Catalytic Models of Tyrosinase: Reactivity Differences between Systems Based on Mono- and Binucleating Ligands

Article abstract of DOI:10.1002/ejic.201500029

A new tyrosinase model based on the binucleating ligand L_py2 is synthesized and characterized. The ligand L_py2 contains a combination of an imine and a pyridine function in the sidearms, which are bridged by a flexible alkyl spacer. As shown by UV/Vis and NMR spectroscopy, the Cu₂L_py2 complex catalyzed the conversion of the monophenol 2,4-di-tert-butylphenol (DTBP-H) into the o-quinone 3,5-di-tert-butylquinone (DTBQ) with a turnover number (TON) of 18. The dicopper complex of L_py2 thus shows monophenolase activity that is comparable to that of the recently developed L_py1 model of tyrosinase, which is based on a known mononucleating ligand (M. Rolff, J. Schottenheim, G. Peters, F. Tuczek, Angew. Chem. Int. Ed. 2010, 122, 6583). The electron-poor substrate 4-hydroxybenzoic acid methyl ester (MeBA-OH), in contrast, is converted by Cu₂L_py2 into the semiquinone. For both substrates, the oxygenation reactions were also conducted in a stoichiometric fashion to obtain information on the intermediates involved. For the substrate MeBA-OH, we detected a binuclear μ-catecholato copper(II) complex by high-resolution ESI mass spectrometry. These studies were complemented by investigations of deactivation mechanisms that could be invoked to explain the limitation of the TON. To this end, a bis-μ-hydroxido L_py2 dicopper(II) complex as well as a semiquinone L_py2 complex were prepared. Both complexes may represent decay products of the catalyst. A tyrosinase model based on the binucleating ligand L_py2 was developed and characterized. The ligand L_py2 contains a combination of an imine and a pyridine function in the sidearms that are bridged by a flexible alkyl spacer. The Cu₂L_py2 complex catalyzes the conversion of monophenol DTBP-H into the o-quinone DTBQ (TON = 18). An electron-poor substrate is converted into the semiquinone.

Full text of DOI:10.1002/ejic.201500029

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DOI:10.1002/ejic.201500029
Catalytic Models of Tyrosinase: Reactivity Differences
between Systems Based on Mono- and Binucleating
Ligands
Julia Schottenheim,[a] Claus Gernert,[b] Benjamin Herzigkeit,[a]
Jan Krahmer,[a] and Felix Tuczek*[a]
Keywords: Bioinorganic chemistry / Metalloproteins / Enzyme models / Enzyme catalysis / Copper
A new tyrosinase model based on the binucleating ligand
poor substrate 4-hydroxybenzoic acid methyl ester (MeBA-
Lpy2 is synthesized and characterized. The ligand Lpy2 con- OH), in contrast, is converted by Cu2Lpy2 into the semi-
tains a combination of an imine and a pyridine function in
the sidearms, which are bridged by a flexible alkyl spacer.
quinone. For both substrates, the oxygenation reactions were
also conducted in a stoichiometric fashion to obtain infor-
mation on the intermediates involved. For the substrate
MeBA-OH, we detected a binuclear μ-catecholato copper(II)
complex by high-resolution ESI mass spectrometry. These
studies were complemented by investigations of deactivation
mechanisms that could be invoked to explain the limitation
of the TON. To this end, a bis-μ-hydroxido Lpy2 dicopper(II)
complex as well as a semiquinone Lpy2 complex were pre-
pared. Both complexes may represent decay products of the
catalyst.
As shown by UV/Vis and NMR spectroscopy, the Cu2Lpy
2
complex catalyzed the conversion of the monophenol 2,4-di-
tert-butylphenol (DTBP-H) into the o-quinone 3,5-di-tert-
butylquinone (DTBQ) with a turnover number (TON) of 18.
The dicopper complex of Lpy2 thus shows monophenolase
activity that is comparable to that of the recently developed
Lpy1 model of tyrosinase, which is based on a known mono-
nucleating ligand (M. Rolff, J. Schottenheim, G. Peters, F. Tu-
czek, Angew. Chem. Int. Ed. 2010, 122, 6583). The electron-
Introduction
Pigmentation is one of the most obvious phenotypic
properties of living beings. It is based on melanin, which is
one of the most common biopigments, and contributes to
the richness of color in a variety of bacteria, fungi, plants,
and animals. Melanins are heterogeneous polyphenolic bio-
polymers with a complex structure, the color of which var-
ies from yellow to black.[1,2] The biosynthesis of melanin
starts with conversion of the amino acid tyrosine into dopa-
quinone, which is catalyzed by tyrosinases.[2–4] Type 3 cop-
per enzyme tyrosinase mediates the o-hydroxylation of
monophenols to catechols and the subsequent two-electron
oxidation to o-quinones (monophenolase activity,
Scheme 1a),[5] but also shows diphenolase activity, which in-
volves the two-electron oxidation of catechols to o-quinones
(Scheme 1b).[6]
Scheme 1. (a) Monophenolase activity and (b) diphenolase activity
of tyrosinase.
The first X-ray crystallographic characterization of a
tyrosinase active site was performed for the bacterium
Streptomyces castaneoglobisporus.[7] In analogy to the other
two important Type 3 copper proteins hemocyanin and
catechol oxidase, it contains a binuclear copper center,
whereupon each copper is coordinated by three histidines.
Bonding of dioxygen leads to the characteristic Cu2 μ-
η2:η2-peroxide structure,[8] which mediates the o-hydroxyl-
ation and two-electron oxidation of diphenolic substrates
(see Scheme 2).
