Peroxygenase-Catalysed Epoxidation of Styrene Derivatives in Neat Reaction Media

Article abstract of DOI:10.1002/cctc.201901142

Biocatalytic oxyfunctionalisation reactions are traditionally conducted in aqueous media limiting their production yield. Here we report the application of a peroxygenase in neat reaction conditions reaching product concentrations of up to 360 mM.

Full text of DOI:10.1002/cctc.201901142

Accepted Article
Title: Peroxygenase-catalysed epoxidation of styrene derivatives in
neat reaction media
Authors: Marine C. R. Rauch, Florian Tieves, Caroline E Paul, Isabel
WCE Arends, Miguel Alcalde, and Frank Hollmann
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ChemCatChem
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COMMUNICATION
Peroxygenase-catalysed epoxidation of styrene derivatives in
neat reaction media
Marine C. R. Rauch,[a] Florian Tieves, Caroline E. Paul, Isabel W. C. E. Arends, Miguel Alcalde,
[a]
[a]
[b]
[c]
Frank Hollmann *[a]
Abstract: Biocatalytic oxyfunctionalisation reactions are traditionally
conducted in aqueous media limiting their production yield. Here we
report the application of a peroxygenase in neat reaction conditions
reaching product concentrations of up to 360 mM.
catalysed epoxidation reactions using hydrogen peroxide or
organic hydroperoxides as oxidants, albeit in aqueous reaction
media thereby limiting the reagent concentration to the lower
millimolar range.
Klibanov and coworkers reported peroxidase-reactions under
non-aqueous conditions.[10] Unfortunately, these contributions
have not yet found widespread attention in the biocatalysis
community.
Epoxides are important building blocks in organic synthesis.
The ring opening of epoxides leads to useful ɑ- or β-substituted
alcohols.[1] As a result, a broad range of catalytic methods for the
epoxidation of C=C-double bonds have been established.[2]
Compared to this variety, only few biocatalytic methods are known.
The chemoenzymatic epoxidation of alkenes using lipase-borne
peracids for example is receiving tremendous interest but yields
racemic products. Amongst the stereospecific epoxidation
methods the use of flavin-dependent styrene monooxygenases[4]
and P450 monooxygenases[5] are most prominent.
[3]
The latter approaches rely on reductive activation of
molecular oxygen using reduced nicotinamide cofactors
(
NAD(P)H) as source of reducing equivalents. This not only
implies complicated and vulnerable electron transport chains but
also, due to the exclusive water-solubility of the cofactors, largely
limits these processes to aqueous reaction media.
The majority of the alkenes of interest are however
hydrophobic, limiting the final product titres to the lower millimolar
range. This is inacceptable from an economic and an
environmental point-of-view. Current solutions focus around two-
liquid-phase-system approaches (2LPS).[6]
Ideally, (bio)catalytic epoxidation reactions should occur in
organic media (even neat) to enable high product concentrations.
In this respect, peroxygenases represent a promising solution. [7]
Peroxygenases are heme-thiolate enzymes enabling P450
monooxygenase-like oxyfunctionalisation reactions. In contrast to
monooxygenases, peroxygenases do not rely on (water-soluble)
redox partners but on (organic) peroxides, enabling their potential
application in non-aqueous media. Pioneering works by Pu, Wang
and Zhang[8] and Hofrichter[9] have established peroxygenase-
Scheme 1. Comparison of biocatalytic epoxidation reactions.
[
a]
M.C.R. Rauch, Dr. F. Tieves, Dr. C.E. Paul, Prof. Dr. F. Hollmann*
Department of Biotechnology
Delft University of Technology
Van der Maasweg 9, 2629HZ Delft (The Netherlands)
E-mail: f.hollmann@tudelft.nl
Prof. Dr. I.W.C.E. Arends
Faculty of Science
Utrecht University
Budapestlaan 6, 3584CD Utrecht (The Netherlands)
Prof. Dr. M. Alcalde
We therefore set out to establish peroxygenase-catalysed,
selective oxyfunctionalisation reactions in neat reaction media. As
model peroxygenase we chose an evolved recombinant
peroxygenase from Agrocybe aegerita (rAaeUPO) to catalyse
the epoxidation of styrene and its derivatives.[9]
[
[
b]
c]
[11]
t
As oxidant, we chose tert-butyl hydroperoxide ( BuOOH) due
to its high solubility in hydrophobic media.
Department of Biocatalysis
Institute of Catalysis, CSIC
To employ rAaeUPO in neat reaction media, we first
immobilised it covalently on an epoxide-modified polyacrylic
matrix (Immobead IB-COV-1). Under non-optimised conditions,
28049 Madrid (Spain)
Supporting information for this article is given via a link at the end of
the document.
