Synthesis of natural product precursors by Baeyer-Villiger oxidation with cyclohexanone monooxygenase from Acinetobacter

Article abstract of DOI:10.1055/s-2001-13394

The Baeyer-Villiger oxidation of the 2-substituted ketones 1 and 3 with the coupled system cyclohexanone monooxygenase from Acinetobacter NCIMB 9871/formate dehydrogenase from Pseudomonas sp. 101 provides the lactones (R)-2 and (R)-4 with high enantiomeric excess which are precursors in the synthesis of lipoic acid. The symmetrically trisubstituted ketone 5 was oxidised to the lactones 6a and 6b in a ratio of approx. 3:1. The absolute configuration of 6a and 6b was determined by hydrolysis of the racemic lactone with PLE yielding the hydroxycarboxylic acid (-)-7 with known absolute configuration.

Full text of DOI:10.1055/s-2001-13394

SPECIALTOPIC
947
Synthesis of Natural Product Precursors by Baeyer–Villiger Oxidation with
Cyclohexanone Monooxygenase from Acinetobacter
Synthesis of
N
atur
l
a
l
Prod
r
uc
t
Prec
u
i
rsors
b
c
y Enzymat
h
ic
B
aeyer–Villige
rS
Oxidation chwarz-Linek,1a,a Andreas Krödel,a Friedrich-Alexander Ludwig,a Alexander Schulze,a Sebastian Rissom,b
Udo Kragl,1b,b Vladimir I. Tishkov,c Marina Vogel*a
a
University of Leipzig, Department of Chemistry and Mineralogy, Johannisalle 29, 04103 Leipzig, Germany
Fax +49(341)9736599; E-mail: vogel@organik.chemie.uni-leipzig.de
b
Institute of Biotechnology, Research Center Jülich, Germany
c
Department of Chemical Enzymology, M. V. Lomonossov Moscow State University, 119899 Moscow, Russian Federation,
Fax +7(95)9392742; E-mail: vit@enzyme.chem.msu.ru
Received 1 March 2001
trations of enzyme and substrate. However, this method
Abstract: The Baeyer–Villiger oxidation of the 2-substituted ke-
has a considerable disadvantage; it requires an effective
tones 1 and 3 with the coupled system cyclohexanone monooxyge-
nase from Acinetobacter NCIMB 9871 / formate dehydrogenase
from Pseudomonas sp. 101 provides the lactones (R)-2 and (R)-4
NADPH regeneration. The latter problem has probably
been the reason for the fact that, barring one example,19 no
with high enantiomeric excess which are precursors in the synthesis Baeyer–Villiger oxidations using CHMO on a preparative
of lipoic acid. The symmetrically trisubstituted ketone 5 was oxi-
dised to the lactones 6a and 6b in a ratio of approx. 3:1. The abso-
scale have been reported. In contrast to NADH, regenera-
tion of NADPH by enzymatic methods is much more dif-
lute configuration of 6a and 6b was determined by hydrolysis of the
ficult.20 Tishkov et al. developed a protein-engineered
racemic lactone with PLE yielding the hydroxycarboxylic acid (–)-
formate dehydrogenase (FDH) from Pseudomonas sp.
7 with known absolute configuration.
101 by multiple site-directed mutagenesis which accepts
Key words: oxidation, enzymes, kinetic resolution, hydrolysis,
NADP+ to an extent making it suitable for NADPH regen-
natural products
eration.21,22
Until now, three examples of application of this new
promising enzyme are known.23–25 FDH was first used for
In a recent review, Hoberg pointed out the biological and
chemical relevance of seven-membered oxacycles.2 One
of the most powerful and elegant methods for expanding
six-membered rings is the well-known Baeyer–Villiger
oxidation.3,4 Unfortunately, there are only very few exam-
ples for achieving at least some enantioselectivity when
oxidising prochiral or racemic ketones using chiral metal
complexes,5 chiral auxiliaries,6 or the Sharpless epoxida-
tion system.7 However, to our knowledge there is no
method that could rival with the enzymatic Baeyer–Vil-
liger oxidation concerning the stereoselectivities as well
as the spectrum of substrates.8–10 Among several Baeyer–
Villiger monooxygenases, cyclohexanone monooxygena-
se from Acinetobacter NCIMB 9871 (CHMO; EC
1.14.13.22) has been studied most intensively.11–13 Some
interesting applications for the synthesis of precursors of
natural products like azadirachtin14 and ionomycin15 were
published.