[a] Institut für Anorganische Chemie, Christian-Albrechts-
Universität zu Kiel,
Max-Eyth-Staße 2, 24118 Kiel, Germany
E-mail: ftuczek@ac.uni-kiel
http://www.ac.uni-kiel.de/tuczek
In 2009, the isolation and crystallization of a prophenol
oxidase from the moth Manduca sexta was achieved, which
is the inactive precursor of phenoloxidase in insects.[9] Since
then more crystal structures of tyrosinase were obtained.[10]
[b] Institut für Physikalische Chemie, Christian-Albrechts-
Universität zu Kiel,
Max-Eyth-Staße 1, 24118 Kiel, Germany
Supporting information for this article is available on the
WWW under http://dx.doi.org/10.1002/ejic.201500029.
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Scheme 2. Monophenolase cycle of tyrosinase.
The publications include the structures of enzymes from the
fungus Agaricus bisporus and the bacterium Bacillus
megaterium, and also studies with tyrosinase inhibi-
tors.[10a,10b]
Numerous examples of small-molecule model systems re-
producing the mono- and diphenolase activities of Type 3
copper proteins have been published.[11] However, the
number of tyrosinase models exhibiting catalytic mono-
phenolase remains very limited.[5] The first catalytic model
systems of tyrosinase were based on binuclear copper com-
plexes.[12–14] The functionalized sidearms in the ligands
were bridged by comparatively rigid aromatic spacers such
as xylyl for the L-66 system of Casella et al. or biphenyl for
the BiPh(impy)2 system of Réglier et al.[13,14] Bulkowski et
al., in contrast, employed dicopper systems supported by
macrocyclic ligands that contained flexible alkyl spacers.[12]
Scheme 3. Mononucleating ligands for model complexes: (a) Lpy1,
Lbzm1, Lhpz1, and Lhpz2 developed by our research group, and
(b) the HC(3-tBuPz)2(Py) ligand of Herres-Pawlis et al.
During the last few years, our group has synthesized a (CH3CN)2]PF6 in dichloromethane after 6.5 hours.[16] In
family of copper complexes based on mononucleating li- the meantime, another catalytic model was published that
gands that show catalytic monophenolase activity.[15–17] The was based on a mononucleating ligand. The HC(3-tBuPz)2-
ligands contain an imine function and a variable N-hetero- (Py) ligand developed by Herres-Pawlis, Stack et al. con-
cyclic part, which was pyridine for Lpy1, benzimidazole for tains pyridine and pyrazole functions (Scheme 3b). The de-
L
bzm1, and pyrazole for Lhpz1 and Lhpz2 (Scheme 3a).
The copper(I) complex [CuILpy1(CH3CN)2]PF6 was exhibits catalytic monophenolase activity toward a variety
shown to generate 3,5-di-tert-butylquinone (DTBQ) upon of monophenolic substrates.[18]
rived copper(I) complex forms a stable dioxygen adduct and
addition of 50 equiv. 2,4-di-tert-butylphenol (DTBP-H),
In this paper, we combine a flexible alkyl spacer with
100 equiv. NEt3 and molecular oxygen with a turnover pyridine-imine-functionalized sidearms to obtain a new bi-
number (TON) of 18 after 8 hours.[15] By replacing the pyr- nucleating ligand, Lpy2 (Scheme 4). The bridging of the
idine function with benzimidazole (Lbzm1) or pyrazole sidearms should mainly contribute to stabilize the inter-
(Lhpz1, Lhpz2) (Scheme 3a), both the reaction rate and turn- mediates of the catalytic cycle. In particular, we wanted to
over number of the catalytic conversion of monophenol further characterize the catecholato species that is com-
DTBP-H into the o-quinone DTBQ could be in- parable to the D-met form of the enzymatic system
creased.[15–17] The highest TON of 31 was achieved by cata- (Scheme 2). Moreover, the performance of the Lpy2 system
lytic oxygenation of a 500 μm solution of [CuILbzm1- with respect to the catalytic conversion of monophenols
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into o-quinones has been determined. Regarding this by the evolution of the 407 nm absorption band (Figure 1).
activity, it is of interest to evaluate the influence of the
bridge, which is present in Lpy2 and absent in Lpy1.
Furthermore, the activity of the Lpy2 system with respect
to an electron-rich (DTBP-H) and an electron-poor sub-
strate 4-hydroxybenzoic acid methyl ester (MeBA-OH) were
compared. Finally, studies on the deactivation of the cata-
lyst complexes were conducted to understand the limitation
of the turnover number of the models.
Figure 1. Catalytic oxygenation of a 500 μM solution of Cu2Lpy
2
in dichloromethane in the presence of DTBP-H (100 equiv.) and
NEt3 (200 equiv.) during the first 3 h at room temperature. The
solution was diluted before each measurement to a concentration
of 200 μM of Cu2Lpy2; l = 1 mm. (Inset) Turnover number per di-
copper unit (black circle) and turnover frequency per minute (gray
triangle) as a function of time over 7 h of oxygenation.
Based on an ε-value of 1830 Lmol–1 cm–1, the system
produces 16 TON per dicopper unit during the first 3 h.[13]
The rate of DTBQ formation decreases with time and, after
4.5 h, the yield reaches a maximum value of 18 turnovers
(Figure 1 inset).
During the first 5 min of oxygenation, the turnover fre-
quency (TOF) was approximately 0.8 turnovers per minute
and this was halved within 30 min. With further reaction
time, the TOF decreases continuously until it becomes al-
most zero after 7 h (Figure 1 inset).
Scheme 4. Synthesis of ligand Lpy
copper(I) complex Cu2Lpy2.
2 and the corresponding
Results and Discussion
The Cu2Lpy2 complex is thus able to catalytically mediate
the conversion of monophenol DTBP-H into o-quinone
DTBQ with a TON that is comparable to that of the Lpy1
system.[15,16] The bridging of the pyridine-imine units by the
flexible alkyl chain of the Lpy2 system therefore does not
hinder the catalytic monophenolase activity. On the con-
trary, the Lpy2 system is faster at the beginning of oxygen-
ation than its Lpy1 counterpart, and it remains stable for
longer after reaching the maximum TON (see below).
