Baeyer-Villiger oxidation of bridged endo-tricyclic ketones with engineered Escherichia coli expressing monooxygenases of bacterial origin

Article abstract of DOI:10.1055/s-2005-918938

Whole-cell biotransformations using engineered strains of Escherichia coli expressing cyclopentanone (CPMO) and cyclohexanone monooxygenases (CHMO) of various bacterial origins have been tested for substrate acceptance on tricyclic ketones. Based on the s

Full text of DOI:10.1055/s-2005-918938

LETTER
2751
Baeyer–Villiger Oxidation of Bridged endo-Tricyclic Ketones with Engineered
Escherichia coli Expressing Monooxygenases of Bacterial Origin
Baeyer–Villiger
Oxida
a
tion of Brid
r
ged end
k
o-Tricyclic
K
o
etones D. Mihovilovic,* Radka Snajdrova, Alexander Winninger, Florian Rudroff
Vienna University of Technology, Institute for Applied Synthetic Chemistry, Getreidemarkt 9/163, 1060 Vienna, Austria
Fax +43(1)5880115499; E-mail: mmihovil@pop.tuwien.ac.at
Received 6 September 2005
O
O
Abstract: Whole-cell biotransformations using engineered strains
of Escherichia coli expressing cyclopentanone (CPMO) and cyclo-
hexanone monooxygenases (CHMO) of various bacterial origins
have been tested for substrate acceptance on tricyclic ketones.
Based on the stereopreference of the biocatalytic Baeyer–Villiger
oxidation, our recent clustering of this library of enzymes into two
distinct groups based on protein sequence was confirmed. Together
with short and facile reaction sequences for the production of the
substrate ketones, microbial biooxidation enables access to antipo-
dal product lactones as versatile building blocks in natural product
and bioactive compound synthesis.
O
Baeyer–Villiger monooxygenase
Baeyer–Villiger monooxygenase
(CH2)n
(CH2)n
1a,b
2a,b
O
O
O
1c,d
2c,d
Scheme 1 Whole-cell biotransformation of tricyclic ketones
Key words: biocatalysis, biooxidation, recombinant whole-cell
biotransformation, Baeyer–Villiger oxidation, monooxygenase,
stereoselectivity
(n = 1, 2).
Tricyclic bridged ketones 1a,b were prepared according
to the literature12 with minor modifications in a short and
efficient sequence: initial Diels–Alder cyclization of the
appropriate alkenes with activated diene 3 to 5a,b was
followed by reductive removal of the chlorines (6a,b),
acid-catalyzed hydrolysis of ketal (7a,b), and subsequent
hydrogenation using Pd/C (Scheme 2).
The stereoselective Baeyer–Villiger (BV) oxidation to
chiral lactone intermediates has received considerable at-
tention in recent years.1 Both organometal complexes2
and enzymes (BV monooxygenases – BVMOs)3 are avail-
able as enantioselective catalysts fulfilling the require-
ments of sustainable and green chemistry strategies.
The synthetic route to compounds of type 1c,d utilized
carbic anhydride 8 to generate diol 9.13 Subsequent trans-
formations involved activation of the hydroxyls (10) and
Kolbe nitrile formation (11).14 Cyclization to ketone 1c
was performed via diacid 12, following a route recently
developed by our group for the preparation of fused bicy-
cloketones (Scheme 3).6c Alkene 1c was easily converted
to compound 1d by atmospheric pressure hydrogenation.
Very recently, we introduced a platform of recombinant
strains of Escherichia coli overexpressing BVMOs of
various microbial origin.4,5 This small library of enzymes
possesses overlapping substrate specificity for the
biooxidation of various ketones and displays
enantiocomplementary6 and regio-divergent biotrans-
formations.7 The utilization of recombinant whole-cells
minimizes potential enzymatic side reactions8 and simul-
taneously allows for a facile application of cofactor de-
pendent biocatalysts.9 Our current research program aims
at providing an easy-to-use biocatalytic toolbox of renew-
able catalytic entities for stereoselective BV oxidations10
to organic chemists and to demonstrate its potential in nat-
ural product synthesis.
Only related bicyclo-substrates of type 1a,b were previ-
ously oxidized by a cyclohexanone monooxygenase
(CHMO) using isolated enzyme.15 The stereopreference
of monooxygenases originating from Acinetobacter
(CHMOAcineto),16
Arthrobacter
(CHMOArthro),17
Brevibacterium
Brachymonas
(CHMOBrachy),18
(CHMOBrevi1, CHMOBrevi2),19 Comamonas (CPMOComa),20
17
and Rhodococcus (CHMORhodo1, CHMORhodo2
)
species
Enzyme-mediated BV biooxidation of tricyclic bridged
ketones of type 1 as prochiral precursors potentially
generates four new chiral centers (2) in a single desymme-
trization step (Scheme 1).11
in whole-cell mediated BV oxidations utilizing E. coli as
host organism was investigated in this study for the bio-
oxidation of endo-tricyclic ketones 1a–d.21
Protein expression of BVMOs at high level was triggered
by addition of isopropyl-b-D-galactopyranoside to the
growing culture of recombinant E. coli strains. Fermenta-
tions with substrates 1a,b were carried out according to
our previously reported procedure on 100 mg scale in
Erlenmeyer flasks.4,22 Biooxidation of ketones 1c,d was
carried out in parallel format using 12- and 24-well plastic
dishes.23 A crucial aspect of such a mini-scale screen for
SYNLETT 2005, No. 18, pp 2751–2754
Advanced online publication: 12.10.2005
DOI: 10.1055/s-2005-918938; Art ID: D26905ST
© Georg Thieme Verlag Stuttgart · New York
0
3
.1
1
.2
0
0
5

