Tyrosinase-like reactivity in a CuIII2(??-O) 2 species

Article abstract of DOI:10.1002/chem.200800229

A study was conducted to demonstrate a bis(??-oxo)dicopper(III) species that binds and ortho-hydroxylates phenolates for reactivity of tyrosinase. The study characterized a metastable species from the low-temperature reaction of sodium p-chloro-phenolate (p-Cl-C₆H₄ONa) with a bis(??-oxo)dicopper(III) species. The study found that the addition of 10 equivalents of the sodium salt of p-chlorophenol at -90?°C can cause bleaching of the spectral features. High performance liquid chromatography (HPLC) analysis showed that 4-chlorocatechol was formed in 76% yield. The study used UV/UVis monitoring for reaction in acetone to determine the initial features to the bis(??-oxo) species and found that these features disappear after phenolate addition. The study observed that the activation parameters for the monophenolase reaction catalyzed by tyrosinase.

Full text of DOI:10.1002/chem.200800229

COMMUNICATION
DOI: 10.1002/chem.200800229
Tyrosinase-Like Reactivity in a CuIII
2(m-O)2 Species
G
Anna Company,[a] Sara Palavicini,[b] Isaac Garcia-Bosch,[a] Ruben Mas-BallestØ,[c]
Lawrence Que, Jr.,*[c] Elena V. Rybak-Akimova,*[d] Luigi Casella,*[b] Xavi Ribas,[a] and
Miquel Costas*[a]
Understanding the mechanisms by which the oxidizing po-
tential of the O2 molecule is used in the oxidation of organic
substrates is a formidable task with fundamental interest in
technology and biochemistry.[1] Nature usually uses transi-
tion-metal ions to overcome kinetic barriers associated with
spin multiplicity of the O2 molecule.[2] For example, Type 3
copper proteins use a bimetallic site to bind and/or activate
Scheme 1. Biological function of tyrosinase towards the final synthesis of
melanins.
O2. Among the enzymes that contain this type of active site
structure, tyrosinase is a particularly interesting enzyme that
catalyzes the ortho-hydroxylation of phenols to catechols
and further oxidation to the corresponding quinones
(Scheme 1).[3]
arising from these studies proposes that a (m-h2:h2-peroxo)-
dicopper(II) species is responsible for the hydroxylation
step via an electrophilic attack over the aromatic ring. That
Recent X-ray crystallographic studies of this enzyme have
shown the presence of a rather flexible active site that ac-
commodates large changes in the Cu···Cu distance during its
catalytic cycle,[4] and they confirm previous spectroscopic
studies implicating a side-on (m-h2:h2-peroxo)dicopper(II)
species preceding the hydroxylation of the phenol moiety.[5]
Extensive work has been done to unravel the reaction mech-
anism of this enzyme.[6] The generally accepted mechanism
ꢀ
ꢀ
is, O O cleavage occurs after or along with C O bond for-
mation.[3,7] Nevertheless, this proposal has been challenged
by the observation that the (m-h2:h2-peroxo)dicopper(II)
core is usually in nearly degenerate equilibrium with its bis-
A
N
this species may be the actual executor of the arene hydrox-
ylation step. Along these lines, Tolman and co-workers have
shown that bis
C
N
Stack and co-workers have reported that [CuII (m-h2:h2-O2)-
[a] A. Company, I. Garcia-Bosch, Dr. X. Ribas, Dr. M. Costas
Departament de Química, Universitat de Girona
Campus de Montilivi, 17071, Girona (Spain)
Fax : (+34)972-418150
2
(DBED)]2+
undergoes an intermolecular reaction at ꢀ1208C with phe-
E-mail: miquel.costas@udg.edu
nolates that proceeds by initial phenolate binding and subse-
III
ꢀ
[b] S. Palavicini, Prof. L. Casella
Department of General Chemistry, University of Pavia
27100 Pavia (Italy)
quent O O bond breakage to form [Cu
2(m-O)2(phenolate)-
(DBED)]+, which further decays by electrophilic ortho-hy-
droxylation of the phenolate to give the corresponding cate-
chol.[10] These observations firmly establish that the bis
(m-
oxo)dicopper(III) core is capable of eliciting tyrosinase-like
E-mail: bioinorg@unipv.it
ACHTREUNG
[c] Dr. R. Mas-BallestØ, Prof. L. Que, Jr.
