Microbial Baeyer-Villiger oxidation of prochiral polysubstituted cyclohexanones by recombinant whole-cells expressing two bacterial monooxygenases

Article abstract of DOI:10.1002/ejoc.200400676

The microbial Baeyer-Villiger oxidation of prochiral 3,5- dimethylcyclohexanones bearing various functionalities with recombinant E. coli cells overexpressing cyclohexanone monooxygenase from Acinetobacter sp. NCIMB 9871 and cyclopentanone monooxygenase f

Full text of DOI:10.1002/ejoc.200400676

FULL PAPER
Microbial Baeyer–Villiger Oxidation of Prochiral Polysubstituted
Cyclohexanones by Recombinant Whole-Cells Expressing Two Bacterial
Monooxygenases
Marko D. Mihovilovic,*[a] Florian Rudroff,[a] Birgit Grötzl,[a] and Peter Stanetty[a]
Keywords: Baeyer–Villiger oxidation / Biocatalysis / Biotransformations / Ketones / Oxygenases
The microbial Baeyer–Villiger oxidation of prochiral 3,5-di-
methylcyclohexanones bearing various functionalities with
recombinant E. coli cells overexpressing cyclohexanone
monooxygenase from Acinetobacter sp. NCIMB 9871 and
cyclopentanone monooxygenase from Comamonas sp.
NCIMB 9872 has been investigated. A distinct difference in
substrate specificity and stereoselectivity of the two enzymes
was observed, and enantiocomplementary products were ob-
tained in some cases. The biocatalytic systems enabled ac-
cess to chiral lactones as valuable intermediates for the total
synthesis of various natural compounds. Substituents with
varying lipophilicity and hybridization have been prepared
by a diastereoselective synthetic route and subsequently bio-
transformed for the investigation of conformational and elec-
tronic effects on the biooxidation,.
(© Wiley-VCH Verlag GmbH & Co. KGaA, 69451 Weinheim,
Germany, 2005)
oxygen-transfer process requires NADPH and FAD as co-
factors.
Introduction
Baeyer–Villiger oxidation of cyclic ketones offers a facile
entry to chiral lactones, which are interesting intermediates
in enantioselective chemistry and are frequently encoun-
tered as precursors in natural compound synthesis.[1] Over
recent years, asymmetric, metal-catalyzed Baeyer–Villiger
oxidations have been continuously improved and represent
CHMO is currently the best studied Baeyer–Villigerase,
and its remarkably broad substrate specificity has been es-
tablished in several studies.[4,5,8] One of the most important
advantages of the enzyme is its ability to perform this trans-
formation in an enantioselective way with nonnatural sub-
strates. In addition, the enzyme is capable of performing
stereoselective oxidation of heteroatoms, with the conver-
sion of thioethers to optically pure sulfoxides being an espe-
cially valuable biotransformation.[9]
In order to provide a simple and convenient catalytic sys-
tem for synthetic chemists, we designed an E. coli-based
overexpression system for CHMO[10,11] as a second-genera-
tion recombinant version of the previously reported “de-
signer yeasts”.[12–14] By performing whole-cell fermenta-
tions, all required cofactors, especially NADPH, are recy-
cled by the organism and it is not necessary to use artificial
regeneration systems.[15]In addition the laborious process of
enzyme isolation, which is complicated by protein stability,
especially in the case of CHMO, can be avoided by whole-
cell fermentations. It is the aim of this approach to take
away the nimbus of using “exotic reagents” and to make
recombinant cells an ordinary tool in synthetic chemistry.
Whole-cell fermentation protocols have previously been
optimized for CHMO, with promising results reported with
nongrowing recombinant cells.[16,17] The first dynamic kin-
etic resolution of a racemic precursor was reported using a
high-pH-tolerant recombinant strain.[18] Very recently, sig-
nificant progress has been made to scale-up monooxygen-
ase-mediated biooxidations to large-scale synthetic applica-
tions.[19,20]
a promising strategy for future process developments.[2]
A
complementary methodology is offered by biocatalysis. This
“green chemistry” alternative usually gives products with
optical purities that are still unattainable by artificial cata-
lytic entities.
Flavin-dependent
Baeyer–Villiger
monooxygenase
(BVMO)-catalyzed biotransformations play a key role in
degradative pathways of microorganisms.[3–6] The action of
such enzymes in cell metabolism is restricted to a single
compound, but a remarkable acceptance for nonnatural
substrates has been observed. Together with high enantiose-
lectivity, this protein family represents a powerful catalytic
system for chiral synthesis.
