Mechanistic insights into phenol oxidation by a copper(II) complex of a pyridine- And amide-containing copolymer in an aqueous medium

Article abstract of DOI:10.1002/ejic.201402778

A Cu^II complex of a pyridine- and amide-containing copolymer (Cu-P1) exhibits effective activity toward phenol hydroxylation in 50% aqueous methanol solution at apparent pH 8.0 and 25°C. The complex shows significant first-order rate accelerations of 4.2 × 10⁵ and 1.4 × 10⁵ relative to phenol autoxidation in air and in the presence of 20 mM H₂O₂, respectively. The reaction mechanisms with H₂O₂ and air are different on the basis of (a) the different activity profiles for Cu binding, (b) the different deuterium kinetic isotope effects (2.8 in 20 mM H₂O₂ and 1.2 aerobically), and (c) the formation of a dinuclear substrate-(Cu-P1) complex with H₂O₂ but a mononuclear one aerobically. The mechanism in the presence of H₂O₂ is consistent with that of the type-3 dicopper tyrosinase. Although oxidized di-Cu^II tyrosinase can only use H₂O₂ for phenol hydroxylation, Cu^II-P1 can use either air or H₂O₂ for this process. The studies herein introduce a versatile chemical system for the further exploration of Cu-oxygen chemistry and other types of metal-centered chemistry and for the aerobic degradation of aromatic compounds in environmental and green chemistry.

Full text of DOI:10.1002/ejic.201402778

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DOI:10.1002/ejic.201402778
Mechanistic Insights into Phenol Oxidation by a
Copper(II) Complex of a Pyridine- and Amide-Containing
Copolymer in an Aqueous Medium
Vasiliki Lykourinou[a,b] and Li-June Ming*[a]
Keywords: Enzyme models / Copper / Oxidation / Dinuclear complexes / Metallopolymers
A CuII complex of a pyridine- and amide-containing copoly-
mer (Cu-P1) exhibits effective activity toward phenol hydrox- mononuclear one aerobically. The mechanism in the pres-
of a dinuclear substrate–(Cu-P1) complex with H2O2 but a
ylation in 50% aqueous methanol solution at apparent pH
8.0 and 25 °C. The complex shows significant first-order rate
accelerations of 4.2ϫ105 and 1.4ϫ105 relative to phenol
ence of H2O2 is consistent with that of the type-3 dicopper
tyrosinase. Although oxidized di-CuII tyrosinase can only use
H2O2 for phenol hydroxylation, CuII-P1 can use either air or
H2O2 for this process. The studies herein introduce a versa-
tile chemical system for the further exploration of Cu–oxygen
chemistry and other types of metal-centered chemistry and
for the aerobic degradation of aromatic compounds in envi-
ronmental and green chemistry.
autoxidation in air and in the presence of 20 m
M H2O2,
respectively. The reaction mechanisms with H2O2 and air are
different on the basis of (a) the different activity profiles for
Cu binding, (b) the different deuterium kinetic isotope effects
(2.8 in 20 m
M H2O2 and 1.2 aerobically), and (c) the formation
C–H bond. Biomimetic chemical systems may be more
available, stable, and catalytically versatile than enzymes;
thus, they may have wide applications. Understanding the
activity and mechanism of Cu-containing chemical systems
can provide information for a better understanding of O2
utilization by copper enzymes,[5,9,10] and these systems have
possible applications in the fields of environmental and
green chemistry,[11] aging, cancer research, and oxidative
stress-associated apoptosis.[12–14]
In spite of the inhomogeneous chemical and physical na-
ture of copolymers, some bulk properties of polymers can
be well controlled, such as their hydrophobicity and hydro-
philicity, their optical, magnetic, electrochemical, and me-
chanical properties,[15] as well as their physical shapes;
therefore, they have a wide variety of applications. Several
metal-binding polymers have been prepared and utilized as
catalysts for different types of metal-centered reactions.[16]
Moreover, those with side chains designed for specific inter-
actions exhibit high affinities toward target proteins and
have association constants in the order of 105–107 m–1,[17]
which are attributed to the “induced-fit” in the recognition.
