Microbial Baeyer-Villiger oxidation of terpenones by recombinant whole-cell biocatalysts - Formation of enantiocomplementary regioisomeric lactones

Article abstract of DOI:10.1039/b703175k

Recombinant whole-cell expression systems for Baeyer-Villiger monooxygenases of various bacterial origin were utilized in the regiodivergent biooxidation of cyclic terpenones enabling access to enantio- and regioisomeric lactones on preparative scale. This journal is The Royal Society of Chemistry.

Full text of DOI:10.1039/b703175k

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Microbial Baeyer–Villiger oxidation of terpenones by recombinant whole-cell
biocatalysts—formation of enantiocomplementary regioisomeric lactones†
ˇ
Petra Cernuchova´ and Marko D. Mihovilovic*
Received 1st March 2007, Accepted 30th March 2007
First published as an Advance Article on the web 25th April 2007
DOI: 10.1039/b703175k
Recombinant whole-cell expression systems for Baeyer–Villiger monooxygenases of various bacterial
origin were utilized in the regiodivergent biooxidation of cyclic terpenones enabling access to enantio-
and regioisomeric lactones on preparative scale.
Normal lactones of a-substituted cyclohexanones can be
Introduction
also synthesized by chemical Baeyer–Villiger oxidation using
m-chloroperoxybenzoic acid, trifluoroperoxyacetic acid, peroxy-
acetic acid and hydrogen peroxide as oxidants. However, starting
from unsaturated ketones such as dihydrocarvone, competitive
oxidation of the carbon–carbon double bond can take place.
Within non-chiral reactions, utilization of Sn-beta zeolites and
platinum complexes in H2O2 or PSA (monopersuccinic acid)
in water enabled complete control of the parallel oxidation
processes, ultimately giving selective Baeyer–Villiger oxidation
upon reaction of bifunctional substrates.10
Baeyer–Villiger oxidation of cyclic ketones to chiral lactones
allows access to highly interesting and versatile intermediates in
the synthesis of bioactive or natural products.1–4 The biocatalytic
approach to realize this transformation with high regio- and
enantioselectivity takes advantage of the high promiscuity of
flavin dependent monooxygenases to accept a large diversity of
non-natural substrates. Moreover, this “green chemistry” strategy
utilizes molecular oxygen for the oxidation process as most
sustainable oxidant. An increasing number of such Baeyer–Villiger
monooxygenases (BVMOs) has become available in recent years.
Due to the application of recombinant overexpression systems for
the production of biocatalyst as well as the direct implementation
of such systems in whole-cell mediated biotransformations, the
microbial Baeyer–Villiger biooxidation has become a highly
attractive methodology to access optically pure lactones in both
kinetic resolution and desymmetrization processes.4
While the regioselectivity of the Baeyer–Villiger oxidation is
governed predominantly by electronic effects leading to preferred
migration of the more nucleophilic and higher substituted carbon
center, stereoelectronic effects can override this rule-of-thumb
in reactions catalyzed by salen-type complexes with Zr as the
central atom5 or, more frequently, in enzymatic transformations.6
In particular, in the series of fused bicycloketones incorporating
a cyclobutanone structural motif, regiodivergent biooxidation of
the antipodal substrate ketones to enantiomerically pure “normal”
and “abnormal” lactone regioisomers was observed as one of the
remarkable behaviors of BVMOs.7
Herein, we are reporting a preliminary study on regiodivergent
and enantiocomplementary biooxidations in the cyclohexanone
series. Utilizing a collection of BVMO producing recombinant E.
