A new macrocyclic heterobinuclear Cu(II)-Zn(II) complex: synthesis, crystal structure, phosphate hydrolysis, and DNA binding studies

Article abstract of DOI:10.1080/00958972.2019.1608439

A new macrocyclic heterobinuclear Cu(II)-Zn(II) complex was synthesized and characterized by elemental analysis, FT-IR, ES-MS, and single-crystal X-ray diffraction. Five-coordinate geometry for the new complex is proposed. The copper…zinc distance bridged by two phenolic oxygens and a acetate ligand is 2.9508 ?. The phosphate ester hydrolysis activity and the DNA binding ability of the complex were studied. The results showed that the present complex has an efficient catalytic activity of phosphoester bond cleavage. The catalytic rate constant k_cat for the hydrolysis of 4-nitrophenyl phosphate disodium salt hexahydrate (pNPP) by the synthesized complex is 2.69 × 10^?4 s^?1 and 10⁵ times faster than the spontaneous hydrolysis of the phosphate monoester. The complex shows a good binding ability to calf thymus (CT-DNA) and the corresponding binding constant is 1.9 × 10⁵ M^?1. The linear Stern-Volmer quenching constant obtained by the fluorescent spectroscopic is 6.3 × 10⁴ M^?1.

Full text of DOI:10.1080/00958972.2019.1608439

Journal of Coordination Chemistry
ISSN: 0095-8972 (Print) 1029-0389 (Online) Journal homepage: https://www.tandfonline.com/loi/gcoo20
A new macrocyclic heterobinuclear Cu(II)-Zn(II)
complex: synthesis, crystal structure, phosphate
hydrolysis, and DNA binding studies
Huizhi Kou, Yang Wang, Peipei Ding, Jianfen Li & Baoxian Shi
To cite this article: Huizhi Kou, Yang Wang, Peipei Ding, Jianfen Li & Baoxian Shi (2019):
A new macrocyclic heterobinuclear Cu(II)-Zn(II) complex: synthesis, crystal structure,
phosphate hydrolysis, and DNA binding studies, Journal of Coordination Chemistry, DOI:
10.1080/00958972.2019.1608439
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JOURNAL OF COORDINATION CHEMISTRY
https://doi.org/10.1080/00958972.2019.1608439
A new macrocyclic heterobinuclear Cu(II)-Zn(II) complex:
synthesis, crystal structure, phosphate hydrolysis, and
DNA binding studies
Huizhi Kou, Yang Wang, Peipei Ding, Jianfen Li and Baoxian Shi
School of Chemical and Environmental Engineering, Wuhan Polytechnic University, Wuhan, China
ABSTRACT
ARTICLE HISTORY
Received 25 November 2018
Accepted 30 March 2019
A new macrocyclic heterobinuclear Cu(II)-Zn(II) complex was syn-
thesized and characterized by elemental analysis, FT-IR, ES-MS,
and single-crystal X-ray diffraction. Five-coordinate geometry for
the new complex is proposed. The copper … zinc distance
bridged by two phenolic oxygens and a acetate ligand is 2.9508
Å. The phosphate ester hydrolysis activity and the DNA binding
ability of the complex were studied. The results showed that the
present complex has an efficient catalytic activity of phosphoester
bond cleavage. The catalytic rate constant kcat for the hydrolysis
of 4-nitrophenyl phosphate disodium salt hexahydrate (pNPP) by
the synthesized complex is 2.69ꢀ 10ꢁ4 sꢁ1 and 105 times faster
than the spontaneous hydrolysis of the phosphate monoester.
The complex shows a good binding ability to calf thymus (CT-
KEYWORDS
Macrocyclic heterobinuclear;
crystal structure; phosphate
ester hydrolysis; DNA
binding; viscosity
DNA) and the corresponding binding constant is 1.9 ꢀ 105 Mꢁ1
.
The linear Stern-Volmer quenching constant obtained by the
fluorescent spectroscopic is 6.3ꢀ 104 Mꢁ1
.
1. Introduction
Over the past decades, the rational design and construction of macrocyclic ligands
and their complexes have attracted a great deal of interest due to their fascinating
CONTACT Baoxian Shi
wyydcc@163.com
ß 2019 Informa UK Limited, trading as Taylor & Francis Group

