Dinuclear Cu(II) complexes based on p-xylylene-bridged bis(1,4,7-triazacyclononane) ligands: Synthesis, characterization, DNA cleavage abilities and evaluation of superoxide dismutase- and catalase-like activities

Article abstract of DOI:10.1002/aoc.4297

Three new dinuclear Cu(II) complexes with the formulas [Cu₂(pxdmbtacn)Cl₄] (1), [Cu₂(pxdmbtacn)Cl_0.7(NO₃)_1.3(OH)₂(H₂O)_1.3]?6H₂O (2) and [Cu₂(pxdiprbtacn)Cl₄] (3) together with one previously reported complex, [Cu₂(pxbtacn)Cl₄] (4), were obtained from Cu(II) salts with three p-xylylene-bridged bis-tacn ligands bearing pendant alkyl substituents or without pendant group. Complex 2 was structurally characterized as a centrosymmetric dinuclear molecule with each metal center being coordinated to some labile ligands in addition to one tacn ring. Based on the results of mass spectrometry and UV–visible spectroscopy, complexes 1 and 3 are capable of existing in aqueous solution as dinuclear species but 4 can partially form a dimer of the original dinuclear motif. Complexes 1, 3 and 4 can all effectively cleave supercoiled DNA oxidatively in the presence of hydrogen peroxide. The superoxide dismutase (SOD) activities of 1 and 3 measured under physiological conditions are comparable to that of the native CuZnSOD enzyme but the enzymatic activity of 4 is about three- to fourfold lower. Furthermore, complexes 1, 3 and 4 demonstrate moderate scavenging effect on hydrogen peroxide and their catalase activities are in the decreasing order of 3 > 1 > 4.

Full text of DOI:10.1002/aoc.4297

Received: 30 October 2017
DOI: 10.1002/aoc.4297
Revised: 2 January 2018
Accepted: 5 January 2018
F U L L P A P E R
Dinuclear Cu(II) complexes based on p‐xylylene‐bridged
bis(1,4,7‐triazacyclononane) ligands: Synthesis,
characterization, DNA cleavage abilities and evaluation of
superoxide dismutase‐ and catalase‐like activities
Qi Tang† | Ji‐Qing Wu† | Hong‐Yan Li | Yan‐Fang Feng | Zhong Zhang
| Yu‐Ning Liang
Key Laboratory for Chemistry and
Three new dinuclear Cu(II) complexes with the formulas [Cu2(pxdmbtacn)Cl4]
(1), [Cu2(pxdmbtacn)Cl0.7(NO3)1.3(OH)2(H2O)1.3]⋅6H2O (2) and
Molecular Engineering of Medicinal
Resources (Ministry of Education of
China), School of Chemistry and
[Cu2(pxdiprbtacn)Cl4] (3) together with one previously reported complex,
[Cu2(pxbtacn)Cl4] (4), were obtained from Cu(II) salts with three p‐xylylene‐
bridged bis‐tacn ligands bearing pendant alkyl substituents or without pendant
group. Complex 2 was structurally characterized as a centrosymmetric dinu-
clear molecule with each metal center being coordinated to some labile ligands
in addition to one tacn ring. Based on the results of mass spectrometry and UV–
visible spectroscopy, complexes 1 and 3 are capable of existing in aqueous solu-
tion as dinuclear species but 4 can partially form a dimer of the original dinu-
clear motif. Complexes 1, 3 and 4 can all effectively cleave supercoiled DNA
oxidatively in the presence of hydrogen peroxide. The superoxide dismutase
(SOD) activities of 1 and 3 measured under physiological conditions are compa-
rable to that of the native CuZnSOD enzyme but the enzymatic activity of 4 is
about three‐ to fourfold lower. Furthermore, complexes 1, 3 and 4 demonstrate
moderate scavenging effect on hydrogen peroxide and their catalase activities
are in the decreasing order of 3 > 1 > 4.
Pharmacy of Guangxi Normal University,
Guilin, People's Republic of China
Correspondence
Zhong Zhang and Yu‐Ning Liang, School
of Chemistry and Pharmacy of Guangxi
Normal University, Key Laboratory for
Chemistry and Molecular Engineering of
Medicinal Resources (Ministry of
Education of China), Guilin, People's
Republic of China.
Email: zhangzhong@mailbox.gxnu.edu.cn;
liangyuning008@163.com
Funding information
National Natural Science Foundation of
China, Grant/Award Number: 21361004;
Guangxi Natural Science Foundation of
China, Grant/Award Numbers:
2017GXNSFAA198125 and
2014GXNSFAA118044; Natural Science
Foundation of Key Laboratory for Chem-
istry and Molecular Engineering of
Medicinal Resources (Guangxi Normal
University), Ministry of Education of
China, Grant/Award Number:
KEYWORDS
catalase mimic, copper(II) complex, nuclease activity, SOD mimic, triazacyclononane
CMEMR2012‐A08, CMEMR2013‐C08
1 | INTRODUCTION
in aging as well as in a number of diseases associated with
oxidative stress.[1–7] For these reasons, the elimination of
Reactive oxygen species (ROS) such as superoxide radical
(O2•−) and hydrogen peroxide (H2O2), generated from the
direct reduction of molecular O2 during the respiratory
event, are known to induce cellular damage at elevated
concentrations and have been proved to play a vital role
excess amounts of these reactive species from organisms
is of great importance to prevent the occurrence of
various diseases including neurological disorders and
cancer. Superoxide dismutases (SODs) and catalases are
the chief members of the natural antioxidant defense
system helping to limit the accumulation of the deleteri-
•−
†These authors contributed equally to this work.
ous O2 and H2O2. The former plays an essential role
Appl Organometal Chem. 2018;e4297.
wileyonlinelibrary.com/journal/aoc
Copyright © 2018 John Wiley & Sons, Ltd.
1 of 15
https://doi.org/10.1002/aoc.4297

