Tyrosinases in Organic Chemistry: A Versatile Tool for the α-Arylation of β-Dicarbonyl Compounds

Article abstract of DOI:10.1002/ejoc.201800188

A tyrosinase-mediated arylation towards a variety of different building blocks is presented. Utilizing phenol or simple substituted phenols, the corresponding quinones are synthesized in a two-step procedure by an enzyme-catalyzed oxidation (tyrosinase from Aspergillus oryzae). The activated intermediates undergo a 1,4-addition with selected β-dicarbonyl compounds. Starting from phenol, yields of isolated product for the hydroxylation-oxidation-arylation sequence range from 43–77 %, whereas substituted acceptors provided 9–55 %, only. Different substitution patterns on phenol revealed that electron donating functionalities are preferentially accepted to electron withdrawing ones, whereas ortho-substituted phenols are not accepted at all.

Full text of DOI:10.1002/ejoc.201800188

A Journal of
Accepted Article
Title: Tyrosinases in organic chemistry: a versatile tool for the α-
arylation of β-dicarbonyl compounds
Authors: Roxanne Krug, Dennis Schroeder, Jan Gebauer, Sanel Suljic,
Yuma Morimoto, Nobutaka Fujieda, Shinobu Itoh, and Joerg
Pietruszka
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To be cited as: Eur. J. Org. Chem. 10.1002/ejoc.201800188
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10.1002/ejoc.201800188
European Journal of Organic Chemistry
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Tyrosinases in organic chemistry: a versatile tool for the
α-arylation of β-dicarbonyl compounds
Roxanne Krug,[a] Dennis Schröder,[b] Jan Gebauer,[c] Sanel Suljić,[a] Yuma Morimoto,[c] Nobutaka
Fujieda,[c] Shinobu Itoh,[c] Jörg Pietruszka*[a,b]
Abstract: A tyrosinase-mediated arylation towards a variety of
different building blocks is presented. Utilizing phenol or simple
substituted phenols, the corresponding quinones are synthesized in a
two-step procedure by an enzyme-catalyzed oxidation (tyrosinase
from Aspergillus oryzae). The activated intermediates undergo a 1,4-
addition with selected β-dicarbonyl compounds. Starting from phenol,
yields of isolated product for the hydroxylation-oxidation-arylation
sequence range from 43-77%, while substituted acceptors provided
9-55%, only. Different substitution patterns on phenol revealed that
electron donating functionalities are preferentially accepted to
electron withdrawing ones, whereas ortho-substituted phenols are not
accepted at all.
dihydroxyphenylalanine (2) (L-DOPA) followed by its oxidation to
the corresponding o-dopaquinone (3). Interestingly, later
investigations on the biosynthesis by Cánovas and co-workers
revealed a strong dependence on the pH whether the o-
dopaquinone (3) is transformed to melanin via dopachrome (4)
intermediates (under physiological pH values) or if it proceeds via
a p-topaquinone (5) intermediate (pH < 4). Thereby, the synthesis
of o-dopaquinone (3) is irrespective of the pH value.[5]
Introduction
Tyrosinases, alongside with laccases, belong to the enzyme class
of oxidoreductases. These type III binuclear copper oxidases are
present in all domains of life, e.g., in fungi, plants and bacteria.[1]
The fact that they use molecular oxygen for their oxidation on
phenolic compounds accompanied with the release of water
makes them an interesting and versatile tool as catalysts in
organic chemistry. In nature, tyrosinases are responsible for
browning of fruits and vegetables and are essential for
melanisation. The latter comes along with UV protection, which is
also related to pathogenesis and defensive roles in
microorganisms.[2, 3] Based on the work of Raper, Mason and
Lerner,[4] the proposed melanin biosynthesis starts with the amino
acid tyrosine. Since tyrosinases - per definition - exhibit the
capability to perform a hydroxylation of phenolic compounds
Scheme 1. Proposed biochemical pathway towards melanin.
The fact that the tyrosinase is able to perform two subsequent
reactions turns them into an attractive tool also from a chemical
point of view, since oxidation of aromatic compounds using
molecular oxygen as only stoichiometric reagent would seem
ecologically beneficial.[6] More conventional alternatives have
been reported: Bogle and co-workers showed a few examples of
arylation reactions with a polymer supported periodate as oxidant
for substituted phenols.[7] Another example is given in the
synthesis of epicolactone in which the catechol derivatives are
oxidized by potassium ferrocyanide.[8] Moreover, the application
of electrochemical tools for the oxidation of catechols and
methylcatechols in presence of benzoylacetonitrile has recently
been reported as well.[9] As alternative, “green” oxidants, laccases
and tyrosinases have already found their way into the fields of
organic chemistry.[10, 11] Laccases are known to oxidize catechols
to quinones, which may act as Michael acceptor in 1,4-addition
reactions. For instance, Beifuss et al. provided an application of
the laccase from Agaricus bisporus to mediate the reaction of
diverse pyrazolones with catechols yielding benzofuranones.[11a]
Furthermore, Tozzi et al. presented a tyrosinase catalysed
reaction towards dibenzoazocanes.[11b] Relating to these
biocatalysts, our institute presented a variety of arylation
(phenolase activity) followed by
a subsequent oxidation
(catecholase activity), the initial steps of the melanin synthesis are
the
hydroxylation
of
L-tyrosine
(1)
to
L-3,4-
[a]
[b]
MSc. R. Krug, MSc. J. Gebauer, Dr. S. Suljić, Prof. Dr. J. Pietruszka
Institut für Bioorganische Chemie, Heinrich-Heine Universität
Düsseldorf located at the Forschungszentrum Jülich
Prof. Dr. J. Pietruszka, D. Schröder
Institut für Bio- und Geowissenschaften (IBG-1: Biotechnologie),
Forschungszentrum Jülich
52425 Jülich, Germany
E-mail: j.pietruszka@fz-juelich.de
http://www.iboc.uni-duesseldorf.de/
[c]
Prof. Dr. S. Itoh, Dr. Y. Morimoto, Dr. N. Fujieda
Department of Material and Life Science, Division of Advanced
Science and Biotechnology
Graduate School of Engineering, Osaka University
2-1 Yamada-oka, Suita, Osaka 565-0871, Japan
Supporting information for this article is given via a link at the end of
the document.((Please delete this text if not appropriate))
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10.1002/ejoc.201800188
European Journal of Organic Chemistry
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reactions with different laccases.[12] The current paper presents
an update on the latter mentioned laccase mediated arylation
reactions being subject in several publications in the past few
years.[12] The thereby synthesised products, possessing a
the ethyl ester 6b did not exceed the 55% (Scheme 2, entry 1).