2752
M. D. Mihovilovic et al.
LETTER
O
O
O
O
O
O
Cl
Cl
Cl
Cl
i)
ii)
(CH2)n
Cl
Cl
Cl
Cl
(CH2)n
(CH2)n
6a,b
3
4a (n = 2)
4b (n = 1)
5a,b
iii)
O
O
iv)
(CH2)n
(CH2)n
7a,b
1a,b
Scheme 2 (i) D, 82–85%; (ii) Na, t-BuOH, THF, reflux, 67–88%; (iii) p-TSA, dry acetone, r.t., 73–82%; (iv) H2 (5 bar), Pd/C, EtOAc, r.t.,
85–87%.
i)
iii)
ii)
CN
CN
O
OH
OH
OTs
OTs
O
O
10
8
9
11
iv)
vi)
v)
COOH
COOH
O
O
1d
1c
12
Scheme 3 (i) LAH, THF, reflux, 75–95%; (ii) TsCl, pyridine, –35 °C, 75%; (iii) NaCN, DMSO, 150 °C, 56%; (iv) KOH, H2O, EtOH, reflux,
46%; (v) pyridine, Ac2O, 1 N HCl, reflux, 71%; (vi) H2 (1 bar), Pd/C, r.t., 84%.
this study was the comparability of the multi-well determine physical and spectral data together with
experiment with fermentation results in shake-flasks. Our specific optical rotation of isolated lactones 2c,d. Ra-
optimization efforts took advantage of previous studies cemic reference material of lactones 2a, 2b, and 2d was
for the use of microscale processing technologies in prepared by chemical oxidation of the corresponding
kinetic studies and bioprocess design.24
ketones with MCPBA.
The parallel screening format was designed to provide an Results of the biotransformations of substrates 1a–d to
easy-to-use methodology for the assessment of recombi- lactones 2a–d with all eight overexpression systems of E.
nant whole-cell biocatalysts, implementing reproducible coli are summarized in Table 1.25
and optimized conditions for cell growth, expression of
recombinant biocatalyst, and the biotransformation itself.
Substrates 1a and 1b were oxidized by all BVMOs and
gave the respective lactones 2a,b in excellent stereoselec-
Standardized experiments were carried out with 0.5 mg of
tivity. Our recently discovered trend for enantiodivergent
substrate per mL of broth in 12- or 24-well dishes in the
biotransformations was also observed for this compound
type, with CPMOComa and CHMOBrevi2 (‘CPMO group’)
presence of 1 equivalent of b-cyclodextrin to facilitate
biooxidation of slowly converted substrates. Transforma-
providing antipodal products to the ‘CHMO group’.4
tions were analyzed after 24 hours of fermentation time at
In the case of tricyclo-ketones 1c and 1d the BVMOs dis-
played a selective acceptance of biooxidation precursors.
Compound 1c was essentially no substrate for CHMO-
24 °C by extraction of the sample with EtOAc supple-
mented by an internal standard. This fermentation tem-
perature was determined as optimum value to achieve
type enzymes, with conversion rates below 10%. In con-
good protein expression and to minimize loss of bio-
trast, CPMO-type proteins readily gave the expected lac-
catalyst due to limited enzyme stability.
tone 2c. With respect to stereopreference, CPMOComa and
CHMOBrevi2 produced (–)-2c in moderate to poor stereo-
selectivity, while trace amounts of lactones obtained from
These screening conditions provided conversion results
that were comparable with shake-flask experiments.
Single preparative biotransformations were carried out to
Synlett 2005, No. 18, 2751–2754 © Thieme Stuttgart · New York

