Angewandte
Chemie
DOI: 10.1002/anie.201403537
ꢀ
C H Oxidation
Enantioselective Allylic Hydroxylation of w-Alkenoic Acids and Esters
by P450 BM3 Monooxygenase**
Katharina Neufeld, Birgit Henßen, and Jçrg Pietruszka*
ꢀ
Abstract: Chiral allylic alcohols of w-alkenoic acids and
derivatives thereof are highly important building blocks for the
synthesis of biologically active compounds. The direct enan-
allylic C H oxidation of simple linear compounds remain
a challenge as asymmetry-inducing factors are missing.[7] The
enantioselective allylic acetoxylation of terminal olefins using
a combination of a PdII/bis(sulfoxide) system with benzoqui-
none as the terminal oxidant and CrIII(salen) as a chiral Lewis
acid cocatalyst reported by White and co-workers in 2008 was
a milestone and ee values up to 63% were achieved; the
desired products were obtained in 69–89% yield and 50–
57% ee as roughly 5:1 mixtures with the corresponding linear
alcohols.[8] We envisioned that a biocatalytic approach might
be preferable to conventional chemistry, since enzymes
ensure high selectivity by furnishing completely chiral reac-
tion environments. The well-known and highly promising
NADPH-dependent bacterial cytochrome P450 BM3 mono-
oxygenase from Bacillus megaterium was the probe system of
choice due to its soluble and self-sufficient character, its
superior catalytic features compared to other monooxyge-
nases, and the availability of large-scale production and
purification protocols.[9,10] The native function of P450 BM3 is
supposed to be the subterminal hydroxylation of fatty acids by
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tioselective C H oxidation of linear terminal olefins offers the
shortest route toward these compounds, but known synthetic
methods are limited and suffer from low selectivities. Described
herein is an enzymatic approach using the P450 BM3
monooxygenase mutant A74G/L188Q, which catalyzes allylic
hydroxylation with high to excellent chemo- and enantioselec-
tivities providing the desirable secondary alcohols.
C
hiral (wꢀ2)-hydroxy-w-alkenoic acids as well as their
esters, vinyl lactones, and protected alcohols are pivotal
structural elements abundantly present in diverse natural and
biologically active compounds including decanolides, oxy-
lipins, antibiotics, and pheromones.[1] Extensive research
toward the enantioselective synthesis of these moieties has
resulted in multistep approaches comprising prefunctionali-
zation and protection group chemistry as well as functional
group interconversions and oxidation state manipulations.[2]
A chemoenzymatic route with an alcohol dehydrogenase
catalyzed selective reduction as the key step was reported by
our group and represents arguably the most efficient access
nowadays (2 or 3 steps, 73–77% overall yield, > 99% ee, both
enantiomers accessible).[3] Significant streamlining could be
achieved by the direct asymmetric oxidation of readily
available “unactivated” olefins at the allylic position, provid-
ing a highly atom-economic single-step process.[4] In this
ꢀ
insertion of one oxygen atom from O2 into C H bonds;
however, the enzymeꢀs high versatility and evolvability have
been proven by protein engineering.[9,11] P450 BM3 mutants
were reported to hydroxylate alkanes at the 3-position with
enantioselectivity of up to 46% S-ee and preferentially
catalyze the allylic hydroxylation of terminal alkenes and
terminally unsaturated fatty acids over epoxidations; neither
the preparative feasibility nor the enantioselectivity of the
latter transformations have yet been examined.[12,13] De-
scribed herein is an enzymatic access to S-configured wꢀ2-
hydroxy-w-alkenoic acids and esters through P450 BM3
catalyzed allylic hydroxylation. This approach shows the
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context, significant success in the highly topical field of C H
activation was achieved by Agudo et al. and Li et al. regard-
ing the enzymatic oxidation of cyclic and linear molecules,
respectively.[5,6] However, methods for the enantioselective
ꢀ
highest enantioselectivity observed to date for the allylic C H
[*] B.Sc. K. Neufeld, B. Henßen, Prof. Dr. J. Pietruszka
Institut fꢀr Bioorganische Chemie der Heinrich-Heine-Universitꢁt
Dꢀsseldorf im Forschungszentrum Jꢀlich
Stetternicher Forst, Geb. 15.8, 52426 Jꢀlich (Germany)
and
oxidation of terminal linear olefins and simultaneously
addresses key issues of modern organic synthesis like the
use of mild reaction conditions, aqueous media, sustainable
catalysts, and O2 as a “green” oxidant (Figure 1).[14]
A P450 BM3 library of 65 variants was constructed by
mutating active-site residues R47, Y51, A74, F87 and L188, all
of which are known determinants of activity and selectivity of
this enzyme (for details, see the Supporting Information:
S2).[9a,15] This set of mutants served as a versatile catalyst pool
for assaying the ability of P450 BM3 to catalyze the allylic
hydroxylation of the model substrate ethyl 6-heptenoate (1a).
Analytical-scale reactions were performed in 96-well plates
and analyzed by GC with regard to conversion of starting
material and the chemo- and enantioselectivity of product
formation. Consistent with previous reports on the P450 BM3
catalyzed oxygenation of terminal olefins,[13] all mutants
tested gave the S-configured branched allylic alcohol (2a)
Institut fꢀr Bio- und Geowissenschaften (IBG-1: Biotechnologie),
Forschungszentrum Jꢀlich
52425 Jꢀlich (Germany)
E-mail: j.pietruszka@fz-juelich.de
[**] We thank the Ministry of Innovation, Science and Research of the
German federal state of North Rhine-Westphalia and the Heinrich
Heine University Dꢀsseldorf (scholarship within the CLIB Graduate
Cluster Industrial Biotechnology for K.N.). We are further grateful to
Dr. Martina Bischop and Dr. Thomas Fischer for providing the
reference compounds and Prof. Dr. Ulrich Schwaneberg for the
P450 BM3 plasmids.
Supporting information for this article is available on the WWW
Angew. Chem. Int. Ed. 2014, 53, 1 – 6
ꢀ 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
1
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