The same is true for the tert-butyl ester 6c (51%, Scheme 2, entry
1). In a next step, indanone esters 6d and 6e were investigated.
The tert-butyl derivative 6e gave 43% yield of compound 8e
whereas the ethyl ester 6d resulted in the formation of arylated
product 8d in moderate yield (61%; Scheme 2, entry2).
quaternary stereogenic center, represent building blocks for a
13]
variety of biologically interesting compounds.[12,
Instead of
laccases and catechols, we herewith present an alternative
starting from phenols as precursor by using the purified (trypsin
activated) fungal tyrosinase melB from Aspergillus oryzae.[14] The
advantage is obvious, since the tyrosinase can perform the
necessary hydroxylation towards the catechols and subsequently
perform the oxidation to the desired o-quinones, which undergo
the previously mentioned 1,4-addition reaction. With this
biocatalyst in hand, it is possible to start from readily available and
cheap precursors. Compared to the laccase, we are able to
perform two steps instead of one. We hereby present our recent
results on tyrosinase mediated arylation reactions, touching the
subject of substrate scope and limitations.
Results and Discussion
In order to establish the application of the tyrosinase from
A. oryzae for the presented arylation, we chose the commercially
available cyclopentanone carboxylate 6a (R = OMe; Scheme 2)
and phenol (7a) as substrates for initial studies and optimizations
of the reaction conditions. The following basics of the published
reaction conditions[12] for laccases were applied for comparison:
a) Performance of the reaction at room temperature and
maximum duration of three days. b) The use of acetonitrile as co-
solvent and buffer was adopted (here in a ratio of 1:1.1 instead of
the published 1:2 ratio and a change of KPi to Tris-HCl buffer).
Since the initial experiments with the tyrosinase (43 U/mL)
resulted in a full conversion of the ester 6a into the product 8a,
this volumetric activity was kept for all further experiments. In case
of the exemplarily chosen cyclopentanone methyl ester 6a the
maximum duration of three days resulted in a decrease of product
yield and presumably increase of polymerization products.
Interestingly, this compound seems to be far more reactive than
the other products. Already after 3.5 h full conversion was
achieved and 65% of the desired product 8a could be isolated
after purification via silica chromatography (Scheme 2, entry 1).
This result depends also on the applied equivalents of the starting
materials. Initial experiments with equimolar ratios of both starting
materials or in case of an excess of the Michael donor 6 did not
lead to full conversion. Not until the phenol (7a) was used in
excess, the desired product yields could be accomplished.
However, the yield of isolated product could not be increased
further, presumably due to the formation of unknown polymeric
product visible at the baseline of the TLC and column
chromatography, respectively. Confident that the established
reaction conditions are adequate for the tyrosinase, since the
published arylation product 8a was obtained in the same yield of
66% with the laccase from P. ostreatus,[12a] further variations on
the donor molecule were investigated (Scheme 2). Unfortunately,
changes on the ester functionality of the cylopentanone derivative
resulted in a slight decrease in yield. Despite several attempts,
Scheme 2: Tyrosinase-mediated 1,4-additions of donor substrates 6a-6i to
phenol (7a) providing highly substituted compounds 8a-i [a) full conversion of
the donor after 3.5 h].
Changing to isocoumarins 6f and coumarins 6g,h as donors did
also proof possible and the desired products 8f-h could be
isolated in 70-77% yield, respectively (Scheme 2, entry 3 and 4).
This is in accordance with the published results using the laccase
from A. bisporus for the corresponding oxidation-arylation-
sequence using catechols (63-71% yield).[12d] Last, also
conversion of oxindole 6i was shown to be a suitable starting
material and resulted in 53% yield of the desired arylation product
8i along with the formation of structurally unassigned side-
products (Scheme 2, entry 5).
Next to these initial experiments, comparative experiments in
cases of substrate 6b were performed in the presence of catechol
9a, to investigate whether the tyrosinase accepts preferentially
the phenol or catechol (Scheme 3). Furthermore, the effect of an
additional amount of H2O2 as well as a change in buffer were
tested.
Additional comparative kinetics emphasize the experimental
results revealing that the tyrosinase from A. oryzae shows a
higher reactivity towards catechol 9a than in presence of phenol
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10.1002/ejoc.201800188
European Journal of Organic Chemistry
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7a as substrate, demonstrated by the over 500 times higher
turnover number kcat (Fig. 1, A). Likewise, the application of
catechol 9a in the arylation reaction resulted in an increase of
yield about 16% (Scheme 3, entry 3) compared to the application
of phenol (Scheme 3, entry 1). Furthermore, the KM-value in
MeCN and Tris-HCl (1:1), when catechol 9a is the substrate, is
approximately 30 times higher than in presence of the phenol 7a.
Thus, demonstrating that the phenol is indeed rather accepted by
the enzyme, presumably due to its similarity towards the natural
substrate, but catechol is more readily oxidized.
The lower catalytic efficiency in case of phenol 7a emphasizes the
assumption that the hydroxylation is the rate-determining step in
the reaction sequence. Consequentially, the oxidation of catechol
9a towards the corresponding o-quinone is preferred. With regard
to the kinetics in KPi-buffer systems one can see similar relations
(Fig. 1, B; for further details on the kinetics, see supporting
information). Remarkably, the oxidation of the catechol is even
faster in KPi-buffer, leading to the assumption that the Tris-HCl
buffer might inhibit the reaction.[15] Despite this known fact, the
choice of buffer does not affect the outcome in yield of the desired
arylation products.
In a last step, after variation of the donors, the substrate
acceptance of the tyrosinase concerning different phenolic
acceptors was investigated. Therefore, substituted phenols 7b-f
were used, bearing either electron withdrawing or donating
groups (Scheme 3). The electronic effects have a distinct impact
on the catalyzed reactions. The tyrosinase from A. oryzae accepts
electron-donating groups on the phenolic derivatives rather than
electron-withdrawing ones. In general, it becomes clear that the
introduction of a substituent, irrespective of its position, comes
along with a decrease of product formation. Electron-withdrawing
groups, such as a nitro- (7c) or a trifluoromethyl (7d) group in
ortho, meta, or para position, did not result in any product
formation. Only the simple fluorinated derivative o-, m- and p-7b
showed traces of a product 8j, but with the low yield of the crude
product (~1-2%) it was not worth investigating or analysing it in
more detail (Scheme 3). In order to verify, whether it is a single
fact of our tyrosinase from A. oryzae, which does not accept these
compounds, the commercially available mushroom tyrosinase
(from Sigma Aldrich[16]) was tested and did also not show any
conversion of one of the above mentioned substrates (7b-d)
bearing electron-withdrawing groups.
