Whole-cell mediated Baeyer-Villiger oxidation of functionalized bicyclo[3.3.0]ketones by recombinant E. coli

Article abstract of DOI:10.1055/s-2002-25341

Recombinant whole cells of Escherichia coli overexpressing Acinetobacter sp. NCIMB 9871 cyclohexanone monooxygenase (E.C. 1.14.13.22) have been utilized for the Baeyer-Villiger oxidation of functionalized bicyclo[3.3.0]ketones. Prochiral substrates were d

Full text of DOI:10.1055/s-2002-25341

LETTER
703
Whole-cell Mediated Baeyer–Villiger Oxidation of Functionalized
Bicyclo[3.3.0]ketones by Recombinant E. coli
Baeyer-Villiger
Oxidaa
r
tionalize
k
d Bicyclo[3.
o
3.0]ketones D. Mihovilovic,*a Bernhard Müller,a Margaret M. Kayser,b Jon D. Stewart,c Peter Stanettya
a
Vienna University of Technology, Institute of Applied Synthetic Chemistry, Getreidemarkt 9/163-OC, 1060 Vienna, Austria
Fax +43(1)5880115499; E-mail: mmihovil@pop.tuwien.ac.at
b
c
University of New Brunswick, Department Physical Sciences, Saint John, N.B., E2L 4L5, Canada
University of Florida, Department Chemistry, Gainesville, FL 32611, USA
Received 27 February 2002
compounds were developed based on intermediates pre-
pared via enzymatic Baeyer–Villiger oxidation.17–25
Abstract: Recombinant whole cells of Escherichia coli overex-
pressing Acinetobacter sp. NCIMB 9871 cyclohexanone monooxy-
genase (E.C. 1.14.13.22) have been utilized for the Baeyer–Villiger
oxidation of functionalized bicyclo[3.3.0]ketones. Prochiral sub-
strates were designed to probe the impact of polarity and spatial
configuration of the functional groups on conversion and enantiose-
lectivity. The data give additional insight into the steric require-
ments of the active site of the enzyme for high optical purity. The
synthetic utility of the engineered E. coli strain is demonstrated and
contributions to the non-natural substrate profile available so far are
provided.
Monooxygenases are dependent on cofactors, which com-
plicates their utilization in organic chemistry and has
hampered widespread use among the synthetic communi-
ty. NAD(P)H has to be recycled to enable a cost-efficient
biotransformation on preparative scale. This can be per-
formed by the implementation of a second enzymatic re-
action using, for example, glucose-6-phosphate
dehydrogenase from Leuconostoc mesenteroides26 or a
mutant formate dehydrogenase from Pseudomonas sp.
11027,28 as reported recently for the CHMO system.29,30
Key words: biocatalysis, recombinant whole-cell biotransforma-
tion, cyclohexanone monooxygenase, Baeyer–Villiger oxidation,
substrate profiling, enantioselectivity
Another approach to overcome the cofactor obstacle is the
utilization of living whole cells, which provide natural re-
cycling systems for all factors needed. To minimize un-
wanted side reactions catalyzed by competing enzymes in
the organism with overlapping substrate acceptance, a re-
combinant overexpression system can be used in place of
a natural isolate. Production of the required protein at high
levels can often be accomplished by a strong promoter
and this was successfully demonstrated for Acinetobacter
cyclohexanone monooxygenase in a Saccharomyces cer-
evisiae based recombinant strain.31 More recently, we
have developed an efficient E. coli expression system for
the same enzyme [BL21(DE3)(pMM4)]32 and successful-
ly used the strain for the first biotransformations of
prochiral33 and heterocyclic ketones34 with recombinant
whole-cells.35,36
Chiral Baeyer–Villiger oxidation of ketones represents a
powerful entry to asymmetric lactones as valuable inter-
mediates in organic chemistry and frequently encountered
precursors in enantioselective synthesis.1 While ap-
proaches utilizing organometallic reagents have been con-
tinuously improved over recent years,2–9 efficient and
highly selective preparation of this class of compounds is
still the domain of biocatalytic methods.10–14 Flavin de-
pendent monooxygenases catalyzing Baeyer–Villiger re-
actions are usually part of degradative pathways that
allow microorganisms to use non-carbohydrate com-
pounds as alternative sources of carbon and cell energy.15
Although the physiological role of these enzymes is relat-
ed to the degradation of a specific compound in most cas-
es, these biocatalytic entities often display a remarkably
broad acceptance for non-natural substrates. This feature
together with high enantioselectivity are prerequisites for
a powerful catalytic system for chiral synthesis.
