How well should the active site and the specific recognition be defined for proficient catalysis? - Effective and cooperative polyphenol/catechol oxidation and oxidative dna cleavage by a copper(II)-binding and H-bonding copolymer

Article abstract of DOI:10.1002/ejic.200800012

Despite the mainly inhomogeneous and unstructured nature of linear polymers, the Cu^II complex of a vinylpyridine-acrylamide copolymer exhibits very efficient 2-electron catalysis toward the oxidation of catechol and derivatives to form quinones with and without 80 mM (0.27%) H ₂O₂, showing remarkable (0.114-2.67) × 10 ⁵ and (2.83-9.60) × 10⁴-fold rate enhancements, respectively, in terms of first-order rate constant relative to auto-oxidation of the substrates in an aqueous environment under mild conditions. Metal-binding profiles suggest the presence of cooperativity in the catalysis. The oxidation catalysis is inhibited by the di-copper tyrosinase specific kojic acid. Moreover, electron paramagnetic resonance spectra reveal magnetic interaction of the Cu^II ions. On the basis of the results, the catalysis by this Cu^II-polymer seems to be consistent with the mechanism of type-3 di-copper oxidases. This complex also shows effective single- and double-stranded DNA cleavage in the presence of 1.0% H₂O₂. These studies suggest this Cu^II-polymer complex can serve as a unique chemical nuclease and a versatile chemical system for further exploration of Cu-oxygen chemistry. Wiley-VCH Verlag GmbH & Co. KGaA, 2008.

Full text of DOI:10.1002/ejic.200800012

FULL PAPER
DOI: 10.1002/ejic.200800012
How Well Should the Active Site and the Specific Recognition Be Defined for
Proficient Catalysis? – Effective and Cooperative Polyphenol/Catechol
Oxidation and Oxidative DNA Cleavage by a Copper(II)-Binding and
H-Bonding Copolymer
[
a]
[a,b]
[a]
[a]
[a]
Vasiliki Lykourinou, Ahmed I. Hanafy,
Giordano F. Z. da Silva, Kirpal S. Bisht,
[a]
[c]
[d]
Randy W. Larsen, Brian T. Livingston, Alexander Angerhofer, and Li-June Ming*
Keywords: Catechol oxidase / Copper / Dinuclear catalysis / Polymers / Oxidation
Despite the mainly inhomogeneous and unstructured nature
of linear polymers, the Cu complex of a vinylpyridine-acryl-
amide copolymer exhibits very efficient 2-electron catalysis
toward the oxidation of catechol and derivatives to form quin-
nase specific kojic acid. Moreover, electron paramagnetic
II
II
resonance spectra reveal magnetic interaction of the Cu
II
ions. On the basis of the results, the catalysis by this Cu -
polymer seems to be consistent with the mechanism of type-3
di-copper oxidases. This complex also shows effective single-
ones with and without 80 m
2 2
M (0.27%) H O , showing re-
5
4
markable (0.114–2.67)ϫ10 and (2.83–9.60)ϫ10 -fold rate
enhancements, respectively, in terms of first-order rate con-
stant relative to auto-oxidation of the substrates in an aque-
ous environment under mild conditions. Metal-binding pro-
files suggest the presence of cooperativity in the catalysis.
The oxidation catalysis is inhibited by the di-copper tyrosi-
and double-stranded DNA cleavage in the presence of 1.0%
II
H
2
O
2
. These studies suggest this Cu -polymer complex can
serve as a unique “chemical nuclease” and a versatile chemi-
cal system for further exploration of Cu–oxygen chemistry.
(© Wiley-VCH Verlag GmbH & Co. KGaA, 69451 Weinheim,
Germany, 2008)
Introduction
characterizable active site may not be a key issue in poly-
mer-based catalysis (which nevertheless is not obtainable in
polymers). Thus, the question “How well should the active
site and the specific recognition be defined for proficient ca-
talysis?” in metallopolymers should also be raised which
awaits answers in order to further the applications of
metallopolymers in catalysis. Since polymers can be easily
molded into different physical forms such as films and po-
rous particles, their further applications in heterogeneous
catalysis can be envisioned.
Most linear polymers are known for their inhomogen-
eous physical and chemical nature, such as variation in their
chain lengths, random sequence in subunit composition of
copolymers, disordered polymeric main chains, and flexibil-
ity of their side chains. Such inhomogeneous nature renders
detailed investigation of their structures very difficult, and
mostly impossible for copolymers. Nevertheless, bulk prop-
erties of polymers can be quite well controlled, including
their hydrophobicity and hydrophilicity, optical, magnetic,
and electrochemical properties,[1] and physical shapes,
which render their wide applications. Despite the disordered
and random sequence in copolymers, a number of metal-
binding copolymers have recently been prepared and uti-
The copper-dioxygen chemistry is relevant to the utiliza-
tion of an oxygen donor, such as O and H O , in the oxi-
2
2
2
dative transformation of organic molecules by Cu com-
[3,4]
plexes that mimic the action of Cu-containing oxidases.
These biomimetic chemical systems may be better access-
ible, more stable, and more catalytically versatile than en-
zymes, thus may have wider applications and provide chem-
[
2]
lized as catalysts for various metal-centered reactions.
hypothesis can thus be raised that a crystallographically
A
[
3,4]
ical insight into the mechanisms of enzymes.
