Laccase initiated C-C couplings: Various techniques for reaction monitoring

Article abstract of DOI:10.1016/j.procbio.2015.05.011

In this study the fungal laccase from Myceliophthora thermophila (Novozym 51003) was investigated for oxidative C-C couplings of phenolic compounds. This enzyme requires only molecular oxygen as oxidant, which represents a great advantage for oxidative coupling reactions. However, such couplings are typically considered as highly unselective, due to the radical reaction mechanism of laccases. Based on different analytical techniques we gained detailed insights into laccase initiated coupling reactions. A preselection of potential substrates was realized via oxygen measurement during laccase initiated oxidations. Furthermore in situ FT-IR spectroscopy facilitated analyses in-depth, due to feasibility to detect dissolved as well as solid compounds. This technique allowed the detection of the grade of couplings of various laccase substrates, depending on different conditions. As a result oxidative dimerizations of 2,6-disubstituted phenols could be identified as highly selective and were scaled-up to multigram scale. Thereby the oxidation product of 2,6-diisopropyl phenol can be easily reduced to the corresponding biphenol, the antibacterial agent dipropofol. The presented techniques open up new biocatalytical approaches receiving quinoid units as key elements of many natural compounds and pharmaceuticals.

Full text of DOI:10.1016/j.procbio.2015.05.011

Accepted Manuscript
Title: Laccase initiated C-C Couplings: Various Techniques
for Reaction Monitoring
Author: Claudia Engelmann Sabine Illner Udo Kragl
PII:
S1359-5113(15)00261-5
DOI:
Reference:
http://dx.doi.org/doi:10.1016/j.procbio.2015.05.011
PRBI 10426
To appear in:
Process Biochemistry
Received date:
Revised date:
Accepted date:
2-4-2015
12-5-2015
14-5-2015
Please cite this article as: Engelmann C, Illner S, Kragl U, Laccase initiated C-C
Couplings: Various Techniques for Reaction Monitoring, Process Biochemistry (2015),
http://dx.doi.org/10.1016/j.procbio.2015.05.011
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Highlights
Laccase initiated C-C Couplings: Various
Techniques for Reaction Monitoring
Claudia Engelmann, Sabine Illner and Udo Kragl*
Department of Chemistry
University of Rostock
Albert Einstein Str. 3a, D-18059, Rostock (Germany)
Fax: (+49)381 498 6452
E-mail: udo.kragl@uni-rostock.de
Highlights:
A pathway to identify selective laccase (Novozym®51003) mediated coupling reactions is
investigated.
In situ FT-IR analysis facilitates new insights in laccase initiated oxidative couplings.
Different analytical methods gained detailed insights resulting in a synthesis of 7 g of pure
diquinone.
1
Page 1 of 40

Page 2 of 40

Laccase initiated C-C Couplings: Various
Techniques for Reaction Monitoring
Claudia Engelmann, Sabine Illner, and Udo Kragl*
2
Page 3 of 40

1
Abstract
2
3
In this study the fungal laccase from Myceliophthora thermophila (Novozym®51003) was investigated for
oxidative C-C couplings of phenolic compounds. This enzyme requires only molecular oxygen as oxidant,
which represents a great advantage for oxidative coupling reactions. However, such couplings are
typically considered as highly unselective, due to the radical reaction mechanism of laccases. Based on
different analytical techniques we gained detailed insights into laccase initiated coupling reactions. A
preselection of potential substrates was realized via oxygen measurement during laccase initiated
oxidations. Furthermore in situ FT-IR spectroscopy facilitated analyses in-depth, due to feasibility to
detect dissolved as well as solid compounds. This technique allowed the detection of the grade of
couplings of various laccase substrates, depending on different conditions. As a result oxidative
dimerizations of 2,6-disubstituted phenols could be identified as highly selective and were scaled-up to
multigram scale. Thereby the oxidation product of 2,6-diisopropyl phenol can be easily reduced to the
corresponding biphenol, the antibacterial agent dipropofol. The presented techniques open up new
biocatalytical approaches receiving quinoid units as key elements of many natural compounds and
pharmaceuticals.
4
5
6
7
8
9
10
11
12
13
14
15
16
17 Keywords:
18 Biocatalysis, Oxidative coupling, Quinone, Real-time reaction monitoring, Multigram Scale
19
3
Page 4 of 40

