The first catalytic tyrosinase model system based on a mononuclear copper(I) complex: Kinetics and mechanism

Article abstract of DOI:10.1002/anie.201000973

Ready... steady... go! The copper(I) complex 1 not only catalyzes the oxygenation of di-tertbutylphenol (DTBP-H) to di-tert-butylquinone (DTBQ) in a tyrosinase-like fashion, but also allows the reactive cycle to be studied in a stepwise and controlled manner. This feature opens new insights into the individual stages of the tyrosinase reaction, phenol hydroxylation, and release of the product as quinone. The implications for the enzymatic reaction are discussed. Copyright

Full text of DOI:10.1002/anie.201000973

Communications
DOI: 10.1002/anie.201000973
Copper Monooxygenases
The First Catalytic Tyrosinase Model System Based on a Mononuclear
Copper(I) Complex: Kinetics and Mechanism**
Malte Rolff, Julia Schottenheim, Gerhard Peters, and Felix Tuczek*
Dedicated to Professor Martin Jansen on the occasion of his 65th birthday
Tyrosinase is a ubiquitous enzyme mediating the o-hydroxyl-
ation of monophenols to catechols and the subsequent two-
electron oxidation to quinones.[1] Its physiological function is
the conversion of tyrosine into dopaquinone, which consti-
tutes the first step of melanine synthesis.[2,3] As evident from
spectroscopic data and a recent X-ray crystal-structure
determination,[4] the active site of tyrosinase contains a
binuclear copper center coordinated by six histidine residues.
This type of active site (type 3) is
nolic substrates has more and more shifted into the focus of
interest.[18–24] An additional challenge in this research area has
been the synthesis of catalytic tyrosinase-model systems.
Apart from a patent,[25] however, there are only two reports
referring to this point. In 1990, Rꢀglier et al. synthesized the
ligand bis-2,2’-[2-(pyrid-2-yl)ethyl]iminobiphenyl (BiPh-
(impy)2) which contains two pyridylethylimine sidearms
bridged by a biphenyl spacer (Scheme 1).[21,26] It was shown
analogous to the active sites of
hemocyanin and catechol oxidase.[3]
All of these copper proteins in their
oxy states bind dioxygen in a char-
acteristic side-on bridging structure
whereby the CuI centers in the
deoxy states are converted into CuII
in the oxy state. Starting from the
oxy state of tyrosinase, monophe-
nols are converted into o-diphenols
(monophenolase reaction), presum-
ably in an electrophilic substitution
reaction.[5] Subsequently, the o-
Scheme 1. Ligand BiPh(impy)2 by Rꢀglier et al., ligand L66 and ligand L by Casella et al.
diphenol (catechol) intermediates
are converted into o-quinones. A
second reactivity of the oxy site is
the two-electron oxidation of external catechol substrates to
o-quinones (diphenolase reactivity). Whereas tyrosinase
exhibits both mono- and diphenolase reactions, the enzymatic
activity of catechol oxidase is restricted to diphenolase
reactivity.[6]
The reactivity of tyrosinase towards phenolic or aromatic
substrates has successfully been reproduced with small-
molecule copper complexes.[7–12] Whereas initially mainly
aromatic hydroxylation reactions of the ligand framework by
m-h2:h2-peroxo and bis-m-oxo dicopper cores were investi-
gated,[13–17] the hydroxylation of external mono- and diphe-
that a mixture of this ligand with two equivalents of CuI salt,
100 equivalents of 2,4-di-tert-butylphenol (DTBP-H), and
200 equivalents of triethylamine (NEt3) leads to the catalytic
generation of quinone with a turnover number (TON) of 16,
monitored by the appearance of the optical absorption band
of 3,5-di-tert-butyl-o-quinone (DTBQ) at approximately
400 nm. After one hour, the reaction stopped, presumably
by formation of an oxo-bridged complex. A mechanism was
proposed for this reaction, involving the formation of a
binuclear peroxo complex and a catechol-bridged intermedi-
ate. Shortly afterwards Casella et al. described a catalytic
model system of tyrosinase based on the ligand a,a’-bis{bis[2-
(1-methylbenzimidazol-2-yl)ethyl]amino}-m-xylene
(L66,
[*] M. Rolff, J. Schottenheim, G. Peters, F. Tuczek
Institut fꢁr Anorganische Chemie
Christian-Albrechts-Universitꢂt zu Kiel
Max-Eyth-Strasse 2, 24098 Kiel (Germany)
Fax: (+49)431-880-1520
Scheme 1).[18] In a stoichiometric mode (TON = 1) the
system was found to lead to hydroxylation of carbometh-
oxyphenol to carbomethoxycatechol. With phenol groups
containing electron-donating substituents such as two tert-
butyl groups, TONs greater than 1 were observed.