Cyclohexanone monooxygenase (CHMO) is a bacterial
flavoprotein, which was found in Acinetobacter sp. NCIMB
9871 by Donoghue and Trudgill in 1975.[7] By applying se-
lective growth conditions in cyclohexanol, a degradative
pathway was triggered involving the Baeyer–Villiger oxi-
dation of cyclohexanone to ε-caprolactone by CHMO. The
[a]
Vienna University of Technology, Institute of Applied Synthetic
Chemistry,
Getreidemarkt 9/163-OC, 1060 Vienna, Austria
Fax: +43-1-58801-15499
E-mail: mmihovil@pop.tuwien.ac.at
Eur. J. Org. Chem. 2005, 809–816
DOI: 10.1002/ejoc.200400676
© 2005 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
809

Marko D. Mihovilovic, F. Rudroff, B. Grötzl, P. Stanetty
FULL PAPER
Scheme 1. Biotransformation of functionalized cyclohexanone systems with CHMO-expressing recombinant cells
Scheme 2. Lactone 3a as a key intermediate for the synthesis of tirandamycin and calyculin A
With the recent construction of a whole-cell overexpres- effect of polarity and steric strain on the substrate specific-
sion system for cyclopentanone monooxygenase (CPMO) ity of CHMO and CPMO. Although a crystallographic
[21]
from Comamonassp. NCIMB 9872 (EC 1.14.13.16),[22] structure determination has been reported very recently for
we started a comparative substrate-profiling program. a rather distantly related BVMO from a Thermobifida sp.
CPMO has received considerably less attention in biocata- with a very different substrate-acceptance pattern,[35] so far,
lytic Baeyer–Villiger oxidations, as a biocatalytic behavior active-site models for enzymes that convert cycloketones are
similar to CHMO was initially assumed.[23,24] However, re- only available based on substrate profiling.[36–40] The qual-
cent studies have revealed some differences in substrate ac- ity of predictions derived from such “super-substrate mod-
ceptance as well as stereospecificity of the two enzymes, els” depends heavily on the size and diversity of the data
with the enantiodivergent biooxidation of some ketones be- set they are based on.
ing the most prominent example.[25–27]
The functionalities contained in substrate ketones 1a–g
In our previous studies with recombinant whole cells ex-
pressing CHMO we observed a significant dependence of
conversion and enantioselectivity on conformational as-
pects of the substrate.[26,28,29] These findings prompted us
to reinvestigate such effects on polysubstituted cyclohexa-
nones. Ketones 1a–g (Scheme 1) are highly interesting
model compounds for such a study, since the corresponding
lactones 2/3 are versatile building blocks for subsequent
natural compound synthesis: Taschner and coworkers have
established 3a, which is derived from the intermediate lac-
tone 2a by a rearrangement process after a biotransforma-
tion with isolated CHMO,[30] as a key intermediate for the
synthesis of calyculin (Scheme 2).[31] This compound has
also been utilized in approaches towards tirandamycin
A.[32–34]
involve both hydrophilic (R = OH) and lipophilic groups
(R = Cl) in cis (1a/c) and trans substitution patterns (1b/d).
Since we have observed a significant effect of hybridization
on the enantioselectivity of CHMO when transforming pro-
chiral bicyclo[3.3.0]ketones,[26,29] both sp3 (1a–d) and sp2
centers (1e) at position 4 were studied. We also became in-
terested in a 4,4-disubstituted substrate (1f), superimposing
the structural features of cis- and trans-ketones 1a–e. In or-
der to compare the effect of substitution at position 4 with
the unsubstituted case, we included ketone 1g (R = H) in
the study as a reference point.[41]
Access to the model substrates proceeded in a straight-
forward manner. The mono-protected quinone 4 is a readily
available precursor for the subsequent functionalization.[42]
Reduction of the double bonds by catalytic hydrogenation
using palladium on charcoal gave a mixture of cis- and
trans-isomers 5 (Scheme 3). This mixture of diastereomers
was converted into the thermodynamically favored cis con-
Results and Discussion
This study focuses on the 3,5-dimethyl ketones 1, bearing former, with both methyl groups adopting an equatorial po-
various substituents in position 4, as probes to study the sition, by base-promoted epimerization. Taking advantage
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Microbial Baeyer–Villiger Oxidation of Prochiral Polysubstituted Cyclohexanones
FULL PAPER
Scheme 3. (i) H2, Pd/C, 5 bar; (ii) Na/MeOH, room temp., 69% (overall); (iii) NaBH4, dry MeOH, 0 °C, 90%; (iv) 1% H2SO4/acetone,
(1:1), room temp. to 40 °C, 88–92%; (v) PPh3, NCS, THF, 61–80%; (vi) MePPh3+Br–, BuLi, dry THF, 91%; (vii) ZnEt2, CH2I2,
CF3COOH, CH2Cl2, 0 °C, then 2 n HCl, room temp., 50% (overall)
of the acidity of the α-protons adjacent to the carbonyl Smith reaction conditions using the Cu/Zn couple[28,47] or
group, repeated deprotonation and reprotonation in a pro- activated Zn[42] failed to give reproducible yields. Conse-
[48]
tic solvent[43] gave cis-5 in greater than 95% purity as a key quently, we changed to ZnEt2
to generate the required
intermediate for subsequent functionalization. The hydro- carbene species, and best results were obtained with
genation and epimerization was performed without purifi- CF3COOH as additive.[49] Due to the high volatility of the
cation. The final step gave pure intermediate 5 in an overall ketal intermediate, we preferred to convert precursor 6e di-
yield of 69% after column chromatography.
rectly into ketone 1f in a single operation.