coli strains we were able to provide access to all regio- and enan-
tiomeric lactones for the first time starting from terpenone precur-
sors. In particular we have studied the biooxidation of pure enan-
tiomers of trans- and cis-dihydrocarvone 1a/b, carvomenthone
1c and menthone 1d (Scheme 1). We compared substrate accep-
tance profiles and stereopreferences of cyclohexanone (CH) and
cyclopentanone (CP) monooxygenases originating from Acineto-
bacter (CHMOAcineto),11 Arthrobacter (CHMOArthro),12 Brachymonas
(CHMOBrachy),13 Brevibacterium (CHMOBrevi1
,
CHMOBrevi2),14
Rhodococcus (CHMORhodo1
,
CHMORhodo2),12 and Comamonas
However, such a behavior was found only in a few cases of
the cyclohexanone series. Alphand and Furstoss reported that
wild-type cells of Acinetobacter NCIMB 9871 or the mutant
strain Acinetobacter TD63 convert (−)-trans-dihydrocarvone to
the expected normal lactone, while (+)-trans-dihydrocarvone
afforded abnormal lactone.8 In contrast, metabolism of carveol
and dihydrocarveol in Rhodococcus erythropolis DCL14 displayed
transformation of (+)-trans and (−)-cis-dihydrocarvone to normal
lactones while opposite enantiomers led to the formation of the
abnormal lactones.9
Institute of Applied Synthetic Chemistry, Vienna University of Technol-
ogy, Getreidemarkt 9/163-OC, 1060 Vienna, Austria. E-mail: mmihovil@
pop.tuwien.ac.at; Fax: +43-1-58801-15499; Tel: +43-1-58801-15420
† Electronic supplementary information (ESI) available: GC, GC-MS, 1H
and 13C NMR spectra. See DOI: 10.1039/b703175k
Scheme 1 Baeyer–Villiger oxidations of ketones 1a–d to “normal”
lactones 2a–d and “abnormal” lactones 3a–d.
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15
(CPMOComa
)
species in whole-cell mediated Baeyer–Villiger oxi-
enzymes (CHMOAcineto, CHMOArthro, CHMOBrachy, CHMORhodo1,
dations with recombinant E. coli as the host organism.16
CHMORhodo2) led to the formation of “normal” lactones 2a,c with
good to excellent conversions. In contrast, the opposite enan-
tiomers (+)-1a and (+)-1c were transformed exclusively into “ab-
normal” lactones 3a,c with “CHMO”-type proteins. Biooxidation
with “CPMO”-type enzymes (CHMOBrevi2, CPMOComa) showed
in both cases preferred formation of “abnormal” lactones 3a,c,
however, with low conversions. A somewhat different outcome
was observed for the biotransformation of (+)-1a and (+)-1c with
CHMOBrevi1, where both “normal” and “abnormal” lactones were
obtained in ratios of 55 : 45 and 41 : 59.
Biooxidation of cis-dihydrocarvone 1b was also investigated:
“CHMO”-type enzymes converted (−)-1b to “normal” lactone
(−)-2b with excellent conversions, while “abnormal” lactone (−)-
3b was generated with “CPMO”-type enzymes, however in low
conversions. While these findings are in line with biooxidations
of (−)-1a,c, the opposite enantiomer (+)-1b was only poorly to
moderately converted by all studied strains displaying decreased
regioselectivities.
Host strain mediated reduction to the corresponding alcohol
(10–30%) was observed whenever BVMO mediated biooxidation
was sluggish; this side reaction became even more prominent when
replacing LB media with TB media. Another possible side reaction
was observed for the cis-substrates: these compounds can undergo
chemical isomerization to trans-products and the presence of cells
seems to further promote this effect (below 20% of side product).
It is important to point out that both lactones (“normal” and
“abnormal”) can be prepared selectively by our BVMO collection
from each enantiomer of trans- and cis-dihydrocarvone as well as
carvomenthone.
Results and discussion
Enantiomerically pure biooxidation substrates were either pur-
chased (menthone 1d) or prepared according to literature pro-
tocols: reduction of (−)- and (+)-carvone was performed with
Zn dust to give cis- and trans-isomers of enantiomerically pure
dihydrocarvone in a ratio of 15 : 85, which were separated by
flash column chromatography; reduction with a mixture of zinc
dust and nickel chloride hexahydrate led to enantiomerically pure
carvomenthone.17
The regiopreference of Baeyer–Villiger biooxidation was first
investigated for (−)- and (+)-trans-dihydrocarvone with all eight
overexpression systems of E. coli. Initial mini-scale screening
experiments using 24-well plastic dishes previously standardized
in our laboratory18 gave results with only very low reproducibility
because of the high volatility of substrates, hence making baffled
flask biotransformations mandatory. The optimized experiments
were carried out with 10 mg of substrate in 25 cm3 of medium.
