Kinetic resolution of phenyl methyl sulfoxides by mammalian methionine sulfoxide reductase A

Article abstract of DOI:10.1016/j.tetlet.2017.11.022

Chiral sulfoxides are widely used in organic synthesis as chiral auxiliaries. There are numerous strategies for the preparation of enantiomerically pure sulfoxides, based either on the enantioselective oxidation of sulphides or the enantiospecific reduction of sulfoxides. For both cases, bioconversion techniques have been developed and proposed for large-scale synthesis. Methionine sulfoxide reductase enzymes (MsrA and MsrB) catalyse the stereoselective conversion of methionine sulfoxide to methionine. MsrA can also catalyse the reduction of other exogenous sulfoxides, including p-tolyl methyl sulfoxide. However, the stereoselectivity towards this type of substrate is not yet well characterized. The activity and enantioselectivity of MsrA toward several aryl methyl sulfoxides is presented in this paper.

Full text of DOI:10.1016/j.tetlet.2017.11.022

Accepted Manuscript
Kinetic resolution of phenyl methyl sulfoxides by mammalian methionine sulf-
oxide reductase A
Cesare Achilli, Annarita Ciana, Giampaolo Minetti
PII:
S0040-4039(17)31425-9
https://doi.org/10.1016/j.tetlet.2017.11.022
TETL 49465
DOI:
Reference:
Tetrahedron Letters
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Revised Date:
Accepted Date:
27 September 2017
6 November 2017
10 November 2017
Please cite this article as: Achilli, C., Ciana, A., Minetti, G., Kinetic resolution of phenyl methyl sulfoxides by
mammalian methionine sulfoxide reductase A, Tetrahedron Letters (2017), doi: https://doi.org/10.1016/j.tetlet.
2017.11.022
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Tetrahedron Letters
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Kinetic resolution of phenyl methyl sulfoxides by mammalian methionine sulfoxide
reductase A
Cesare Achillia , Annarita Cianaa , Giampaolo Minettia,∗
a University of Pavia, Department of Biology and Biotechnology, Laboratories of Biochemistry, via Bassi 21, 27100 Pavia, Italy
ARTICLE INFO
ABSTRACT
Article history:
Received
Chiral sulfoxides are widely used in organic synthesis as chiral auxiliaries. There are numerous
strategies for the preparation of enantiomerically pure sulfoxides, based either on the
enantioselective oxidation of sulphides or the enantiospecific reduction of sulfoxides. For both
cases, bioconversion techniques have been developed and proposed for large-scale synthesis.
Methionine sulfoxide reductase enzymes (MsrA and MsrB) catalyse the stereoselective
conversion of methionine sulfoxide to methionine. MsrA can also catalyse the reduction of other
exogenous sulfoxides, including p-tolyl methyl sulfoxide. However, the stereoselectivity
towards this type of substrate is not yet well characterized. The activity and enantioselectivity of
MsrA toward several aryl methyl sulfoxides is presented in this paper.
Received in revised form
Accepted
Available online
Keywords:
Aryl methyl sulfoxide
Kinetic resolution
Chiral auxiliary
2009 Elsevier Ltd. All rights reserved.
Methionine sulfoxide reductase
Enzyme
In the last two decades, enantiomerically pure chiral
sulfoxides have found many applications in organic synthesis,
mainly because they can act as excellent chiral auxiliaries,
capable of guiding carbon-to-carbon bond formation in an
enantioselective manner, but also in the reduction of carbonyl