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TANG ET AL.
in conversion of superoxide anion to hydrogen peroxide
and oxygen under physiological conditions, and the latter
is able to catalyze the decomposition of hydrogen perox-
ide into water and oxygen.[8–12]
Cl0.7(NO3)1.3(OH)2(H2O)1.3]⋅6H2O (2), [Cu2(pxdiprbtacn)
Cl4] (3) and [Cu2(pxbtacn)Cl4] (4), were prepared from
p‐xylylene‐bridged ditopic bis‐tacn derivatives bearing ste-
ric alkyl substituents (pxdmbtacn = 1,4‐bis(4,7‐dimethyl‐
1,4,7‐triazacyclonon‐1‐ylmethyl)benzene, pxdiprbtacn =
1,4‐bis(4,7‐diisopropyl‐1,4,7‐triazacyclonon‐1‐ylmethyl)
benzene) or no pendant group (pxbtacn = bis(1,4,7‐
triazacyclonon‐1‐ylmethyl)benzene) (Figure 1). The
magnetic, redox and spectroscopic properties of com-
plexes 1, 3 and 4 were investigated in detail. All three
complexes can oxidatively degrade supercoiled pBR322
DNA with the help of H2O2. Furthermore, these
For a variety of diseases related to a deficiency of these
native antioxidant enzymes, the use of exogenous SODs
and catalases has been firstly considered as a feasible
therapeutic approach.[13–15] However, the immunogenic
response, short lifetime and lower tissue permeability of
these high‐molecular‐mass materials make them difficult
to be used in vivo for therapeutic application.[16,17] This
spurred researchers to look for biomimetic small‐mole-
•−
cule compounds which can degrade O2 or H2O2 effec-
complexes also exhibit catalytic activity towards the
tively as promising substitutes of native enzymes.[18–22]
Recently, numerous and diverse redox‐active transition
metal complexes based on N‐donor ligands have been
reported to exhibit marked catalytic effect on the dispro-
dismutation of O2
and H2O2 functioning as dual
•−
SOD/catalase mimics. From the results obtained from
these investigations, the different enzymatic activities
of these complexes were related to the structural fea-
tures of the bis‐tacn ligands and the solution behavior
of their complexes.
portionation of reactive O2 or H2O2 in vitro[23–27] and
•−
some pentaazamacrocyclic Mn(II) compounds as antioxi-
dant SOD mimics have found biomedical applications in
preclinical or clinical trials.[28–31] As a facially coordinated
tridentate macrocycle, 1,4,7‐triazacyclononane (tacn)
and its derivatives are apt to construct thermodynamically
stable and kinetically inert metal complexes, and some of
these compounds have manifested an ability to cleave
DNA or protein.[32–37] In the past two decades, several
Cu(II) or Mn(II) complexes containing N‐functionalized
mono‐tacn ligands with unsaturated or labile binding
sites have been reported to show a considerable antioxi-
dant effect on the scavenging of superoxide radical in
the physiological environment.[38–46] It is anticipated that
more efficacious model compounds of SODs will be
obtained from bis‐tacn (or multi‐tacn) ligands which
possess two or more active centers for the binding of
substrate. Nevertheless, a mononuclear Cu(II) complex
of a bis‐tacn ligand bearing a flexible ethylene spacer
has been prepared by Li et al., and it exhibited only a
low SOD activity due to the saturated coordination
environment around the central metal furnished by
macrocyclic nitrogen atoms.[47] This result hints that rigid
or semi‐rigid spacers should be introduced into the
bis‐tacn (or multi‐tacn) ligands to facilitate the formation
of dinuclear (or polynuclear) metal compounds. Since
SOD mimics usually produce toxic hydrogen peroxide
during the catalytic cycles by a ping‐pong mechanism,
the mimicking compounds exhibiting both SOD‐ and
catalase‐like activities are intensively pursued which
demonstrate special advantage over those merely having
SOD activity.[48–50] Therefore, there is a growing require-
ment to develop small molecular mimics showing dual
SOD/catalase activity to be used as antioxidant drugs.
In the work reported herein, four dinuclear Cu(II)
complexes, [Cu2(pxdmbtacn)Cl4] (1), [Cu2(pxdmbtacn)
2 | EXPERIMENTAL
2.1 | Materials and Methods
All reagents were obtained from commercial sources
and used without further purification. 1,4,7‐
Triazacyclononane
was
purchased
from
Ltd
Wuxi
(China).
Tongchuang Biotechnology
Co.
Tris(hydroxymethyl)aminomethane, 4‐(2‐hydroxyethyl)
piperazine‐1‐ethanesulfonic acid (HEPES), plasmid
pBR322 DNA, loading buffer (10×), agarose, ethidium
bromide (EB), xanthine, xanthine oxidase, nitroblue tetra-
zolium (NBT) and superoxide dismutase (CuZnSOD)
from bovine erythrocytes were purchased from Sigma.
All buffer solutions were prepared using triple‐distilled
water. All stock solutions were stored at 4 °C and used
within 2 days. The H2O2 stock solution was prepared by
diluting a commercial 30% aqueous solution and its
concentration was determined by iodometric titration.[51]
Elemental analyses for C, H and N were conducted
with an Elementar Vario Micro Cube elemental analyzer.
Fourier transform infrared (FT‐IR) spectra were recorded
with a PerkinElmer Spectrum One spectrometer as KBr
pellets in the 400–4000 cm−1 spectral region. Electrospray
ionization mass spectrometry (ESI‐MS) was performed
FIGURE 1 Structures of three p‐xylylene‐bridged bis‐tacn ligands
(R = H, CH3, CH(CH3)2)

TANG ET AL.
3 of 15
with a Bruker Esquire 3000 mass spectrometer. UV–visi-
ble spectra were acquired with a Varian CARY‐100
spectrophotometer. Powder X‐ray diffraction (PXRD) pat-
terns for the as‐synthesized samples were recorded using
a Rigaku D/max‐2500 V diffractometer with Cu Kα radia-
tion (λ = 1.54056 Å) at 293 K in the 2θ range from 3° to
65°. Magnetic measurements were performed with a
Quantum Design MPMS‐XL7 magnetometer in the
temperature range 2–300 K under a magnetic field of
2000 Oe. Diamagnetic corrections for sample and sample
holder were applied using Pascal's constants. Electron
spin resonance (ESR) spectra of powdered samples were
recorded at room temperature with a Bruker EMX10/12
spectrometer operating at the X‐band frequency
(9.8 GHz) and magnetic field modulation of 100 kHz.
cleavage. The experiment was repeated three times to
ensure consistency of the results.
2.4 | SOD‐Like Activity Assays
The SOD‐like activity was assessed by an indirect method
originally developed by Beauchamps and Fridovich with
some modifications.[52] Superoxide anions were generated
by the xanthine–xanthine oxidase system and measured
using the NBT method. The solutions containing xanthine
(1.0 × 10–3 M), NBT (2.5 × 10–3 M) and various concentra-
tions of the complexes were prepared with phosphate
buffer (pH = 7.4, 0.01 M) at 25 °C. Sufficient xanthine oxi-
dase was added to the solutions to produce a change in
absorbance of 0.025 min−1 at 550 nm. The NBT reduction
rate was measured in the presence and absence of various
concentrations of the investigated complexes. Each com-
plex was run in three parallel tests. The IC50 value (the
concentration of the complex to cause 50% inhibition of
NBT reduction) of each complex was acquired from the
concentration‐dependent inhibition curves and the rela-
tive activity represents the ratio of the measured activity
of the native CuZnSOD to that of each complex under
the same experimental conditions.
2.2 | Electrochemical Experiments
Cyclic voltammetry experiments were performed using a
CHI 660D system under a nitrogen atmosphere in a
three‐electrode cell with a glassy carbon working
electrode, a platinum wire auxiliary electrode and a
saturated Ag/AgCl reference electrode. The complexes
were dissolved in triple‐distilled water and their concen-
trations were adjusted to 1.0 × 10−3 M. Tetra‐n‐
butylammonium bromide (0.1 M) was used as the
supporting electrolyte and the pH of the solution was
close to 6.0. The half‐wave potential (E1/2) was calculated
as the midpoint of the anodic (Epa) and cathodic (Epc)
peak potentials: E1/2 = (Epa + Epc)/2.
2.5 | Catalase‐Like Activity Assays
The catalase‐like activity was determined using the
volumetric method. The dismutation reaction of H2O2
catalyzed by each complex was carried out at room
temperature in a three‐necked flask (250 ml) equipped
with a magnetic stirrer and closed with a rubber septum.
The flask was connected to an inverted graduated burette
filled with water. Under stirring, the required volume of
H2O2 stock solution was injected into an aqueous solution
of the complex through the septum with a microsyringe.
Volume of oxygen evolved was measured at certain time
intervals and the experimental data were plotted as a
curve describing the volume of oxygen evolved as a func-
tion of time. The initial rate of dioxygen evolution was
expressed as mol(O2) s–1 and obtained from the slope of
the curve. The turnover number kcat, the Michaelis
constant KM and kcat/KM were determined from the
double‐reciprocal Lineweaver–Burk plot obtained at a
constant catalyst concentration. The experiment was
repeated three times to ensure consistency of the results.
2.3 | DNA Cleavage Experiments
The DNA cleavage activities of the complexes were evalu-
ated via agarose gel electrophoresis, which was conducted
using the following procedure. Plasmid pBR322 DNA in
HEPES buffer (pH = 6.8) was mixed with varying concen-
trations of complexes in the absence and presence of
H2O2. The resulting mixture was incubated at 37 °C for
2 h in the dark, after which 2 μl of buffer was added to
quench the reaction. Then the DNA samples were loaded
on 2% agarose gel and electrophoresed at 70 V for 2 h in
TAE buffer. The gel was stained with 0.1% EB, visualized
by UV light and photographed. Control experiments were
carried out using Cu(II) salts or the free ligands at a
concentration of 12–24 μM in the absence and presence
of H2O2, respectively.
2.6 | X‐ray Data Collection and Structure
Determination
The cleavage mechanism of pBR322 DNA induced by
the complexes was investigated in the presence of various
radical scavengers, namely C2H5OH, dimethylsulfoxide
(DMSO), KI, NaN3, L‐histidine and SOD. All samples
were treated by the same method described for DNA
Diffraction data were collected at 296 K with graphite‐
monochromated Mo Kα radiation (λ = 0.71073 Å) using
a Bruker Smart Apex II CCD diffractometer. The SAINT