After 1 h of oxygenation, a small portion of the reaction
mixture was diluted to a concentration of 25 μM Cu2Lpy2
in dichloromethane. This solution was treated with 6 M
hydrochloric acid and extracted with dichloromethane to
eliminate any copper species. The solvent was removed un-
der reduced pressure and the resulting residue was investi-
Synthesis of the Ligand Lpy2 and the Copper(I) Complex
Cu2Lpy2
The synthesis of Lpy2 (Scheme 4) started with oxidative
ring opening of cylohexene oxide to give hexane-1,6-dial.[19]
The imine condensation of 2-(pyrid-2-yl)ethylamine
(2 equiv.) and hexane-1,6-dial provided Lpy2, which was
converted into the corresponding copper(I) complex
Cu2Lpy2 by addition of tetrakis(acetonitrile)copper(I) hexa-
fluorophosphate (2 equiv.) under anaerobic conditions. All
compounds were obtained in satisfactory yields and purities
(see Exp. Sect.).
Catalytic Monophenolase Activity of the Lpy2 Model
System
1
gated by H NMR spectroscopy (Figure 2).
To test the catalytic activity of the new model system
Integration of the respective signals in the 1H NMR
involving Lpy2, a catalytic oxygenation was performed and spectrum (Figure 2) showed that after 1 h of oxygenation,
monitored by in situ UV/Vis spectroscopy at room tempera- 77% of the starting amount of DTBP-H remained, 9% was
ture. A mixture of DTBP-H (100 equiv.) and triethylamine converted into the C–C coupling product 3,3Ј,5,5Ј-tetra-
(200 equiv.) was added to a solution of Cu2Lpy2 (500 μM) tert-butyl-2,2Ј-biphenol,[20,21] and 14% of DTBP-H was
in dichloromethane under anaerobic conditions, then mo- converted into o-quinone DTBQ, in accordance with the
lecular oxygen was bubbled through the solution. The Lpy2 TON of 14 determined spectrophotometrically after that
system catalytically generated DTBQ, which was detected reaction time.
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1
Figure 2. H NMR spectrum of the organic phase after HCl quench (1 h oxygenation) with detected compounds (blue: DTBP-H, green:
DTBQ; red: C–C coupling product).
Scheme 5. Proposed mechanistic cycle of the Lpy2 model system.
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In comparison to the mononuclear models (Lpy1, Lbzm1),
Clearly, the electron-withdrawing effect of the methyl
the formation of the C–C coupling product, and thus the ester group in the substrate MeBA-OH hinders its complete
unwanted free-radical side reaction, could be significantly oxidation to the o-quinone. Therefore, only the semi-
reduced in the Lpy2 system. After 1 h of catalytic oxygena- quinone MeBASQ is generated, which presumably is
tion, only 9% of the coupling product was found; at that formed by one-electron oxidation of the catechol resulting
reaction time, 22% of coupling product was produced by from the o-hydroxylation of MeBA-OH. We assume that in
the Lpy1 and even 47% by the Lbzm1 system.[15,16] Suppres- the catalytic formation of the semiquinone MeBASQ (anal-
sion of the free-radical side reaction may be attributed to ogous to Scheme 5), the semiquinone is released from inter-
better shielding of the peroxide moiety in the side-on-per- mediate 4 by one-electron oxidation of the catechol. The
oxo intermediate (see Scheme 5) through the alkyl-bridged resulting mixed-valent CuI–CuII complex is reduced to the
ligand Lpy2. This shielding, in particular, prevents the H- CuI–CuI form by one-electron reduction from the “phenol
atom abstraction reaction of phenols leading to phenoxyl pool” of the reaction mixture.
radicals, which represents the initial step of the free-radical
pathway.[20,21] In addition, steric factors may prevent the
recombination of two phenoxyl radicals, leading to the C–
C coupling product.
Stepwise Performance of the Catalytic Cycle for the Lpy2
Model System
Due to the close analogy to the Lpy1 system, we propose
the mechanistic cycle shown in Scheme 5 for the new Lpy2
model of tyrosinase. To confirm this mechanistic cycle, a
stoichiometric oxygenation was performed with the
standard substrate DTBP-H. The resulting absorption
spectra were very similar to those obtained by stoichio-
metric oxygenation of DTBP-H with the Lpy1 system (Fig-
ure 4).[15]
Catalytic Oxygenation of the Substrate MeBA-OH
The catalytic oxygenation was also carried out by using
the substrate MeBA-OH, which has an electron-with-
drawing methyl ester function instead of the electron-donat-
ing tert-butyl groups. A mixture of MeBA-OH (100 equiv.)
and triethylamine (200 equiv.) was added to a 250 μM
solution of Cu2Lpy2 in dichloromethane under anaerobic
conditions, then molecular oxygen was bubbled through the
solution for 5 h. The resulting UV/Vis spectra are shown in
Figure 3.
Figure 4. Stoichiometric oxygenation of a 250 μM solution of
Cu2Lpy2 in dichloromethane with the substrate DTBP-H; l =
10 mm.
At first a 250 μM solution of Cu2Lpy2 was prepared
under a nitrogen atmosphere and the UV/Vis spectrum was
measured. This is the precursor 1 of the catalytically active
species 2 (cf. Scheme 5), and does not exhibit significant
absorbance in the visible region (Figure 4, black line). In
the next step, the phenolato complex [CuI2Lpy2(DTBP)2-
(NEt3)2] (2) is prepared by reaction of Cu2Lpy2 with
sodium-2,4-di-tert-butylphenolate (Na-DTBP; 2 equiv.) and
Figure 3. Catalytic oxygenation of a 250 μM solution of Cu2Lpy
2
in dichloromethane in the presence of MeBA-OH (100 equiv.) and
NEt3 (200 equiv.) during the first 5 h at room temperature; l =
1 mm.