Although
O is readily available in the atmosphere, its use as an oxi-
2
[
[
a] Department of Chemistry, University of South Florida,
dation agent in multi-cell organisms requires transpor-
tation, storage, and activation specifically carried out by
4
202 Fowler Avenue, Tampa, FL 33620, USA
Fax: +01-813-974-1733
E-mail: ming@shell.cas.usf.edu
[5]
different proteins and enzymes in living organisms. Cu-
b] Department of Chemistry, Faculty of Science, Al-Azhar Uni-
versity,
[6]
containing oxygenases, such as tyrosinase and polyphenol
Nasr City, Cairo, Egypt
oxidase, are ubiquitous. They use O to carry out hydroxyl-
2
[
[
c] Department of Biology, University of South Florida,
Florida, USA
ation and/or oxidation reactions. Hydroxylation processes
of hydrocarbons to yield alcohols or phenols are spin-for-
d] Department of Chemistry, University of Florida,
Gainesville, FL 32611, Florida, USA
bidden, wherein the paramagnetic triplet-state O needs to
2
2584
© 2008 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Eur. J. Inorg. Chem. 2008, 2584–2592

Oxidation Catalysis by a CuII Polymer
be activated to the singlet state to effectively insert into the not observed when the metal alone or the metal with the
singlet C–H bond, and are thus very challenging chemical monomers 4Vp and Ac are used as the control under the
and industrial tasks. Moreover, copper-based oxidative “ar- same conditions.
tificial nucleases” have received considerable attention ow-
ing to their mechanistic flexibility and applications in mo-
lecular biology such as DNA footprinting.[ Understand-
7,8]
ing the activity and mechanism of these Cu-centered chemi-
cal systems can provide information about oxidative dam-
age to nucleic acids and other biomolecules and gain better
[
6,9,10]
understanding of the mechanisms of copper enzymes,
with possible applications in the fields of environmental
[
11]
green chemistry,
aging, cancer research, and oxidative
stress-associated apoptosis.[
12,13–14]
Design aspects of biomimetic systems rely on the struc-
tural features of their biological templates, such as the
Figure 1. (a) Rate for the oxidation of THB in the presence of 0,
metal-binding sites in metalloenzymes.[
15,16]
We previously 1.0, 2.0, 5.0, 40, and 80 m H O (from bottom) and the rate
2 2
2 2
prepared and investigated a metal-binding copolymer (P1) (ϫ1/10) for the oxidation of DTBC in the presence of 50 m H O
(
∆) and fitted with pre-equilibrium kinetics (dashed) and the Hill
equation (dotted). (b) Rate for the production of o-quinone from
.0 m THB (᭹), 10 m DTBC ( ), and 4 m catechol ( ) by 10 µ
of 4-vinylpyridine (4Vp) and acrylamide (Ac) with an
average repeating unit (RU) of 4Vp Ac to further explore
3
1
8
᭿
᭡
the versatility of metallopolymers. P1 was demonstrated to
II
Cu -P1(RU) and a higher concentration (10 m) of catechol (ϫ)
by 10 µ Cu in the presence of 3 equiv. of 4-Vp and 1 equiv. of
Ac. All the experiments were performed in 1:1 methanol/25 m
MES buffer at pH 6.0 and 25 °C.
[
17]
II
bind metal ions with a stoichiometry of metal/RU = 1:1.
This polymer can bind metal ions through the pyridine ring
and potentially form H-bonds with its amide group, and
may thus loosely mimic the active-site environment of vari-
ous metalloenzymes. Despite the poorly defined “active
site” in these bio-mimicking metallopolymer complexes,
Fitting the rates of THB oxidation in the presence of
[
19]
they have been demonstrated to exhibit significant hydro- 0.27% H O (80 m) as a function of [THB] to Equation
lytic activities. Herein, we reveal the oxidative activity of (2) yields a first-order rate constant kcat = 0.152 s (t1/2
Cu -P1 and discuss its mechanism for the oxidations of 4.6 s), an apparent dissociation constant KЈ = 2.40 m, and
polyphenol and catechol and oxidative cleavage of DNA in the catalytic efficiency kcat/KЈ = 63.3  s
2
2
[
17]
–1
=
II
–1 –1
(᭡; Figure 1a).
the presence of the green oxidizing agents, H O and the This catalysis afford a significant rate enhancement of
2
2
4
air, in aqueous environment under mild conditions.
2.83ϫ10 -fold in terms of k /k , i.e., under saturating sub-
cat o
strate conditions, wherein the slow background oxidation
–
6 –1
of THB was determined to give k = 5.38ϫ10
s
for the
o
Results and Discussion
rate law rate = k [THB]. Despite the structural randomness
o
of copolymers, the results indicate that metallo-active sites
in P1 seem to properly assemble upon Cu binding to per-
Polyphenol/Catechol Oxidation
II
Although H O is a widely used oxidation reagent, it re- form this effective oxidative catalysis of THB in the pres-
2
2
quires further activation by a catalyst for effective oxidation ence of the green oxidation reagent H O .
