Catalytic phenol hydroxylation with dioxygen: Extension of the tyrosinase mechanism beyond the protein matrix

Article abstract of DOI:10.1002/anie.201301249

A new catalyst (see structure) hydroxylates phenols with O₂ via a stable side-on peroxide complex, which is similar to the active site of tyrosinase in terms of the ligand environment and its spectroscopic properties. The catalytic oxidation of phenols to quinones proceeds at room temperature in the presence of NEt₃ and even non-native substrates can be oxidized catalytically. The reaction mechanism is analogous to that of the enzyme-catalyzed reaction. Copyright

Full text of DOI:10.1002/anie.201301249

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DOI: 10.1002/anie.201301249
Tyrosinase Model
Catalytic Phenol Hydroxylation with Dioxygen: Extension of the
Tyrosinase Mechanism beyond the Protein Matrix**
Alexander Hoffmann, Cooper Citek, Stephan Binder, Arne Goos, Michael Rꢀbhausen,
´
´
Oliver Troeppner, Ivana Ivanovic-Burmazovic, Erik C. Wasinger, T. Daniel P. Stack,* and
Sonja Herres-Pawlis*
A pinnacle of bio-inorganic chemistry is the ability to
leverage insights gleaned from metalloenzymes toward the
design of small analogues capable of effecting catalytic
reactivity outside the context of the natural system.[1,2]
Structural mimicry of active sites is an attempt to insert
a synthetic catalyst into an enzymatic mechanism. Such
a mechanism evolves by selection pressures for efficiency and
traverses an energetic path, with barriers and wells neither
too high nor too deep in energy—a critical factor of catalytic
turnover.[3] An advantage of metalloenzymes over small
metal complexes is the site-isolation of the metal center in
the protein matrix and the attendant ability to attenuate
destructive decay processes—reaction sinks. This protection
provides access to thermal regimes that allow barriers and
wells to be traversed. Synthetic complexes too must avoid any
deleterious reactions, often necessitating the deliberate
incorporation of protective superstructures.[4,5] Such limita-
tions make reproducing enzymatic catalytic reactivity in
a synthetic complex with native substrates a significant
challenge, as evidenced by the dearth of good examples,
despite decades of effort.
oxygen-atom insertion reactions. Tyrosinase is a ubiquitous
dinuclear copper enzyme that catalyzes the hydroxylation of
phenols to catechols and the subsequent oxidation of
catechols to quinones by activating dioxygen in the form of
a side-on-bonded peroxide dicopper(II) species, crystallo-
graphically[10] and spectroscopically identified.[11,12] The seem-
ingly simple, regiospecific transformation mediated by tyro-
sinase is not reproduced easily by synthetic methods, though
its importance has been acknowledged by recent efforts to
move beyond stoichiometric oxidants,[13] as these sometimes
multistep syntheses are often low to moderate yielding and
frequently unselective.[14–17] Limited examples of stoichiomet-
ric phenolate hydroxylation exist with synthetic, tyrosinase-
like side-on peroxide complexes[18–22] formed by oxygenation
of copper(I) complexes. Only two complexes achieve signifi-
cant catalytic phenol hydroxylation using dioxygen: a dinu-
cleating, polydentate imine complex reported by Rꢁglier
et al.,[23] and a mononucleating analogue reported by Tuczek
et al.[24] both capable of roughly 16 turnovers in the presence
of triethylamine. Neither study could identify an operative
oxygenated copper species, presumably precluding a more
detailed mechanistic analysis of the key substrate hydroxyl-
ation step. The possibility of a synthetic side-on peroxide as
a catalytic oxidant is suggested by the work of Casella et al.
with a dinucleating copper benzimidazole complex, achieving
1.2 turnovers with readily oxidized phenols.[21,25]
The intellectual investment of translating a catalytic
mechanism from an active site to a synthetic system is
justified by a comparison of Natureꢀs dexterity with dioxygen
to perform catalytic monooxygenase-type chemistry[6–9] and
the dependence of synthetic chemists on exotic reagents for
[*] Dr. A. Hoffmann, Prof. Dr. S. Herres-Pawlis
Department of Chemistry
[**] A.H., S.B., A.G., M.R., and S.H.-P. thank the Deutsche For-
schungsgemeinschaft (FOR 1405) for financial support. S.H.-P.