Continuing our substrate profiling studies of CHMO, we
became interested in bicyclo[3.3.0]ketones 1 as useful
precursors for asymmetric Baeyer–Villiger oxidations
(Scheme 1). The resulting lactones of type 2 have been
used in various natural product syntheses.37–39 Due to the
prochiral nature of compounds 1, asymmetrization gener-
ates optically active products with a theoretical yield of
100%, whereas a kinetic resolution of racemic samples
leads only to a maximum 50% yield of enantiopure prod-
uct.
The best studied Baeyer–Villigerase to date is cyclohex-
anone monooxygenase (CHMO) from Acinetobacter sp.
NCIMB 9871 (E.C. 1.14.13.22).16 The enzyme oxidizes
more than 100 non-natural substrates and generally per-
forms desymmetrization of prochiral ketones as well as
kinetic resolution of racemic precursors with good enanti-
oselectivity.12,13 Synthetic routes to a number of natural
Furthermore, we considered substrates 1 as interesting
probes to study the influence of polarity of functional
groups at the bicyclic core on the whole-cell biotransfor-
mation. Insight into steric requirements of the enzyme’s
active site might be gained by comparing the bioconver-
Synlett 2002, No. 5, 03 05 2002. Article Identifier:
1437-2096,E;2002,0,05,0703,0706,ftx,en;D04502ST.pdf.
© Georg Thieme Verlag Stuttgart · New York
ISSN 0936-5214

704
M. D. Mihovilovic et al.
LETTER
nation using Mitsunobu conditions and ZnCl2 as chlorine
donor43 gave clean stereoinversion to compounds 6 and 7,
respectively. However, we observed substantial formation
of elimination product 8. This obstacle could be overcome
by a modified protocol utilizing triphenylphosphine and
NCS,44,45 which decreased the undesired elimination to ca.
10%. Substrate ketones 1d and 1e were prepared by sub-
sequent deprotection as outlined.
Scheme 1 Whole-cell biotransformations of functionalized bi-
cyclo[3.3.0]ketones.
In the construct BL21(DE3)(pMM4) the gene for CHMO
is under the control of a T7 promoter.32 Protein expression
utilizes a two step procedure46 and is induced by addition
of isopropyl- -D-thiogalactopyranoside (IPTG). This trig-
gers the production of T7-RNA polymerase, which selec-
tively recognizes the T7 promoter region upstream of the
gene for CHMO on plasmid pMM4.
sion of exo- and endo-substrates 1 with respect to enanti-
oselectivity.
Starting from key intermediate 340 the unsubstituted sub-
strate 1a was accessible via a modified Huang–Minlon re-
action followed by deprotection (Scheme 2).41 Compound
1a was used to establish substrate acceptance of this class
of compounds in general.
Results of the biotransformations of substrates 1a–e
(Scheme 2) with whole-cells of BL21(DE3)(pMM4) are
summarized in the Table. All ketones were converted to
the corresponding lactones. Starting from prochiral sub-
strates, three asymmetric centers were introduced in one
biocatalytic step. Conversion was improved by addition of
-cyclodextrin, a known effect by cyclic sugars of this
type,47 and was particularly necessary in the case of meth-
oxy substrates 1b and 1c to obtain acceptable substrate
consumption. We attribute the decreased bioconversion to
the higher polarity of these compounds compared to 1a or
the chloro derivatives. Both chloro substrates 1d and 1e
were readily converted to the corresponding lactones by
the recombinant strain in high chemical yields.