2
H. KOU ET AL.
Scheme 1. Synthesis of H2L and the title complex.
complex structures and their potential applications in catalysts, medicine, sensors,
electrochemistry and magnetism [1–6]. Great efforts have been devoted to synthe-
size a large variety of macrocyclic ligands containing different kinds of donor heter-
oatoms such as nitrogen, oxygen, and sulfur. Schiff bases have drawn much
attention because of the convenience in preparation and structural flexibility. The
macrocyclic Schiff bases can selectively chelate certain metal ions according to the
number, type and position of the donor atoms, the ionic radii of the metal centers
and the coordinating property of the counter ions [7]. These functionalized macro-
cyclic complexes exhibit favorable biological activities including antibacterial, anti-
fungal, antidiabetic, antiproliferative, anticancer, herbicidal, and antiinflammatory
activities [8–12]. There are also some examples with phosphatase-like activity [13,
14] and DNA cleavage ability [15]. Our previous research showed that Schiff-base
metal complexes have better biological activities and the hydrolysis activity to
pNPP [16–18].
In this article, the precursor ligand 3,3’-(ethane-1,2-diylbis(benzylazanediyl))bis(me-
thylene)bis(2-hydroxy-5-methyl-benzaldehyde) (H2L) is synthesized. A new heterobinu-
clear Cu(II)-Zn(II) complex is designed with H2L, 1,3-propanediamine, and different
metal ions; its phosphoesterase hydrolysis activity and the DNA binding ability are
studied. The routes for the synthesis of ligand and macrocyclic complex are shown in
Scheme 1.
2. Experimental
All commercial solvents and chemicals were of analytical grade and used without puri-
fication. Agarose gel and calf-thymus DNA were purchased from Sigma. IR spectra
were performed using KBr disc on a vector 22 FI-IR spectrophotometer. Elemental
analyses were measured on a Perkin-Elmer 240 analyzer. Electrospray ionization mass
spectrum (ESI-MS) was carried out on a Finnigan LCQ ES-MS mass spectrograph using
methanol as a mobile phase with a sample concentration of 1.0 mM. UV-vis spectra
were recorded on an UV-2450 spectrophotometer. Fluorescence spectroscopic studies
were determined on an F-7000 FL Spectrophotometer. Circular dichroism (CD) spectra
were measured on a Jasco J-810 chromatographic instrument. The crystal structure of
the complex was measured on a Bruker AXS SMART diffractometer (Mo-Ka radiation
monochromator).