4 of 15
TANG ET AL.
program was used for integration of the diffraction pro-
files[53] and an empirical absorption correction was
applied with the SADABS program.[54] The structure
was solved by direct methods using the SHELXS program
of the SHELXTL package and refined by full‐matrix least
squares on F2 with the SHELXL program.[55] All non‐
hydrogen atoms were located in successive difference
Fourier syntheses and refined with anisotropic thermal
parameters on F2. All of the hydrogen atoms attached to
carbon atoms were placed in calculated positions and
treated as riding atoms during the refinement. The hydro-
gen atoms attached to oxygen atoms were located from
difference Fourier maps with O―H bond distances
restrained to 0.85 Å and refined isotropically with Uiso(H)
= 1.5Ueq(O). CCDC 1582386 contains the supplementary
crystallographic data for complex 2. Crystallographic data
and structural refinement details are summarized in
Table 1. Selected bond lengths and angles are collected in
Table 2. Hydrogen bond data are presented in Table S1.
2.00 g) and isopropyl bromide (25.6 mmol, 3.18 g)
sequentially. The mixture was then refluxed for 20 h
under a nitrogen atmosphere. After cooling the mix-
ture to ambient temperature, the generated KBr and
excess K2CO3 were removed by filtration. The filtrate
was washed with chloroform (2 × 50 ml), and the
combined filtrates were dried over anhydrous Na2SO4.
Evaporating the solvent under reduced pressure
yielded a pale yellow oil. Yield: 1.26 g, 74%. FT‐IR
(KBr, cm–1): 3435(m), 2962(s), 2927(s), 2871(m),
2816(m), 1663(s), 1462(m), 1384(m), 1359(m), 1300(m),
1170(m), 1116(m), 1086(m), 1057(m), 1019(m), 838(w),
760(w), 705(w), 577(w), 492(w). ESI‐MS: m/z = 529.6
[M + H]+.
TABLE 1 Crystal data and structure refinement of complex 2
Complex
2
Empirical formula
Formula weight
Temperature (K)
Wavelength (Å)
Crystal system
Space group
a (Å)
C24H60.59Cl0.70Cu2N7.30O13.18
2.7 | Synthesis of Ligands
814.36
296
2.7.1 | Bis(1,4,7‐triazacyclonon‐1‐
ylmethyl)benzene (pxbtacn)
0.71073
Monoclinic
P21/n
The ligand pxbtacn was obtained according to the
established method.[56]
8.987(3)
13.289(4)
16.446(6)
90
2.7.2 | 1,4‐Bis(4,7‐dimethyl‐1,4,7‐
triazacyclonon‐1‐ylmethyl)benzene
(pxdmbtacn)
b (Å)
c (Å)
α (°)
The oily precursor pxbtacn (2.0 mmol, 1.16 g) was
dissolved in a mixture of formic acid (10 ml) and formal-
dehyde (10 ml) with stirring. The resulting solution was
refluxed for 20 h and then cooled to room temperature.
Subsequently, 20 ml of water was poured into the solution
and its pH value was adjusted to about 12 by adding
solid NaOH. After filtration, the filtrate was extracted
with chloroform (5 × 50 ml) and the combined extracts
were dried over anhydrous Na2SO4. The solvent was
evaporated under reduced pressure to give a yellow oil.
Yield: 4.60 g, 86%. FT‐IR (KBr, cm–1): 3422(s), 2943(m),
2852(m), 2813(m), 2703(m), 1663(s), 1464(s), 1382(s),
1294(m), 1184(m), 1117(m), 1079(m), 1062(m), 1032(m),
998(m), 898(w), 856(w), 829(w), 741(w), 596(w), 556(w),
499(w). ESI‐MS: m/z = 417.3 [M + H]+.
β (°)
101.827(6)
90
γ (°)
Volume (Å3)
1922.4(11)
2
Z
Calculated density (g cm−3
Absorption coefficient (mm−1
)
1.407
)
1.22
F(000)
862
Limiting indices
−11 ≤ h ≤ 11
−16 ≤ k ≤ 16
−20 ≤ l ≤ 20
Reflections collected
Independent reflections
Data/restraints/parameters
Goodness‐of‐fit on F2
R indices [I > 2σ(I)]
16 688
3769
3769/13/238
1.13
R1 = 0.0735
wR2 = 0.1991
2.7.3 | 1,4‐Bis(4,7‐diisopropyl‐1,4,7‐
triazacyclonon‐1‐ylmethyl)benzene
(pxdiprbtacn)
R indices (all data)
R1 = 0.0999
wR2 = 0.2157
To a solution of pxbtacn (3.2 mmol, 1.16 g) in dry
acetonitrile (50 ml) was added solid K2CO3 (14.5 mmol,
Largest diff. peak and hole (e Å−3
)
0.731 to −0.511