The spectra differ greatly from those of the catalytic oxy- NEt3 (2 equiv.) in dichloromethane under Schlenk condi-
genation of the electron-rich substrate DTBP-H. In particu- tions.
lar, they show a peak at 402 nm and two shoulders at 385
In addition, we were able to synthesize the phenolato
and 430 nm, indicating the formation of semiquinones.[23,24] copper(I) complex 2 directly and characterized it by NMR
Given that semiquinones have extinction coefficients of spectroscopy (see Exp. Sect.). The corresponding absorp-
approximately 7500 Lmol–1 cm–1,[24] the Lpy2 system was tion spectrum (Figure 4, green line) clearly shows a shoul-
calculated to generate MeBASQ catalytically in nine turn- der at approximately 330 nm, which can be associated with
overs per dicopper unit after 5 h.
a phenolate-to-copper(I) ligand-to-metal charge transfer
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(LMCT) transition.[25,26] While molecular oxygen is intro- 340 nm and the appearance of a shoulder at 430 nm.[27,28]
duced into the reaction mixture of the stoichiometric oxy- This spectrum is in agreement with the features of the cate-
genation for 30 min, the absorbance at 330 nm decreases cholato species of the Cu2L-66 system.[26]
(Figure 4, blue line) and the color of the solution changes
from yellow to brown. In case of the CuLpy1 system, the
formation of a μ-catecholato complex 4py (“compound 4”)
could be evidenced at this stage of the reactive cycle.[15,16]
We assume that an analogous intermediate, 4DTBP, is
formed in case of the Cu2Lpy2 system, which should have a
higher stability because of the alkyl spacer. However, the
change in the absorption spectrum is rather insignificant
(Figure 4, blue line).
Subsequently, catalytic conditions were re-established by
addition of DTBP-H (100 equiv.) and NEt3 (200 equiv.),
followed by further oxygenation for 60 min. The resulting
spectrum (Figure 4, red line) shows the known absorbance
at 407 nm, which is caused by DTBQ. The catalytic oxygen-
ation can be continued for a few hours with a steady in-
crease of the band at 407 nm. After 2 h, three turnovers
of DTBQ were generated. This shows that the catecholato
intermediate 4 is also catalytically competent, although to
Figure 5. Stoichiometric oxygenation of a 250 μM solution of
Cu2Lpy2 in dichloromethane with the substrate MeBA-OH; l =
10 mm.
a lesser degree than the copper(I) complex 2, which
produces 16 turnovers under comparable conditions (see
Figure 1).
After addition of MeBA-OH (100 equiv.) and NEt3
(200 equiv.) and oxygenation for another 60 min, the spec-
trum shown in Figure 5 (red line) was obtained. Import-
antly, the spectrum shows a peak at 402 nm and two shoul-
ders at 385 and 430 nm, which indicates the formation of
semiquinone.[23,24]
Direct catalytic oxygenation of a mixture of Cu2Lpy2,
MeBA-OH (100 equiv.) and triethylamine (200 equiv.) in
dichloromethane generated the same spectrum (see Fig-
ure 3). This confirms that with the electron-poor substrate
MeBA-OH, the o-quinone cannot be formed and only the
semiquinone MeBASQ is generated. This final product
evidently results from one-electron oxidation of the
catecholato intermediate formed by o-hydroxylation of
MeBA-OH (see Figure 5).
Unfortunately, no direct observation of a peroxo adduct
was possible in our system. With respect to this point, it
is known that binuclear copper(I) complexes supported by
binucleating ligands containing flexible alkyl spacers also
form intermolecular peroxo species.[22] To address this
point, kinetic measurements were carried out by varying the
concentration of the catalyst (see Exp. Sect.). From the fact
that the rate of product formation scales linearly with the
concentration of the binuclear copper(I) precursor Cu2Lpy2
(see the Supporting Information, Figure S1), we conclude
that dioxygen predominantly binds in an intramolecular
fashion and that the side-on-peroxo intermediate 3, shown
in Scheme 5, is in fact the catalytically active species.
A stoichiometric oxygenation reaction was also per-
formed by using the substrate MeBA-OH, which has an
electron-withdrawing methyl ester function instead of the
Catecholato Intermediates 4DTBP and 4MeBA
The spectroscopic changes that occurred during the
electron-donating tert-butyl groups of DTBP-H. The stoichiometric oxygenation with MeBA-OH suggest a
electron-withdrawing effect hinders the oxidation of bound sufficient stability of the corresponding catecholato inter-
catecholate to o-quinone (see above) and thus should con- mediate 4 in the Lpy2 system to allow its direct synthesis
tribute to a further stabilization of the catecholato species and isolation.
4MeBA [26]
.