Reduction of compound 5 with sodium borohydride at
In general, deprotection was carried out under transketa-
0 °C led to cis- and trans-alcohols 6a and 6b in a ratio of lization conditions using a mixture of acetone and 1% sul-
1:3 as the first model substrates for the substrate profiling. furic acid at 25–40 °C to give ketones 1a–e.
The two diastereomers could be separated by flash column
Expression of CHMO or CPMO in growing cultures of
chromatography. Since both compounds were required for recombinant E. coli strain BL21(DE3)(pMM4) or
the subsequent substrate acceptance studies, we preferred DH5α(pCMO206) was triggered by addition of isopropyl-
this rapid route to both compounds to a selective reduction β-d-thiogalactopyranoside (IPTG). Fermentations were
to 6a using l-selectride[44] and subsequent Mitsunobu inver- carried out according to our previously reported procedure
sion, a protocol recently utilized by us on similar sys- on a 250 mL scale in Erlenmeyer flasks;[26] β-cyclodextrin
tems.[26]
was added to facilitate the biotransformations.[50] Racemic
The introduction of a chloro substituent as representative reference samples of lactones were prepared by chemical
nonpolar substrate for the subsequent biotransformations oxidation with m-CPBA where applicable (2c/d/f/g, 3a/b).
was performed by a nucleophilic substitution of the pro-
tected alcohols under Mitsunobu-type conditions in the with recombinant whole-cells are summarized in Table 1.
presence of triphenylphosphane and NCS.[26,45] The trans-
All substrates 1a–g were converted into the correspond-
formation gave clean inversion of the stereocenter to pro- ing lactones (2c–g, 3a/b) by CHMO-expressing cells with
ducts 6c and 6d in good to excellent yields. good to very good yields and excellent enantiomeric excess.
The results of the biotransformations of substrates 1a–g
Introduction of the exocyclic methylene group towards The biotransformation of cis-ketone 1a with recombinant
compound 6e was performed under standard Wittig condi- whole-cells gave the rearranged lactone (–)-{4S-[4α,5β(S*)]}-
tions.[46] We preferred this two-step route to the method re- 3a similar to previous reports on biooxidation with the iso-
ported in the literature,[42] since reproducing the in situ epi- lated enzyme.[30] The seven-membered-ring intermediate
merization under the basic conditions of the Wittig reaction was not observed, and intramolecular attack of the 4-
was troublesome.
hydroxy group gave the corresponding γ-lactone exclusively.
The isomeric trans substrate 1b showed similar behavior,
Access to the cyclopropyl spiro system was investigated
from compound 6e. Standard heterogeneous Simmons– and the rearranged compound (+)-3b was isolated as the
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Marko D. Mihovilovic, F. Rudroff, B. Grötzl, P. Stanetty
Table 1. Biotransformation of functionalized cyclohexanone substrates with CHMO- and CPMO-expressing recombinant cells
FULL PAPER
Substrate
R
cis-OH
trans-OH
cis-Cl
Product
3a
Enzyme
Yield[a]
ee[b]
[α]2D0
CHMO
CPMO
CHMO
CPMO
CHMO
CPMO
CHMO
CPMO
CHMO
CPMO
CHMO
CPMO
CHMO
CPMO
77%
n.c.[c]
80%
n.c.[c]
53%
n.c.[c]
40%
n.c.[c]
54%
63%
57%
n.c.[c]
65%
58%
99%
n.a.[d]
96%
–24.5 (c = 1.71, CHCl3)
n.a.[d]
1a
1b
1c
1d
1e
1f
+53.7 (c = 1.76, CHCl3)
3b
n.a.[d]
99%
n.a.[d]
–67.1 (c = 0.76, CHCl3)
n.a.[d]
2c
n.a.[d]
Ͼ99%
n.a.[d]
92%
–81.5 (c = 1.30, CHCl3)
trans-Cl
=CH2
2d
n.a.[d]
+8.4 (c = 0.80, CHCl3)
–8.6 (c = 0.64, CHCl3)
+73.0 (c = 1.00, CHCl3)
n.a.[d]
–12.7 (c = 2.0, CHCl3)
+7.7 (c = 1.3, CHCl3)
2e
99%
Ͼ99%
n.a.[d]
Ͼ99%
91%
cyclopropyl
H
2f
1g
2g
[a] Yield of product isolated after purification by column chromatography. [b] Enantiomeric excess was determined by chiral-phase gas
chromatography; racemic reference material was prepared by oxidation of ketones 1a–d and 1f/g with m-CPBA. [c] No conversion. [d]
Not applicable.
only product of the biooxidation. Biotransformation of the vey of a group of prochiral 3,4,5-trisubstituted cyclohexa-
lipophilic substrates bearing a chloro substituent gave the nones for the microbial oxidation to the corresponding lac-
expected lactones (–)-2c/d, also in high optical purity and tones. A diastereoselective route to substrates with both hy-
acceptable yields. Only olefin 1e, which bears an sp2 center drophilic (1a/b) and lipophilic (1c–f) substituents has been
in position 4, gave lactone (+)-2e in slightly decreased op- developed.
tical purity. However, enzymatic oxidation was highly
chemoselective for the Baeyer–Villiger process and no unde-
sired attack at the double bond was observed.