Biotransformations were stopped after 24 hours of fermentation
◦
time at 24 C and analyzed by chiral phase GC after extraction
of the sample with EtOAc supplemented by an internal standard.
Results of this screening program are summarized in Table 1.
Recently, we have proposed the existence of two distinct
sub-clusters of cycloketone oxidizing BVMOs based on protein
sequence alignment, substrate acceptance, and stereopreference.19
Baeyer–Villiger oxidations of (−)-trans-dihydrocarvone (−)-1a
and (−)-carvomenthone (−)-1c catalyzed with “CHMO”-type
Table 1 Screening for Baeyer–Villiger oxidations of ketones 1a–d by recombinant E. coli cells producing BVMOs of bacterial origin
trans-Dihydrocarvone
cis-Dihydrocarvone
Carvomenthone
Menthone
Conversiona
Strain
“normal” : “abnormal” lactone ratio
CHMOAcineto
CHMOArthro
CHMOBrachy
CHMOBrevi1
CHMOBrevi2
CPMOComa
CHMORhodo1
CHMORhodo2
+++
100 : 0
++
100 : 0
+++
100 : 0
+++
100 : 0
+
0 : 100
+
0 : 100
+++
100 : 0
+++
100 : 0
+++
0 : 100
+++
0 : 100
++
0 : 100
++
55 : 45
+(traces)
0 : 100
+
0 : 100
++
0 : 100
+++
0 : 100
+++
100 : 0
+++
100 : 0
+++
100 : 0
+++
100 : 0
+
0 : 100
+
0 : 100
+++
100 : 0
++
+
++
100 : 0
+
100 : 0
+++
100 : 0
++
100 : 0
+(traces)
0 : 100
+
0 : 100
++
100 : 0
+++
100 : 0
++
0 : 100
+
0 : 100
++
0 : 100
++
41 : 59
n.c.
—
++
0 : 100
++
n.c.
—
n.c.
—
n.c.
—
n.c.
—
n.c.
—
n.c.
—
n.c.
—
n.c.
—
+++
100 : 0
+++
100 : 0
+++
100 : 0
+++
100 : 0
n.c.
—
n.c.
—
+++
100 : 0
+++
100 : 0
64 : 36
+(traces)
0 : 100
+
57 : 43
+
100 : 0
n.c.
—
+
37 : 63
+
27 : 73
+
0 : 100
+++
0 : 100
100 : 0
28 : 72
a Conversion according to GC: +++: >90%, ++: 50–90%, +: <50%.
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Table 2 Baeyer–Villiger oxidations of ketones 1a–d on preparative scale
Yield (%)a
[a]2D0 (CHCl3)
2 (normal)
Substrate
(−)-1a
(+)-1a
(−)-1b
(+)-1b
(−)-1c
(+)-1c
Strain
Time/h
2 (normal)
3 (abnorm.)
3 (abnorm.)
CHMOAcineto
CPMOComa
CHMOAcineto
CHMOBrevi1
CHMOAcineto
CPMOComa
CHMOBrevi1
CHMOBrachy
CHMOBrevi1
CPMOComa
CHMOBrevi1
CHMOBrachy
CHMOAcineto
19
72
19
24
22
53
72
48
24
46
44
24
24
77 (80)8
—
—
34
77
—
60
—
71
—
18
+45.5 (c 1.48)
—
—
−42.1 (c 1.12)
−3.7 (c 3.78)
—
+3.3 (c 0.98)
—
—
+34.1 (c 0.46)
−34.6 (c 0.90)
−36.2 (c 0.73)
—
70 (73)8
28
—
28
—
7
—
60
44
76
−45.8 (c 0.62)
—
—
—
+16.9 (c 1.15)
—
—
34
—
+34.3 (c 2.35)
−35.1 (c 1.36)
−33.9 (c 1.55)
—
−17.2 (c 1.04)
—
(+)-1d
82 (90)8
—
+19.9 (c 1.58)
a Isolated yield after FCC.