This enzymelike property may also play an important role
in catalysis by flexible polymer catalysts. To further explore
the versatility of metallopolymers for specific recognition
and as potential catalysts, we have prepared and investi-
gated a copolymer (P1) of 4-vinylpyridine and acrylamide
with an average repeating unit (RU) of (4-vinyl pyridine)3-
(acrylamide)1, which can bind to metal ions through the
pyridine rings and may possibly exhibit recognition through
H bonding with the amide group. Thus, metal complexes of
P1 may loosely mimic the metal-binding, substrate recogni-
Introduction
The use of dioxygen[1] as an oxidation agent in biological
systems requires its specific activation by different en-
zymes,[2] including Fe- and Cu-containing oxidases and
oxygenases such as di-Cu tyrosinase and polyphenol oxid-
ase. An oxygen donor can be involved in the oxidative
transformation of synthetic organic compounds and bio-
molecules when it is activated by Cu complexes or Cu-con-
taining oxidases.[3,4] Di- and multinuclear Cu oxidases and
oxygenases are ubiquitous[5] and can use O2 and H2O2 for
the hydroxylation or oxidation reactions of phenol and
polyphenol derivatives. The hydroxylation of tyrosine by ty-
rosinase yields the neurologically significant DOPA, which
can be further oxidized to yield o-dopaquinone and ulti-
mately forms melanin pigments.[6,7] Tyrosinase has also
been used in biosensors for oxygen and phenol detection,
as a catalyst for the degradation of pollutant phenols, in
the selective synthesis of o-quinones and related polymers,
and in many other applications.[8]
Oxygenation and hydroxylation processes are challenging
chemical and industrial tasks, as the hydroxylation of
hydrocarbons to yield alcohols and phenols is a difficult
spin-forbidden process, wherein the triplet-state dioxygen
needs to be activated to effectively insert into the singlet
[a] Department of Chemistry, University of South Florida,
4202 Fowler Avenue, Tampa, FL 33620, USA
E-mail: ming@mail.usf.edu
http://chuma.cas.usf.edu/~ming/
[b] Current address: Department of Chemistry & Chemical Bio-
logy, Northeastern University, 360 Huntington Ave., 102 Hurtig
Hall, Boston, MA 02115, USA
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tion, and metal-centered catalytic properties of metalloen-
zymes and have demonstrated significant hydrolytic[18,19]
and oxidative activities.[19,20] Herein, we report that the CuII
complex of P1 (CuII-P1; 1 with the average RU shown in
Scheme 1) can effectively catalyze the hydroxylation and
oxidation of phenol and tyramine, but not tyrosine, with air
or H2O2 as green oxidizing agents in an aqueous medium
by a mechanism analogous to that of dicopper hydroxylases
and oxidases. The results further address the question we
previously raised, “How well should the active site and the
specific recognition be defined for proficient catalysis?”,[20]
and reveal that the assembly of a metal-binding active site,
the recognition of substrates, and the stabilization of the
transition states in various reactions may all become pos-
sible through induced-fit, despite the randomness in the se-
quence of copolymers. The results also point to a new direc-
tion for the further exploration and application of metal-
mediated reactions by metallopolymers.
Figure 1. (a) Initial rate of phenol oxidation by 50 μm RU of CuII-
P1 in 50% methanol/0.1 m HEPES buffer solution at pH 8.0 and
25 °C in the presence of 0.0, 2.0, 5.0, and 20.0 mm H2O2 (from
bottom) and fit of the data to Equation (2). (b) Rate for phenol
oxidation (12.0 mm) as a function of [H2O2] at pH 8.0 and 25 °C
catalyzed by 50 μm RU of Cu-P1. (c) Hanes plots of the data from
(a). (d) Hanes plots of the data from (a) in the presence of 2, 4,
and 8 mm phenol (from top).