cofactor regeneration in a dehydrogenase-catalysed re-
duction of acetophenone.23 The coupling of CHMO with
the NADP+-dependent FDH in the Baeyer–Villiger oxida-
tion of 4-methylcyclohexanone was the first application
of this recombinant enzyme together with a monooxygen-
ase.24 The corresponding chiral -lactone had been syn-
thesised with high chemical and enantiomeric purity (ee
>99%) in repetitive mode on a preparative scale. Oxygen
delivery turned out to be the rate-limiting factor of the
conversion. In order to avoid problems connected with
oxygen feeding by bubbling or intensive stirring on such
a scale, bubble-free aeration through a thin-walled silicon
rubber tube26,27 was used.
In addition to this first example, the Baeyer–Villiger oxi-
dation of the ketones 1, 3 and 5 with the coupled enzyme
system was investigated to yield lactones, which are inter-
esting precursors of natural products.
In principle, there are two possibilities to carry out
CHMO-catalysed Baeyer–Villiger oxidations: with whole
cells or with isolated and enriched enzymes, respectively.
The first method often suffers from over-metabolism and
side reactions. This is also true if recombinant strains like
a cyclohexanone monooxygenase expressing baker’s
yeast16 or Escherichia coli17,18 are used. The second ap-
proach has some advantages. The reaction process can be
executed in a simpler manner using well-defined concen-
The Baeyer–Villiger oxidation of ketone 1 was described
for the first time using a monooxygenase MO2 from
Pseudomonas putida NCIMB 10007.28 In a kinetic resolu-
tion, lactone (R)-2 was formed with considerable enantio-
meric excess (93%) in good yield (30%). Starting from
(R)-2, a useful intermediate for a synthesis of lipoic acid
could be produced by ring-opening.29
Firstly, the oxidation of ketone 1 with the coupled system
CHMO/FDH (Scheme 1) was carried out on analytical
scale. The conversion was followed using chiral GC. Dif-
ferent ketone concentrations were tested (10, 20, 40, and
Synthesis 2001, No. 6, 04 05 2001. Article Identifier:
1437-210X,E;2001,0,06,0947,0951,ftx,en;C02201SS.pdf.
© Georg Thieme Verlag Stuttgart · New York
ISSN 0039-7881

948
U. Schwarz-Linek et al.
SPECIALTOPIC
Scheme 1
80 mM). The formation of only one lactone isomer was
obtained during the whole reaction period in all cases.
Figure 1 Formation of the lactone (–)-2 in a repetitive batch on a
preparative scale
The best result was obtained using a 20 mM substrate con-
centration (see experimental, Table 2). Hence, this con-
centration was used for the conversion of 1 on a
preparative scale in the reactor with integrated bubble-
free aeration.24 Ketone 1 was oxidised in two consecutive
batches successfully (Figure 1).25,30 Kinetic resolution of
1 proceeded nearly completely yielding only one lactone
with high enantiomeric purity (>99% GC). The specific
In their investigations on a new synthesis of (+)-lipoic ac-
id, Willetts et al. oxidised ketone 3 using several mono-
oxygenases, but not the CHMO from Acinetobacter.31,32
All enzymes produced the lactone (R)-4 with good to sat-
isfactory yields. Only a monooxygenase from Pseudomo-
nas putida NCIMB 9872 formed the (S)-lactone with low
enantiomeric excess (42%) which is a direct precursor of
(+)-lipoic acid.33
25
rotation was determined to be [ ]D –27.2° (c 2.29,
CHCl3). A third batch was not successful, probably due to
the irreversible inhibition of the monooxygenase by the In the oxidation of ketone 3 with CHMO/FDH only one
lactone. The absolute configuration of the (–)-lactone was lactone was preferentially formed and very fast. In con-
proposed to be (R)-2 only by comparison with the elution trast to 1, the other ketone enantiomer was also accepted,
behavior of the chemically synthesised lactone ( )-2 with but it was oxidised quite slowly (Figure 2). The conver-
the structurally similar compound ( )-4 using the same sion can be followed easily and quickly by chiral GC.