LETTER
Baeyer–Villiger Oxidation of Bridged endo-Tricyclic Ketones
2753
Table 1 Biotransformations of endo-Tricyclic Ketones with Whole-Cells of Recombinant E. coli Expressing Bacterial BVMOs
2a
2b
2c
2d
Strain
Yield (%),a
eec (+/–, %)
Yield (%),a
eec (+/–, %)
Conv. (%),b
eec (+/–, %)
Conv. (%),b
eec (+/–, %)
CHMOAcineto
CHMOArthro
CHMOBrachy
CHMOBrevi1
CHMOBrevi2
CPMOComa
CHMORhodo1
CHMORhodo2
63
97 (–)
47
97 (–)
n.c.d
n.a.
3
92 (+)
46
99 (–)
40
99 (–)
6
49
93 (+)
92 (+)
46
99 (–)
42
99 (–)
n.c.
n.a.
12
94 (+)
56
93 (–)
49
94 (–)
n.c.
n.a.
n.c.
n.a.
78
94 (+)
67
92 (+)
68a
74 (–)
3
74 (–)
57
83 (+)
45
91 (+)
73a
36 (–)
62a
23 (–)
58
99 (–)
50
99 (–)
2
63a
95 (+)
91 (+)
54
63
4
26
99 (–)
99 (–)
90 (+)
95 (+)
a Isolated yield after chromatographic purification.
b Conversion of substrate in parallel screening according to GC.
c The ee values were determined by chiral phase GC; the sign of specific rotation is given; racemic reference material prepared by MCPBA
oxidation of ketones 1a,b,d.
d Abbreviations: n.c. = no conversion; n.a. = not applicable.
biooxidations with CHMO-type enzymes showed good The obtained compounds represent versatile building
selectivity for the generation of (+)-lactone 2c. The high blocks for natural and bioactive compound synthesis. In
chemoselectivity of the enzymatic BV oxidation should particular, lactone 2c bearing an additional alkene func-
be noted, as no indication of epoxidation at the C=C dou- tionality seems suitable as precursor for the total synthesis
ble bond was observed in the biotransformations.
of various prostaglandines.26 Currently, the further
elaboration of the chiral products obtained is addressed in
our laboratories together with the determination of their
absolute configuration.
Substrate 1d was more readily accepted by CHMO-type
BVMOs and CHMORhodo1 gave good isolated yields of
(+)-2d in excellent optical purity. Again, CPMO-type bio-
catalysts produced the antipodal lactone, however, either We consider our approach to utilize the natural diversity
with poor conversion (CHMOBrevi2) or in low stereoselec- of BVMOs in enantiodivergent transformations as highly
tivity (CPMOComa).
complementary to recent advances in the random27 and
knowledge-based28 modification of these enzymes based
on the previous publication of the first structure of this
protein family.29 Taken together, these strategies are
aiming at the design of a toolbox for BV biooxidations,
which should ultimately become a standard technique in
day-to-day synthetic chemistry.
In summary, the present study demonstrates the potential
of a library of recombinant whole-cell expression systems
for bacterial BVMOs to produce antipodal lactones with a
tricyclic structural core. Both the stereopreference of the
enzymatic oxidation together with the substrate accep-
tance is in good agreement with our previous hypothesis
of two BVMO groups, which is based on protein sequence
and biocatalyst performance.4 Furthermore, we have
optimized our previous efforts in characterizing novel
BVMOs as versatile biocatalysts by using a parallel
screening system for the rapid determination of substrate
profiles and stereopreference.
Acknowledgment
This project was funded by the Austrian Science Fund (FWF,
Projects No. P16373 & I19-B10). The authors thank Dr. Pierre E.
Rouviere (E.I. DuPont Company) for supporting this project by the
generous donation of six E. coli expression systems for BVMOs,
and Prof. Margaret M. Kayser (University of New Brunswick) for
providing the strain for CPMOComa
.
Synlett 2005, No. 18, 2751–2754 © Thieme Stuttgart · New York

