Multigram Scale Enzymatic Synthesis of (R)-1-(4′-Hydroxyphenyl)ethanol Using Vanillyl Alcohol Oxidase

Article abstract of DOI:10.1002/adsc.201800197

The enantioselective oxyfunctionalisation of C?H bonds is a highly interesting reaction, as it provides access to chiral alcohols that are important pharmaceutical building blocks. However, it is hard to achieve using traditional methods. One way in which it can be achieved is through the action of oxidative enzymes. Although many reports of the oxyfunctionalisation capabilities of enzymes at an analytical scale have been published, reports on the use of enzymes to achieve oxyfunctionalisation on a synthetically relevant scale are fewer. Here, we describe the scale-up of the conversion of 4-ethylphenol to (R)-1-(4′-hydroxyphenyl)ethanol using the flavin-dependent enzyme vanillyl alcohol oxidase. The process was optimised by testing different reaction media and substrate and enzyme concentrations and by performing it under an oxygen atmosphere. Under optimised reaction conditions, 4.10 g (R)-1-(4′-hydroxyphenyl)ethanol at 97% ee was obtained from 10 g 4-ethylphenol (isolated yield 36%). These results highlight some of the challenges that can be encountered during scale-up of an enzymatic oxyfunctionalisation process to a synthetically relevant scale and will be of use for the development of enzymatic processes for the synthesis of industrially relevant compounds. (Figure presented.).

Full text of DOI:10.1002/adsc.201800197

Title: Multigram Scale Enzymatic Synthesis of (<i>R</i>)-1-(4ʹ-
Hydroxyphenyl)ethanol Using Vanillyl Alcohol Oxidase
Authors: Tom Ewing, Jasmin Kuehn, Silvia Segarra, Marta Tortajada,
Ralf Zuhse, and Willem van Berkel
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To be cited as: Adv. Synth. Catal. 10.1002/adsc.201800197
Link to VoR: http://dx.doi.org/10.1002/adsc.201800197

Advanced Synthesis & Catalysis
10.1002/adsc.201800197
UPDATE
DOI: 10.1002/adsc.201((will be filled in by the editorial staff))
Multigram Scale Enzymatic Synthesis of (R)-1-(4ʹ-
Hydroxyphenyl)ethanol Using Vanillyl Alcohol Oxidase
a
b
c
c
b
Tom A. Ewing , Jasmin Kühn , Silvia Segarra , Marta Tortajada , Ralf Zuhse ,
a*
Willem J. H. van Berkel
a
Laboratory of Biochemistry, Wageningen University & Research, Stippeneng 4, 6708 WE Wageningen, The
Netherlands
Phone: +31 317 482861, Fax: +31 317 484801, E-mail: willem.vanberkel@wur.nl
Chiracon GmbH, Biotechnologiepark, 14943 Luckenwalde, Germany
Biopolis S. L., Parc Científic de la Universitat de València, Edificio 2, C/Catedrático Agustín Escardino 9, 46980
b
c
Paterna, Spain
Received: ((will be filled in by the editorial staff))
Supporting information for this article is available on the WWW under http://dx.doi.org/10.1002/adsc.201######.((Please
delete if not appropriate))
Abstract. The enantioselective oxyfunctionalisation of C- introducing oxygen atoms, which often display
H bonds is a highly interesting reaction, as it provides exquisite regio- and enantioselectivity and are active
access to chiral alcohols that are important pharmaceutical under mild reaction conditions. Well-known classes
building blocks. However, it is hard to achieve using of oxygenating enzymes include heme-dependent
traditional methods. One way in which it can be achieved is
through the action of oxidative enzymes. Although many
reports of the oxyfunctionalisation capabilities of enzymes
at an analytical scale have been published, reports on the
use of enzymes to achieve oxyfunctionalisation on a
synthetically relevant scale are fewer. Here, we describe
the scale-up of the conversion of 4-ethylphenol to (R)-1-
cytochrome P450 enzymes and peroxygenases, non-
heme iron oxygenases and flavin-dependent
[3–7]
oxygenases . Some of these enzymes have been
utilised to obtain valuable products from preparative-
[8–12]
scale transformations
. For many others, however,
our knowledge of their oxyfunctionalisation
capabilities is limited to the analytical scale. In order
to stimulate the use of oxidoreductases in industrial
chemical synthesis, it is crucial to demonstrate that
they can be used to obtain compounds on a
(
4′-hydroxyphenyl)ethanol using the flavin-dependent
enzyme vanillyl alcohol oxidase. The process was
optimised by testing different reaction media and substrate
and enzyme concentrations and by performing it under an
oxygen atmosphere. Under optimised reaction conditions,
[13]
synthetically relevant scale
.
