Catalytic Cooperativity, Nuclearity, and O2/H2O2 Specificity of Multi-Copper(II) Complexes of Cyclen-Tethered Cyclotriphosphazene Ligands in Aqueous Media

Article abstract of DOI:10.1002/ejic.201700811

Three ligands L1, L2, and L3 with 2, 4, and 6 1,4,7,10-tetraazacyclododecane (cyclen) moieties attached to a cyclotriphosphazene core, respectively, were synthesized, and oxidation activities of their Cu^II complexes were investigated. Aerobic o

Full text of DOI:10.1002/ejic.201700811

A Journal of
Accepted Article
Title: Catalytic Cooperativity, Nuclearity, and O2/H2O2 Specificity
of Multi-Copper(II) Complexes of Cyclen-Tethered
Cyclotriphosphazene Ligands in Aqueous Media
Authors: Le Wang, Yong Ye, Vasiliki Lykourinou, Junliang Yang,
Alexander Angerhofer, Yufen Zhao, and LJ Ming
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To be cited as: Eur. J. Inorg. Chem. 10.1002/ejic.201700811
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10.1002/ejic.201700811
European Journal of Inorganic Chemistry
FULL PAPER
Catalytic Cooperativity, Nuclearity, and O2/H2O2 Specificity of
Multi-Copper(II) Complexes of Cyclen-Tethered
Cyclotriphosphazene Ligands in Aqueous Media
Le Wang,[a,b,c] Yong Ye,*[b] Vasiliki Lykourinou,[c],‡ Junliang Yang,[b] Alexander Angerhofer,*[d] Yufen
Zhao,*[b,e] and Li-June Ming*[c]
Abstract: Three ligands L1, L2, and L3 with 2, 4, and 6, respectively, Introduction
1,4,7,10-tetraazacyclododecane (cyclen) moieties attached to a
cyclotri-phosphazene core were synthesized, and oxidation activities
of their Cu(II) complexes investigated. Aerobic oxidation of catechol
by these complexes follows an intramolecular dinuclear pathway
with significant cooperativity (i.e.,   1.5 out of a maximum of 2 for
two potential substrate binding sites) and kinetic constants (i.e., kcat
= 17.5  10−3 s−1, Km = 2.8 mM, and quite remarkable catalytic
specificity kcat/Km 12.5 M−1 s−1 per di-Cu center), while that by
untethered Cu(II)–cyclen follows a bimolecular dinuclear pathway
Multi-domain and multi-subunit proteins are known to exhibit
cooperativity, such as tetrameric hemoglobin but not monomeric
myoglobin. Likewise, synthetic molecules with multi-domain
structures might also exhibit such unique property as observed
in dendrimers with identical metal-binding arms that exhibit
significant metal-binding cooperativity, which is not seen in
untethered ligands.[1] Cyclotriphosphazene (Cpz) is a versatile
scaffold for incorporating up to six moieties to exhibit various
chemical and biochemical properties,[ 2 ] which can serve as
prototypical multi-domain molecules for investigation of metal-
binding chemical cooperativity, protein-like ligand-binding
cooperativity, and enzyme-like catalytic cooperativity. Metal
complexes of various Cpz-containing ligands exhibit diverse
structural features and physical and chemical properties.[3,4] The
complexes of Cpz-containing ligands with tethered imidazole or
other N-heterocyclic groups exhibit activities toward cleavage of
plasmid DNA under physiological conditions.[5] Its Cu complexes
exhibit significantly more effective and specific hydrolytic activity
toward a phosphomonoester (Kd = 5 M) than a phosphodiester,
showing significant cooperativity with  = 1.5 for a maximum of 2
without noticeable cooperativity ( = 0.96) and 4-fold lower kcat
,
despite their similar dinuclear mechanisms. The proximity of Cu(II)
centers is suggested by EPR spectra and relaxations, showing a
broad spectral component particularly in Cu6L3. Thermodynamic
parameters also indicate the significance of multi-Cu(II) sites in the
oxidative catalysis. Air is a more specific oxidation agent for the
representative complex Cu2L1, showing 3.2-fold higher catalytic
specificity kcat/Km than H2O2 toward a catechol substrate. The
research provides further molecular basis for better understanding of
O2/H2O2-specific oxidation by multi-domain Cu complexes.
for two potential binding sites and following
a dinuclear
mechanism that mimics alkaline phosphatase.[6]
In addition to hydrolytic reactions, dioxygen activation and
oxidation processes mediated by transition metal centers play
essential roles in biological systems (e.g., the respiratory chain,
animal pigmentation, and browning of fruits and vegetables),[7]
environmental chemistry (e.g., degradation of inert organic
hazards),[8] medicinal chemistry,[9] and industrial processes.[10]
Redox-active Type-3 dinuclear copper enzymes, such as
catechol oxidase and tyrosinase, and their corresponding
biomimetic complexes have been extensively studied.[7, 11 , 12 ]
Many di-Cu complexes can bind and activate O2 and/or H2O2 to
hydroxylate or oxidize catechol, phenol, and their derivatives,
presumably by following the same dinuclear pathways as the
type-3 enzymes.