E-mail: ftuczek@ac.uni-kiel.de
[**] The authors acknowledge synthetic and/or spectroscopic support
from Ursula Cornelissen, Stephanie Pehlke, Marianne Karbstein,
and Franziska Wendt.
Stoichiometric hydroxylation of phenols has also been
shown for a mononuclear CuI complex supported by L, the
tridentate analogue of the ligand L66 (Scheme 1). In this
reaction an involvement of the binuclear m-h2:h2 peroxo
complex was demonstrated.[27] The question therefore arises
Supporting information for this article (all syntheses, analytical
data, and spectroscopic measurements) is available on the WWW
under http://dx.doi.org/10.1002/anie.201000973.
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as to whether mononuclear copper(I) complexes are also
capable of mediating the phenol hydroxylation reaction in a
catalytic fashion.
To address this problem we synthesized the ligand L1
(Scheme 2) which represents one half of the binucleating,
tetradentate ligand BiPh(impy)2 with the imine function
To eliminate absorptions from copper species, the reaction
mixture was quenched after 8 h of oxygenation with an excess
of HCl and extracted with CH2Cl2. The UV/Vis spectrum of
the organic phase clearly exhibits the optical spectrum of
DTBQ (Figure 1, solid line). The organic phase was inves-
tigated by NMR spectroscopy, which indicated a mixture of
DTBQ (15% of the starting amount of DTBP-H), DTBP-H
ꢀ
(52%), 3,5-di-tert-butyl-catechol (DTBC-H2, 3%) and the C
C coupling product 4,4’,6,6’-tetra-(tert-butyl)-2,2’-biphenol
(30%). This result is in good agreement with the TON for
the formation of quinone determined from the 407 nm
absorbance (Figure 1, inset).[29,30] In a control experiment a
1:1 mixture of DTBP-H/DTBC-H2 was oxygenated in the
presence of NEt3 with subsequent HCl quench, leading to a
1:1 mixture of DTBP-H/DTBQ; the optical absorption and
NMR spectra of DTBQ were found to be identical to those
resulting from the copper-catalyzed oxygenation of DTBP-H.
To check the role of the mixed pyridine/imine donor set of
L1, we additionally synthesized the ligands L2 and L3
containing two imine and two pyridine functions, respectively
(Scheme 2).[28] Oxygenation of mixtures of [Cu(L2)]+ or
[Cu(L3)]+ with 50 equivalents DTBP-H and 100 equivalents
NEt3, however, did not lead to the appearance of the 407 nm
band, indicating that these complexes are catalytically
inactive with respect to the oxygenation of monophenols.
Clearly, only the combination of pyridine and imine is
successful whereas either two pyridine or two imine donor
functions per metal center do not provide the proper
coordination for a catalytic action of the copper complex.
To study the mechanism of the Cu(L1)-mediated oxygen-
ation of DTBP-H to DTBQ, the reaction was also run in a
Scheme 2. Ligand set L1–L3 used in this study.[28]
terminated by a tert-butyl group. The CuI bis(acetonitrile)
complex derived from L1, [CuI(L1)(CH3CN)2](PF6) (1) is also
converted into the phenolato complex by the procedure
developed by Casella;[18] that is, 1 was treated with one
equivalent of DTBP-H and two equivalents of NEt3, leading
to a 1:1 mixture (2) of the neutral phenolato-triethylamine
complex [CuI(L1)(DTBP)(NEt3)] and HNEt3PF6.