2754
M. D. Mihovilovic et al.
LETTER
CDCl3): d = 1.30–1.55 (m, 2 H), 1.70–1.90 (m, 2 H), 2.05–
References
2.35 (m, 2 H), 2.70–2.95 (m, 4 H), 6.00–6.15 (m, 2 H). 13
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NMR (50 MHz, CDCl3): d = 39.7 (d), 41.0 (t), 47.0 (d), 49.7
(t), 136.1 (d), 219.9 (s).
endo-Tricyclo[5.2.1.02,6]decan-4-one (1d): colorless
amorphous solid; mp = 97–100 °C. 1H NMR (200 MHz,
CDCl3): d = 1.16–1.59 (m, 6 H), 2.03–2.40 (m, 6 H), 2.51–
2.74 (m, 2 H). 13C NMR (50 MHz, CDCl3): d = 22.1 (t), 38.6
(d), 39.3 (t), 40.6 (t), 41.3 (d), 221.0 (s).
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(23) Typical Procedure for Screening Experiment in Multi-
Well Dishes (12 or 24 Wells).
Each well was charged with LB-amp medium (2 mL/12-well
format or 1 mL/24-well format) and inoculated with 1% of
an overnight preculture of recombinant E. coli strains. A
plate was incubated at 120 rpm at 37 °C on an orbital shaker
for 2 h. IPTG was added (final concentration of 0.025 mM)
together with substrate (1 mg or 0.5 mg) and b-cyclodextrin
(1 equiv). The plate was shaken at r.t. for 24 h and then
analyzed by chiral phase GC after extraction of the sample
with EtOAc.
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(25) Physical and Spectroscopic Data of Lactones 2a–d.
endo-9-Oxatricyclo[6.2.2.02,7]dodecan-10-one (2a): beige
crystals, mp 76–78 °C. 1H NMR (200 MHz, CDCl3):
d = 1.09–2.06 (m, 14 H), 2.39–2.41 (q, J = 2.7 Hz, 1 H),
4.36–4.41 (q, J = 3.3 Hz, 2 H). 13C NMR (50 MHz, CDCl3):
d = 16.9 (t), 19.3 (t), 19.5 (t), 20.0 (t), 20.5 (t), 20.8 (t), 32.3
(d), 36.2 (d), 39.8 (d), 78.9 (d), 177.3 (s). Specific optical
rotation (CHMOBrachy): [a]D20 –31.8 (c 0.95, CHCl3); 99%
ee.
endo-8-Oxatricyclo[5.2.2.02,6]undecan-9-one (2b):
colorless crystals, mp 78–80 °C. 1H NMR (200 MHz,
CDCl3): d = 1.66–1.90 (m, 12 H), 2.55 (q, J = 3.1 Hz, 1 H),
4.55 (q, J = 3.7 Hz, 1 H). 13C NMR (50 MHz, CDCl3):
d = 16.7 (t), 20.6 (t), 27.7 (t), 27.9 (t), 28.1 (t), 37.7 (d), 39.2
(d), 41.2 (d), 78.9 (d), 177.3 (s). Specific optical rotation
(CHMORhodo2): [a]D20 –17.3 (c 1.70, CHCl3); 99% ee.
endo-5-Oxatricyclo[6.2.1.02,7]undec-9-en-4-one (2c):
colorless oil. 1H NMR (200 MHz, CDCl3): d = 1.45 (d,
J = 8.0 Hz, 1 H), 1.64 (d, J = 8.0 Hz, 1 H), 1.93–2.98 (m, 6
H), 3.70 (m, 1 H), 4.30 (m, 1 H), 6.05–6.28 (m, 2 H). 13
C
NMR (50 MHz, CDCl3): d = 33.4 (t), 35.9 (d), 38.6 (d), 44.2
(d), 46.0 (d), 50.6 (t), 69.9 (t), 134.9 (d), 136.3 (d), 173.7 (s).
Specific optical rotation (CHMOBrevi2): [a]D20 –12.7 (c 1.72,
CHCl3); 74% ee.
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endo-5-Oxatricyclo[6.2.1.02,7]undecan-4-one (2d):
colorless solid, mp 78–80 °C. 1H NMR (200 MHz, CDCl3):
d = 1.30–1.59 (m, 6 H), 2.12–2.55 (m, 6 H), 4.02–4.32 (m, 2
H). 13C NMR (50 MHz, CDCl3): d = 21.9 (t), 23.3 (t), 30.4
(t), 35.9 (d), 37.0 (d), 39.0 (d), 40.7 (d), 41.2 (t), 68.2 (t),
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(21) endo-Tricyclo[6.2.1.02,7]undecan-11-one (1a): yellow oil.
1H NMR (200 MHz, CDCl3): d = 0.81–1.89 (m, 14 H), 2.06–
2.14 (m, 2 H). 13C NMR (50 MHz, CDCl3): d = 17.1 (t), 18.1
(t), 19.5 (t), 32.9 (d), 43.4 (d), 216.7 (s).
20
174.3 (s). Specific optical rotation (CHMORhodo1): [a]D
+32.2 (c 3.53, CHCl3); 95% ee.
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Natl. Acad. Sci. U.S.A. 2004, 101, 13157.
endo-Tricyclo[5.2.1.02,6]decan-10-one (1b): yellow oil. 1H
NMR (200 MHz, CDCl3): d = 1.63–1.75 (m, 12 H), 2.46 (m,
2 H). 13C NMR (50 MHz, CDCl3): d = 27.7 (t), 29.2 (t), 29.9
(t), 38.2 (d), 43.6 (d), 214.9 (s).
endo-Tricyclo[5.2.1.02,6]dec-8-en-4-one (1c): beige
amorphous solid; mp = 97–99 °C. 1H NMR (200 MHz,
Synlett 2005, No. 18, 2751–2754 © Thieme Stuttgart · New York