In the case of CHMO-mediated biooxidations, the dif-
ferent stereochemistry of the substrates does not have a sig-
nificant effect on the efficiency of the biooxidation or the
enantioselectivity of the enzymatic reaction. In the case of
trisubstituted cyclohexanone substrates, CHMO seems to
be less susceptible to conformational aspects than we had
previously found for a series of bicyclo[3.3.0]ketones.[26]
Even the presence of a quaternary center with spiro-disub-
stitution at position 4 does not influence the efficiency or
stereoselectivity of the enzymatic oxidation. This might pro-
vide valuable insights to further refine existing active-site
models. Position 4 is particularly tolerant towards various
types of hybridization, as substituents can adopt axial and
equatorial positions for sp3 centers. Occupation of both
substitution sites does not hamper transformation by the
whole-cell expression system, even in the presence of two
additional substituents in the vicinity. The steric flexibility
of the active site also allows the presence of an sp2 methyl-
ene group.
While substrate acceptance of CHMO and CPMO was
observed to significantly overlap in previous studies using
recombinant whole-cells, the latter expression system dis-
plays some restrictions for the conversion of polysubstituted
cyclohexanones in the present survey. Only sterically less
challenging substrates were transformed, with enantiocom-
plementary selectivity compared to CHMO. This further
extends the array of ketones oxidized to enantiodivergent
lactones and underscores the high potential of this enzyme
pair for future applications.
Substrate 1f combines structural features of lipophilic ke-
tones 1c and 1d by bearing two substituents in the 4-posi-
tion. This compound was readily accepted by CHMO and
was converted into (+)-2f with excellent enantioselectivity.
Biotransformation of 1g has previously been carried out
using isolated CHMO and gave good yields using the
whole-cell fermentation protocol. The enantioselectivity
was comparable to literature reports and the absolute con-
figuration of (–)-2 was assigned as (4S,6R).[30]
Biooxidations with CPMO were only successful in two
cases to yield the expected lactones: ketones 1e and 1g were
readily converted by recombinant CPMO-producing cells.
Obviously, in the case of 3,5-dimethyl precursors only lim-
ited flexibility of the active site of CPMO seems to exist.
Substitution at the axial or equatorial position at carbon 4
of the substrate prevents conversion by the enzyme. This is
the case for both lipophilic and hydrophobic groups. Only
a methylene substituent seems to be accommodated within
the active site simultaneously with the presence of groups
in the 3- and 5-positions of the substrate. Consequently, the
4-unsubstituted ketone 1g is also oxidized by CPMO.
It is remarkable that the biooxidation with CPMO leads
to antipodal lactones 2e/g as obtained in CHMO-mediated
transformations. Both microbial reactions proceed in excel-
lent stereoselectivity. This extends the number of substrates
where enantiodivergent transformation is observed for this
pair of enzymes.
The complementary biocatalytic performance of CHMO
and CPMO with respect to stereoselectivity is similar to a
recently discovered pair of BVMOs from Brevibacterium,
Conclusions
Based on an initial study by Taschner et al. for which also display enantiodivergent biotransformation for
CHMO,[30] this work represents an extended systematic sur- several structurally diverse substrate ketones.[51] Further
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Microbial Baeyer–Villiger Oxidation of Prochiral Polysubstituted Cyclohexanones
FULL PAPER
tion. Subsequently, pure ketone 5 was added slowly with a syringe.
After 12 h stirring at room temp., GC indicated complete conver-
sion. The mixture was hydrolyzed with saturated NH4Cl solution,
extracted three times with diethyl ether, and washed with brine. The
combined organic layers were dried with sodium sulfate and the
solvent was evaporated. The very volatile product 6e (1.80 g, 91%,
colorless volatile liquid) was used directly for the subsequent trans-
formation without further purification. 1H NMR spectroscopic
data identical to those described in the literature.[42]13C NMR
(CDCl3): δ = 18.0 (q), 34.7 (d), 44.7 (t), 64.2 (t), 64.4 (t), 102.5 (t),
108.8 (s), 156.0 (s) ppm.
substrate profiling of the recombinant expression systems
for these enzymes is currently underway in our laboratories
in order to establish a toolbox of BVMO biocatalysts for a
broad range of synthetic applications.
