Enantioselective Sulfoxidation of Thioanisole by Cascading a Choline Oxidase and a Peroxygenase in the Presence of Natural Deep Eutectic Solvents

Article abstract of DOI:10.1002/cplu.201900751

A bienzymatic cascade for selective sulfoxidation is presented. The evolved recombinant peroxygenase from Agrocybe aegeritra catalyses the enantioselective sulfoxidation of thioanisole whereas the choline oxidase from Arthrobacter nicotianae provides the

Full text of DOI:10.1002/cplu.201900751

Accepted Article
Title: Enantioselective Sulfoxidation of Thioanisole by Cascading a
Choline Oxidase and a Peroxygenase in the presence of Natural
Deep Eutectic Solvents
Authors: Yongru Li, Yunjian Ma, Peilin Li, Xizhen Zhang, Doris
Ribitsch, Miguel Alcalde, Yonghua Wang, and Frank
Hollmann
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Enantioselective Sulfoxidation of Thioanisole by Cascading a Choline
Oxidase and a Peroxygenase in the presence of Natural Deep Eutectic
Solvents
Yongru Li,[a] Yunjian Ma,[a] Peilin Li,[a] Xizhen Zhang,[b] Doris Ribitsch,[c] Miguel Alcalde,[d] Frank
Hollmann,*[e] and Yonghua Wang*[a]
Abstract: A bienzymatic cascade for selective sulfoxidation is
presented. The evolved recombinant peroxygenase from Agrocybe
aegeritra catalyses the enantioselective sulfoxidation of thioanisole
whereas the choline oxidase from Arthrobacter nicotianae provides
the H2O2 necessary via reductive activation of ambient oxygen. The
reactions are performed in choline chloride-based deep eutectic
solvents serving as co-solvent and stoichiometric reductant at the
same time. Very promising product concentrations (up to 15 mM
enantiopure sulfoxide) and catalyst performances (TNs of 150000 and
2100 for the peroxygenase and oxidase, respectively) have been
achieved.
One issue that needs to be addressed in biocatalytic
sulfoxidation reactions is the poor solubility of most thiols in
aqueous media, preferred in biocatalysis. Thioanisole, for
example, is soluble in to approximately 4 mM. As a consequence,
the majority of biocatalytic reports on thioanisole sulfoxidation use
such low reagent concentrations, which is not attractive neither
from an economic nor an environmental point-of-view.[7]
In recent years, (natural) deep eutectic solvents ((NA)DES)
have enjoyed an increasing popularity in biocatalysis.[8]
Particularly, their often stabilizing effect on enzymes but also their
solubilisation of less hydrophilic reagents are of interest.
Especially choline chloride (ChCl) is the basis of many (NA)DES.
Recently, we have demonstrated that ChCl can be used not only
as solvent component but also as sacrificial electron donor for the
reductive activation of molecular oxygen by the enzyme choline
oxidase from Arthrobacter nicotianae (AnChOx) to promote
Chiral sulfoxides are widely found in drugs, chiral intermediates
as well as chiral ligands in catalysis.[1] Since the first report by
Märcker in 1865 on the HNO3-promoted sulfoxidation aromatic
sulfides,[2] many further synthetic methodologies have been
added to the portfolio including transition metal catalysis, peracids
and hydrogen peroxide or hypohalites.[3] Nevertheless, the issue
of overoxidation to the (mostly undesired) sulfone remains to be
solved satisfactorily. More importantly, enantioselective methods,
highly desired especially for products in the pharmaceutical and
agrochemical sectors are still scarce.[3c, 4] To address the latter
challenge, biocatalysis is of particular interest due to the often
inherent enantioselectivity of enzymes.[5] Therefore, it is not very
astonishing that chiral sulfoxidation (of benzyl methyl sulfide,
thioanisole) is a common model reaction for monooxygenases
and peroxidases.[6]
perhydrolase-[9]
and
peroxygenase-catalysed
oxyfunctionalisation reactions.[10] Inspired by the recent
contribution by Brenna and coworkers on the peroxygenase-
catalysed sulfoxidation of thiols[6] we became interested in
expanding this reaction concept to ChCl-based DES exploiting
the dual functionality of these DES as co-solvent and as co-
substrate (Figure 1 and Figure 2).
[a]
Yongru Li, Dr. Yunjian Ma, Peilin Li and Prof. Dr. Yonghua Wang
School of Food Science and Engineering
South China University of Technology, Guangzhou 510640, China
E-mail: yonghw@scut.edu.cn (Yonghua Wang)
Xizhen Zhang
School of Biology and Biological Engineering
South China University of Technology, Guangzhou 510640, China
Dr. Doris Ribitsch
[b]
[c]
[d]
Austrian Centre for Industrial Biotechnology (ACIB)
Konrad Lorenz Straße 22, 3430 Tulln, Austria
Prof. Dr. Miguel Alcalde
Department of Biocatalysis
Institute of Catalysis, CSIC
28049 Madrid (Spain)
[e]
Prof. Dr. Frank Hollmann
Department of Biotechnology
Delft University of Technology
Van der Maasweg 9, 2629HZ, Delft, The Netherlands
E-mail: F.Hollmann@TUDelft.nl
Supporting information for this article is given via a link at the end of
the document.