Aerobic PhOH oxidation by CuII-P1 exhibits a signifi-
cant rate acceleration kcat/ko of 4.2ϫ105 with respect to
PhOH autoxidation without the catalyst (ko
=
3.3ϫ10–9 s–1) under the same conditions. This aerobic
phenol oxidation activity is comparable to those of the CuII
complex of the Alzheimer-disease-related β-amyloid[21] with
kcat = 3.90ϫ10–3 s–1, KЈ = 1.23 mm, and kcat/KЈ =
3.17 m–1 s–1 and a [RuVIL(O)2]2+ complex with k = 11.0 and
2.36 m–1 s–1 in water and MeCN, respectively.[23] The oxi-
dation rate is also higher than catalytic wet air oxidations
(CWAOs) for PhOH degradation by homogeneous CuII and
heterogeneous CuCeOx catalysts at 150 °C and ca. 9 atm
PO2 with k = 0.168 and 0.0338 m–1 s–1, respectively; how-
ever, this process follows a free radical pathway.
Scheme 1.
Results and Discussion
A noticeable aerobic oxidation of phenol by CuII-P1 was
The rate of PhOH oxidation for the production of o-
observed in the absence of H2O2 (Figure 1a, ᭹ and at quinone by CuII-P1 in the presence of a certain amount of
[H2O2] = 0 in Figure 1, b) and reached a plateau at high H2O2 reaches a plateau at high [PhOH] (Figure 1, a); this
PhOH concentrations; this suggests that the process is suggests that the process is metal-centered and follows en-
metal-centered and that enzymelike kinetics apply [Equa- zymelike kinetics [Equations (1) and (2)]. For instance, the
tions (1) and (2), see Exp. Sect.]. The rate of oxidation as a reaction in the presence of saturating amount of H2O2
function of [PhOH] can be fitted to Equation (2) to yield (20.0 mm, 0.068%, which is much lower than the concentra-
the rate constants kcat = 1.27ϫ10–3 s–1 and KЈ = 1.02 mm tion of off-the-shelf 3% H2O2 solution from drug stores in
and the catalytic efficiency (ce ) kcat/KЈ = 1.25 m–1 s–1. The the United States) yields kcat = 3.6ϫ10–3 s–1, KЈ = 2.6 mm,
O2
rate is significantly higher than the autoxidation rate of and catalytic efficiency (ceH O ) kcat/KЈ = 1.4 m–1 s–1, which
2
PhOH in the absence of CuII-P1 under the same conditions. decreases to 0.90 m–1 s–1 when2the aerobic oxidation contri-
O2 in the air serves as the oxidizing agent, and this provides bution to kcat is considered. Here, the KЈ value characterizes
a rare example of the catalytic hydroxylation of (poly)- the apparent dissociation of PhOH from a presumed inter-
phenol by a CuII center with O2 as the oxidizing agent.[21,22] mediate ternary complex H2O2–(CuII-P1)–PhOH. Phenol
Conversely, the oxidized met-form (di-CuII) tyrosinase is peroxidation by CuII-P1 in the presence of 20.0 mm H2O2
not active with O2 but requires reduction to afford the exhibits a significant rate acceleration; kcat/ko is 1.4ϫ105
di-CuI center, which then binds O2 to yield the active times higher with respect to PhOH autoxidation without
(di-CuII)–peroxo intermediate.[31] Accordingly, a transient the catalyst (ko = 3.3ϫ10–8 s–1) under the same conditions.
CuI center in P1 must form, probably substrate-induced, to For comparison, the rate constants are 0.038 s–1 for a CuII–
bind O2 and form an active Cu–(per)oxo center for PhOH polymer complex with salen metal-binding sites (2 in
hydroxylation and oxidation.