TANG ET AL.
5 of 15
TABLE 2 Selected bond lengths (Å) and angles (°) of complex 2
826(m), 792(m), 744(m), 626(w), 579(w), 541(w), 464(w),
426(w). ESI‐MS: m/z = 703.2 [Cu2(pxdmbtacn)(NO3)2Cl]+,
728.2 [Cu2(pxdmbtacn)(NO3)3]+.
Cu1–O1
2.064(6)
2.792(12)
2.042(6)
2.295(5)
87.3(4)
Cu1–O2
Cu1–N1
Cu1–N3
1.907(8)
2.194(4)
2.069(6)
Cu1–O4
Cu1–N2
Cu1–Cl1a
2.8.3 | [Cu2(pxdiprbtacn)Cl4] (3)
O1–Cu1–O2
O1–Cu1–N1
O1–Cu1–N3
O2–Cu1–N1
O2–Cu1–N3
O4–Cu1–N2
N1–Cu1–N2
N2–Cu1–N3
N1–Cu1–Cl1a
N3–Cu1–Cl1a
O1–Cu1–O4
O1–Cu1–N2
O2–Cu1–O4
O2–Cu1–N2
O4–Cu1–N1
O4–Cu1–N3
N1–Cu1–N3
O1–Cu1–Cl1a
N2–Cu1–Cl1a
92.6(3)
176.9(2)
77.7(4)
90.1(4)
166.2(3)
85.2(3)
84.5(2)
77.7(2)
99.2(2)
A methanolic solution (5 ml) of CuCl2⋅2H2O (0.20 mmol,
0.0341 g) was added dropwise to a methanol solution
(5 ml) of pxdiprbtacn (0.10 mmol, 0.0528 g). The resulting
reaction mixture was kept at reflux for 3 h with stirring.
After cooling, the solution was left at room temperature
for slow evaporation and a green powder was precipitated
after one day. The solid product was filtered off, washed
several times with ether and dried in air. Yield: 0.062 g
(78% based on metal precursor). FT‐IR (KBr, cm–1):
3437(s), 2970(m), 2835(m), 1639(m), 1489(m), 1446(m),
1385(m), 1294(w), 1215(w), 1140(m), 1099(m), 1075(m),
1015(m), 983(m), 949(w), 865(w), 786(m), 731(m),
97.5(2)
95.6(3)
112.0(3)
162.8(3)
85.1(3)
85.1(2)
86.4(3)
155.7(2)
119.6(2)
aThe minor part of the molecule.
622(m), 526(m), 438(w). ESI‐MS: m/z
=
363.1
[Cu2(pxdiprbtacn)Cl2]2+, 761.3 [Cu2(pxdiprbtacn)Cl3]+.
2.8 | Synthesis of Cu(II) Complexes
2.8.1 | [Cu2(pxdmbtacn)Cl4] (1)
2.8.4 | [Cu2(pxbtacn)Cl4] (4)
The complex was prepared from CuCl2⋅2H2O (0.20 mmol,
0.0341 g) and pxbtacn (0.10 mmol, 0.0360 g) following a
reported procedure.[57] Yield: 0.054 g (86% based on metal
precursor). FT‐IR (KBr, cm–1): 3456(s), 3267(s), 2933(m),
2848(m), 1631(m), 1489(m), 1453(m), 1377(m), 1348(m),
1283(m), 1235(w), 1150(w), 1096(m), 1004(s), 943(m),
893(m), 863(m), 827(m), 766(m), 731(m), 628(m),
A methanol solution (5 ml) of CuCl2⋅2H2O (0.20 mmol,
0.0341 g) was mixed with a methanol solution (5 ml) of
pxdmbtacn (0.10 mmol, 0.0416 g) with stirring. Then the
mixture was stirred for a further 2 h under reflux to give a
clear blue solution. The solution was filtered, and the
filtrate was allowed to evaporate under room temperature
until a green powder was obtained. The solid product was
washed several times with ether and dried in air. Yield:
0.051 g (74% based on metal precursor). FT‐IR (KBr, cm–1):
3517(s), 3434(s), 2923(m), 2866(m), 2823(m), 1598(s),
1457(m), 1345(m), 1215(w), 1158(w), 1069(m), 1002(m),
862(m), 787(m), 702(m), 526(m), 507(m), 424(m). ESI‐
581(m), 493(w), 428(w). ESI‐MS: m/z
=
521.1
[Cu2(pxdtacn‐2H)Cl]+, 593.1 [Cu2(pxdtacn)Cl3]+, 1223.1
[Cu4(pxdtacn)2Cl7]+. The phase purity of the as‐prepared
product was confirmed by the good match of its experi-
mental PXRD pattern with that simulated from the
reported crystal structure (Figure S1).
MS: m/z = 307.1 [Cu2(pxdmbtacn)Cl2]2+
.
3 | RESULTS AND DISCUSSION
3.1 | Synthesis
2.8.2 | [Cu2(pxdmbtacn)
Cl0.7(NO3)1.3(OH)2(H2O)1.3]⋅6H2O (2)
Upon mixing CuCl2⋅2H2O (0.20 mmol, 0.0341 g) and
pxdmbtacn (0.10 mmol, 0.0416 g) in a mixture of metha-
nol (5 ml) and water (5 ml), aqueous NaOH (2 M) was
added to adjust the pH of the mixture to 7.0. The reaction
mixture was stirred for a further 3 h under reflux,
followed by the addition of four equivalents of KNO3.
The solution was filtered and left to evaporate under
ambient conditions. Blue plate‐shaped single crystals suit-
able for X‐ray analysis were grown within two weeks.
Yield: 0.048 g (61% based on metal precursor). FT‐IR
(KBr, cm–1): 3448(s), 2929(m), 2874(m), 1635(m),
1384(s), 1158(w), 1126(w), 1062(m), 998(m), 901(w),
The powder products of three chloride complexes were
prepared by treating CuCl2⋅2H2O and bis‐tacn ligands in
methanol using a molar ratio of 2:1. Unfortunately, no
single crystals of these complexes suitable for X‐ray struc-
tural analysis could be successfully grown by evaporating
their methanolic solutions in air. In order to facilitate the
crystallization of dinuclear Cu(II) complexes, four equiva-
lents of KNO3 were introduced into the solutions of three
chloride complexes separately and only blue plate crystals
of complex 2 containing partially occupied chloride and
nitrate groups were separated from the mother liquid.
The physical properties and elemental analysis data for