JOURNAL OF COORDINATION CHEMISTRY
3
2.1. Synthesis of 3,3’-(ethane-1,2-diylbis(benzylazanediyl))
bis(methylene)bis(2hydroxy-5-methyl-benzaldehyde) (H2L)
2.1.1. Preparation of N,N’-dibenzylethane-1,2-diamine
Samples of benzaldehyde (11.1 g, 0.11 mol) and ethylenediamine (3.00 g, 0.05 mol)
were dissolved in 150 mL absolute methanol and refluxed for 4 h. After removal of the
solvent, the crude was recrystallized from ethanol to give light yellow powders. The
product was dissolved in absolute methanol, NaBH4 (3.78 g, 0.1 mol) was added under
the condition of ice bath, then stirred at room temperature. The reaction mixture was
extracted with CH2Cl2 (3 ꢀ 40 mL). After filtration and evaporation, the light yellow oily
liquid was obtained.
2.1.2. Preparation of 3-(bromomethyl)-2-hydroxy-5-methylbenzaldehyde
Samples of 5-methylbenzaldehyde (54.4 g, 0.4 mol), paraformaldehyde (18.2 g, 0.06 mol)
and 40% hydrobromic acid (540.5 g, 2.8 mol) were mixed in a 1 L round bottom flask,
refluxed for 4 h, then cooled to ambient temperature and extracted with CH2Cl2
(3 ꢀ 150 mL). The crude was recrystallized with ethanol and dried in vacuum for 8 h.
2.1.3. Preparation of H2L
13.0 g of 3-(bromomethyl)-2-hydroxy-5-methylbenzaldehyde and 20.7 g of K2CO3 were
dissolved in 150 mL THF, then a THF solution of N,N’-dibenzylethane-1,2-diamine was
added dropwise, stirred 12 h at room temperature and then filtered. The mixture was
extracted with CH2Cl2 (3 ꢀ 150 mL). The obtained aqueous phase was made alkaline by
addition of a saturated solution of NaHCO3 and the resultant mixture was extracted
with dichloromethane (3 ꢀ 150 mL). The combined CH2Cl2 fractions were dried over
with anhydrous Na2SO4. After filtration and evaporation, the residue was recrystallized
from acetone ethanol to give white solid (yield: 46.2%). 1H NMR (400 Hz, CDCl3),
d(ppm): 10.16 (s, 2H), 7.17–7.35 (m, 14H), 3.59 (s, 4H), 3.51 (s, 4H), 2.65 (s, 4H). 13C
NMR (100 Hz, CDCl3), d(ppm): 193.1, 158.6, 137.2, 129.3, 129.2, 128.3, 127.4,124.9, 121.6,
58.5, 54.9, 53.9, 50.1, 20.2.
2.2. Preparation of [CuZnL(l-OAc)2](ClO4)
0.107 g of H2L and 0.040 g of Cu(OAc)2ꢂH2O were dissolved in 10 mL absolute metha-
nol, respectively. Then the solution of Cu(OAc)2ꢂH2O was added to the solution of H2L.
The mixture was stirred vigorously while a methanol solution containing 0.0422 g of
1,3-diaminopropane was added dropwise over a period of 3 h; the resulting solution
was stirred for another 4 h at room temperature. Then the zinc perchlorate hexahy-
drate (0.0680 g) was added to above solution and stirred for 4 h. The resulting precipi-
tate was filtered from the solution; the green crystals were collected. The green block
crystals suitable for the X-ray measurement were obtained by evaporation of the fil-
trate at room temperature for 1 week. Yield: 68%, Anal. found (%): C, 55.6; H, 5.9; N,
7.2. Calcd (%): C 55.5; H 6.0; N 7.3. IR (KBr, ꢀ/cmꢁ1): 1626 ꢀ(C ¼ N), 1107, 621 ꢀ(ClO– ),
4
1402, 1580, 1313 ꢀ(OAc–).

4
H. KOU ET AL.
2.3. Hydrolysis of pNPP
The hydrolysis activity of the complex toward 4-nitrophenylphosphate disodium salt
hexahydrate (pNPP) was examined by monitoring the increase in the visible absorb-
ance at 406 nm and 25 ꢃC due to the release of the 4-nitrophenolate anion in DMF/
Tris-HCl buffer (2:3, v:v) solution at pH ¼ 7.2. pNPP stock solutions were freshly pre-
pared before performing the kinetic measurement and the ionic strength was main-
tained at I ¼ 0.10 M with potassium chloride. The pseudo-first-order rate constant (kobs
(sꢁ1)) of the hydrolysis of pNPP was obtained by the initial rate method (<5% conver-
sion). The concentration of 4-nitrophenolate was calculated from the extinction coeffi-
cient (14975 Mꢁ1cmꢁ1).
2.4. DNA-binding experiments
The DNA-binding experiments were performed by an UV-vis spectrometer. The stock
solution of CT-DNA was prepared by dissolving CT-DNA in Tris-HCl buffer (100 mL,
50 mM Tris-HCl, 50 mM NaCl, pH ¼ 7.2) and stored at 4 ꢃC not more than 4 days. The
CT-DNA concentration in terms of nucleotide was determined by employing a molar
extinction coefficient of 6600 Mꢁ1cmꢁ1 (nucleotide)ꢁ1 at 260 nm [19]. The spectra
were recorded in the condition of keeping the concentration of complex constant (50
mM) and varying the concentration of CT-DNA (0–50 mM). In the fluorescence quench-
ing experiments, DNA was pretreated with ethidium bromide (3,8-diamino-5-ethyl-6-
phenylphenanthridinium bromide, EB) and Tris-HCl buffer. The fluorescence intensities
were recorded with increasing amounts of complex concentration (0–200 mM). Circular
dichroism (CD) spectra of CT-DNA were recorded in the absence and presence of the
complex, sweep range of 240–320 nm. Viscosity measurements were performed using
a capillary viscometer at 25.0 0.1 ꢃC. Each set of data were measured in triplicate,
averages are presented as (g/g0)1/3 versus the ratio of complex to DNA, where g is the
viscosity of CT-DNA in the presence of the complex and g0 is the viscosity of CT-DNA
alone. Viscosity values are calculated from the observed flow time of CT-DNA contain-
ing solutions corrected for the flow time in buffer alone (t0), g ¼ (t – t0) [20].
3. Results and discussion
3.1. Characterization of the complex
The complex itself is a green block-shaped crystal and stable in the atmosphere. It is
easily soluble in DMF and DMSO and moderately soluble in methanol and ethanol.
3.2. IR spectra
In IR spectra of the complex, the sharp C ¼ N bond stretching vibration is observed at
1626 cmꢁ1, indicating formation of the macrocyclic complex [21]. In addition, the two
peaks at 1580 and 1313 cmꢁ1 for the complex can be ascribed to the asymmetric and
symmetric stretching vibration of carboxylate groups [22]. The absorption bands at
1107 and 621 cmꢁ1 in the complex are attributed to perchlorate ions, which indicates