19
20 Introduction
21
22
23
24
25
26
27
28
29
Biphenol and quinoid units are important building blocks for the synthesis of industrially relevant
pharmaceuticals, herbicides, natural products, as well as engineering materials. The syntheses are often
performed with transition metal catalysts and complex ligand systems, examples are the Suzuki or
Ullmann coupling.[1-4] Herein laccases offer an interesting and competitive alternative for the synthesis
of biphenols or their oxidized forms in contrast to classical metal catalyzed reactions (Scheme 1).[5, 6]
However, such biocatalytic oxidations, followed by regioselective couplings to pure diquinones or
biphenols, are rarely executed at larger scale. The radical mechanism of this biotransformation,[7-9] not
yet completely clarified, may be one reason for the lack of effective coupling processes using this
enzyme.
Biocatalysis
O2
2 H2O
R
R
R
R
R
R
Laccase
2
O
O
OH
0.01 mol%, 23°C, 2 - 4 h
*
R
R
R
R
R
R
R
H
H
2
O
O
HO
OH
70 - 97%
R
selective oxidative
Homocoupling
R = Me, OMe, iPr
Keto-form
Enol-form
Suzuki
Pd-catalyst
Toluol, Base
X
B
0.005 - 5 mol%
ca. 100°C, 4 - 24 h
R1
R2
R1
R2
moderate to high yields
selective Homo- and Heterocoupling
X = leaving group: I, Br, (Cl)
Ullmann
Cu
(ca. 200°C)
2
X
+ CuX2
10 - 100 mol%
R
R
R
moderate yields
selective Homocoupling
30
31
32
Scheme 1. Potential synthetic routes of selective aryl-aryl couplings. *These quinones are easily reduced to aromatic
biphenols.[10, 11]
33
34
35
36
37
38
Laccases, well known multicopper oxidases (E.C. 1.10.3.2.), catalyze one-electron oxidations of phenolic
compounds, typically followed by various C-C, C-N or C-O coupling reactions.[12-15] A few phenolic
compounds also act as mediators to oxidize more complex non-phenolic substrates.[9, 16-18] In such
biocatalytic oxidative C-C couplings only O2, as the cheapest, “greenest”, and most abundant oxidant is
used. The only byproduct water is certainly also advantageous in relation to environmental
considerations.[12, 19] Laccases are highly stable enzymes, mostly fungal and readily accessible from
4
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39
40
41
42
43
44
45
46
47
numerous sources. They are already applied in industrial processes such as wastewater treatment and
degradation of dyes in textile industry or for bleaching of pulp and paper via delignification.[8, 20-22]
Nowadays, laccase-related research is mainly focused on selective enzymatic synthesis of organic
compounds and biosensor development.[22-24] Especially biosynthesis of fine chemicals is very
promising as a “green” and cheap alternative for the use of transition metal catalysis.[25] In laccase
mediated reactions neither activated substrates like organic boron compounds nor the previous
syntheses of complex catalyst systems are required. Noteworthy, Wellington et al. reported a direct
laccase mediated synthesis of drugs with anticancer activity [26, 27] and Beifuss et al. used laccases for
various oxidation reactions yielding carpanones, benzopyrans and trimerization of sesamol.[5, 28]
48
49
50
51
We have been interested in the development of preparative biocatalytic, eco-friendly and easily
applicable processes for a selective synthesis of pure quinones. A major drawback for the application of
laccases in synthesis of highly functionalized products is the occurrence of non-specific polymerization
through mono-electronic oxidation (Scheme 2).[7, 9, 16]
OH
R
R
R
R
R = Me, OMe, iPr, ...
R
O2
laccase
2 H2O
homocoupling
+ substrate
heterocoupling
+ nucleophile
O
OH
O
O
O
OH
Nu
OH
O
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
or
R
R
R
R
R
R
R
R
or
or
Nu
coupling with
nucleophiles
cross-coupling
R
R
R
R
O
OH
dimerization
polymerization
52
53
Scheme 2. Laccase initiated oxidation of phenolic compounds and possible follow-up reactions.[17, 29-35]
54
55
56
57
58
A comprehensive reaction monitoring of laccase initiated couplings of phenolic derivates using
oxygen measurements, HPLC and in situ FT-IR spectroscopy is demonstrated. The main aim was to
gain detailed insights into these oxidative couplings depending on different conditions. We established
basic steps to identify appropriated substrates realizing atom-efficient, selective and fast reactions at
multigram scale.
59
5
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60
61 Experimental Section
62
63
64
65
66
67
68
General. All commercially available chemicals were obtained in high purity and used without further
purification, if not stated otherwise. Hydroquinone (HQ), 3-methylcatechol (3-MC), 4-tert-butylcatechol
(4-tert-BC), 2,5-dihydroxybenzoic acid (25-DHBAcid), 2,6-tert-butylphenol (2,6-tert-BP), 2,6-
diisopropylphenol (2,6-DIPP) and 2,4-dihydroxybenzaldehyde (2,4-DHBAldehyde) were purchased from
Sigma-Aldrich (Schnelldorf, Germany); 2,6-dimethylphenol (2,6-DMP) from Alfa Aesar (Karlsruhe,
Germany); 4-methylcatechol (4-MC) from Merck (Darmstadt, Germany); 2,6-dimethoxyphenol (2,6-
DMOP) from Acros (Geel, Belgium). Solvents for extraction and purification were distilled prior to use.
69
70
71
72
73
Laccase Novozym® 51003 from Myceliophthora thermophila (N51003), produced by genetically modified
Aspergillus oryzae, was a gift from Novozymes A/S (Bagsvaerd, Denmark). This enzyme was provided
as a crude extract with 1,000 U g-1 with respect to the conversion of syringaldazine at pH 7.5 and 30 °C.
In this study all enzyme activities were determined according to the standard ABTS assay (further
information is given in the supporting information), resulting in an activity of 270 U mL-1.
74
75
76
77
78
79
80
81
82
Substrate screening. Screening of laccase substrates was performed by online measurement of the
oxygen consumption via an optical oxygen sensor system (PreSens GmbH, Germany) integrated in a
bubble column with a sintered glass bottom (ø 3 cm). Before each measurement the reaction volume of
30 mL was degassed with argon and gassed with pressured air for calibration. All diphenolic compounds
were examined in a potassium phosphate buffer (pH 7) containing 20% (v/v) methanol, to ensure a
complete solubility of 5 mM for all substrates at room temperature. Before starting the measurement the
solution was gassed to get an oxygen content of 100%. To initiate the reaction laccase N51003 (27 U
according to the ABTS assay) was added. The reaction mixture was stirred continuously with an
overhead stirrer during all screening experiments at 300 rpm.
83
84
85
Reaction monitoring. Reaction progress was monitored using an in situ FT-IR spectrometer (ReactIRTM
15, Mettler Toledo) and in certain cases complementary via HPLC analysis (see below). Experiments
were performed in water/methanol mixtures at pH 6 (adjusted with acetic acid) using substrate
6
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86
87
88
89
90
91
92
concentrations of 20 - 50 mM. Substrates were investigated using a methanol content of 20 - 70% (v/v).
Subsequently 135 - 405 U N51003 were added to a continuously aerated (compressed air supply of 100
mL min-1) and intensively stirred solution. The in situ FT-IR spectrometer with an AgX FibreConduit
diamond ATR probe was used in a specific range from 2000 – 700 cm-1 with a spectral resolution of 4
cm-1. For reaction monitoring the iC IRTM 3D software was used without baseline corrections. A
difference spectrum, obtained by subtraction of a reference spectrum after one minute, allowed a
background elimination including the absorption of the solvent.
93
94
95
96
97
98
99
Samples for HPLC (Knauer, UV detection) were taken in defined intervals and analyzed with a
Discovery® HS F5 column (Supelco, 15 mm x 4 mm ID, 3 µm) at 30 °C and a flow rate of 0.4 mL min-1. A
linear gradient of 10% methanol and 90% of 0.1% aqueous formic acid up to 100% methanol over 20
min was used. The separation was continued with 100% methanol for 10 min and afterwards a linear
gradient up to 10% methanol was applied. This eluent composition was used again for 5 min, whereby
the analysis took 45 min overall. 3-Methylcatechol and hydroquinone were detected at a wavelength of
220 nm with retention times of 14.0 and 10.2 min, respectively.
100
101
102
103
104
105
106
107
Biotransformations of 2,6-disubstituted phenolic compounds. Oxidations of 2,6-disubstituted
phenolic compounds were performed in a bubble column (air flow 100 mL min-1) with integrated oxygen
measurement. A water/methanol mixture (50%, v/v) was acidified with acetic acid (pH 6). The reaction
was started by adding 1.5 mmol substrate (50 mM) and 100 U of N51003 to 30 mL reaction mixture. The
process was monitored via oxygen consumption with continuous stirring (overhead stirrer, 300 rpm) at
room temperature. After reaction the resulting precipitate was filtered and initially washed with water to
remove the protein. In addition, the crystalline product was rinsed with 3 x 5 mL cold methanol to remove
substrate residues.
108
109
110
111
112
Synthesis at multigram scale was performed in a similar manner using 10 g (56.1 mmol) of 2,6-DIIP and
2700 U of N51003 in 500 mL of a water/methanol mixture (50%, v/v). The reaction was stopped after 4 h
with an excess of acetone to inactivate the enzyme. The reaction mixture was filtrated for protein
separation and the resulting solution was partially evaporated under reduced pressure. In total 7 g of
reddish-purple crystals (70% isolated yield) were obtained.
7
Page 8 of 40