Chiral alcohols, which are often important
intermediates in the synthesis of active
4
.10 g (R)-1-(4′-hydroxyphenyl)ethanol at 97% ee was
obtained from 10 g 4-ethylphenol (isolated yield 36%).
These results highlight some of the challenges that can be
encountered during scale-up of an enzymatic
oxyfunctionalisation process to a synthetically relevant
pharmaceutical ingredients, present an interesting
target for these preparative-scale transformations[14].
One class of alcohols that are important
scale and will be of use for the development of enzymatic pharmaceutical building blocks are chiral α-aryl
processes for the synthesis of industrially relevant alcohols
compounds.
[15,16]
. A number of biocatalytic approaches
for their synthesis have been described, including
enantioselective reduction of a prochiral ketone,
kinetic resolution or deracemisation of racemic
alcohols and the enantioselective hydration of
Keywords: Alcohols; Alkylphenols; Enantioselectivity;
Flavoprotein; Hydroxylation; Oxidoreductases
[17–22]
vinylphenol derivatives
. Another potential
enzymatic synthesis route for chiral α-aryl alcohols is
the direct oxyfunctionalisation of alkyl-substituted
aromatic molecules. An enzyme that can be used for
this purpose is vanillyl alcohol oxidase from
Introduction
The selective oxyfunctionalisation of C-H bonds
under mild reaction conditions is of great interest to
synthetic organic chemists. In recent years, much
effort has been put into developing metal-based
catalysts that can introduce oxygen atoms using
environmentally benign oxidants such as molecular
oxygen[ . An attractive alternative to traditional
chemical catalysts is the use of enzymes capable of
Penicillium simplicissimum (VAO, EC 1.1.3.38),
which is capable of catalysing the enantioselective
hydroxylation of 4-alkylphenols. Here, we describe
the development of the multigram scale enzymatic
synthesis of (R)-1-(4ʹ-hydroxyphenyl)ethanol using
VAO. This compound may be of use for the synthesis
of bioactive molecules containing 1-(4′-
1,2]
hydroxyphenyl)ethanol moieties. For example,
1
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Advanced Synthesis & Catalysis
10.1002/adsc.201800197
racemic 1-(4′-hydroxyphenyl)ethanol has been
employed as an intermediate in the synthesis of
artemisinin derivatives that display
immunosuppressive activity, though the bioactive
enantiomer was not defined[
.
23]
VAO catalyses the oxidation of a wide range of
para-substituted phenols at their Cα atom using
[24,25]
molecular oxygen as an electron acceptor
. VAO
displays a broad substrate scope, catalysing the
oxidation of benzylic alcohols or amines, the
oxidative demethylation of 4-(methoxymethyl)phenol
and the hydroxylation of 4-allylphenols. In addition,
VAO oxidises phenols bearing unfunctionalised alkyl
chains as the para-substituent. This leads to mixtures
of hydroxylated and dehydrogenated products, with
the ratio between the two being determined by the
[26,27]
Scheme 2. Mechanism of the enantioselective
hydroxylation of 4-ethylphenol by VAO. First, 4-
ethylphenol binds in the enzyme’s active site in its
phenolate form. Following this, a hydride is transferred
from the Cα atom of its ethyl group to the N5 atom of FAD,
yielding a para-quinone methide intermediate and reduced
FAD. Subsequently, a water molecule is activated by Asp-
size of the alkyl chain
and the composition of the
reaction medium[ . The hydroxylation of short chain
28]
4
-alkylphenols proceeds with high regio- and
enantioselectivity. The conversion of 4-ethylphenol,
4
-propylphenol or 2-methoxy-4-propylphenol by
VAO yields the corresponding 1-(4′-
hydroxyphenyl)alcohols as the sole hydroxylation
product, in addition to more minor amounts of vinylic
side-products from dehydrogenation of the substrate
1
70 and attacks the Cα atom of the para-quinone methide
intermediate, yielding the alcohol product. Stopped-flow
kinetics analysis has demonstrated that this occurs after (or
possibly concomitant to) the reoxidation of FAD by
and ketone side-products from further oxidation of the
formed alcohols (Scheme 1)[29]. The hydroxylation
[
31]
molecular oxygen . The position of Asp-170 relative to
the planar para-quinone methide intermediate explains the
preferential formation of the (R)-enantiomer of 1-(4ʹ-
hydroxyphenyl)ethanol.