Experimental Section
Unless otherwise noted, chemicals and microbial growth media
were purchased from commercial suppliers and used without fur-
ther purification. All solvents were distilled prior to use (LP: light
petroleum, EtOAc: ethyl acetate). Flash column chromatography
was performed on silica gel 60 from Merck (40–63 µm). Melting
points were determined with a Kofler-type Leica Galen III micro
hot stage microscope and are uncorrected. NMR spectra were re-
corded from CDCl3 or [D6]DMSO solutions on a Bruker AC 200
(200 MHz) or Bruker Avance UltraShield 400 (400 MHz) spec-
trometer and chemical shifts are reported in ppm relative to TMS
as internal standard. Combustion analysis was carried out in the
Microanalytical Laboratory of the University of Vienna. Enantio-
meric excess was determined by GC using a BGB 175 column (30 m
× 0.25 mm ID, 0.25 µm film) on a HP 6890 Series chromatograph.
Biotransformation progress and conversion control was performed
with a ThermoQuest Trace GC 2000 using a standard capillary
column DB5 (30 m × 0.32 mm ID). Specific rotation [α]2D0 was de-
termined with a Perkin–Elmer Polarimeter 241.
Reduction with NaBH4 in dry MeOH: Compound 5 (500 mg,
2.70 mmol) was dissolved in dry MeOH (20 mL) and cooled to
0 °C. Then, NaBH4 (2 equiv.) was added in portions whilst main-
taining the temperature at 0 °C. After complete addition of NaBH4
the reaction went to completion within 1 h. Unreacted NaBH4 was
slowly hydrolyzed by addition of 0.1 n NaOH and the aqueous
layer was extracted three times with diethyl ether. The combined
organic phases were washed with brine, dried over sodium sulfate,
and the solvent was evaporated. The two diastereomers formed
were separated by column chromatography (LP/EtOAc, 10:1, silica
gel).
(7α,8α,9α)-7,9-Dimethyl-1,4-dioxaspiro[4.5]decan-8-ol (6a): Re-
duction according to the above procedure gave pure alcohol 6a[44]
(113.2 mg, 23%) as colorless crystals after chromatographic separa-
tion. M.p.: 68–72 °C.
cis-7,9-Dimethyl-1,4-dioxaspiro[4.5]decan-8-one (5): 7,9-Dimethyl-
1,4-dioxaspiro[4.5]deca-6,9-dien-8-one (4; 9.11 g, 50.5 mmol) and
Pd/C catalyst (1 g, 10%) suspended in EtOAc (200 mL) were placed
into a Parr apparatus. Reduction was carried out under a hydrogen
pressure of 5 bar at room temp. overnight. Once GC indicated com-
plete conversion, the catalyst was removed by filtration through a
bed of Celite® and the solvent was evaporated. A cis/trans mixture
of 5 was obtained as a yellow oil and used without purification in
the epimerization step.
The crude cis,trans-7,9-dimethyl-1,4-dioxaspiro[4.5]decan-8-one (5)
from the above reduction (7.16 g, 38.9 mmol) was added to a solu-
tion of NaOMe, prepared from sodium (447 mg, 19.5 mmol) by the
usual method, in dry MeOH (40 mL) and the reaction mixture was
stirred at room temp. for 12 h. The progress of the epimerization
was determined by GC. The reaction mixture was poured into
water and extracted three times with EtOAc. The combined organic
layers were dried with sodium sulfate and the solvent was evapo-
rated. Flash column chromatography (LP/EtOAc, 5:1, NEt3 im-
pregnated silica gel) gave pure cis-5 as a yellowish liquid (6.42 g,
overall yield: 69%). The 1H NMR spectroscopic data were identical
to reports in the literature.[44]13C NMR (CDCl3): δ = 14.1 (q), 41.0
(d), 43.6 (t), 64.6 (t), 107.2 (s), 213.1 (s) ppm. trans-5 (spectroscopic
data obtained as a mixture with cis-5). 1H NMR (CDCl3): δ = 1.01
(d, J = 6.7 Hz, 3 H), 1.14 (d, J = 6.6 Hz, 3 H), 1.61–1.90 (m, 2 H),
2.00–2.17 (m, 2 H), 2.59–2.87 (m, 2 H), 3.95–4.12 (m, 4 H) ppm.
13C NMR (CDCl3): δ = 16.0 (q), 40.0 (d), 41.1 (t), 64.2 (t), 107.7
(s), 215.6 (s) ppm.
(7α,8β,9α)-7,9-Dimethyl-1,4-dioxaspiro[4.5]decan-8-ol (6b): Pure al-
cohol 6b (339.4 mg, 67%) was isolated as colorless crystals after
separation by column chromatography. M.p.: 65–68 °C. H NMR
(CDCl3): δ = 1.02 (d, J = 6.1 Hz, 6 H), 1.25–1.44 (m, 3 H), 1.52–
1.80 (m, 4 H), 2.75 (t, J = 9.3 Hz, 1 H), 4.93 (s, 4 H) ppm. 13C
NMR (CDCl3): δ = 18.5 (q), 36.7 (t), 42.0 (d), 64.2 (t), 64.4 (2 t),
80.9 (d), 108.1 (s) ppm.
1
Chlorination with PPh3/NCS: Triphenylphosphane (2 equiv.) in dry
THF (10% solution) was treated dropwise with N-chlorosuccinim-
ide (1 equiv.) in dry THF (5% solution). Alcohol 6a/b (1 equiv.) in
dry THF (10% solution) was added to the resulting suspension.