Importantly, oxygenation of a mixture 1 or 2 with
50 equivalents of DTBP-H and 100 equivalents of NEt3 in
CH2Cl2 was found to catalytically lead to generation of
DTBQ, as shown by the appearance of an absorption band of
DTBQ at 407 nm (Figure 1). Based on an e value of
1830mꢀ1 cmꢀ1, a TON of 18 per dicopper unit (see below)
was calculated, almost identical to the result of Rꢀglier et al.
However, the reaction was found to be much slower,
concomitant with a longer lifetime: whereas for the BiPh-
(impy)2 system the reaction is completed in 1 h, the first
16 cycles for the Cu(L1) system take 10 h and 18 cycles are
completed after 1 day.
+
stoichiometric fashion. To avoid the presence of HNEt3 in
the solution, the parent copper phenolato triethylamine
complex was prepared from 1 and NaDTBP/NEt3, leading
to a mixture (2’) of [Cu(L1)(NEt3)(DTBP)] and NaPF6.
A solution of 2’ in CH2Cl2 was oxygenated for 30 min
without adding further NEt3 or DTBP-H. The absorption
spectrum of 2’ exhibits a shoulder at approximately 350 nm,
corresponding to a phenolate!Cu transition (Figure 2,
curve a). After oxygenation an absorption spectrum with a
shoulder at 340 nm (Figure 2, curve b) is observed. The
corresponding spectrum is similar to that of the catecholato
complex of the L55 system by Casella et al.[31] The resulting
intermediate (4) thus may contain catecholate coordinated to
a binuclear copper complex. This assumption was supported
by quenching the reaction mixture with HCl, and subsequent
extraction with CH2Cl2. NMR spectroscopic analysis of the
organic phase lead to a roughly 5:4:1 mixture of DTBP-H/
DTBQ/DTBC-H2, suggesting the presence of one catecholate
and one phenolate in intermediate 4.[32] Compound 4 was also
isolated and characterized by elemental analysis, magnetic
susceptibility (c), and mass spectrometry. Elemental analysis
is compatible with
a binuclear formulation of 4 as
[Cu2(L1)2(OH)(DTBC)(DTBP)]·2NaPF6. The c versus T
data, on the other hand, indicate only a weak antiferromag-
netic interaction, suggesting that compound 4, upon isolation,
has split into two CuII monomers, [Cu(L1)(DTBC)] +
[Cu(L1)(DTBP)(OH)] (compound 4’; see Supporting Infor-
mation). Further evidence for this constitution is provided by
Figure 1. UV/Vis spectroscopic monitoring of the catalytic formation of
DTBQ by oxygenation of a 20 mm solution of 1 in CH2Cl2 in the
presence of 50 equivalents DTBP-H and 100 equivalents NEt3 during
the first 6 h (dashed lines) and after 8 h and subsequent HCl quench/
CH2Cl2 extraction (solid line); l=5 cm. Inset: Turnover number per
dicopper unit as a function of time.
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Oxygenation of the phenolato triethylamine CuI complex
2 initially forms the binuclear m-h2:h2 peroxo complex 3
containing two coordinated phenolates. In this complex only
one of the bound phenolates can be hydroxylated, leading to
an asymmetrically bridged m-catecholato-m-hydroxo com-
plex,[33] intermediate 4. Hydroxylation is thereby mediated
by the s*-orbital of the coordinated peroxide.[16] In a
stoichiometric mode, the reaction stops at the level of 4.
Importantly, addition of an excess of DTBP-H/NEt3 to 4
under anaerobic conditions leads to release of the substrate as
quinone (Scheme 3 and Figure 2, curve c). Moreover, upon
oxygenation of this mixture, the catalytic action of the system
is resumed, as evident from the growing of the 407 nm band of
DTBQ with time. This result indicates that both compound 2
and intermediate 4 are catalytically competent.
To gain further insight into the mechanism of the
hydroxylation step, the conversion of 2ꢀ into 4 was also
studied kinetically. The reaction sequence can be described
with the following kinetic scheme [Eq. (1)–(3)](see Support-
ing Information).