Department of Chemistry and Center for Metals in Biocatalysis
University of Minnesota
AHCTREUNG
reactivity when an aromatic ring is placed in close proximity
to it. These results contrast with the radical-type reactivity
207 Pleasant Str SE Mpls, MN55545 (USA)
E-mail: larryque@umn.edu
exhibited by most bis
U
G
[d] Prof. E. V. Rybak-Akimova
Department of Chemistry, Tufts University
Medford, MA 02155 (USA)
ꢀ
cal question arising from these studies is whether O O
bond breakage necessarily occurs only after phenolate bind-
ing, thus suppressing the radical-type reactivity until the aro-
matic ring is in close proximity to the core. Here we report
E-mail: erybakak@granite.tufts.edu
Supporting information for this article is available on the WWW
under http://www.chemistry.org or from the author.
Chem. Eur. J. 2008, 14, 3535 – 3538
ꢀ 2008 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
3535

the first example of a bis
binds and then ortho-hydroxylates phenolates, thus mimick-
ing the reactivity of tyrosinase. We have trapped and
A
N
Nevertheless, addition of 10 equivalents of the sodium
salt of p-chlorophenol at ꢀ908C caused immediate bleach-
ing of the spectral features associated with 2. Acidic work-
up and HPLC analysis revealed that 4-chlorocatechol was
formed in 67% yield with respect to the initial dicopper
complex. The identity of the product was further confirmed
spectros
ing from the low-temperature reaction of sodium p-chloro-
phenolate (p-Cl-C6H4ONa) with a bis(m-oxo)dicopper(III)
copically characterized a metastable species result-
1
species preceding phenolate hydroxylation. Finally, kinetic
analysis of the hydroxylation reaction has been performed
to obtain activation parameters that can be compared with
those found for tyrosinase.[12]
by isolation with preparative HPLC and H NMR analysis.
ꢀ
ꢀ
Neither quinone nor C C or C O coupling products were
obtained. Similar reactions with p-carbomethoxyphenolate
and p-cyanophenolate also show the formation of the corre-
sponding catechol as the sole oxidation product. Therefore,
We recently found that the reaction of [CuI2(m-
XYLMeAN)]
A
2 constitutes a rare example of a bis
N
N
species capable of performing the phenolate hydroxylation
to form a catechol, thus mimicking the activity exhibited by
tyrosinase.
Insight into the reaction mechanism was obtained by trap-
ping at very low temperature (ꢀ908C) a metastable reaction
intermediate formed after reaction of 2 with sodium p-
chloro
reaction in acetone showed that the initial features corre-
sponding to the bis(m-oxo) species (2) immediately disap-
A
AHCTREUNG
pear after phenolate addition (Figure 1, top). Concomitantly,
new spectral features appear at 390 and 563 nm correspond-
ing to a new species 3Cl. The latter is thermally very sensitive
and rapidly decomposes (t1/2 ꢁ 20 s) at ꢀ908C. Neverthe-
less, resonance Raman experiments (Figure 1, bottom) of
frozen solutions of 3Cl with laser excitation at 407 nm show a
resonance enhanced feature at 597 cmꢀ1 that experiences a
ꢀ26 cmꢀ1 shift when 18O2 is used in the generation of 2. This
feature is not enhanced when 568 nm laser excitation is used
in the experiment. Moreover, no isotope-sensitive features
that could be assigned to a (m-h2:h2-peroxo)dicopper(II) spe-
cies were observed in the 700–770 cmꢀ1 region.[6a] On the
other hand, laser excitation at 568 nm shows intense peaks
at 1264, 1409, and 1642 cmꢀ1, characteristic of phenolate vi-
bration modes.[15] These vibrational features are not affected
by the use of 18O2, and they are not enhanced with laser ex-
citation at 407 nm. The Raman data thus provide direct evi-
dence for phenolate binding to the Cu2O2 core in 3Cl.