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Table 1. Substrate scope of the epoxidation of styrene derivatives with
immobilised rAaeUPO. Data are an average of duplicates and corrected from
potential substrate evaporation.
(
Figure S1). Further development will have to focus on optimised
immobilisation procedures yielding higher activity.
Nevertheless, having the immobilised enzyme in hand, we
first explored its substrate scope (Table 1). Pleasingly, all of the
styrene derivatives tested were converted with satisfactory to
excellent turnover numbers for the biocatalyst. In accordance
with previous reports[9, 12] wild-type rAaeUPO converted the
majority of styrenes non-stereoselectively giving (near racemic)
epoxides, one notable exception being cis-ß-methyl styrene,
which was converted highly stereoselectively into (1R,2S)-cis-β-
methylstyrene oxide. It is also interesting to note that in some
cases, the desired epoxide was not stable and spontaneously
rearranged into the corresponding carbonyl compound (for
further details see SI section 5.3).
Epoxy
product
Carbonyl
product
Nevertheless, very significant product concentrations of up
to 100 mM were achieved. The catalytic performance of
rAaeUPO in terms of turnover numbers (TON = amount of
product divided by the amount of enzyme, [mol mol ]) was
excellent.
ee
Time
(h)
Substrate
TON[b]
(%)
(mM(%))[
a]
(mM)
-1
1
a
a
16
12
30
2 (12)
42
86
3203
2
80
9
47 (37)
1 (11)
22598
To identify parameters influencing the productivity of the
reaction, we systematically varied the biocatalyst loading and
the tBuOOH feeding rate in the epoxidation of cis-β-
methylstyrene (Figure 1). The initial rate of the epoxidation
3a
35
86
1779
4a
5a
6a
36
59
24
>99
9
2 (4)
21
60
62
6762
14235
22420
t
reaction correlated directly with the dosing rate of BuOOH. This,
21 (26)
102 (81)
however not necessarily translated in higher product titres. Most
probably, increasing feed rates of the oxidant also increased the
50
[13]
undesired oxidative inactivation of the enzyme’s active site.
This is supported by the observation that the robustness of the
reactions (i.e. the duration of product accumulation) inversely
correlated with the BuOOH feed rate (Table S2).
7
8
9
a
a
a
10
8
39
12
17
15
15
90 (90)
22 (73)
12 (43)
22 (59)
25 (69)
49
62
42
22
42
17794
5338
4982
6584
6406
t
16
15
11
6
4
2
0
60
40
20
0
10a
11a
12a
3
42
1 (23)
42
712
1
3a
4a
14
36
-c
60 (81)
59 (62)
62
97
13167
16904
1
28
15a
4
6
25 (86)
49
5160
1
.25
2.5
5
7.5
10
20
5
10
16a
17a
18a
none
136
-
42
-
-
-
0
mM/h mM/h mM/h mM/h mM/h mM/h mM/h mM/h
5
.62 µM
11.24 µM
147 (52)
33 (100)
108
69
50356
5872
none
t
t
Figure 1. Characterisation of BuOOH feeding rate and enzyme concentration
comparing the initial reaction rates (black diamonds) and final product
concentrations (grey squares). General conditions: room temperature, 20 rpm
in overhead rotator. Data presented are an average of duplicates and
corrected from potential substrate evaporation (see SI.section 5.1)
Reaction conditions: [rAaeUPO] = 5.62 µM, BuOOH dosing rate = 5 mM/h,
room temperature, 20 rpm in overhead rotator.
a] The concentrations of carbonyl product were calculated using the calibration
curves of the epoxides. Carbonyl products are aldehyde or ketone in β position
[
from the ring opening of the epoxides
[
[
b] TON = [product]/[enzyme].
c] n.d. = not determined
As mentioned above, epoxides are versatile building blocks
for the synthesis of a broad range of products. Amino alcohols, for
example, are common structural motifs in many pharmaceutically
7
2.8 % of the enzyme was immobilised (for further details see SI
section 4). The remaining catalytic activity, however was only 3%
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COMMUNICATION
active ingredients.[14] We therefore envisioned a chemoenzymatic
methylamine resulting in pseudoephedrine (58.2%), ephedrine
(7%) and isoephedrine (34.8%) (Scheme S2).
cascade
reaction
comprising
the
rAaeUPO-catalysed,
stereoselective epoxidation of cis-β-methylstyrene followed by the
chemical oxirane-opening with methyl amine yielding
Overall, with this contribution we have demonstrated that
peroxygenase-catalysed epoxidations can be performed under
neat reaction conditions. This opens up new possibilities for the
preparative scale-application of this promising class of enzymes.