Scheme 3: Comparative experiments towards arylated product 8b using
either phenol (7a), o-catechol (9a) and/or H2O2 as additive
Figure 1: Michaelis-Menten kinetics of the tyrosinase from A. oryzae [A in
presence of phenol 7a (grey) or catechol 9a (black) as substrate; in a 1:1 ratio
of MeCN and Tris*HCl buffer; B same kinetics in MeCN and KPi-buffer (1:1).
For comparison, specific activities for phenol 7a and catechol 9a have been
normalized to the specific activity for the natural substrate L-tyrosine; further
comparative kinetics between commercially available and purified tyrosinase
are shown in the supporting information]
Scheme 4: Oxidation and arylation of cyclopentanone 6a with phenols 7b-7f.
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10.1002/ejoc.201800188
European Journal of Organic Chemistry
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ceric ammonium molybdate solution followed by heating. NMR spectra
were recorded on a Bruker Advance/DRX 600 instrument (1H at 600 MHz;
13C at 151 MHz). Chemical shifts (δ) are reported relative to deuterated
chloroform (1H: 7.26 ppm; 13C: 77.2 ppm) or acetone (1H: 2.05 ppm; 13C:
In contrast, electron-donating groups, e.g., such as in cresols (7e)
and methoxy phenols 7f showed results that are more promising.
Although the ortho position is still not favored and thus does not
lead to any detectable products o-8m and o-8n, the methylated
phenolic compounds m-7e and p-7e could be transformed into the
desired product m-8m successfully. In comparison to the meta-
substrate m-7e, phenol p-7e gave higher yields (26%, Scheme 4).
It is worth emphasizing that m-7e and p-7e (same for m-7f and p-
7f) resulted in the same regioisomeric product. Consequently, the
conversion of the starting materials differs because of the
substitution pattern, thus resulting in different yields of arylation
product. For the methoxy-compounds, again the para-substituted
phenol p-7f gave the highest yield (55%) of product m-8n.
29.8 ppm). Infrared spectra were recorded using
a Perkin–Elmer
SpectrumOne IR spectrometer. Melting points were measured with a
Büchi melting point B-540 device.
Starting materials
The starting materials 6a and 6b are commercially available. Compound
6f has been prepared as reported previously[12b] starting from the
commercially available chromone-3-carboxylic acid. In a second step,
compound 6f was reduced according to the published method using the
H-Cube from Thales Nano. The same procedure was applied in case of
compounds 6g-h,[12c] starting from the commercially available ethyl
coumarin-3-carboxylate and 3-acetylcoumarin.
Conclusions
Synthesis of tert-butyl 2-oxocyclopentane-1-carboxylate (6c): i)
According to Bunnage et al.[18] of adipoyl chloride (600 mg, 3.3 mmol, 1 eq)
in Et2O (1.3 mL) was added dropwise to a stirred solution of tert-butanol
(790 mg, 10.7 mmol, 3.25 eq) and N,N-dimethylaniline (1.25 g, 10.3 mmol,
3.15 eq) in Et2O (2.5 mL) at 0 °C. After complete addition, the reaction was
stirred at room temperature for 16 h. Afterwards the reaction mixture was
diluted with H2O and the organic phase was extracted five times with a 3 M
HCl solution, two times with NaHCO3 and once with brine. It was dried
(MgSO4) and purified via column chromatography. The desired product
(colourless oil) was isolated in 70% yield (590 mg, 2.3 mmol). Rf (80/20
PE/EtOAc): 0.62. 1H-NMR (600 MHz, CDCl3) [ppm] δ 1.44 [s, 18 H, 2
C(CH3)3], 1.60 (m, 4 H), 2.22 (mc, 4 H). ii) NaH (31 mg, 60% oil
suspension) is stirred in toluene (1.5 mL) and heated to 60 °C. To the
suspension, 8 mg (total amount: 200 mg, 0.8 mmol) of the di-tert-
butyladipoyl ester from i) were added and stirred for 30 min. The remaining
starting material was added and the reaction mixture heated for 3 h at
100 °C. Compound 6c could be isolated in 64% yield (90 mg, 0.5 mmol).
Rf (80/20 PE/EtOAc): 0.50. 1H-NMR (600 MHz, CDCl3) [ppm] δ 1.46 [s, 9
H, (CCH3)3], 1.79-1.87 (m, 1 H, 3-H or 4-H), 2.07-2.13 (m, 1 H, 3-H or 4-
H), 2.22-2.29 (m, 4 H, 5-H + 3-H or 4-H), 3.03 (t, J = 8.6 Hz, 1 H, 1-H). 13C-
NMR (151 MHz, CDCl3) [ppm] δ 21.1 (C-3 or C-4), 27.6 (C-5), 28.2
[C(CH3)3], 38.2 (C-4 or C-3), 55.9 (C-1), 81.8 [C(CH3)3], 168.9 (C-1‘ or C-
2‘), 213.0 (C-2‘ or C-1‘). HRMS: m/z = 183.1028 [M-H]+, calculated for
To sum up, we successfully established a tyrosinase-initiated
(using purified and trypsin activated tyrosinase from A. oryzae)
one-pot hydroxylation-oxidation-arylation sequence forming
products (8a-8i), all possessing a quaternary carbon atom.[12]
HPLC measurement of 8a revealed no selectivity coming from the
tyrosinase (chromatogram see supporting information).