2
2
to take place. For example, the oxidation of the polyphenol
Similar to the polyphenol THB, the commonly used cate-
1
,2,3-trihydroxybenzene (THB) by H O in an aqueous en- chol substrate 3,5-di-tert-butylcatechol (DTBC) was effec-
2
2
II
vironment (1:1 methanol/25 m MES buffer at pH 6.0 and tively oxidized by Cu -P1 in the presence of 50 m H O
2
2
2
5 °C) is indeed a very slow process, affording a pseudo- (ϫ1/10; ∆; Figure 1a). The catalysis follows the same pre-
–
6
–1
–1
first-order rate constant of k = 3.45ϫ10
law rate = k [H O ] toward 8 m THB which represents a 0.447 s), KЈ = 2.45 m, and kcat/KЈ = 633  s (dashed
s
for the rate equilibrium kinetics to give kcat = 1.55 s (t
of only
o
1/2
–1
–1
o
2
2
half-life (t1/2) of 55.8 h. Conversely, THB oxidation by H O
is dramatically enhanced by 10 µ RU of Cu -P1 under the ficiency kcat/k of 2.67ϫ10 -fold was obtained with respect
trace; Figure 1a). A considerable first-order catalytic pro-
2
2
II
5
o
–
6 –1
above conditions which reaches a plateau at high [THB] to k of 5.80ϫ10
s
under the same conditions. There
Figure 1a). Such kinetic pattern indicates an enzyme-like seems to be a slight cooperativity with a Hill coefficient θ
pre-equilibrium kinetic pathway [i.e., THB = S in Equation = 1.56 (dotted trace) for the oxidation of DTBC; while
1) and Equation (2); Experimental Section], which also re- THB gives a small θ = 1.05. The cooperativity is probably
flects that the polyphenol substrate binds directly to the partially due to interactions/influences among metal centers
o
(
(
II
II
metal center in Cu -P1 to afford the S-Cu -P1 intermedi- during catalysis (discussed later).
ate [Equation (1)]. A free-radical-initiated oxidation would
A plot of the rate of oxidation of 8.0 m THB in aque-
not show such pre-equilibrium kinetics and the substrate- ous 1:1 methanol/25 m MES buffer at pH 6.0 and 25 °C
II
bound intermediate, wherein second-order kinetics is ex- by Cu -P1 as a function of [H O ] is also non-linear which
2
2
[
18]
–1
pected with much higher reaction rate and less selectivity.
can be fitted to Equation (2) to give k = 0.241 s , KЈ
On the contrary, such a pre-equilibrium kinetic pattern is = 9.70 m, and kcat/KЈ = 24.8  s
cat
–1 –1
(᭹; Figure 1b) and a
Eur. J. Inorg. Chem. 2008, 2584–2592
© 2008 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.eurjic.org
2585

L.-J. Ming et al.
FULL PAPER
4
significant rate enhancement of 6.99ϫ10 -fold in terms of a bi-substrate reaction. The data were analyzed with the
kcat/k . Likewise, the oxidation of 10 m DTBC as a func- Hanes plots [Equation (3), Figure 2a], which are consistent
o
–
1
[28]
tion of [H O ] (
᭿
; Figure 1b) yields k = 0.65 s , KЈ = with a random bi-substrate mechanism.
The secondary
2
2
cat
–
1
–1
5
6
.58 m, and kcat/KЈ = 98.8  s , affording 1.1ϫ10 -fold plots from the Hanes plots derived from Figure 1a (i.e., A
rate enhancement with respect to k = 5.80ϫ10
–
6 –1
s
for = THB and B = H O ; Figure 2a) as a function of 1/[H O ]
o
2
2
2
2
DTBC auto-oxidation. The more inert substrate catechol according to Equation (3) yield KЈTHB = 6.37 m, KЈH2O2
=
(
4 m) can also be oxidized into quinone in the presence of 15.4 m, and KЈiTHB = 0.76 m. The intrinsic dissociation
–
1
H O , showing k = 0.12 s and KЈ = 3.8 m (
᭡
; Fig- constant KЈiTHB is close to the value of 1.02 m for THB
2
2
cat
ure 1b). The rate is higher than the oxidation of catechol oxidation without H O , which confirms the validity of this
2
2
II
at a higher concentration (10 m) catalyzed by Cu in the data process according to the bi-substrate mechanism. The
presence of 3 equiv. of 4-Vp and 1 equiv. of Ac (ϫ; Fig- KЈiTHB/KЈTHB ratio of 0.12 is much smaller than 1.0 which
ure 1b). The observation of saturation kinetics herein indi- indicates the binding of H O to the active center signifi-
2
2
II
cates that H O binds to the Cu center during the oxidat- cantly decreases THB binding affinity to afford a relatively
ive catalyses, following the pathways shown in Equation (1). larger KЈTHB value (i.e., in the case of oxidation of THB
2
2
[
28]
II
A noticeable rate of THB oxidation by 10 µ Cu -P1 in the presence of H O ). A similar Hanes-plot analysis for
2
2
was detected in the absence of H O which was significantly the rate-vs.-[H O ] plots at various [THB] [i.e., with A and
2
2
2
2
higher than its auto-oxidation rate. Herein, O in the air B switched in Equation (3)] yields KЈiH2O2/KЈH2O2 = 0.103
2
serves as the oxidation agent which is only 0.27 m at 25 °C (Figure 2b), indicating the binding of THB also decreases
for 100% air saturated water. This aerobic oxidation of H O affinity to the metal center, suggesting some exclusive
2
2
II
THB by Cu -P1 also follows pre-equilibrium kinetics nature of the polyphenol THB and the oxidant H O in
2
2
[
0
1
Equation (1), Equation (2)], giving rate constants k
=
metal binding.