113
114
Analytical details including product quantification and purity control via UV/Vis measurements, DSC,
IR, NMR spectra are given in the supporting information.
115 Results and Discussion
116
Substrate screening
117
118
119
120
121
122
123
As mentioned above, laccases oxidize a broad range of phenolic substrates under consumption of
molecular oxygen as oxidant. Thus the in situ oxygen measurements allow an easy evaluation of
conversion rates of different substrates. The results of the screening of some interesting laccase
substrates, monitored by a non-invasive optical oxygen sensor, are presented in Table 1. 2,5-
Dihydroxybenzoic acid (2,5-DHBAcid) was only slowly and meta-substituted 2,4-dihydroxybenzaldehyde
(2,4-DHBAldehyde) was not oxidized. The measurements confirm the suitability of ortho- and para-phenolic
as
well
as
monophenolic
compounds
as
substrates
for
N51003.[5,
36]
8
Page 9 of 40

124
125
126
Table 1. Substrate screening via online oxygen monitoring.
Entry Substrate MeOH [a] Structure
[% (v/v)]
Initial oxygen Conversion
consumption rate[b] [molsub
[mg L-1∙min-1] molcat-1 min-1]
OH
HO
1
3-MC
20
6.7
207.9
OH
HO
2
3
4-MC
20
20
5.3
9.4
164.4
291.6
OH
OH
4-tert-BC
O
2,4-
OH
4
5
6
20
20
20
0
0
HO
DHBAldehyde
HQ[c]
7.4; 0.14
1.7
229.6; 37.2
52.7
HO
OH
OH
O
2,5-
HO
DHBAcid
HO
OH
20
5.7
4.9
353.7
60.8
O
O
7
8
9
2,6-DMOP
50*
OH
2,6-tert-
50*
0.02
0.3
BP
OH
OH
2,6-DIPP
50*
50*
0.6
1.4
7.4
10 2,6-DMP
17.4
[a] Buffered solution (pH 7; 30 mL) containing different volumes methanol
as co-solvent, 5 mM substrate and varying enzyme quantities (nCat, 20% MeOH
= 0.06 µmol; *nCat,
MeOH= 0.30 µmol). [b] Substrate conversion was
50%
calculated on the basis of the reduction of one mol oxygen for concomitant
oxidation of two or four substrate molecules concerning the number of O-H
groups and the used mol of the enzyme. The concentration of enzyme in
the crude extract was determined by Gel electrophoresis and BCA assay
(data shown in SI). [c] HQ showed two different slopes during one
measurement.
9
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126
127
128
129
130
131
132
133
134
135
136
137
The bond dissociation energy (BDE) of the phenolic O-H bond is often used as an explanation for its
oxidation sensitivity. Quideau et al. described a preferred oxidation of hydroxyl groups in ortho-position
by the linkage of hydrogen bridges resulting in a stabilization effect for temporarily formed phenoxy
radicals; while hyper conjugation and resonance effects compensate electronic vacancy.[3, 18] A
presence of alkyl or alkoxy groups in ortho- or para-positions drastically decreased the BDE of O-H
bonds.[3] In contrast, molecules with electron-donating substituents showed low DO–H values and were
oxidized with higher reaction rates by laccases.[18] In summary, BDEs are of importance for the
oxidizability of laccase substrates, but also the number of OH-groups and the position of the residues
have a great influence on the reaction rate. A final prediction of reactivity is not possible; partly due to the
different sources of the laccases (fungi, plants, insects, microorganisms, etc.) and their respective
specific redox potentials.[37]
138
139
140
141
142
143
144
145
146
147
Consequently, the initial consumption of dissolved oxygen by laccase was used for comparison of the
oxidation rate for different substrates (Table 1). The wide range of tested mono- and diphenolic
compounds allows a clear distinction between the substrates depending on number and position of
hydroxyl groups. N51003 exhibited highest oxidation rate for 2,6-dimethoxyphenol (2,6-DMOP), MC, 4-
tert-butylcatechol (4-tert-BC) and HQ. The most rapid oxygen consumption was measured for 4-tert-BC
regarding the initial reaction rate. In case of 2,6-DMOP, also known as Syringol, oxidation was drastically
improved by a formation of intramolecular hydrogen bridges. The reaction rate of 2,6-DMOP decreases
significantly by a factor of 6 using 50% (v/v) methanol, although methanol itself has only a negligible
effect on N51003 (data shown in SI). Consequently the reaction medium and here especially its solvation
capacity has a decisive influence on the stabilization of the phenoxy radical.[38, 39]
148
149
150
151
Based on the first results ortho- and para-dihydroxylated phenols like 3-MC and HQ as well as the
monophenolic compound 2,6-DMOP were chosen for further investigations. 3-MC was used instead of 4-
tert-BC despite its lower conversion rate, due to earlier investigations with regard to solvent stability and
coupling behavior.[40, 41]
152
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153
154
In situ FT-IR reaction monitoring
155
156
157
158
159
160
161
162
163
164
165
Laccase intiated homo- and heteronuclear coupling reactions are occasionally very unselective and
additional products precipitate during the reaction e.g. due to dimer-, oligo- and polymerizations
(Scheme 2).[5, 8, 42] Consequently, analysis and downstream processing of the reactants represent a
major challenge, especially with respect to low solubilities of the resulting products. In this study
representative couplings were followed with an in situ FT-IR spectrometer observing the range of
occurring reactions. FT-IR spectroscopy as a suitable non-invasive technique detects all compounds in
either liquid, gaseous or solid phase without sampling, in comparison to classical analytical methods like
HPLC.[43] This real-time process monitoring is still relatively uncommon for biocatalytic reactions,
although this technique is already established in bioprocesses.[44-46] So far, merely small-scale
biotransformations are examined concerning lipase-catalyzed esterifications [47, 48] or nitrile
biotransformations.[49]
166
0.20
2,6-DMOP
B
HQ
3-MC
0.16
0.12
0.08
0.04
0.00
C=O
C=C
A
0.20
0.15
0.10
0.05
0.00
0
25 50 75 100 125 150
Time / min
1750 1500 1250 1000 750
-1
Wavenumber / cm
167
168
169
170
171
Figure 1. In situ FT-IR reaction monitoring of the oxidation of 3-MC, HQ and 2,6-DMOP. (A) FT-IR spectra at the end of reaction.
(B) Changes in the C=O absorption (1615 – 1630 cm-1) during oxidation. Conditions: 50 mL water/methanol (30:70, v/v) acidified
with acetic acid (pH 6), 50 mM 2,6-DMOP/ HQ, 20 mM 3-MC, 25 °C, 135 U of N51003, 100 mL min-1 aeration.
172
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173
174
175
176
177
178
179
180
181
182
183
184
185
186
187
During the reaction the respective hydroxyl groups are oxidized to carbonyl groups, which results in a
conversion into a quinoidic structure. As a result C=O and C=C vibration bands within the range of 1615
– 1660 cm-1 and 1540 - 1600 cm-1 increased.[50-52] In Figure 1 typical in situ FT-IR difference spectra of
selected laccase initiated oxidations are presented showing relevant differences in the fingerprint region
from about 1500 to 750 cm-1.[53] For 3-MC several wide vibration bands indicate very complex series of
molecule specific absorptions suggesting non-specific homo- and heteronuclear couplings.[54] In
contrast intense vibration bands only occurred around 1660 cm-1 (overlapping) in the measured
difference spectrum during oxidation of para-substituted HQ. Intermediary semiquinones or different
oxidized forms of the substrate were not visible via FT-IR spectroscopy. Based on this lack of traceability