reaction is highly enantioselective, with the (R)-
enantiomers of the alcohol product being formed at
94% ee in all cases.
quinone methide intermediate, which is subsequently
hydrated in the enzyme‘s active site. The
enantioselectivity of the hydration is determined by
the presence of an active site base (Asp-170) that
activates water for attack at one side of the planar
para-quinone methide intermediate. Accumulation of
the (R)-enantiomer is further enhanced by the fact that
the (S)-enantiomer of 1-(4′-hydroxyphenyl)ethanol is
more easily oxidised further to 4-
hydroxyacetophenone than the (R)-enantiomer is[26].
A VAO variant (D170S/T457E) where the active site
base was moved to the other side of the substrate
Scheme 1. Conversion of 4-ethylphenol (1) by VAO. displayed reversed enantioselectivity, converting 4-
Thickness of the arrows gives a qualitative indication of the ethylphenol to (S)-1-(4′-hydroxyphenyl)ethanol at
[33]
relative rates at which the depicted reactions occur. VAO 80% ee . Its ability to catalyse regio- and
readily
hydroxyphenyl)ethanol
converts
4-ethylphenol
to
with
(R)-1-(4′- enantioselective hydroxylations using only molecular
(S)-1-(4ʹ- oxygen as a cosubstrate makes VAO an interesting
(2),
hydroxyphenyl)ethanol (3) and 4-vinylphenol (4) being candidate for biocatalytic applications.
formed as side-products. The formed alcohol can
The synthetic utility of VAO at small scale has
subsequently be oxidised further to
4- been demonstrated previously. The conversion of 4-
hydroxyacetophenone (5), with the (S)-enantiomer being ethylphenol and 2-methoxy-4-propylphenol was
more readily converted than the (R)-enantiomer. Scheme performed at 100 mg scale, yielding 33.2 mg (28%)
adapted from[
26]
.
and 52 mg (45%) respectively of the corresponding
[29]
purified alcohols . To evaluate the potential of VAO
to be used as a biocatalyst for the synthesis of chiral
The catalytic mechanism of VAO provides an
explanation for this regio- and enantioselectivity[30–32]. alcohols on a more synthetically relevant scale, we
Binding of the phenolate form of the substrate to the
scaled up the conversion of 4-ethylphenol to the 10 g
active site is followed by hydride transfer from the Cα scale.
atom of the substrate to the N5 atom of the FAD
cofactor (Scheme 2). This yields a planar para-
2
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Advanced Synthesis & Catalysis
10.1002/adsc.201800197
Table 1. Products obtained from the conversion of 4-ethylphenol (1) by VAO in the presence of organic solvents. 5 mg 1
at a concentration of 2.9 mM) and 0.4 U VAO were incubated in 84 mM potassium phosphate buffer, pH 7.5, at 30 °C in
(
the presence of 10% acetone or 10% acetonitrile. After the incubation period, reaction products were extracted and
analysed by GC. Compounds are numbered as in Scheme 1. GC chromatograms are shown in Fig. S1. The given numbers
are the amount of a compound as a percentage of the total present upon analysis.
Reaction conditions
0% acetone, 4 h
0% acetonitrile, 22h
1 (%) 2 (%) 3 (%) 2-ee (%) 4 (%) 5 (%)
1
-
83
76
0.7
2.5
98
94
14
14
3.0
1.3
1
5.9
Results and Discussion
Therefore, 23 mM substrate with 0.02 U VAO/mg
substrate was chosen as the conditions for scale-up.