[a]
[b]
Dr. L. Wang
Department of Chemical Engineering
Shanghai University of Engineering Science
Shanghai 201620, PRC
Dr. L. Wang, Prof. Dr. Y. Ye,* and Prof. Dr. Y. Zhao*
Department of Chemistry
Zhengzhou University
Zhengzhou 450052, PRC
E-mails: yeyong@zzu.edu.cn, yfzhao@xmu.edu.cn
Dr. V. Lykourinou‡ and Prof. Dr. L.-J. Ming*
Department of Chemistry
University of South Florida
Tampa, FL 33620-5250, USA
E-mail: ming@usf.edu
Prof. Dr. A. Angerhofer*
Department of Chemistry
[c]
[d]
University of Florida
Gainesville, FL 32611, USA
E-mail: alex@chem.ufl.edu
Prof. Dr. Y. Zhao*
Department of Chemical Biology
Xiamen University
Xiamen 361005, PRC
Current address: Department of Chemistry & Chemical Biology,
Northeastern University, Boston, MA 02115, USA
A few approaches have been followed in mechanistic studies of
the oxidation reactions by di-Cu–peroxo/oxo complexes, such as
the assembly of mononuclear metal centers and the use of
ligands with multi-metal binding capability with a hydroxo, alkoxo,
phenoxo, oxo, or peroxo group as a bridging ligand.[12– 14 ]
Moreover, specific recognition[ 15 ] and a general acid/base[ 16 ]
have been verified to be significant factors for effective catalysis
by metal complexes. Macrocyclic polyamine ligands such as
[e]
‡
Supporting information for this article is given via a link at the end of
the document.((Please delete this text if not appropriate))
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10.1002/ejic.201700811
European Journal of Inorganic Chemistry
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1,4,7-triazacyclononane,
1,5,9-triazacyclododecane,
and
can be well fitted to a simplified equilibrium M + cy’
M-cy’, in
1,4,7,10-tetraazacyclododecane (cyclen) and their derivatives
can form multinuclear complexes[17] that exhibit various chemical
activities,[18] and can be covalently linked to various scaffolds
such as Cpz for building new multinuclear metal complexes.
With the aim of further development of metal complexes
possessing hydrolytic, oxidative, or other chemical activities
under physiological conditions, three multi-domain ligands L1–
L3 with different numbers of cyclen linked to Cpz via a phenol-
phosphoester bond were synthesized. The oxidation activities
and the reaction mechanisms of catechol-type substrates by the
Cu(II) complexes of these ligands were investigated and
presented herein.
which the cyclen arms cy’ of the ligands L’s are considered
completely independent and act like a free cyclen ligand. The
fitting affords apparent affinity constants of (3.0 ± 2.1) × 105, (4.0
± 1.1) × 105, and (4.7 ± 1.1) × 105 M–1. The similar magnitude of
the affinity constants indicates Cu(II) binding to the multi-dentate
ligands in a similar fashion. The lack of a sigmoidal shape
before reaching the equivalents of 2, 4, and 6 indicates absence
of detectable interactions between/among the cyclen arms that
may potentially cause binding cooperativity. Such metal-binding
cooperativity has previously observed for multi-domain ligands
such as dendrimers,[1] which is more pronounced in proteins
such as metal binding to dinuclear aminopeptidase.[26]
L1
L2
L3
Results and Discussion
Cu(II) binding. Upon Cu(II) binding to the multi-dentate ligands
L1, L2, and L3, a d-d transition at 600 nm is detected which is
consistent with a type-2 Cu(II) center of a strong ligand field due
to its all-N ligand environment. This transition is similar to those
of Cu(II)-cyclen (599 nm)[19] and the Cu(II) complexes of several
cyclen-containing ligands such as N,N'-meta-xylylenebis(cyclen)
and its para-isomer (both at 594 nm),[20] which adopt a square
Figure 1. Optical titration of Cu(II) into L1 (▼, 0.10 mM), L2 (○, 0.05 mM), and
L3 (●, 0.05 mM) in 50% methanol/HEPES solution (100 mM at pH 7.0 and 25
C), monitored at max = 600 nm. The solid traces are the best fits to 2:1, 4:1,
pyramidal coordination geometry with the four N atoms at the
[20, 21]
and 6:1 metal-to-ligand stoichiometry.
The electronic spectra of the
basal positions
and an axial group (even a very weak
−
−
complexes have similar features with molar absorptivity of 203  25 M−1 cm−1
per Cu, and follow similar spectral change upon Cu(II) binding to L2 (inset).
ligand such as NO3 and ClO4 ) that may be involved in further
coordination and/or H-bonding with a cyclen NH group based on
crystallographic studies.[22] A molar absorptivity of ~200 M−1
cm−1 per Cu is consistent with a coordinated external ligand,
such as OH− or Cl−.[19b] Analogous Cu-complexes with all-N
square planar coordination geometry shows a transition at <600
nm, while all-N distorted octahedral geometry shows at >600 nm
and a tetrahedral coordination is even lower in energy at >700
nm.[ 23 ] Despite our attempts, crystal structures of the CuL
complexes were not obtained which might be due to the
significant flexibility of the cyclen-arms of the complexes.
Nevertheless, a square planar/pyramidal coordination geometry
can be expected for the metal centers in these CuL complexes
on the basis of the structures of the other cyclen-containing Cu-
complexes. N-rich coordination sphere of different geometries in
metalloproteins follows the same trend, such as the all-N
(distorted tetrahedral 3 His’) active sites of Cu(II)-substituted
Although the electronic spectra of these Cu(II) complexes are
similar, the EPR spectra of the complexes are significantly
different despite their identical metal-binding cyclen sites. The
EPR spectrum of Cu1L1 at 5 K shows prototypical features of a
2
type-2 Cu(II) center with a dx2–y ground state in a distorted
square pyramidal coordination environment,[ 27 ] which can be
simulated with g// = 2.202, A// = 550 MHz, g = 2.055, and A =
55 MHz (Fig. 2B) similar to that of the Cu(II)-cyclen complex with
g// = 2.202, A// = 554 MHz, g = 2.055, and A = 45 MHz (Fig.