an overlap of several decreasing and increasing vibration bands occur during the oxidation of substrates.
However, an absorption change to zero indicates a complete conversion, especially visible in the
spectrum of the monophenolic compound 2,6-DMOP (Figure 1). Surprisingly, throughout oxidation of
2,6-DMOP very sharp bands increased, while a precipitate with a purple metallic gloss is formed
immediately. This suggests the dimeric quinoid form of 2,6-DMOP, confirmed by several analytic
methods and indirect evidence via a simple reduction (further details in the SI).
188
189
190
191
192
193
194
195
196
197
198
199
Further investigations included the oxidation of the ortho-substituted diphenolic 3-MC to gain a better
understanding complementary to previously published work.[40, 41] Without any coupling partner, the
laccase mediated oxidation of 3-MC resulted in several wide vibration bands in the fingerprint region for
3-MC (Figure 1A). The following section highlights the polymerization grade of 3-MC in relation to
substrate concentration (Figure 2) and enzyme activity (data shown in SI) in difference spectra. Figure 2
illustrates the laccase initiated oxidation of 20 mM or 30 mM 3-MC under otherwise identical conditions.
Both spectra show similar intensities for C=O (around 1654 cm-1) and C=C vibration (1596 cm-1), but in
the fingerprint region (1500 – 750 cm-1) the absorption intensities differ significantly. In this section of the
spectrum diverse C-C, C-O and C-H vibration bands occurred.[54] Higher intensities of complex bending
vibrations within the molecule at 30 mM suggested an increase in polymerization at higher substrate
concentrations consistent with the results of previous work.[41] The polymerization was also preferred
with lower enzyme activity due to longer reaction times (data shown in SI).
12
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200
201
202
203
Figure 2. In situ FT-IR reaction monitoring varying the concentration of 3-MC. (A) 20 mM of 3-MC (270 min reaction time). (B)
30 mM of 3-MC (150 min). Conditions: 50 mL water/methanol (30:70, v/v) acidified with acetic acid (pH 6), 25 °C, 337.5 U of
N51003, 100 mL min-1 aeration.
204
205
206
207
208
Additional, conventional HPLC measurements were executed to confirm the found FT-IR results. In
Figure 3 the conversion of 3-MC and HQ (HPLC) is compared with the product formation evaluated by
specific C=O or C=C vibration bands (FT-IR spectroscopy). The correlations between the two different
methods confirm the suitability of in situ FT-IR spectroscopy for reaction monitoring. Both analytical
methods show also the time lag and similar reaction rates (Figure 3).
0.10
0.08
0.06
0.04
0.02
0.00
100
80
60
40
20
0
0.6
0.5
0.4
0.3
0.2
0.1
0.0
100
80
60
40
20
0
B
A
HPLC
IR (1594 cm-1)
HPLC
IR (1659 cm-1)
0
50 100 150 200 250 300
Time / min
0
50
100
Time / min
150
200
209
210
211
212
213
Figure 3. Correlation of the substrate conversion measured by HPLC (red) with product formation measured by in situ FT-IR
spectroscopy (black). (A) 3-MC. (B) HQ. Conditions: 50 mL water/methanol (30:70, v/v) acidified with acetic acid (pH 6), 50 mM
substrate, 25 °C, 135 U of N51003, 100 mL min-1 aeration.
214
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215
216
217
No maximum in FT-IR absorption was observed in contrast to a minimum in the conversion of HQ
visible around 0.01 A.U.. These results indicate further reactions such as dimerization or polymerization
subsequent to the intended laccase initiated oxidation of 3-MC or HQ.
218
219
220
221
222
223
224
In addition to the evaluation of suitable substrates via O2-measurements, further informations about
potential products are available. In situ FT-IR spectroscopy provides insights into the selectivity through
occurrence of sharp and intense absorption bands in the difference spectra, due to high symmetrically
coupling products. Furthermore the maximum concentration without an occurrence of significant
polymerization can be easily determined to ensure optimized reaction conditions. Consequently
unselective homo- and heterocouplings can be kept to a minimum at relatively high enzyme
concentrations and thereby short reaction times.
225
Selective oxidative C-C coupling reactions
226
227
228
229
230
Recently, selective biocatalytic oxidative couplings of substituted phenols gained a strong research
interest since they represent an alternative to classical transition metal catalysis.[19, 55] Due to the fact
that 2,6-disubstituted phenols are readily converted and show no significant byproduct formation
demonstrated in O2- and FT-IR measurements in the sections above, this group of substrates was
chosen for detailed investigations to scale up selective couplings.
231
232
233
234
235
236
237
238
239
240
To the best of our knowledge this is the first time that a selective oxidation of several 2,6-disubstituted
phenol derivatives was performed in this manner using laccases. In 1990 the biocatalytic oxidation of
hindered phenols using 50,000 U tyrosinase has already been studied by Panday et al..[29] With long
reaction times of 9 up to 60 hours only poor selectivities and yields were achieved. However, in our study
the same oxidative couplings of the 2,6-disubstituted phenol derivatives were performed with a maximum
of 405 U of N51003 in significantly smaller reaction times, whereby the pure coupling products easily
precipitated from solution. Nevertheless, only few reports investigated the obtained product spectrum,
due to the very poor solubility in common solvents. Moreover, the reported screening experiments mostly
consider only substrate conversions and products were merely characterized by UV/Vis and/or IR
spectroscopy.[31, 37]
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241
242
243
244
245
246
Figure 4. In situ FT-IR reaction monitoring of the oxidation of 2,6-disubstituted phenol derivatives. (A) 2,6-DMOP (143 min
reaction time). (B) 2,6-DMP (127 min). (C) 2,6-DIPP (78 min). Conditions for 2,6-DMOP: 50 mL water/methanol (30:70, v/v)
acidified with acetic acid (pH 6), 50 mM of 2,6-DMOP, 25 °C, 270 U of N51003, 100 mL min-1 aeration. Conditions for 2,6-
DMP/2,6-DIPP: 50 mL water/methanol (50:50, v/v) acidified with acetic acid (pH 6), 50 mM of 2,6-DMP and 2,6-DIPP, 25 °C,
405 U of N51003, 100 mL min-1 aeration.
247
248
249
250
251
252
253
254
255
256
A usage of laccases does not require expensive combinations of (transition) metals and ligand
complexes or even activated substrates. Additionally the oxidative C-C coupling is carried out at neutral
pH and room temperature in e.g. 50% (v/v) water/methanol mixtures. The high stability in methanol up to
70% (v/v) of N51003 is of great benefit, since most laccase substrates are often low water soluble. In
Figure 4 very sharp bands are found in the FT-IR spectra during the oxidation of 2,6-disubstituted
phenols. As mentioned in the previous section, these sharp bands indicate highly symmetric coupling
products through laccase initiated regioselective oxidative dimerization of 2,6-dimethylphenol (2,6-DMP),
2,6-DMOP and 2,6-DIPP. Furthermore this analytical method allows an easy in situ reaction control of
forming highly insoluble precipitates. During this specific homocoupling no side reactions were found
(data shown in SI).
257
15
Page 16 of 40