The mixture was stirred at room temp. until the solution became
clear (1–3 h). After evaporation of the volatiles the residue was
taken up in water and extracted with diethyl ether. The organic
layer was washed with brine, dried over anhydrous sodium sulfate
and then the solvents were evaporated. The crude product was puri-
fied by flash chromatography on silica gel (LP/EtOAc, 10:1).
(7α,8α,9α)-8-Chloro-7,9-dimethyl-1,4-dioxaspiro[4.5]decane
(6c):
trans-6b (100 mg, 0.50 mmol) was converted according to the above
procedure into 6c. Colorless liquid (180 mg, 80%). 1H NMR
(CDCl3): δ = 1.02 (d, J = 6.1 Hz, 6 H), 1.22–2.28 (m, 6 H), 3.94
(br. s, 4 H), 4.11 (br. s, 1 H) ppm. 13C NMR (CDCl3): δ = 19.0 (q),
35.4 (d), 36.3 (t), 64.2 (2t), 71.8 (d), 110.6 (s) ppm.
(7α,8β,9α)-8-Chloro-7,9-dimethyl-1,4-dioxaspiro[4.5]decane
(6d):
cis-6a (930 mg, 5.00 mmol) was converted following the above pro-
cedure to give 6d as a colorless liquid (625 mg, 61%). 1H NMR
(CDCl3): δ = 1.12 (d, J = 6.3 Hz, 6 H), 1.27–1.44 (m, 2 H), 1.75–
1.88 (m, 2 H), 1.91–2.10 (m, 3 H), 3.13 (t, J = 10.5 Hz, 1 H), 3.94
(br. s, 4 H) ppm. 13C NMR (CDCl3): δ = 20.3 (q), 38.1 (d), 42.8
(t), 64.3 (t), 64.5 (t), 73.1 (d), 107.4 (s) ppm.
cis-7,9-Dimethyl-8-methylene-1,4-dioxaspiro[4.5]decane (6e): Meth-
yltriphenylphosphonium bromide(6.00 g, 16.8 mmol) was washed
with dry diethyl ether and dried in vacuo for about 15 min. Freshly
washed Wittig salt (5.80 g, 16.3 mmol) was suspended in dry THF
(50 mL) under an argon atmosphere and cooled to –5 °C. Then,
nBuLi (16.8 mL, 2.14 m solution in hexane) was added dropwise at
such a rate to keep the reaction temperature between –5 °C and
0 °C. After complete addition of nBuLi the temperature was in-
creased to room temp. and the mixture was stirred for 1 h. During
this period the reaction mixture became an orange-red clear solu-
General Procedure for Ketal Deprotection: The corresponding ketal
was stirred at the specified temperature in a 1:1 mixture (20% solu-
tion) of 1% sulfuric acid and acetone until complete conversion
(as determined by GC). The reaction mixture was hydrolyzed with
saturated sodium hydrogen carbonate solution and then extracted
Eur. J. Org. Chem. 2005, 809–816
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© 2005 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
813

Marko D. Mihovilovic, F. Rudroff, B. Grötzl, P. Stanetty
FULL PAPER
three times with diethyl ether. The combined organic layers were
washed with brine, dried with sodium sulfate, and the solvent was
evaporated. Pure ketones were obtained after purification by col-
umn chromatography.
recombinant E. coli in a baffled Erlenmeyer flask. The culture was
incubated at 120 rpm at 37 °C on an orbital shaker for 2 h, and
then IPTG was added to a final concentration of 0.025 mm. The
substrate (3–6 mm) was added neat along with β-cyclodextrin
(1 equiv.). The culture was incubated at room temp. until GC
showed complete conversion of the ketone (24–96 h). The biomass
was removed by centrifugation, the fermentation broth was filtered
through a bed of Celite®, saturated with sodium chloride, and re-
peatedly extracted with the corresponding solvent (EtOAc, diethyl
ether). The combined organic layers were dried with sodium sulfate,
filtered, and the solvent was removed in vacuo.
(3α,4α,5α)-4-Hydroxy-3,5-dimethylcyclohexanone (1a): Ketal 6a
(50 mg, 0.27 mmol) gave 35.6 mg (92%) of 1a as a colorless oil after
24 h at room temp.[30]1H NMR (CDCl3): δ = 1.09 (d, J = 6.7 Hz,
6 H), 1.87–2.16 (m, 4 H), 2.45 (t, J = 13.7 Hz, 2 H), 2.54 (br. s, 1
H), 3.68 (br. s, 1 H) ppm. 13C NMR (CDCl3): δ = 18.1 (q), 37.6
(d), 42.9 (t), 72.7 (d), 212.5 (s) ppm.
