Microbial Baeyer-Villiger oxidation of bicyclo[4.3.0]ketones by two recombinant E. coli strains. A novel access to indole alkaloids

Article abstract of DOI:10.1055/s-2002-25340

Recombinant Escherichia coli overexpressing Pseudomonas sp. NCIMB 9872 cyclopentanone monooxygenase (CPMO; E.C. 1.14.13.16) and Acinetobacter sp. NCIMB 9871 cyclohexanone monooxygenase (CHMO; E.C. 1.14.13.22) have been utilized in whole-cell biotransforma

Full text of DOI:10.1055/s-2002-25340

700
LETTER
Microbial Baeyer–Villiger Oxidation of Bicyclo[4.3.0]ketones by Two
Recombinant E. coli Strains. A Novel Access to Indole Alkaloids
Microbial Baeyer–Vill
a
iger Oxida
t
r
ion of Bi
k
c
yclo[4.3.0]k
o
etone
s
D. Mihovilovic,*a Bernhard Müller,a Margaret M. Kayser,b 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
University of New Brunswick, Dept. Phys. Sciences, Saint John, N.B., E2L 4L5, Canada
Received 27 February 2002
gio-, and enantioselectivities as observed for the isolated
enzyme.5
Abstract: Recombinant
Escherichia
coli
overexpressing
Pseudomonas sp. NCIMB 9872 cyclopentanone monooxygenase
(CPMO; E.C. 1.14.13.16) and Acinetobacter sp. NCIMB 9871 cy-
clohexanone monooxygenase (CHMO; E.C. 1.14.13.22) have been
utilized in whole-cell biotransformations of prochiral bicyc-
lo[4.3.0]ketones. The lactones produced in a biocatalytic Baeyer–
Villiger oxidation represent key intermediates for the synthesis of
several indole alkaloids. The two over-expression systems demon-
strated a tendency for the formation of opposite enantiomers with
CPMO giving (+)-lactones in good yields and excellent enantiomer-
ic excess.
While more than 100 substrates for CHMO were identi-
fied in several studies,4,5 CPMO from Pseudomonas sp.
NCIMB 9872 (CPMO; E.C. 1.14.13.16)20–22 received
considerably less attention in biocatalytic applica-
tions,23,24 probably because a substrate profile similar to
CHMO was assumed. Just recently, an overexpression
system has been constructed and utilized for the biotrans-
formation of representative cyclohexanones.25
Key words: biocatalysis, recombinant whole-cell biotransforma-
tion, monooxygenase, Baeyer–Villiger oxidation, indole alkaloids,
enantioselectivity
In this publication we are expanding our substrate-profil-
ing studies of recombinant E. coli expression systems for
Baeyer–Villiger monooxygenases26,27 to bicycloketones 1
as precursors for chiral lactones 2 (Scheme 1).28,29 Com-
pounds 2 represent key intermediates in the total synthesis
of several members of the indole alkaloid family. The use
of prochiral substrate ketones in a biocatalytic asymmetri-
zation step is highly advantageous, since it generates op-
tically active products with a theoretical yield of 100%,
while kinetic resolution of racemic samples leads only to
a maximum of 50% enantiopure product.
The utilization of enzymatic systems offers efficient ac-
cess to enantiomerically pure lactones via Baeyer–Vil-
liger oxidation1 of the corresponding cyclic ketones2–6 and
is considered a ‘green chemistry’ alternative to organome-
tallic catalysts.7–14 Such chiral compounds are interesting
precursors in enantioselective natural compound synthe-
sis.
Lactone (–)-2a has been used by Riva and coworkers,30 in
a total synthesis of (–)-alloyohimbane 3, a member of the
yohimbine–respirine alkaloid group. This class of com-
pounds has interesting pharmacological properties, acting
as 2-adrenoceptor antagonists, serotonin and dopamine
receptor antagonists, as well as an aphrodisiac.31 Repre-
sentatives of this class have been detected in three plant
families: Apocynaceae, Rubiaceae and Loganiaceae. Re-
cently, new bisindoles of the yohimbine–gambarine type
have been found also in different Uncaria species.32 The
antipodal (+)-2a was utilized by Danieli in the total syn-
thesis of (–)-antirhine 4,33 another indole alkaloid origi-
nally isolated from Antirhea putaminosa.34
The widespread use of flavin-dependent monooxygenases
as catalytic entities for these transformations throughout
the synthetic community was hampered by the fact that
cofactor recycling is essential for this class of enzymes,
which makes the application of such systems more diffi-
cult. While the situation has been improved by the devel-
opment of efficient and more robust recycling systems for
NADPH required by these enzymes in recent years,15,16
another way to circumvent this obstacle is to use whole-
cells instead of isolated enzymes.
Advances in molecular biology mean that recombinant
systems can be developed with significantly improved se-
lectivity for the desired biotransformation and with higher
efficiency compared to the native strains. Recently, we
designed overexpression systems for the most extensively
studied Baeyer–Villigerase to date, cyclohexanone mono-
oxygenase (CHMO) from Acinetobacter sp. NCIMB
9871 (E.C. 1.14.13.22).17 Both Saccharomyces
cerevisiae18 and Escherichia coli19 were used as hosts and
the new organisms demonstrated the same chemo-, re-
Since both enantiomeric lactones 2 have been fully char-
acterized they are interesting model compounds for us to
establish the absolute configuration of the fermentation
products. In addition, the compounds served as key inter-
mediates in natural compound syntheses, demonstrating
applications of our expression systems.
Our biocatalytic approach to lactones 2 via microbial
Baeyer–Villiger oxidation required rapid and efficient ac-
cess to ketone 1. We adapted35 and optimized protocols36
for the tosylation of diol 5 and conversion to the corre-
sponding dinitrile, followed by hydrolysis to diacid 6
Synlett 2002, No. 5, 03 05 2002. Article Identifier:
1437-2096,E;2002,0,05,0700,0702,ftx,en;D04402ST.pdf.
© Georg Thieme Verlag Stuttgart · New York
ISSN 0936-5214