cat
–
1
.012 s (t1/2 = 58 s), KЈ = 1.02 m, and kcat/KЈ =
–
1 –1
1.8  s (the lowest trace , Figure 1a). This catalysis af-
᭹
4
fords 3.12ϫ10 -fold rate enhancement in terms of kcat/k ,
o
wherein the auto-oxidation rate constant k under the same
o
–
7 –1
conditions is 3.85ϫ10
s
(t = 21 d). Similarly, aerobic
oxidation of DTBC by Cu -P1 at pH 6.0 yields k
.065 s and KЈ = 0.16 m. The dramatic decrease in the
1/2
II
=
cat
–
1
0
KЈ value results in a significant catalytic efficiency kcat/KЈ =
–
1
–1
4
4
06  s . The catalysis affords k /k = 9.60ϫ10 with k
cat o o
–
7 –1
determined to be 6.77ϫ10
s
under the same conditions,
representing a dramatic decrease in t1/2 from 11.9 d to
0.7 s. The significant influence of H O on KЈ reflects a
mutual exclusion of the two substrates, THB/DTBC and
1
2
2
II
Figure 2. Hanes plot of THB oxidation by Cu -P1 in the presence
of 0, 1.0, 2.0, 5.0, 40, and 80 m H (a; from top) and 0.4, 0.8,
.0, and 4.0 m THB (b; from top), from which the dissociation
2
O
2
H O , showing much higher KЈ values for THB and DTBC
2
2
2
in the presence of H O .
2
2
constants in Equation (3) can be obtained.
The rate constants in the studies herein are comparable
or higher than those of a number of structurally defined
II
III
[20–25]
Cu complexes and Fe -porphyrin systems
toward
Because cooperativity is an important biological prop-
the oxidation of catechol and derivatives, and are only ca. erty, possible cooperativity of the metal centers was further
II
1
50–350 times lower than that of horseradish peroxidase investigated, wherein Cu was added to P1 in the presence
[25]
II
toward THB oxidation.
dation of catechol is in the range of (1.6–2.2)ϫ10
Cu -macrocycle complexes
and (3.98–13.2)ϫ10
For example, kcat for the oxi- of the redox-inactive Zn at a constant total concentration
–
4
–1
II
II
s
by of ([Cu ] + [Zn ]) and monitored with the oxidative ac-
tivity. A proportional increase in the activity with the mol
by some dinuclear Cu com- fraction of Cu (XCu) should be observed until 1 equiv. of
II
[20]
–4 –1
and (2.75–5.13)ϫ10
s
–
3
–1
II
II
s
[
21,22]
II
II
II
plexes;
oxidation of DTBC in methanol or mix solvent Cu is added for a simple Cu binding to the copolymer if
–1[23,26]
by di-Cu complexes shows k = 0.0553–1.81 s
and there is no cooperativity during catalysis since isolation of
cat
–1
–1 [24]
III
II
II
kcat/Km = 60–140  s ; oxidation of THB by the Fe - Cu ions from each other by Zn ions should not affect
heme complex of a catalytic antibody gives k = 0.83 s–1 the activity in this case (dashed fittings, Figure 3). On the
cat
–
1 –1
and kcat/Km = 210  s in the presence of 5 mM H O
contrary, sigmoidal activity profiles were observed toward
at pH 8.0 and kcat = 42 s and kcat/Km = 9100  s by the oxidation of THB with and without 15 m H O , giving
2
2
–
1
–1 –1
2
2
[25]
horseradish peroxidase.
Moreover, DTBC oxidation by Hill coefficients θ of 1.4 and 2.8, respectively (solid fittings,
II
Cu -P1 without H O shows a significant 1.2% activity rel- Figure 3). The results suggest a possible presence of cooper-
2
2
ative to that of gypsywort catechol oxidase[ in terms of ativity among Cu centers for polyphenol oxidation by
27]
II
II
the second-order rate constant kcat/Km.
Cu -P1. This cooperativity may be a consequence of dinu-
Because both THB and H O can bind to the Cu center clear catalysis of the 2-electron oxidation of polyphenol and
as shown by the pre-equilibrium kinetic pattern [Figure 1a, catechol. Herein, Cu is well isolated by Zn ions at lower
b; Equation (1)], the catalysis by Cu-P1 is thus considered XCu
II
2
2
II
II
.
2586
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© 2008 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Eur. J. Inorg. Chem. 2008, 2584–2592

Oxidation Catalysis by a CuII Polymer
The fitting of the EPR spectrum is not perfect probably
II
because of the presence of magnetically coupled Cu clus-
ters (see below). The line-broadening in the spectrum sug-
gests the presence of magnetic interactions between/among
II
II
Cu ions. When 1 equiv. of Cu is added to P1 without
II
Zn dilution, the spectrum becomes much broader and
shows complete loss of the hyperfine features in the gЌ re-
gion (Figure 4b) with a small anisotropy of gЌ = 2.200, gЌ
=
2.125 and a peak-to-peak width of 200 G, noticeably
II
larger than that in the sample with 75% Zn dilution
120 G from the simulation; Figure 4a). A weak and broad
Figure 3. Oxidative activity of Cu-P1 toward THB in the absence
(
II
(᭹
) and presence (o) of 15 m H
2
O
2
as a function of Cu mol
signal at g = 4.3 in Figure 4b (inset), which is missing in
the magnetically diluted sample (inset, Figure 4a), is likely
II
II
fraction (XCu) at a fixed total concentration of Cu and Zn . The
traces are fittings to the Hill equation (solid traces) or a simple 1:1
metal-to-ligand binding (dashed traces).