258
Table 2. Laccase initiated oxidation of 2,6-disubstituted phenolic
compounds.
259
Substrate[a]
2,6-DMOP
2,6-DMP
Characteristic
bands[b] [cm-1]
Time
[min]
Yield[c]
[%]
STY
TOF[d]
[g L-1 h-1] [h-1]
260
1624, 1609, 1555,
1263, 1183
40
97
77
90
11.2
2.8
6252
2265102
1589, 1468, 1183
(overlapping)
100
2,6-DIPP
1584 (overlapping) 130
3.7
2
1926
6
2
[a] Conditions: mixture of 15 mL water acidified with acetic acid (pH 6) and
15 mL methanol, 50 mM of the substrates, 25 °C, 162 U N51003, 1 mL
263
00
min-1 aeration. [b] Intense absorption bands (in situ FT-IR spectrometer).
[c] Isolated yield. [d] TOF was determined from molSub molcat-1 h-1.
264
265
266
267
268
269
270
271
272
273
274
275
276
277
278
279
280
Finally the selected 2,6-disubstituted phenols were selected to investigate the assumed homocoupling
(Table 2) and the reaction mechanism. The hypothesized, selective pathway of the laccase initiated
oxidation of 2,6-disubstituted phenol derivatives is shown in Scheme 3, which based on the findings of
Yamamura and Nishiyama.[56] The presented FT-IR based investigations indicated an initial and
selective formation of product 2 coupled in para-position. This product exists in equilibrium with the enol
form 3, which is much more stable than keto form 2, due to preserved aromaticity. The broad substrate
spectrum of laccases was decisive for the possibility that biphenolic compound 3 represents a suitable
substrate. The subsequent oxidation of compound 3 into the final oxidized form 4 occurred. Noteworthy,
intermediates 2 and 3 are also found in the in situ FT-IR spectra. For example, initially formed carbonyl
bonds of 2 resulted in an increase of the C=O vibration band. Within the reaction this band temporarily
decreased, due to keto-enol tautomerism, while the last oxidation step of biphenol 3 to DQ 4 caused
again an increase of the C=O band. Under the chosen conditions these shifts of vibration bands are
visible via in situ FT-IR reaction monitoring, e.g. in Figure 4C. Confirming comparatively lower
conversion rate (Table 1) the intermediates of 2,6-DIPP were detectable in the spectra. Due to the
analysis of dissolved as well as solid compounds, this special in situ technique provides useful insights
to clarify the reaction mechanism.
281
16
Page 17 of 40

O
OH
O
R
R
R
R
R
R
R
R
R
R
R
R
OH
Keto-enol
tautomerism
R
R
Laccase
Laccase
H
H
2
0.5 O2 H2O
0.5 O2 H2O
1
O
OH
O
2
3
4
R
=
Me, OMe, iPr
282
283
Scheme 3. Reaction of the oxidation of 2,6-disubstituted substrate mediated through laccase.
284
285
286
287
288
The high selectivity of these fast reactions and the easy subsequent reduction of 4 to biphenolic
compound 3 gives access to highly functionalized biaryls for a wide range of applications. For example,
2,6-DMOP-DQ 4 was obtained as deep purple crystals with a yield of 97% in less than one hour and the
quinoidic product was simply reduced with acetic acid and zinc powder according to Purohit et al. (data
shown in SI).[10]
289
Scale up of the oxidation of 2,6-diisopropylphenol
290
291
292
293
294
295
296
With the detailed information from previous investigations 2,6-disubstituted phenolic compounds were
subjected to biocatalytic oxidative coupling at larger scale. 2,6-DIPP was selected due to good solubility
in organic solvents and the interesting reduction product of 2,6-DIPP-DQ (Scheme 4). The
corresponding biphenol is the antibacterial agent dipropofol.[11, 57] For biotransformation of 2,6-DIPP
(10 g, 56.1 mmol) we used a small stirred-tank reactor with constant aeration. Due to low oxygen
solubility in water (0.25 mmol/L, 25 °C) adequate gas delivery ensures maximum oxygen concentration
in the reaction mixture and minimizes limitations throughout the reaction.
297
298
17
Page 18 of 40

299
300
301
302
Scheme 4. Biocatalytic oxidative C-C coupling of 2,6-DIPP using 0.01 mol% N51003 (270 U g-1substrate according to the standard
activity assay, cf. SI). The observed color change of reaction mixture during reaction time and the isolated product crystals are
shown on the left side.
303
304
305
306
307
308
309
310
The biocatalytic reaction was performed at room temperature using only 0.01 mol% biocatalyst in
water/methanol mixtures. During the oxidation the dimer precipitated allowing full conversion of the
substrate after 4 hours. Simple filtration and crystallization yielded 70% of pure diquinone in crystalline
form. This laccase initiated reaction with high product concentration (14 g L-1) represents an interesting
alternative for the production of 2,6-disubstituted diquinones. Potential product applications are colorful
pigments including colorants for paints, hair, cosmetics or coatings. By an ordinary reduction highly
functionalized biphenols are readily available as building blocks for further syntheses e.g. of
pharmaceuticals or liquid crystals.
311
312 4. Summary & Conclusion
313
314
315
In this study we found appropriate substrates for selective laccases initiated oxidative reactions with
the commercially available N51003 using different analytical methods. Furthermore, we realized an
atom-efficient, selective and fast oxidative coupling in a multigram scale.
316
317
318
Herein a substrate preselection was performed via measurement of oxygen consumption observing that
ortho- and para-diphenolic as well as selected monophenolic derivatives were suitable substrates for
N51003. In situ FT-IR analysis provided insights into the coupling behavior of various phenolic
18
Page 19 of 40

319
320
321
322
323
324
325
326
327
328
329
compounds. An evaluation of the reactions with this method depending on substrate concentrations and
enzyme activities is possible even if the products precipitate. Moreover, this online technique enabled
the monitoring of polymerizations during laccase mediated oxidations. Especially high symmetrically
dimerizations were detected and identified as selective coupling reactions initiated by N51003, so that
selective oxidative homocouplings of 2,6-disubstituted phenols were investigated in detail. All tested
reactions formed sharp and intense FT-IR bands indicating regioselective synthesis of symmetrical
diquinones in comparably short periods. As a result yields up to 97% were achieved with high purities
using methanol tolerating N51003. In exemplary manner the simple reduction of quinones to biaryls was
also shown with good yields. On the basis of the previously results a multigram scale was realized for
the oxidative dimerization of 2,6-DIPP with 7 g isolated product at only 0.01 mol% biocatalyst and high
productivity.
330
331
332
333
334
In comparison to reported transition metal catalysts enabling C-C couplings of a broad substrate
spectrum, this biocatalytic approach is readily feasible using mild reaction conditions, low catalyst costs
and high space-time yields. Air as most abundant oxidant and water as byproduct demonstrate the high
atom efficiency of these selective oxidative couplings. The investigations represent the great potential of
this commercially available laccase in research and industry for new synthetic routes.
335
336
337
338
339
340
341
342
343
344
19
Page 20 of 40