LETTER
Microbial Baeyer–Villiger Oxidation of Bicyclo[4.3.0]ketones
701
Scheme 1 Access to lactones 2 via microbial Baeyer–Villiger oxidation as key intermediates for some indole alkaloids.
(Scheme 2). One-pot cyclization in acetic anhydride/pyri- Fermentations of substrates 1 with the CHMO expression
dine and decarboxylation of compound 8 represents a system proceeded rather slowly, with substantial amounts
shortcut to ketone 1a compared to the usual Dieckmann of starting material remaining after 24–36 hours when
condensation route.37,38 Catalytic hydrogenation of olefin biocatalytic activity of this system came to a halt. We at-
1a afforded compound 1b.
tribute this to the size and steric arrangement of the com-
pounds reaching the spatial limitations of the active site.
Biotransformations of 1a and 1b gave the corresponding
lactones in almost racemic mixtures (reference material of
rac-2b was prepared by chemical oxidation with mCP-
BA) with a slight preference for the (–)-4aS,8aS configu-
ration. Absolute configuration was established by
comparing the sign of the specific rotation to literature da-
ta.30,33,40
Biocatalytic Baeyer–Villiger oxidations were performed
with two recombinant E. coli strains: In both the CHMO-
system BL21(DE3)(pMM4) and the CPMO construct
DA5a(pCMP201) the production of the corresponding
monooxygenase is induced by addition of isopropyl- -D-
thiogalactopyranoside (IPTG).
Results of the biotransformations of substrates 1 with
whole-cells expressing CHMO and CPMO are summar-
ized in the Table. All ketones were converted to the corre-
sponding lactones with high chemoselectivity. The double
bond in substrate 1a was not oxidized under fermentation
conditions. Conversion was improved by addition of -
cyclodextrin, a known effect by cyclic sugars of this
type.39
Biotransformations with the CPMO-expression system
proceeded generally to completion after 48 hours incuba-
tion at room temperature. Both yields and enantioselectiv-
ities were superior to the CHMO strain. Two stereogenic
centers were established in the course of the biotransfor-
mation with preference for the (+)-4aR,8aR configuration.
Table Whole-cell Biotransformations of Bicyclo[4.3.0]ketones
20
Substrate
Strain
Product
2a
Yielda
eeb
[ ]D
Abs. config.
(–)-4aS,8aS
(+)-4aR,8aR
(–)-4aS,8aS
(+)-4aR,8aR
1a
1a
1b
1b
CHMO
CPMO
CHMO
CPMO
33% (85%)
76%
~ 5%
> 99%
~ 3%
99%
–0.7 c 0.36, CH2Cl2
+24.5 c 1.0, CHCl3
–1.2 c 0.46, EtOH
+39.1 c 1.0, CHCl3
2a
2b
21% (65%)
83%
2b
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 ketone 1b.
Scheme 2 (i) TsCl, pyridine, 83%; (ii) NaCN, EtOH, reflux, 88%; (iii) 6 N KOH, then H3PO4, 92%; (iv) Ac2O, pyridine, then 0.5 N HCl,
61%; (v) Pd/C, H2 81 psi, THF, 77%.
Synlett 2002, No. 5, 700–702 ISSN 0936-5214 © Thieme Stuttgart · New York

