Effect of coordination sphere of the copper center and Cu-?Cu distance on catechol oxidase and nuclease activities of the copper complexes

Article abstract of DOI:10.1002/aoc.3138

To explore the effect of Cu-?Cu distance in the structure of copper complexes on their catechol oxidase and nuclease activity, six copper complexes with a similar coordination sphere but different Cu-?Cu distances were synthesized and characterized with elemental analysis, single-crystal X-ray diffraction, molar conductivity measurements, IR and UV-visible spectroscopy. Complex 1 is a binuclear copper complex and complex 4 is a polynuclear complex with a Z-chain structure, as evidenced by their crystal structures. Complementary characterizations showed that complexes 2 and 3 have a similar binuclear structure to the complex 1; and complexes 5 and 6 are analogous to complex 4. The catechol oxidase activity of complexes 1, 2, 3 is quite akin to that of 4, 5, 6, suggesting that the catechol oxidase activity of the complexes was determined by the coordination environment of the copper center, when Cu-?Cu distance is large. In contrast, DNA cleavage activity of the complexes 1, 2 and 3 are much higher than that of 4, 5 and 6, indicating that the planar ligand structure in the complexes 4, 5 and 6 is more critical than the copper coordination sphere and the Cu-?Cu distance for their nuclease activity.

Full text of DOI:10.1002/aoc.3138

Full Paper
Received: 20 November 2013
Revised: 12 February 2014
Accepted: 12 February 2014
Published online in Wiley Online Library: 3 April 2014
(wileyonlinelibrary.com) DOI 10.1002/aoc.3138
Effect of coordination sphere of the copper
center and Cu―Cu distance on catechol
oxidase and nuclease activities of the
copper complexes
Bin Zheng, Hui Liu*, Jie Feng and Jingyan Zhang*
To explore the effect of Cu―Cu distance in the structure of copper complexes on their catechol oxidase and nuclease activity,
six copper complexes with a similar coordination sphere but different Cu―Cu distances were synthesized and characterized
with elemental analysis, single-crystal X-ray diffraction, molar conductivity measurements, IR and UV–visible spectroscopy.
Complex 1 is a binuclear copper complex and complex 4 is a polynuclear complex with a Z-chain structure, as evidenced by their
crystal structures. Complementary characterizations showed that complexes 2 and 3 have a similar binuclear structure to the
complex 1; and complexes 5 and 6 are analogous to complex 4. The catechol oxidase activity of complexes 1–3 is quite akin
to that of 4–6, suggesting that the catechol oxidase activity of the complexes was determined by the coordination environment
of the copper center, when Cu―Cu distance is large. In contrast, DNA cleavage activity of the complexes 1, 2 and 3 are much
higher than that of 4, 5 and 6, indicating that the planar ligand structure in the complexes 4, 5 and 6 is more critical than
the copper coordination sphere and the Cu―Cu distance for their nuclease activity. Copyright © 2014 John Wiley & Sons, Ltd.
Additional supporting information may be found in the online version of this article at the publisher’s web-site.
Keywords: copper complexes; catechol oxidase; nuclease; intermetallic distance; coordination sphere
and co-workers found that a complex with copper(II) ion-bound
CysGly dipeptides linked by an azobenzene shows better DNA
Introduction
Metal complexes are being widely studied as model complexes
of enzymes in understanding the roles of metal ions, reaction
mechanisms of enzymes and geometries of metal centers, etc.
For example, many copper complexes have been synthesized
to mimic the catalytic reaction of catchol oxidase, and to investi-
gate the reaction mechanism of nuclease.[1–8] Through extensive
studies of the model complexes, it has been believed that the
distance between two adjacent copper ions in catechol oxidase
has a significant impact on its catalytic activity.[9–16] Ennio and
co-workers have reported that binuclear copper complexes with
two tridentate Schiff base ligands that have an intermetallic
distance close to 2.9 Å show higher catechol oxidation activity than
those mononuclear copper complexes or binuclear complexes with
an intermetallic distance longer or shorter than 2.9 Å.[17] Many
studies have also indicated that binuclear or multinuclear
copper complexes showed higher catechol oxidase or chemical
nuclease activities than those of mononuclear ones, which
might be due to the synergistic interaction between the two
copper centers.[1–3,5–8,18] On the other hand, recent studies have
shown that some multinuclear copper complexes could cleave
DNA with higher efficiency or selectivity than those of the
mononuclear complexes.[19–21] A binuclear copper complex
containing a bidentate ligand with two tris(2-pyridylmethyl)
amine units covalently linked in their 5-pyridyl positions by a
―CH2CH2― bridge is more efficient than its mononuclear
complex in DNA cleavage.[19] In addition, it was also reported that
the activities of DNA cleavage are different for binuclear metal
complexes that have different intermetallic distances.[22,23] Hirota
cleavage activity in cis type (Cu―Cu distance ~8 Å) than that of
trans type (Cu―Cu distance ~12 Å).[22] However, all these model
complexes of catechol oxidase or nuclease have different Cu―Cu
distances and different coordination spheres for copper centers; thus
it is not easy to compare their catechol oxidase and nuclease activi-
ties in terms of Cu―Cu distance and structure of the complexes.