GC conditions.31,32
Thus, if the reaction is stopped after 30 minutes, a lactone
with high enantiomeric purity can be isolated. (Table 1).
The absolute configuration of the preferentially formed
lactone was proposed to be (R)-4 (Scheme 1) by compar-
ison with the elution sequence of the chemically synthe-
sised racemic compound ( )-4 with published results31
(Table 1).
The yield of (R)-2 (39%) was satisfactory, whereas the
yield of the residual ketone (15%) was unsatisfactory.
This could be due to the use of the thin-walled silicon rub-
ber tube in the reactor. Although such silicon tubes pos-
sess a high porosity for oxygen, organic compounds will,
however, be adsorbed well onto the surface of the tubes.
Thus, a more suitable material has to be found for a gen- The racemic seven-membered lactone 6 is an interesting
eral application of such reactor types in organic synthesis. precursor in the synthesis of (–)-lardolure, the aggregation
pheromone of the acarid mite Lardoglyphus konoi.34
Because of the tube problem, the oxidations of the ketones
3 and 5 were executed only on an analytical scale.
(–)-Lardolure was prepared in a thirteen-step synthesis.35
Table 1 Conversion of the Ketones 1, 3 and 5 on an Analytical Scale
Time (min)
60a
Ketone
Yieldb (%)
eeb (%)
64
Configurationc Lactone
Yieldb (%)
eeb (%)
>99
Configurationc
1
3
5
60
66
–
Sd
Se
2
4
6
39
Rd
Re
7S
30a
76
34
>99
1440
100
48
a At this time the reaction should be stopped to obtain a high enantiomeric excess.
b Determination by chiral GC (Lipodex E).
c Absolute configuration.
d The configuration was proposed only by comparison with the results published in Lit.28 The determination of the configuration is under in-
vestigation.
e The absolute configuration of lactone 4 was dedicated by comparison with the elution sequence of the racemic lactone in GC [(R)-4: tR = 15.6
min, (S)-4: tR = 16.3 min, column temp 150°C] with published data [(R)-4: tR = 14.8 min, (S)-4: tR = 15.3 min] which were obtained under sim-
ilar conditions (Lipodex E, 25 m, column temp 160°C).31
Synthesis 2001, No. 6, 947–951 ISSN 0039-7881 © Thieme Stuttgart · New York

SPECIALTOPIC
Synthesis of Natural Product Precursors by Enzymatic Baeyer–Villiger Oxidation
949
first general method to obtain optically pure medium ring
lactones with excellent enantiomeric excess using esteras-
es.36 Seven-membered lactones with longer-chain alkyl
substituents neighboured to the ring-oxygen were also hy-
drolysed with the same esterases successfully.37 One of
these enzymes, the Guinea pig liver esterase (PLE), was
used for the kinetic resolution of ( )-6 (Scheme 3). The
reaction was followed by chiral GC. The conversion rate
of ( )-6, controlled by the addition of NaOH, was >50%.
For the first time, an enantiomerically pure trisubstituted
lactone was isolated (tR = 6.7 min) and characterised by
the specific rotation [ 23 +22.2° (c 5.7, CHCl3)). The hy-
D
droxycarboxylic acid, which was formed, was optically
active but not enantiomerically pure. An optical rotation
23
of [
–2.0° (c 1, CHCl32)3was found. Comparing with
D
published data [Lit.35
[
D
–4.2° (c 8.69, CHCl3)], it
could be deduced that (–)-7 possesses the (2R,4S,6S) con-
figuration. Therefore, the carboxylic acid (–)-7 was
formed from the lactone (3R,5S,7S)-6a (tR = 6.2 min).