In fact, we succeeded in preparing the catecholato inter-
The stoichiometric oxygenation with MeBA-OH was mediate 4MeBA by the following route. A solution of
monitored by UV/Vis analysis, and the results are shown in Cu2Lpy2 (31.6 mg, 35.0 μmol) in dichloromethane (3 mL)
Figure 5. A 250 μM solution of Cu2Lpy2 was prepared was prepared under Schlenk conditions. A solution of
under a nitrogen atmosphere and the corresponding MeBA-OH (10.7 mg, 70.0 μmol) and NEt3 (14.2 mg,
UV/Vis spectrum is given by the black line. The phenolato 140 μmol) in dichloromethane (3 mL) was added slowly.
species [CuI2Lpy2(MeBA-O)2(NEt3)2] (2) was generated by Molecular oxygen was introduced into the solution for
adding MeBA-OH (2 equiv.) and NEt3 (4 equiv.). The 30 min, which changed the color from light-brown to dark-
spectrum exhibits a strong absorption at approximately green. After the oxygenation the reaction mixture was over-
310 nm, which can be attributed to a phenolate-to- laid with diethyl ether (20 mL) and it was allowed to stand
copper(I) LMCT transition (Figure 5, green line).[25,26] for 30 min at room temperature. The solution was decanted
While molecular oxygen is introduced into the solution for from the precipitated gray-green solid, which was dried in
30 min, the catecholato copper(II) intermediate 4MeBA is air for 1 d to yield intermediate 4MeBA (19 mg, 70%) as a
formed, as evident from an increase of the absorbance at gray-green solid.
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Figure 6. Segment of the HRMS (ESI) of 4MeBA: simulated and measured isotope pattern of the complex [M + 2Cl – OH – OCH3]+ as
shown top right.
The reaction product could be characterized as a solid intermediates 4, HCl quenching was performed by using
with UV/Vis spectroscopy (see the Supporting Information, 250 μM solutions of 4DTBP and 4MeBA, respectively, in
Figure S2) and by high-resolution ESI mass spectrometry CH2Cl2 (Figure 7). The spectra before the quenches show
(for the full spectrum see the Supporting Information, Fig-
ure S3). The simulated and measured isotope patterns
correspond to 4MeBA in the form of [M + 2Cl – OH –
OCH3]+ (Figure 6). This is the complete binuclear cop-
per(II) complex with a coordinated catecholate. However,
an exchange of hydroxide and methoxide with chloride
occurred during the measurement, for which dichlorometh-
ane was used as solvent. Specifically, the bridging OH
ligand was replaced by a μ-chlorido ligand and the OCH3-
group of the methyl ester function of the catecholate was
substituted by chloride (see Figure 6 and Scheme 6).
{HRMS (ESI; CH2Cl2, CH3OH): m/z = 653.023 [M + 2Cl –
OH – OCH3]+; calcd for C27H29Cl2Cu2N4O3: m/z =
635.020}.
Scheme 6. Formation of the complex [M + 2Cl – OH – OCH3]+
during the MS (ESI) measurements of the intermediate 4MeBA; HCl
derives from the solvent dichloromethane.
Isolation and mass-spectrometric investigation of the
catecholato adduct was also performed for the electron-rich
substrate DTBP-H. However, in this case, the MS data were
Figure 7. UV/Vis spectra of the intermediates (a) 4DTBP and (b)
less clear.
4MeBA in dichloromethane before and after HCl quench (l =
To further confirm that both substrates (DTBP-H and
10 mm); OPh = DTBP (a) and MeBA-O (b). Left scales refer to
MeBA-OH) are coordinated as catecholate in the respective the initial, right scales refer to the final spectra.
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the known absorption bands of the catecholato species to synthesize the bis-μ-hydroxido-copper(II) complex
(cf. Figures 4 and 5, blue lines). After quenching, the CuLpy2-OH independently (see Exp. Sect.)[29] and charac-
characteristic absorbances of DTBQ (407 nm) or semi- terize it by HRMS (ESI) analysis (for the full spectrum see
quinone MeBASQ were detected (see Figure 7).[23,24] By the Supporting Information, Figure S5). The simulated and
addition of HCl, coordinated 3,5-di-tert-butyl catecholate measured isotope patterns show very good agreement (Fig-
(DTBC) is therefore released as o-quinone DTBQ, whereas ure 8).
in the case of MeBA-OH, only the semiquinone is formed.
Deactivation of the Catalytically Active Complex
Deactivation of the catalytic system is evident, inter alia,
from an intense blue coloration of the oxygenation mixture,
which occurs in the Lpy1 and Lbzm1 systems already after
15 h but for the Lpy2 model is only clearly visible after
two days. With respect to the formation of this decay prod-
uct, the Lpy2 system thus appears to be more stable than
its Lpy1 counterpart. The blue species shows a strong ab-
sorbance at 589 nm with an ε-value of approximately
1000 Lmol–1 cm–1 (see the Supporting Information, Figure
S4).
The blue coloration of the reaction mixture and the
deactivation of the catalytically active complex go hand in
Figure 8. Segment of the HRMS (ESI) of CuLpy2-OH: simulated
and measured isotope pattern of the complex [M + 2H]+ as shown
top right.
hand. To obtain more information about these processes,
two possible inactive complexes that could be formed in the
oxygenation mixture were explored. One possibility is the
formation of a stable bis-μ-hydroxido dicopper(II) complex
through reaction with water, which accumulates in solution
as the oxygenation proceeds (see Scheme 7). It was possible
Solutions with different concentrations of the complex
CuLpy2-OH in acetone were prepared and UV/Vis spectra
were measured (see the Supporting Information, Figure
S6). They show an absorption at 611 nm, but with an
extinction coefficient (ε = 72 Lmol–1 cm–1) that is too small
in comparison with the blue species. Nevertheless, the bis-
μ-hydroxido-copper(II) complex CuLpy2-OH could be de-
tected by ESI mass spectrometry in the blue oxygenation
mixture of the catalytic oxygenation after two days.