6 of 15
TANG ET AL.
three bis‐tacn ligands and their dinuclear Cu(II) com-
plexes are presented in Table 3.
is thus distorted square pyramidal with one chloride
group occupying the basal plane. The Cu1―Cl1 bond
length of 2.295(5) Å is normal for such a bond in bis‐tacn
complexes[60,61] but longer than those of other bonds
around Cu(II). The whole dinuclear unit is centrosym-
metric with the inversion center at the midpoint of the
p‐xylylene spacer. Due to the anti conformation assumed
by the ditopic ligand pxdmbtacn, two Cu(II) ions within
a dinuclear entity are separated from each other by a dis-
tance of 11.741 Å, which is close to the Cu⋅⋅⋅Cu separation
of 11.81 Å found in the structure of [Cu2(pxbtacn)Cl4].[57]
3.2 | Crystal Structure of Complex 2
Single crystals of complex 2 belong to the monoclinic
system, space group P21/n. The asymmetric unit of the
complex is composed of one half of a dinuclear unit and
three crystallization water molecules. As depicted in
Figure 2, each of the two Cu(II) ions is facially coordi-
nated with three nitrogen atoms (N1, N2 and N3) from
tacn backbone and the remaining binding sites around
Cu(II) are completed by one hydroxide ion (O1) together
with one or more other labile ligand/ligands (either an
aqua ligand (O2) and a nitrate ion or a chloride group
(Cl1) with an occupancy ratio of 0.65:0.35). When a
hydroxide ion, a water molecule and a nitrate ion reside
at the labile sites, the coordination geometry around
Cu(II) can be regarded as axially elongated octahedral in
which the basal plane is defined by two methyl‐
substituted tacn nitrogen atoms as well as two oxygen
atoms provided by both hydroxide and aqua ligands. A
tacn nitrogen atom connected to a bulky p‐xylylene group
and a weakly bound nitrate ion occupy the two apical
positions. The bond lengths between central Cu(II) and
apically coordinated N1 and O4 atoms are 2.194(6) and
2.792(6) Å, respectively. Compared with them, the basal
bond distances are much shorter with an average value
of 2.021 Å. Except for the markedly larger Cu―O (nitrate)
bond length, other corresponding Cu―N and Cu―O
bond lengths fall in the normal range expected for octahe-
dral Cu(II) complexes with tacn ligands.[58,59] The cis and
trans bond angles about the Cu(II) center range from
77.7(4)° to 112.0(3)° and from 162.8(3)° to 176.9(2)°,
respectively. Some bond angles deviate significantly from
the ideal octahedral angles of 90° and 180°, revealing the
moderate distortion of the Cu(II) sphere. While a chloride
group acts as the labile ligand bound at Cu(II) in place of
a water molecule and a nitrate, the geometry about Cu(II)
3.3 | ESR Spectroscopy and Magnetic
Properties
The ESR spectrum and magnetic properties of complex 4
have been thoroughly investigated by Spiccia and co‐
workers,[57] and thus these magnetic characterizations
were only conducted for complexes 1 and 3. The solid‐
state X‐band ESR spectra of complexes 1 and 3 recorded
at room temperature are shown in Figure 3. Both spectra
exhibit two broad signals in the ΔMs = 1 region and no
half‐field transition could be detected at ca 1600 G. The
analysis of the resonance spectra yields the following
ESR parameters: g|| = 2.183, g⊥ = 2.077 and g|| = 2.189,
g⊥ = 2.091 for 1 and 3, respectively. These spectra are of
axial type (g|| > g⊥ > 2.0023) with the unpaired electron
2
2
localized mainly in the dx −y orbital of Cu(II) in square
pyramidal geometry. The observed g|| values for the two
complexes are less than 2.3, in agreement with the cova-
lent character of the metal–ligand bonds.[62]
Magnetic susceptibility measurements were per-
formed for complexes 1 and 3 using powdered samples
in the temperature range 2–300 K. The magnetic behav-
iors in the form of χm and χmT versus T plots are shown in
Figure 4. The observed χmT value of 1 at 300 K is
0.905 cm3 K mol–1, which is larger than the theoretical
spin‐only value of 0.75 cm3 K mol–1 expected for two
TABLE 3 Physical properties and elemental analysis of three bis‐tacn ligands and their dinuclear Cu(II) complexes
Calcd (found)
Yield
(%)
μeff
(BM)
Compound
Formula
Color
C (%)
H (%)
N (%)
pxdmbtacn
C24H44N6
86%
74%
62%
74%
61%
78%
86%
Yellow
Yellow
Yellow
Green
Blue
—
69.23 (69.15)
72.67 (72.64)
66.63 (66.87)
42.05 (41.95)
35.40 (35.32)
48.18 (48.18)
38.16 (38.49)
10.58 (10.63)
11.44 (11.40)
10.06 (10.34)
6.47 (6.79)
7.50 (7.41)
7.58 (7.46)
5.76 (5.91)
20.19 (20.64)
15.89 (15.95)
23.31 (23.16)
12.26 (12.03)
12.56 (12.49)
10.53 (10.61)
13.35 (13.18)
pxdiprbtacn
C32H60N6
—
pxbtacn
C20H36N6
—
1
2
3
4
C24H44Cl4Cu2N6
C24H60.59Cl0.70Cu2N7.30O13.18
C32H60Cl4Cu2N6
C20H36Cl4Cu2N6
2.69
—
Green
Green
2.59
2.47

TANG ET AL.
7 of 15
The obtained values for each complex compare well with
those reported for complex 4.[57] The magnetic coupling
seems impossible to be transferred intramolecularly
across the long p‐xylylene spacer but more likely to be
mediated intermolecularly via a hydrogen bonding path-
way between neighboring molecules.
FIGURE 2 Coordination environments of Cu(II) atoms in
complex 2. All hydrogen atoms except those on the coordinated
hydroxide groups and water molecules are omitted for clarity. The
dashed lines represent the minor part of the disordered components
(symmetry code: A 1 − x, 1 − y, 2 − z)
3.4 | FT‐IR Spectra
The FT‐IR spectra of three bis‐tacn ligands and their dinu-
clear Cu(II) complexes are shown in Figure S2 and some
significant absorption bands are listed in Table 4, where
the characteristic vibrations of the ligands before and after
complexation are compared. The medium intensity bands
observed for the free ligands at 1079–1113 cm–1 can be
assigned to ν(C―N) stretching. In the spectra of the
complexes, these bands are shifted towards the lower
wavenumber side by about 10–20 cm–1, showing the coor-
dination of the ligands to Cu(II) ions occurs through the
macrocyclic nitrogen donors. This is further supported
by the appearance of new bands at 424–438 cm–1, which
can be attributed to ν(M―N) vibrations.[64] For the free
uncoupled S = 1/2 Cu(II) ions. As the temperature is
lowered, the χmT value decreases smoothly above 14 K
and then drops abruptly to 0.223 cm3 K mol–1 at 2 K. Such
a behavior indicates the existence of an antiferromagnetic
coupling in 1. Similar magnetic behavior was also
observed for 3. Its χmT value is 0.837 cm3 K mol–1 at room
temperature and decreases to a minimum value of
0.205 cm3 K mol–1 at 2 K. The magnetic susceptibility data
for both complexes were fitted by a modified Bleaney–
Bowers equation including
a correction term for
paramagnetic impurity[63] based on the spin Hamiltonian
H = −2JS1S2:
À
Á
χm ¼ 2Ng2β2 ð1−ρÞ=kT½3 þ expð−2 J=kTÞꢀ
À
Á
þ Ng2β2 ρ=2kT þ TIP
(1)
where N, g, β, k and T have their usual meanings, 2 J is the
coupling constant between two Cu(II) ions, ρ is the molar
fraction of monomeric impurity and TIP denotes the tem-
perature‐independent paramagnetism. The best fit of the
χmT data led to g = 2.11, 2 J = −5.20 cm–1, ρ = 0.17, TIP
= 2.68 × 10–4 cm3 mol–1 for 1 and g = 2.06, 2 J =
−7.40 cm–1, ρ = 0.17, TIP = 1.83 × 10–4 cm3 mol–1 for 3.
FIGURE
3
X‐band ESR spectra of powdered samples of
FIGURE 4 Plots of χm (▪) and χmT (•) versus T for complexes 1
(a) and 3 (b), where the solid lines represent the fitted data
complexes 1 and 3 measured at room temperature