Functionalization of precursor 3 was performed by diaste-
reoselective reduction of the carbonyl group to give endo-
alcohol 4. Subsequent methylation and deprotection gave
endo-methoxy 1b as representative polar substrate. Com-
pound 4 served as key intermediate to access the exo se-
ries: inversion of the alcohol by a Mitsunobu protocol
using nitrobenzoic acid as nucleophile afforded com-
pound 5, which was converted to exo-methoxy substrate
1c via the above procedure. This approach is superior in
yield to a previously reported method via alcohol tosyla-
tion and inversion.42
For the introduction of a less polar chloro-substituent we
used alcohols 4 and 5 again as key intermediates: Haloge-
Scheme 2 (i) Ref.41; (ii) NaBH4, MeOH, 94%; (iii) NaH, DMS, THF, 90%; (iv) 0.1 M H2SO4, H2O, acetone, 66%; (v) PPh3, DEAD, p-nitro-
benzoic acid, Et2O, 77%; (vi) KOH, quant.; (vii) NaH, DMS, THF, 98%; (viii) 0.1 M H2SO4, H2O, acetone, 80%; (ix) PPh3, NCS, THF, 52%;
(x) 0.1 M H2SO4, H2O, acetone, 68%; (xi) PPh3, NCS, THF, 50%; (xii) 0.1 M H2SO4, H2O, acetone, 58%.
Synlett 2002, No. 5, 703–706 ISSN 0936-5214 © Thieme Stuttgart · New York

LETTER
Baeyer–Villiger Oxidation of Functionalized Bicyclo[3.3.0]ketones
705
Table Whole-cell Biotransformations of Functionalized Bicyclo[3.3.0]ketones
20
Substrate
X
Product
2a
Config.
–
Yielda
eeb
[ ]D
Abs. config.c
4aS-cis
1a
1b
1c
1d
1e
H
50%
89%
9%
–4.7 c 0.34, CDCl3
–3.0 c 0.16, EtOAc
–36.2 c 1.0, CDCl3
–23.7 c 1.0, CDCl3
–38.9 c 0.5, CDCl3
MeO
MeO
Cl
2b
endo
exo
24% (71%)
40% (81%)
75% (98%)
78% (97%)
4aS-(4a ,6 ,7a )
4aS-(4a ,6 ,7a )
2c
96%
80%
>99%
2d
endo
exo
4aR-(4a ,6 ,7a )d
4aR-(4a ,6 ,7a )d
Cl
2e
a Isolated yield after chromatographic purification; yield in brackets is based on consumed starting material.
b Ee determined by chiral phase gas chromatography; racemic reference material prepared by mCPBA oxidation of ketones 1.
c Based on results obtained with bicyclo[4.3.0]ketones.48
d Note, that the chloro substituent requires a change in priority of numbering, which leads to a reversal of the R/S assignment; in all cases, the
sense of chirality was the same.
passed through a bed of Celite®, and the product was isolated by re-
Stereochemistry at the 6-position substantially influenced
peated extraction with EtOAc. The combined organic layers were
the enantioselectivity of the biocatalyst: endo-lactones 2b
and 2d displayed a significantly decreased optical purity
compared to the moderate 89% ee observed for the unsub-
dried over sodium sulfate and concentrated. Lactones 2a–e49 were
purified by flash column chromatography.
stituted system 2a. By contrast, exo-ketones 1c and 1e
were oxidized with excellent enantioselectivities. The na- Acknowledgement
ture of the group X seems to have only a minor effect on
the chirality of the conversion for the exo-substrates. Ab-
This project was funded by the Oesterreichische Nationalbank
(grant no. JF-7619). Support by Baxter Immuno Austria and Novar-
solute configuration was assigned by comparison to re-
sults obtained for the microbial Baeyer–Villiger oxidation
of biocyclo[4.3.0]ketones.48
tis Donation and Sponsoring is gratefully acknowledged. We thank
Dr. Erwin Rosenberg (Vienna University of Technology) for his as-
sistance in the determination of enantiomeric purity.