In this article, we designed and synthesized six Cu(II) complexes
with N-(2-pyridylmethyl)-amino acids, N-(2-pyridylmethyl)-amino
ethanol and their derivatives as ligands[24–27] (Fig. S1, supporting in-
formation) to understand the effect of intermetallic distance and
complex structure on their catechol oxidase and nuclease activities.
The copper complexes have two categories of structure – one is a
binuclear structure and the other is a polynuclear Z-chain structure –
but the copper centers in both cases have a very similar coordina-
tion sphere. We demonstrated that the catechol oxidase activities
of the complexes are not determined by the Cu―Cu distance when
it is above 3.4 Å, while, for the nuclease activities of the complexes
with a similar coordination sphere, the planar structure feature is a
more critical factor than the Cu―Cu distance.
*
Correspondence to: Jingyan Zhang and Hui Liu, State Key Laboratory of Bioreactor
Engineering, Shanghai Key Laboratory of New Drug Design, School of Pharmacy,
East China University of Science and Technology, Shanghai, 200237. People’s
Republic of China. E-mail: jyzhang@ecust.edu.cn; hliu@ecust.edu.cn
State Key Laboratory of Bioreactor Engineering, Shanghai Key Laboratory of
New Drug Design, School of Pharmacy, East China University of Science and
Technology, Shanghai 200237, People’s Republic of China
Appl. Organometal. Chem. 2014, 28, 372–378
Copyright © 2014 John Wiley & Sons, Ltd.

Influence of structure on the catalytic activities of copper complexes
Cu(L6)Cl (6)[25]
Experimental
Yield 0.29 g (82%). IR (KBr, cmꢀ1) 3117 (N―H), 1612, 1386 (CO2ꢀ);
Anal. Calcd for C15H15ClCuN2O2: C 50.85, N 7.91, H 4.27; found: C
50.70, N 7.66, H 4.44.
Materials and Measurements
All reagents and organic solvents used in this study were reagent
grade and used as received. Calf thymus DNA (CT-DNA) was
purchased from Sigma Aldrich Company, and others from
SinoPharm Chemical Reagent Co. Ltd. Solvents were dried
according to the standard procedure and distilled prior to use.
Electronic absorption spectra of the catalytic products and
reactions were recorded on a Cary 50 spectrophotometer
(Varian, USA). 1H NMR spectra were recorded on an Avance 500
analyzer (Bruker, Germany). Elemental analyses were performed
with an Elementar Vario ELIII analyzer (Germany) .Conductivity
measurements were carried out with an HI8733 conductivity
meter using methanol as solvent. X-ray crystallographic data of
the compounds were collected on a SMART diffractometer
(Bruker, USA) using Mo-Ka radiation (λ = 0.71073 Å).
Catechol Oxidase Activity Assay
Reactivity studies were performed in a mixed-solvent composition
of methanol and 50 mM phosphate buffer in pH 8.0 (volume ratio
6:4), at room temperature. The reaction proceedings were followed
by UV–visible spectroscopy at 405 nm. The reaction rates were
determined by UV–visible spectroscopy and the kinetic data of
oxidation were analyzed using the Michaelis–Menten model.
DNA Binding and Cleavage Activity
DNA binding experiments were performed in Tris–HCl/NaCl
buffer (5 mM Tris, 50 mM NaCl, pH 7.2) using an aqueous solution
of the complexes. CT-DNA in Tris–HCl buffer gave a ratio of UV
absorbance at 260 and 280 nm of 1.9:1, indicating that the DNA
was sufficiently free of protein. The concentration of CT-DNA
was measured from its absorption intensity at 26ꢀ01nm using
Synthesis of Ligands
Ligands L1–L6 were known; they were characterized by comparing
their proton NMR spectra (see supporting information) with those
found in the literature.[24–27]
the molar absorption coefficient value of 6600
M
cmꢀ1 [28]
.
