Enzymatic kinetic resolution of chiral sulfoxides-an enantiocomplementary approach

Article abstract of DOI:10.1039/c9cc05470g

A new enzymatic assay for the preparation of chiral sulfoxides that is enantiocomplementary to the known (S)-enantiomer-reducing activity of methionine sulfoxide reductase A (MsrA) is described. To this end, we have utilized the enzyme DMSO reductase (DmsABC), recently discovered by us being highly upregulated in stationary phase E. coli bacteria.

Full text of DOI:10.1039/c9cc05470g

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DOI: 10.1039/C9CC05470G
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Enzymatic Kinetic Resolution of Chiral Sulfoxides - An
Enantiocomplementary Approach
Vladimír Nosek and Jiří Míšek *
Received 00th January 20xx,
Accepted 00th January 20xx
DOI: 10.1039/x0xx00000x
A new enzymatic assay for the preparation of chiral sulfoxides that sulfoxide.18,19 We envisaged that the existence of this MsrB
is enantiocomplementary to the known (S)-enatiomer-reducing would allow us to develop an enantiocomplementary enzymatic
activity of methionine sulfoxide reductase A (MsrA) is described. To process for asymmetric preparation of (S)-sulfoxides. However,
this end, we have utilized the enzyme DMSO reductase (DmsABC), the low activity and high substrate specificity of natural MsrBs
recentlly discovered by us being highly upregulated in the we tested, prevented us from the development of a practical
stationary phase E. coli bacteria.
and general method for kinetic resolution of sulfoxides. This
phenomenon of lower activity and high substrate specificity of
natural MsrBs as compared to MsrAs was observed previously
and seems to be general.18 During our research into the
stereochemical aspects of oxidative stress in E. coli we
developed chiral fluorescent probes (S)- and (R)-Sulfox-1.20 By
employing these probes, we have discovered a new (R)-
sulfoxide-reducing activity in E. coli under stress conditions. The
underlying cause of the activity was unambiguously assigned to
DMSO reductase (DmsABC).21 E. coli DMSO reductase is a known
enzyme but its role in protection against oxidative stress has not
been reported before.22,23 As far as its stereoselectivity of
reduction is concerned, there were reports on enantioselective
reduction of sulfoxides in whole E. coli cells, where DMSO
reductase was suspected for the activity, however no direct
evidence was provided.24–26 The observed very high activity and
enantioselectivity of DMSO reductase with our probes under
specific condition prompted us to test this enzyme for
enantiocomplementary kinetic resolution of racemic sulfoxides.
Herein, we report on the new enzymatic protocol for
efficient and general kinetic resolution of chiral sulfoxides that
is enantiocomplementary to the known method using MsrA
enzyme.
Biocatalysis has become one of the essential modalities in
modern asymmetric catalysis along with the transition metal
catalysis and organocatalysis.1–5 The use of enzymes for
preparation of enantioenriched chiral molecules can be highly
efficient in terms of activity and enantioselectivity.
Nevertheless, the common drawback of natural enzymes is their
high substrate specificity and the lack of natural
enantiocomplementary counterparts. These issues can now be
addressed by methods of directed evolution of enzymes that
were pioneered by Arnold in the nineties and has since become
an indispensable tool for generation of tailor-made enzymes for
production of high-value chemicals in both academia and
industry.6–14 It has been shown that natural enantioselective
enzymes can be laboratory-evolved to exhibit opposite
enantioselectivity.15,16 However, such a process is relatively
tedious, and the resulting mutants do not necessarily have the
same level of activity and selectivity as the wild type.
We have previously reported a general chemoenzymatic
method for deracemisation of chiral sulfoxides.17 This method
utilizes methionine sulfoxide redutase A (MsrA), a natural
enzyme capable of highly efficient kinetic resolution of racemic
sulfoxides (Scheme 1). MsrA reacts exclusively with (S)-
enantiomers of an exceptionally wide range of racemic
sulfoxides and thus provids enantiomerically pure (R)-
sulfoxides. There exists
a natural enantiocomplementary
enzyme methionine reductase B (MsrB) that reduces the (R)-
Department of Organic Chemistry, Faculty of Science, Charles University in Prague
Hlavova 2030/8, 12843 Prague 2, Czech Republic. E-mail: misek@natur.cuni.cz
† Footnotes relating to the title and/or authors should appear here.