The other possible deactivation scenario involves the for-
mation of a copper(II) semiquinone complex. It is known
that o-quinones and, especially, DTBQ form these com-
plexes in the presence of copper(I) species and chelate li-
gands.[30–34] We were able to generate a blue semiquinone
copper(II) complex with the binucleating ligand Lpy2 di-
rectly in solution and detect it in situ by UV/Vis spec-
troscopy. For this experiment, a solution of Cu2Lpy2
(5 mM, 13.6 mg, 15.0 μmol) in tetrahydrofuran (3 mL) was
prepared under anaerobic conditions. Upon addition of
DTBQ (6.60 mg, 30.0 μmol), the light-yellow solution
immediately became dark-blue. The solution was diluted to
record UV/Vis spectra (see the Supporting Information,
Figure S7). They show an intense absorption at 736 nm
with an extinction coefficient of ε = 1255 Lmol–1 cm–1 that
is comparable to the ε-value of the blue species. However,
the wavelength of the absorption band is more than 100 nm
too high. The oxygenation mixture contains many species
that may act as additional ligands to further stabilize a
copper(II) semiquinone complex. At present, we cannot
determine the exact identity of the copper(II) semiquinone
complex, but the above experiment shows that its formation
is possible under the respective conditions and, in fact, may
Scheme 7. Influence of water on the catalytic cycle and formation
of the inactive CuLpy2-OH complex during oxygenation.
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cause the intense blue coloration of the reaction mixture
after long reaction times.[30]
These studies were complemented by investigations of
the deactivation mechanisms of the employed catalyst to
explain the limitation of the TON. In this regard, we were
able to obtain a high-resolution ESI mass spectrum of the
Lpy2 bis-μ-hydroxido copper(II) complex, which may be
one of the dead-end species of the catalytic system. Another
possibility is the formation of a copper(II) semiquinone
complex, which accounts for the intense-blue coloration of
the reaction mixture after prolonged reaction times. With
respect to this deactivation mechanism, the Lpy2 system
appears to be more stable than its Lpy1 analogue, although
this apparently does not lead to a higher turnover number.
Conclusions
Based on the binucleating ligand Lpy2, the synthesis of a
new model system of tyrosinase has been achieved. Cata-
lytic oxygenation of DTBP-H is mediated with a turnover
number that is similar to that obtained with the mono-
nuclear Lpy1 model (Scheme 8).[15,16] Bridging of the pyr-
idine-imine units by the flexible alkyl chain therefore does
not hinder the catalytic monophenolase activity. On the
contrary, the Lpy2 system is faster at the beginning of oxy-
genation than its Lpy1 counterpart, and the unphysiological
free-radical side-reaction, which leads to the biphenolic
C–C coupling product, is significantly reduced.
Experimental Section
Materials and Techniques: All reagents were used as received from
Sigma–Aldrich and Abcr GmbH & Co. KG. Solvents were all used
as reagent grades. Dichloromethane, toluene, and acetonitrile were
purified further by heating to reflux over calcium hydride and
distilled under nitrogen atmosphere. Diethyl ether and tetra-
hydrofuran were purified by heating to reflux over lithium-
aluminum hydride and methanol over magnesium methoxide and
distilled under nitrogen atmosphere. Elemental analyses were per-
formed with a Euro Vector CHNS-O-element analyzer (Euro EA
3000) and the samples were burned in sealed tin containers in an
oxygen stream. The content of chloride was determined according
to Schöniger[36] with a potentiograph E536 from Metrohm using
ion selective electrodes. The NMR spectra were recorded at 300 K
with a Bruker Avance 400 Pulse Fourier Transform spectrometer
operating at a 1H frequency of 400.13 MHz and a 13C frequency
of 100.62 MHz and were referenced on residual protons of the
solvent. Optical absorption spectra were recorded in solution with
an Agilent Technologies 8453 UV/Vis spectrophotometer (l = 1 mm
and l = 10 mm). High-resolution (ESI) mass spectra were measured
Scheme 8. Comparison of the Lpy1 und Lpy2 systems.
with an APEX
Daltonics (7.05 T magnet).
3 FT-ICR mass spectrometer from Bruker
Besides the standard substrate DTBP-H, it was also
possible to catalytically oxygenate the electron-poor sub-
strate MeBA-OH by using the Lpy2 system. In this case,
however, the corresponding semiquinone MeBASQ is ob-
tained.
The bridging alkyl spacer stabilizes the intermediates
without changing the mechanism. In this respect, the Lpy1
and Lpy2 pair of ligand systems corresponds to, for
example, the L-6 and L-66 ligands of Casella and co-
workers.[35] As for the Lpy1 system, the relevant intermedi-
Hexane-1,6-dial: suspension of sodium periodates (1.50 g,
A
7.01 mmol) in a mixture of toluene (60 mL) and distilled water
(8 mL) was stirred for 15 min at room temperature. Cyclohexene
oxide (310 mg, 3.16 mmol) was added and the reaction mixture was
stirred for 4 d at room temperature. The resulting solid was filtered
off and the solution was allowed to stand for 2 d. The formed solid
was filtered off again, the phases were separated, and the aqueous
phase was extracted with toluene (2 ϫ 20 mL). The combined or-
ganic phases were washed with distilled water (20 mL) and with
ates of the Lpy2 catalytic cycle could be generated in a step- brine (20 mL). After drying over magnesium sulfate, the solvents
were removed under reduced pressure to give a light-yellow oil,
which crystallized after a few weeks to a colorless solid, yield
103 mg (29%); 1H NMR (400 MHz, CDCl3): δ = 9.73 (t, 2 H,
-CHO), 2.44 (m, 4 H, CH2CHO), 1.62 (m, 4 H,
CH2CH2CHO) ppm. 13C NMR (100 MHz, CDCl3): δ = 201.84
(CH, 2 C, CHO), 43.53 (CH2, 2 C, CH2CHO), 21.46 (CH2, 2 C,
CH2CH2CHO) ppm; C6H10O2 (114.14): calcd. C 63.14, 8.83, 0.00;
found C 63.75, H 8.65, N 0.00.
wise fashion and detected by UV/Vis spectroscopy, showing
defined spectral changes, especially when using MeBA-OH
as substrate. Moreover, the copper(I) phenolato intermedi-
ates 2 were characterized by NMR spectroscopy, and the
catecholato intermediate 4MeBA was detected by high-
resolution ESI mass spectrometry. Unfortunately, we were
unable to detect the side-on-peroxo bridged intermediates
3, also at low temperatures, but it could be shown by means
of kinetic experiments that no intermolecular peroxo
species are formed.