UV–visible absorption titration experiments were conducted
by adding CT-DNA solution to the complexes, keeping the
concentration of complexes constant while the concentration
of CT-DNA was varied. The binding constant Kb was determined
using the following equation:[29,30]
Synthesis of Metal Complexes
Cu2(L1)2Cl22H2O (1)[24]
½DNAꢁ=ðεa ꢀ εbÞ ¼ ½DNAꢁ=ðεb ꢀ εf Þ þ 1=Kbðεb ꢀ εf Þ
A methanol solution (10 ml) of L1 (1 mmol, 0.18 g) was added to
an aqueous solution (10 mL) of CuCl2.2H2O (1 mmol, 0.17 g). The
reaction mixture was stirred for 30 min and filtered. The filtrate
was kept at room temperature for several days, yielding dark-
blue crystals suitable for X-ray analysis. Yield 0.21 g (78%). IR
(KBr, cmꢀ1) 3443 (O―H), 3216 (N―H), 1650, 1375 (COꢀ2 ); Anal.
Calcd for C18H22Cl2Cu2N4O4 · 2H2O: C 36.49, N 9.46, H 4.42; found:
C 35.93, N 9.03,H 4.66.
Complexes 2–6 were synthesized using a similar method, ex-
cept the copper chloride dihydrate was replaced by the copper
acetate monohydrate and copper perchloride hexahydrate for
complexes 4 and 5, respectively.
where [DNA] is the concentration of DNA in base-pairs, εa, εb and
εf are the extinction coefficients of complex bound to DNA at a
definite concentration, the complex completely bound to DNA
and the free complex in solution, respectively. The Kb value is
determined by the plot [DNA] / (εa ꢀ εb) versus [DNA].
DNA cleavage activity was carried as follows. The complex and
supercoiled PSICOR-GFP DNA (0.5 μg) in 50 mM Tris 18 mM/NaCl
pH 7.2 buffer were first mixed together. The mixture was then
incubated at 37 °C for 4 h. The reaction was quenched by adding
2 μl of a loading buffer solution (0.05% bromophenol blue, 1%
SDS and 50% glycerol, pH 8.0) and then subjected to electropho-
resis on 0.6% agarose gel containing 50 μg ethidium bromide
(EB) in 40 ml TBE buffer (89 mM Tris, 89 mM boric acid and 2 mM
EDTA, pH 8.3) at 80 V for approximately 1 h. The cleavage effi-
ciency was measured by determining the ability of the complex
to convert the supercoiled DNA (band I) to nicked circular form
(band II) or linear DNA (band III). Agarose gel electrophoresis
was carried out with DYY-6C electrophoresis apparatus (Liuyi
Instrumental Co., China). The gels were visualized and digitized
with a Tanon-2500 gel image analysis system and analyzed using
Tanon Gis software.
Cu2(L2)2Cl2 (2)[24]
Yield 0.16 g (62%). IR (KBr, cmꢀ1) 3236 (N―H), 1642, 1381 (CO2ꢀ);
Anal. Calcd for C16H18Cl2Cu2N4O4: C 36.37, N 10.60, H 3.43; found:
C 35.83, N 10.67, H 4.16.
Cu2(L3)2Cl4 (3)[26]
Yield 0.15 g (60%). IR (KBr, cmꢀ1) 3369 (O―H), 3204 (N―H); Anal.
Calcd for C16H24Cl4Cu2N4O2: C 33.52, N 9.77, H 4.22; found: C
33.58, N 9.71, H 4.22.
Cu(L4)(CH3COO) (4)
Results and Discussion
Yield 0.32 g (75%). IR (KBr, cmꢀ1) 3427 (O―H), 3180 (N―H), 1615,
1381 (CO2ꢀ); Anal. Calcd for C17H18CuN2O5: C 51.84, N 7.11, H 4.61;
found: C 51.49, N 7.46, H 4.45.
Structures of the Complexes
The crystallographic data and details of the refinement of the
crystal structures of compounds 1 and 4 are summarized in
Table 1.The selected bond distances and angles are listed in
Table 2. The crystal structures of the 1 and 4 are shown in Fig. 1.