to be attributed to the forbidden |∆M
to magnetic coupling frequently observed in dinuclear Cu
| = 2 transition owing
S
II
[
26,32]
II
complexes.
dashed trace in Figure 4a) by considering the presence of
5% magnetically coupled system from (b). The results
The Zn -diluted sample can be better fitted
Cooperativity is a common property of multi-domain
proteins/enzymes. The presence of catalytic cooperativity
was revealed in a recent study of dendrimer-based chemical
(
1
II
[
29]
indicate that Cu ions can be close to each other to form
magnetically coupled centers in Cu -P1. The proximity of
Cu ions in the polymer can thus result in dinuclear cataly-
model system,
presumably attributing to the proximity
II
of the active centers on the dendritic arms. Cooperativity of
dendrimers toward metal binding has also been previously
II
[30]
sis and cooperativity.
demonstrated in a few metal-binding dendrimers,
show-
II
The d–d transition of Cu -P1 at 615 nm (Figure 5) re-
ing a distinct difference in metal-binding when the dendritic
arms were isolated units. It is worth noting that the simple
flects a quite strong ligand field, most likely attributing to
its all-N ligand environment with a distorted octahedral ge-
ometry consistent with the EPR results. For comparison,
II
metallopolymer Cu -P1 described herein also exhibits coo-
perativity in metal binding and catalysis, which is mostly
likely attributed to the proximity of the metal centers due
to folding of the polymer chains. The observation of coo-
perativity suggests that metallopolymers can serve as rea-
sonable functional models for metalloenzymes.
II
the all-N(His) ligated Cu -substituted carbonic anhydrase
B absorbs at 750 nm with gЌ = 2.31 and gЌ = 2.06[ and
33]
Cu,Zn-superoxide dismutase absorbs at 680 nm with g
=
Ќ
[
34]
2.268 and gЌ = 2.087.
II
The EPR spectrum of Cu -P1 under the conditions for
the kinetic studies was obtained in the presence of
II
0
.75 equiv. of Zn per RU for the purpose of magnetic di-
lution, which shows an axial magnetic environment with gЌ
2.270, gЌ = 2.085, and AЌ = 553 and AЌ = 35 MHz
dotted trace; Figure 4a). These values are consistent with
=
(
II
those reported for a Cu center with a nitrogen-rich ligand
environment. The spectral features can be attributed to a
typical tetragonally distorted Cu center, i.e., an elongation
[
31]
II
along the z axis due to the Jahn–Teller effect.
II
Figure 5. Electronic spectrum of 0.5 m RU of Cu -P1 under the
conditions for the oxidative catalysis.
The oxidative catalysis is further investigated with the di-
Cu oxidase specific inhibitor kojic acid (KA) [5-hydroxy-2-
(
hydroxymethy1)-γ-pyrone] (structure shown in Fig-
[
35]
ure 6a). The α-ketoenolate moiety of KA mimics the cat-
echol group during oxidation. The inhibition of Cu -P1 by
II
KA toward THB oxidation in the absence of H O follows
2
2
a competitive pattern (Figure 6a), with an inhibition con-
stant of K = 0.34 m. KA inhibition of di-Cu enzyme is
i
much more specific, e.g., K was found to be 3.4 µ toward
i
II
[36]
Figure 4. X-band EPR spectra of Cu -P1 with (a) and without (b) Streptomyces antibioticus tyrosinase
and the IC50 is
II
magnetic dilution by Zn (75%). The insets are low-field region 0.28 m toward lobster polyphenol oxidase with catechol
that show the weak forbidden transition due to magnetic coupling.
The dotted trace in (a) is the fitted spectrum for an axial species.
While (b) is fitted to show only slight anisotropy (dashed), (a) is
best fitted (dashed) as a mixture of 85% of the axial species (dot-
ted) and 15% of the magnetically coupled species (b).
[35c]
as the substrate,
yphenol oxidases from various sources exhibit a competitive
or a mixed pattern with K values in a range of 0.02 to
and the inhibition toward several pol-
i
[
35b]
0.7 m.
Eur. J. Inorg. Chem. 2008, 2584–2592
© 2008 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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2587

L.-J. Ming et al.
FULL PAPER
served.[38] The results presented herein suggest that a dinu-
II
clear active center seems to assemble in Cu -P1 to afford
the high oxidative activity, despite the nature of structural
and conformational randomness of copolymers.
Figure 6. a) Kojic acid (with the structure shown as inset) inhibi-
tion (from bottom, 0, 0.5, 2.0, 3.0, 6.0 m) toward THB oxidation
II
by 10 µ Cu -P1 at 100 m H
2
O
2
in 0.1  MES buffer-methanol
solution, showing a competitive inhibition pattern. b) Inhibition of
THB oxidation (2.0 m) by kojic acid (from bottom, 0, 0.5, 1.0,
2
.0 m) at varying amounts of [H
2 2
O ], showing a mixed inhibition
pattern against H
2
2
O .