Scheme 1) in 30% H2O2 at 80 °C;[24] (1.83–3.80)ϫ10–3 s–1
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for CuII complexes of some pyrrole–imine ligands (3 in ternary complex H2O2–(CuII-P1)–PhOH and the intrinsic
Scheme 1)[25] at different pH values with 1:1 PhOH/30% constants Ki(PhOH) = 0.52 mm and Ki
= 0.97 mm for
(H2O2)
H2O2; 0.18 mLmin–1 g–1 (converted to 2.81ϫ10–4 –1 s–1) the dissociation of phenol and H2O2 from the complexes
m
for a recycling continuous flow reactor of pillared clay con- (CuII-P1)–PhOH and H2O2–(CuII-P1), respectively. The ra-
taining iron;[26] 1.23 s–1 for CuII(picolinato)2 (4 in Scheme 1) tio K/Ki is 2.5 for phenol and 2.6 for H2O2; therefore, the
encapsulated in zeolite Y under 280 W microwave irradia- binding of one substrate slightly decreases the affinity of
tion in the presence of 30% H2O2;[27] kcat = 0.213 s–1, KЈ the other.[29,30] The mechanism for this hydroxylation reac-
= 1.31 mm, and kcat/KЈ = 163 s–1 m–1 for CuII–β-amyloid at tion in the presence of H2O2 presumably follows the “perox-
Ͼ50 mm H2O2;[21] and kcat = 548 s–1, Km = 2.19 mm, and ide shunt” pathway in the action of tyrosinase with a ran-
kcat/KЈ = 2.5ϫ105 s–1 m–1 for horseradish peroxidase C.[28]
A control reaction with p-quinone and the trapper 3- ter.[31]
dom binding manner of PhOH and H2O2 to the active cen-
methyl-2-benzothiazolinone hydrazone (MBTH) under the
The gradual replacement of the CuII ions in Cu-P1 by
same conditions and time frame of ca. 5–10 min did not redox-inactive ZnII ions is expected to serve as a practical
show a noticeable change in the absorption of p-quinone method to address the nature of active-center cooperativity
for a possible Schiff base adduct. There is also no signifi- by steady deactivation of the catalyst through ZnII dilution.
cant residual absorption at λ ≈ 400 nm attributable to a A linear correlation between the activity and the extent of
quinone after the o-quinone produced from PhOH by ZnII dilution should be observed with a constant amount
H2O2/(CuII-P1) is removed by the formation of an adduct of [CuII] + [ZnII] and P1 if the CuII centers in Cu-P1 act
with MBTH. These results indicate the predominant forma- independently as mononuclear centers without cooperativ-
tion of o-quinone and exclude significant radical-initiated ity. Quite the opposite, a sigmoidal activity profile was ob-
hydroxylation of PhOH, which would also produce the para served as a function of the mol fraction of CuII toward
isomer, and, therefore, corroborate a selective metal-cen- PhOH oxidation in the presence of H2O2 (Figure 2, ᭹). The
tered ortho-hydroxylation reaction.
data can be fitted to the Hill equation with a Hill coefficient
CuII-P1 exhibits 40% higher catalytic efficiency toward θ = 3.80. However, the activity profile for PhOH oxidation
PhOH hydroxylation/oxidation for air as the oxidizing without H2O2 is less sigmoidal (Figure 2, ᭺) and gives a
agent than for 20 mm H2O2 as the oxidizing agent, that is, smaller θ value of 1.62. This observation suggests that the
ceO /ceH O = 1.4. Conversely, CuII–β-amyloid is ca. 50-fold rate-determining steps in the presence and absence of H2O2
2
2
2
more effective for peroxidation than for aerobic hydroxyl- may be different. Presumably, a cooperative di-CuII active
ation/oxidation of PhOH with ceO /ceH O = 0.019. The center is present in the oxidation of PhOH by CuII-P1 in
threefold higher rate enhancemen2t kcat/2ko for aerobic the presence of H2O2, consistent with a dinuclear catalytic
phenol oxidation than peroxidation by CuII-P1 also indi- cycle as observed in the di-Cu enzymes tyrosinase and cate-
cates that this metallopolymer is more effective toward chol oxidase.[5,31] Such a cooperative dinuclear center does
PhOH hydroxylation/oxidation with air than with H2O2 as not seem to be present in the aerobic oxidation of PhOH
the oxidizing agent. The results suggest that CuII-P1 can by CuII-P1 in the absence of H2O2; therefore, the pathway
serve as a model for the further design of metallopolymers is probably mononuclear.