1
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Figure 1. Chemoenzymatic sulfoxidation of thioanisole using AnChOx as in situ
Figure 2. Bienzymatic sulfoxidation of thioanisole using AnChOx as in situ H2O2
generation catalyst and rAaeUPO as selective sulfoxidation catalyst.
Conditions: 1 mL reaction mixture containing [AnChOx] = 5 μM, [rAaeUPO] =
100 nM, [Methyl phenyl sulfide] = 35 mM, [ChCl] = 100 mM in buffer system and
50% v/v NADES in NADES system. NaPi buffer (pH 7.0, 50 mM) was used in
buffer system. 500 rpm, 24 h, 30°C.
H2O2 generation catalyst. Conditions:
1 mL reaction mixture containing
[AnChOx] = 5 μM, [Methyl phenyl sulfide] = 35 mM, [ChCl] = 100 mM in buffer
system and 50% v/v NADES in NADES system. NaPi buffer (pH 7.0, 50 mM)
was used in buffer system. 500 rpm, 24 h, 30°C.
In a first set of experiments we used AnChOx in various DES
to generate H2O2, which then spontaneously oxidised thioanisole
(Figure 1).
Pleasingly, in the bienzymatic system, enantiomerically pure
(R)-sulfoxide was formed in all experiments (ee > 99%, Table S4).
Nevertheless, conversions in this system never exceeded approx.
50-60%.
Especially urea- and propanediol-containing DES gave the
highest conversions. Interestingly, these were not necessarily
those solvents in which AnChOx exhibits its highest activity.[10] In
carbohydrate-based DES, the overall sulfoxidation activity was
the lowest. Possibly, carboxylate groups activate the in situ
formed H2O2 for the spontaneous sulfoxidation reaction.[11] Overall,
this procedure appeared to be a viable method for the preparation
of racemic sulfoxides. However, especially if biological activity of
the sulfoxide products is desired, enantioselective sulfoxidation is
preferred. Therefore, we also evaluated peroxygenase-catalysed,
enantioselective oxygenation of thioanisole (Figure 2).[6]
As the best results were obtained with Urea-containing DES,
we further characterised the influence of several relevant reaction
parameters of the efficiency of the bienzymatic sulfoxidation
reaction.
First, we evaluated the influence of the water content in the
DES reaction mixture (Figure 3). In accordance to our previous
findings with bienzymatic hydroxylation and epoxidation
reactions,[10] no sulfoxidation activity was found in neat DES and
at least 25% (v/v) of water (buffer) were necessary. This is,
however, in stark contrast to previous findings on the
perhydrolase-catalysed reactions in DES.[9, 12] In the latter case,
significant enzyme activity was found also in neat DES. For all
further experiments, a 1:1 volumetric ratio of buffer and DES was
used.
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whereas in the presence of 50% (v/v) of ChCl-Urea the overall
productivity steadily increased over the entire AnChOx
concentration range investigated. This may be attributed to the
reduced activity of AnChOx in the presence of DES.[10] In any case,
it can be asserted that the H2O2 generation rate (under these
conditions) was overall rate limiting (vide infra).
Previous studies using hydrolases in DES had established a
stabilising effect of DES on the biocatalyst.[12] We therefore also
compared the influence of the reaction temperature on the
bienzymatic sulfoxidation reaction in buffer and DES-media
(Figure 5). Very much to our surprise, no stabilising effect of the
DES was observed.
Figure 3. Influence of the water content in the reaction medium on the product
formation of the bienzymatic sulfoxidation reaction. Conditions: [AnChOx] = 7
μM, [rAaeUPO] = 100 nM, [Methyl phenyl sulfide] = 25 mM. 0-75% (v/v %)
NADES was added in NaPi buffer (pH 7.0, 50 mM). 500 rpm, 24 h, 30°C.
Increasing the concentration of the H2O2-generation catalyst
(AnChOx) influenced the overall product formation in a saturation-
type manner (Figure 4).
Figure 5. Comparison of the influence of the reaction temperature on the
product formation of the bienzymatic sulfoxidation reaction performed in buffer
() and in DES (). General conditions: [AnChOx] = 7 μM in NADES system,
[AnChOx] = 3 μM in phosphate buffer system, [rAaeUPO] = 100 nM, [Methyl
phenyl sulfide] = 25 mM. In NaPi buffer (pH 7.0, 50 mM) system, [ChCl] = 100
mM, and in NADES system, 50% (v/v %) NADES was added in NaPi buffer (pH
7.0, 50 mM). 500 rpm, 24 h, 20-50°C.
Investigating the pH dependency of both reaction systems
(Figure 6) we found no sharp optimum; the highest reaction rates
were observed in alkaline media.