8 of 15
TANG ET AL.
TABLE 4 Significant absorption peaks in the FT‐IR spectra of three bis‐tacn ligands and their dinuclear Cu(II) complexesa
Compound
ν(N―H)
δ(N―H)
ν(NO3)
ν(C―N)
ν(M―O)
ν(M―N)
pxdmbtacn
1079m
1086m
1113m
1069m
1062m
1075m
1096m
pxdiprbtacn
pxbtacn
3290b
1492m
1
2
3
4
424m
426w
438w
428w
1384s
541w
3267s
1489m
ab, broad; s, strong; m, medium; w, weak.
ligand pxbtacn, the characteristic absorption bands at
3290 and 1490 cm–1, due to the N―H stretching and
bending vibrations of the secondary amine groups, respec-
tively, are shifted to 3267 and 1489 cm–1 in the spectrum
of its complex. This observation also indicates the involve-
ment of the macrocyclic nitrogen atoms in coordination.
The presence of nitrate ion in 2 is evidenced by a strong
peak centered at 1384 cm–1; meanwhile the observation
of ν(M―O) band at 541 cm–1 confirms the binding of
H2O/OH– group to the central Cu(II) ions in this
complex.[65]
Since the biological activities of the metal complexes
are strongly dependent on their existing forms in solution,
the solution stability of complexes 1 and 3 in phosphate
buffer at pH = 7.4 was evaluated using UV–visible spec-
troscopy. As depicted in Figure S3, within a 48 h period,
the intensity of the d–d transition band of Cu(II) for both
complexes decreases slightly and no shift of the absorp-
tion maximum occurs, providing evidence that for these
complexes the coordination sphere around central Cu(II)
remains intact in the neutral buffer medium. Further-
more, the stability of complex 3 in alkaline solution was
determined by monitoring its UV–visible spectra in the
pH range from 7 to 12. With the increase of basicity, the
absorption band due to the d–d transition of Cu(II)
undergoes a minor hypsochromic shift from 652 to
641 nm accompanied by an irregular change of the band
intensity. The slight change of the metal‐centered band
implies that there are no significant structural and elec-
tronic properties changes in Cu(II) centers. Therefore, it
is expected that the complex can still exist in alkaline
solution as a dinuclear species although the removable
small molecules around Cu(II) might be varied. This
3.5 | UV–Visible Spectra
By examining the solid‐state UV–visible spectra of the
three dinuclear Cu(II) complexes 1, 3 and 4 with those
of their respective ligands (Figure 5), a medium strong
absorption band is observed in the spectral region from
240 to 400 nm for each of the three complexes (277, 271
and 258 nm for 1, 3 and 4, respectively) but which is not
conspicuous in the spectra of the respective ligands. Thus,
this band is interpreted as the ligand‐to‐metal charge
transfer process from either the chloride or the macrocy-
clic nitrogen donors to the square pyramidal Cu(II) ion.
The characteristic d–d transition of the central d9 Cu(II)
ion, as expected, emerges at 675, 711 and 657 nm for 1,
3 and 4, respectively. The UV–visible spectra of complexes
1 and 3 recorded in water are similar to their solid‐state
spectra although the ligand‐to‐metal charge transfer band
and the Cu(II) d–d transition are separately shifted to lon-
ger wavelengths (288 and 298 nm for 1 and 3, respec-
tively) and shorter wavelengths (632 and 660 nm for 1
and 3, respectively). However, on dissolving in water the
two bands of 4 are blue‐shifted to 252 and 639 nm, respec-
tively. The shift of the electronic absorption bands can
probably be ascribed to the different coordination envi-
ronment of Cu(II) in aqueous solution compared to the
solid state, which results from the substitution of chloride
bound to Cu(II) by other small ligands such as water or
hydroxide ion.
FIGURE
5
Solid‐state UV–visible absorption spectra of
complexes 1, 3 and 4 and their respective ligands

TANG ET AL.
9 of 15
conclusion was further confirmed by the mass spectro-
metric analysis.
The ratio of the peak currents deviates from unity
and the peak‐to‐peak separations are larger than
300 mV, indicative of the irreversibility of these two redox
processes. Complex 3 undergoes two stepwise reductions
in the potential range from 1.6 to −0.8 V. The first wave
with E1/2 of 0.218 V (versus Ag/AgCl) seems to be
quasi‐reversible with a separation between the potential
peaks of 171 mV, whereas the second one with E1/2 of
−0.283 V (versus Ag/AgCl) is characteristic of an irrevers-
ible electrochemical process since the peak‐to‐peak
separation approaches 400 mV. The determined redox
potentials for these two Cu(II) complexes compare well
with those found for other dinuclear Cu(II) complexes.[66]
The observed two redox processes might be attributed
to the successive one‐electron transfer reduction
of the dinuclear Cu(II) moieties given in the form of
equations 2 and 3:
3.6 | ESI‐MS Analysis
ESI‐MS was used to investigate the structural stability of
the dinuclear motif of complexes 1, 3 and 4 in their aque-
ous solutions. Only one major peak at m/z = 307.09
(100%) is observed in the positive‐ion mode ESI‐MS spec-
trum of complex 1, which can be assigned to the dication
[Cu2(pxdmbtacn)Cl2]2+. The mass spectrum of 2 shows
two major singly charged ions [Cu2(pxdmbtacn)
(NO3)2Cl]+ and [Cu2(pxdmbtacn)(NO3)3]+ at m/z =
703.17 (100%) and 728.17 (79%), respectively. Complex 3
dissolved in water exhibits two dominant peaks at m/z =
363.14 (100%) and 761.25 (34%), which correspond to
the dinuclear fragment ions [Cu2(pxdiprbtacn)Cl2]2+
and [Cu2(pxdiprbtacn)Cl3]+, respectively. When the pH
of this solution was adjusted to 10 by adding
aqueous NaOH, both peaks disappear and new
signals are seen at m/z = 410.15 (100%) and 797.35
(49%). These peaks can be attributed to the doubly
charged ions [Na2Cu2(pxdiprbtacn))(CO3)2]2+ and
[NaCu2(pxdiprbtacn))(CO3)2]+ each containing two
CO32– anions, where one CO32– ion is in place of two chlo-
ride anions and involved in chelation to one central Cu(II)
CuIICuII þ e−→CuIICuI
CuIICuI þ e−→CuICuI
(2)
(3)
Since at least one of the half‐wave potentials of the
two redox processes for complexes 1 and 3 lies between
2–
ion. The source of CO3 may be tentatively ascribed to
•−
capture of atmospheric CO2 by the alkaline solution.
The experimental isotopic distribution patterns of these
peaks match well with the calculated ones. For complex
4, four monocationic peaks are observed in the m/z range
500–800 with the dominant peak centered at m/z = 593.06
(100%) being the dinuclear cation [Cu2(pxbtacn)Cl3]+.
One further peak with lower intensity due to the dimeric
species [Cu4(pxbtacn)2Cl7]+ can be seen at m/z = 1223.09
(64%). The result indicates that 4 may partially dimerize
into a tetranuclear species in aqueous solution in addition
to existing as a dinuclear molecule. The mass spectra of
complexes 1, 3 and 4 are presented in Figure S4 and
the insets show the experimental and calculated
isotopic distribution patterns of some major ions in the
respective spectra.
the reduction potentials corresponding to O2/O2
(+0.02 V versus Ag/AgCl) and O2•−/H2O2 (+1.11 V versus
Ag/AgCl) redox couples in neutral solution, these
complexes are considered to be competent SOD mimick-
ing systems capable of cleaning the superoxide radical.[10]
3.8 | DNA Cleavage
The chemical nuclease activities of complexes 1 and 3
towards supercoiled pBR322 DNA were investigated by
gel electrophoresis in the absence and presence of H2O2
under near‐physiological conditions (pH = 6.8). Although
complex 4 cannot exist merely as dinuclear species in
aqueous solution, for a comparison the plasmid DNA
scission activity of complex 4 in HEPES buffer solution
was also evaluated, with its initial concentration and
other experimental conditions being identical to those
used in the assays for other two complexes. The DNA
cleavage efficiency was assessed by determining the abil-
ity of these complexes to convert the plasmid DNA from
the original supercoiled form (Form I) to the open circular
(nicked) form (Form II) and the linear form (Form III).
The extent of cleavage for pBR322 DNA in the
presence of the three complexes without the redox agent
could be ignorable, which indicates that all three com-
plexes do not exhibit detectable nuclease activity towards
3.7 | Cyclic Voltammetry
The redox behavior of complexes 1 and 3 was investigated
via cyclic voltammetry in a three‐electrode system
equipped with saturated Ag/AgCl reference electrode.
Figure S5 shows the representative voltammograms of
two complexes in aqueous solution at room temperature.
The cyclic voltammetry curve of 1 reveals two reduction
waves with E1/2 of 0.659 and −0.862 V versus Ag/AgCl
electrode during the cathodic scan from 2.0 to −1.0 V.