Introducing substituted phenolic compounds revealed that the
tyrosinase from A. oryzae is able to convert phenols with electron
donating substituents (OMe, Me) rather than electron withdrawing
ones (F, NO2, CF3). In both cases the ortho-substituted phenols
are not accepted. This might be due to steric hindrance in the
active site of the tyrosinase. Since the phenolic compounds have
to enter the active site in case of the tyrosinase to reach the
binuclear copper center the substituents in ortho position might
have a steric influence. The – albeit limited - acceptance of the
fluorinated substrate o-7b appeared to be an exception. However,
since fluorine is obviously relatively small, it might fit better into
the pocket. This fact is different to laccases, which lack the
phenolase activity, and they are tunnelling the electrons from the
substrates to the corresponding copper atoms utilizing the
oxidase (catecholase) activity.[17] The enzymatic kinetics revealed
that the hydroxylation is the rate-determining step in the reaction
sequence towards the arylation products. Therefore, the present
tyrosinase can be also considered as alternative oxidizing
enzyme, since catechols are converted in a higher catalytic
efficiency. Mild conditions and low-cost as well as readily
accessible starting materials makes this procedure an appealing
alternative to more conventional methods.
-
C10H15O3 : 183.1027. All analytical data are in full agreement to those
published.[18]
Synthesis of ethyl 1-oxo-2,3-dihydro-1H-indene-2-carboxylate (6d):
Referring to a protocol of Brown et al.,[19] indanone (1.03 g, 7.8 mmol) in
diethyl carbonate (27.6 g, 234 mmol, 30 eq) was added to a suspension of
NaH (623 mg, 15.6 mmol, 60% suspension in oil, 2 eq) in diethyl carbonate
(27.62 g, 234 mmol, 30 eq) via syringe. The mixture was heated to reflux
for 5 min (formation of a green solid). Another 12.5 mL of diethyl carbonate
was added and heated to reflux for further 15 min. The mixture was
allowed to cool to room temperature before it was acidified with 1 M HCl
until pH 1. The mixture was extracted four times with EtOAc and the
organic phases dried over MgSO4. Purification occurred via column
chromatography (90/10 PE/EtOAc). Compound 6d could be isolated as
brown oil in 53% yield (812 mg, 4.0 mmol). Rf (80/20 PE/EtOAc): 0.52. 1H-
NMR (600 MHz, CDCl3) [ppm] δ 1.31 (t, J = 7.1 Hz, 3 H, 3‘-H), 3.38 (dd, J
= 17.2, 8.2 Hz, 1 H, 3-Ha), 3.56 (dd, J = 17.2, 4.1 Hz, 1 H, 3-Hb), 3.71 (dd,
J = 8.3, 4.1 Hz, 1 H, H-2), 4.25 (q, J = 7.2 Hz, 2 H, 2‘-H), 7.40 (dd, J =
7.7 Hz, 1 H, 6 or 7-H), 7.50 (d, J = 7.7 Hz, 1 H, 5 or 8-H), 7.62 (dd, J =
7.4 Hz, 1 H, 7 or 6-H), 7.78 (d, J = 7.7 Hz, 1 H, 8 or 5-H). 13C-NMR
(151 MHz, CDCl3) [ppm] δ 14.4 (C-3‘), 30.5 (C-3), 53.5 (C-2), 61.9 (C-2‘),
124.8 (C-5 or C-8), 126.7 (C-8 or C-5), 127.9 (C-6 or C-7), 135.4 (C-4a or
C-4b), 135.5 (C-7 or C-6) 153.8 (C-4b or C-4a), 169.3 (C-1‘), 199.7 (C-1).
Experimental Section
Chemicals and Methods
All chemicals used were purchased from Sigma Aldrich, Alfa Aesar, VWR
International/Merck and Fischer Scientific. The acetonitrile, used for the
enzymatic reactions, was purchased from Fischer Scientific as HPLC
grade. Further solvents were either distilled or taken from the solvent
purification system (MBraun, SPS-800). Thin layer chromatography (TLC)
was conducted on POLYGRAMÔ SIL G/UV254 plates with fluorescence
indicator. Detection occurred either by UV absorption or treatment with
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10.1002/ejoc.201800188
European Journal of Organic Chemistry
FULL PAPER
HRMS: m/z = 205.0859 [M+H]+, calculated for C12H13O3: 205.0859. All
H or Ph-H), 7.94 (d, J = 8.2 Hz, 1 H, Ar-H or Ph-H). 13C-NMR (151 MHz,
CDCl3) [ppm] δ 28.2 [C(CH3)3], 52.7 (C-3), 84.6 [C(CH3)3], 115.3 (C-Ar),
124.7 (C-Ar), 125.2 (C-Ar), 127.5 (C-4a/b or C-Ph), 128.0 (CH), 128.8 (C-
Ph), 129.1 (C-Ph), 136.4 (Cquart. C-4a/b or C-Ph), 140.6 (C-4a/b or C-Ph),
149.5 (C-1‘), 174.0 (C-2). IR ṽ 2982, 1768, 1725, 1608, 1479, 1464, 1394,
1370, 1346, 1288, 1252, 1202, 1146, 1089, 1048, 1024, 990, 845, 758,
723, 697, 675 cm-1. HRMS: m/z = 309.1438 [M+H]+, calculated for
C19H20NO3: 309.1438. All analytical data are in full agreement to those
published.[21]
analytical data are in full agreement to those published.[19]
Synthesis of tert-butyl 1-oxo-2,3-dihydro-1H-indene-2-2carboxylate
(6e): To the solution of indanone (300 mg, 2.3 mmol) in THF (2.3 mL),
109 mg of NaH (60% oil suspension, 4.5 mmol) in THF (9 mL) were added
at room temperature. The mixture was refluxed under the addition of
pyrrol-tert-butyl-carboxylate (775 mg, 4.5 mmol) in THF (1.1 mL). After
completion of the reaction, the mixture was cooled to 0 °C and quenched
with 1 N HCl. The solution was extracted four times with Et2O and washed
one with NaCl. Two column chromatography afforded 11% of the desired
product 6e (58 mg, 0.2 mmol). Rf (80/20 PE/EtOAc): 0.64. 1H-NMR
(600 MHz, CDCl3) [ppm] δ 1.49 [s, 9 H, C(CH3)3], 3.33 (dd, J = 17.1, 8.2 Hz,
1 H, 3-Ha), 3.49 (dd, 17.2, 4.0 Hz, 1 H, 3-Hb), 3.62 (dd, J = 8.2, 4.0 Hz, 1
H, 2-H), 7.34 (dd, J = 7.4 Hz, 1 H, 6-H or 7-H), 7.49 (d, J = 7.7 Hz, 1 H, 5-
H or 8-H), 7.60 (dd, J = 7.4 Hz, 1 H, 7-H or 6-H), 7.75 (d, J = 7.7 Hz,1 H,
8-H or 5-H). 13C-NMR (151 MHz, CDCl3) [ppm] δ 28.15 [C(CH3)3], 30.46
(C-3), 54.40 (C-2), 82.17 [C(CH3)3], 124.68 (C-5 or C-8), 126.64 (C-8 or C-
5), 127.79 (C-6 or C-7), 135.33 (C-7 or C-6), 135.59 (C-4a or C-4b), 153.80
(C-4b or C-4a), 168.45 (C-1 or C-1’), 200.13 (C-1 or C-1’). All analytical
data are in full agreement to those published.[20]
Arylation products
The compounds 8a-n have been synthesized according to the following
general procedure. The analytical data of products 8a,b,e and m-8m[12a]
as well as 8f[12b] and 8g.[12c] are in full agreement to those published.