compounds and olefins.1 There are three factors behind the
success of these molecules as chiral auxiliaries: their high optical
stability, their efficiency in transferring chiral information, and
the availability of both enantiomers in optically pure form. The
main procedures for the large-scale preparation of optically pure
sulfoxides by oxidation of the respective sulphide are based on
the modified Sharpless method or on the use of chiral
oxaziridines. In recent years, bioconversion techniques based on
oxidative enzymes such as monooxygenase and chloroperoxidase
have been increasingly developed.2
An alternative to obtaining optically pure sulfoxides is the
kinetic resolution of a racemic mixture, which can be achieved by
means of an enantiospecific reaction involving the transformation
of only one of the enantiomers. For this purpose the alternatives
are oxidation to the sulfone or reduction to the sulphide.3 More
recently, the ability of the bacterial enzyme dimethyl sulfoxide
reductase to catalyze the reduction of a wide range of aryl alkyl
sulfoxides has been discovered, in some cases showing high
enantiospecificity.4 Another class of enzymes that has
biologically evolved the ability to reduce the sulfoxide group is
methionine sulfoxide reductase (Msr).5 The Msrs differ for their
stereospecificity towards the two diastereomeric forms of
methionine sulfoxide that may result from aberrant oxidation of
methionine (as the free amino acid or inserted into proteins)
(SS)sulfoxide, whereas MsrBs reduce (SC)methionine-
(RS)sulfoxide. The physiological electron donor for Msrs is the
thioredoxin system which, in vitro, can be replaced by 1,4-
dithiothreitol (DTT) or 1,4-dithioerythritol (DTE).6 Moskovitz
and co-workers have shown that mammalian MsrA has, in vitro,
high enzymatic activity against (SS)p-tolyl methyl sulfoxide,
while the substitution of methyl with the phenyl group
completely suppresses the catalytic activity, probably due to the
excessive steric hindrance of the substrate.7 However, the activity
towards the RS enantiomer has not been tested, and it is not
possible assume a priori that an enzyme also maintains its natural
stereospecificity towards synthetic substrates. While the present
article was under review, data were reported on the asymmetric
resolution of a series of chloro-substituted phenyl methyl
sulfoxides by recombinant MsrA from Pseudomonas montelii
overexpressed in Escherichia coli and used in a whole cell
system. An enantioselectivity towards the S enantiomer was
observed, with enantiomeric excesses of 93.2% (ortho), 61.2%
(meta) and 97.0% (para).8
Herein, we have evaluated the enantiospecificity of
mammalian MsrA towards p-tolyl methyl sulfoxide. In addition,
the performance in the enantiospecific reduction of a series of
phenyl methyl sulfoxides has also been tested. Mammalian MsrA
was obtained by recombinant DNA technology, the substrates
were in the racemic form and DTT was used as an electron
donor. Direct reduction of the sulfoxide to sulphide by DTT
alone was found to be negligible under our experimental
conditions. The degree of conversion was determined by reverse
phase HPLC analysis and the enantioselectivity was checked by
HPLC analysis on a column packed with cellulose tribenzoate-
MsrAs reduce (SC)methionine-
d—ur—ing—cellular metabolism.
∗ Corresponding author. Tel.: +39-0382-987891; fax: +39-0382-987240; e-mail: minetti@unipv.it

