Selective Oxyfunctionalisation Reactions Driven by Sulfite Oxidase-Catalysed In Situ Generation of H2O2

Article abstract of DOI:10.1002/cctc.201902297

H₂O₂ can be accepted by several peroxygenases as a clean oxidant, able to supply both the necessary electrons and oxygen atom at the same time. The biocatalysts, in turn, are able to catalyse an array of interesting oxygen insertion reactions at enantio- and regio-selectivities hard to attain with classical chemical methods. The sensitivity of most peroxygenases towards H₂O₂, however, requires this oxidant to be generated in situ. Here, we suggest the application of (modified) sulfite oxidases to couple the oxidation of sulfites to the reduction of oxygen. This enables us to use calcium sulfite, an industrial waste product from scrubbing flue gases, as an electron donor to reduce oxygen. This will supply the required peroxide in a controlled manner and enables us to perform these challenging reactions at the expense of simple salts.

Full text of DOI:10.1002/cctc.201902297

Accepted Article
Title: Selective oxyfunctionalisation reactions driven by sulfite oxidase-
catalysed in situ generation of H2O2
Authors: Morten Martinus Cornelis Harald van Schie, Alexander T.
Kaczmarek, Florian Tieves, Patricia Gómez de Santos,
Caroline E. Paul, Isabel W.C.E. Arends, Miguel Alcalde,
Günter Schwarz, and Frank Hollmann
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To be cited as: ChemCatChem 10.1002/cctc.201902297
Link to VoR: http://dx.doi.org/10.1002/cctc.201902297
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Selective oxyfunctionalisation reactions driven by sulfite oxidase-
catalysed in situ generation of H2O2
M.M.C.H. van Schie,*[a] A.T. Kaczmarek,[b] F. Tieves,[a] P. Gomez de Santos,[c] C.E. Paul,[a] I.W.C.E.
Arends,[d] M. Alcalde,[c] G. Schwarz,[b] F. Hollmann[a]
Abstract: H2O2 can be accepted by several peroxygenases as a
clean oxidant, able to supply both the necessary electrons and
oxygen atom at the same time. The biocatalysts, in turn, are able to
catalyse an array of interesting oxygen insertion reactions at enantio-
and regio-selectivities hard to attain with classical chemical methods.
The sensitivity of most peroxygenases towards H2O2, however,
requires this oxidant to be generated in situ. Here, we suggest the
application of (modified) sulfite oxidases to couple the oxidation of
sulfites to the reduction of oxygen. This enables us to use calcium
sulfite, an industrial waste product from scrubbing flue gases, as an
electron donor to reduce oxygen. This will supply the required
peroxide in a controlled manner and enables us to perform these
challenging reactions at the expense of simple salts.
to sulfite as stoichiometric reductant, as these salts are a by-
product from flue gas treatment and are thus produced at large
amounts.[8]
To use sulfite as stoichiometric reductant for the in situ
generation of H2O2, we envisioned the application of sulfite
oxidases (SO), which are ubiquitously found in all kingdoms of life.
SO play a central role in the sulfur metabolism of living cells.[9] As
the name implies, SO oxidizes sulfite to sulfate using a unique
pterin-based molybdenum cofactor (MoCo) in the active site.[10] In
vertebrates, SO localizes into the intermembrane space of
mitochondria and consists of three distinguishable domains, the
N-terminal heme domain, incorporating a cytochrome b5-type
heme cofactor, followed by the MoCo-binding domain, and the C-
terminal dimerization domain. Upon oxidation of sulfite, the
molybdenum in the active site is reduced from MoVI to MoIV. The
two electrons are subsequently transferred via an intra-molecular
electron transfer from the MoCo to the heme domain and finally
to cytochrome c.[11] Alternatively, sulfite-derived electrons can be
transferred to nitrite, forming the radical nitric oxide.[12] In contrast,
in plants, SO consists only of the MoCo and dimerization domain
and localizes in peroxisomes.[13] Here, the redox balance is closed
through reaction with molecular oxygen, forming superoxide,
which rapidly dismutates to H2O2. When the heme domain in the
mammalian SO is deleted, similar behavior of H2O2 formation is
observed.[14] So far, SO have been extensively studied from
fundamental and therapeutic points of view. Furthermore, these
enzymes have also been immobilized on electrodes in biosensors
and fuel cells.[15] In this work, we envision a biocatalytic purpose
for this H2O2 generation system in the application of
peroxygenase reactions (Scheme 1).