345 ACKNOWLEDGMENT
346
347
348
The authors thank the Deutsche Forschungsgemeinschaft (DFG, KR 2491/10-1) for financial support
and Günter Maier from Novozymes Switzerland AG (Switzerland) for a kind donation of the laccase
Novozym® 51003.
349 ABBREVIATIONS
350
351
352
353
354
2,6-DMOP - 2,6-Dimethoxyphenol; 2,6-DMOP-DQ – 2,6-Dimethoxyphenol-diquinone; 2,6 DMP - 2,6-
Dimethylphenol; 2,6-DIPP - 2,6-Diisopropylphenol; 2,6-DIPP-DQ – 2,6 Diisopropylphenol-diquinone; HQ
- Hydroquinone; 3-MC - 3-Methylcatechol; 4-MC - 4-Methylcatechol; 4-tert-BC - 4-tert-Butylcatechol; 2,4-
DHBAldehyde – 2,4-Dihydroxybenzaldehyde; 2,5-DHBAcid – 2,5-Dihydroxybenzoeic acid; 2,6-tert-BP – 2,6-
tert-Butylphenol; ABTS - 2,2'-Azino-bis(3-ethylbenzothiazoline-6-sulphonic acid).
355
356 REFERENCES
357
358
359
360
361
362
363
364
365
366
367
368
369
370
371
372
373
[1] Suzuki A. Cross-Coupling Reactions Of Organoboranes: An Easy Way To Construct C-C Bonds
(Nobel Lecture). Angew. Chem., Int. Ed. 2011;50:6722.
[2] Bonrath W, Eggersdorfer M, Netscher T. Catalysis in the industrial preparation of vitamins and
nutraceuticals. Catal. Today 2007;121:45.
[3] Quideau S, Deffieux D, Douat-Casassus C, Pouységu L. Plant Polyphenols: Chemical Properties,
Biological Activities, and Synthesis. Angew. Chem., Int. Ed. 2011;50:586.
[4] Ullmann F, Bielecki J. Ueber Synthesen in der Biphenylreihe. Ber. Dtsch. Chem. Ges.
1901;34:2174.
[5] Constantin M-A, Conrad J, Beifuss U. An unprecedented oxidative trimerization of sesamol
catalyzed by laccases. Tetrahedron Lett. 2012;53:3254.
[6] Mogharabi M, Faramarzi MA. Laccase and Laccase-Mediated Systems in the Synthesis of Organic
Compounds. Adv. Synth. Catal. 2014;356:897.
[7] Lu J, Huang Q. Removal of Acetaminophen Using Enzyme-Mediated Oxidative Coupling Processes:
II. Cross-Coupling with Natural Organic Matter. Environ. Sci. Technol. 2009;43:7068.
[8] Jeon J-R, Baldrian P, Murugesan K, Chang Y-S. Laccase-catalysed oxidations of naturally occurring
phenols: from in vivo biosynthetic pathways to green synthetic applications. Microb. Biotechnol.
2012;5:318.
20
Page 21 of 40

374
375
376
377
378
379
380
381
382
383
384
385
386
387
388
389
390
391
392
393
394
395
396
397
398
399
400
401
402
403
404
405
406
407
408
409
410
[9] Torres-Duarte C, Roman R, Tinoco R, Vazquez-Duhalt R. Halogenated pesticide transformation by
a laccase-mediator system. Chemosphere 2009;77:687.
[10] Purohit CS, Parvez M, Verma S. Kinetic characterization of a bioinspired, heterogeneously active
macromolecular catalyst for phenol oxidation and coupling reactions. Appl. Catal., A 2007;316:100.
[11] Ogata M, Tutumimoto Sato K, Kunikane T, Oka K, Seki M, Urano S, Hiramatsu K, Endo T.
Antibacterial Activity of Dipropofol and Related Compounds. Biol. Pharm. Bull. 2005;28:1120.
[12] Abdel-Mohsen HT, Conrad J, Beifuss U. Laccase-catalyzed synthesis of catechol thioethers by
reaction of catechols with thiols using air as an oxidant. Green Chem. 2014;16:90.
[13] Hollmann F, Arends I, Buehler K, Schallmey A, Buhler B. Enzyme-mediated oxidations for the
chemist. Green Chem. 2011;13:226.
[14] Rulíšek L, Ryde U. Theoretical studies of the active-site structure, spectroscopic and
thermodynamic properties, and reaction mechanism of multicopper oxidases. Coord. Chem. Rev.
2013;257:445.
[15] Burton SG. Laccases and Phenol Oxidases in Organic Synthesis - a Review. Curr. Org. Chem.
2003;7:1317.
[16] Cañas AI, Camarero S. Laccases and their natural mediators: Biotechnological tools for sustainable
eco-friendly processes. Biotechnol. Adv. 2010;28:694.
[17] Jeon J-R, Chang Y-S. Laccase-mediated oxidation of small organics: bifunctional roles for versatile
applications. Trends Biotechnol. 2013;31:335.
[18] Medina F, Aguila S, Baratto MC, Martorana A, Basosi R, Alderete JB, Vazquez-Duhalt R. Prediction
model based on decision tree analysis for laccase mediators. Enzyme Microb. Technol. 2013;52:68.
[19] Roduner E, Kaim W, Sarkar B, Urlacher VB, Pleiss J, Gläser R, Einicke W-D, Sprenger GA, Beifuß
U, Klemm E, Liebner C, Hieronymus H, Hsu S-F, Plietker B, Laschat S. Selective Catalytic Oxidation of
C-H Bonds with Molecular Oxygen. ChemCatChem 2013;5:82.
[20] Kunamneni A, Ballesteros A, Plou FJ, Alcalde M. Fungal laccase - versatile enzyme for
biotechnological applications. In: Méndez-Vilas A, editor. Communicating Current Research and
Educational Topics and Trends in Applied Microbiology, 2007.
[21] Fernandez-Fernandez M, Sanroman MA, Moldes D. Recent developments and applications of
immobilized laccase. Biotechnol. Adv. 2013;31:1808.
[22] Rodríguez Couto S, Toca Herrera JL. Industrial and biotechnological applications of laccases: A
review. Biotechnol. Adv. 2006;24:500.
[23] Imran M, Asad MJ, Hadri SH, Mehmood S. Production and industrial applications of laccase
enzyme. J. Cell Mol. Biol. 2012;10:1.
[24] Witayakran S, Ragauskas AJ. Synthetic Applications of Laccase in Green Chemistry. Adv. Synth.
Catal. 2009;351:1187.
[25] Wenda S, Illner S, Mell A, Kragl U. Industrial biotechnology-the future of green chemistry? Green
Chem. 2011;13:3007.
21
Page 22 of 40