General procedure for the arylation reaction with the tyrosinase from
A. oryzae: In a 10 mL flask, 0.15 mmol of compound 6a-6i and 0.18 mmol
of phenol 7a-7f (1.2 eq) were diluted in 1.0 mL acetonitrile. 1100 µL of the
purified and activated enzyme solution in 10 mM Tris*HCl buffer pH 6
(43 U/mL; see activation of tyrosinase) were added to the reaction mixture
and stirred at room temperature up to three days. Afterwards, the reaction
mixture was acidified with a few drops of 1 M HCl, diluted with dH2O and
EtOAc and the aqueous phase was extracted four times with EtOAc. All
combined organic phases were washed once with sat. NaCl solution and
dried over MgSO4. Purification via silica column chromatography
(PE/EtOAc 70/30 – 80/20) resulted in the desired products 8a-8n.
Problems to remove solvent residues kept in the, to some extent, oily and
resinous products have been overcome with several “washing steps” with
deuterated chloroform.
Synthesis of tert-butyl 2-oxo-3-phenlindoline-1-carboxylate (6i): i) The
synthesis of 6i was performed according to a published procedure by
Hamashima et al.[21] To a solution of isatin (1.0 g, 6.8 mmol) in THF
(30.0 mL) at -40 °C, a solution of PhMgBr in THF (1 M, 13.6 mL) was added.
The mixture was allowed to warm to room temperature. The reaction
mixture was diluted with diethyl ether, cooled to 0 °C, and quenched with
1 M HCl. The aqueous phase was extracted four times with ether and the
combined organic layers were washed once with water and brine. Then,
dried over MgSO4. After removal of the solvent under reduced pressure,
the crude mixture was directly used in the second step. ii) The crude
mixture as obtained in i) (theoretical amount: 1.5 g, 10.2 mmol) was diluted
in CH2Cl2 (100 mL). After addition of DMAP (124 mg, 1.0 mmol) and
Boc2O (2.5 eq, 5.56 g, 25.0 mmol) the reaction was stirred for 3 h at room
temperature. The mixture was diluted with NH4Cl and CH2Cl2 and the
aqueous phase was extracted four times with CH2Cl2 and washed once
with brine. The organic phase was dried over MgSO4 and the solvent
evaporated under reduced pressure. The product (white, crystalline) was
isolated after two column chromatographic purification steps (PE/EtOAc
90/10) in 22% yield (648 mg, 1.5 mmol, over 2 steps). Rf (PE/EtOAc
70/30): 0.67. Mp: 135-136 °C. 1H-NMR (600 MHz, CDCl3) [ppm] δ 1.38 [s,
9 H, C(CH3)3], 1.61 [s, 9 H, C(CH3)3], 7.24 (t, J = 7.5 Hz, 1 H, 6-H or 7-H),
7.29-7.34 (m, 6 H, Ph + 5-H or 8-H), 7.45 (t, J = 7.9 Hz, 1 H, 7-H or 6-H),
7.98 (d, J = 8.2 Hz, 1 H, 8-H or 5-H). 13C-NMR (151 MHz, CDCl3) [ppm] δ
27.7 [C(CH3)3], 28.2 [C(CH3)3], 81.8 (C-3), 84.1 [C(CH3)3], 84.7 [C(CH3)3],
115.6 (C-5 or C-8), 124.2 (C-8 or C-5), 125.1 (C-6 or C-7), 126.9 (C-Ph),
127.5 (C-4a/b or C-Ph), 128.7 (C-Ph), 129.3 (C-Ph), 130.5 (C-7 or C-6),
136.1 (C-4a/b or C-Ph), 140.8 (C-4a/b or C-Ph), 149.3 (C-2 or C-1‘ or C1‘‘),
151.1 (C-2 or C-1‘ or C1‘‘), 171.7 (C-2 or C-1‘ or C1‘‘). IR ṽ 2981, 1799,
1747, 1730, 1611, 1471, 1396, 1371, 1346, 1314, 1284, 1247, 1147, 1113,
1101, 1087, 1036, 1003, 971, 940, 924, 907, 854, 839, 800, 771, 760, 744,
General procedure for the arylation reaction with the commercially
available tyrosinase (from mushroom): In a 10 mL flask, 0.15 mmol of
compound 6a and 0.18 mmol of the substituted phenol p-7b-7d (1.2 eq)
were diluted in 1.0 mL acetonitrile. After addition of 1.1 mL Tris*HCl buffer
(10 mM, pH 6), 0.17-0.28 mg (2687 U/mg) of the commercially available
tyrosinase[16] were added to the reaction mixture and stirred at room
temperature up to three days. Since no conversion was detected (TLC),
the reaction mixture was discarded.