1,6-Bis[(2-pyrid-2-yl)iminoethylene]hexane (Lpy2): Hexane-1,6-dial
(172 mg, 1.51 mmol) was filled into a Schlenk flask fitted with a
Dean–Stark trap and flushed with nitrogen for 30 min. The
aldehyde was diluted in anhydrous toluene (40 mL) and 2-(pyrid-
2-yl)ethylamine (573 mg, 4.69 mmol) was added dropwise. The
mixture was stirred and heated to reflux with removal of water for
6 h. After cooling to room temperature, the solution was decanted
By HCl quenching, the bound catecholates of com-
pounds 4DTBP and 4MeBA could be released, leading to
quinone for the electron-rich substrate DTBP-H and semi-
quinone for the electron-poor substrate MeBA-OH.
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from the residue, which was discarded. The solvent was removed
immediately under reduced pressure to obtain a dark brown oil,
yield 475 mg (97%). 1H NMR (400 MHz, CDCl3): δ = 8.46 (dq,
2 H, Py H-6), 7.53 (td, 2 H, Py H-4), 7.10–7.04 (m, 6 H, imine H,
atmosphere by dilution of Cu2Lpy2 (5.65 mg, 6.25 μmol) in di-
chloromethane (25 mL). An aliquot (3 mL) of this solution was
taken and placed in a cuvette (10 mm) and a UV/Vis spectrum was
measured (Figure 4, black line). The remaining solution contained
Py H-3, Py H-5), 3.05 (t, 4 H, Py-CH2-CH2), 2.86 (t, 4 H, Py-CH2), 4.97 mg (5.50 μmol) of Cu2Lpy2 in 22 mL of dichloromethane. To
2.04 (m, 4 H, imine-CH2), 1.18 (m, 4 H, imine-CH2-CH2) ppm. this solution was added triethylamine (11.1 mg, 11.0 μmol) and Na-
13C NMR (100 MHz, CDCl3): δ = 184.36 (CH, 2 C, imine C), DTBP (2.51 mg, 11.0 μmol) under a nitrogen atmosphere, and an
159.04 (Cq, 2 C, Py C-2), 148.36 (CH, 2 C, Py C-5), 135.35 (CH, aliquot (3 mL) was again placed in a cuvette (10 mm) and a UV/
2 C, Py C-4), 122.39 (CH, 2 C, Py C-3), 120.27 (CH, 2 C, Py C-5), Vis spectrum was measured (Figure 4, green line). Molecular oxy-
40.72 and 40.82 (CH2, 2 C, imine-CH2), 38.79 (CH2, 2 C, Py-CH2), gen was then introduced into the remaining solution (19 mL) and,
29.49 (CH2, 2 C, aldehyde-CH2), 28.68 (CH2, 2 C, aldehyde-CH2- after 30 min, another UV/Vis spectrum was recorded (Figure 4,
CH2) ppm; C20H26N4 (322.45); calcd. C 74.50, H 8.13, N 17.38; blue line). Subsequently, the catalytic conditions were re-established
found C 74.32, H 8.27, N 16.91.
by addition of DTBP-H (98.0 mg, 475 μmol) and NEt3 (96.1 mg,
950 μmol), followed by further oxygenation. After 60 min, a UV/
Vis spectrum was measured (Figure 4, red line).
[Cu2Lpy2(CH3CN)4](PF6)2 (Cu2Lpy2): Under Schlenk conditions, a
solution of tetrakis(acetonitrile)copper(I) hexafluorophosphate
(187 mg, 504 μmol) in acetonitrile (10 mL) was added dropwise to
a solution of Lpy2 (85.6 mg, 255 μmol) in acetonitrile (10 mL) and
the mixture was stirred for 30 min at room temperature. The re-
sulting residue was collected by filtration trough a Schlenk frit and
dried under reduced pressure to give a light-brown solid, yield
Stoichiometric Oxygenation of Cu2Lpy2 with the Substrate MeBA-
OH: A solution of Cu2Lpy2 (250 μM) was prepared under a nitro-
gen atmosphere by dilution of Cu2Lpy2 (5.65 mg, 6.25 μmol) in
dichloromethane (25 mL). An aliquot (3 mL) of this solution was
taken and placed in a cuvette (10 mm) and a UV/Vis spectrum was
measured (Figure 5, black line). The remaining solution contained
4.97 mg (5.50 μmol) of Cu2Lpy2 in 22 mL of dichloromethane. To
this solution was added triethylamine (22.2 mg, 22.0 μmol) and
MeBA-OH (1.67 mg, 11.0 μmol) under a nitrogen atmosphere, and
an aliquot (3 mL) was again placed in a cuvette (10 mm) and a
UV/Vis spectrum was measured (Figure 5, green line). Molecular
oxygen was then introduced into the remaining solution (19 mL)
and, after 30 min, another UV/Vis spectrum was recorded (Fig-
ure 5, blue line). Subsequently, the catalytic conditions were re-es-
tablished by addition of MeBA-OH (72.3 mg, 475 μmol) and NEt3
(96.1 mg, 950 μmol), followed by further oxygenation. After
60 min, a UV/Vis spectrum was measured (Figure 5, red line).