2A). These parameters are consistent with those of cyclen-
based Cu-complexes in a square planar coordination sphere.[23]
The spectrum of Cu2L1 is quite different from that of Cu1L1 and
is best simulated with three components: 48% of component A
with g// = 2.202, A// = 554 MHz, g = 2.056, and A = 47 MHz
similar to the spectrum of Cu1L1, 12% of component B with g// =
2.416, A// = 389 MHz, g = 2.0832, and A = 14.5 MHz, and 40%
24
]
carbonic anhydrase at 750 nm[
and Cu,Zn-superoxide
dismutase at 680 nm (distorted square planar 4 His‘).[25]
of a broad component C with g// = 2.262, A// = 178 MHz, g
2.0480, and A = 7.5 MHz and H-strain broadenings in the order
of 400 MHz (Fig. 2C). The second component with relatively
=
Plotting the change in absorption as a function of [Cu(II)] affords
metal-to-ligand stoichiometry of 2:1 (▼, Fig. 1), 4:1 (o), and 6:1
(), respectively, for Cu(II) binding to L1, L2, and L3. The results
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10.1002/ejic.201700811
European Journal of Inorganic Chemistry
FULL PAPER
large g// would indicate coordination to four oxygen ligands
according to the Peisach-Blumberg rules[ 28 ] if it could be
considered to have only four ligands but this is unlikely for
Cu(II)–cyclen complexes as seen from recent structural data of
similar complexes.[27, 29] It only accounts for 12% of the spectral
intensity and was included in the simulation because it was quite
pronounced in the echo-detected field sweep spectra of the
same sample (cf. Fig. 3). In case of metalloproteins, the three-
N(His) coordination sphere in Cu(II)-substituted carbonic
anhydrase[24] shows g// = 2.31, and g = 2.06 and the four-N(His)
coordination sphere in Cu,Zn-superoxide dismutase exhibits g// =
2.268, and g = 2.087.[25, 30]
cases. The spectral features suggest a common coordination
environment of the metal centers (i.e., component A) in all the
complexes with a tetragonally distorted octahedral geometry and
a
nitrogen-rich coordination environment.[28]
The loss in
resolution of hyperfine coupling with increasing amounts of
Cu(II) may be attributed to magnetic interactions among the
multiple Cu(II) centers within the complexes.
Spin-echo-detected EPR (se-EPR) spectra are sensitive to
changes in electronic relaxation times and only relatively slow-
relaxing signals can be better revealed. The se-EPR spectrum
of Cu1L1 (Fig. 3B) is similar to that of Cu–cyclen (Fig. 3A) and
can be fitted with similar spectral parameters of g// = 2.202 (vs.
2.002), A// = 554 MHz, g = 2.055, and A = 45 MHz, which
concludes their similar geometric and magnetic properties. The
se-EPR spectra of Cu2L1 (Fig. 3C) and Cu4L2 (Fig. 3D) are
similar to each other, but significantly different from that of
Cu1L1, and can be reasonably simulated with the two
components A and B already from their cw-EPR spectra (Fig.
3C), with component A resembling the spectrum of Cu1L1.
Although the EPR spectra of these complexes are different, the
Cu(II) sites exhibit similar electronic spectra (Fig. 1) and kinetic
parameters (see next section) with respect to each Cu(II) site
which suggests that the different Cu(II) centers undergo fast
exchange at room temperature to render one similar average
site for all the complexes. The se-EPR spectra of Cu2L1 (Fig.
2C) and Cu4L2 (Fig. 2D) can be simulated with two components
without the broad component, whereas that of Cu6L3 cannot.
Further fitting of this complex was not attempted since
anisotropy of the relaxation times can skew the shape of the
underlying EPR spectra.
Figure 2. EPR spectra of Cu–cyclen (A), Cu1L1 (B), Cu2L1 (C), Cu4L2 (D),
and Cu6L3 (E) in DMSO/100 mM HEPES buffer at pH 7.5 and 50 K, their
simulated spectra (dashed traces), and the three components for the
simulation in spectra C and D (dotted traces).
Notably, component C at 40% of the total intensity has to be
introduced to the spectrum of Cu2L1 in order to generate a good
fit. This component is practically absent in the echo-detected
EPR spectrum indicating that its relaxation is too fast for echo
detection. Together with the lack of clear features, component C
reflects the signature of possible magnetic coupling due to
proximity of the two Cu(II) centers in Cu2L1. The spectra of
Cu4L2 and Cu6L3 were simulated to some degree with the same
three components in Cu2L1, i.e., 56% of A, 14% of B, and 30%
of C for Cu4L1, and 24% of A, 20% of B, and 56% of C for
Cu6L3 (Figs. 2D,E). It should be noted that Cu6L3 presented the
most difficulty for a reasonable fit with variations in the
distribution of the components, possibly indicating changes in
the broad complex spectrum that were not seen in the other
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10.1002/ejic.201700811
European Journal of Inorganic Chemistry
FULL PAPER
Figure 3. Spin-echo-detected EPR spectra of Cu–cyclen (A), Cu1L1 (B),
Cu2L1 (C), Cu4L2 (D), and Cu6L3 (E) in DMSO/100 mM HEPES buffer at pH
7.5, simulated spectra (dashed traces), and the components (dotted traces).
Catalytic activities and cooperativity: The Cu(II) complexes of
L1, L2, and L3 exhibit significant activities toward catechol
oxidation in 50% methanol aqueous solution of 100 mM HEPES
at pH 7.0 and 25 °C, showing an enzyme-like saturation kinetic
pattern which can be fitted to Eq. (2) to yield the kinetic
constants kcat and K’ (Fig. 4A, Table 1) and significant first-order
catalytic proficiencies (kcat/ko)[31] of (0.75–2.1)  105 relative to
catechol auto-oxidation (ko). The kcat values follows the order of
Cu2L1 < Cu4L2 < Cu6L3, but are close to each other after
normalization with respect to Cu(II) concentrations, i.e., 0.0175 ±
0.0012 s–1 (Table 1); whereas the K’ values remain close to
each other (2.8 ± 0.3 mM). This observation indicates that the
higher activities of Cu4L2 and Cu6L3 are attributed to their
higher metal contents, and the similar kcat and K’ values per Cu
center of these complexes are indicative of their similar catalytic
mechanism.
Table 1. Kinetic parameters for catechol oxidation by the Cu–L complexes[a]
Complex
kcat
K’
(mM)
kcat/K’
CP[b]
(kcat/ko)
θ
(10−3 s–1
)
(M–1 s–1
)
Cu2L1
Cu4L2
35.5 (17.8c)
73.8 (18.5c)
97.8 (16.3c)
4.16
2.89
3.06
12.3
24.1
39.4
8.08
7.49 × 104
1.56 × 105
2.06 × 105
8.80 × 103
1.5
1.7
Cu6L3
2.48
1.5
Cu(cyclen)
0.515
0.96
[a] In 50% methanol/100 mM HEPES buffer at pH 7.0 and 25 °C. [b] The
background auto-oxidation rate constant ko = 4.74 × 10–7 s–1 [32]
[c] Turn-
over number per Cu(II) center.