As the costs associated with protein purification
could be a major hurdle for the use of enzymatic
conversions in an industrial setting, we subsequently
tested whether we could eliminate this step by
performing the reaction using a cell-free extract of
Escherichia coli expressing an N-terminally His-
tagged variant of VAO. The plasmid encoding for this
His-tagged VAO, pJ404-His-VAO, contains a version
of the gene encoding for VAO that is codon-
Optimisation of Reaction Conditions
As a first step in the scale-up process, we optimised
the reaction conditions for the conversion of 4-
ethylphenol (1) at an analytical scale by testing the
effect of the addition of organic solvents to the
reaction medium. To this end, the conversion of 5 mg
1
was performed in 84 mM potassium phosphate
buffer, pH 7.5, in the presence of 10% acetone or
acetonitrile. The composition of reaction mixtures
after the reaction as determined by gas
optimised for expression in E. coli. This construct
allows VAO to be expressed at higher levels than the
[35]
chromatography is given in Table 1. Using acetone as
a cosolvent gave the best results, with complete
conversion of 1 and 83% of the formed product being
previously used plasmid, pBC11 , which contains
the vaoA gene from P. simplicissimum. Using this
cell-free extract, the conversion of 100 mg 1 gave a
similar result to that using the purified enzyme (Table
2, reaction c). Apparently, the use of E. coli cell-free
extract did not lead to any undesired side reactions
taking place. Therefore, the cell-free extract was used
for further scale-up reactions.
(
R)-1-(4′-hydroxyphenyl)ethanol (2) after 4 h. The
formation of 2 is underestimated, as part of the 1-(4ʹ-
hydroxyphenyl)ethanol undergoes elimination of
water during the gas chromatographic analysis,
yielding 4-vinylphenol (4) [as confirmed by GC
analysis of racemic pure 1-(4′-
hydroxyphenyl)ethanol]. The addition of acetone
likely improves the solubility of the aromatic
substrate and products. In addition, the solubility of
oxygen in acetone is increased as compared to in
water [277 mg/L in acetone at 25 °C and 44 mg/L in
Scale-up to 10 g Scale
Having established the optimised reaction conditions
for the conversion of 1 to 2, we next set out to scale
up the reaction to a more synthetically relevant scale
of 10 g of substrate. First, the reaction was scaled up
to 1 g of substrate using the same reaction conditions
as for 100 mg. This led to almost complete
[34]
water at 20 °C at 1 bar pure oxygen atmosphere ],
preventing oxygen depletion from becoming limiting
for the reaction. Although the solubility of the
substrates may be increased further by using even
higher concentrations of acetone, its presence led to
significant inhibition of the enzyme. The rate of
conversion of 2 mM vanillyl alcohol to vanillin by
VAO in 50 mM potassium phosphate buffer, pH 7.5,
conversion (93%) of 1 and to similar formation of 2
as was obtained at 100 mg scale (Table 2, reaction d).
1
Analysis of the reaction products by H-NMR
allowed quantification of the products without being
hindered by the decomposition of 1-(4′-
-1
at 25 °C dropped from 2.2 s in the absence of
hydroxyphenyl)ethanol to 4 observed during GC
-1
acetone to 0.73 s in the presence of 10% acetone and analysis, revealing that the enzymatic reaction yields
-1
0
.12 s in the presence of 20% acetone. With this in
less than 3% of 4 as a side-product (Table 2, reaction
d). To obtain pure 2, it was purified from the crude
reaction mixture by recrystallisation, yielding 244.3
mg (21%) pure 2 at 99% ee (Fig. S3+S4).
Subsequently, the reaction was scaled up to our
final target scale of approximately 10 g of starting
material. However, using the same reaction conditions
with 13.4 g 1 led to lower conversion of the substrate
[63% (Table 2, reaction e)]. TLC analysis revealed
that the reaction was essentially complete after 9 h,
with no further product formation being observed
mind, scale-up was performed using the reaction
medium containing 10% acetone.
Scaling up the reaction to 100 mg scale with
increased substrate concentrations revealed that
almost complete conversion (97%) could be achieved
using a substrate concentration of 23 mM and 2 U
VAO (Table 2, reaction a). Increasing the substrate
concentration further to 45 mM led to lower
conversion (87%) even though the same enzyme to
substrate ratio was used (Table 2, reaction b).