In the menthone series (1d) the findings by the group of
Furstoss8 for CHMOAcineto could be generalized for the (+)-
substrate: “CHMO”-type enzymes give “normal” lactone 2d with
excellent conversions while “CPMO”-type enzymes did not accept
this precursor. None of the enzymes expressed within this strain
collection accepted (−)-1d.
substrate ketones was recently reported for the biooxidation of b-
substituted cyclopentanones and -hexanones determining migra-
tory preference to yield proximal or distal lactone products.21 The
CHMO originating from Brevibacterium (CHMOBrevi1) displayed
a distinctly different biooxidation profile, which underscores its
“borderline” position in the phylogenetic relationship relative to
“CHMO”-type and “CPMO”-type enzymes of this collection.
Considering the obtained results, we can conclude that both
“normal” and “abnormal” terpene derived lactones can be pre-
pared by enzyme mediated Baeyer–Villiger oxidation. Currently,
additional studies to determine the scope and limitation of this
behavior by BVMOs as well as the influence of various functional
substituents are being carried out by our group.
To confirm the results of the screening, some representative
biotransformations were also performed on preparative scale in
order to isolate and characterize the biooxidation products; results
are summarized in Table 2.
This set of experiments confirms the principal access to both
regioisomers of the biooxidation process, in enantiocomplemen-
tary form, of the trans-dihydrocarvone and carvomenthone series
for the first time. In the cis-dihydrocarvone series three out of
the possible four isomers are accessible on preparative scale. In
these cases the biooxidation represents the only means to access
the “abnormal” products via a Baeyer–Villiger process. Moreover,
the enzymes are highly chemoselective and tolerate the presence
of olefinic substituents, which would undergo epoxidation upon
chemical oxidation.
While the obtained yields (18–82%) are not optimized, advanced
fermentation techniques have recently been applied in similar
cases to improve the overall performance of the whole-cell
mediated biooxidation reaction.20 Adaptations of these techniques
to counter the high volatility of these precursors as the major
challenge of this substrate class are currently underway in our
laboratories.
Experimental
Unless otherwise noted, chemicals and microbial growth media
were purchased from commercial suppliers and used without
further purification. All solvents were distilled prior to use. Shake
flask fermentations were performed in a Gerhard THO5 orbital
thermoshaker. Flash column chromatography was performed on
silica gel 60 from Merck (40–63 lm). NMR spectra were recorded
from CDCl3 solutions on a Bruker AC 200 (200 MHz) spectrome-
ter and chemical shifts are reported in ppm using TMS as internal
standard. Enantiomeric purity was determined by chiral phase GC
using a BGB 175 column (30 m × 0.25 mm ID, 0.25 lm film) on a
ThermoFinnigan Trace or Focus chromatograph and compared to
reference material obtained by chemical m-chloroperoxybenzoic
acid oxidation where applicable. GC-MS analyses were carried
out on a GC-MS Voyager 8000Top with standard capillary column
DB5 (30 m × 0.32 mm ID, 1.0 lm film). Specific rotation [a]2D0 was
determined using a Perkin Elmer Polarimeter 241.
Conclusions
The observed substrate acceptance pattern and stereopreference
of the BVMOs investigated within this study is in good agree-
ment with our recently published hypothesis of two groups
of cycloketone oxidizing BVMOs.19 Within this class of sub-
strates “CHMO”-type enzymes displayed very good substrate
acceptance, while “CPMO”-type BVMOs usually gave poorer
conversions. “CHMO”-Type enzymes showed regiodivergent ox-
idations to both “normal” and “abnormal” lactones depending
on the absolute configuration of the ketone precursor; “CPMO”-
type BVMOs usually yielded “abnormal” lactone products. A
similar influence of the absolute configuration of enantiopure
Since no comprehensive characterization of the “normal” and
“abnormal” lactones of cis- and trans-dihydrocarvone, carvomen-
thone and menthone has been published until now, we provide
experimental data of all synthesised lactones.
Typical procedure for screening experiment
Precultures were inoculated with a single colony from a plate and
incubated at 37 ◦C on an orbital shaker at 120 rpm overnight.