Figure 2 Conversion/time curves of the oxidation of ketone 3 on an
analytical scale
A key step was the preparation of the enantiomerically
pure 2,4-dimethyl-6-hydroxyheptanoic acids (+)-7 and (–
)-7 by ring-opening of the racemic lactone ( )-6 with (S)-
prolinol and separation of the formed diastereomers. An
enzymatic Baeyer–Villiger oxidation of the meso-ketone
5 using the enzyme system CHMO/FDH could be an al-
ternative way (Scheme 2).
Scheme 3
The conversion of 5 is the first example for the enzymatic
Baeyer–Villiger oxidation of a symmetrically trisubstitut-
ed ketone. Lactone 6a was found to be the main product.
This was an expected result because the CHMO preferen-
tially oxidises 2- and 4-alkyl substituted cyclohexanones
and also 2,6-disubstituted cyclohexanones, respectively,
to lactones with (S)-configuration.13 Hence, the high
amount of the enantiomeric lactone 6b was an unexpected
result.
Scheme 2
In summary, with the coupled enzyme system CHMO/
FDH, a system is available to carry out enzymatic Baey-
er–Villiger oxidations also on a laboratory scale. Using a
reactor with bubble-free aeration, CHMO/FDH catalysed
the formation of a precursor of the natural product (+)-li-
poic acid on this scale. For the first time, a symmetrically
trisubstituted ketone was oxidised with CHMO success-
fully. The determination of the absolute configurations of
the lactones however, is one of the main problems. In the
case of the trisubstituted lactone, a PLE-catalysed hydrol-
ysis was used to form a hydroxycarboxylic acid with
known absolute configuration.
Figure 3 Conversion/time curve of ketone 5 on an analytical scale
Ketone 5 was oxidised very fast. It was converted com- Optical rotations were measured using a Perkin Elmer 241 pola-
rimeter, and a Polartronic D polarimeter (Schmidt & Haensch).
Mass spectra were obtained using a HP 5890 Series II with MSD
pletely, but not enantioselectively. The lactones 6a and 6b
were formed with a constant ratio of approx. 3:1 (48% ee)
during the whole reaction period (Figure 3). For the deter-
mination of the absolute configuration of the lactones, an
indirect method had to be applied because the lactones are
non-crystalline compounds. Rousseau et al. developed the
5972 (70 eV) and a HP-5 column (30 m). IR spectra were recorded
1
using a Genesis FT-IR-Spectrometer, ATI Mattson. H and 13C
NMR spectra were recorded using a Varian Gemini 200 spectrom-
eter. Gas chromatograms were recorded using a HP 5890 Series II
with a CP-Chirasil-Dex column (25 m, Chrompack) or a Lipodex E
Synthesis 2001, No. 6, 947–951 ISSN 0039-7881 © Thieme Stuttgart · New York

950
U. Schwarz-Linek et al.
SPECIALTOPIC
column (25 m, Macherey-Nagel), injector temperature 250°C, de-
tector temperature 250°C. The enzymatic hydrolysis was monitored
with a pH meter Präcitronic MV 870.
ter centrifugation, a sample of the organic layer (1 L) was analysed
using dodecane as internal standard. Products were identified in
comparison with the chemically synthesized lactones. For the re-
sults, see Table 1. The conversion curves of ketones 3 and 5 are re-
ported in Figures 2 and 3.
CHMO was prepared from Acinetobacter NCIMB 9871 following
a known procedure11,12 which was simplified because there was no
need to purify the enzyme to homogeneity.38 Two enzyme fractions
Lactone 2 in a Repetitive Batch
1
1
CHMO 1 (6.6 U mL ) and CHMO 2 (46 U mL ) in SH-buffer (21
mM phosphate buffer with 10 mM mercaptoethanol, pH 7.2) were
The reaction was carried out in a reactor with integrated bubble-free
aeration.24
1
used. The FDH (10 U mL in phospate buffer, 100 mM, pH 7.0,
which contained 10 mM EDTA and (NH4)2SO4 of approx. 5% of
saturation.) was a gift of Prof. Tishkov. Guinea pig liver esterase
(PLE, acetone powder) was purchased from Sigma. Ketone 1 was
purchased from Aldrich. Ketone 3 and the racemic lactone 4 were
prepared,31 just as ketone 5 and the racemic lactone 6.35
To a solution of ketone 1 (110 mg, 0.6 mmol) in Tris/HCl buffer
(pH 8, 20.3 mL) were added sodium formate (6 mL, 1 M in Tris/HCl
buffer, pH 8, 6 mmol), NADP+ (1.5 mL, 8 mM in Tris/HCl buffer,
1
12 mol), FDH (18 U, 1.8 mL, 0.6 U mL ), and CHMO (6 U, 273
1
L, 0.2 U mL ). The mixture was kept at 30°C and stirred gently.