There are two copper centers in the crystal structure of com-
plex 1. Each Cu(II) ion is coordinated by two nitrogen atoms from
Cu(L5)ClO4 (5)[27]
Yield 0.18 g (43%). IR (KBr, cmꢀ1) 3177 (N―H), 1608, 1383 (CO2ꢀ);
Anal. Calcd for C12H13ClCuN4O6: C 35.30, N 13.72, H 3.21; found: C
35.44, N 13.77, H 4.06.
Appl. Organometal. Chem. 2014, 28, 372–378
Copyright © 2014 John Wiley & Sons, Ltd.
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B. Zheng et al.
Table 1. Crystallographic parameters for complexes 1 and 4
1
4
Empirical formula
Formula weight
Temperature (K)
Wavelength (Å)
Crystal system
Space group
a (Å)
C9H12ClCuN2O3
295.20
C18H21CuN2O6
424.91
293(2)
293(2)
0.71073
0.71073
Monoclinic
C2/c
Orthorhombic
P212121
15.764(3)
7.8575(16)
12.817(3)
b (Å)
11.433(2)
c (Å)
12.781(3)
19.047(4)
β (Å)
99.88(3)
90
Volume (Å3)
Z
2269.4(8)
1918.1(7)
8
4
Calculated density (Mg mꢀ3
Absorption coefficient (mmꢀ1
F(000)
)
1.728
1.471
)
2.152
1.175
1200
800
Crystal size (mm)
0.20 × 0.20 × 0.20
3.24–25.01
6582 / 2004 [0.0640]
99.7
0.20 × 0.20 × 0.20
3.04–25.01
11546 / 3375 [0.0477]
99.7
Theta range for data collection (°)
Reflections collected / unique [Rint
Completeness to theta (%)
Data/restrains / parameters
Goodness-of-fit on F2
]
2004 / 0 / 149
1.271
3375 / 0 / 248
1.118
Final R indices
R1 = 0.0696, wR2 = 0.1319
R1 = 0.0977, wR2 = 0.1409
0.774 and ꢀ0.528
R1 = 0.0381, wR2 = 0.0815
R1 = 0.0432, wR2 = 0.0835
0.287 and ꢀ0.209
R indices (all data)
Largest diff. peak and hole (eÅꢀ3
)
Table 2. Selected bond lengths (Å) and bond angles (°) for complexes 1and 4
Bond length
Bond angle
1
4
Cu(1)―O(2)
Cu(1)―O(2)#
Cu(1)―N(1)
Cu(1)―N(2)
Cu(1)―Cl(1)
1.966(4)
2.750(4)
1.990(5)
2.011(4)
2.265(2)
O(2)―Cu(1)―N(1)
N(1)―Cu(1)―N(2)
N(1)―Cu(1)―Cl(1)
O(2)―Cu(1)―O(2)#
N(2)―Cu(1)―O(2)#
164.98(19)
82.96(19)
100.77(15)
87.77(13)
89.48(18)
O(2)―Cu(1)―N(2)
O(2)―Cu(1)―Cl(1)
N(2)―Cu(1)―Cl(1)
N(1)―Cu(1)―O(2)#
82.11(17)
94.18(13)
176.22(14)
90.43(16)
91.15(11)
Cl(1)―Cu(1)―O(2)#
Cu(1)―O(3)
Cu(1)―N(1)
Cu(1)―N(2)
Cu(1)―O(1)
Cu(1)―O(2)#
1.935(2)
1.982(3)
2.004(3)
1.949(2)
2.311(2)
O(3)―Cu(1)―O(1)
O(1)―Cu(1)―N(1)
O(1)―Cu(1)―N(2)
O(3)―Cu(1)―O(2)#
N(1)―Cu(1)―O(2)#
94.85(10)
164.39(11)
83.26(10)
94.45(10)
91.61(11)
O(3)―Cu(1)―N(1)
O(3)―Cu(1)―N(2)
N(1)―Cu(1)―N(2)
O(1)―Cu(1)―O(2)#
N(2)―Cu(1)―O(2)#
97.18(11)
172.49(12)
83.53(12)
97.34(10)
93.00(10)
the secondary amine and pyridine of one ligand, and one coordi-
nated oxygen atom from carboxylate groups of the ligand. With
one chlorine atom, a four-coordinating planar copper center
was formed. The two copper ions were bridged by two oxygen
atoms from the carboxylate groups of two ligands, as shown in
Fig. 1. The Cu2O2 core is also in one plane, which is perpendicular
to the plane of the CuN2OCl as revealed by the bond angles listed
in Table 2. The distance between two copper centers is 3.4 Å,
which is larger than that of native catechol oxidase and its many
binuclear model copper complexes.[10,17,31] The Cu―O length in
the copper coordination plane is 2.0 Å, and 2.7 Å for the axial
bond; the latter is much longer than that in many copper com-
plexes,[10,31] suggesting that this Cu―O is a weak bond.