JOURNAL OF COORDINATION CHEMISTRY
5
Figure 1. The ESI-MS spectra of the complex (inset: the isotopic distribution of the peak at m/
z ¼ 759.08; left: experimental pattern; right: calculated pattern).
that the perchlorate ions are not coordinated to the metal ions and are present as
counter ions in the crystal lattice.
3.3. ES-MS spectra
The formation of the compound was further confirmed through ESI-MS spectra
method. The ESI-MS spectrum of the complex in methanol solution is given in
Figure 1, together with isotope distributions chart. The spectra of the complex shows
a major peak at m/z 759.08 corresponding to the [CuZn(OAc)L]þ ion (calc. 759.72),
illustrating that the complex is very stable in methanol solution, and no other splitting
ion peaks exist indicating that the complex has high purity. The results are consistent
with the theoretical and experimental isotope distributions.
3.4. Crystal structure of complex
The perspective view of [CuZn(OAc)L]þ is shown in Figure 2 (the ClO4 anion and
–
hydrogen atoms are ignored to facilitate observation), together with the atom num-
bering schemes. The significant crystallographic data are listed in Table 1. Selected
bond lengths and angles relevant are presented in Table 2.
Each unit of the complex shows that the Zn atom resides at the N(imine)2O2 site
and the Cu atom at the N(amine)2O2 site with both phenyl ligands found on the same
side of the mean molecular plane. Both of the metal ions coordinate with the ligand
by a pentacoordinate manner, and the Cu-donor bond distances are in the range of
1.980–2.100 Å, while the Zn-donor bond distances are in the range of 2.002–2.045 Å.

6
H. KOU ET AL.
Figure 2. Perspective view of [CuZn(OAc)L]þ.
Table 1. Crystal data and structure refinement for the complex.
Empirical
C39H43ClCuN4ZnO8
Formula weight
Crystal system
Space group
a (Å)
860.l3
Monoclinic
P2(1)/n
10.827(4)
13.68(5)
27.303(10)
90
b (Å)
c (Å)
a (ꢃ)
b (ꢃ)
92.042(7)
c (ꢃ)
90
4043(2)
4
1.413
1.240
Volume (Å3)
Z
D(calc) (g/cm3)
l (Mo-Ka) (nm)
F(000)
1780
Crystal size (mm)
Temperature (K)
Mo-Ka radiation (Å3)
Range (ꢃ)
Nref, Npar
0.16 ꢀ 0.12 ꢀ 0.1
298
0.71073
2.05, 25.01
7105, 499
Tot., uniq., data R(int)
Observed data [I > 2.0sigma(I)]
R, wR2, S
22,073, 7872, 0.058
5207
0.0691, 0.1662, 1.026
–0.457, 0.744
Min. and max. resd. dens. (e/Å3)
The electron-donating group connected to Zn(II) or Cu(II) can be described as a dis-
torted square-pyramidal, in which the apical position is occupied by the oxygen atom
arising from the acetate ions linker [Zn1-O3 ¼ 2.039(4) Å] and [Cu1-O4 ¼ 1.980(4) Å],
respectively. The basal plane is formed by two imine nitrogen atoms arising from the
Schiff base ligand and two phenoxo oxygen atoms. The copper … zinc distance
bridged by two phenolic oxygens is 2.9508 Å with Cu1-O1-Zn1 angle of 91.75ꢃ and
Cu1-O2-Zn1 angle of 94.73ꢃ, respectively, which are in the range of appropriate metal-
… metal distances 3.0–5.4 Å of the binuclear complexes for phosphoesterase mimics
[23–25]. Therefore, the complex is predicted to have phosphatase activity.