gratefully acknowledges the Fonds der Chemischen Industrie for
a Liebig fellowship, the Bundesministerium fꢁr Bildung und
Forschung initiative MoSGrid (01IG09006) for computational
support, and Prof. Dr. K. Jurkschat for valuable advice. I.I.-B. and
O.T. gratefully acknowledge support through the “Solar Technolo-
gies Go Hybrid” initiative of the State of Bavaria. Financial support
was provided by the U.S. National Institutes of Health (NIH;
GM50730) for T.D.P.S. and the William R. and Sara Hart Kimball
Stanford Graduate Fellowship for C.C. E.C.W. thanks the CSU Chico
College of Natural Sciences for financial support and release time to
support this work. Portions of this research were carried out at the
Stanford Synchrotron Radiation Laboratory, a national user facility
operated by Stanford University on behalf of the U.S. Department of
Energy, Office of Basic Energy Sciences.
Ludwig-Maximilians-Universitꢀt Mꢁnchen
81377 Munich (Germany)
and
Fakultꢀt Chemie, Technische Universitꢀt Dortmund
44227 Dortmund (Germany)
E-mail: sonja.herres-pawlis@cup.uni-muenchen.de
C. Citek, Prof. Dr. T. D. P. Stack
Department of Chemistry, Stanford University
Stanford, CA 94305 (USA)
E-mail: stack@stanford.edu
Dr. S. Binder, A. Goos, Dr. M. Rꢁbhausen
Institute for Applied Physics and Center for Free Electron Laser
University of Hamburg (Germany)
´
´
O. Troeppner, Prof. Dr. I. Ivanovic-Burmazovic
Department of Chemistry and Pharmacy
University of Erlangen-Nuremberg (Germany)
Supporting information for this article (including materials and
methods, synthetic procedures, Raman measurements, XAS spec-
troscopy details, TD-DFTdetails, and optimized coordinates of 1) is
available on the WWW under http://dx.doi.org/10.1002/anie.
201301249.
Prof. Dr. E. C. Wasinger
Department of Chemistry and Biochemistry
California State University, Chico, CA 95929 (USA)
5398
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Here we report a synthetic catalyst capable of hydrox-
ylating a wide variety of phenols using dioxygen that proceeds
through a room-temperature(RT)-stable analogue of oxy-
genated tyrosinase—a side-on peroxide complex, which
possesses ligation and spectroscopic attributes similar to
those of the enzymatic active site. Efficient stoichiometric
oxidation of phenolates to catecholates at ꢀ788C is shown, as
well as catalytic oxidation of phenols to quinones at room
temperature with triethylamine, through a reaction pathway
consistent with the generally accepted enzymatic mecha-
nism.[11] Conditions are described in which catalytic turnover
is halted and restored at a proposed catecholate–product
adduct. This investigation demonstrates that simple structural
mimicry suffices to not only transfer the inherent enzymatic
catalytic reactivity into a synthetic complex, but that bio-
inspiration is a viable stategy of pursuing selective trans-
formations of substrates beyond scope of the enzyme.[26]
Oxygenation of [CuI{bis(3-tert-butyl-pyrazolyl)pyridyl-
methane}]SbF6 in dichloromethane at ꢀ788C results in
near-quantitative formation (vide infra) of [Cu2O2(HC(3-
tBuPz)2(Py))2](SbF6)2 (1), a side-on peroxide dicopper(II)
complex (Figure 1A), as evidenced by the characteristic O–O
stretch in its resonance Raman spectrum at 750 cmꢀ1, which
shifts by 39 cmꢀ1 upon 18O2 substitution (Figure 1C).[27,28] The
expected mass signature and isotope pattern observed in the
cryo-ESI-TOF mass spectrum also shifts appropriately upon
oxygenation with 18O2 (Figure S3 in the Supporting Informa-
tion). The ligand-to-metal-charge-transfer (LMCT) features
at 350 nm (20 mmꢀ1 cmꢀ1) and 550 nm (1 mmꢀ1 cmꢀ1) have
a 20:1 intensity ratio,[22,27] similar to those of oxy-tyrosinase
and oxy-hemocyanin,[29] and the feature near 412 nm
(0.9 mmꢀ1 cmꢀ1) is assigned tentatively to a pyrazole/pyridine
p*!dxy charge-transfer transition, based on a natural tran-
sition orbital analysis of a TD-DFT calculated spectrum (TD-
DFT= time-dependent density functional theory; Fig-
ure 1B).[22,30] The DFT-optimized structure of 1 predicts
a planar Cu2O2 core with a Cu–Cu separation of 3.57 ꢂ, in
line with the 3.51 ꢂ distance determined by Cu K-edge
extended X-ray absorption fine structure models (Table S2
and Figure S5 in the Supporting Information).[27,28] Taken
together, these data fully support the structural homology
between tyrosinase and 1.