Product concentrations of up to 360 mM have been achieved
representing one of the highest product titres obtained with
oxidoreductase catalysis[17] and certainly the highest product
concentration with isolated enzymes.[6a, 6b, 18]
(
pseudo)ephedrine (Scheme 2).
Scheme 2. Envisioned chemoenzymatic cascade to obtain
Nevertheless, some issues remain to be solved en route to a
truly preparatively useful system. First and foremost, more active
immobilisates of rAaeUPO need to be found. The activity recovery
of the peroxygenase needs to be improved to obtain more active
catalysts.[19] We are confident that from the wealth of
immobilisation methods available today, a suitable method will
be found in our ongoing research. Also, more enantioselective
rAaeUPO versions are highly desirable to broaden the scope of
the reaction.
(
pseudo)ephedrine from cis-ß-methyl styrene.
The epoxidation reaction was performed on a 10 mL scale
t
with gradual BuOOH feed (Figure 2). Although a conservative
tBuOOH feed rate of 5 mM h-1 was applied, inactivation of the
biocatalyst represented a major challenge for the reaction,
necessitating further provision of the reaction with fresh enzyme
[
20]
(
indicated by arrows in Figure 2). It is also interesting to note that
in contrast to previous experiments using rAaeUPO in aqueous
reaction media using H
,[15] the peroxide utilisation efficiency
2
O
2
was only approximately 50%. It will be interesting to further
investigate this increased catalase activity of rAaeUPO.
Experimental Section
Enzyme preparation. Recombinant expression and purification of the
evolved unspecific peroxygenase mutant from A. aegerita in P. pastoris
was performed following a previously described procedure.[9]
Immobilisation protocol. Immobeads (IB-COV-1) from ChiralVision was
used to immobilise rAaeUPO. The beads were washed before usage and
then mixed with rAaeUPO . Immobilisation was carried out for 5 hours
using overhead rotator. After 5 hours, the immobilisation mixture was
stored at 6 °C for 12 hours without stirring or shaking. After overnight
incubation, the supernatant was removed and the beads 3 times washed.
The washing fractions were pooled. The peroxygenase concentration was
determined via CO difference spectra in the supernatant and the washing
fraction to calculate the amount of immobilised peroxygenase. A detailed
description of the immobilisation of the enzymes is available in the
Supporting Information.
Enzymatic reaction conditions. Reactions were performed in GC vial of
1.5 mL at room temperature. Immobilised rAaeUPO was first weighed in
the vial according to the concentration of enzyme wanted, then pure
substrate was added to the vial. Before each samples were taken, the vial
was weighed in order to estimate the loss of substrate by evaporation.
t
BuOOH was added in the vial via a tube connected to a syringe pump. An
Figure 2. Time-course of the rAaeUPO-catalysed epoxidation of cis-β-
methylstyrene. Conditions: 10 mL cis-β-methylstyrene, [rAaeUPO@IB-COV-
overhead rotator from neoLab was mixing the reactions at 20 rpm. At
intervals, aliquots were withdrawn, extracted with ethyl acetate, dried over
MgSO and analysed by chiral gas chromatography. Details of gas
4
chromatography and temperature profiles are shown in Supporting
Information.
1]
total = 41.8 µM (added in equal portions at the times indicated by the arrows),
t
room temperature. BuOOH was added continuously according to the feed
profile shown as a dashed grey line.
Chemical ring opening. 10 mg of pure epoxide were diluted in 200 µL of
From this experiment, 360 mM of (1R,2S)-β-methylstyrene
oxide were obtained. The turnover number of the enzyme was
more than 8500. Next to the desired product, the reaction mixture
also contained some benzaldehyde, originating from rAaeUPO-
catalysed C=C-bond cleavage.[16] To avoid negative influences of
°
MeNH
2
(40% in water). The reaction was mixed during 20 hours at 60 C.
The reaction was then extracted with dichloromethane and analysed on
NMR.
this
by-product,
the desired product
was
purified
Acknowledgements
chromatographically and subjected to chemical ring-opening with
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Financial support by the European Research Council (ERC
Consolidator Grant No. 648026) is gratefully acknowledged.
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Keywords: Biocatalysis • Epoxidation • Peroxygenases • Neat
reaction conditions • Cascade reactions
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Entry for the Table of Contents
Layout 2:
COMMUNICATION
Marine C. R. Rauch, Florian Tieves,
Caroline E. Paul, Isabel W. C. E.
Arends, Miguel Alcalde, Frank Hollmann
*
Page No. – Page No.
Peroxygenase-catalysed epoxidation
of styrene derivatives in neat reaction
media
Neat! Peroxygenases catalyse epoxidation of C=C-double bonds under non-
aqueous conditions.
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