JOURNAL OF COORDINATION CHEMISTRY
7
Table 2. Selected bond lengths (Å) and angles (ꢃ) for the complex.
Bonds
Distance
Bonds
Distance
Cu1-O4
Cu1-O1
Cu1-O2
Cu1-N3
Cu1-N4
1.980(4)
2.009(4)
2.066(4)
2.095(5)
2.100(4)
Zn1-O1
2.002(4)
2.010(5)
2.019(5)
2.039(4)
2.045(4)
Zn1-N2
Zn1-N1
Zn1-O3
Zn1-O2
Bond
Angle
Bond
Angle
160.3(2)
88.10(19)
98.8(2)
97.92(18)
O4-Cu1-O1
O4-Cu1-O2
O1-Cu1-O2
O4-Cu1-N3
O1-Cu1-N3
O2-Cu1-N3
O4-Cu1-N4
O1-Cu1-N4
O2-Cu1-N4
N3-Cu1-N4
O4-Cu1-Zn1
O1-Cu1-Zn1
O2-Cu1-Zn1
N3-Cu1-Zn1
N4-Cu1-Zn1
101.89(17)
100.99(16)
75.61(17)
105.55(17)
151.69(19)
92.33(17)
109.91(18)
88.79(17)
147.70(17)
88.34(18)
81.89(11)
42.54(11)
43.85(11)
135.65(13)
131.03(12)
O1-Zn1-N2
O1-Zn1-N1
N2-Zn1-N1
O1-Zn1-O3
N2-Zn1-O3
N1-Zn1-O3
O1-Zn1-O2
N2-Zn1-O2
N1-Zn1-O2
O3-Zn1-O2
O1-Zn1-Cu1
N2-Zn1-Cu1
N1-Zn1-Cu1
O3-Zn1-Cu1
O2-Zn1-Cu1
97.5(2)
107.79(19)
76.23(17)
89.30(19)
150.78(18)
98.80(17)
42.73(11)
130.35(16)
130.03(15)
77.71(12)
44.40(11)
Figure 3. View of one molecular unit depicting the connection via weak C–HꢂꢂꢂO hydrogen bonds
in the complex.
Hydrogen-bond interactions in maintaining the three-dimensional network of the
complex play an important role. As shown in Figure 3, each perchlorate anion acts as
H-bonding acceptors to link two adjacent structural units through the following hydro-
gen bond interactions: C33-H33ꢂꢂꢂO7, C10-H10AꢂꢂꢂO8, C21-H21BꢂꢂꢂO8, C23-H23BꢂꢂꢂO8,
respectively.

8
H. KOU ET AL.
Figure 4. Kinetic experiments for the cleavage of NPP by the complex in DMF/tris–HCl buffer solu-
tion at pH ¼ 7.2, 25 ꢃC and I ¼ 0.10 M (KCl), [complex] ¼ 2 ꢀ 10ꢁ4 M.
3.5. Kinetic studies on phosphate ester hydrolysis
The ability of the complex to promote phosphate ester hydrolysis was tested using the acti-
vated model substrate pNPP. Release of 4-nitrophenolate was monitored in pH ¼ 7.2 buffer
solution by UV-vis spectroscopy. The initial rates of NPP cleavage were determined as a func-
tion of the substrate concentration as depicted in Figure 4. As can be seen from Figure 4, ini-
tially the rate value increases linearly with the increase of NPP concentration but deviates
gradually from linearity and finally tends toward a saturation curve. Michaelis-Menten kinet-
ics has been used to explain the behavior in the system. The kinetic parameters were deter-
mined according to the Lineweaver–Burk method (Figure 5) and given as follows: Vmax
5.37ꢀ 10ꢁ8 M sꢁ1, Michaelis constant Km ¼ 2.81ꢀ 10ꢁ3 M, catalytic rate constant kcat
¼
¼
Vmax/[Complex] ¼ 2.69ꢀ 10ꢁ4 sꢁ1 (R ¼ 0.997 for five points) and second-order rate constant
k ¼ kcat/Km ¼ 9.57 ꢀ 10ꢁ2 Mꢁ1sꢁ1. The kcat of the title complex is 10 times higher than that
observed for the corresponding binuclear copper complex [26] and 105 times faster than the
spontaneous hydrolysis of the phosphate monoester [27]. It suggests that the title complex
has an efficient catalytic activity of phosphoester bond cleavage.
3.6. DNA-binding studies
3.6.1. Absorption spectral studies
The binding ability of the complex with CT-DNA is studied by measuring the electronic
absorption spectroscopy. It is reported that hypochromicity and bathochromic shift