that are from the secondary amine and pyridine of one ligand,
and two coordinated oxygen atoms from the carboxylate groups
of the two ligands, with the other oxygen atom from acetate,
forming a N2O3 pentahedron structure, with one bridge oxygen
in the apex and the other four forming a plane. However, the
acetate is totally out of the plane, and the phenyl ring is almost
perpendicular to the copper-centered plane. The Cu―O distances
in the plane are both 1.9 Å, while the axial Cu―O is 2.3 Å, which is
shorter than that in complex 1. However, because complex 4 has a
Z-chain conformation, the neighboring copper-centered planes
are not parallel as that in 1; the Cu―Cu distance in 4 is thus much
longer than that of 1 (5.3 Å vs. 3.4 Å). A similar complex has
reported in which the copper centers in that complex were
bridged by chlorine atoms instead of the oxygen atoms of carbox-
ylate groups as in 4.[24,32,33]
Compared to the structure of complex 1, X-ray crystal structural
analysis indicates that complex 4 has poly-copper centers with a
Z-chain conformation (Fig. 1). The copper centers are bridged by
the two oxygen atoms of carboxylate groups from one ligand.
Each copper ion is coordinated by five atoms: two nitrogen atoms
In spite of many attempts, we were unable to obtain single
crystals of 2, 3, 5 and 6. Hence the molar conductance of complexes
1–6 was measured to further identify their structures. According to
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Influence of structure on the catalytic activities of copper complexes
and similar IR data was obtained
(Fig. S3, supporting information).
We therefore believe it is from
water molecules. This observa-
tion is consistent with the ele-
ment analysis result that there is a
water molecule in complex 1. The
IR spectrum of 2 is similar to that
of 1 except that no band in the
3443 cmꢀ1 region was observed.
The bands in the 3369 and 1038
cmꢀ1 regions may be ascribed
to νO–H and νC–O vibrations of
Figure 1. Crystal structures of complexes 1 and 4.
the hydroxyl group in 3. This sug-
the literature, for 1:2 electrolytes in methanol solution at room
temperature the conductance is roughly 160–220 s cm2 molꢀ1; for
1:1 electrolytes the conductance is 80–115 s cm2 molꢀ1; for non-
gests that the alcoholic hydroxyl group in 3 is unionized, which is
consistent with the results of molar conductance and element
analysis. The IR spectra of 5 and 6 are similar to that of 4, except
for the band in the 3427 cmꢀ1 region ascribed to νO–H vibrations
of the phenolic hydroxyl group in 4. The similarity in the IR spectra
clearly indicates that the structures of complexes 2 and 3 are
similar to that of 1, and 5 and 6 are similar to that of 4. The bands
in the 1082, 941 and 615 cmꢀ1 regions were ascribed to ClO4ꢀ
vibrations for complex 5. These bands indicate that perchlorate
ion is coordinated in complex 5.
electrolytes, conductance is below 80 s cm2 molꢀ1 [34]
Under the
.
same conditions, the molar conductance of complexes 1–6 in
methanol are 55, 67, 68, 38, 56 and 29 s cm2 molꢀ1, respectively,
suggesting that the six complexes are all non-electrolytes, and the
anions of the complexes are all coordinated to the copper centers.