The formation of 1 is effectively quantitative (> 95%
yield) in a variety of solvents at ꢀ788C, as assessed by
iodometric titrations of the released peroxide after treatment
with trifluoroacetic acid.[22,31] The complex is stable for weeks
in CH2Cl2 at ꢀ788C, yet decays within 1 day in tetrahydro-
furan or acetone. Compound 1 reacts within 60 min with
a wide variety of sodium phenolate salts (5 equiv), both
electron-rich and -deficient; the side-on peroxide oxidant is
efficiently consumed releasing catechol products after an
acidic workup (Table 1A). Using a 1:1 oxidant/phenolate
stoichiometry, impressive catecholate yields (> 90%) are
possible at ꢀ788C, albeit reaction times of nearly 1 week are
required. Expectedly, the mass of the p-methoxy-1,2-catechol
product is shifted by 2 a.u. if 1 is formed with 18O2, indicating
dioxygen as the oxygen atom source.[32]
From kinetic data, the hydroxylation of phenolate to
catecholate by 1 is best understood as a second-order process:
first order in [1] and first order in [phenolate], with a pre-
equilibrium binding event and a rate-limiting oxidation step,
ꢀ
presumably C O bond formation. An intramolecular com-
petitive kinetic isotope effect of 1.2(2) measured at ꢀ788C
ꢀ
with 2-d-4-tert-butylphenolate excludes rate-limiting C H
bond cleavage. The observed rate constants kobs saturate with
respect to added phenolate (Figure 2), consistent with an
initial phenolate-binding equilibrium Keq, followed by an
intramolecular rate-determining oxidation step kox from
a substrate–complex adduct (Table S1 in the Supporting
Information),[18,32,33] as electron-deficient phenolates clearly
react more slowly. A plot of ln(kox) versus sp+ for a variety of
phenolates gives a Hammett parameter 1 = ꢀ0.99 (Figure 2),
consistent with the trend reported for tyrosi-
nase (1 = ꢀ1.8 to ꢀ2.2)[34,35] and in line with an
electrophilic aromatic substitution mecha-
nism.
At room temperature in CH2Cl2, 1 is
formed quantitatively but decays irreversibly
with a half-life of 30 min. However, this rare
stability allows for catalytic hydroxylation of
phenols to quinone products at room temper-
ature. Only two other synthetic side-on per-
oxide species have greater thermal stability,
but exogenous substrate reactivity has not
been reported.[36,37] With 25 equiv of p-
methoxyphenol and 50 equiv of triethylamine
under 1 atm of O2,[23,24,38] 10 equiv of quinone
is formed in 1 h or 15 equiv in 24 h (Table 1B),
as assessed by the characteristic optical feature
of quinone at 400 nm (Figure S1 in the Sup-
porting Information). Slower catalytic reac-
tion rates are observed with more electron-
Figure 1. A) Preparation of the side-on peroxide species 1. B) Absorption spectra of 1;
inset: TD-DFT-predicted optical spectrum of 1. C) Resonance Raman spectra of 1 in
acetone with excitation at 412 nm (red: 16O2, black: 18O2, asterisks (*): solvent peaks);
deficient phenols. At these higher temper-
atures, the oxidation of the catechol to qui-
none and its subsequent release is proposed to
inset: isotopic shift of the feature at 750 cmꢀ1
.