for the effective aerobic hydroxylation of arenes in environ-
2
mental remediation and other applications in oxidation
chemistry. On the other hand, Cu-β-amyloid has been im-
plied to cause more significant damage with H2O2 as the
oxidizing agent than with the air as the oxidizing agent dur-
ing oxidative stress.[21]
The oxidation of 10.0 mm phenol by CuII-P1 as a func-
tion of [H2O2] is nonlinear (Figure 1, b), which indicates
that H2O2 can also bind to the metal center as a substrate.
The data can be fitted to the pre-equilibrium kinetics
[Equation (2)] with S = H2O2 to give kcat = 0.0046 s–1 and
KЈH O = 2.3 mm for the apparent dissociation of H2O2 from
2
2
the presumed ternary complex H2O2–(CuII-P1)–PhOH. As
both PhOH and H2O2 bind to the metal center as the sub-
strates, the data were further analyzed with a Hanes plot to
reveal their relationship in terms of metal binding [Equa-
tions (3) and (4), see Exp. Sect.].[29] The data in Figure 1 (a)
were replotted according to Equation (3) with A or B set to
be H2O2 or phenol (Figure 1, c and d). Plotting of the slope
Figure 2. Oxidation of 8.0 mM PhOH by 60 μm RU of Cu-P1 as a
function of CuII mol fraction XCu at constant [CuII] + [ZnII] = 60
μm in the presence (᭹, left scale) and absence (᭺, right scale) of
10.0 mm H2O2.
The amino acid tyrosine (Tyr) was also used as a sub-
and the y intercept from the Hanes plots versus 1/[H2O2] in strate to gain further insight into the substrate specificity
Equation (3) and versus 1/[phenol] in Equation (4) yields and catalytic versatility of Cu-P1. However, there was no
the apparent constants KPhOH = 1.35 mm and KH O
=
observable oxidation of Tyr under the same conditions and
2
2.4 mm for the dissociation of phenol and H2O2 from 2the on the same time frame. The lack of activity may be attrib-
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uted to Tyr binding to the metal ion through the chelating lyst (ko = 2.1ϫ10–8 s–1 and 2.2ϫ10–7 s–1 in the absence and
α-aminocarboxylate moiety instead of the phenolate moi- presence of 100 mm H2O2, respectively); this also reflects
ety, as previously reported.[32] The removal of the carboxyl- more effective peroxidation than aerobic oxidation, as op-
ate group of Tyr can occur in vivo to afford tyramine (ty). posed to PhOH hydroxylation/oxidation.