tert-Butyl 1-(3,4-dihydroxyphenyl)-2-oxocyclopentane-1-carboxylate
(8c) was obtained as slightly yellow oil; 51% (22 mg, 75 µmol). Rf (70/30
PE/EtOAc): 0.17. 1H-NMR (600 MHz, CDCl3) [ppm] δ 1.42 [s, 9 H,
C(CH3)3], 1.90-2.01 (m, 2 H, 3-H, 4-H or 5-H), 2.29-2.35 (m, 1H, 3-H, 4-H
or 5-H), 2.44-2.50 (m, 2 H, 3-H, 4-H or 5-H), 2.75 (mc, 1 H, 3-H, 4-H or 5-
H), 5.55 (s, OH), 5.80 (s, OH), 6.77-5.80 (m, 2 H, Ar-H), 6.96 (s, 1 H, Ar-
H). 13C-NMR (151 MHz, CDCl3) [ppm] δ 19.5 (C-3, C-4 or C-5), 28.0
[C(CH3)3], 35.0 (C-3, C-4 or 5), 37.8 (CH2), 65.2 (C-1), 82.7 [C(CH3)3],
115.1 (C-Ar), 115.3 (C-Ar), 120.1 (C-Ar), 128.8 (C-6), 143.5 (C-8 or C-9),
143.5 (C-9 or C-8), 170.4 (C-2 or C-1‘), 213.0 (C-2 or C-1‘). IR ṽ 3414,
2957, 2927, 2856, 2358, 2339, 1724, 1602, 1457, 1369, 1258, 1151, 1124,
1074, 743. 309 cm-1. HRMS: m/z = 315.1203 [M+Na], calculated for
716, 693 cm-1. HRMS: m/z
=
448.1731 [M+Na]+, calculated for
+
C16H20O5Na: 315.1208; 293.1383 [M+H]+, calculated for C16H21O5
:
C24H27O6NNa+: 448.1731; 443.2180 [M+NH4]+, calculated for
C24H27O6NNH4+: 443.2177. iii) The product (150 mg, 0.4 mmol) from ii)
was dissolved in methanol (7.1 mL) and Pd/C (71 mg, 0.7 mmol) was
added to the solution. The mixture was stirred at room temperature under
a H2-atmosphere (balloon) for 5 h. Afterwards the reaction mixture was
filtrated over celite to remove the catalyst and washed with ether. After
removal of the solvent under reduced pressure, the desired product (white
solid) could be isolated via column chromatography in 57% yield (62 mg,
0.2 mmol). Rf (PE/EtOAc 70/30): 0.66. Mp: 102-106 °C. 1H-NMR (600 MHz,
CDCl3) [ppm] δ 1.64 [s, 9 H, C(CH3)3], 4.73 (s, 1 H, 3-H), 7.16-7.17 (m, 2
H, Ar-H or Ph-H), 7.19-7.21 (m, 2 H, Ar-H or Ph-H), 7.29-7.28 (m, 4 H, Ar-
293.1384.
Ethyl
2-(3,4-dihydroxyphenol)-1-oxo-2,3-dihydro-1H-indene-2-
carboxylate (8d) was obtained as colorless solid, 61% yield (29 mg,
92 µmol). Rf (70/30 PE/EtOAc): 0.10. Mp.: 142-143 °C. 1H-NMR (600 MHz,
Aceton-d6) [ppm] δ 1.15 (t, J = 7.1 Hz, 3 H, 3‘-H), 3.67 (d, J = 17.4 Hz, 1
H, 3-Ha), 4.07 (d, J = 17.4 Hz, 1 H, 3-Hb), 4.14 (q, J = 7.1 Hz, 2 H, 2‘-H),
6.76 (s, 2 H, Ar-H), 6.97 (s, 1 H, Ar-H), 7.47 (dd, J = 7.4 Hz, 1 H, 6-H or 7-
H), 7.62 (d, J = 7.8 Hz, 1 H, 5-H or 8-H), 7.70-7.74 (m, 2 H, Ar-H). 13C-
NMR (151 MHz, Aceton-d6) [ppm] δ 14.3 (C-3’), 41.1 (C-3), 62.1 (C-2’),
This article is protected by copyright. All rights reserved.

10.1002/ejoc.201800188
European Journal of Organic Chemistry
FULL PAPER
65.3 (C-2), 115.8 (C-Ar ), 116.1 (C-Ar ), 119.8 (C-Ar), 125.1 (C-5, C-6, C-
7 or C-8), 127.4 (C-5 or C-8), 128.8 (C-6 or C-7), 130.8 (C-4a/b or C-Ar),
135.9 (C-4a/b or C-Ar), 136.3 (C-5, C-6, C-7 or C-8), 145.4 (C-Ar), 145.6
(C-Ar), 153.1 (C-4a/b or C-Ar), 171.5 (C-1’), 200.7 (C-1). IR ṽ 3448, 3261,
2984, 1713, 1686, 1604, 1590, 1517, 1469, 1441, 1365, 1328, 1264, 1214,
1190, 1158, 1139, 1118, 1083, 1050, 1027, 1013, 957, 921, 881, 863, 845,
812, 788, 766, 742, 692, 678, 659 cm-1. HRMS: m/z = 313.1073 [M+H]+,
calculated for C18H17O5+: 313.1071.
Salts for the different buffer systems were purchased from VWR
Chemicals, AppliChem Panreac ITW Companies and Carl ROTH®.
Trypsin from bovine pancreas as well as trypsin inhibitor were purchased
from Sigma Aldrich. Donor organism for the used plasmid, containing the
desired tyrosinase, was Aspergillus oryzae. The accession number of the
tyrosinase gene is BD165761, DNA Data Bank of Japan, Mishima-shi. No
synthetic gene was used. For further specific details, see ref. 14. Acceptor
organism is E. coli BL21 (DE3). The initial vector was a pET-30(+) vector
from Novagen. Complete description of the final GVO is: E.coli
c]
BL21/pETHmelB.[14a,
Expression of the tyrosinase in E. coli was
3-Acetyl-3-(3,4-dihydroxyphenyl)chroman-2-one (8h) was obtained as
colourless solid, 77% yield (34 mg, 115 µmol). Rf (70/30 PE/EtOAc): 0.12.
Mp: 242-243 °C (turned black from 220 °C on). 1H-NMR (600 MHz,
acetone-d6) [ppm] δ 2.03 (s, 3 H, 2‘-H), 3.60 (d, J = 16.3 Hz, 1 H, 3-Ha),
3.75 (d, J = 16.3 Hz, 1 H, 3-Hb), 6.68 (dd, J = 8.3, 2.4 Hz, 1 H, 14-H), 6.79
(d, J = 2.4 Hz, 1 H, 10-H), 6.82 (d, J = 8.3 Hz, 1 H, 13-H), 6.98 (d, J =
8.1 Hz, 1 H, 8-H), 7.08 (dd, J = 7.5 Hz, 1 H, 6-H), 7.24 (dd, J = 7.7 Hz, 1
H, 7-H), 7.33 (d, J = 7.6 Hz, 1H, 5-H), 8.00 (bs, OH), 8.13 (bs, OH). 13C-
NMR (151 MHz, aceton-d6) [ppm] δ 27.0 (C-2’), 32.3 (C-3), 63.6 (C-2),
115.6 (C-10),116.5 (C-13), 116.6 (C-8), 119.8 (C-14), 123.1 (C-4a or C-4b),
125.4 (C-6), 126.5 (C-9), 129.2 (C-7), 129.4 (C-5), 146.2 (C-11 or C-12),
146.3 (C-12 or C-11),152.1 (C-4a or C-4b), 168.0 (C-1’ or C-1), 203.7 (C-1
or C-1’). IR ṽ 3462, 3211, 1762, 1683, 1607, 1542, 1533, 1488, 1457, 1437,
1360, 1339, 1284, 1256, 1234, 1299, 1182, 1144, 1108, 1084, 1030, 979,
947, 916, 869, 834, 809, 795, 774, 761, 680 cm-1. HRMS m/z = 299.0915
[M+H]+, calculated for C17H15O5+: 299.0914; 321.0735 [M+Na], calculated
for C17H14O5Na: 321.0739.