3
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Advanced Synthesis & Catalysis
10.1002/adsc.201800197
Table 2. Products obtained from the conversion of 4-ethylphenol (1) by VAO. 1 and VAO or His-VAO cell-free extract
were incubated in 88 mM potassium phosphate buffer, pH 7.5, containing 10% acetone at 30 °C for 24 h. Subsequently,
1
reaction products were extracted and analysed by GC or H-NMR. Compounds are numbered as in Scheme 1. GC
1
chromatograms are shown in Fig. S2, H-NMR spectra in Fig. S4. The given numbers are the amount of a compound as a
percentage of the total present upon analysis. CFE: cell-free extract.
Reaction
a
Substrate
100 mg
Enzyme
2 U VAO
1 (%) 2 (%) 3 (%) 2+3 (%) 2-ee (%) 4 (%) 5 (%)
GC
GC
3.1
83
58
74
83
4.0
4.1
2.1
1.3
91
87
95
97
8.3
1.5
1.0
0.9
0.8
1.8
1.1
1.0
1.8
2
3 mM
100 mg
5 mM
100 mg
3 mM
1.0 g
b
c
d
2 U VAO
13
24
4
GC
2 U His-VAO
CFE
20 U His-VAO
CFE
0.8
6.5
7.5
37
23
2
GC
8.6
2.6
9.2
6.5
2.7
2
3 mM
1H-NMR
GC
88
89
e
13.4 g
268 U His-VAO
CFE
202 U His-VAO
CFE
51
82
1.9
4.1
93
90
2
3 mM
f [a]
GC
10.0 g
6.0
6.3
2
3 mM
1H-NMR
[
a]
2
Reaction vessel was kept under pure O atmosphere for the first 9 h
after this time. Possibly, the reaction does not proceed mixture (6.51 g crude product obtained from 10 g
further due to inactivation of the enzyme. As the large starting material) and during recrystallisation. The
volume used for this reaction could lead to oxygen
depletion becoming rate-limiting, we performed the
reaction with 10 g 1 under identical conditions, but
with the reaction vessel under oxygen atmosphere for
the first 9 h of the reaction to try and increase the
conversion of the substrate. Under these conditions,
the substrate was almost completely converted after
loss of material during the extraction procedure may
be attributable to the relatively high polarity of the
product making its extraction into organic solvents
inefficient. This may be solved in future by testing
other solvents for extraction or by performing more
rounds of extraction. From the GC data, it can be
calculated that the crude compound obtained after
extraction contains 5.34 g 2, which corresponds to a
crude yield of 47%. This means that a further 23% of
the product was lost during its purification by
24 h [94% (Table 2, reaction f)]. TLC analysis again
revealed that the reaction was essentially complete
after 9 h under oxygen atmosphere. Extraction of the
reaction mixture with ethyl acetate yielded 6.51 g
crude product. Based on the 82% formation of 2
recrystallisation. Although this loss is significant,
recrystallisation is likely the most efficient method to
observed in the GC experiments, a crude yield of 5.34 increase the ee of the product by removing the (S)-1-
g (47%) 2 can be estimated. After recrystallisation,
pure 2 was obtained in 36% isolated yield and 97% ee
(4′-hydroxyphenyl)ethanol formed as a side-product.
1-(4′-Hydroxyphenyl)ethanol can also be
(
Fig. S3+S4). The observation that maintaining
biocatalytically synthesised by the hydration of 4-
vinylphenol using phenolic acid decarboxylase
sufficient oxygen levels is crucial to allow the
reaction to proceed is in agreement with previous
studies regarding biotransformations using
[20,22]
(PAD)
. All tested PADs preferentially yielded
the (S)-enantiomer of 1-(4′-hydroxyphenyl)ethanol,
providing a potential route to the opposite enantiomer
than that formed by VAO.
