt
dt
According to the Lambert-Beer, the relationship of absorbance and the concentration
just as below:
A  bc(b 1cm)
The concentration change of 4-nitrophenolate (4N) which is the product of the
substrate can be expressed as:
1
d[NP]  dA

So the hydrolysis rate is：
d[NP] 1 dA
V 

dt
 dt
The molar extinction coefficient of 4N has been measured which equal to 14975
mol·L-1·cm-1 at 420 nm. The rate of hydrolysis has been measured from the ration
of slop from the plot of -In(A - At) versus time (Figure. 4) by initial rate method.
2.3
2.2
2.1
2.0
1.9
1.8
200
300
400
500
600
Time(second)
Figure 4. The kinetics study of the complex reacts with pNPP as times change
From Figure. 4, the ability of the systems to hydrolyze pNNP is described by the
slope and equal to 1.17×10-4 s-1 at pH=7.2. The initial rate is higher than the similar
structure of mononuclear Cu and Ni complex [17,32]. The difference mainly assign to
the distorted structure and the mental-mental synergic combination of the complex.

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Figure 5. The hydrolysis rate of complex changes with the concentration of pNPP, [complex] =
200 µM. Inset shows the plot of Lineweaver-Burk.
A plot of initial rate versus substrate concentration is shown in Figure 5. The
variation of hydrolysis rate dependents on the concentration of substrate, the reaction
rate is almost linearly changed when the substrate phosphate concentration is low,
while with the increase of substrate phosphate concentration, the reaction rate
changed from linear to saturation curve. The profile indicates the kinetic model is in
great agreement with Michaelis-Menten-like behavior, typical for native
metalloenzymes in the system. The initial rates obtained in for the range of pNPP
concentrations used were linearized by means of the Lineweaver–Burk method (inset
Figure 5)
1
K
m
1
1


(2)
V
V max [S] V max
From the equation (2), the kinetic parameters were determined and given as follows:
Vmax = 2.65×10-9 mol· L-1 ·s-1, Michaelis constant KM = 1.35×10-2 mol·L-1, the
catalytic rate constant kcat = Vmax/[Complex]) =1.33×10-5 s-1, second-order rate
constant kNPP (= kcat / KM) = 9.85×10-4 mol-1 L s-1. Compared with the pNPP
uncatalyzed hydrolysis rate at pH 7.2 of 2×10-9 s-1 [33], the title complex at the same
pH provides about four orders of magnitude rate acceleration for hydrolysis of
phosphate ester. The reactivity of complex is higher than the other similar binuclear
systems may due to the macrocyclic ring size decreasing and the distorted structure of
the title complex [34].
DNA binding activity

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Absorption spectral studies
Electronic absorption spectroscopy is an effect way to investigate the binding
characteristics of metal complex with DNA. In general, intercalative binding of a
complex to DNA results in hypochromism and red shift due to a strong stacking
interaction between the aromatic chromophore of ligand and the base pairs of DNA
and the extent of hypochromism gives a measure of the intercalative binding strength
[35]. The UV-vis absorption spectra of Cu(Ⅱ) Ni(Ⅱ) complex in the absence and
presence of increasing amounts of CT-DNA(0-100 µM) are shown in Figure 6.
Figure 6. Absorption spectra of the Cu(Ⅱ)Ni(Ⅱ) complex in the absence and presence of
increasing amounts of CT-DNA(0~100 µM) at 25℃ in 50 mM Tris-HCl/NaCl, pH=7.2. The arrow
shows the absorbance changing upon increasing the DNA concentrations. Inset shows the plot of
[DNA]/Eap vs [DNA] for absorption titration of CT-DNA with complex.
By monitoring the changes of absorbance at 355 nm, upon increasing the
concentration of DNA to the complex, great hypochromism (27.1%) together with
large bathochromic shift were observed which indicated that the interaction mode of
the complex with DNA is typical intercalative binding. The intrinsic binding constant
(Kb) can be calculated from the equation 3 [36]:
[DNA]/Eap= [DNA]/E+1/KbE
(3)
where [DNA] is the DNA concentration in M (per nucleotide), Eap= εa-εf, E = εb-εf,
and εa,εb and εf are the molar extinction coefficients of solutions containing both
complex and DNA, free complex, and the complex fully bounded to DNA,

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respectively. Kb is the intrinsic binding constant. Absorption curves were established
for from the change in the absorption intensity as a function of [DNA]. Each set of
data was fitted to the above equation. The intrinsic binding constant Kb was obtained
from the ratio of slop to intercept of the plot of [DNA]/Eap vs [DNA] and given as
1.29×105 M-1 , which is higher than the previous reported symmetric dinuclear
nickel(Ⅱ ) [37] and heternuclear Zn(Ⅱ)Ni(Ⅱ) complex[34], but lower than that of
classical intercalators such as EB whose binding constants have been found to be in
order of 106-107 M-1 [38]. The complex mentioned here provided more aromatic
moiety to overlap with the stacking base pairs of the double-helix DNA through
intercalator binding results in hypochromism and bathochromism while the pyridyl
moiety of the complex may just prefer major groove binding and explained for the
minor red shift [39].
Fluorescence spectral studies
The fluorescence spectroscopy provides insight into the changes taking place in the
DNA microenvironment on the addition of the complexes [40]. From the previous
investigation, no fluorescence was observed for the metal complex either in the buffer
or in the presence of DNA, while EB emits intense fluorescence in the presence of
DNA due to its strong intercalation between the adjacent DNA base pairs. To elicit
the interactions between the complex and CT-DNA, the fluorescence spectral
variation of CT-DNA bounded with EB was titrated with different concentration of
complex (Figure 7).