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Table 1: A) Reaction of phenolic substrates and 1 under N2 atmosphere at ꢀ788C.[a] B) Catalytic oxidation of phenols to quinones.[b]
drive the reduction of the CuII centers back to a CuI state,
permitting re-oxygenation to 1 (Scheme 1).
results in the return of unreacted phenol at ꢀ788C even after
a reaction time of hours. After phenol deprotonation, the
resultant triethylammonium cation presumably provides the
necessary proton for the protonation of hydroxide to water as
catecholate reduces CuII to CuI.
Beyond the ability to catalytically oxidize a tyrosinase
substrate, N-acetyltyrosine methyl ester,[39] to its dopaquinone
form, the oxidative reactivity of 1 can be extended to more
complicated phenols, outside the typical substrate scope of
the enzyme (Table 1B). Estrone, an estrogenic hormone, is
regioselectively ortho-hydroxylated to 3,4-estrone-o-catechol
in > 90% yield at ꢀ788C in less than 5 min when 5 equiv of its
sodium salt are added relative to 1 (Table 1A). The 3,4-
estrone-o-quinone is formed under the catalytic conditions at
258C with 4 turnovers.[32] While mushroom tyrosinase itself
Three conditions appear to be necessary for catalytic
turnover and support operation through a tyrosinase mech-
anism: ambient reaction temperatures (258C), proton man-
agement, and excess dioxygen. Under catalytic reaction
conditions at ꢀ788C, rapid formation of a green species is
observed, tentatively assigned as a CuII catecholate complex
(Scheme 1), as one equiv of p-methoxy-1,2-catechol is formed
as the sole product upon an acidic quench at this temperature.
If warmed to 258C, this green species yields a quinone rather
than a catecholate product, suggesting catecholate-binding
inhibition at low temperatures (Scheme 1). Phenol deproto-
nation and binding to a copper center is essential in the
catalytic oxidation process, as exclusion of triethylamine
Figure 2. Reactivity of 1 at ꢀ788C in CH2Cl2. Left: Substrate-binding
kinetics of the stoichiometric hydroxylation reaction with 4-fluorophe-
nolate. Right: Hammett plot for the stoichiometric hydroxylation
reaction with 1–20 equiv of various p-substituted phenolates.
Scheme 1. Proposed catalytic mechanism of phenol oxidation by 1 in
the presence of triethylamine.
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can oxidize estrone,[40] 8-hydroxyquinoline is not a viable
substrate,[32] possibly due to the steric demands of the fused
ring structure positioned ortho to the phenol oxygen
(Table 1B).[26] Compound 1 oxidizes 8-hydroxyquinoline
both stoichiometrically to the catechol (7,8-dihydroxyquino-
line) at ꢀ788C, and catalytically to its quinone at 258C
(quinoline-7,8-dione; 8 turnovers; Table 1B), extending the
substrate scope beyond that possible in the enzymatic
system.[2] The substrate flexibility of 1 may thus make it
a potentially useful reagent for the efficient stoichiometric
conversion of a wide variety of phenolates to catechol or
a catalyst for the multi-turnover oxidation of phenols by
dioxygen to quinones, which are readily reduced back to
catechols.[17]
Oxygen-insertion reactions that use dioxygen directly are
extremely limited, despite the indisputable advantages to
using Earthꢀs ready supply of dioxygen. The difficulty lies in
directing the oxidative power of dioxygen and in assuring that
reactions profiles do not have insurmountable barriers or
thermodynamically overstabilized intermediates. In this
work, the essence of the tyrosinase enzyme mechanism,
founded on the oxidizing power of dioxygen, is translated
from its protein environment into a small complex through
only approximate structural mimicry of the oxygenated active
site, yet selective and efficient catalytic ortho-hydroxylation
reactivity results. This strategy of copying pre-existing reac-
tivity from Nature opens the door not only to challenging
organic transformations, but for developing useful synthetic
tools with substrate scopes beyond those of biological
systems.
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Received: February 12, 2013
Published online: April 22, 2013
Keywords: bioinorganic chemistry · copper · dioxygen ·
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homogeneous catalysis · tyrosinase
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