Conversely, the inhibition of KA toward THB oxidation Figure 7. Proposed random bi-substrate mechanism for the oxi-
II
as a function of [H O ] exhibits a mixed pattern (Figure 6b) dation of THB by Cu -P1 in the presence (steps D–G or A,H,G)
and close to a non-competitive pattern with inhibition con-
2
2
2 2 2 2
and absence (A–C and E–G) of H O . The production of H O
under reduction conditions (I) is also consistent with this mecha-
nism. The substrate complexes at steps E and F are also shown in
Figure 4. The kinetic parameters can be obtained from the second-
stants of Kic = 4.2 m and Kiu = 5.9 m, for the dissoci-
II
ation of KA from the ternary complexes KA-(THB-Cu -
II
P1) and KA-(THB/H O -Cu -P1), respectively. The mixed ary plots of the Hanes equation.
2
2
inhibition pattern indicates that KA and H O are not com-
2
2
pletely exclusive, which also reflect that the binding natures
of the substrate and H O may be different (however, their
2
2
The sequence and structure of a copolymer chain is ran-
dom in nature and best described in terms of RU. The coor-
bindings to the metal center are observed herein to affect
each other; Figure 2). The inhibition results suggest that
II
II
dination sphere of Cu -P1 is explored with molecular me-
Cu -P1 catalysis has catechol/polyphenol oxidase charac-
[
39]
chanics calculations (MM3 force field,
BioCAChe 6.0,
teristics and is likely to follow a dinuclear mechanism for
the 2-electron oxidation reaction of catechol and pol-
yphenol as the enzymes.
Fujitsu, Beaverton, OR), which reveal that the best coordi-
nation sphere is formed by pyridine ligands from different
RUs, i.e., pyridines separated by more than 3 monomeric
units. Metal binding to pyridines from the same RU of
4
Vp Ac causes significant distortion of metal–pyridine co-
3 1
Proposed Mechanism and Modeling
ordination. Metal binding to pyridines from different poly-
The 2-electron oxidation catalyses herein with and with- mer chains afford a more preferred binding mode
out H O are consistent with the mechanism of dinuclear (≈ 400 kcal/mol lower), which can result in crosslink of the
2
2
[
6,41]
catechol oxidase.
A random bi-substrate mechanism is polymer chains and coagulation of the metallopolymer
proposed (Figure 7) based on the kinetic studies described complexes at higher concentrations as we observed pre-
[
17]
herein and according to the catalytic pathways of catechol viously.
The complex is soluble under the conditions
oxidase. In the absence of H O , THB is bound to and oxi- herein for kinetic studies.
2
2
II
II
dized by Cu -P1 (᭹; Figure 1a) in form of a dinuclear Cu
center by inner-sphere 2-electron transfer to yield Cu 2 and with a dinuclear catalysis, the active oxygenated intermedi-
Because the results from the kinetic studies are consistent
I
o-quinone product (steps A, B). The dinuclear center can ate is modeled with a dinuclear center analogous to that
II
[40]
be formed from two Cu centers on the same polymer chain of hemocyanin with a pseudo-C2h symmetry (Figure 8a).
separated by three RU or on different polymer chains. The This oxy/peroxy dinuclear site allows substrate binding to
inner-sphere electron transfer is followed by dioxygen bind- one of the metal ions from the top or bottom of the Cu–
II
ing (C) to form a dinuclear Cu -peroxo center or its isoelec- O –Cu plane without distorting the overall coordination
2
[
3,4]
tronic forms
which can also be formed upon peroxide sphere. A catechol substrate can be docked into the dinu-
II
binding (D and Figure 1b) analogous to the peroxide shunt clear site of peroxide-bound Cu -P1 without significant
pathway in cytochrome P450 mechanism.[ THB can bind distortion (Figure 8b), in which catechol is bound to one
to the dinuclear active site through two pathways (D–G and Cu center through a deprotonated hydroxy while the other
37]
II
A, H, G) and oxidized by the dinuclear Cu -peroxo center hydroxy forms a H-bond with the bridging peroxide and a
to complete the catalytic cycle. Under reduction conditions, proximal amide group from the polymer. Upon deproton-
II
the Cu -peroxo intermediate is expected to undergo ation and binding of the second hydroxy group to the other
through the pathway I to produce H O as previously ob- Cu center (Figure 8c), the energy is lowered relatively by ca.
2
2
2588
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© 2008 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Eur. J. Inorg. Chem. 2008, 2584–2592

Oxidation Catalysis by a CuII Polymer
240 kcal/mol. This binding mode has been suggested to be This difference suggests that additional pathway(s) may be
the case in the enzyme-substrate complex of catechol ox- present for the disappearance of the ln form, such as further
[
6,41]
idase.
However, the coordination is significantly dis- cleavage to yield small fragments that cannot be revealed
torted from the original, in which the Cu–O –Cu plane is on the gel.
2
puckered to the opposite direction.