The solution was aerated by pumping the reaction mixture through
a thin-walled silicon tube (4 mm i.d. 40 cm) at a rate of 5 mL
Lactone ( )-2
1
Peracetic acid (40%, 10.3 mL, 54 mmol) was added dropwise to an
ice-cooled solution of ketone 1 (1 g, 5.4 mmol) and NaHCO3 (0.91
g, 10.8 mmol) in CH2Cl2 (40 mL) during 20 min. The mixture was
stirred for 2 h under ice-cooling and then for 6 h at r.t. Then
NaHSO3 (H2O–sat. NaHSO3, 2:1; 45 mL) was added dropwise un-
der ice-cooling during 20 min. After stirring for 10 min, the organic
layer was separated. The aqueous layer was extracted with CH2Cl2
(2 10 mL). The combined organic layers were washed with H2O
(2 10 mL), sat. NaHCO3 (2 10 mL), dried (Na2SO4), and con-
centrated in vacuo. Distillation of the crude product (1.05 g) in a
Kugelrohr ball-tube distillation apparatus (Büchi GKR-51) yielded
( )-2 (266 mg, 24%); bp 127°C (1.7 10 1 mbar).
min 1 with an exterior air flow of about 14.5 L h . The reaction was
stopped after 150 min (48% conversion of 1) by ultrafiltration
through an Amicon PM 10 membrane. The concentrate (the enzyme
solution) was washed with Tris/HCl buffer (2 20 mL) and concen-
trated again. This enzyme solution (6.8 mL) was ready for use in the
second batch.
The combined aqueous filtrates were extracted with EtOAc (4 20
mL), dried (Na2SO4), and concentrated in vacuo. After flash chro-
matography (PE–EtOAc, 2:1) of the crude product, 42.0 mg (35%)
of the lactone ( )-2, [ 25 –27.2° (c 2.29, CHCl3), ee >9%, tR = 23.9
D
25
min; and 16.1 mg (15%) of the residual ketone 1, [
–23.6° (c
D
2.29, CHCl3), ee = 80%, tR = 7.2 min (Lipodex E, 120°C), were iso-
GC: tR = 20.2 min and 20.9 min (CP-Chirasil-Dex, 150°C) and
tR = 23.9 min and 24.4 min (Lipodex E, 130°C), for (–)-2 and ( )-2,
respectively.
lated.
Enzymatic Hydrolysis of Lactone ( )-636
In a vial (3.5 mL) were placed the buffer NaH2PO4 (0.1 M adjusted
to pH 7.2; 1 mL), the lactone ( )-6 (100 mg, 0.64 mmol), and the
crude enzyme PLE (100 mg). The mixture was stirred gently at r.t.
The reaction started immediately, and the pH value decreased. It
was corrected by the addition of NaOH (2 M). After a consumption
of NaOH (180 L) corresponding to about 60% conversion, a sam-
ple was taken from the reaction mixture, extracted with EtOAc (60
L), acidified with 2 N HCl (3 L) and analysed in GC (programme:
120°C, 4 min, 10°C/min to 180°C). A ratio of 6b (tR = 6.7 min) to
6a (tR = 6.2 min) of 15:1 was found after 3.5 h. NaOH (30 L) was
added and the mixture was stored in a refrigerator overnight. The
solution was extracted with Et2O (7 2 mL). The combined organic
layers were washed with 10% NaHCO3 (3 5 mL), dried (Na2SO4),
and concentrated in vacuo to give 39 mg (39%) of lactone 6b (>99%
1
IR (neat): = 2980, 2937, 2865, 1733 (C=O) cm .