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LBamp (25 cm3: 1% peptone, 0.5% yeast extract, 1% NaCl in dion.
water, supplemented by 200 lg mL−1 of ampicillin) was inoculated
with the overnight preculture (250 lL) and incubated for 2–3 hours
dH(200 MHz; CDCl3; Me4Si) 1.22 (3 H, d, J 6.7 Hz), 1.50–2.07
(4 H, m), 1.82 (3 H, s), 2.22–2.34 (1 H, m), 2.68–2.85 (1 H, m),
4.15–4.18 (2 H, m) and 4.72–4.83 (2 H, m); dC(50 MHz; CDCl3;
Me4Si) 18.4 (q), 21.8 (q), 31.8 (t), 34.1 (t), 37.2 (d), 46.4 (d), 71.5
(t), 111.1 (t), 145.7 (s) and 177.7 (s); m/z 168 (M+, 0.4%), 138 (23),
110 (100), 95 (46), 68 (89).
◦
at 37 C. After reaching an optical density OD590 = 0.6, 4 lL of
IPTG stock solution was added (final concentration of 0.134 mM)
to start the expression of the corresponding monooxygenase,
followed by addition of 10 mg of substrate. Transformations were
analyzed after 24 hours of fermentation time at 24 ◦C by extraction
of the sample with EtOAc supplemented by an internal standard.
7-Methyl-4-isopropenyl-2-oxo-oxepanone 2b.
(4S,7R)-(−). (−)-cis-Dihydrocarvone 1b (100 mg in 250 cm3
of LBamp) was oxidized with CHMOAcineto according to the general
procedure. The crude product was purified by column chromatog-
raphy (silica gel, PE : Et2O = 3 : 1) and (−)-2b9 was obtained as a
colorless oil (85 mg, 77%). [a]2D0 −3.7 (c 3.78, CHCl3).
Typical procedure for biotransformation on preparative scale
Fresh LBamp medium (1% peptone, 0.5% yeast extract, 1% NaCl
in dion. water, supplemented by 200 lg mL−1 of ampicillin) was
inoculated with 1% of an overnight preculture of the correspond-
ing recombinant E. coli strain in a baffled Erlenmeyer flask. The
culture was incubated at 120 rpm at 37 ◦C on an orbital shaker for
2–3 hours. After reaching an optical density OD590 = 0.6, IPTG
stock solution was added to a final concentration of 0.134 mM.
The substrate (3–6 mM) was added neat along with b-cyclodextrin
(1 equiv.). The culture was incubated at rt for 19–72 hours. The
biomass was removed by centrifugation, saturated with sodium
chloride, and repeatedly extracted with the corresponding solvent
(EtOAc, diethylether or dichloromethane). The combined organic
layers were dried over sodium sulfate, filtered, and the solvent was
removed in vacuo. The crude material was purified by standard
flash column chromatography.
(4R,7S)-(+). (+)-cis-Dihydrocarvone 1b (50 mg in 125 cm3 of
LBamp) was oxidized with CHMOBrevi1 according to the general
procedure. The crude product was purified by column chromatog-
raphy (silica gel, PE : Et2O = 3 : 1) and (+)-2b was obtained as a
colorless oil (33 mg, 60%). [a]2D0 +3.3 (c 0.98, CHCl3).
dH(200 MHz; CDCl3; Me4Si) 1.37 (3 H, d, J 6.4 Hz), 1.72–2.05
(4 H, m), 1.79 (3 H, s), 2.52–2.59 (1 H, m), 2.75–3.04 (2 H, m),
4.44–4.55 (1 H, m) and 4.84–4.86 (2 H, m); dC(50 MHz; CDCl3;
Me4Si) 21.3 (q), 21.8 (q), 29.7 (t), 33.3 (t), 38.4 (t), 38.6 (d), 75.5
(d), 111.4 (t), 146.3 (s) and 173.8 (s); m/z 168 (M+, 6%), 139 (13),
108 (98), 81 (39), 67 (100).
3-Methyl-6-isopropenyl-2-oxo-oxepanone 3b9.
(3R,6R)-(−). (−)-cis-Dihydrocarvone 1b (95 mg in 250 cm3
of LBamp) was oxidized with CPMOComa according to the general
procedure. The crude product was purified by column chromatog-
raphy (silica gel, PE : Et2O = 3 : 1) and (−)-3b was obtained as a
colorless oil (30 mg, 28%). [a]2D0 −45.8 (c 0.62, CHCl3).
7-Methyl-4-isopropenyl-2-oxo-oxepanone 2a.