Figure 9. Time course (10, 20, 40, 60, 90, 120 min, lanes 1–6 and
II
Figure 8. Stereo plots of a) Coordination structure of a putative
dinuclear site in Cu -P1 calculated with the MM3 force field proto-
1Ј–6Ј, respectively) of plasmid cleavage by Cu -P1 in the presence
of 1.0% H (lanes 1–6, 50.0 µ RU; , nc form; , ln form; and
theoretical fittings, solid traces) and by free Cu /H
II
2
O
2
᭹
᭡
II
col. The di-copper site in hemocyanin (dark ball-and-stick) is su-
perimposed onto the structure to show their similarity. The Cu ions
are shown as gray spheres and the bridging peroxo as dark spheres.
b) Docking of catechol (dark ball and stick) to the dinuclear site
2 2
O (50 µ,
lanes 1Ј–6Ј; o, nc form; ∆, ln form; and theoretical fitting, dotted
traces) at pH 8.0 and room temperature, stained with ethidium bro-
mide. The standard DNA ladder starts with 1 kbp from the bottom
with 1 kbp increment upward. R is the plasmid DNA pQE30Xa of
ca. 3.5 kbp without treatment. The data are fitted with the kinetic
models discussed in the text.
II
of Cu -P1 reveals a favorable binding configuration, in which cate-
II
chol is bound to one Cu through a deprotonated hydroxy while
the other hydroxy is still protonated and H-bonding with the bridg-
ing peroxide and a proximal amide group from the polymer. c)
Catechol is bound to the two metal ions upon deprotonation of
II
CuII ions can cleave plasmids in the presence of an oxi-
dation agent, thus serve as the reference herein (lanes 1Ј–
the second hydroxy group, which causes distortion of the di-Cu
center.
[42]
II
6
Ј). Under the same conditions, Cu cleaves the sc and nc
II
forms into ln form less effectively than Cu -P1. The ln form
is not apparent until about 20 min later (lane 2Ј), resulting
in accumulation of the nc form. The buildup of nc concen-
Oxidative Plasmid DNA Cleavage
II
Cu -P1 shows a significant activity toward oxidative tration (due to single-stranded ss-DNA cleavage) and slow
cleavage of plasmid DNA which displays unique reaction appearance of ln (due to ds-DNA cleavage) is consistent
features. Both the activity and the DNA cleavage pattern with a random ss cleavage in which about 1% ds cleavage
II
[43]
by the complex are different from bare Cu ions (Figure 9), is expected statistically for every ss cleavage.
A simulta-
wherein nicking of the supercoiled (sc) plasmid by 50 µ neous fitting of the time-dependent changes of both nc and
II
RU of Cu -P1 rapidly occurs within 10 min (lane 1) to yield ln forms to a consecutive kinetic pattern, i.e., scǞncǞln
both nicked circular (nc) and linear (ln) forms of the plas- reveals that the formation of nc and ln forms up to 60 min
mid. The nc form can also be cleaved into ln form, but still can be reasonably fitted (dotted traces, Figure 9) without
retains a significant amount after two hours (lane 6). The considering ds cleavage of the sc form (however, the disap-
nc form shows a pseudo-first-order decay with a rate con- pearance of the sc form cannot be clearly traced within the
–
1
stant k = 0.0095 min
(᭹, Figure 9), suggesting possible time frame). A drastic change after 60 min (lanes 4Ј–6Ј) on
1
double-stranded (ds) cleavage of the nc form to yield the ln both forms may reflect their quick degradation due to ran-
form in a single step. However, the formation of the ln form dom multiple-site nicking to yield small fragments as shown
follows a bi-exponential kinetics (
᭡
, Figure 9), which can be by the smeared gel pattern.
II
deconvoluted into a fast initial increase with a pseudo-first-
The results reveal that Cu -P1 shows an apparent prefer-
–
1
order rate constant 0.066 min in the first 20 min and a ence toward nicking and cleavage of sc ds-DNA (to afford
slower rate with a pseudo-first-order rate constant the same nc and ln forms, respectively) as well as cleaving nc form
–1
as that for the decay of the nc form (0.0095 min ). The into ln form, rather than random cleavage toward all forms
former is probably also partially attributed to the cleavage of plasmid as in the case of DNA cleavage by hydroxy radi-
of the sc form. The initial concentration of the nc form is cals which cannot be described by the kinetic patterns
II
slightly greater than the final concentration of the ln form. herein. The cleavage pattern by Cu -P1 is reminiscent of
Eur. J. Inorg. Chem. 2008, 2584–2592
© 2008 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.eurjic.org
2589

L.-J. Ming et al.
FULL PAPER
II
[44]
and a few chemical model systems[45] nomenon may also play a role in catalysis associated with
Cu -bleomycin
II
that have been demonstrated to exhibit ds-DNA cleavage. the flexible chains of Cu -P1 which can form H-bonding
The apparent ds-DNA cleavage activity of Cu -P1 may be via its amide groups. The easy synthesis of P1 and its metal
II
attributed to that this macromolecular complex does not complexes suggests that it can serve as a template for fur-
quickly diffuse away from the reaction site after one DNA ther design of metallopolymer systems for various chemical,
[
50]
strand is cleaved due to its large size and a possible involve- biological, and environmental applications.