Tyramine can be hydroxylated/oxidized by CuII-P1 to form
The rate of hydroxylation/oxidation of PhOH is sup-
its corresponding quinone with significantly different kcat posed to be affected by its deuteration if the C–H bond
values of 9.0ϫ10–5 s–1 (KЈ = 7.4 mm and kcat/KЈ = cleavage is the rate-determining step. The kinetic isotope
0.012 m–1 s–1) and 2.9ϫ10–3 s–1 (8.0 mm and 0.36 m–1 s–1) in effects (KIEs) for the phenol oxidation are 1.19 and 2.80 in
the absence and presence of 100 mm H2O2, respectively the absence and presence of 20 mm H2O2 (Figure 4), respec-
(Figure 3, a); this represents a 30-fold higher catalytic effi- tively, in terms of kcat. The KIE values become 1.20 and
ciency for the peroxidation than the aerobic oxidation of ty. 1.78, respectively, in terms of kcat/KЈ owing to an increase
The similar KЈ (k–1/k1 + kcat/k1) but much larger kcat value in KЈ upon deuteration; therefore, the KIE is influenced by
for 100 mm H2O2 indicates that there is less dissociation of substrate binding. The smaller KIE in the absence of H2O2
ty from the active metal center (k–1/k1) in the presence of reflects that C–H bond breakage may not be the rate-de-
H2O2. Conversely, the similar kcat and KЈ values for PhOH termining step. The small difference in the KIE indicates
hydroxylation/oxidation in the presence and absence of a that the rate-determining step with and without H2O2 may
saturating amount of H2O2 (Figure 1) reflect a similar affin- be different (discussed later), consistent with the results
ity of PhOH to the active metal center. The different activi- from the ZnII dilution studies (Figure 2).
ties among the various substrates indicate the presence of
catalytic specificity, which is a key characteristic of enzyme
catalysis. The nonlinear rate profile of [H2O2] follows pre-
equilibrium kinetics with kcat = 2.0ϫ10–3 s–1 and KЈ =
13 mm for the oxidation of 12.0 mm ty (Figure 3, b) and
indicates its direct binding to the active metal site.
Figure 4. Phenol (᭺) and deuterated phenol (᭹) oxidation by 50
μm RU of Cu-P1 in 50% methanol/HEPES buffer aqueous solution
at pH 8.0 in the absence (A) and presence (B) of 50 mm H2O2.
To reveal the nuclearity in the reactions in the presence
and absence of H2O2, a “mechanistic Job plot” was con-
ducted.[19,33] The classic optical Job plot (absorption vs. mol
fraction)[34] determines the stoichiometry of a metal com-
Figure 3. (A) Initial rate of tyramine oxidation by 50 μm RU of
plex that is formed as M + nL w MLn, wherein the maxi-
mum absorption affords the stoichiometry, as observed in
some metal-drug binding.[35] For the pre-equilibrium kinet-
ics shown in Equation (1), the intermediate (Cu-P1)–S is
the determinant for the activity, that is, rate = kcat[(Cu-P1)–
S], and is under equilibrium with the reactants, analogously
Cu2+-P1 in the presence of 0.0, 5.0, 11.8, 50.0, and 100.0 mm H2O2
(from bottom) and fit of the data to Equation (2). (B) Hanes plot
of the data from (A). (C) Rates for the oxidation of 12 mm tyramine
by 50 μm RU of Cu-P1 as a function of [H2O2]. Conditions: 50%
methanol/0.1 m HEPES buffer solution at apparent pH 8.0 and
25 °C.
The apparent and intrinsic dissociation constants from to the equilibrium above for the formation of MLn. Herein,
the Hanes plots [Equations (3) and (4)] are Kty = 3.2 mm the rate serves as the output rather than the optical density
(cf. KЈ = 8.0 mm at 100 mm H2O2, Figure 3, a), K
=
in the classic Job plot. Thus, the maximum activity in the
H2O2
15.5 mm (cf. KЈH O = 13 mm at 12 mm ty, Figure 3, c), Ki(ty) mechanistic Job plot reflects the stoichiometry of (CuII-P1)/
2
2
= 28.2 mm (cf. KЈ = 7.4 mm without H2O2, Figure 3, a), and S, that is, a maximum activity at XCu-P1 = 0.5 reflects the
Ki
= 17.6 mm. The K/Ki ratio is 0.12 for ty and 0.67 formation of a 1:1 (CuII-P1)/S complex, whereas a maxi-
(H2O2)
for H2O2, which indicates that the binding of one substrate mum activity at XCu-P1 = 0.67 indicates the formation of a
decreases the dissociation of the other; this is consistent dinuclear active center as (CuII-P1)2–S. The difference in
with the analysis of the kcat and KЈ values described above stoichiometry of the intermediate with and without H2O2
but is opposed behavior for PhOH. The significant decrease is clearly revealed with the maximums at XCu-P1 = 0.67 and
in Kty for the dissociation of ty from the ty–(Cu-P1)–H2O2 0.5 (Figure 5, a), respectively. The results indicate a di-CuII
ternary complex relative to its intrinsic dissociation Ki(ty) active center may be associated with the rate-determining
from the ty–(Cu-P1) complex might be attributed to elec- step in the presence of H2O2, whereas a mononuclear CuII
trostatic interactions between the positive amine group of ty active center is more likely without H2O2. The results are
and a bound peroxide ligand in close proximity. Tyramine consistent with the ZnII dilution results (Figure 2), wherein
oxidation affords rate accelerations by factors of 4.3ϫ103 the apparent cooperativity in the presence of H2O2 hints at
and 3.2ϫ105 in the absence and presence of 100 mm H2O2 a possible dinuclear active center during catalysis, which is
when compared to the autoxidation of ty without the cata- not as clear without H2O2.