JOURNAL OF COORDINATION CHEMISTRY
9
Figure 5. Lineweaver-Burk plot for the complex.
are considered as evidence of intercalative mode of binding between the aromatic
chromophore of the compound and the base pairs of DNA [28]. The absorption spec-
tra of H2L and newly synthesized complex in the absence and presence of CT-DNA are
shown in Figure 6. As can be seen from Figure 6 the binding of free ligand and the
complex to CT-DNA result in a decrease in the absorption intensities with a small
amount of red-shift in the UV-vis absorption spectra. The observed changes in the
absorption spectra clearly suggest that the binding of H2L and the complex to DNA
double-helix is by an intercalative mode. After intercalating the base pairs of DNA, the
ꢄ
p -orbital of the intercalated ligand can couple with the p-orbital of the base pairs,
ꢄ
thus decreasing the p–p transition energy and resulting in the bathochromism [29].
On the other hand, the coupling p-orbital is partially filled by electrons, thus decreas-
ing the transition probabilities and concomitantly resulting in hypochromism [30]. The
smaller extent of hypochromism may be because of the stronger intercalation
between the complex and DNA. The binding ability of the compound was calculated
by intrinsic binding constant Kb using Equation (1) [31]:
½
ꢅ
½
ꢅ
ð
DNA = e ꢁ e
¼
DNA = e ꢁ e þ 1=K e ꢁ e
fÞ bð fÞ
(1)
ð
fÞ
a
b
b
where [DNA] is the concentration of DNA, ea is the extinction coefficient of complex in
the presence of CT-DNA, ef is the extinction coefficient of free complex, and eb is the
extinction coefficient of complex in fully bound to DNA. The slope of the plot gives
the value of 1/(eb – ef), while intercept is equal to 1/Kb(eb – ef). The intrinsic binding
constant Kb was calculated from the ratio of the slope to the intercept and found to
be 1.05 ꢀ 105 Mꢁ1 (R ¼ 0.991 for five points) and 1.93 ꢀ 105 Mꢁ1 (R ¼ 0.987 for five

10
H. KOU ET AL.
Figure 6. Absorption spectra of (a) free H2L and (b) the complex in tris–HCl buffer (pH ¼ 7.2) solu-
tion upon addition of DNA. [complex] ¼ 50 mM, [DNA] ¼ 0–50 mM. Arrows show the absorption
changes upon increasing concentration of the DNA. Inset shows the plot of [DNA]/(ea – ef) versus
[DNA] for the absorption titration.
points) for H2L and the complex, respectively. Hence, the binding affinity of the com-
plex is higher than that of the free ligand. The Kb value for the complex was obviously
stronger than similar binuclear Cu-Cu and Zn-Zn complexes that have been published
[30, 32, 33], may be due to the benzyl pendant-arms of the titled complex enhanced
the intercalation to the base pairs of CT-DNA.
3.6.2. Fluorescence spectra
Fluorescence spectroscopy is another effective technique for the study of DNA-inter-
action with the synthesized complex. It is well-known that free EB has no fluorescence
characteristic, but it displays intense fluorescence signal after intercalating into DNA.
This enhanced fluorescence can be quenched by the addition of the complex [30]. For
all the compounds, no emission was observed either alone or in the presence of CT-
DNA in the buffer. The addition of the ligand does not provoke any significant
changes of the intensity or position of the emission band of the DNA-EB system, indi-
cating that free ligand cannot displace EB from the DNA-EB complex. The emission
spectra of EB bound to DNA in the absence and presence of the synthesized complex
are shown in Figure 7. From Figure 7, it is apparent that the addition of the complex
to DNA pretreated with EB causes a gradual decrease in fluorescence intensity, indicat-
ing that the synthesized complex competes with EB to bind to DNA by intercalation.
Fluorescence quenching constant can be calculated by the Stern-Volmer equation [34]:
½ ꢅ
I0=I ¼ 1 þ K Q
where I0 and I are the intensity of fluorescence in the absence and presence of the
complex, respectively, K is quenching constant and [Q] is concentration of quencher.
In the plot of I0/I versus [complex]/[DNA] (inset Figure 7), K is obtained by the ratio of
the slope to intercept. So the K value for the complex was calculated as 6.3 ꢀ 104 Mꢁ1
(R ¼ 0.988 for five points). The interaction mode of DNA with the macrocyclic complex
is related with the structure of macrocyclic complex, the positive charge on the metal
ion and hydrophobicity [34]. The positive charge on the metal ion can interact with