Scheme 2. The reactions described in this work.
THF resulted in the fast formation of a deep yellow species
2 that is characterized by two intense UV/Vis features at
lmax =308 nm
(20000mꢀ1 cmꢀ1)
and
lmax =413 nm
The accumulated data can be interpreted with two differ-
(28000mꢀ1 cmꢀ1).[13] Resonance Raman experiments of
frozen acetone solutions using laser excitation at 413 nm re-
vealed a characteristic Cu2O2 breathing vibration peak at
600 cmꢀ1 that showed a downshift of 23 cmꢀ1 when 18O2 was
ent scenarios. The first is that 3Cl is actually a mixture of re-
sidual bis
phenolate species. Alternatively, 3Cl may be formulated as
[CuIII2(m-O)2(p-Cl-C6H4O)(m-XYLMeAN)]+, where bis
(m-oxo)
A
N
A
ACHTREUNG
used. These are common spectral features for a CuIII
A
and phenolate vibrations are uncoupled. We favor the latter
hypothesis on the basis of the following observations. Kinet-
ic analysis (vide infra) indicates that reaction of p-Cl-
C6H4ONa is fast even for stopped-flow methodology and no
residual 2 should be present under the experimental condi-
tions used to prepare the resonance Raman sample. Also,
the UV/Vis spectrum of 3Cl does not change upon varying
the concentration of phenolate. Furthermore, the features
core[11,14] that led us to formulate 2 as [CuIII
ACHTREUNG
[13]
XYLMeAN)]2+
.
Use of several solvents (THF, diethyl ether,
ꢀ
ꢀ
CH2Cl2, or acetone) and counterions (ClO4 , CF3SO3 ,
ꢀ
BArFꢀ, and SbF6 ) did not change the nature of 2, and no
experimental evidence for the isomeric (m-h2:h2-peroxo)di-
copper(II) species was observed. This was further substanti-
ated by DFT calculations at the B3LYP level, which indicat-
ed that the peroxo form was 35.5 kJmolꢀ1 higher in
energy.[13] Extraction and analysis of the ligand after thermal
decay of 2 did not show any evidence for ligand modifica-
tion, indicating that intramolecular aromatic hydroxylation
does not take place in this meta-xylyl-based system.
associated with the bis(m-oxo) core (390 nm) and with the
A
phenolate (563 nm), decay with the same kinetic behavior
(as monitored by UV/Vis spectroscopy). In addition, we
have observed negligible perturbations in the energy of the
Cu2O2 breathing mode in the resonance Raman spectra of
3536
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ꢀ 2008 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Chem. Eur. J. 2008, 14, 3535 – 3538

Tyrosinase-Like Reactivity in a CuIII
2(m-O)2 Species
A
COMMUNICATION
Plotting the rate of decay of 3X against the corresponding
Hammett substituent constants (s+) affords a linear correla-
tion (R2 =0.99) that gives a 1 value of ꢀ1.9 for the hydroxyl-
ation step (Figure 2), indicative of an electrophilic oxidizing
Figure 2. Hammett plot for the thermal decay of 3X at ꢀ808C in acetone
(X=F, Cl, CO2Me and CN). [2]=0.050 mm, [p-X-C6H4ONa]=0.075 mm.
species that attacks the aromatic ring. This value is in close
proximity to the value reported for [CuIII
2(m-O)2(phenolate)-
A
(DBED)]+ (1=ꢀ2.2),[10] selected model compounds with
(m-h2:h2-peroxo)dicopper(II) cores (1=ꢀ1.8 to ꢀ2.1),[11b,17]
and to that found in tyrosinase (1=ꢀ2.4) (Table 1).[12]
Figure 1. Top: UV/Vis spectra of 2 (dashed line) and 3Cl (solid line) in
acetone at ꢀ908C. Experimental conditions: Complex 3Cl was generated
by reaction of a 0.2 mm solution of 2 with 1.5 equivalents of p-Cl-
C6H4ONa in acetone at ꢀ908C. Bottom: Resonance Raman spectra of
3Cl generated with 16O2 (a) and 18O2 (b) with laser excitation at 407 nm.