10 of 15
TANG ET AL.
supercoiled DNA individually. Due to the redox activity of
Cu(II) ion, most of the reported Cu(II)‐based DNA
cleavage agents act via an oxidative mechanism. In the
oxidative degradation of plasmid DNA, H2O2 is com-
monly used as a reducing reagent to generate ROS, i.e.
hydroxyl radical, singlet oxygen, superoxide anion as well
as reactive metal–oxo adducts, under the mediation of
Cu(II) complexes. One or more of them is/are responsible
for the subsequent chemical cleavage of the supercoiled
plasmid DNA. Given those finding, the nuclease activities
of the three Cu(II) complexes were assayed with the aid of
H2O2. As shown in Figure 6, complexes 1 and 3 can
effectively cleave supercoiled pBR322 DNA to open circu-
lar and linear forms even if their concentrations are as
low as 1 μM, but the cleavage of DNA by 4 only can be
observed when its concentration is higher than 2 μM.
The supercoiled DNA (Form I) can almost be completely
converted into degraded forms catalyzed by one of the
three complexes at a concentration of 8, 16 and 20 μM
for 1, 3 and 4, respectively. Meanwhile linear DNA (Form
III) begins to appear when the concentration of 1 is 6 μM
and also as it is raised to 12 and 20 μM for 3 and 4,
respectively. It is evident that complex 1 exhibits higher
nuclease efficiency than 3 and 4. Furthermore, the
plasmid DNA cleavage processes mediated by the three
complexes are all concentration‐dependent ones.
To determine which ROS is responsible for the oxida-
tive degradation of plasmid DNA in the H2O2‐containing
cleavage systems discussed above, various ROS scaven-
gers,[67] namely diffusible hydroxyl radical scavengers
(DMSO and ethanol), singlet oxygen scavengers (L‐histi-
dine and NaN3), superoxide scavenger (SOD) and H2O2
scavenger (KI), were introduced into the reaction systems
before the addition of the said complexes under the same
cleavage conditions. As can be seen in Figure 7, the cleav-
age activity of all complexes is inhibited markedly in the
presence of H2O2 scavenger (KI), making clear the crucial
role of H2O2 in such oxidative cleavage. Addition of L‐his-
tidine into the cleavage system also inhibits the DNA
cleavage to some degree for all catalysts, evidencing the
possibility that singlet oxygen species may be involved in
the cleavage process. For 1 and 4, the superoxide
FIGURE 6 Cleavage of pBR322 DNA mediated by complexes 1
(a), 3 (b) and 4 (c) at various concentrations in HEPES buffer after
2 h incubation at 37 °C. (a) Lanes 1–7: DNA + 0.50 mM H2O2 + 1
([1] = 1, 2, 4, 6, 8, 10 and 12 μM); lane 8: DNA + 0.50 mM H2O2 + 12
μM CuCl2; lane 9: DNA + 0.50 mM H2O2 + 12 μM pxdmbtacn; lane
10: DNA control. (b) Lanes 1–7: DNA + 0.50 mM H2O2 + 3 ([3] = 1,
2, 4, 8, 12, 16 and 20 μM); lane 8: DNA + 0.50 mM H2O2 + 20 μM
CuCl2; lane 9: DNA + 0.50 mM H2O2 + 20 μM pxdiprbtacn; lane 10:
DNA control. (c) Lanes 1–7: DNA + 0.50 mM H2O2 + 4 ([4] = 2, 4, 8,
12, 16, 20 and 24 μM); lane 8: DNA + 0.50 mM H2O2 + 24 μM
CuCl2; lane 9: DNA + 0.50 mM H2O2 + 24 μM pxbtacn; lane 10:
DNA control
FIGURE 7 Cleavage of pBR322 DNA by 20 μM of complexes 1
(a), 3 (b) and 4 (c) and different inhibitors in HEPES buffer after
2 h incubation at 37 °C. (a) Lanes 1–6: DNA + 0.50 mM H2O2 + 1 +
different inhibitors (1 mM KI, 1 mM C2H5OH, 1 mM NaN3, 1 mM
DMSO, 20 U ml–1 SOD and 1 mM L‐histidine); lane 7: DNA control.
(b) Lanes 1–6: DNA + 0.50 mM H2O2 + 3 + different inhibitors
(1 mM KI, 1 mM C2H5OH, 1 mM NaN3, 1 mM DMSO, 20 U ml–1
SOD and 1 mM L‐histidine); lane 7: DNA control. (c) Lanes 1–6:
DNA + 0.50 mM H2O2 + 4 + different inhibitors (1 mM KI, 1 mM
C2H5OH, 1 mM NaN3, 1 mM DMSO, 20 U ml–1 SOD and 1 mM L‐
histidine); lane 7: DNA control

TANG ET AL.
11 of 15
3.9 | SOD‐Like Activity
The SOD activities of the three Cu(II) complexes were
measured following an indirect method by utilizing NBT
as a superoxide anion indicator,[68] which is based on
the kinetic competition between the mimic and NBT for
reacting with O2•–. In this indirect method, SOD activity
is inversely related to the amount of formazan formed
by the reaction of superoxide anion with NBT, which is
spectrally detected at 550 nm. The reduction of NBT was
examined after the introduction of each of the three
Cu(II) complexes to the reaction system under physiolog-
ical conditions (pH = 7.4) and the plots of absorbance at
550 nm against time in the presence of varying concentra-
tions of Cu(II) complexes are shown in Figure 8. The
inhibition percentage of NBT reduction was determined
spectrophotometrically for each complex and the catalytic
activity was evaluated by the inhibitor concentration for
50% inhibition of NBT reduction (IC50) (Figure 9). It is
clear that complexes 1, 3 and 4 can efficaciously inhibit
the reduction of NBT at physiological pH and the
SOD‐like activities of these Cu(II) complexes are found
to decrease in the order: 3 > 1 > 4. The IC50 values of
the three mimic complexes are summarized in Table 5
and compared to those of native CuZnSOD, and some
azamacrocyclic mononuclear and dinuclear Cu(II) com-
pounds reported in the literature. It can be seen that the
first two complexes described in the present work are
among the most active Cu(II) mimic compounds known.
Their remarkable SOD‐like activities may be ascribed to
the convenient substitution of labile Cl– or other small
•–
ligands bound at Cu(II) active sites by the substrate O2
during the catalytic process. The SOD‐like activity of 4 is
about 3–4 times lower than those of complexes 1 and 3.
The lower activity of 4 is due to the fact that the complex
FIGURE 8 Absorbance values (Abs550) as a function of time
plotted for varying concentrations of complexes 1 (a), 3 (b) and 4 (c)
scavenger (SOD) also prevents the DNA degradation par-
tially; thus the involvement of superoxide anion could not
be ruled out for the oxidative cleavage system containing
1 or 4. From these results, we can concluded that the
DNA cleavage mediated by these complexes may follow
an oxidative mechanism in which reactive Cu(I) species
deriving from the Cu(II) precursors is likely favorable
for the generation of ROS.
FIGURE
9
Inhibition percentage of NBT reduction with
increasing concentration of complexes 1, 3 and 4