1
185 mg (79%); H NMR (400 MHz, CDCl3): δ = 8.47 (br. s, 2 H,
Py H-6), 7.95 (t, 2 H, Py H-4), 7.55–7.42 (m, 6 H, imine H, Py H-
3, Py H-5), 3.73 (br. s, 4 H, Py-CH2-CH2), 3.28 (br. s, 4 H, Py-
CH2), 2.24 (s, 12 H, CH3CN), 2.05 (br. s, 4 H, imine-CH2), 1.25
(br. s, 4 H, imine-CH2-CH2) ppm. 13C NMR (100 MHz, CDCl3): δ
= 183.99 (CH, 2 C, imine C), 160.42 (Cq, 2 C, Py C-2), 149.07 (CH,
2 C, Py C-5), 139.10 (CH, 2 C, Py C-4), 124.98 (CH, 2 C, Py C-3),
123.19 (CH, 2 C, Py C-5), 119.90 (Cq, 2 C, CN), 67.17 (CH2, 2 C,
imine-CH2), 50.38 (CH2, 2 C, Py-CH2), 37.21 (CH2, 2 C, aldehyde-
CH2), 25.27 (CH2, 2 C, aldehyde-CH2-CH2), 0.83 (CH3, 2 C,
CH3CN) ppm.C28H38Cu2F12N8P2 (903.68): calcd. C 37.21, H 4.24,
N 12.40; found C 37.03, H 4.37, N 12.49.
[Cu2Lpy2(DTBP)2(NEt3)2]·2 NaPF6 (2): A solution of triethylamine
(22.3 mg, 220 μmol) and Na-DTBP (50.3 mg, 220 μmol) in di-
chloromethane (15 mL) was added under Schlenk conditions to a
solution of Cu2Lpy2 (95.0 mg, 110 μmol) in dichloromethane
(10 mL). The reaction mixture was stirred under nitrogen for
30 min at room temperature. The resulting residue was collected by
filtration trough a Schlenk frit und dried under reduced pressure
Kinetic Measurements: Concentration dependence of the Catalyst
Cu2Lpy2 on the product formation was measured as follows. Solu-
tions with different concentrations of Cu2Lpy2 in dichloromethane
(10 mL) were prepared under a nitrogen atmosphere (500, 250, 125,
and 50 μM). A mixture of DTBP-H (100 equiv.) and triethylamine
(200 equiv.) was added to each of the solutions with different con-
centrations of Cu2Lpy2 in dichloromethane under anaerobic condi-
tions, and then molecular oxygen was bubbled through the solu-
tion. The catalytic formation of DTBQ was detected by the evol-
ution of the 407 nm absorption band by recording UV/Vis spec-
troscopy during the first 15 min of oxygenation (see the Supporting
Information, Figure S1).
1
to obtain a brown solid, yield 90 mg (58%). H NMR (400 MHz,
CDCl3): δ = 8.28 (d, 2 H, Py H-6), 8.01 (br. s, 2 H, imine H), 7.86
(t, 2 H, Py H-4), 7.49 (d, 2 H, Py H-3), 7.32 (m, 2 H, Py H-5), 7.23
(d, 2 H, dtbp H-3), 6.99 (dd, 2 H, dtbp H-5), 6.71 (d, 2 H, dtbp H-
6), 3.80 (t, 4 H, Py-CH2-CH2), 3.59 (m, 8 H, Py-CH2, imine-CH2),
3.28 (m, 4 H, imine-CH2-CH2), 3.45 (q, 12 H, triethylamine CH2),
1.75 (m, 18 H, triethylamine CH3) ppm.
Acknowledgments
[Cu2Lpy2(OH)2](ClO4)2 (CuLpy2-OH): Under Schlenk conditions,
a light-blue solution of copper(II)perchlorate hexahydrate (140 mg,
387 μmol) in methanol (3 mL) was added dropwise to a solution
of Lpy2 (61.0 mg, 189 μmol) in methanol (3 mL) and the mixture
was stirred for 30 min at room temperature. The reaction mixture
became dark-green and was overlaid with diethyl ether (30 mL) and
allowed to stand for 2 d at 5 °C. The precipitated light-brown solid
was filtered off and discarded. The filtrate was concentrated com-
pletely under reduced pressure to obtain a dark-green solid, yield
130 mg (100%). UV/Vis (acetone, 10 mm): λmax (ε, L·mol–1·cm–1)
= 364, 405, 611 (72) nm. HRMS (ESI; CH2Cl2, CH3OH): m/z calcd
for C20H30Cu2N4O2 [M + 2H]+ 484.096; found: 484.093 (intensity:
3.18·107); C20H28Cl2Cu2N4O10 (682.46): calcd. C 35.20, H 4.14, N
8.21, Cl 10.39; found C 35.03, H 4.37, N 7.79, Cl 9.95.
The authors would like to thank Prof. Dr. J. Grotemeyer for the
mass spectrometric measurements. The authors also thank the
Deutsche Forschungsgemeinschaft (DFG), CAU Kiel, and COST
CM1003 for financial support of this research.
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Received: January 13, 2015
Published Online: ᭿
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Tyrosinase Models
J. Schottenheim, C. Gernert, B. Herzigkeit,
J. Krahmer, F. Tuczek* ................... 1–12
A tyrosinase model based on the binucleat-
ing ligand Lpy2 was developed and charac-
terized. The ligand Lpy2 contains a combi-
nation of an imine and a pyridine function
in the sidearms that are bridged by a flex-
ible alkyl spacer. The Cu2Lpy2 complex ca-
talyzes the conversion of monophenol
DTBP-H into the o-quinone DTBQ (TON
= 18). An electron-poor substrate is con-
verted into the semiquinone.
Catalytic Models of Tyrosinase: Reactivity
Differences between Systems Based on
Mono- and Binucleating Ligands
Keywords: Bioinorganic chemistry
/
Metalloproteins / Enzyme models / En-
zyme catalysis / Copper
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