JOURNAL OF COORDINATION CHEMISTRY
11
Figure 7. Emission spectra of EB bound to DNA in the absence (1) and presence (2–5) of the com-
plex; inset: Stern-Volmer quenching plots of EB bound to DNA. [EB] ¼ 10 mM, [DNA] ¼ 25 mM,
[complex] 2–5 ¼ 0-200 mM; kex ¼ 520 nm.
the negative charged phosphate units in the DNA helix. The four-phenyl-containing
structure also facilitates DNA-binding.
3.6.3. CD spectral studies
Circular dichroism (CD) is a very useful technique to monitor the DNA-morphology
changes, which is sensitive to small variations in the chiral conformation of DNA. The
CD spectrum of CT-DNA exhibits a positive band at 275 nm due to base stacking and
a negative band at 245 nm because of right-handed helicity of DNA [35]. The CD spec-
tra of CT-DNA in the absence and presence of the ligand and complex are given in
Figure 8. It is observed from Figure 8 that the positive CD band signal increased with
addition of the ligand and complex to CT-DNA. The increased extent of the complex is
higher than that of the ligand. The changes in the CD spectra in the presence of the
complex indicated that the CT-DNA was unwinded after the intercalation binding with
the base pairs of DNA and induced conformational changes from B-DNA to Z-
DNA [32].
3.6.4. Viscosity study
Viscosity study is known to be a powerful hydrodynamic quantification technique to
support an intercalation model. In order to further clarify the binding mode of the
complex with DNA, a viscosity measurement was studied. In an intercalation model,
the aromatic groups in the complex stack between the base pairs and lengthen DNA-

12
H. KOU ET AL.
Figure 8. CD spectra of (a) free DNA, and in the presence of the (b) ligand and (c) complex.
([DNA] ¼ 250 mM, [complex] ¼ 250 mM).
Figure 9. Effect of increasing amounts of the complex on the relative viscosity of CT-DNA at
25.0 0.1 ꢃC; [DNA] ¼ 200 mM.

JOURNAL OF COORDINATION CHEMISTRY
13
helix, leading to increase of the DNA viscosity [36]. The effect of the ligand and com-
plex on the viscosity of DNA is shown in Figure 9. The results indicate that the
absence and presence of the compound have an obvious effect on the viscosity of
DNA. The viscosity of the DNA increases evidently with the addition of increasing
amounts of the complex and free ligand. The results confirm that the binding mode
of the complex with DNA is intercalation, as also indicated by the spectro-
scopic studies.
4. Conclusion
A new macrocyclic heterobinuclear Cu(II)-Zn(II) complex was synthesized and structur-
ally characterized by different methods. The phosphate ester hydrolysis activity and
the DNA-binding ability of the complex were explored. The results showed that the
present complex has an efficient catalytic activity of phosphoester bond cleavage. The
studies of UV-vis absorption, fluorescence spectroscopy, circular dichroism and viscos-
ity measurements of the mixture of the complex and DNA suggested that the complex
has high DNA-binding ability. It is evident that the binding mode between the aro-
matic chromophore of the complex and the base pairs of DNA is achieved by a mod-
erate intercalation.
Disclosure statement
No potential conflict of interest was reported by the authors.
Funding
The authors are grateful for the financial support of the Technical Innovation Major Project of
Hubei Province (no. 2017ABA155), and the Central Committee Guides Local Science and
Technology Development Special Project of Hubei Province (no. 2018ZYYD062).
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