ment of H-bonding interaction between the complex and
DNA. As a consequence, subsequent cleavage of the second
strand can proceed effectively and become apparent in the
Experimental Section
early stage of the reaction (lanes 1 and 2). Moreover, the
II
II
All the chemicals in this report are commercially available. The
local Cu concentration in Cu -P1 is high which may result
in localized cleavage. Herein, the plasmid shows no observ-
able migration retardation in the gel that can be justified to
be attributable to interaction with the metallopolymer. If
there is any, it must be transient during the fast reaction
which cannot be revealed with the gel method. The copoly-
synthesis of the copolymer P1 follows the literature pro-
cedures.[
17,51]
The formation of the polymer and its RU stoichiome-
1
try are confirmed with H NMR, wherein the spectrum of the poly-
mer exhibit broad signals as reported which are distinct from the
sharp features from the monomers.
samples Ͻ1.0 m prepared with Cu(CH COO ) and the polymer
3 2
[17]
Electronic spectra of the
–
mer itself and H O separately did not exhibit noticeable in aqueous methanol solution were obtained with a Varian Cary50
2
2
effect on the plasmid in two hours under the same condi- spectrophotometer. Electron paramagnetic resonance (EPR) ex-
tions.
periments were performed with a Bruker Elexsys E580 cw/pulsed
X-band spectrometer. For a typical cw EPR spectrum at ca. 5–6 K,
the field was set wide enough to reveal a possible low-field transi-
tion for magnetically coupled systems with a microwave frequency
of 9.4 GHz, field modulation typically around 2 G, and time con-
stant of 40–80 ms. The g and A tensors were obtained with numeri-
cal fitting program provided by Bruker.
The oxidative DNA cleavage by this metallopolymer is
quite effective compared to other systems.[
46–48]
For in-
stance, linearization and further cleavage of plasmid DNA
was observed in 30 min by a dinuclear Cu complex of 5 µ
in the presence of 3-mercapto-propionic acid, while only
partial nicking of sc plasmid was observed by a mononu-
clear Cu complex under the same conditions;
percoiled plasmid was nicked by 20-µg Cu -polymer com-
plex in 10 min in the presence of monoperoxyphthalate;
linearization of plasmid DNA by 10-µM Cu in the pres-
ence of 25-µM 2-tert-butyl 1,4-hydroquinone was reported
II
II
[46]
The initial rates of the oxidation of catechol and analogs by 10-
su-
II
II
µM Cu -P1(RU) in buffered aqueous methanol solution (50%) at
2 2
pH 6.0 with fixed amounts of H O were determined with a Varian
Cary50 spectrophotometer. The oxidation of the polyphenol 1,2,3-
[
47]
II
trihydroxybenzene (THB) can be directly monitored at 420 nm (ε
–1
–1
–1
–1 [52]
=
4583  cm ) and DTBC 420 nm (ε = 1910  cm ). Alter-
[
48]
II
to occur in 30 min.
The results show that Cu -P1 can
natively, 3-methyl-2-benzothiazolinone hydrazone (MBTH) can
4
–1
–1 [53]
serve as an efficient and selective “artificial DNase” toward trap o-quinone to form a red adduct (ε500 = 3.25ϫ10  cm ).
the cleavage of sc and nc ds-DNA. Since ds-DNA cleavage
Pre-equilibrium kinetics can be described as the binding of a sub-
[14b]
is a key to trigger cell apoptosis,
ds-DNase activity of Cu -P1 points a direction for rational
the observation of the
strate to the catalytic metal center to form an intermediate P1-
II
II
Cu -S complex, followed by the conversion of the bound substrate
design of DNA-targeting drugs for therapeutic purposes,
into products [Equation (1)]. The rate law for this reaction mecha-
e.g., clustered redox-metal centers with a high local metal nism is expressed as in Equation (2), with KЈ = (k + kcat)/k as
–
1
1
II
concentration to afford a high ds-DNase activity.
the apparent dissociation constant of the P1-Cu -S complex, as-
suming that the concentration of the complex is much lower than
that of unbound substrate. Herein, the initial rates for the oxidation
of a catechol substrate S at different concentrations are determined
and fitted to Equation (2) to yield the rate constant kcat and KЈ.
Conclusions
II
Oxidation catalysis by the metallopolymer Cu -P1 in the
presence of the green oxidation agents H O and/or air to-
2
2
ward the oxidation of catechol/polyphenol and DNA cleav-
age is quite proficient. Moreover, the presence of coopera-
tive catalysis by Cu -P1 is also very unique among chemi-
(1)
II
cal systems. Despite the fact that polymers are considered
mostly unstructured, the “active center” in metallopolymers
herein seems to be able to assemble into a dinuclear site to
exhibit a significant catechol oxidase-like 2-electron oxidat-
ive activity toward polyphenol and catechol. Linear copoly-
mers with specifically designed side chains have recently
been demonstrated to exhibit high affinities toward target
(
2)
For a random bi-substrate reaction, the rate of catalysis of one
substrate S1 is systematically determined at various concentrations
of the other substrate S2 (i.e., rate vs. [S1] at various [S2]) and the
data analyzed by the use of the Hanes plots [Equation (3)], wherein
5
proteins with association constant in the order of 10 –
7
–1 [49]
1
0  ,
wherein the “induced-fit” theory has been sug-
gested to play a role in the recognition of the target proteins A can be fixed to either S1 or S2 and B is the other variable once
by the flexible linear copolymers. Such enzyme-like phe- A is fixed.[
28]
2590
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Eur. J. Inorg. Chem. 2008, 2584–2592

Oxidation Catalysis by a CuII Polymer
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[
[
19] For comparison, over-the-counter H O solution for household
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