411
412
413
414
415
416
417
418
419
420
421
422
423
424
425
426
427
428
429
430
431
432
433
434
435
436
437
438
439
440
441
442
443
444
445
446
447
[26] Wellington KW, Kolesnikova NI. A laccase-catalysed one-pot synthesis of aminonaphthoquinones
and their anticancer activity. Bioorg. Med. Chem. 2012;20:4472.
[27] Wellington KW, Qwebani-Ogunleye T, Kolesnikova NI, Brady D, de Koning CB. One-Pot Laccase-
Catalysed Synthesis of 5,6-Dihydroxylated Benzo[b]furans and Catechol Derivatives, and Their
Anticancer Activity. Arch. Pharm. (Weinheim) 2013;346:266.
[28] Constantin M-A, Conrad J, Merişor E, Koschorreck K, Urlacher VB, Beifuss U. Oxidative
Dimerization of (E)- and (Z)-2-Propenylsesamol with O2 in the Presence and Absence of Laccases and
Other Catalysts: Selective Formation of Carpanones and Benzopyrans under Different Reaction
Conditions. J. Org. Chem. 2012;77:4528.
[29] Pandey G, Muralikrishna C, Bhalerao UT. Mushroom tyrosinase catalysed coupling of hindered
phenols : A novel approach for the synthesis of diphenoquinones and bisphenols. Tetrahedron Lett.
1990;31:3771.
[30] Mikolasch A, Schauer F. Fungal laccases as tools for the synthesis of new hybrid molecules and
biomaterials. Appl. Microbiol. Biotechnol. 2009;82:605.
[31] Benfield G, Bocks SM, Bromley K, Brown B. Studies of fungal and plant laccases. Phytochemistry
1964;3:79.
[32] Solano F, Lucas-Elı́o P, López-Serrano D, Fernández E, Sanchez-Amat A. Dimethoxyphenol
oxidase activity of different microbial blue multicopper proteins. FEMS Microbiol. Lett. 2001;204:175.
[33] Kohnen S, Halusiak E, Mouithys-Mickalad A, Deby-Dupont G, Deby C, Hans P, Lamy M, Noels AF.
Catalytic activation of copper (II) salts on the reaction of peroxynitrite with propofol in alkaline medium.
Nitric Oxide 2005;12:252.
[34] Wan Y, Du Y, Miyakoshi T. Enzymatic catalysis of 2, 6-dimethoxyphenol by laccases and products
characterization in organic solutions. Sci. China, Ser. B: Chem. 2008;51:669.
[35] Nematollahi D, Momeni S, Khazalpour S. Different strategies in electrochemical synthesis of new
mono and di-substituted hydroquinone and benzoquinone. Electrochim. Acta 2014;147:310.
[36] Wellington KW, Steenkamp P, Brady D. Diamination by N-coupling using a commercial laccase.
Bioorg. Med. Chem. 2010;18:1406.
[37] Tadesse MA, D'Annibale A, Galli C, Gentili P, Sergi F. An assessment of the relative contributions of
redox and steric issues to laccase specificity towards putative substrates. Org. Biomol. Chem.
2008;6:868.
[38] Elsler B, Schollmeyer D, Dyballa KM, Franke R, Waldvogel SR. Metal- and Reagent-Free Highly
Selective Anodic Cross-Coupling Reaction of Phenols. Angew. Chem., Int. Ed. 2014;53:5210.
[39] Kirste A, Elsler B, Schnakenburg G, Waldvogel SR. Efficient Anodic and Direct Phenol-Arene C-C
Cross-Coupling: The Benign Role of Water or Methanol. J. Am. Chem. Soc. 2012;134:3571.
[40] Herter S, Mikolasch A, Michalik D, Hammer E, Schauer F, Bornscheuer U, Schmidt M. C-N coupling
of 3-methylcatechol with primary amines using native and recombinant laccases from Trametes
versicolor and Pycnoporus cinnabarinus. Tetrahedron 2011;67:9311.
22
Page 23 of 40

448
449
450
451
452
453
454
455
456
457
458
459
460
461
462
463
464
465
466
467
468
469
470
471
472
473
474
475
476
477
478
479
480
481
482
483
484
485
[41] Illner S, Plagemann R, Saling P, Kragl U. Eco-Efficiency Analysis as a reaction-engineering tool -
case study of a laccase-initiated oxidative C-N coupling. J. Mol. Catal. B: Enzym. 2014;102:106.
[42] Adelakun OE, Kudanga T, Green IR, le Roes-Hill M, Burton SG. Enzymatic modification of 2,6-
dimethoxyphenol for the synthesis of dimers with high antioxidant capacity. Process Biochem.
2012;47:1926.
[43] Andanson J-M, Baiker A. Exploring catalytic solid/liquid interfaces by in situ attenuated total
reflection infrared spectroscopy. Chem. Soc. Rev. 2010;39:4571.
[44] Vojinović V, Cabral JMS, Fonseca LP. Real-time bioprocess monitoring: Part I: In situ sensors.
Sens. Actuators, B 2006;114:1083.
[45] Roychoudhury P, Harvey LM, McNeil B. The potential of mid infrared spectroscopy (MIRS) for real
time bioprocess monitoring. Anal. Chim. Acta 2006;571:159.
[46] Kornmann H, Rhiel M, Cannizzaro C, Marison I, von Stockar U. Methodology for real-time,
multianalyte monitoring of fermentations using an in-situ mid-infrared sensor. Biotechnol. Bioeng.
2003;82:702.
[47] Mueller JJ, Baum S, Hilterhaus L, Eckstein M, Thum O, Liese A. Simultaneous Determination of
Mono-, Di-, and Triglycerides in Multiphase Systems by Online Fourier Transform Infrared Spectroscopy.
Anal. Chem. 2011;83:9321.
[48] Müller JJ, Neumann M, Scholl P, Hilterhaus L, Eckstein M, Thum O, Liese A. Online Monitoring of
Biotransformations in High Viscous Multiphase Systems by Means of FT-IR and Chemometrics. Anal.
Chem. 2012;82:6008.
[49] Dadd MR, Sharp DCA, Pettman AJ, Knowles CJ. Real-time monitoring of nitrile biotransformations
by mid-infrared spectroscopy. J. Microbiol. Methods 2000;41:69.
[50] Breton J, Boullais C, Burie J-R, Nabedryk E, Mioskowski C. Binding Sites of Quinones in
Photosynthetic Bacterial Reaction Centers Investigated by Light-Induced FTIR Difference Spectroscopy:
Assignment of the Interactions of Each Carbonyl of QA in Rhodobacter sphaeroides Using Site-Specific
13C-Labeled Ubiquinone. Biochemistry (Mosc.) 1994;33:14378.
[51] Song Y, Xie J, Song Y, Shu H, Zhao G, Lv X, Xie W. Calculation of standard electrode potential of
half reaction for benzoquinone and hydroquinone. Spectrochim. Acta, Part A 2006;65:333.
[52] Nonella M, Mathias G, Tavan P. Infrared Spectrum of p-Benzoquinone in Water Obtained from a
QM/MM Hybrid Molecular Dynamics Simulation. J. Phys. Chem. A 2003;107:8638.
[53] Dunkers J, Ishida H. Vibrational assignments of 3-alkyl-3,4-dihydro-6-methyl-2H-1,3-benzoxazines
in the fingerprint region. Spectrochim. Acta, Part A 1995;51:1061.
[54] Coates J. Interpretation of infrared spectra, a practical approach. In: Meyers RA, editor.
Encyclopedia of Analytical Chemistry. Chichester: John Wiley & Sons Ltd, 2000.
[55] Jiang Q, Sheng W, Tian M, Tang J, Guo C. Cobalt(II)–Porphyrin-Catalyzed Aerobic Oxidation:
Oxidative Coupling of Phenols. Eur. J. Org. Chem. 2013;2013:1861.
[56] Yamamura S, Nishiyama S. Anodic Oxidation of Phenols Towards the Synthesis of Bioactive
Natural Products. Synlett 2002;2002:533.
23
Page 24 of 40