Electronic Supplementary Information (ESI) available: [details of any supplementary
information available should be included here]. See DOI: 10.1039/x0xx00000x
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DmsABC reductase turned out to be strongly dependent on the
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DOI: 10.1039/C9CC05470G
process of preparation of the membrane fraction, which led to
Our previous work
a rather low reproducibility. Another advantage of the whole
cell format is that no external reductant is required as the
intracellular NADH drives the reduction. We used stationary
phase growth E. coli population for the reaction as the activity
of DmsABC reductase is naturally upregulated under this
condition.21 It has been shown that natural activity of
enantiocomplementary MsrA is also upregulated at the
stationary phase.31 Our previous measurement with Sulfox-1
probes indicated that stationary phase E. coli activity of MsrA is
singnificantly lower than that of DmsABC reductase but still
measurable. Thus, we used MsrA knock-out strain for kinetic
resolution experiments in order to prevent this undesired
activity. The preliminary experiment was performed with 1a as
a model substrate. E. coli cells (MsrA knock out – KEIO collection
ID JW4178)32 were harvested at the stationary phase (OD600=3),
resuspended in M9 minimal buffer (OD600=70; 2 ml) and
MsrA (0.1 mol%)
O-
S+
O-
S+
(whole E. coli cells)
+
S
aqueous buffer/decane
R1
R2
R1
R2
R1
R2
rac-1
(R)-1
2
up to s >100
This study
DmsABC (0.2 ppm)
(whole E. coli cells)
O-
S+
O-
S+
+
S
aqueous buffer/decane
R1
R2
R1
R2
R1
R2
rac-1
(S)-1
up to s >100
Scheme 1 Methods for enzymatic kinetic resolution of chiral sulfoxides.
2
Our quest for an efficient enantiocomplementary method substrate 1a (65 mol; 8 ml M9 minimal buffer) and decane (5
for kinetic resolution of sulfoxides commenced with the vol %) were added and the resulting mixture was incubated at
recombinant expression of MsrBs with highest reported 37 °C. Conversion and enantiomeric excess of the sulfoxide 1a
activities (see the ESI).27–29 Among those, MsrB from A. thaliana were monitored at various timepoints by HPLC. Delightfully,
turned out to be the most active and enantioselective (Scheme after two hours the observed conversion was 51% and ee >99%
2). However, as compared with MsrA, the model reaction for the (S)-enantiomer giving the selectivity factor s >100. In
employing sulfoxide 1h required ten-fold excess of the enzyme order to assess the amount of DmsABC reductase in the reaction
MsrB (1 mol%) compared to MsrA (0.1 mol%) in order to reach mixture, proteomic analysis of the harvested cells was
a reasonable conversion (44%) in 44 hours. The enantiomeric performed. The relative intensity of all proteins was related to
excess of the isolated product was 63% giving the selectivity the total protein concentration. Based on this analysis the
factor of 17 (calculated according to the formula s=ln[(1-c)(1- amount of DmsABC reductase in the reaction mixture was
ee)]/ln[(1-c)(1+ee)] where
c is conversion and ee is estimated to be 0.00002 mol% or 0.2 ppm. This level of activity
enantiomeric excess).30 This experiment indicated rather poor is in agreement with the previously reported data for the
performance of the enzyme in the kinetic resolution of purified enzyme and documents the exceptional catalytic
sulfoxides. At this point, we concluded that the known MsrBs performance enzymes may possess.22
are unlikely to be the ideal choice for development of a general
and efficient enantiocomplementary method.
Next, we assessed the substrate scope of the reaction. A
wide range of aryl alkyl and alkyl alkyl sulfoxides 1a-1m were
successfully resolved with excellent enantioselectivity (s factor
>100)(Scheme 3). Similarly to MsrA, DmsABC reductase can
efficiently resolved various aryl melthyl/ethyl sulfoxides. Also,
chiral sulfoxides bearing two adjacent sp3 carbons, which are
often difficult substrates for chemical catalysis can be resolved
with high enantioselectivity. Propyl tolyl sulfoxide 1e that is
unreactive with MsrA can react with DmsABC reductase but the
enantioselectivity is very low (s=3). Isomeric isopropyl tolyl
sulfoxide 1f is even less reactive suggesting the sensitivity of the
enzyme to the branching at the alpha carbon of the sulfoxide
moiety. Interestingly, chloromethyl tolyl sulfoxide 1g that is also
unreactive with MsrA proved to be a good substrate for DmsABC
reductase affording the product with high enantioselectivity
(s>100). This chloromethyl substitution enables further
synthetic modifications by means of nucleophilic substitution or
reactions with organometallic species and thus further expands
the scope of accessible chiral sulfoxides in an enantiomerically
pure form.33–36 As the enzyme provides (S)-enatiomers of
sulfoxides, we also tested Omeprazole as a substrate in order to
obtain the blockbuster drug Esomeprazole (1n). Gratifyingly,
racemic Omeprazole was successfully resolved with an excellent
selectivity factor s >100. This rather surprising reactivity
O-
S+
O-
S+
MsrB (A. thaliana)
(1 mol%)
S
+
phosphate buffer
DTT (4 eq.)
Br
Br
Br
rac-1h
(S)-1h
2h
conv. 44%
ee 63%
s =17
Scheme 2 Kinetic resolution of a model substrate 1h with methionine sulfoxide
reductase B from A. thaliana.