Peroxygenases (E.C. 1.11.2.1, UPO for unspecific
peroxygenases) are powerful catalysts for the selective
oxyfunctionalisation of (inert) C-H and C=C-groups.[1] These are
reactions which are still hard to perform using classic chemical
methods. Especially the high catalytic activity, robustness and
simplicity of application of these enzymes makes them potentially
useful catalysts for organic oxyfunctionalisation chemistry.
Unfortunately, as heme-enzymes, UPOs also suffer from a
pronounced instability towards hydrogen peroxide, the
stoichiometric oxidant in UPO-reactions.[2] This issue is met by in
situ generation of H2O2 at rates, which maximize productive UPO-
turnover while minimizing the undesired oxidative inactivation of
the enzymes.[3] Today, a broad range of in situ H2O2 generation
systems are available, ranging from biocatalytic,[4] heterogeneous
[6]
catalysis,[5] photochemical
and electrochemical methods.[7]
Now we raise the question about the environmental impact of
such systems, especially with large-scale applications in mind,
and hypothesize if we can use waste products from other
processes for this purpose. In this respect, we drew our attention
[a]
[b]
Dr. M.M.C.H. van Schie, Dr. F. Tieves, Dr. C.E. Paul, Prof. Dr. F.
Hollmann
Department of Biotechnology, University of Technology Delft van
der Maasweg 9, 2629HZ Delft (The Netherlands)
E-mail: m.m.c.h.vanschie@tudelft.nl
Dr. A.T. Kaczmarek, Prof. Dr. G. Schwarz
Institute of Biochemistry, Department of Chemistry, CMMC,
University of Cologne
Scheme 1. The cascade of sulfite oxidase and peroxygenases.
Zuelpicher Str. 47, D-50674 Cologne (Germany)
P. Gomez de Santos, Prof. Dr. M. Alcalde
Department of Biocatalysis, Institute of Catalysis, CSIC
28049 Madrid (Spain)
Prof. Dr. I.W.C.E. Arends
Faculty of Science, University of Utrecht
Utrecht (The Netherlands)
To test the viability of the proposed scheme, we chose the
evolved, recombinant peroxygenase from Agrocybe aegerita
(rAaeUPO) as model enzyme for the selective hydroxylation of
ethylbenzene to (R)-1-phenylethanol.[16] Five SO enzymes from
different sources (all recombinantly expressed in Escherichia coli)
were tested; one plant SO (from Arabidopsis thaliana) and two
mammalian SO, from mouse and human, either in their natural
configuration containing the heme moiety (full) or a shortened
variant, devoid of the heme-subunit (MoCo, Figure 1).
[c]
[d]
Supporting information for this article is given via a link at the end of
the document.
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Increased sulfite concentrations corresponded to improved
reaction continuity, but also a decreased reaction rate (figure 2B).
Furthermore, we saw less product formation than expected with
the amount of sulfite added. When adding 5 mM of Na2SO3, for
instance (figure 2B, black time course), only 3.1 mM of product
was obtained. Similar observations were made for the human
MoCo variant (figure S1). These effects are most likely related to
the spontaneous reaction between sulfite and H2O2 yielding
17]
sulfate and water.[13b,
The direct oxidation of sulfite by
molecular oxygen is also possible, which would also decrease
oxygen availability.[18] The reaction with peroxide has been
reported to follow first order kinetics; the rate is thus linearly
dependent on the in situ concentration of both sulfite as peroxide.