Spectra of 3Cl generated with 16O2 (c) and 18O2 (d) with laser excitation at
568 nm.
Finally, kinetic analysis of the thermal decay of 3Cl in the
ꢀ88.58C to ꢀ608C temperature range affords activation pa-
rameters for the hydroxylation step. Activation parameters
for the reaction are characterized by a negative activation
entropy and a relatively small activation enthalpy. Interest-
ingly, the DS° value is significantly smaller than that report-
ed for intermolecular phenol hydroxylation by [CuII -
2
the bis
C
A
ꢀ
ular arene hydroxylation exhibited by (m-h2:h2-peroxo)di-
copper(II) complexes supported by m-xylyl-bridged bis(2-
the similarity between the resonance Raman enhanced vi-
brations of the bis
U
pyridylethyl)amine [CuII2(R-XYL)(O2)]2+[19] and iPrTACN
[CuII (m-XYLiPr4)(O2)]2+ chelates.[20] Presumably, phenolate
ported for the [CuIII
binding and hydroxylation are not completely independent
recently described by Stack and co-workers.[10] Therefore,
reactions in the kinetic study of [CuII2(MeL66)(O2)]2+ and
T
we conclude that 3Cl is best described as the phenolate
thus kinetic parameters for the hydroxylation are likely to
adduct of the bis(m-oxo) species 2 (Scheme 2).
A
Formation and decay of 3Cl were studied by UV/Vis stop-
ped-flow methods. The reaction between 2 and the pheno-
late to form 3Cl is very fast (k> 106 mꢀ1 sꢀ1), too fast even for
stopped-flow techniques at very low temperatures (ꢀ888C),
and neither precise reaction rates nor activation parameters
could be obtained for this process. On the other hand, kinet-
ic analysis indicates that the decay of 3Cl is a first-order pro-
cess. The analogous species 3X (X=F, CO2Me and CN)
were generated by addition of 1.5 equivalents of p-X-
C6H4ONa to 2 at ꢀ808C in acetone (see Supporting Infor-
mation for UV/Vis spectral features), and their correspond-
ing decay rates were studied by UV/Vis and fitted to a
single exponential function by nonlinear regression methods.
Table 1. Kinetic parameters for the hydroxylation of phenols by tyrosi-
nase and selected model compounds.
¼
¼
Compound[a]
1
DH
[kJmolꢀ1
DS
Ref.
G
]
[JKꢀ1 molꢀ1
]
3Cl
tyrosinase
ꢀ1.9
ꢀ2.4
ꢀ2.2
ꢀ1.8
ꢀ1.8
ꢀ2.1
–
37.1ꢂ0.5
ꢀ55ꢂ3
–
[12]
61ꢂ9
ꢀ24/+21ꢂ11
[b]
[b]
[10]
A
U
[17,18]
[12a]
[19]
G
2(MeL66)(O2)]2+
29.1ꢂ3.0
ꢀ115ꢂ15
E
ACHTREUNG
n.d.[c]
n.d.[c]
N
C
50ꢂ1[d]
50.1ꢂ0.2
ꢀ35ꢂ2[d]
ꢀ50.4ꢂ0.9
[20]
AHCTREUNG
[a] See Supporting Information for a structural diagram of the complexes.
[b] An Arhenius plot affords Ea =42.7 kJmolꢀ1, A=9”1011. [c] Not deter-
mined. [d] R=H.