coated silica, which is able to resolve the two enantiomers of
these sulfoxides, as previously demonstrated9 (experimental
details are provided in the ESI). Mammalian MsrA displayed an
elevated enantiospecificity for the (SS)p-tolyl methyl sulfoxide,
confirming what is already known with respect to the natural
substrate (Table 1). The same level of activity and the same
enantiospecificity were also observed for the other substrates that
were tested (Table 1). The behavior towards p-chlorophenyl
methyl sulfoxide was consistent with that recently observed for
MsrA from Pseudomonas monteilii.8 The stereospecificity of
mammalian MsrA did not seem to depend on the identity of the
substituents in the para position of the aromatic ring. It was also
observed that mammalian MsrA exhibited a negligible activity
against phenyl ethyl sulfoxide according to what was already
reported in the literature, where substituting methyl with larger
moieties, such as phenyl, caused a decrease in the enzymatic
activity of mammalian MsrA, whereas the other substituent
adjacent to the reaction center had only a minor effect on the
enzyme activity.7
Table 1. Values of enantiomeric excess of RS and degree of conversion obtained after the reduction of aryl methyl sulfoxides by
mammalian MsrA
Substrate
Enantiomeric excess of RS (%)
Degree of conversion (%)b
phenyl methyl sulfoxide
p-tolyl methyl sulfoxide
90
96
85
91
92
46
44
42
41
51
p-fluorophenyl methyl sulfoxidea
p-chlorophenyl methyl sulfoxidea
p-bromophenyl methyl sulfoxidea
a These sulfoxides were not commercially available, therefore they were prepared by oxidation of the respective sulphides with peroxydisulphate, and purified by
recrystallization as previously described.10
b Degree of conversion is expressed as the ratio between the moles of sulphide and the moles of the initial sulfoxide.
4. Hanlon, S. P.; Graham, D. L.; Hogan, P. J.; Holt, R. A.; Reeve, C.
D.; Shaw, A. L.; McEwan, A. G. Microbiology 1998, 144, 2247-
2253
racemic mixtures of aryl methyl sulfoxide, yielding the RS
5. Achilli, C.; Ciana, A.; Minetti, G. Biofactors 2015, 41, 135-152
In light of the data reported herein, mammalian MsrA
represents a new enzymatic method for the kinetic resolution of
enantiomer with a good degree of optical purity. Mammalian
6. Minetti, G.; Balduini, C.; Brovelli, A. Ital. J. Biochem. 1994, 43,
273-283
MsrA, thanks to recombinant DNA technology, can be expressed
in prokaryotic cells at very high levels and could find application
in large-scale strategies of bioconversion using raw cell extracts
or directly into intact cultured cells. The preference for substrates
having a methyl group adjacent to the sulfoxide moiety could
also be exploited to reduce complex molecules containing more
sulfoxide units, since mammalian MsrA would be able to select
the reaction site based on the different steric hindrance. The
behavior of other MsrA isoforms remains to be explored, as it is
the ability of members of the MsrB family (with the noteworthy
presence of a selenoenzyme11) to reduce unnatural sulfoxides,
and whether they are specific towards the RS enantiomer.
7. Moskovitz, J.; Weissbach, H.; Brot, N. Proc. Natl. Acad. Sci.
U.S.A. 1996, 93, 2095-2099
8. Yang J.; Yuan Z.; Zhou Y.; Zhao J.; Yang M.; Cheng X.; Ou G.;
Chen Y. J. Mol. Catal. B Enzym. 2017 In Press
9. Abo, M.; Okubo, A.; Yamazaki, S. Tetrahedron-Asymmetr. 1997,
8, 345-348
10. Arumugan, A.; Srinivasan, C.; Kuthalingam, P. Ind. J. Chem.
1978 16, 478-480
11. Achilli,C.; Ciana, A.; Rossi, A.; Balduini, C.; Minetti, G. J.
Leukoc. Biol. 2008 83, 181-189
Acknowledgments
This work was supported with FAR funds from the University
of Pavia to GM. Authors would like to thank Prof. Herbert
Weissbach for having kindly provided the recombinant bacteria
for the overexpression and purification of MsrA.
The authors declare that they have no conflict of interest.
References and notes
1. Pellissier, H. Tetrahedron 2006, 62, 5559-5601
2. Fernàndez, I.; Khiar, N. Chem. Rev. 2003, 103, 3651-3705
3. O’Mahony, G. E.; Kelly, P.; Lawrence, S. E.; Maguire, A. R.
Arkivoc. 2011, 2011, 1-110

Graphical Abstract
Kinetic resolution of phenyl methyl
sulfoxides by mammalian methionine
sulfoxide reductase A
Leave this area blank for abstract info.
Cesare Achilli, Annarita Ciana, Giampaolo Minetti

4
Tetrahedron
Chiral sulfoxides are of great interest as chiral
auxiliaries in organic synthesis
An enzymatic kinetic resolution of aryl methyl
sulfoxides is proposed
Methionine sulfoxide reductase A (MsrA) reduces
in vivo Sc-Met-Ss-sulfoxide to Sc-Met
We show here that MsrA reduces the S enantiomer
of some aryl methyl sulfoxides
MsrA is suggested for the large-scale preparation of
optically pure sulfoxides