This is also an explanation for why, during experiments at
increased sulfite concentration (figure 2B, green and purple time
courses), the reaction rate increased after 16 hours; at this point
the Na2SO3 concentration had significantly decreased. We
therefore set out to limit the concentration of SO32- in the reaction
mixture using CaSO3, which is poorly soluble in water (up to 0.45
mM)[19] and could form a solid phase in the reaction acting as a
sulfite reservoir. Advantageously, CaSO3 is also the primary
product from flue gas desulfurisation with lime stone, and can thus
be considered to be a waste product from industry, available in
large quantities.[8] Very pleasingly, this strategy indeed enhanced
the product accumulation significantly (figure 3).
Figure 1. Comparison of different sulfite oxidases to drive the rAaeUPO-
catalysed hydroxylation of ethylbenzene. General conditions: [SO] = 100 nM,
[rAaeUPO] = 500 nM, [Na2SO3]0 = 10 mM, [ethylbenzene]0 = 100 mM in a 50
mM Bis-Tris buffer at pH 7.0. 250 µl reactions were performed in duplicates in
a thermos shaker at 30 °C and 500 rpm for 24 hours.
Pleasingly, all SO tested enabled rAaeUPO-catalysed
hydroxylation of ethylbenzene. The heme-depleted SO gave
higher activities (i.e. overall product concentrations) than the
heme-containing ones, which is most likely due to the faster direct
aerobic reoxidation at the MoIV centers. For all further experiments
we focused on the plant SO (PSO). To get further insights into the
parameters influencing the performance of the SO-rAaeUPO-
oxyfunctionalisation system we systematically varied some
reaction parameters such as pH, enzyme concentrations and
sulfite concentrations (figure 2 and supporting information).
A
B
Figure 3. Comparison of Na2SO3 (white) and CaSO3 (black) on the PSO –
rAaeUPO cascade. General conditions: [PSO] = 100 nM, [rAaeUPO] = 500 nM,
2-
Figure 2. Influence of the reaction pH (A) and sulfite concentration (B) on the
plant-SO – rAaeUPO cascade performance. General conditions: [PSO] = 100
nM, [rAaeUPO] = 500 nM, [Na2SO3]0 = 10 mM, [ethylbenzene]0 = 100 mM in a
50 mM Bis-Tris buffer at pH 7.0. Reactions were performed in duplicates, with
reaction volumes of 250 µl, in a thermos shaker at 30 °C and 500 rpm. A:
performance at pH 6.5 (black), pH 7.0 (red), pH 7.5 (blue) or pH 8.0 (green). B:
Sodium sulfite concentration of 5 mM (black), 10 mM (red), 20 mM (blue), 50
mM (green) or 100 mM (purple).
[SO3
]
0 = 50 mM, [ethylbenzene]0 = 100 mM in a 50 mM Bis-Tris buffer at pH
7.0. 250 µl reactions were performed in duplicates at 30 °C and 500 rpm.
Reactions with Na2SO3 were performed in a thermoshaker, while the samples
with CaSO3 were mixed by a magnetic stirrer in a water bath. Mixing (and thus
oxygen transfer) by the thermoshaker was actually more efficient than with the
magnetic stirring bar (figure S6).
Encouraged by these results, we next decided to explore
the scope of the proposed sulfite-driven oxidation reaction. Here,
we tested several classical oxyfunctionalisation reactions
applying different peroxygenases in the cascade with PSO (table
1). The requirement of the CaSO3 slurry to be homogeneously
dispersed in solution did, in some occasions, induce significant
standards deviations.
The reaction functioned optimally at neutral pH values,
which appears to be a compromise between the preferred
conditions for SO (basic) and the peroxygenase (slightly acidic).
Changing the ratio between the two enzymes showed careful
balance of the two catalysts is required as an excess of sulfite
oxidase activity will presumably result in accumulation of the
hydrogen peroxide, which deactivates the biocatalysts (figure
S2A). When the PSO concentration was limiting, a turnover
frequency of 4.4 (± 0.5) s-1 for the oxidase could be determined.