.
The rates of catechol oxidation by the three Cu2nL complexes
are clearly sigmoidal and are better fitted to the Hill’s equation
with Hill coefficients θ of ~1.5 (dashed traces, Fig. 4A; Table 1)
out of a maximum of 2 for two metal sites of potential dinuclear
catalysis (discussed later). The close proximity of the Cu centers
in the complexes with multiple domains may have rendered the
significant cooperativity in the catalysis. Note that the Hill
coefficient is around 2.5 in hemoglobin of four subunits, out of a
The rate for the oxidation of 6 mM catechol by the complex
Cu1L1 with one-half of the metal-binding sites occupied (2.2
10–7 M/min) is about 7-fold slower than those of the other
complexes with the metal-binding sites fully occupied, e.g., a
rate of 1.5  10–6 M/min per Cu by Cu2L1 under the same
conditions. The EPR spectrum of Cu1L1 is similar to that of the
untethered Cu-cyclen complex (Figs. 2A,B & 3A,B), indicating
their similar Cu environments in frozen state. These results
suggest proximity of Cu sites in the Cu2nL1 complexes may be
important for catalysis, possibly rendering dinuclear catalysis.
maximum of
4 for the four possible binding sites. For
comparison, the untethered Cu(II)-cyclen complex shows about
4-fold lower kcat relative to the Cu2nL complexes per Cu center
without noticeable catalytic cooperativity (θ ≈ 0.96; ♦, Fig. 4A
and Table 1). Similar to multi-domain/subunit proteins, synthetic
molecules with multi-domain structures may also exhibit the
unique property, such as the significant metal-binding
cooperativity among the dendritic arms of dendrimers that is not
seen in untethered arms.[1] The CuL complexes herein do not
exhibit metal-binding cooperativity (Fig. 1) but catalytic
cooperativity (Fig. 4A), suggesting that metal binding to the
cyclen sites in these complexes is independent and random
whereas the catalysis follows cooperative dinuclear reaction
mechanism which is further discussed later.
Substrate specificity: The specificity of the prototypical complex
Cu2L1 was further investigated with various catechol-containing
substrates, 3,5-di-t-butylcatechol (DTBC) and the catecholamine
neurotransmitters dopamine, epinephrine, and norepinephrine.
These substrates are effectively oxidized by this complex with
high catalytic proficiencies of 155 to 75,000-fold higher than
uncatalyzed reactions (Table 2), which however do not seem to
show significant cooperativity (cf. Fig. 5A). The 2-order smaller
catalytic proficiencies for the three catecholamines relative to
catechol and DTBC seem to correlate with their biological roles
as neurotransmitters to be stable enough to resist non-regulated
oxidative transformation. Nevertheless, their metal-mediated
oxidation reflects that they may be victimized by oxidative stress
Figure 4. (A) Plots of the initial oxidation rates of catechol by Cu–cyclen (♦, 10
μM), Cu2L1 (▼, 5 μM), Cu4L2 (○, 5 μM), and Cu6L3 (●, 5 μM) in 100 mM
HEPES (50% methanol) at pH 7.0 and 25 °C, and fitted to equation 2 (solid
traces). The dashed traces are fittings to the Hill’s equation to obtain the
cooperativity coefficients. (B) Profiles of kcat for oxidation of catechol by H2O2
() and DTBC (○) by Cu2L1 in 100.0 mM HEPES/menthol (1:1) at pH 7.0 and
25 °C, and fitted to pre-equilibrium kinetics Eqs. 1–2.
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10.1002/ejic.201700811
European Journal of Inorganic Chemistry
FULL PAPER
caused by misregulated redox-active metal centers, such as the
oxidation chemistry of the Alzheimer’s disease-related Cu-β-
amyloid.[ 32 ] In the proposed mechanism for di-Cu catechol
oxidase,[ 33 ] the catechol substrate is oxidized by the met-di-
Cu(II) form of the enzyme via two-electron transfer to afford the
quinone product and reduced di-Cu(I)-enzyme, followed by
binding of O2 to form an active (di-Cu)-O2 center which binds
and oxidizes the second substrate. The aerobic oxidation of
catechol and its derivatives by the Cu(II) complexes may follow
the same dinuclear pathway as catechol oxidase, detailed in a
later section.
which are the rate constants with saturating amounts of
catechol/DTBC and H2O2.
This kind of H2O2-saturation kinetic pattern was previously
suggested to fallow an alternative pathway with a possible
change in the rate determining step in the presence of H2O2
from first-order to zero-order kinetics with respect to H2O2 in
which the oxidation of the substrate by the di-Cu(II) center is
considered the rate-limiting step. [36] Regardless, the proposed
pathway has the same kinetic pattern as saturation kinetics with
a reversible H2O2 binding (instead of H2O2 dissociation at path a
in reference [36]) followed by DTBC oxidation by the bound
H2O2 (path c in reference [36]). If the change in rate would be
attributed to the previously described mechanism, the
mechanism would not necessarily follow a bi-substrate pathway
with both the catechol substrate S and H2O2 bound to the metal
center to form the ternary complex peroxo–[Cu2L1]–S, but
regenerate the di-Cu(II) center via oxidation of the di-Cu(II)
center by H2O2 after the oxidation of the substrate36 which
however is inconsistent with the systems herein as discussed
below. Both kcat and K’ in catechol oxidation increase
significantly with [H2O2] (Table 2, Fig. 4B), resulting in no net
gain in catalytic specificity kcat/K’. Conversely, the catalytic
specificity of DTBC oxidation is much smaller in the presence of
saturating amounts of H2O2, indicating Cu2L1 is more specific
toward DTBC oxidation by air than that by H2O2.
Table 2. Oxidation of catechol-containing substrates by Cu2L1[a], unless as
noted.
