The biological Baeyer-Villiger oxidation of a racemic ketone does not have to
follow the ‘classic’ kinetic resolution format as described above, but can proceed via a
‘nonclassic’ route involving oxidation of both enantiomers with opposite
regioselectivity. Thus, oxygen insertion occurs on the two opposite sides of the ketone
at each of the enantiomers. As shown in Scheme 2.164, both enantiomers of the
bicyclo[3.2.0]heptenones were microbially oxidized, but in an enantiodivergent manner [1366, 1367]. Oxygen insertion on the (5R)-ketone occurred as expected, adjacent
to C7, forming the 3-oxabicyclic lactone. On the other hand, the (5S)-ketone
underwent oxygen insertion in the ‘wrong sense’ towards C5, which led to the
2-oxabicyclic species. The synthetic utility of this system has been proven by the
large-scale oxidation using an E. coli designer bug harboring cyclohexanone
monooxygenase together with a suitable NADPH-recycling enzyme [1368, 1369]. In
order to minimize product toxicity, in-situ substrate-feeding product removal (SFPR)
was applied [1370, 1371].
It has been shown that the molecular reasons of enantiodivergent BaeyerVilliger reactions [1372, 1373] can either be the docking of the substrate in a single
enzyme in two opposite modes or due to the presence of different monooxygenases
present in the microbial cells [1374].
In order to overcome problems associated with whole-cell Baeyer-Villliger
oxidations, an impressive number of bacterial ‘Baeyer-Villigerases’ possessing
opposite stereopreference [1375, 1376] were purified, characterized [1377–1380]
and cloned into a suitable (nonpathogenic) host, such as baker’s yeast [1381–1385]
or E. coli [1386].
The majority of Baeyer-Villigerases are NADPH-dependent, but several candidates (e. g. from Pseudomonas putida) accept NADH, which is more easily
recycled [1387]. In order to facilitate cofactor recycling, a selfsufficient fusion
protein consisting of a Baeyer-Villigerase and a phosphite dehydrogenase unit for
NADH-recycling were designed [1388]. The overall performance of the fusionprotein was comparable to that of the single (non-fused) proteins. A clever concept
of internal cofactor recycling for isolated Baeyer-Villigerases was developed using
a coupled enzyme system [1389]. Thus, the substrate ketone is not used as such, but
is rather produced by enzymatic oxidation of the corresponding alcohol (at the
expense of NADP
+ or NAD
+
, resp., Sect. 2.3.1) using a dehydrogenase from
Thermoanaerobium brockii or from Pseudomonas sp. In a second step, the
O
R
R
O
R
R
R
R
O
O
O
R
R
O
Acinetobacter or
Pseudomonas sp.
5
7
2
3
+
e.e. >96%
e.e. >94%
R = H, CH 3
rac
[O]
[O]
O 2
+
Scheme 2.164 Enantiodivergent microbial Baeyer-Villiger oxidation involving ‘nonclassic’
resolution
192
2 Biocatalytic Applications
follow the ‘classic’ kinetic resolution format as described above, but can proceed via a
‘nonclassic’ route involving oxidation of both enantiomers with opposite
regioselectivity. Thus, oxygen insertion occurs on the two opposite sides of the ketone
at each of the enantiomers. As shown in Scheme 2.164, both enantiomers of the
bicyclo[3.2.0]heptenones were microbially oxidized, but in an enantiodivergent manner [1366, 1367]. Oxygen insertion on the (5R)-ketone occurred as expected, adjacent
to C7, forming the 3-oxabicyclic lactone. On the other hand, the (5S)-ketone
underwent oxygen insertion in the ‘wrong sense’ towards C5, which led to the
2-oxabicyclic species. The synthetic utility of this system has been proven by the
large-scale oxidation using an E. coli designer bug harboring cyclohexanone
monooxygenase together with a suitable NADPH-recycling enzyme [1368, 1369]. In
order to minimize product toxicity, in-situ substrate-feeding product removal (SFPR)
was applied [1370, 1371].
It has been shown that the molecular reasons of enantiodivergent BaeyerVilliger reactions [1372, 1373] can either be the docking of the substrate in a single
enzyme in two opposite modes or due to the presence of different monooxygenases
present in the microbial cells [1374].
In order to overcome problems associated with whole-cell Baeyer-Villliger
oxidations, an impressive number of bacterial ‘Baeyer-Villigerases’ possessing
opposite stereopreference [1375, 1376] were purified, characterized [1377–1380]
and cloned into a suitable (nonpathogenic) host, such as baker’s yeast [1381–1385]
or E. coli [1386].
The majority of Baeyer-Villigerases are NADPH-dependent, but several candidates (e. g. from Pseudomonas putida) accept NADH, which is more easily
recycled [1387]. In order to facilitate cofactor recycling, a selfsufficient fusion
protein consisting of a Baeyer-Villigerase and a phosphite dehydrogenase unit for
NADH-recycling were designed [1388]. The overall performance of the fusionprotein was comparable to that of the single (non-fused) proteins. A clever concept
of internal cofactor recycling for isolated Baeyer-Villigerases was developed using
a coupled enzyme system [1389]. Thus, the substrate ketone is not used as such, but
is rather produced by enzymatic oxidation of the corresponding alcohol (at the
expense of NADP
+ or NAD
+
, resp., Sect. 2.3.1) using a dehydrogenase from
Thermoanaerobium brockii or from Pseudomonas sp. In a second step, the
O
R
R
O
R
R
R
R
O
O
O
R
R
O
Acinetobacter or
Pseudomonas sp.
5
7
2
3
+
e.e. >96%
e.e. >94%
R = H, CH 3
rac
[O]
[O]
O 2
+
Scheme 2.164 Enantiodivergent microbial Baeyer-Villiger oxidation involving ‘nonclassic’
resolution
192
2 Biocatalytic Applications