when employing either HAPMO or PAMO (entries 12 and
13). In both cases (S)-8b was obtained with complete
enantioselectivity and high conversion. Since (S)-8b contains
three functional groups, it represents an interesting starting
material for additional transformations.
The progress of the PAMO-catalyzed biooxidation of rac-
8a was studied (Figure 2). The DKR was very fast, leading to
50% conversion after only 4 hours. Enantiomeric excess of
excellent enantioselectivities and conversions in most of the
cases. Indeed, as a result of the presence of acidic hydrogen in
the substrate structure, its racemization can be performed by
working at slightly basic pH, resulting in the dynamic kinetic
resolution of the starting material, affording the final products
with conversions close to 100%. Furthermore, it illustrates
that HAPMO and PAMO can accept not only aromatic but
also aliphatic substrates. In general, higher yields are obtained
by increasing the ester alkyl chain up to the isopropyl (even
benzyl) group. These reactions were performed at larger scale
(50 mg) to obtain the enantiopure products with moderate to
high yields. By employing an organic cosolvent in these
biocatalyzed processes, both the activity and selectivity of the
enzyme could be improved, also allowing the use of a higher
substrate concentration.
Experimental Section
General procedure for the biocatalyzed oxidation of the racemic a-
alkyl-b-ketoesters rac-1–10a employing purified BVMOs: The cor-
responding racemic a-alkyl-b-ketoester (50 mg, 0.22–0.32 mmol) was
dissolved in Tris-HCl buffer (50 mm, pH 9.0, 13 mL) containing 1%
DMSO. Then, NADPH (0.2 mm), glucose-6-phosphate (40 mm),
glucose-6-phosphate dehydrogenase (75 units), and PAMO (15
units) were added. The mixture was shaken at 250 rpm at 308C.
Reactions were stopped after 24 or 36 h by extraction with EtOAc
(3 ꢀ 10 mL). The organic layer was dried over Na2SO4, the solvent was
evaporated under reduced pressure, and the conversions were
measured by GC analysis. No additional purification was required,
except for compounds 4, 5b, for which flash chromatography on silica
gel was employed, using hexanes/EtOAc (9:1) as the eluent. Diesters
1–10b were obtained in enantiopure form (except for (S)-9b,
achieved with ee = 50%) with yields ranging from 60 to 76%.
Figure 2. Time-dependent conversion of rac-8a into (S)-8b using
~
purified PAMO. Optical purity of 8a: , conversion of 8b: ꢃ, optical
^
purity of (S)-8b:
.
the final diester (S)-8b was excellent during the whole
oxidative process. Initially, the oxidation mainly proceeded as
a kinetic resolution, with optical purities of the starting
material (R)-8a close to 20% ee at conversions of 50%. After
4 hours, the optical purity of 8a diminished and the ee value of
the final diester remained constant. After 24 hours, total
conversion to enantiopure (S)-8b was achieved.
Received: May 16, 2011
Published online: July 15, 2011
Finally, racemic substrates having aromatic rings in their
structure were also analyzed. Isopropyl rac-2-benzyl-3-oxo-
butanoate (rac-9a) was not oxidized by HAPMO, but it could
be converted with complete conversion using PAMO
(entry 14). For this biocatalyst, moderate optical purity was
obtained in the synthesis of (S)-9b. Much better results can be
achieved in the biotransformation of rac-10a, which has the
aromatic ring attached to the ester group. For both HAPMO
and PAMO, diester (S)-10b was recovered with total con-
version and complete selectivity (entries 15 and 16).
Once we could obtain several a-acylated hydroxy esters
with excellent conversions and selectivities, the next step was
the synthesis of the corresponding optically active a-hydroxy
esters. Initially, we tested a set of commercially available
hydrolases to obtain the selective hydrolysis of the acetyl or
propionyl moiety (see the Supporting Information). For all
the biocatalysts tested, either no hydrolysis or poor regiose-
lectivity was observed. Thus, chemical hydrolysis was per-
formed by treatment of the starting diesters with the
corresponding alcohol in the presence of a catalytic amount
of hydrochloric acid. By using this approach, the enantiopure
hydroxy esters (S)-1–9c were obtained with high yields (60–
85%).
Keywords: biocatalysis · enantioselectivity · enzyme catalysis ·
dynamic kinetic resolution · oxidation
.
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This study demonstrates that BVMOs can be used to
catalyze the oxidation of a set of a-alkyl-b-ketoesters with
Angew. Chem. Int. Ed. 2011, 50, 8387 –8390
ꢀ 2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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