Substrate
kcat
K’
(mM)
kcat/K’
CP[b]
(kcat/ko)
ko
(10−3 s–1
)
(M–1 s–1
)
(s−1
)
Catechol
Catechol[b]
Catechol[e]
DTBC
35.5
944
2.89
84.2
12.3
11.2
16.2
94.6
29.4
2.74
0.660
0.635
7.49 × 104 4.74×10–7[c]
2.79×105 3.38×10–6[d]
1.76104 4.74×10–7[c]
1.41×104 1.50×10–5[c]
1.16×104 9.95×10–5[d]
8.32
212
0.515
2.24
DTBC[b]
1160
1.72
0.266
0.372
39.4
Dopamine
Epinephrine
Norepinephrine
0.628
0.403
0.586
195
205
155
8.8×10–6[c]
1.3×10–6[c]
2.4×10–6[c]
To determine the patterns and affinities for the binding of the
catechol substrate and H2O2 to the Cu(II) center, the rate
constants of DTBC under different concentrations of H2O2 were
determined (Fig. 5A) and analyzed with the Hanes plots (Eq. 3,
Figs. 5B,C).[9, 37 ] The secondary plots of the slopes and y-
intercepts as functions of 1/[B] (Fig. 5D) from the Hanes plots
[a] In 100 mm HEPES buffer/methanol (1:1) solution at pH 7.0 and 25 C.
[b] In the presence of saturating amounts of H2O2 extrapolated from the
fittings in Fig. 4B. [c] The background rates constants for the oxidation of
the substrates, Reference [32]. [d] The auto-oxidation rate of the
substrates in the presence of 100 mM H2O2. [e] Kinetic parameters for
catechol oxidation by Cu(cyclen) normalized to 2[Cu(cyclen)] under the
same conditions as in [a].
(Fig. 6B) yield KDTBC = 5.15 mM, KH2O2 = 28.5 mM, and KiDTBC
=
2.47 mM with A = DTBC and B = H2O2 in Eq. (3). The intrinsic
dissociation constant KiDTBC = 2.47 mM obtained from the Hanes
analysis is close to K’ = 2.24 mM for DTBC oxidation without
H2O2 (Table 2), corroborating the bi-substrate mechanism. The
ratio KDTBC/KiDTBC = 2.08 (>1.0) indicates the binding of H2O2 to
the metal center slightly decreases DTBC binding affinity to
exhibit some exclusive nature between the two substrates H2O2
and DTBC.
Catalytic specificity and mechanism: Hydrogen peroxide is an
34
]
environmentally clean oxidation agent,[
which however
frequently requires further activation in order to perform effective
oxidation reactions by various chemical and biochemical
systems, such as peroxidases, the met-forms (di-CuII) of
A similar Hanes analysis of the rate-vs.-[H2O2] plots at various
[DTBC]s (Fig. 5C; with A and B switched in Eq. 3) and its
secondary plots yields KDTBC = 6.10 mM, KH2O2 = 37.5 mM, and
KiH2O2 = 16.4 mM. The ratio KH2O2/KiH2O2 = 2.29 indicates the
binding of DTBC also decreases H2O2 affinity to the metal center,
corroborating the slightly exclusive nature of their binding to the
metal. The results are consistent with a random bi-substrate
mechanism, wherein H2O2 and catechol bind to the metal
centers in the CunL complexes independently and slightly
exclusively to form the active ternary complexes DTBC–(CunL)–
H2O2.
35
]
catechol oxidase and tyrosinase,[33,
and di-Cu(II)
complexes.[36] The kcat values for H2O2-mediated oxidation of
catechol and DTBC by Cu2L2 were determined at certain fixed
H2O2 concentrations. A plot of kcat as a function of [H2O2] follows
a saturation kinetic pattern (Fig. 4B), reflecting direct H2O2
binding to the metal center in the presence of the substrate S to
afford a ternary peroxo–([di-CuII]–S) intermediate. Fitting of the
kcat values as a function of [H2O2] to Eq (2) yields the overall kcat
= 0.944 and 1.16 s–1, K’ = 84.2 and 39.4 mM, and catalytic
proficiencies (kcat/ko) of 2.79  105 and 1.16  104, respectively,
for oxidation of catechol and DTBC by H2O2 (Fig. 4B; Table 2),
Likewise, Hanes analysis of H2O2-mediated oxidation of catechol
by Cu2L1 yields KCA = 4.17 (and 3.79) mM, KH2O2 = 200 (and
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10.1002/ejic.201700811
European Journal of Inorganic Chemistry
FULL PAPER
158) mM, KiCA = 1.69 mM (relative to K’ = 2.89 mM in the
absence of H2O2, Table 2), and KiH2O2 = 42.7 mM (Table 3). The
ratios KCA/KiCA = 2.46 and KH2O2/KiH2O2 = 3.70 indicate the
binding of catechol to the metal center slightly decreases H2O2
binding and vice versa, as observed in DTBC oxidation
discussed above.
solvents, pHs, and/or temperatures. Some best performing
catechol oxidase models showed turnover numbers of 2.75–9.83
s–1 toward catechol oxidation,[40] while the catechol oxidase from
Ipomoea batatas41 exhibits kcat = 2,293 s−1 and Km = 2.5 mM that
is expectedly not closely matched by any of the model
complexes. Notably, the activities of the CunL complexes herein
are comparable to the catechol oxidase activity of hemocyanin
(which has a similar di-Cu coordination site as catechol oxidase)
from various sources,[42] i.e., kcat = 0.0035–0.183 s–1, Km = 2.6–
250 mM, and kcat/Km = 0.2–9.0 M–1 s–1.
The catalytic activity of Cu2L1 toward dopamine oxidation (kcat
=
1.72  10–3 s–1 and K’ = 0.628 mM) is close to those of the Cu(II)
complexes of β-amyloid peptides (kcat = (0.748–28.0) × 10–3 s–1
and K’ = 0.27–0.90 mM at pH 7.0 and 25 °C)[32b] and the Cu(II)
complex of an octapeptide fragment of prion[43] (kcat = 1.1 × 10−3
s−1 and K’ = 0.25 mM in the presence of 73 mM H2O2 in 80%
methanol aqueous solution at pH 7.5 and 30 °C), but expectedly
much less than that of tyrosinase from the cephalopod Illex
argentinus (kcat = 120 and 393 s–1 and Km = 1.3 and 0.39 mM).[44]
Although the di-Cu(II) met-form of tyrosinase exhibits H2O2-
mediated oxidation catalysis, it does not catalyze
oxidation/oxygenation by the use of air as the oxidation agent;
whereas the complexes herein perform effective aerobic
oxidation of catechol and derivatives.