Figure 4. Influence of the H2O2-generation rate (of the oxidase concentration)
on the performance of the bienzymatic sulfoxidation reaction in aqueous buffer
() and in DES (). General conditions: [AnChOx] = 0.35-7 μM, [rAaeUPO] =
100 nM, [Methyl phenyl sulfide] = 25 mM. In NaPi buffer (pH 7.0, 50 mM) system,
[ChCl] = 100 mM, and in NADES system, 50% (v/v %) NADES was added in
NaPi buffer (pH 7.0, 50 mM), 500 rpm, 24 h, 30°C.
Interestingly, in buffer, the highest product formation was
observed already at AnChOx concentrations as low as 3 μM
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Figure 6. Comparison of the influence of the reaction pH on the product
formation of the bienzymatic sulfoxidation reaction performed in buffer () and
in DES (). General conditions: [AnChOx] = 7 μM in NADES system, [AnChOx]
= 3 μM in phosphate buffer system, [rAaeUPO] = 100 nM, [Methyl phenyl sulfide]
= 25 mM. In NaPi buffer (pH 7.0, 50 mM) system, [ChCl] = 100 mM, and in
NADES system, 50% (v/v %) NADES was added in NaPi buffer (pH 5.0-9.0, 50
mM). 500 rpm, 24 h, 30°C.
Figure 7. Influence of the rAaeUPO concentration on the rate of the bienzymatic
sulfoxidation reaction in buffer () and in DES (). General conditions: The total
reaction volume is 1 mL and the final conditions of this reaction were: [AnChOx]
= 7 μM in NADES system, [AnChOx] = 3 μM in phosphate buffer system,
[rAaeUPO] = 50-200 nM, [Methyl phenyl sulfide] = 25 mM. In NaPi buffer (pH
7.0, 50 mM) system, [ChCl] = 100 mM, and in NADES system, 50% (v/v %)
NADES was added in NaPi buffer (pH 7.0, 50 mM). 500 rpm, 24 h, 30°C.
Finally, we also varied the rAaeUPO concentration to
investigate its effect on the initial rate and the final product
concentration (Figure 7). Interestingly, varying the peroxygenase
concentration between 50 nM and 200 nM had no significant
effect in both reaction media, confirming our previous assumption
that the rAaeUPO-catalysed sulfoxidation is not the overall rate-
limiting step of the bienzymatic cascade. Interestingly, while the
rate of the overall reaction in the DES-system was approximately
half of the buffer system (in accordance with the lower AnChOx
activity under these conditions Figure 4) the final product
concentration in all experiments was essentially identical (13.9
mM  1.5 mM corresponding to 55% conversion, Table S5-6).
Overall, in the current contribution we have demonstrated that
sulfoxidation reactions are feasible in ChCl-based DES. By using
AnChOx as in situ H2O2 generation catalyst, we could use the
DES not only as a (co-)solvent but also as a reagent for the
reaction. Recently, there is an increasing interest in this dual use
of DES.[9-10, 13]
Using AnChOx alone, full conversion of thioanisole was
achieved albeit producing racemic products. If combined with
rAaeUPO, enantiomerically pure sulfoxide was obtained
explusively, albeit at the expense of significantly reduced yields.
Today, we are lacking a plausible explanation for this observation.
One possible reason may be inhibition of AnChOx by the (R)-
sulfoxide, but further experiments will be necessary to identify the
molecular reason, alleviate it and bring the production system to
preparative applicability. It is worth mentioning that in none of our
experiments the corresponding overoxidation product (sulfone)
was observed.
Another interesting outcome of this study is that in case of the
proposed bienzymatic sulfoxidation reaction, no specific benefit of
DES was found. The presence of ChCl-based DES did not
improve the stability or the activity of the biocatalysts. This is in
[9, 12, 14]
stark contrast to previous findings on hydrolases in DES
or even the AnChOx-rAaeUPO-system if used for hydroxylation
and epoxidation reactions.[10] This discrepancy calls for further
studies aiming at more systematic investigation and
understanding of the effects of DES on biocatalysts!
Acknowledgements
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This work was supported by the National Key R&D Program of
China (2018YFC0311104), Key Program of Natural Science
Foundation of China (31930084), National Outstanding Youth
Science Foundation of China (31725022). F.H. gratefully
acknowledges funding by the European Research Commission
(ERC consolidator grant, No.648026) and the Netherlands
Organization for Scientific Research (VICI grant, No.
724.014.003).
Keywords: biocatalysis • enantioselectivity • natural deep
eutectic solvents • peroxygenase • sulfoxidation
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Entry for the Table of Contents
Natural deep eutectic solvents (NADES) based on choline chloride were used as cosolvents for the bienzymatic sulfoxidation of
thioanisole comprising a choline oxidase (AnChOx) for the in situ generation of H2O2 to promote the peroxygenase (rAaeUPO)-
catalysed, stereoselective sulfoxidation. Hence, the NADES had a double function as cosolvent and as stoichiometric reductant for
the in situ H2O2 generation. The preliminary results presented here hint towards practical usefulness of this approach.
6
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