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Table 1. Substrate scope for the reaction of PSO coupled to various
peroxygenases. General conditions: [SO] = 200 nM, [rAaeUPO] = 500 nM,
[CaSO3]0 = 100 mM, [substrate]0 = 50 mM in a 50 mM Bis-Tris buffer. 500 µl
reactions were performed in triplicates at 30 °C with magnetic stirrers mixing at
500 rpm for 24 hours. For dextromethorphan, the substrate concentration was
10 mM. For thioanisole, 100 U ml-1 of CPO from Caldariomyces fumago was
added as the peroxygenase. ee: enantiomeric excess.
TTN [a]
SO /
(Peroxygenase)
Conversion
[mM]
ee
[%]
Product
Catalyst
77 000
/
(30 800)
15.4
(± 1.8)
rAaeUPO
98.2
83 500
/
(33 400)
16.7
(± 4.6)
rAaeUPO
rAaeUPO
-
5.8
-
70 000
/
(28 000)
14.0
(± 0.41)
26 350
/
(10 540)
rAaeUPO
(Solo) [b]
5.72
(± 0.52)
Figure 4. Chemoenzymatic transformation of styrene into styrene oxide (full) or
1-phenyl-2-hydroxyl-ethanesulfonate (open), depending on the sulfite salt used.
2-
67 000
/
(n.d.)
General conditions: [SO] = 200 nM, [rAaeUPO] = 500 nM, [SO3
] = 100 mM,
0
13.4
(± 0.16)
CfuCPO
CiVCPO
98.4
[styrene]0 = 50 mM in a 50 mM Bis-Tris buffer. 500 µl reactions were performed
in triplicates at 30 °C with magnetic stirrers mixing at 500 rpm.
In a final set of experiments, we scaled up the reaction to
semi-preparative scale in order to show the applicability of the
suggested system. As the reactions generally stopped within 24
hours, we added more biocatalyst at regular intervals. Spiking
experiments showed only the PSO needed to be added every 24
hours, as the UPO remained active for over 3 days under the
applied reaction conditions (figure S2B). During these initial
experiments, over prolonged time and at a larger reaction volume,
a decrease in reaction rate was observed as compared to the
small scale experiments. We ascribed this to a decrease in
oxygen transfer rates. We therefore supplied pure oxygen to the
larger reaction via a balloon. Furthermore, we observed no
inhibition of activity for the cascade in the presence of the sulfate
that would be produced over time (figure S2C). At the end of the
preparative scale reaction, after 96 hours, 47.4 mM of R-
phenylethanol was produced with an enantiomeric excess of
97.8%. After isolation, 155 mg of product was obtained which
contained, next to the phenylethnol, 23% of the overoxidated
acetophenone. The overoxidation was approximately ten times
higher than the one observed during the reactions at smaller scale,
which can stem from the higher phenylethanol concentrations
towards the end of the reaction. The acetophenone produced
requires two oxidation steps, we can deduce that the rAaeUPO
and the PSO made 142 000 and 180 000 turnovers, respectively.
As the reaction rate showed no regression over time (figure S2D),
we assume even higher turnovers can be achieved.
-
0.0
-
[a] TTN: mol product mol-1cat. [b] Via O-dealkylation of dextromethorphan
As shown in table 1, a range of classical peroxygenase reactions
could be promoted by the proposed SO3-H2O2 generation system
ranging from the (stereospecific) hydroxylation of sp3-C-H-bonds
via oxidative demethylation[20] and epoxidation[21] to sulfoxidation
reactions.[22] Conversions and total turnover numbers were in the
same order of magnitude, with the exception for
dextromethorphan, where the rAaeUPO catalysed reaction was
probably limiting. We also evaluated SO for chemoenzymatic
halogenations reaction based on hypohalites. These should be
generated in situ from halides using the vanadium-dependent
haloperoxidase from Curvularia inaequalis;[23] albeit without
success. We ascribe this observation to the deactivation of SO by
the bleach formed by VCPO. This phenomenon has been
observed for other oxidases before and was also confirmed by us
in a control reaction where 10 µM NaClO nearly fully inhibited the
SO.[24] Sulfite itself did not seem to affect the activity of VCPO.