On the basis of the ligand similarity in complexes 2, 3 and 1 and
in 5, 6 and 4, the crystal structures of complexes 1 and 4, elemental
analysis and their molar conductance, the structures of 2, 3, 5 and 6
were proposed as shown in Fig. 2. Similar UV–visible spectra of
complexes 1–6 further support the proposed structures (Fig. S2,
supporting information). There is an absorbance band at 256 nm
for the all complexes, which contributes to the pyridine group in
the ligand. The absorption of 5 at 256 nm is larger than the others,
which may be due to the imidazole group in the ligand. The bands
at ~690 nm are assigned to copper d–d transition in octahedral
geometry structure.[10,17]
To further identify their structures, the complexes were also char-
acterized by IR spectroscopy. The IR bands of the complexes are
given in Table 3. The bands in the 3117–3236, 1608–1650 and
1375–1386 cmꢀ1 regions were ascribed to νN–H, νas(CO2ꢀ) and νs
(CO2ꢀ) vibrations, respectively. There is an extra band at 3443
cmꢀ1 of complex 1, which may be assigned to νO–H vibrations of
water molecules. To further determine the speculation, we
performed IR measurement for complex 1 after rigorous drying,
Catechol Oxidase Activity
Catechol oxidase activities of complexes 1–6 were first investigated
using 3,5-di-tert-butylcatechol (3,5-DTBC) as a substrate, because
its oxidation product 3,5- di-tert-butyl-o-quinone (3,5-DTBQ)
is stable and shows strong absorption at 405 nm (ε = 1900 Mꢀ1
cmꢀ1). Thus the catechol oxidase activity of the complexes
can be evaluated by detecting the change in absorption at
405 nm of 3,5-DTBQ. All the complexes showed similar cate-
chol oxidase activities under the same reaction conditions as
shown in Fig. 3. With reaction processing, the substrate–catalyst
adduct breaks down, resulting in a decrease in absorption. At
the same time, the product starts to form, leading to absorp-
tion recovery. The kinetics of 3,5-DTBC oxidation by the
complexes were determined by plots of the initial reaction
rates versus substrate concentration. Figure 4 displays the
Figure 2. Structures of the complexes 1-6.
Appl. Organometal. Chem. 2014, 28, 372–378
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B. Zheng et al.
Table 3. IR absorption bands (cmꢀ1) for complexes 1–6
νO–H νN–H νas νs
νC–O
Compound (cmꢀ) 1 (cmꢀ1 (CO2ꢀ) (cmꢀ1) (CO2ꢀ) (cmꢀ1
)
)
(cmꢀ1
)
1
2
3
4
5
6
3443
3216
3236
3204
3180
3177
3117
1650
1642
1375
1381
3369
3427
1038
1615
1608
1612
1381
1383
1386
dependence of initial rate on the concentration of 3,5-DTBC
catalyzed by complex 1 as an example. The maximum velocity
(Vmax), catalytic constant (Kcat), Michaelis binding constant (Km)
and catalytic efficiency parameter (Kcat/Km) of the six com-
plexes were evaluated and the data are summarized in Table 4.
All six complexes showed comparable activity. When compared
in terms of the copper center, complexes 1–3 showed a
slightly higher activity, which may due to their binuclear struc-
ture, since many works reported that the catechol oxidase
activity of binuclear copper complexes is higher than that
of mononuclear copper complexes.[35] Although the Cu―Cu
distance of complexes 1–3 is shorter than that of complexes
4–6, the distance is still much larger than that of the native
enzyme (the Cu―Cu distance in catechol oxidase met form is
2.9 Å); hence the activity is relatively lower than the many
reported binuclear compounds and catechol oxidase it-
self.[17,36] The Cu―Cu distance in complexes 4–6 is much
larger and thus they can be regarded as a mononuclear com-
plex in the catalysis. It is likely that the copper centers in these
six complexes actually act as a mononuclear complex; thus
their activity is comparable. Taking these results together, the
activity of the binuclear and polynuclear copper complexes is
determined by the coordination sphere of the copper center
instead of the Cu―Cu distance when it is longer than 3.4 Å.
This result is consistent with the literature in that the activity
will be lower if the Cu―Cu distance is larger than 4.0 Å.[10]
However, the key issue regarding the activity of these com-
plexes is whether they still use their binuclear or polynuclear
core structures as catalytic species in solution. It was reported
in the literature that oxygen-bridged binuclear copper
complex could maintain its binuclear state in solutions of
different pH.[37] To clarify this question, the mass spectra of
Figure 4. Plot of reaction rate versus concentration of 3,5-DTBC cata-
lyzed by complex 1. Inset shows the reciprocal Lineweaver–Burk plot.
some binuclear and polynuclear compounds were recorded.
The m/z 519.3 peak was observed in complex 1 assigned as a
(Cu2(L1)2Cl)+) species; similarly, in complex 2, a binuclear
species Cu2(L)2Cl)+ was observed at m/z 491.2. Similarly for
polynuclear complex 6, a binuclear and trinuclear species were
observed at m/z 745.8, and 1113.7, respectively, corresponding
to [Cu2L2(OH)]+ and [Cu3(HL2)2L2(H2O)]+. These results revealed
that binuclear complexes 1 and 2 maintained their binuclear
structure intact in solution, and that polynuclear complex 6
was in a polynuclear state in solution also.