Cu2L1 shows a significantly higher catalytic specificity for
aerobic oxidation than oxidation by H2O2 toward DTBC with a
ratio Rcs = CSO2/CSH2O2 = 322% (CSO2 and CSH2O2 are the
catalytic specificities of aerobic oxidation and H2O2-mediated
oxidation), while the Rcs ratio of 110% for catechol is only slightly
in favor of oxidation by air than by H2O2. Conversely, the Cu(II)
complex of a 4-vinylpyridine-co-acrylamide copolymer (CuP1)
shows Rcs = 64% toward DTBC oxidation by air/H2O2 (1:1
Figure 5. Oxidation of DTBC in the presence of 0, 10, 20, 40, 80, and 120 mM
H2O2 (from top) at 25 °C and pH 7 by 2 μM Cu2L1 (A) and the Hanes plot
against [DTBC] (B); (C) Hanes plot of (A) against [H2O2] with 0.2, 0.4, 0.8, 2.0,
and 4.0 mM DTBC (from top); and (D) the slope (○) and y-intercept (●) from
(B) as a function of 1/[H2O2].
methanol/25 mM MES buffer at pH 6.0 and 25 °C) and Rcs
=
48% toward 1,2,3- trihydroxybezene;[45] and Cu-β-amyloid(1–20)
exhibits Rcs = 440/1510 = 29% toward DTBC oxidation by
air/H2O2 (25 mM H2O2 in 100 mM HEPES buffer at pH 7.0).[32a]
Table 3. Hanes plot constants for DTBC, catechol (CA), and H2O2 from Eq. 3
A = DTBC
A = H2O2
A = CA
A = H2O2
B = H2O2
B = DTBC
B = H2O2
B = CA
The axial ligand in some Cu-cyclen complexes forms a H-bond
with an amine-NH group of cyclen.[22] The higher O2-specificity
of the CuL complexes might be contributable to H-bonding
between a bound O2 and the NH group. Such stabilization of a
bound O2 via H-bonding is best known in hemoglobin and
myoglobin, in which the bound O2 forms a H-bond with the NH
group of the distal His side chain. A bound H2O2 in the form of
OOH− may also form H-bonding with an acceptor such as an
carbonyl group found in CuP1 (i.e., the amide group) and Cu-β-
amyloid (i.e., various amino acid residues and the peptide bond)
which can enhance oxidation activity by H2O2, whereas a bound
KB (mM)
KA (mM)
KiA (mM)
KA/KiA
28.5
5.15
2.47
2.08
6.10
37.5
16.4
2.29
200
4.17
1.69
2.46
3.79
158
42.7
3.70
Comparison with other systems: The oxidative activities of the
Cu-L complexes toward catechol and DTBC with kcat = 0.036–
1.16 s−1 and K’ = 0.52–39 mM are comparable to or much better
than many Cu(II) complexes of macrocyclic or multidentate
ligands with kcat in the range of (0.33–2.5) ×10–4 to 1.5 s–1 and K’
= 0.071–2.0 mM.[36, 38 ] Several Cu(II)-containing multinuclear
complexes catalyze DTBC oxidation with kcat = 0.038–9.0 s−1
and K’ = 0.35–5.0 mM.[11a, 14a, 18a, 39] Detailed comparison with
various model complexes is not quite feasible as the reactions
were conducted under different conditions, e.g., different
2−
peroxide O2 can form H-bonds with an NH group. The CuL
complexes herein show higher Rcs toward O2 than H2O2,
suggesting that a bound O2 may form H-bonding with the cyclen
ligand better than H2O2. Owing to their high specificity toward
oxidation by air than by H2O2 of catechol substrates, these CunL
complexes may thus serve as prototypes for further design of
catalysts using air as an oxidation agent.
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10.1002/ejic.201700811
European Journal of Inorganic Chemistry
FULL PAPER
complexes, which affords the stoichiometry of [Cu–
cyclen]:catechol = 2:1 (▼, Fig. 6), reflecting a dinuclear catalytic
pathway by assembling two Cu centers to form the intermediate
[Cu–cyclen]2–catechol.
Catalytic mechanism and nuclearity: If all the Cu(II) centers in
these complexes are equivalent and follow a similar pathway
toward catechol oxidation, Cu1L1 would show 50% activity of
Cu2L1. However, the rate of oxidation of 6-mM catechol by
Cu1L1 is 6.8-fold lower than that by Cu2L1, suggesting that the
mononuclear Cu1L1 complex does not contain the same
structural motif as Cu2nL complexes for an effective catalysis
Thermodynamic Study: The kcat values for the oxidation of
catechol by the cyclen-containing CuL complexes were acquired
at different temperatures and the activation energy Ea
determined according to the Arrhenius equation kcat = A exp(–
Ea/RT) which yields the enthalpy of activation ∆H‡ (= Ea – RT)
with the trend Cu6L3 < Cu4L2 < Cu2L1 < Cu(cyclen). The Gibbs
free energy of activation ∆G‡ was obtained from kcat according to
the Eyring–Polanyi equation kcat = (kBT/h) exp(–∆G‡/RT) with kB
being the Boltzmann constant and h the Planck constant, from
which the entropy of activation ∆S‡ was calculated, following the
trend Cu6L3 > Cu4L2 > Cu2L1 > Cu(cyclen) for the –∆S‡ values
(Table 4). The “normalized kcat values” (thus the “normalized
∆G‡ values”) with respect to a single di-Cu center in the three
Cu2nL complexes are similar, resulting in more pronounced –∆S‡
values for Cu4L2 and Cu6L3 (Table 4). The observation of pre-
equilibrium kinetics for catechol oxidation by these complexes
indicates formation of the enzyme-like Michaelis complex [S–
CuL] prior to product formation. The negative ∆S‡ values and
their trend indicate that the transition state TS‡ is better
organized than the prior step, probably due to the involvement of
more metal sites in Cu4L2 and Cu6L3 to stabilize and organize
the substrate than in Cu2L1 and Cu(cyclen).
that presumably follows a dinuclear pathway.