(4S,7S)-(+). (−)-trans-Dihydrocarvone 1a (60 mg in 125 cm3
of LBamp) was oxidized with CHMOAcineto according to the general
procedure. The crude product was purified by column chromatog-
raphy (silica gel, petroleum ether (PE) : Et2O = 6 : 1) and (+)-2a8
was obtained as a colorless oil (51 mg, 77%). [a]2D0 +45.5 (c 1.48,
CHCl3) (lit.,8 +46.2, c 1.1, CHCl3).
dH(200 MHz; CDCl3; Me4Si) 1.20 (3 H, d, J 6.7 Hz), 1.64–2.14
(4 H, m), 1.80 (3 H, s), 2.40–2.50 (1 H, m), 2.76–2.93 (m, 1H),
4.32–4.49 (2 H, m) and 4.88–4.90 (2 H, m); dC(50 MHz; CDCl3;
Me4Si) 18.0 (q), 21.6 (q), 28.6 (t), 29.5 (t), 36.7 (d), 43.2 (d), 69.0
(t), 112.3 (t), 144.1 (s) and 177.2 (s); m/z 168 (M+, 3%), 153 (9),
138 (12), 110 (98), 95 (51), 68 (100).
(4R,7R)-(−). (+)-trans-Dihydrocarvone 1a (92 mg in 250 cm3
of LBamp) was oxidized with CHMOBrevi1 according to the general
procedure. The crude product, a mixture of normal and abnormal
lactones, was separated by column chromatography (silica gel, PE :
Et2O = 6 : 0.5; 6 : 1) and (−)-2a9 was obtained as a colorless oil
(35 mg, 34%). [a]2D0 −42.1 (c 1.12, CHCl3)
7-Methyl-4-isopropyl-2-oxo-oxepanone 2c22.
(4R,7S)-(+). (−)-Carvomenthone 1c (50 mg in 125 cm3 of
LBamp) was oxidized with CHMOBrevi1 according to the general
procedure. The crude product was purified by column chromatog-
raphy (silica gel, PE : EtOAc = 9 : 1) and (+)-2c was obtained as
a colorless oil (39 mg, 71%). [a]2D0 +16.9 (c 1.15, CHCl3).
dH(200 MHz; CDCl3; Me4Si) 1.37 (3 H, d, J 6.4 Hz), 1.58–1.97
(4 H, m), 1.73 (3 H, s), 2.24–2.35 (1 H, m), 2.58–2.80 (2 H, m),
4.41–4.54 (1 H, m) and 4.74–4.76 (2 H, m); dC(50 MHz; CDCl3;
Me4Si) 20.1 (q), 22.6 (q), 34.3 (t), 35.8 (t), 40.2 (t), 41.8 (d), 76.2
(d), 110.1 (t), 148.4 (s) and 174.6 (s); m/z 168 (M+, 6%), 125 (33),
108 (43), 81 (34), 67 (100).
(4S,7R)-(−). (+)-Carvomenthone 1c (93 mg in 250 cm3 of
LBamp) was oxidized with CHMOBrevi1 according to the general
procedure. The crude product, a mixture of normal and abnormal
lactone, was separated by column chromatography (silica gel, PE :
EtOAc = 9.5 : 0.5) and (−)-2c was obtained as a colorless oil
(36 mg, 34%). [a]2D0 −17.2 (c 1.04, CHCl3).
3-Methyl-6-isopropenyl-2-oxo-oxepanone 3a.
(3S,6R)-(+). (−)-trans-Dihydrocarvone 1a (95 mg in 250 cm3
of LBamp) was oxidized with CPMOComa according to the general
procedure. The crude product was purified by column chromatog-
raphy (silica gel, PE : Et2O = 6 : 1) and (+)-3a9 was obtained as a
colorless oil (19 mg, 18%). [a]2D0 +34.1 (c 0.46, CHCl3).