1H NMR (200 MHz, CDCl3): = 1.26 (t, 3H, J = 7.1 Hz), 1.40 1.90
(m, 6H), 2.30 2.80 (m, 4H), 4.16 (q, 2H, J = 7.1 Hz), 4.74 (m, 1H).
13C NMR (50 MHz, CDCl3): = 14.5, 23.2, 28.4, 34.6, 35.0, 41.4,
61.1, 76.9, 170.4, 175.2.
MS (70 eV): m/z = 200 (1), 155 (3), 143 (10), 130 (40), 113 (17), 84
(50), 67 (24), 55 (100), 41 (67).
Enzymatic Baeyer–Villiger Oxidation on an Analytical Scale;
General Procedure
In a vial (2 mL) were placed the ketones in Tris/HCl buffer
(Table 2), sodium formate (200 L, 1 M in Tris/HCl buffer),
NADP+ (125 L, 8 mM in Tris/HCl buffer), and FDH (2 U, 200 L).
The mixture was warmed up to 30°C under gentle stirring. The re-
action was started by the addition of the CHMO (Table 2). The con-
version was followed by chiral GC (Lipodex E); samples of the
reaction mixture (100 L) were extracted with EtOAc (200 L). Af-
GC; tR = 6.7 min) as a colourless liquid; ee >99%; [ 23 +22.2° (c
D
5.7, CHCl3).
The aqueous layers were combined with the washing solution, acid-
ified with 2 N HCl and extracted with Et2O (7 5 mL), dried
(Na2SO4), and concentrated to give the optically active hydroxy
acid 7; yield: 60 mg (54%); colourless needles from hexane–Et2O
(10:1), mp 81 83°C (Lit.35 mp 83.5 85°C); ee 50% (GC,
tR = 11.5 min, shoulder detection; the enantiomers could not be sep-
Table 2 Reaction Conditions of the Enzymatic Baeyer–Villiger
Oxidation of Ketones 1, 3 and 5 on an Analytical Scale
arated.); [ 23 –2.0° (c, CHCl3) [Lit.35
[
23 –4.2° (c 8.69, CHCl3)].
D
D
1H NMR (200 MHz, CDCl3): = 0.87, (d, 3H), 1.12 (d, 6H), 1.03
1.70 (m, 4H), 2.57 (m, 1H), 2.50 2.65 (m, 1H), 3.80 3.94 (m, 1H),
5.70 6.05 (br s, 2H).
13C NMR (50 MHz, CDCl3): = 18.1, 19.5, 24.3, 27.8, 38.8, 42.0,
46.9, 66.0, 182.5.
Ketone
Amount c (mM)
(mg)
Tris/HCl CHMO
Time (min)
( L)
398
440
452
( L)
1
3
5
3.68
2.00b
0.78
20
10
5
77a
1260
1020
1440
22a
22c
Acknowledgement
a CHMO 1.
We wish to thank Prof. U. Kragl (University of Rostock) for his
fruitful co-operation. Especially, we want to thank Prof. V. I. Tish-
kov (Moscow State University, Russia) for the generous gift of the
b Ketone 3 was dissolved in EtOH (1% v/v).
c CHMO 2.
Synthesis 2001, No. 6, 947–951 ISSN 0039-7881 © Thieme Stuttgart · New York

SPECIALTOPIC
Synthesis of Natural Product Precursors by Enzymatic Baeyer–Villiger Oxidation
951
formate dehydrogenase. We thank Prof. P.Welzel (University of
Leipzig) for his support and useful discussions. This work has been
supported in the Graduate College “Mechanisms and Applications
of Non-Conventional Oxidation Reactions“ by the German Rese-
arch Council.
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Willetts, A. J. Mol. Catal. B: Enzymatic 1996, 1, 127.
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Synthesis 2001, No. 6, 947–951 ISSN 0039-7881 © Thieme Stuttgart · New York