Since the
substrate-bound intermediate [CuL]–S in Eq. (1) is the
catalytically active form, the mechanistically important
stoichiometry of the intermediate can be determined by means
of the “mechanistic Job plot”[6, 46b, 47 ] to reveal the nuclearity
during the catalysis. The oxidation of catechol by Cu2L1, Cu4L2,
and Cu6L3 exhibit a maximum at XCuL ~ 0.5 (■, Fig. 6), 0.33 (○),
and 0.25 (●) in their mechanistic Job plots, affording the
CuL:catechol ratios of 0.5:0.5, 0.33:0.67, and 0.25:0.75 (i.e., 1:1,
1:2, and 1:3), respectively. Since the complexes Cu2L1, Cu4L2,
and Cu6L3 contain 2, 4, and 6 metal centers, the ratios of 1:1,
1:2, and 1:3 reflect 2-to-1 metal-to-substrate stoichiometry in the
intermediates [CuL]–S for the oxidation of catechol by these
three complexes, indicating a dinuclear catalytic pathway.
Table 4. Activation parameters for catechol oxidation by CuL complexes at
298 K[a]
Ea
H≠
(kJ/mol)
G≠
(kJ/mol)
S≠
(J/mol K)
(kJ/mol)b
Cu2L1
Cu4L2
29.2
21.7
26.7
19.2
81.2
−183
79.5
−202
(81.2c)
(−208c)
Cu6L3
17.6
43.4
15.1
40.5
78.7
−214
(81.5c)
(−223c)
Cu(cyclen)
(86.6c)
(−155c)
Figure 6. “Mechanistic Job plots” for the oxidation of catechol by Cu2L1 (■),
Cu4L2 (○), and CuL3 (●) at a total concentration ([complex] + [catechol]) of
100 μM and by Cu–cyclen (▼) at a total concentration of 500 μM, and fitted to
complex-to-catechol ratios of 1:1, 1:2, 1:3, and 2:1, respectively (solid traces).
The dashed traces are fittings to a complex-to-catechol ratio of 1:1.
[a] In 100 mM pH 7.0 HEPES buffer/methanol (1:1) solution. [b] obtained
from temperature range 25–55 C. [c] Normalized based on a di-Cu center
relative to Cu2L1.
For comparison, the activity of Cu(II)-cyclen, which resembles a
metal-binding arm in the ligands L1–L3, was determined under
the same conditions. Cu(II)–cyclen catalyzes aerobic catechol
oxidation with kcat = 4.16  10–3 s–1 and kcat/Km = 8.08 s–1 M–1 per
Cu center under the same conditions which are significantly
smaller than those of Cu2L1 (Fig. 4A; Table 1). Moreover,
Cu(II)–cyclen does not seem to show catalytic cooperativity
toward catechol oxidation as the three Cu2L complexes (Fig. 4A).
The mechanistic Job plot of catechol oxidation by Cu–cyclen
shows a maximum at X[Cu–L] ~ 0.67, opposite to those of the CuL
Concluding Remarks
We describe herein the activities, mechanism, and catalytic
specificities of the copper(II) complexes of the multidentate
ligands L1, L2, and L3 toward oxidation of catechol and
derivatives. The studies reveal the involvement of intramolecular
dinuclear Cu(II) centers during catalysis in the Cu2nL complexes
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10.1002/ejic.201700811
European Journal of Inorganic Chemistry
FULL PAPER
for epinephrine and norepinephrine).[ 53 ] The oxidation of 3,5-di-t-
butylcatechol (DTBC) is directly monitored at 420 nm (ε = 1,910 M–1
cm–1).[54] The para-quinone isomer does not generate a significant 500-
nm absorption in the experimental time frame to interfere the detection of
the ortho isomer under the same conditions.
In enzyme-like pre-equilibrium kinetics, the substrate S binds to the
catalytic Cu center of the CuL complexes to form the intermediate S–CuL
complex (analogous to the enzyme-substrate ES complex) which is
followed by conversion to products as described in Equation (1). The
rate law for this reaction mechanism is expressed as Equation (2), with
K’= (k–1 + kcat)/k1 being the apparent dissociation constant of the S–CuL
complex analogous to the Michaelis constant Km in enzyme catalysis,
assuming that the concentration of this complex is much lower than that
of the unbound substrate. The initial rates for the oxidation of the
substrate S at different concentrations are fitted to Equation (2) with non-
linear regression to find the rate constants kcat and K’.
with much higher activities than the mono-Cu(II) complexes
Cu1L1 and Cu(cyclen). Although many dinuclear Cu(II)
complexes show oxidation activity by dioxygen and/or H2O2, it is
still challenging to obtain complexes that incorporate catalytic
cooperativity, specific substrate recognition, and O2/H2O2
specificity. The complexes herein possess all these properties.
Moreover, the Cpz core is quite versatile for (a) systematic
studies of the nature of the ligands (such as imidazole[6] and
cyclen-containing arms herein), (b) assembling various di-Cu
centers (such as Cu2L1, Cu4L2, and Cu6L3), (c) building
substrate specificity (described herein and the specific
phosphomonoester recognition and hydrolysis[6]), (d) rendering
catalytic cooperativity in oxidation (Fig. 5A) and hydrolysis,[6] (e)
exhibiting more specific oxidation reactions by O2 than by H2O2
(Table 2), (f) possibly building a potential substrate recognition
site in the proximity of the metal center, and (g) exploration of
other activities by various metal complexes of Cpz-containing
ligands in the future.