(3R,6S)-(−). (+)-trans-Dihydrocarvone 1a (40 mg in 125 cm3
of LBamp) was oxidized with CHMOAcineto according to the general
procedure. The crude product was purified by column chromatog-
raphy (silica gel, PE : Et2O = 6 : 1) and (−)-3a8 was obtained as a
colorless oil (31 mg, 70%). [a]2D0 −34.6 (c 0.9, CHCl3) (lit.8 −35.8,
c 1.6, CHCl3).
dH(200 MHz; CDCl3; Me4Si) 0.87 (3 H, d, J 6.7 Hz), 0.90 (3 H,
d, J 6.7 Hz), 1.35 (3 H, d, J 6.4 Hz), 1.41–1.96 (6 H, m), 2.46–2.49
(1 H, m) and 4.36–4.50 (1 H, m); dC(50 MHz; CDCl3; Me4Si) 18.5
(q), 18.8 (q), 22.6 (q), 31.3 (t), 33.5 (d), 35.8 (t), 38.1 (t), 40.3 (d),
76.6 (d) and 175.7 (s); m/z 171 (M+, 0.4%), 155 (1), 126 (58), 83
(100), 69 (29), 55 (52).
3-Methyl-6-isopropyl-2-oxo-oxepanone 3c.
(3S,6R)-(+). (−)-Carvomenthone 1c (90 mg in 250 cm3 of
LBamp) was oxidized with CPMOComa according to the general
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procedure. The crude product was purified by column chromatog-
raphy (silica gel, PE : EtOAc = 9 : 1) and (+)-3c was obtained as
a colorless oil (60 mg, 60%). [a]2D0 +34.3 (c 2.35, CHCl3).
(3R,6S)-(−). (−)-Carvomenthone 1c (50 mg in 125 cm3 of
LBamp) was oxidized with CHMOBrachy according to the general
procedure. The crude product was purified by column chromatog-
raphy (silica gel, PE : EtOAc = 9 : 1) and (−)-3c was obtained as
a colorless oil (42 mg, 76%). [a]2D0 −33.9 (c 1.55, CHCl3).
dH(200 MHz; CDCl3; Me4Si) 0.90 (3 H, d, J 6.8 Hz), 0.91 (3 H,
d, J 6.8 Hz), 1.19 (3 H, d, J 6.8 Hz), 1.50–1.89 (6 H, m), 2.70–2.78
(1 H, m) and 4.03–4.20 (2 H, m); dC(50 MHz; CDCl3; Me4Si) 18.4
(q), 19.1 (q), 19.4 (q), 31.0 (d), 31.1 (t), 31.8 (t), 37.0 (d), 44.6 (d),
71.6 (t) and 178.1 (s); m/z 171 (M+, 0.4%), 140 (3), 125 (5), 98
(100), 82 (14), 69 (30), 55 (32).
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(4S,7R)-(+)-4-Methyl-7-isopropyl-2-oxo-oxepanone 2d. (+)-
Menthone 1d (50 mg in 125 cm3 of LBamp) was oxidized with
CHMOAcineto according to the general procedure. The crude
product was purified by column chromatography (silica gel, PE :
EtOAc = 9 : 0.5) and (+)-2d8 was obtained as a colorless oil
(45 mg, 82%). [a]2D0 +19.9 (c 1.58, CHCl3) (lit.,8 +20.5, c 1.4,
CHCl3).
dH(200 MHz; CDCl3; Me4Si) 0.97 (3 H, d, J 6.8 Hz), 0.98 (3 H,
d, J 6.8 Hz), 1.04 (3 H, d, J 6.6 Hz), 1.20–1.99 (6 H, m), 2.42–2.62
(2 H, m), 4.04 (1 H, dd, J 9.0 and 4.4 Hz); dC(50 MHz; CDCl3;
Me4Si) 17.0 (q), 18.3 (q), 23.9 (q), 30.3 (d), 30.9 (t), 33.2 (d), 37.3
(t), 42.4 (t), 84.6 (d) and 174.9 (s); m/z 171 (M+, 2%), 127 (93), 99
(85), 81 (100), 69 (96), 55 (86).
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16 Expression systems for CHMO from Acinetobacter (CHMOAcineto) in
E. coli: G. Chen, M. M. Kayser, M. D. Mihovilovic, M. E. Mrstik,
C. A. Martinez and J. D. Stewart, New J. Chem., 1999, 23, 827–832;
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two-enzyme system, see: S. Rissom, U. Schwarz-Linek, M. Vogel,
V. I. Tishkov and U. Kragl, Tetrahedron: Asymmetry, 1997, 8, 2523–
2526.
Acknowledgements
Funding for this research by the Austrian Science Fund (FWF,
Project No. P16373) is gratefully acknowledged.
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