Figure 2. 500 mm solution of 1 in CH2Cl2 containing 1 equivalent
NaDTBP and 1 equivalent NEt3 (a, 2’), after oxygenation for 30 min (b,
4) and subsequent addition of 200 equivalents NEt3 and 100 equiva-
lents DTBP-H under argon atmosphere (c); l=1 cm. Inset: Kinetics of
the oxygenation of 2’ to 4 at l=310 nm.
k1
20 þ O2 Ð ½CuIIðL1ÞðOPhÞðO2ꢀÞꢁ þ NEt3
ð1Þ
k
ꢀ1
MALDI-TOF mass spectrometry (Figure 3, bottom); the
high-resolution structure of the signal at approximately
m/z 477 is excellently reproduced by a 1:1 superposition of
the signals of [{Cu(L1)(DTBC)} + 3H]+ and [{Cu(L1)(OH)-
(DTBP)} + 3H]+ (Figure 3, top) generated upon ionization of
k2
½CuIIðL1ÞðOPhÞðO2ꢀÞꢁ þ 20 Ð 3 þ NEt3
ð2Þ
ð3Þ
k
ꢀ2
k3
3 ! 4
4’. The splitting of the m-catecholato m-hydroxo CuII dimer
If both the superoxo complex [CuII(L1)(OPh)(O2 )] and
the peroxo complex 3 are highly reactive, kꢀ1 and kꢀ2 can be
assumed to be small compared to k1, k2, and k3, and the
product formation follows the rate law [Eq. (4)]:
ꢀ
2
into two monomers is related to the copper dibenzyl ethylene
diamine (DBED) system where the corresponding intermedi-
ate was found to split into a CuI aquo and a CuII semiquinone
complex upon addition of H+.[23]
It thus has been shown by two methods (workup and
NMR spectroscopic analysis of a solution containing com-
pound 4 as well as direct isolation/analysis of compound 4)
that the product (catechol) yield in a stoichiometric run
(phenol/Cu 1:1) is 50%. This result strongly supports a
binuclear reaction course as shown in Scheme 3:
d½4ꢁ
¼ k1½20ꢁpðO2Þ
ð4Þ
dt
This result is what is observed experimentally (Figure 2,
inset). Although the hydroxylation reaction proceeds in a
binuclear fashion, the reaction rate exhibits a first-order
dependence on the copper concentration.[27] The described
kinetics also accounts for the fact that no peroxo intermediate
could be detected in our system.
The derived mechanistic cycle (Scheme 3) has also an
interesting consequence with respect to the enzymatic
reaction. In general, it is assumed that phenols bind to the
oxy form of tyrosinase.[2] It has been difficult to understand,
however, why addition of (protonated) phenols to synthetic
binuclear peroxo complexes always leads to non-physiolog-
ical radical-coupling products whereas the correct hydroxyl-
ation reaction is only observed after addition of (deproton-
ated) phenolates.[34] The question therefore arises as to
whether phenols may become deprotonated in the enzyme
prior to coordination to the oxy site. However, there is no
residue available in the immediate vicinity of the binuclear
copper center which could perform such a deprotonation,
with the exception of a noncoordinating histidine that is
sequentially positioned directly adjacent to a His residue
coordinating to CuB (in Streptomyces castaneoglobisporus
tyrosinase His215 and His216, respectively).[4,35] The way this
Figure 3. High resolution MALDI-TOF spectrum of 4’ (bottom) and
calculated isotope pattern for the two fragments of [4’+6H]2+ (top;
see text and Supporting Information for further details).
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Cu(L1)/NEt3 system thus enables both a
complete mechanistic understanding of the
tyrosinase reaction and should lead to the
development of further catalytic tyrosinase
model systems. Detailed spectroscopic and
quantum-chemical investigations will now
be performed to obtain further insight into
the elementary steps involved in the de-
scribed catalytic cycle.
Received: February 16, 2010
Revised: June 2, 2010
Published online: July 22, 2010
Keywords: bioinorganic chemistry · enzymes ·
.
homogeneous catalysis ·
type 3 copper proteins · tyrosinases
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Communications
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