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emission spectra of EB bound to DNA in the absence (1) and presence (2-6) of
Figure 7. The
the complex, insect: Stern–Volmer quenching plots of EB bound to DNA. [EB] = 10 µM, [DNA]
= 25 µM, [complex] = 0-200 µM; λex = 520 nm.
In our experiments, upon the addition of the complex, the fluorescent intensity of
the EB-DNA gradually decreased, which indicated that the Cu(Ⅱ) Ni(Ⅱ) complex
competed with EB in bonding to DNA. The reduction of the emission intensity gives
a measure of the affinity of the complex for DNA helix, the fluorescent quenching
curve of EB bound to DNA by the complex is in good agreement with the linear
Stern-Volmer equation,
I0 / I = 1 + KSV [Q]
Where I0 and I is the emission intensity of EB-DNA in the absence and presence of
complex respectively. The Stern-Volmer constant Ksv was obtained as the slope of the
linear plot of I0/I versus [complex]. From the inset in Figure 7 , the Ksv value of the
Cu(Ⅱ) Ni(Ⅱ) complex is 1.25×105 M-1. The value shows that the complex can
replace the EB binding sites from the DNA-EB system, which is usually
determination of the intercalative of complex with DNA. The constant is more or less
equal to the reported intercalators and higher than that of Ni(Ⅱ) complexes [41]. So
we deduce that the Cu(Ⅱ) Ni(Ⅱ) complex exhibit high binding affinity towards the
CT-DNA through intercalative binding modes. The DNA binding affinity with
macrocyclic complexes depends upon the structure of complexes, the positive charge
on the metal ion and hydrophobicity [29]. The complex we reported here showed
higher binding affinity due to the benzyl ring represented more hydrophobic and
interacted with the hydrophobic DNA surface.
Viscosity titration
To further clarify the interaction mode of the Cu(Ⅱ) Ni(Ⅱ) complex with
CT-DNA, a specific viscosity of DNA was carried out by varying amounts of added
complex. The viscosity measurement is the most critical test in evaluating the binding
mode of complex with DNA due to the viscosity of DNA is sensitive to its length [42].
In an intercalation mode, the DNA helix length increase results in the viscosity
enhancement as the base pairs are separated to accommodate the binding ligands,
while moderate intercalation lead to bend the DNA helix would shorten the DNA

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length and reduce the DNA viscosity. In contrast the grooving and electrostatic
binding would exert no such effect on the viscosity. Viscosity data were presented
( / )
0
1/3
expressed as
versus binding ratio [complex]/ [DNA], where η and η0 are
the specific viscosity of DNA bonding with and without the complex, respectively.
The values of η and η0 were calculated from the relation: η =(t-tb)/tb, where t and tb
are the flow time of the buffer in the presence and absence of DNA. The effect of the
ligand and complex on the viscosity of DNA is shown in Figure 8.
Figure 8. Viscosity of the ligand and complex
As shown in Figure 8, as increasing the amount of the complex and free ligand, the
viscosity increased obviously, similar to EB, a well known DNA intercalator. The
ability of the complex to increase the viscosity of DNA depended upon the diimine
ligand. The extended aromatic rings and methyl group substituted response for the
planar rigidity and hydrophobic as a consequence of the untwisting of base pairs and
helical backbone of DNA led to the increase of the viscosity [43]. These results suggest
that the Cu(Ⅱ ) Ni(Ⅱ ) complex bind to DNA through intercalation which was in
accordance with the absorption and emission spectral studies.
Conclusion
In conclusion, we have synthesized and fully characterized a new asymmetrical
bis-phenol pendant-armed macrocyclic heterobinuclear Cu(II)-Ni(II) complex. The
complex showed high phosphate ester hydrolysis activity and antibacterial activity
against E. coli. The DNA binding ability of the complex was also investigated. The UV

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absorption, fluorescence spectral and viscosity studies show that this complex strongly
binding with DNA through intercalation due to the mental-mental synergic combination
of ligand and mental ion. The hydrophobic methyl substituent and extended aromantic
phenyl moiety also correlates with the binding mode.
Acknowledgments:
We sincerely thanks to the the financial support of the technical innovation major
project of Hubei Province (No.2017ABA155) and the financial support from the project
of Hubei Province (No. 2018ZYYD062).
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Highlights
1. A new benzyls pendant-armed macrobicyclic heterbinuclear Cu(Ⅱ)-Ni(Ⅱ)
complex was synthesized.
2. The complex was bridged by two phenolic oxgyens and an acetate radical.
3. The complex showed better hydrolysis, antibacterial activities and binding
properties to DNA.