In the experiments with styrene as the substrate, we noted a
decrease in mass balance and the appearance of a polar
compound on TLC. Based on literature,[25] we deduced this
compound be the hydroxyl sulfonate derivative of styrene. This,
as sulfite is able to act as a nucleophile to ring-open the epoxides.
As for the uncoupling reaction of sulfite with peroxide, this ring
opening reaction is reported to follow first order kinetics.
Therefore, the epoxide is primarily observed if CaSO3 is the sulfite
source. For Na2SO3, only the sulfonated product, mainly the 1-
phenyl-2-hydroxyl-ethanesulfonate, was obtained as previously
described in literature.[25b]
Overall, we provide the proof of principle of using sulfite
oxidases coupled to various peroxygenases, thereby enabling
oxyfunctionalisation reactions up to preparative scale at the
expense of an industrial waste product. The total turnover
numbers achieved for the oxidase are already up to 180 000 for
the arbitrarily chosen sulfite oxidase from Arabidopsis thaliana.
These turnover numbers are in the same order of magnitude as
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[14] A. A. Belaidi, J. Röper, S. Arjune, S. Krizowski, A. Trifunovic, G. Schwarz,
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[18] E. C. Fuller, R. Crist, J. Am. Chem. Soc. 1941, 63, 1644-1650.
[19] H.D. Lutz, “IUPAC-NIST Solubility Database. NIST Standard Reference
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26/SDS-26-pages_187.pdf.
Acknowledgements
The authors gratefully acknowledge funding by The
Netherlands Organization for Scientific Research through a VICI
grant (No.724.014.003). ATK and GS acknowledge funding by
grant CMMC2017-C13 from the Center for Molecular Medicine,
Cologne. CEP acknowledges a VENI grant (No. 722.015.011).
MAG and PGS acknowledge the Spanish Government project
BIO2016-79106-R-Lignolution. PGS thanks the Ministry of
Science, Innovation and Universities (Spain) for her FPI
scholarship (BES-2017-080040)
[20] P. G. de Santos, F. V. Cervantes, F. Tieves, F. J. Plou, F. Hollmann, M.
Alcalde, Tetrahedron 2019, 75, 1827-1831.
[21] M. C. Rauch, F. Tieves, C. E. Paul, I. W. Arends, M. Alcalde, F. Hollmann,
ChemCatChem 2019.
[22] S. G. Allenmark, M. A. Andersson, Chirality 1998, 10, 246-252.
[23] a) J. J. Dong, E. Fernández-Fueyo, J. Li, Z. Guo, R. Renirie, R. Wever,
F. Hollmann, Chem. Commun. 2017, 53, 6207-6210; b) S. Younes, F.
Tieves, D. Lan, Y. Wang, P. Süss, H. Brundiek, R. Wever, F. Hollmann,
ChemSusChem 2019.
[24] A. But, A. van Noord, F. Poletto, J. P. M. Sanders, M. C. R. Franssen, E.
L. Scott, Mol. Catal. 2017, 443, 92-100.
Keywords: Biocatalysis • Sulfite oxidase • Peroxygenase •
Oxyfunctionalisation • Hydrogen peroxide
[25] a) R. T. E. Schenck, S. Kaizerman, J. Am. Chem. Soc. 1953, 75, 1636-
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[27] W. Liu, X.-m. Zhao, H.-b. Zhang, L. Zhang, M.-z. Zhao, Chem. Europ. J.
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M.M.C.H. van Schie,* A.T. Kaczmarek,
F. Tieves, P. Gomez de Santos, C.E.
Paul, I.W.C.E. Arends, M. Alcalde, G.
Schwarz, F. Hollmann
Page No. – Page No.
Selective oxyfunctionalisation
reactions driven by sulfite oxidase-
catalysed in situ generation of H2O2
Sulfite oxidases are able to oxidize CaSO3, an industrial waste product, into CaSO4
using a molybdenum cofactor. The redox balance can subsequently be closed by
reducing oxygen to H2O2, which is required by peroxygenases for their selective
oxyfunctionalisation reactions. We show this biocatalytic cascade can be applied up
to semi-preparative scale.
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