Recently, we have observed that conformational aspects
play a significant role in the recognition and introduction
of chirality by CHMO.33 Based on the above results, high
optical purity is obtained for substituted bicycloketones of
type 1 in a ‘stretched’ geometry (exo), while sterically de-
manding ‘angled’ configurations (endo) decrease the se-
lectivity of the enzyme. Additional substrate profiling
using bicyclic precursors is presently underway in our lab-
oratory.
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LETTER
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Figure 1
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4aS-cis-Hexahydro-cyclopenta[c]pyran-3(1H)-one(2a).
Colorless oil; 1H NMR (200 MHz, CDCl3): = 1.20–2.08
(m, 6 H), 2.25–2.69 (m, 4 H), 4.00 (dd, J = 7 Hz, J = 20 Hz,
1 H), 4.37 (dd, J = 7 Hz, J = 20 Hz, 1 H); 13C NMR (50
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4aS-(4a ,6 ,7a )-Hexahydro-6-methoxy-cyclopenta
[c]pyran-3(1H)-one(2b). Colorless oil; 1H NMR (400 MHz,
CDCl3): = 1.50–1.65 (m, 2 H), 1.82–2.10 (m, 2 H),
2.40–2.65 (m, 4 H), 3.23 (s, 3 H), 3.72–3.83 (m, 1 H, H6),
4.08 (dd, J = 14 Hz, J = 7 Hz, 1 H), 4.31 (dd, J = 14 Hz,
J = 7 Hz, 1 H); 13C NMR (100 MHz, CDCl3): = 32.8 (d),
33.8 (t), 34.9 (t), 35.2 (d), 38.0 (t), 56.4 (q), 69.9 (t), 82.3 (d),
173.8 (s).
4aS-(4a ,6 ,7a )-Hexahydro-6-methoxy-cyclopenta
[c]pyran-3(1H)-one(2c). Colorless oil; 1H NMR (400 MHz,
CDCl3): = 1.24–1.44 (m, 1 H), 1.52–1.68 (m, 1 H),
2.00–2.90 (m, 6 H), 3.29 (s, 3 H), 3.80–3.92 (m, 1 H), 4.10
(dd, J = 14 Hz, J = 7 Hz, 1 H), 4.35 (dd, J = 14 Hz, J = 7 Hz,
1 H); 13C NMR (100 MHz, CDCl3): = 32.1 (d), 34.1 (t),
34.2 (t), 34.4 (d), 38.4 (t), 55.7 (q), 69.7 (t), 81.4 (d), 173.6
(s).
4aR-(4a ,6 ,7a )-6-Chloro-hexahydro-cyclopenta
[c]pyran-3(1H)-one(2d). Colorless crystals, mp 90–92 °C;
1H NMR (400 MHz, CDCl3): = 1.58–1.68 (m, 1 H),
1.70–1.81 (m, 1 H), 2.30–2.70 (m, 6 H), 4.05–4.15 (m, 1 H),
4.20–4.30 (m, 2 H); 13C NMR (100 MHz, CDCl3): = 32.8
(d), 34.2 (t), 35.1 (d), 38.8 (t), 43.1 (t), 56.6 (d), 69.2 (t),
172.7 (s).
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4aR-(4a ,6 ,7a )-6-Chloro-hexahydro-cyclopenta
[c]pyran-3(1H)-one(2e). Colorless crystals, mp 88–90 °C;
1H NMR (400 MHz, CDCl3): = 1.60–1.80 (m, 1 H), 1.92–
2.10 (m, 1 H), 2.20–2.50 (m, 3 H), 2.65–3.20 (m, 3 H), 4.10
(dd, J = 14 Hz, J = 7 Hz, 1 H), 4.45–4.55 (m, 1 H); 13C NMR
(100 MHz, CDCl3): = 32.2 (d), 33.7 (t), 34.5 (d), 39.5 (t),
43.5 (t), 61.4 (d), 69.1 (t), 172.8 (s).
Synlett 2002, No. 5, 703–706 ISSN 0936-5214 © Thieme Stuttgart · New York