486
487
488
[57] Ogata M, Oka K, Seki M, Hoshi M, Takatsu H, Mashino T, Urano S, Endo T. Solubilization of
Dipropofol, an Antibacterial Agent, Using Saccharide and Ascorbic Acid. Biol. Pharm. Bull.
2007;30:1565.
489
490
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Page 25 of 40

490
491 Figure & Scheme Legends
492
493
494
Scheme 1. Possibilities for different selective aryl-aryl couplings according to used catalyst system.
*These quinones can be simple reduced to aromatic biphenol units using Zn/HOAc or
Na2S2O4.[10, 11]
495
496
Scheme 2. Scheme 2. Laccase initiated oxidation of phenolic compounds and possible follow-up
reactions.[17, 29-35]
497
498
499
500
Figure 1.
Figure 2.
Figure 3.
Figure 4.
In situ FT-IR reaction monitoring of the oxidation of 3-MC, HQ and 2,6-DMOP. (A) FT-IR
spectra at the end of oxidation. (B) Changes in the C=O absorption (1615 – 1630 cm-1)
during reaction. Conditions: 50 mL water/methanol (30:70, v/v) acidified with acetic acid (pH
6), 50 mM 2,6-DMOP/ HQ, 20 mM 3-MC, 25 °C, 135 U of N51003, 100 mL min-1 aeration.
501
502
503
504
In situ FT-IR reaction monitoring varying the concentration of 3-MC. (A) 20 mM of 3-MC
(270 min reaction time). (B) 30 mM of 3-MC (150 min). Conditions: 50 mL water/methanol
(30:70, v/v) acidified with acetic acid (pH 6), 25 °C, 337.5 U of N51003, 100 mL min-1
aeration.
505
506
507
508
Correlation of the substrate conversion measured by HPLC (red) with product formation
measured by in situ FT-IR spectroscopy (black). (A) 3-MC. (B) HQ. Conditions: 50 mL
water/methanol (30:70, v/v) acidified with acetic acid (pH 6), 50 mM substrate, 25 °C, 135 U
of N51003, 100 mL min-1 aeration.
509
510
511
512
513
514
In situ FT-IR reaction monitoring of the oxidation of 2,6-disubstituted phenol derivatives. (A)
2,6-DMOP (143 min reaction time). (B) 2,6-DMP (127 min). (C) 2,6-DIPP (78 min).
Conditions for 2,6-DMOP: 50 mL water/methanol (30:70, v/v) acidified with acetic acid (pH
6), 50 mM of 2,6-DMOP, 25 °C, 270 U of N51003, 100 mL min-1 aeration. Conditions for
2,6-DMP/2,6-DIPP: 50 mL water/methanol (50:50, v/v) acidified with acetic acid (pH 6), 50
mM of 2,6-DMP and 2,6-DIPP, 25 °C, 405 U of N51003, 100 mL min-1 aeration.
25
Page 26 of 40

515
Scheme 3. Reaction of the oxidation of 2,6-disubstituted substrate mediated through laccase.
Scheme 4. Biocatalytic oxidative C-C coupling of 2,6-DIPP using 0.01 mol% N51003 (270 U g-1
516
517
substrate
according to the standard activity assay, cf. SI). The observed color change of reaction
mixture during reaction time and the isolated product crystals are shown below the scheme.
518
519
26
Page 27 of 40

519
520
Table 1. Substrate screening via online oxygen monitoring.
Entry Substrate MeOH [a] Structure
[% (v/v)]
Initial oxygen Conversion
521
consumption rate[b] [molsub
[mg L-1∙min-1] molcat-1 min-1]
522
OH
HO
523
207.9
1
3-MC
20
6.7
OH
524
164.4
HO
2
3
4-MC
20
20
5.3
9.4
525
OH
OH
4-tert-BC
291.6
O
2,4-
OH
4
5
6
20
20
20
0
0
HO
DHBAldehyde
HQ[c]
7.4; 0.14
1.7
229.6; 37.2
52.7
HO
OH
OH
O
2,5-
HO
HO
O
DHBAcid
OH
20
5.7
4.9
353.7
60.8
O
7
8
9
2,6-DMOP
50*
OH
OH
OH
2,6-tert-
50*
0.02
0.3
BP
2,6-DIPP
50*
50*
0.6
1.4
7.4
10 2,6-DMP
17.4
[a] Buffered solution (pH 7; 30 mL) containing different volumes methanol
as co-solvent, 5 mM substrate and varying enzyme quantities (nCat, 20% MeOH
= 0.06 µmol; *nCat,
MeOH= 0.30 µmol). [b] Substrate conversion was
50%
calculated on the basis of the reduction of one mol oxygen for concomitant
oxidation of two or four substrate molecules concerning the number of O-H
groups and the used mol of the enzyme. The concentration of enzyme in
the crude extract was determined by Gel electrophoresis and BCA assay
(data shown in SI). [c] HQ showed two different slopes during one
measurement.
27
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525
Table 2. Laccase initiated oxidation of 2,6-disubstituted phenolic
526
compounds.
527
TOF[d]
[g L-1 h-1] [h-1]
Substrate[a]
2,6-DMOP
2,6-DMP
Characteristic
bands[b] [cm-1]
Time
[min]
Yield[c]
[%]
STY
1624, 1609, 1555, 40
1263, 1183
97
77
90
11.2
2.8
6252
2502
1926
1589, 1468, 1183
(overlapping)
100
2,6-DIPP
1584 (overlapping) 130
3.7
[a] Conditions: mixture of 15 mL water acidified with acetic acid (pH 6) and
15 mL methanol, 50 mM of the substrates, 162 U N51003, 100 mL min-1
aeration. [b] Intense absorption bands (in situ FT-IR spectrometer). [c]
Isolated yield. [d] TOF was determined from molSub molcat-1 h-1.
28
Page 29 of 40