Nuclease Activity
The affinity to DNA of the six copper complexes was first compared.
Upon the addition of CT-DNA to complexes 1–6, a decrease in the
molar absorption of the π–π* absorption band of the complexes
was observed, indicating that binding of the complexes to DNA
occurred. The binding constants (Kb) of all the complexes are
summarized in Table 5; the binuclear complexes 1–3 exhibit a
slightly higher affinity to DNA, possibly because their planar
structure that may partially intercalate with DNA. The small differ-
ence in DNA affinity for the six complexes also suggested that
electrostatic attraction between the complexes and DNA may play
a major role in their DNA binding.
DNA cleavage activity of the com-
plexes was then determined by mon-
itoring the conversion of the
supercoiled DNA (band I) to nicked
DNA (band II) and linear DNA (band
III) using agarose gel electrophoresis.
Under the same reaction conditions,
six complexes exhibit different levels
of DNA cleavage activity (Fig. 5). Com-
plexes 1 and 2 show higher DNA
cleavage activity: only 20% and 18%
supercoiled
DNA,
respectively,
remained unreacted, while for com-
plexes 4, 5 and 6 more than 50% of
the supercoiled DNA was left. Nota-
bly, complex 3 exhibits very low
DNA cleavage activity, possibly be-
cause the oxygen atoms coordinated
Figure 3. (a) Oxidation of substrate 3,5-DTBC (5 mM) by the complex 1 (0.1 mM in mononuclear entity).
The spectra were recorded after each 5 min interval as a function of the reaction time upon addition of
3,5-DTBC. (b) Time course of 3,5-DTBC (20 mM) oxidation by complexes 1–6 (0.1 mM in mononuclear en-
tity) at room temperature in phosphate buffer (pH 8.0) monitored at 405 nm.
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Appl. Organometal. Chem. 2014, 28, 372–378

Influence of structure on the catalytic activities of copper complexes
Table 4. Kinetic parameters of complexes 1–6
1
2
3
4
5
6
Km (M)
4.92 × 10ꢀ3
3.76 × 10ꢀ6
3.76 × 10ꢀ2
7.64
1.03 × 10ꢀ2
7.68 × 10ꢀ6
7.68 × 10ꢀ2
7.45
5.68 × 10ꢀ3
6.88 × 10ꢀ6
6.88 × 10ꢀ2
1.42 × 10ꢀ2
8.33 × 10ꢀ6
8.33 × 10ꢀ2
5.87
2.57 × 10ꢀ2
1.48 × 10ꢀ5
1.48 × 10ꢀ1
5.76
9.30 × 10ꢀ3
5.01 × 10ꢀ6
5.01 × 10ꢀ2
5.39
Vmax (M sꢀ1
)
Kcat (sꢀ1
Kcat/Km (Mꢀ1 sꢀ1
)
)
12.1
Table 5. DNA binding constant (Kb) of complexes 1–6
1
2
3
4
5
6
ꢀ1
Kb × 106
M
2.00
3.76
3.66
1.93
2.39
1.34
Figure 7. Gel electrophoresis diagram of the DNA cleavage (38 μM bp)
by complexes 1–6 (50 μM) in the presence of VC (50 μM) in 50 μM Tris/
18 mM NaCl buffer at pH 7.2, 37°C, with an incubation time of 30 min.
oxygen or reducing agents were included during the experiment.
This assumption was supported by cleavage experiments in the
presence of various additives, including DMSO (1.4 M), t-BuOH (1 M),
KI (10 mM), NaN3 (10 mM) (Fig. 6). Hydroxyl radical scavengers DMSO,
t-BuOH and KI show significant inhibition of cleavage activity,
and the singlet oxygen scavenger NaN3 also slightly inhibited the
reaction. The results indicate that diffusible hydroxyl radicals and
singlet oxygen were involved in DNA cleavage by the complexes.
The oxidative cleavage pathway was further supported by
including a reductant – ascorbic acid (VC) – in the cleavage
experiments. Figure 7 indicates that all complexes showed higher
cleavage activity in the presence of VC compared to its absence
(Fig. 5). It is interesting that complex 3 showed almost no DNA
cleavage activity without VC, while it exhibited similar DNA
cleavage activity to complexes 1 and 2 in the presence of VC.