12 of 15
TANG ET AL.
TABLE 5 SOD‐like activities of complexes 1, 3 and 4, native CuZnSOD and some reported azamacrocyclic Cu(II) model complexesa
System
IC50 (μM)
Experimental method
Ref.
1
0.087
0.068
0.25
0.015
0.90
0.76
0.32
3.3
McCord–Fridovich (NBT)
McCord–Fridovich (NBT)
McCord–Fridovich (NBT)
McCord–Fridovich (NBT)
Riboflavin (NBT)
This work
This work
This work
3
4
Native CuZnSOD
[Cu(L1)Cl]ClO4
This work
[39]
[Cu(L2)CH3CN](ClO4)2⋅H2OClO4
[CuCl(L3)]⋅PF6⋅EtOH
[Cu(L4)]⋅(ClO4)2⋅CH3CH2OH
[Cu2HL5Br2](ClO4)3⋅1.5H2O
[(CuimCu)L6]ClO4⋅0.5H2O
Cu2L7
Riboflavin (NBT)
[39]
[43]
[47]
[69]
[70]
[71]
[72]
Riboflavin (NBT)
Riboflavin (NBT)
2.93
0.62
0.11
1.96
McCord–Fridovich (NBT)
McCord–Fridovich (NBT)
McCord–Fridovich (NBT)
Riboflavin (NBT)
[Cu2L8(im)](ClO4)3⋅4H2O
aL1 = 1‐(benzimidazole‐2‐ylmethyl)‐1,4,7‐triazacyclononane, L2 = 1,4‐bis(benzimidazole‐2‐ylmethyl)‐1,4,7‐triazacyclononane, L3 = 1‐carboxylic acid ethyl ester‐
4,7‐bis(R)‐1,4,7‐triazacyclononane, L4 = 1,2‐bis(1,4,7‐triazacyclonon‐1‐yl)ethane, L5 = 6‐[6′‐[3,6,9‐triaza‐1‐(2,6‐pyridina)cyclodecaphan‐6‐yl]‐3‐azahexyl]‐3,6,9‐
triaza‐1‐(2,6‐pyridina)cyclodecaphane, L6
= =
3,6,9,16,19,22‐hexaaza‐6,19‐bis(2‐hydroxyethyl)tricyclo[22,2,2,211,14]triaconta‐1,11,13,24,27,29‐hexaene, L7
3,7,11,15,19,23‐hexaaza‐1(2,6)‐pyridinacyclotetracosaphane, L8
1,11,13,24,27,29‐hexaene.
=
3,6,9,16,19,22‐hexaaza‐6,19‐bis(1H‐imidazol‐4‐ylmethyl)tricyclo[22,2,2,2,11,14]triaconta‐
is subject to dimerization in solution, and the resultant
dimer has fewer labile sites and enhanced steric
hindrance at active centers, which is unfavorable for the
binding of substrate (O2•–). Since the tertiary amine nitro-
gen atoms of the bis‐tacn ligands present in complexes 1
and 3 are respectively substituted by methyl or isopropyl
groups besides xylylene spacer, the dimerization of the
dinuclear components can be effectively blocked in their
solutions due to the steric substituents.
Considering the much lower molecular weights of
complexes 1 and 3 with respect to that of the native
CuZnSOD enzyme and the comparable IC50 values of
0.087 and 0.068 μM of two complexes with that of the
native enzyme (0.015 μM) (relative activity of 16.8 and
21.5% for complexes 1 and 3, respectively), these two
complexes may be regarded as promising SOD mimics.
3.10 | Catalase‐Like Activity
H2O2 is one of the products during the disproportionation
of O2•– catalyzed by SOD mimics and it also exhibits a del-
eterious effect on living organisms in relatively higher
concentrations. It is more attractive if these mimicking
complexes can exhibit both SOD and catalase activities;
thus the catalase‐like activities of the three complexes
FIGURE 10 Initial rate (v0) of H2O2 dismutation as a function of
concentration of complexes 1, 3 and 4 at a constant concentration of
H2O2 (200 mM)
FIGURE 11 Initial rate (v0) of H2O2 dismutation as a function of
concentration of H2O2 at a constant concentration of complexes 1, 3
and 4 (2 mM)

TANG ET AL.
13 of 15
skeleton can effectively hinder the dimerization of the
dinuclear complexes 1 and 3 whereas complex 4 is able
to form a dimeric tetranuclear cluster in aqueous solution.
Complexes 1, 3 and 4 render effective oxidative cleavage
of supercoiled plasmid DNA in the presence of H2O2.
The SOD‐ and catalase‐like activities of complexes 1 and
3 are comparable to that of native CuZnSOD and signifi-
cantly higher than that of complex 4. The lower enzy-
matic activity of complex 4 is derived from the fact that
its dimeric species generated in solution has fewer labile
coordination sites and increasing steric hindrance around
active centers, which are unfavorable for the binding
of substrate.
FIGURE 12 Double reciprocal Lineweaver–Burk plots for
complexes 1, 3 and 4
ACKNOWLEDGMENTS
This work was financially supported by the National Nat-
ural Science Foundation of China (grant no. 21361004),
the Guangxi Natural Science Foundation of China (grant
nos. 2014GXNSFAA118044, 2017GXNSFAA198125) and
the Natural Science Foundation of Key Laboratory for
Chemistry and Molecular Engineering of Medicinal
Resources (Guangxi Normal University), Ministry of Edu-
cation of China (CMEMR2012‐A08, CMEMR2013‐C08).
were investigated in water by volumetric measurement of
the evolved oxygen.
At a set of constant concentrations of complex and
H2O2, the time courses of oxygen evolution in the pres-
ence of complexes 1, 3 and 4 are depicted in Figure S6.
Both complexes 1 and 3 show a moderate catalytic activity
towards the disproportionation of H2O2 but complex 4 is
much less active. Again, this can be interpreted by the fact
that the partial dimerization of this complex leads to
tetranuclear species leaving fewer binding sites for
substrates. When the H2O2 concentration was kept at
200 mM, the initial rate of H2O2 dismutation shows a
linear variation with the concentration of all mimic com-
plexes (Figure 10). While at a constant complex concen-
tration of 2 mM, all complexes exhibit saturation
kinetics with respect to increasing concentration of
H2O2, suggesting a Michaelis–Menten‐type behavior
(Figure 11). The kinetic parameters for H2O2 dispropor-
tionation were estimated by fitting the experimental data
to the Michaelis–Menten equation (Figure 12) and the
values of kcat, KM and kcat/KM are 1.06 s–1, 1.50 M and
0.71 M–1 s–1 for 1, 1.24 s–1, 1.49 M and 0.83 M–1 s–1 for 3
and 0.24 s–1, 1.32 M and 0.18 M–1 s–1 for 4. The catalytic
efficiency of complexes 1, 3 and 4 is higher than those
measured for several reported dicopper(II) mimic com-
pounds in the absence of heterocyclic base.[73,74]
ORCID
Zhong Zhang http://orcid.org/0000-0002-8445-0732
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