monooxygenase-containing whole-cell biocatalysts,
where oxygen depletion was found to be limiting for
[
8,36,37]
the reaction under certain conditions
. Although
we were able to maintain sufficient oxygen levels by
performing the reaction under an oxygen atmosphere, Conclusion
this may not be suitable for an industrial-scale
reaction as the use of pure oxygen poses an explosion
hazard. Use of alternative systems to achieve
sufficient oxygen levels, such as bubble aeration
could provide a solution for industrial-scale
Here, we described the scale-up of the synthesis of
(R)-1-(4ʹ-hydroxyphenyl)ethanol by the
enantioselective hydroxylation of 4-ethylphenol by
VAO. Under optimised reaction conditions, 94%
conversion of 10 g 4-ethylphenol was obtained after
24 h. Purification of the product yielded (R)-1-(4ʹ-
hydroxyphenyl)ethanol in 36% isolated yield and
97% ee. These results highlight some of the issues
that may be encountered during scale-up of an
enzymatic hydroxylation reaction from analytical
transformations[ . One aspect of the process that can
38]
be improved upon is the fact that, despite observing
8
2% formation of 2 in the largest-scale reaction, the
isolated yield is relatively low at 36%. This low yield
is attributable to significant losses of material during
both the extraction of the product from the reaction
4
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Advanced Synthesis & Catalysis
10.1002/adsc.201800197
scale to a synthetically relevant scale and may be of
use in facilitating such scale-up for the synthesis of
chiral pharmaceutical intermediates in an industrial
setting.
methoxybenzyl alcohol) to vanillin (4-hydroxy-3-
methoxybenzaldehyde) at 25 °C in 50 mM potassium
phosphate buffer, pH 7.5, by measuring the absorption of
-
1
-1 [24]
the product at 340 nm (ε = 14,000 M cm
Hewlett Packard 8453 diode array spectrophotometer
Agilent Technologies, Santa Clara, CA, USA).
) using a
(
Experimental Section
Gas Chromatography
Materials
Gas chromatography experiments were performed on a
GC-2010 plus (Shimadzu, Kyoto, Japan) with an FID
detector (260 °C) and a Hydrodex β-6TBDM column (25 m
x 2.45 mm, Machery-Nagel, Düren, Germany). Hydrogen
was used as a carrier gas at a flow rate of 1.5 mL/min and
the injector temperature was 80 °C. Runs were performed
using a 20 min gradient from 80 to 220 °C followed by 10
min at 220 °C.
4
-Ethylphenol and 4-hydroxyacetophenone were from abcr
(
Karlsruhe, Germany). An analytical standard for 1-(4′-
hydroxyphenyl)ethanol was synthesised as a racemic
mixture from 4-hydroxyacetophenone:
A solution of 4-hydroxyacetophenone (3.4 g, 25 mmol)
in MeOH (50 mL) was stirred at 15-20 °C and a solution of
4
NaBH (0.57 g, 15 mmol) in 0.2 M aqueous NaOH (10
mL) was added. Subsequently, the reaction mixture was
stirred at room temperature for 20 h. After this, MeOH was
removed by evaporation and water (10 mL) was added to
the residue. The mixture was extracted with
Analytical-Scale Conversions of 4-Ethylphenol
1
(5 mg, 41 μmol), VAO (0.4 U, 0.8 mL of a 0.5 U/mL
CHCl
over Na
3
/isopropanol (3/1) and the organic layer was dried
SO and the solvent was removed by evaporation.
solution in 50 mM potassium phosphate buffer, pH 7.5) the
specified organic solvent (1.4 mL) and 100 mM potassium
phosphate buffer, pH 7.5, (11.8 mL) were incubated at
2
4
The residue (2.2 g, yellow solid) was chromatographed on
silica gel with dichloromethane/MeOH (50/1), yielding
pure (R,S)-1-(4′-hydroxyphenyl)ethanol (1.45 g, white solid,
3
0 °C. The reaction was performed in a 25 mL flask that
was open to air and the reaction mixture was stirred using a
magnetic stirrer. The stirrer speed was adjusted so as to
form a whirlpool in the solution to achieve maximal
aeration. After the reaction, the solution was concentrated
under reduced pressure. The residue was extracted with
EtOAc (4 x 5 mL). The organic layer was dried over
Na SO and filtered, and the solvent was removed by
2 4
evaporation. The residue was dissolved in MeOH (1 mL)
and analysed by GC.
Scale-up to the 100 mg scale was performed using the
same procedure, but with the volume of the reaction
mixture adapted to achieve the desired substrate
concentration and using 2 U VAO (or His-VAO cell-free
extract). Reactions were performed in 100 mL flasks that
were open to air.
4
2%). The elution times of the enantiomers upon gas
chromatography analysis were assigned by comparison
with 1-(4′-hydroxyphenyl)ethanol formed by conversion of
1
by VAO [primarily (R)-enantiomer].