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CuI center (D) for catechol oxidation by CuII-P1[20] is gen-
erated, followed by O2 binding and a repeat of the cycle (D
to B). When the reaction occurs in air (O2), PhOH binds to
a mononuclear CuII center (E, Figure 6), followed by an
internal electron transfer from the bound phenolate ion to
the CuII center to yield a phenolate radical (F, Figure 6) to
a small extent. This should be the slowest step to exhibit a
Figure 5. (A) Mechanistic Job plot for phenol oxidation by CuII- mononuclear character (cf. Figure 5, a) without a signifi-
P1 in the presence (᭹) and absence (᭺) of 80 mm H2O2 at pH 8.0.
cant KIE (Figure 4, a) and is followed by dioxygen binding
The solid traces are the best fits to (CuII-P1)/phenol ratios of 2:1
·–
and electron transfer to form a CuII–O2 center (G, Fig-
and 1:1, whereas the dashed trace represents a ratio of 1:1 in the
presence of H2O2. (B) Effects of borate (᭡), NaCl (᭹), and NaF
ure 6); this is followed by fast coupling of the bound phenol
radical and the superoxide radical (C) and then C–H bond
breakage to yield the product. The binding and stabilization
of O2 may be assisted by a dinuclear center (G).
(᭺) on the oxidation of 5 mm phenol at pH 9.0.
To determine whether or not catechol is released as an
intermediate during the reaction, the activity was checked
in the presence of sodium borate, which is known to conju-
gate with the syn-diol moiety of catechol.[36] As the oxi-
dation of catechol by CuII-P1[20] is much faster than phenol
hydroxylation (0.12 vs. 0.0046 s–1 for kcat and 31.6 vs. 2.0
m
–1 s–1 for kcat/Km), a bound catechol formed from phenol
hydroxylation can be effectively oxidized. However, if cate-
chol is released after its formation, a decrease in activity
would be observed as it would be removed by coupling with
borate. The hydroxylation rate is not decreased by borate at
Ͻ10 mm, but unexpectedly it is significantly enhanced at
Ͼ10 mm borate with Kd = 22 mm and reaches approxi-
mately five times higher activity at 50 mm (Figure 5, b).
This increase in activity at higher borate concentrations is
not attributed to ionic strength, as both NaCl and NaF
under the same conditions do not noticeably affect the oxi-
dative activity (Figure 5, b). The exact mechanism for such
a significant enhancement by borate is not clear at this stage
and will need future exploration. Regardless, this observa-
tion indicates that catechol is not released as an intermedi-
ate product before the formation of the final quinone prod-
uct.
Figure 6. Proposed mechanism for the oxidation of phenol by CuII-
P1 in the presence (A–D) and absence (E–G and C–D) of H2O2.
The detailed binding pattern of PhOH and H2O2 to the active cen-
ter (A to B) is revealed by the Hanes analysis [Equations (1) and
(3)], and the nuclearity was determined by the mechanistic Job plot.