(3α,4β,5α)-4-Hydroxy-3,5-dimethylcyclohexanone (1b): Ketal 6b
(50 mg, 0.27 mmol) gave 33.6 mg (88%) of 1b as colorless crystals
(4α,5α,6α)-5-Chloro-4,6-dimethyl-2-oxepanone (2c): Microbial oxi-
dation of (3α,4α,5α)-4-chloro-3,5-dimethylcyclohexanone (1c;
100 µL, 92.0 mg, 0.56 mmol) according to the general procedure
with CHMO-producing cells gave the desired lactone 2c (53 mg,
53%) as a colorless oil after purification by column chromatogra-
1
after 24 h at room temp. M.p.: 90–93 °C. H NMR (CDCl3): δ =
1.13 (d, J = 6.5 Hz, 6 H), 1.71–1.94 (m, 3 H), 2.15 (t, J = 13.5 Hz,
2 H), 2.32–2.44 (m, 2 H), 3.18 (t, J = 10.8 Hz, 1 H) ppm. 13C NMR
(CDCl3): δ = 18.9 (q), 39.2 (d), 47.5 (t), 79.7 (d), 209.1 (s) ppm.
C8H14O2 (142.2): calcd. C 67.57, H 9.92; found C 67.34, H 9.69.
1
phy (LP/Et2O, 5:1). H NMR (CDCl3): δ = 1.08 (d, J = 7.0 Hz, 3
H), 1.20 (d, J = 6.4 Hz, 3 H), 2.16–2.43 (m, 3 H), 3.15 (dd, J1 =
11.3, J2 = 14.5 Hz, 1 H), 3.70–3.84 (m, 1 H), 4.21 (br. s, 1 H), 4.47
(dd, J1 = 9.1, J2 = 13.2 Hz, 1 H) ppm. 13C NMR (CDCl3): δ = 7.0
(q), 21.3 (q), 35.2 (t), 35.6 (d), 42.4 (d), 67.1 (t), 72.8 (d), 174.1 (s)
ppm, C8H13ClO2, (176.5): calcd. C 54.40, H 7.42; found C 54.61,
H 7.55.
(3α,4α,5α)-4-Chloro-3,5-dimethylcyclohexanone (1c): Ketal 6c
(100 mg, 0.48 mmol) gave 69.4 mg (90%) of 1c as a colorless oil
after stirring the reaction mixture for 48 h at 40 °C. 1H NMR
(CDCl3): δ = 1.14 (d, J = 6.2 Hz, 6 H), 2.08–2.56 (m, 6 H), 4.18
(br. s, 1 H) ppm. 13C NMR (CDCl3): δ = 19.2 (q), 38.1 (d), 43.1
(t), 71.2 (d), 209.3 (s) ppm, C8H13ClO (160.7): calcd. C 59.81, H
8.16; found C 60.10, H 8.07.
(4α,5β,6α)-5-Chloro-4,6-dimethyl-2-oxepanone (2d): (3α,4β,5α)-4-
Chloro-3,5-dimethylcyclohexanone
(1d;
100 µL,
92.0 mg,
(3α,4β,5α)-4-Chloro-3,5-dimethylcyclohexanone (1d): Ketal 6d
(100 mg, 0.48 mmol) gave 61.7 mg (80%) of 1d as a colorless oil
after stirring the reaction mixture for 24 h at 40 °C. 1H NMR
(CDCl3): δ = 1.23 (d, J = 6.1 Hz, 6 H), 2.00–2.26 (m, 4 H), 2.34–
2.51 (m, 2 H), 3.49 (t, J = 9.9 Hz, 1 H) ppm. 13C NMR (CDCl3):
δ = 20.7 (q), 40.6 (d), 48.2 (t), 70.9 (d), 207.6 (s) ppm. C8H13ClO
(160.7): calcd. C 59.81, H 8.16; found C 60.05, H 8.25.
0.56 mmol) was oxidized with CHMO-producing cells according to
the general procedure. The crude product was purified by column
chromatography (LP/Et2O, 5:1) to give lactone 2d as colorless crys-
tals (40 mg, 40%). M.p.: 96–99 °C. 1H NMR (CDCl3): δ = 1.22 (d,
J = 7.0 Hz, 3 H), 1.30 (d, J = 6.7 Hz, 3 H), 2.09–2.30 (m, 2 H),
2.52–2.79 (m, 2 H), 3.48 (t, J = 8.8 Hz, 1 H), 4.01 (dd, J1 = 8.6
Hz, 1 H), 4.25 (dd, J1 = 2.0, J2 = 13.4 Hz, 1 H) ppm. 13C NMR
(CDCl3): δ = 17.4 (q), 22.1 (q), 37.4 (d), 37.7 (t), 42.4 (d), 70.0 (t),
72.1 (d), 173.4 (s) ppm. C8H13ClO2 (176.5): calcd. C 54.40, H 7.42;
found C 54.70, H 7.62.