1
H-NMR (d
OH), 7.12 (dt, J = 8.02 Hz, 2H, Ar-H), 6.68 (dt, J = 8.69 Hz,
H, Ar-H), 4.85 (d, J = 4.29 Hz, 1H, aliph.-OH), 4.63-4.57
6
-DMSO, 400MHz): δ (ppm): 9.10 (s, 1H, Ar-
2
(
m, 1H, CH), 1.27 (d, J = 6.44 Hz, 3H, CH3).
All other chemicals were from commercial sources and
of the purest grade available.
Expression and Purification of VAO
For small-scale tests with purified enzyme, VAO was
expressed and purified as described previously[39]. For
larger-scale reactions using cell-free extracts, N-terminally
His-tagged VAO was expressed from the pJ404-His-VAO
plasmid, which contains a version of the vaoA gene from P.
simplicissimum that is codon-optimised for expression in E.
coli behind an IPTG-inducible T5 promoter. BL21 E. coli
cells containing this plasmid were grown at 37 °C in LB
medium containing 100 μg/mL ampicillin until the OD600
of the cultures was 0.6. Subsequently, IPTG was added to a
concentration of 0.8 mM and the cells were grown
Synthesis of (R)-1-(4′-Hydroxyphenyl)ethanol at 1
g Scale
1
(1 g, 8.2 mmol) was dissolved in acetone (34.5 mL). 100
mM potassium phosphate buffer, pH 7.5, (296 mL) and
His-VAO cell-free extract (20 U, 14.3 mL of a 1.4 U/mL
solution) were added to the solution. Reactions were
performed in a 1 L flask that was open to air and the
reaction mixture was stirred using a magnetic stirrer. The
stirrer speed was adjusted so as to form a whirlpool in the
solution to achieve maximal aeration. The reaction solution
was stirred for 24 h at 30 °C (water bath) under air
atmosphere. After 24 h, the solution was concentrated
under reduced pressure at 50 °C (water bath). During
concentration, a slimy white solid was formed. The
suspension was filtered and the aqueous phase was
extracted with EtOAc. The organic phase was washed with
overnight at 25 °C. Following this, cells were harvested by
centrifugation (4,000 g, 10 min, 10°C), resuspended in 50
mM potassium phosphate buffer, pH 7.0, containing 0.5
TM
4
mM dithiothreitol, 0.5 mM MgSO and one cOmplete
protease inhibitor pill (Roche, Basel, Switserland) and 1
mg DnaseI (Roche) per 50 mL, and lysed by passing them
through a pre-cooled SLM Aminco French Pressure Cell
three times. Subsequently, cell debris was removed by
centrifugation (39,000 g, 45 min, 4 °C) and the obtained
soluble fractions were used for the reactions.
2 4
brine, dried over Na SO and concentrated under reduced
pressure to give a crude compound (502 mg, green beige
VAO activity was determined by monitoring the
conversion of vanillyl alcohol (4-hydroxy-3-
1
solid). This compound was analysed by GC and H-NMR
to evaluate the success of the conversion. Subsequently, the
5
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Advanced Synthesis & Catalysis
10.1002/adsc.201800197
crude product was purified by recrystallisation. EtOAc (3
mL) was added to the crude product and the suspension
was heated to 80 °C (oil bath) until all solid was dissolved.
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dried under reduced pressure at 50 °C (water bath) to give
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5
0, 13180–13200.
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2
4
[
[
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19]
crude compound (6.51 g, green beige solid). This
1
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Acknowledgements
We would like to thank Prof. Dr. Marco Fraaije (University of
Groningen, The Netherlands) for supplying the pJ404-His-VAO
plasmid and Dr. Caroline Paul (Wageningen University &
Research) for critically reading the manuscript. This work was
funded by the European Union under the INDOX project (FP7-
KBBE-2013-7-613549).
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7
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Advanced Synthesis & Catalysis
10.1002/adsc.201800197
UPDATE
Multigram Scale Enzymatic Synthesis of (R)-1-(4ʹ-
Hydroxyphenyl)ethanol Using Vanillyl Alcohol
Oxidase
Adv. Synth. Catal. Year, Volume, Page – Page
Tom A. Ewing, Jasmin Kühn, Silvia Segarra, Marta
Tortajada, Ralf Zuhse, Willem J. H. van Berkel
8
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