The higher DNA cleavage activity of 1 and 2 may be partially
contributed by the planar structures of their copper centers, as
shown in Fig. 1. The time dependence of the DNA cleavage
activity of complexes with ascorbic acid was also investigated,
as shown in Fig. S6 (supporting information). With increasing
time the cleavage was more complete, as expected.
Taking these results together, the nuclease activity of the
binuclear copper complexes is higher than that of the polynu-
clear Z-chain complexes, though they are all through an oxidative
mechanism. The Cu―Cu distances in these six complexes are
likely too large to have a synergetic effect like that in the native
enzyme, while they are too short to allow each copper center
to bind to one DNA helix at the same time; thus Cu―Cu distance
is not a decisive factor for their difference in nuclease activity. The
activity difference thus may be contributed partially by the copper-
centered planar structure. As seen in the crystal structure of complex
1, the copper center and its coordinated atoms are in an almost
perfect plane, whereas in complex 4 the acetate is totally out
of the copper-centered plane. In addition, the Cu―O distance
(axial bond) in the binuclear complexes is much longer than
the regular Cu―O (oxygen atom from carboxylate) distance
(2.7 Å vs. 1.9 Å), whereas in polynuclear Z-chain complexes the
distance is closer to the regular length (2.3 Å vs. 1.9 Å). The larger
Cu―O distance makes binuclear complexes 1–3 more flexible in
interacting with DNA, while a smaller Cu―O bridge distance in
polynuclear Z-chain complexes 4–6 will have a larger steric
hindrance in their interaction with DNA.
Figure 5. Gel electrophoresis diagram of the DNA cleavage (38 μM bp)
by complexes 1–6 (1 mM) in pH 7.2, 50 mM Tris, 18 mM NaCl buffer at
37°C for 4 h.
to two copper(II) ions in 3 are from hydroxyl groups, instead of
the carboxylic group as in the complexes 1 and 2; thus the cop-
per center is not in a planar structure as in complex 1, which
needs to be further confirmed by its crystal structure. We also
found that the DNA cleavage activity of the complexes is concen-
tration dependent. Figure S4 (supporting information) displays
the DNA cleavage with different concentrations of complex 1.
With the concentration of 1 increasing, cleavage of the
supercoiled DNA was more complete. At a concentration of 2.5
mM, only 7% of the supercoiled DNA was left. Figure S5
(supporting information) shows that the DNA cleavage activity
of complexes is also time dependent. With increasing time, the
DNA cleavage was more complete. After 5 h of incubation, 78%
of the supercoiled DNA was converted to the nicked DNA. How-
ever, the concentration of the complexes used in the cleavage
experiment (in millimoles in the mononuclear entity) is much
higher compared to the complexes reported in the literature, in
which only micromoles were necessary.[38]
The cleavage mechanism by these complexes could be through
an oxidative pathway as in many copper complexes, since no
Figure 6. Gel electrophoresis diagram of the DNA cleavage (38 μM bp)
by complex 1 (2.5 mM) in the presence of various additives with an
incubation time of 4 h.
Appl. Organometal. Chem. 2014, 28, 372–378
Copyright © 2014 John Wiley & Sons, Ltd.
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B. Zheng et al.
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Conclusions
In summary, six new Cu(II)complexes with N-(2-pyridylmethyl)-
amino acids or N-(2-pyridylmethyl)-amino ethanol as ligands were
synthesized. All the complexes exhibit catechol oxidase and
nuclease activity. The catechol oxidase activity of the binuclear
complexes 1–3 is quite similar to that of the 4–6, suggesting that
once the Cu―Cu distance is larger there is no synergetic interac-
tion between Cu centers; thus the activity was determined mainly
by its coordination environment. The nuclease activities of the six
complexes follow the order 1, 2 and 3 > 4, 5 and 6. Although the
DNA cleavage by these complexes is all through the oxidation
pathway, the better copper-centered plane and longer bridge
Cu―O bond in the binuclear complexes compared to that in poly-
nuclear Z-chain complexes make binuclear complexes easier to
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Acknowledgments
The authors gratefully acknowledge the financial support of the
National Natural Science Foundation (Nos. 31070742 and
21001044), the State Key Laboratory of Bioreactor Engineering
(No. 2060204) and the Shanghai Committee of Science and
Technology (No. 11DZ2260600).
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