cis-3,5-Dimethyl-4-methylenecyclohexanone (1e): Crude compound
6e (996 mg, 5.40 mmol) was stirred at 40 °C overnight to give
1
760 mg (90%) of 1e as a colorless oil. H NMR (CDCl3): δ = 1.18
(d, J = 6.3 Hz, 6 H), 2.07 (t, 2 H), 2.36–2.68 (m, 4 H), 4.95 (s, 2
H) ppm. 13C NMR (CDCl3): δ = 18.5 (q), 36.8 (d), 49.9 (t), 105.1
(t), 153.4 (s), 209.9 (s) ppm.
cis-4,6-Dimethyl-5-methylene-2-oxepanone (2e): cis-3,5-Dimethyl-4-
methylenecyclohexanone (1e; 100 µL, 92 mg, 0.66 mmol) was oxid-
ized according to the general procedure with CHMO-producing
cells to give 2e[51] (55 mg, 54%) as a colorless oil after column chro-
matography (LP/EtOAc, 10:1). Biooxidation with CPMO yielded
64 mg (63%) of 2e.
cis-4,8-Dimethylspiro[2.5]octan-6-one (1f): Diethylzinc (13.72 mL,
13.72 mmol, 1 m solution in hexane) was added to dry dichloro-
methane (10 mL). The solution was cooled with an ice-bath and
trifluoroacetic acid (1.57 g, 13.72 mmol) in dichloromethane
(5 mL) was added dropwise to the solution. The mixture was stirred
for 30 min in the ice-bath. Then, methylene iodide (3.67 g,
13.72 mmol) diluted in dichloromethane (5 mL) was added to the
mixture. After stirring for 30 min olefin 6e (1.25 g, 6.86 mmol, in
5 mL of dry dichloromethane) was added to the mixture. Conver-
sion was monitored by GC. Due to the volatility of the intermedi-
ate ketal, deprotection was initiated directly after complete trans-
formation. The reaction mixture was quenched with 2 n HCl
(25 mL) and stirred overnight until deprotection was complete. The
two phases were then separated and the aqueous phase was ex-
tracted with dichloromethane. The combined organic phases were
washed with satd. NaHCO3 solution, brine, and water. The com-
bined organic phases were dried with sodium sulfate and concen-
trated. The crude product was purified by column chromatography
(LP/EtOAc, 12:1) to give 1f as a yellow oil (512 mg, 50%).1H NMR
identical to data described in the literature.[42]13C NMR (CDCl3):
δ = 1.5 (t), 7.6 (t), 17.3 (q), 26.2 (s), 36.7 (d), 48.8 (t), 211.0 (s)
ppm.
cis-4,9-Dimethyl-6-oxaspiro-[2.6]-nonan-7-one
(2f):
cis-4,8-Di-
methylspiro[2.5]octane-6-one (1f; 100 µL, 98 mg, 0.644 mmol) was
oxidized with CHMO-producing cells according to the general pro-
cedure to give 2f (62 mg, 57%) as colorless crystals after column
chromatography (LP/Et2O, 4:1). M.p.: 126–127 °C. 1H NMR
(CDCl3): δ = 0.25–0.43 (m, 2 H), 0.44–0.47 (m, 2 H), 1.12 (d, J =
10 Hz, 8 H), 2.51 (dd, J = 5.9, J = 7.6 Hz, 1 H), 2.97 (dd, J = 2.5,
J = 10.8 Hz, 1 H), 4.06 (dd, J = 4.5, J = 8 Hz, 1 H), 4.44 (d, J =
12.5 Hz, 1 H) ppm. 13C NMR (CDCl3): δ = 11.9 (t), 15.3 (q), 17.3
(t) 18.2 (q), 28.4 (s), 37.7 (d), 39.5 (t), 42.4 (d), 72.1 (t), 174.9 (s)
ppm.
cis-4,6-Dimethyl-2-oxepanone (2g): cis-3,5-Dimethylcyclohexanone
(1g) (200 µL, 184 mg, 1.38 mmol) was oxidized according to the
general procedure with CHMO-producing cells to give 2g[30]
(128 mg, 65%) as a colorless oil after column chromatography (LP/
EtOAc, 4:1). Biooxidation with CPMO yielded 114 mg (58%) of
product.
(4α,5β)-4,5-Dihydro-5-(2-hydroxy-1-methylethyl)-4-methyl-2(3H)-fu-
ranone (3a): Ketone 1a (100 µL, 100 mg, 0.70 mmol) was oxidized
according to the general procedure with CHMO-producing cells
Microbial Baeyer–Villiger Oxidation: Fresh LB-amp medium
(250 mL) was inoculated with 1% of an overnight preculture of
814
© 2005 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.eurjoc.org
Eur. J. Org. Chem. 2005, 809–816

Microbial Baeyer–Villiger Oxidation of Prochiral Polysubstituted Cyclohexanones
FULL PAPER
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to give lactone 3a (85 mg, 77%) as a colorless oil after column
chromatography (LP/EtOAc, 2:1).[30]1H NMR (CDCl3): δ = 0.96
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© 2005 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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Marko D. Mihovilovic, F. Rudroff, B. Grötzl, P. Stanetty
FULL PAPER
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© 2005 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.eurjoc.org
Eur. J. Org. Chem. 2005, 809–816