(1)
(2)
The stoichiometry of a metal complex M–Ln in the equilibrium M + nL
M–Ln can be determined by means of the Job plot, wherein the total
concentration [M] + [L] is kept constant while the metal-to-ligand ratio is
varied and the optical density of the complex is monitored.[46] Likewise,
Experimental Section
the presence of the pre-equilibrium CuL + S
S–CuL in the catalysis
of catechol oxidation allows the use of the “mechanistic Job plot[6, 47]” to
determine the stoichiometry of the substrate-bound complex S–CuL in
the reaction mechanism by monitoring the reaction rate that directly
reflects the concentration of the intermediate (i.e., rate = kcat[S–CuL]),
analogous to the optical density of the complex in the conventional Job
plot. In this case, the total concentration ([CuL] + [S]) is kept constant
while the ratio [CuL]/[S] varies and the oxidation rate of the substrate S is
monitored. The maximum reaction rate in the Job plot reflects the
stoichiometry of the ternary complex, e.g, a maximum at mole fraction
XCu–L = 0.66 reflects that the stoichiometry of the ternary complex S–CuL
is XCu–L:XS = 0.66:0.33 = 2:1 which indicates a dinuclear catalysis.
Materials and Ligand Syntheses are described in the Supporting
Information. The Cu(II) complexes were prepared by mixing
stoichiometric amounts of Cu(II) (99.999%) and the ligands L1–3 in 1:1
menthol/100.0 mM HEPES buffer at pH 7.0 for optical and kinetic studies,
and in DMSO/100 mM HEPES buffer at pH 7.5 for EPR experiments.
The choice of the solvents was partially empirical for better detection and
observation, and for better formation of glass-state at low temperatures
for EPR. All the samples were prepared right prior to measurements.
EPR Measurements. EPR spectra were obtained with a Bruker Elexsys
E580 X-band spectrometer at ~20 K using an Oxford ESR900 cryostat
and the Bruker standard rectangular TE102 resonator, typically with a
microwave frequency at ~9.4 GHz, field modulation around 2 G, and time
constant/conversion time of 40/80 ms. The g and A values were
obtained from spectral simulations performed with the “EasySpin” toolbox
for Matlab.[48] Since a few Cu(II)-substituted metalloproteins[49] and Cu(II)
complexes[50] were determined to have pKa values of around 6–7 for the
coordinated water, it is thus appropriate to choose a pH value away from
the pKa values to avoid possible coexistence of both protonated and
deprotonated forms of the coordinated water.[51] The spectra of Cu(II)-
cyclen and Cu1L1 with a single component demonstrate this viewpoint.
Pulsed EPR spectra were obtained with the same spectrometer and a
Bruker Flexline cryostat at a temperature of 5 to 6 K. Spin-echo-detected
field-swept spectra were obtained using the 2-pulse Hahn echo
sequence 90––180––AQ where ‘AQ’ stands for the observed echo.
Spin lattice relaxation times were obtained with the inversion-recovery
sequence 180–T–90––180––AQ with echo-detection. The traces
were fitted to a first-order relaxation or a combined two-step relaxation to
yield the relaxation times of the Cu(II) centers in the complexes.
For a random bi-substrate catalysis, such as H2O2-mediated oxidation of
catechol by the Cu(II) complexes herein with both H2O2 and catechol as
the substrates,[] the dissociation constants for each substrate can be
determined from the Hanes plots (Eq. 3)[9, 37] by measuring the reaction
rate of one substrate S1 with systematic variation in concentration of the
other substrate S2. The rate law for this catalysis is shown as Eq (3),


[A]

1 KB /[B]
Vmax

KA
KiAK

[A]
1
(3)

B 
Vo
Vmax
[B]KA


in which A can be fixed to either S1 or S2 and B is the other substrate
once A is fixed, KA and KB are apparent dissociation constants for the
ternary complex with both A and B bound to the catalytic center, i.e., A–
(CuL)–B
(CuL)–B + A and A–(CuL) + B, respectively, and KiA is the
CuL + A. A secondary plot
intrinsic dissociation constant in A–(CuL)
of the slope (1 + KB/[B])/Vmax or the y-intercept (KA/Vmax)(1 + KiAKB/KA[B])
as function of [B] yields the dissociation constants. If the binding of one
substrate influences the binding of the other, a significant difference
between KA(B) and KiA(B) would be detected.
Kinetic Measurements. The initial oxidation rates of the substrates by 5
μM Cu(II) complexes of L1, L2, and L3 in buffered aqueous-methanol
solution (50% 100 mM HEPES at pH 7.0) were determined by monitoring
the change in absorption at 500 nm due to the formation of the product
adduct o-quinone-MBTH as previously described[9, 32] (ε = 32,500 M–1
cm–1 for catechol,[52] 27,200 M–1 cm–1 for dopamine, and 27,500 M–1 cm–1
Acknowledgments
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10.1002/ejic.201700811
European Journal of Inorganic Chemistry
FULL PAPER
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Financial support from the National Natural Science Foundation
of China (YY and YZ, grant numbers 20602032, 20732004,
20972143) and the opportunity of a national scholarship from the
Department of Education of China (LW) are acknowledged. The
research on “Catalytic Minimalism from Nature” (LJM, CHE-
0718625) and the research at the University of Florida USA (AA,
CHE-0809725) were sponsored by the National Science
Foundation of USA.
Keywords: catechol oxidase • copper • cyclen • multinuclear •
oxidation
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Entry for the Table of Contents (Please choose one layout)
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Catechol oxidation by three Cu(II)
complexes of 1,4,7,10-tetraaza-
L. Wang, Y. Ye,* V. Lykourinou, J. Yang,
A. Angerhofer,* Y. Zhao,* and L.-J.
Ming*
x5
cyclododecane (cyclen) attached to a
cyclotriphosphazene core follows
enzyme-like saturation kinetics and an
intramolecular dinuclear pathway with
significant cooperativity (  1.5 with a
maximum of 2 for two binding sites),
but not so for the untethered Cu(II)–
cyclen with a lower kcat/Km. The
studies also show that O2 is a more
specific oxidation agent than H2O2 for
catechol oxidation.
Page No. – Page No.
Catalytic Cooperativity, Nuclearity,
and O2/H2O2 Specificity of Multi-
Copper(II) Complexes of Cyclen-
Tethered Cyclotriphosphazene
Ligands in Aqueous Media
Cu(II)
Cu(II)
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