performed in 1 L Fernbach flasks that were tempered and shaken in a New
Brunswick™ Innova® 42 shaker. Harvesting of the cells by centrifugation
was performed via the RC5B centrifuge from Thermo Scientific. Cell
disruption was performed via a Sonoplus or Sonoplus RC6 plus from
Bandelin electronic GmbH & Co.KG, Berlin. pH values of the different
buffer systems were adjusted via the Mikroprozessor-pH-Meter from Knick.
Transformation of E. coli BL21 with the pETH/melB plasmid was
performed by putting 3 µL of the plasmid (negative control with 3 µL of
sterilised water) onto competent cells keeping them for 30 min on ice. Heat
shock occurred for 90 sec at 42 °C. Afterwards 1 mL of LB medium was
added to the cells and incubated for 1 h at 37 °C. After centrifugation, the
supernatant was discarded and the pellet was re-suspended with the
holdover of the supernatant and distributed onto kanamycin agar plates,
which were incubated over night at 37 °C.
Preparatory cultures were prepared by picking one single E. coli
BL21/pETHmelB colony and transferring it into 5 mL LB medium with 5 µL
kanamycin (KAN). Incubation continued over night at 37 °C.
tert-Butyl
3-(3,4-dihydroxyphenyl)-2-oxo-3-phenylindoline-1-
carboxylate (8i) was obtained as slightly yellow solid, 53% yield (32 mg,
77 µmol). Rf (70/30 PE/EtOAc): 0.28. Mp: 87-88 °C. 1H-NMR (600 MHz,
acetone-d6) [ppm] δ 1.61 [s, 9 H, C(CH3)3], 5.50 (bs, OH), 5.75 (bs, OH),
6,55 (dd, J = 8.3, 2.2 Hz, 1 H, 14-H), 6.69 (d, J = 8.4 Hz, 1 H, 13-H), 6.76
(d, J = 2.2 Hz, 1 H, 10-H), 7.13-7.19 (m, 4 H, Ar-H), 7.21-7.32 (m, 4 H, Ar-
H), 7.87 (d, J = 8.2 Hz, 1 H, Ar-H). 13C-NMR (151 MHz, aceton-d6) [ppm]
δ 28.2 [C(CH3)3], 62.5 (C-3), 85.1 [C(CH3)3], 115.2 (C-13), 115.4 (C-Ar),
116.2 (C-10), 121.5 (C-14), 124.9 (C-Ar), 126.3 (C-Ar), 127.6 (C-Ar), 128.6
(2 C-Ar), 128.6 (2 C-Ar), 132.1 (C-Ar), 133.8 (C-Ar), 138.9 (C-Ar), 141.9
(C-Ar), 143.6 (C-Ar), 149.4 (C-2 or C-1‘), 177.0 (C-1‘ or C-2). IR ṽ 3393,
2980, 2930, 1781, 1733, 1603, 1517, 1495, 1478, 1464, 1434, 1395, 1370,
1341, 1309, 1284, 1251, 1143, 1115, 1092, 1034, 1001, 947, 909, 856,
837, 813, 753, 729, 696 cm-1. HRMS: m/z = 418.1649 [M+H]+, calculated
for C25H24NO5+: 418.1649; 440.1468 [M+Na], calculated for C25H23NO5Na:
440.1468.
Expression was performed in 1 L Fernbach flasks. One preparatory
culture was added to 1 L TB media [Terrific-Broth media from Carl
ROTH®; containing casein 12 g/L, yeast extract 24 g/L, K2HPO4 12.54 g/L
and KH2PO4 2.31 g/L, pH ~7.2; 1 mL KAN (50 mg/mL) and 1 M (1.2 mL)
CuSO4]. The expression started at 29 °C for 1 h, followed by 4 h at 36 °C
and again 1 h at 29 °C before induction with IPTG (100 µL, 10 mg/mL).
The culture was further shaken (120 rpm) over night at 21 °C. The cells
were harvested by centrifugation at 4 °C, 12.000 rpm (7084 rcf) for 20 min.
Afterwards the pellets were suspended with 0.85% NaCl solution and
centrifuged a second time before they were stored until further use at
-20 °C.
Purification of the enzyme occurred via a peristaltic pump P-1 from
Pharmacia and a Ni-NTA column. Following buffer systems were used for
protein purification [binding buffer: 0.3 M NaCl, 10 mM imidazole, 10%
glycerol; 20 mM Bis-Tris, pH 7.2; elution buffer I: 0.3 M NaCl, 0.1 M
imidazole, 0.5 mM tris(2-carboxyethyl)phosphine hydrochloride (TCEP),
20 mM Bis-Tris, pH 7.2; elution buffer II: 0.3 M NaCl, 0.5 M imidazole,
0.5 mM TCEP, 20 mM Bis-Tris, pH 7.2]. 5 g of the cell pellet were
suspended ad 20 mL of the binding buffer and disrupted with the sonotrode
for three times 5 min on ice. The Ni-NTA column was washed with binding
buffer (10 mL) before the crude enzyme solution was put on it in flow for
30 min. Afterwards the column was washed again with 5-10 mL of the
binding buffer to elute unbound enzyme. In two steps (15 mL each) the
enzyme was eluted from the column using elution buffer I+II. Purification
of the Ni-NTA occurred with a 2 M imidazole solution, followed by washing
it with dH2O and stored in 20% EtOH.