Catechol is only a transient species (CЈ) as it is not detected in the
presence of borate (Figure 5, B).
The results described herein suggest that the oxidation of
PhOH by CuII-P1 in the presence of H2O2 follows a dinu-
clear mechanism that resembles that of di-Cu tyrosinase,[31]
wherein the addition of H2O2 to a di-CuII center (which
Conclusions
The activity of the metallopolymer CuII-P1 toward the
may or may not contain a bridging ligand, A, Figure 6) hydroxylation and oxidation of phenol with the green oxid-
forms an active μ-η2,η2-peroxo–di-CuII center (B, Figure 6), izing agents H2O2 or aerobic O2 is quite effective and likely
which then performs hydroxylation of PhOH and then oxi- to follow different mechanisms. Although copolymers can
dation (B–D, Figure 6). The ability of a phenolate ion to be mostly unstructured, which is presumably the case for
bind to the CuII–OH center was observed in the Cu-substi- P1, the active metal center of the metallopolymer seems to
tuted endopeptidase astacin and serralysin.[37] In the elec- be able to assemble properly to exhibit a significant cata-
trophilic aromatic substitution mechanism for the phen- lytic activity toward the difficult hydroxylation reaction of
olase and peroxygenase activities of tyrosinase, proton mi- phenol. We previously observed that CuII-P1 also exhibited
gration (C) is a much faster step than C–O bond formation an effective activity and different cleavage pattern toward
(B–C) and exhibits no KIE.[38] In our studies herein, the plasmid DNA from that of free CuII species; this difference
detection of a noticeable deuterium KIE for the peroxi- was partially attributed to the proximity of the metal cen-
dation pathway (A–D) suggests that C–H bond breakage ters in the polymer.[20] Moreover, the FeIII complexes of P1
and proton migration may be part of the rate-limiting step. and its deamidated form exhibit quite different specificities
The lack of borate inhibition (Figure 5, B) indicates that toward phosphoester hydrolysis.[19] The easy syntheses of
catechol is only a transient product (CЈ), which is quickly P1 and its metal complexes and the diversity of their chemi-
oxidized to the final o-quinone product, and the active CuI– cal activities suggest that this pyridine- and amide-contain-
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ing copolymer can serve as a convenient model system for
the design of new metallopolymers and their further chemi-
cal, biological, and environmental applications.
Acknowledgments
The mechanistic studies of oxidation chemistry were partially sup-
ported by the National Science Foundation (NSF), USA (grant
number CHE-0718625).
Experimental Section
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mol fraction of CuII-P1 (XCu–P1) with a constant total concentra-
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(S) to the metal center to form an intermediate (Cu-P1)–S complex,
followed by the conversion of the bound substrate into products
[Equation (1)]. The rate law for this reaction mechanism can be
expressed as Equation (2), wherein KЈ = (k–1 + kcat)/k1 or k–1/k1 +
kcat/k1 is the apparent dissociation constant of the ternary (CuII-
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Received: August 14, 2014
Published Online: ᭿
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Dioxygen Activation
V. Lykourinou, L.-J. Ming* .............. 1–8
A CuII complex of a pyridine- and amide-
containing copolymer catalyzes phenol
hydroxylation/oxidation in aqueous meth-
anol at pH 8.0 and 25 °C. First-order rate
accelerations by factors of 4.2ϫ105 and
1.4ϫ105 and deuterium kinetic isotope ef-
fects of 2.8 and 1.2, respectively, were ob-
served with and without H2O2. The results
suggest that different mechanisms apply for
oxidation by O2 and H2O2.
Mechanistic Insights into Phenol Oxid-
ation by a Copper(II) Complex of a Pyr-
idine- and Amide-Containing Copolymer
in an Aqueous Medium
Keywords: Enzyme models / Copper / Ox-
idation / Dinuclear complexes / Metallo-
polymers
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