Chem. Eur. J. 2008, 14, 3535 – 3538
ꢀ 2008 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemeurj.org
3537

L. Que, Jr., E. V. Rybak-Akimova, L. Casella, M. Costas et al.
be influenced by bimolecular substrate binding. In addition,
although the DS° term is expected to be large and negative
due to immobilization of the copper-bound phenolate in the
transition state, the different structures of the Cu2O2 com-
plexes may involve differences in phenolate binding and in
the degree of immobilization. For instance, the four benz-
Keywords: bioinorganic chemistry · dicopper enzymes ·
ꢀ
model compounds · O O activation · tyrosinase
A
N
to participate in stacking interactions with the phenolate ar-
omatic ring. In our case, phenolate binds rapidly and irrever-
sibly in the first step, and first-order intramolecular decom-
position of 3Cl occurs in the second step (for which the acti-
vation parameters were determined). On the other hand,
the DH° term determined for 3Cl is remarkably similar to
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A
N
(DH° =40.3 kJmolꢀ1, derived from Ea =10.2 kcalmolꢀ1).[10]
Overall, the values obtained for 3Cl are in close proximity to
those reported for aromatic hydroxylations by (m-h2:h2-per-
oxo)dicopper(II) species and also [CuIII
ACHTREUNG
ACHTREUNG
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tion state in all cases. The activation parameters for the
monophenolase reaction catalyzed by tyrosinase are differ-
ent (Table 1).[12] In this case, a large DH° value, and the
analogy with the data for the diphenolase reaction, suggest
ꢀ
that O O cleavage is the main contributor to the enthalpic
barrier, whereas small and substrate-dependent DS° values
indicate strong preorganization and complementarity of the
active site with the transition state configuration of the reac-
tants.[12]
In conclusion, we demonstrate for the first time that a bis-
[9] P. L. Holland, K. R. Rodgers, W. B. Tolman, Angew. Chem. 1999,
111, 1210; Angew. Chem. Int. Ed. 1999, 38, 1139.
A
N
hydroxylating phenolates, and thus mimicking tyrosinase.
Kinetic parameters establish a close similarity between our
system and the (m-h2:h2-peroxo)dicopper(II) species capable
of performing aromatic hydroxylation. Complex 2 differs
from any previously reported system in the fact that exclu-
[10] L. M. Mirica, M. Vance, D. J. Rudd, B. Hedman, K. O. Hodgson,
E. I. Solomon, T. D. P. Stack, Science 2005, 308, 1890.
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sive formation of bis
served, before and after phenolate binding to the Cu2O2 site.
This work further substantiates the notion that the bis(m-
A
G
AHCTREUNG
oxo) core is competent for performing tyrosinase-like activi-
ty.
[15] J. A. Halfen, B. A. Jazdzewski, S. Mahapatra, L. M. Berreau, E. C.
Wilkinson, L. Que, Jr., W. B. Tolman, J. Am. Chem. Soc. 1997, 119,
8217.
Experimental Section
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Full experimental details for the preparation of the complexes, experi-
mental procedures for the phenolate oxidation reactions, resonance
Raman analysis, and kinetic analyses are included as Supporting Informa-
tion.
[18] L. Santagostini, M. Gullotti, E. Monzani, L. Casella, R. Dillinger, F.
Tuczek, Chem. Eur. J. 2000, 6, 519.
Acknowledgements
[19] K. D. Karlin, M. S. Nasir, B. I. Cohen, R. W. Cruse, S. Kaderli, A. D.
Zuberbꢁhler, J. Am. Chem. Soc. 1994, 116, 1324.
Financial support by MEC of Spain through projects CTQ2006–05367/
BQU to M.C., from NIH (GM-38767 to L.Q.) and from DOE (DE-
FG02–06ER15799 to E.R.A.). A.C. and I.G.-B. thank MEC for PhD
grants. R.M.-B. thanks MEC for a post-doctoral grant. L.C. and S.P.
thank a PRINproject of MIUR and the University of Pavia through
FAR project for support.
[20] S. Mahapatra, S. Kaderli, A. Llobet, Y.-M. Neuhold, T. PalanchØ,
J. A. Halfen, V. G. Young, Jr., T. A. Kaden, L. Que, Jr., A. D. Zuber-
bꢁhler, W. B. Tolman, Inorg. Chem. 1997, 36, 6343.
Received: February 5, 2008
Published online: March 17, 2008
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Chem. Eur. J. 2008, 14, 3535 – 3538