Methyl
1-(4,5-dihydroxy-2-methoxyphenyl)-2-oxocyclopentane-1-
carboxylate (m-8n) was obtained as slightly yellow oil, 55% yield (23 mg,
82 µmol). Rf (70/30 PE/EtOAc): 0.07. 1H-NMR (600 MHz, CDCl3) [ppm] δ
1.84-1.91 (m, 1 H, 3-H or 4-H or 5-H), 1.95-2.02 (m, 1 H, 3-H or 4-H or 5-
H), 2.27 (m, 1 H, 3-H or 4-H or 5-H), 2.43-2.50 (m, 2 H, 3-H or 4-H or 5-H),
2.88 (mc, 1 H, 3-H or 4-H or 5-H), 3.67 (s, 3 H, 2‘-H or 7-H), 3.74 (s, 3 H,
7-H or 2‘-H ), 6.44 (s, 1 H, 8-H or 11-H), 6.49 (s, 1 H, 11-H or 8-H). 13C-
NMR (151 MHz, CDCl3) [ppm] δ 19.9 (C-3 or C-4 or C-5), 35.6 (C-3 or C-
4 or C-5), 38.7 (C-3 or C-4 or C-5), 53.1 (C-2‘ or C-7‘), 56.1 (C-2‘ or C-7‘),
64.4 (C-1), 101.3 (C-8 or C-11), 115.6 (C11 or C-8), 119.8 (C-6, C-7, C-9
or C-10), 136.4 (C-6, C-7, C-9 or C-10), 144.7 (C-6, C-7, C-9 or C-10),
151.4 (C-6, C-7, C-9 or C-10), 171.3 (C-2 or C-1‘), 215.1 (C-1‘ or C-2). IR
ṽ 3398, 2955, 1719, 1614, 1574, 1515, 1456, 1429, 1402, 1352, 1306,
1231, 1194, 1171, 1104, 1077, 1036, 1014, 986, 937, 912, 866, 832, 728,
647, 609, 563, 519. HRMS: m/z = 279.0863 [M-H]+, calculated for
For concentration of the protein solutions, Vivaspin 20 (10 kDa MWCO)
or Vivacell 250 from Sartorius Stedim were used. For buffer changes, PD-
10 columns from GE-Healthcare were used (Tris-HCl buffer: 10 mM Tris-
HCl, 10 mM NaCl, 10% glycerol, pH 6). Therefore, the PD-10 columns
were loaded each with 2.5 mL enzyme solution and eluted with 3.5 mL of
the Tris-HCl buffer.
C14H15O6 : 279.0874; 281.1020 [M+H]+, calculated for C14H17O6
281.1020.
:
-
+
Methods in Biology
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10.1002/ejoc.201800188
European Journal of Organic Chemistry
FULL PAPER
DNA- and protein concentrations were measured via Nano Drop 2000c
from Thermo Scientific Corp., MA, USA. Determination of the protein
concentration occurred by using the Bradford assay. As a calibration curve,
a BSA standard curve was taken. Incubation of the enzyme solution with
the Bradford dye took 10 min.
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separated (decrease in molecular weight of about 20 kDa; detected via
MALDI-TOF/MS).[14a] Activation of the tyrosinase occurred via trypsin (from
bovine pancreas) which was added as solution in citrate buffer (100 mM,
pH 6). After 30 min of incubation, trypsin inhibitor (from soybean) was
added. To a 1 mM solution (992 µL) of tyrosine in citrate buffer (100 mM,
pH 6), 8 µL of the activated enzyme solution (turned slightly red) was
added and the reaction rate was monitored at 475 nm.
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Activation of the tyrosinase for the arylation reaction was also performed
with trypsin but this time in Tris-HCl buffer (10 mM; pH 6). In order to
minimize the dilution of the tyrosine during the process of activation via
trypsin and inactivation of the protease, the additives were diluted in a ten-
fold higher concentration so that less volume would be needed for the
activation procedure. Example: 900 µL of the tyrosinase [43 U/mL; 2-
3.2 U/mg (a range of specific activities is given which depends on the
charge of purified enzyme); turnover number: 204-330 min-1 (referring to a
molecular mass of 102 kDa, after digestion)] in Tris-HCl buffer were
activated via the addition of trypsin (100 µL of a ten-times concentrated
solution in Tris-HCl) for 30 min at room temperature. Afterwards trypsin
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inhibitor was added to stop the protease (100 µL of
a ten-times
concentrated solution in Tris-HCl) resulting in a total volume of 1100 µL for
the arylation reactions.
Acknowledgements
This work was supported by the DFG within the IRTG ‘SeleCa-
Selectivity in Chemo- and Biocatalysis’ [grant number IRTG1628].
We gratefully acknowledge the Ministry of Innovation, Science,
and Research of the German federal state of North Rhine-
Westphalia, the Heinrich-Heine-Universität Düsseldorf, the
Forschungszentrum Jülich GmbH, as well as Osaka University for
their generous support of our projects. Furthermore, we thank the
analytical departments of the Forschungszentrum Jülich and the
Heinrich-Heine-Universität Düsseldorf. Last but not least we
would like to thank M. R. Hayes for his patient and uncomplaining
support with the applied biological techniques and methods.
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[16] Commercially available “tyrosinase from mushroom” (Sigma-Aldrich®;
CAS 9002-10-2); lyophilized powder; pH optimum 6-7.
[17] a) C. R. Pudney, A. Guerriero, N. J. Baxter, L. O. Johannissen, J. P.
Waltho, S. Hay, N. S. Scrutton, J. Am. Chem. Soc. 2013, 135, 2512-
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Keywords: 1,4-addition • arylation • biocatalysis • oxidation •
tyrosinase
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10.1002/ejoc.201800188
European Journal of Organic Chemistry
FULL PAPER
This article is protected by copyright. All rights reserved.

10.1002/ejoc.201800188
European Journal of Organic Chemistry
FULL PAPER
Entry for the Table of Contents
FULL PAPER
A tyrosinase mediated
Roxanne Krug, Dennis
arylation reaction is presented.
Starting from a variety of
substituted phenols, the
enzyme generates activated o-
quinones which subsequently
can undergo a 1,4-addition
towards the desired arylation
products, processing a
Schröder, Jan Gebauer, Sanel
Suljić, Yuma Morimoto,
Nobutaka Fujieda, Shinobu Itoh,
Jörg Pietruszka *
Page No. – Page No.
Tyrosinases in organic
chemistry: a versatile tool for
the α-arylation of β-
quaternary center.
dicarbonyl compounds
Key Topic
Domino Reaction
This article is protected by copyright. All rights reserved.