Fortunately, other lines of our research provided us with the
discovery that DmsABC enzyme that is highly upregulated under
stationary phase growth in E. coli possesses the desired
enantiocomplementary activity. DmsABC is a membrane-
associated enzyme consisting of three subunits that are
required for the activity.22 However all our attempts at isolation
of the DmsABC reductase from a membrane fraction led to
significant decrease in activity. Therefore, we resolved to use
the whole E. coli cells format of the reaction. It should be noted
that also the E. coli crude membrane fraction can be utilized for
the kinetic resolution of sulfoxides when supplemented with
NADH as an external reductant. However, the activity of
2 | J. Name., 2012, 00, 1-3
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prompted us to test also substrates 1o and 1p in order to not required in this case. 50 mg of omeprazole was resolved to
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identify the substitution pattern required for high obtain Esomeprazole (1n) in 38% isolatedDyOieI:ld10(.c10o3n9v/eCr9sCioCn0554670%G)
enantioselectivity compared with substrate 1e. These with the enantiomeric excess of 98%. This proof-of-principle
experiments showed that neither of these substrates is resolved experiment was performed in only 100 ml reaction volume
with high enantioselectivity and thus other steric/electronic without any extensive optimisation. For the preparation of
effects must be considered to explain the high enantioselectivity larger quantities of Esomeprazole, an increased reaction volume
for substrate 1n. In order to further demonstrate the utility of and/or further optimisation of the reaction conditions (e.g. flow
the enzymatic kinetic resolution, we resolved Omeprazole into reactor) would be required.
blockbuster drug Esomeprazole on a larger scale. Due to the low
H
N
H
N
O-
S+
O-
S+
DmsABC (2.2 ppm)
aqueous buffer
DmsABC (0.2 ppm)
O-
S+
O-
S+
N
N
(whole E. coli cells)
N
N
MeO
MeO
3h
+
R1
R2
S
R1
R2
R1
R2
aqueous buffer/decane
2h
OMe
OMe
rac-1n
(S)-1n (Esomeprazole)
conversion 56%
isolated yield 38%
98% ee
(S)-1
rac-1
2
O-
S+
O-
S+
O-
S+
O-
S+
Scheme 4 Asymmetric preparation of Esomeprazole by means of DmsABC catalysed
kinetic resolution.
1c[a]
1d[a]
1a
1b
conv. 50%
ee >99%
s >100
conv. 50%
ee >99%
s >100
conv. 49%
ee 98%
s >100
conv. 52%
ee 99%
s >100
In
conclusion,
we
have
developed
a
new
enantiocomplementary enzymatic protocol for highly efficient
kinetic resolution of chiral sulfoxides. Remarkably, a wide range
of substrates can be resolved with an excellent
enantioselectivity, which is a feature that is exceptional even
among other enantioselective enzymes. Although, the principal
enzyme DmsABC reductase is a multidomain, multi cofactor
enzyme complex that is not easily isolated, we came up with a
simple whole-cell format of the reaction that can be easily
utilized for an efficient resolution providing a blockbuster drug
Esomeprazole. Remarkably, the catalyst loading of 0.2 ppm
demonstrates the efficiency that can be achieved with
biocatalysis. In a broader sense, we show that new useful
enzymatic activities can be discovered even in the well-studied
organisms such as E. coli. Thus, we believe, that natural sources
still represent an untapped potential for the discovery of new
reactivities.
O-
S+
O-
S+
O-
S+ Cl
1f[a]
conv. 9%
ee 0%
-
1g[a]
conv. 52%
ee >99%
s >100
1e[a]
conv. 39%
ee 26%
s =3
O-
S+
O-
S+
O-
S+
1j
Br
MeO
NC
1i
1h
conv. 49%
ee 98%
s >100
conv. 52%
ee 99%
s >100
conv. 51%
ee >99%
s >100
O-
S+
O-
S+
O-
S+
1l
1k
1m
We acknowledge support of the Czech Science Foundation (GACR
17-25897Y). We thank Mr. Karel Harant and Mr. Pavel Talacko from
Biocev, Prague, for proteomics and mass spectrometry analysis
(project no. CZ.1.05/1.1.00/02.0109 from the European
Regional Development Fund). We also thank Prof. Pavel
Kočovský for critical reading of the manuscript.
conv. 50%
ee >99%
s >100
conv. 50%
ee 99%
s >100
conv. 52%
ee 99%
s >100
H
N
O-
S+
N
N
MeO
1n (Esomeprazole)[b]
conv. 29%
OMe
ee 40%
s >100
Conflicts of interest
There are no conflicts of interest.
O-
S+
H
O-
S+
N
N
N
MeO
1p[b]
conv. 20%
ee 11%
s =3
1o[b]
conv. 42%
ee 28%
s =3
Notes and references
1
V. Gotor, I. Alfonso and E. García-Urdiales, Eds., Asymmetric Organic
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J. Albarrán-Velo, D. González-Martínez and V. Gotor-Fernández, Biocatal.
solubility of Omeprazole in the reaction buffer, pH was raised to
9. Indeed, this modification of the protocol increased the
solubility of Omeprazole and also suppressed the generation of
undesired side products. Furthermore, decane co-solvent was
Biotransfor., 2018, 36, 102.
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3
Scheme 3 Substrate scope of the DmsABC catalyzed kinetic resolution of chiral
sulfoxides. [a] 0.4 ppm of DmsABC. [b] 2.0 ppm of DmsABC. For details, see the ESI.
This journal is © The Royal Society of Chemistry 20xx
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