702
M. D. Mihovilovic et al.
LETTER
(16) Schwarz-Linek, U.; Krödel, A.; Ludwig, F.-A.; Schulze, A.;
Rissom, S.; Kragl, U.; Tishkov, V. I.; Vogel, M. Synthesis
2001, 947.
(17) Donoghue, N. A.; Norris, D. B.; Trudgill, P. W. Eur. J.
Biochem. 1976, 63, 175.
Hence, CPMO promotes formation of the opposite enan-
tiomer than CHMO for this class of substrates.
In conclusion, we have demonstrated that a recombinant
expression system for CPMO can produce chiral lactones
2a and 2b chemoselectively via microbial Baeyer–Vil-
liger oxidation with high optical purities and in good
yields. Our biotransformation provides a simple and
‘green’ route to (+)-2a, a key intermediate for the synthe-
sis of some indole alkaloids, and represents the first for-
mal total synthesis of (–)-antirhine 4 and (+)-
alloyohimbane 3 using a recombinant strain expressing
CPMO. With respect to both yield and optical purity the
CPMO-system is superior to a recombinant CHMO pro-
ducing strain for this class of bicyclo-ketones.
(18) Kayser, M.; Chen, G.; Stewart, J. Synlett 1999, 153.
(19) Chen, G.; Kayser, M. M.; Mihovilovic, M. D.; Mrstik, M. E.;
Martinez, C. A.; Stewart, J. D. New J. Chem. 1999, 23, 827.
(20) Griffin, M.; Trudgill, P. W. Biochem. J. 1972, 129, 595.
(21) Griffin, M.; Trudgill, P. W. Eur. J. Biochem. 1976, 63, 199.
(22) Trudgill, P. W. Methods Enzymol. 1990, 188, 77.
(23) Bes, M. T.; Villa, R.; Roberts, S. M.; Wan, P. W. H.;
Willetts, A. J. J. Mol. Catal. B: Enzym. 1996, 1, 127.
(24) Adger, B.; Bes, M. T.; Grogan, G.; McCague, R.; Pedragosa-
Moreau, S.; Roberts, S. M.; Villa, R.; Wan, P. W. H.;
Willetts, A. J. Bioorg. Med. Chem. 1997, 5, 253.
(25) Iwaki, H.; Hasegawa, Y.; Lau, P. C. K.; Wang, S.; Kayser,
M. M. submitted to Appl. Environ. Microbiol.
(26) Mihovilovic, M. D.; Chen, G.; Wang, S.; Kyte, B.; Rochon,
F.; Kayser, M. M.; Stewart, J. D. J. Org. Chem. 2001, 66,
733.
Fresh LB-amp medium (1% Bacto-Peptone, 0.5% Bacto-Yeast Ex-
tract, 1% NaCl supplemented by 200 ppm ampicillin) was inoculat-
ed with
a 1/100 aliquot of an overnight preculture of
(27) Mihovilovic, M. D.; Müller, B.; Kayser, M. M.; Stewart, J.
D.; Fröhlich, J.; Stanetty, P.; Spreitzer, H. J. Mol. Catal. B:
Enzym. 2001, 11, 349.
(28) For an alternative E. coli expression system for CHMO see:
Doig, S. D.; O’Sullivan, L. M.; Patel, S.; Ward, J. M.;
Woodley, J. M. Enzyme Microb. Technol. 2001, 28, 265.
(29) Application of the above system for whole-cell
biotransformations: Simpson, H. D.; Alphand, V.; Furstoss,
R. J. Mol. Catal. B: Enzym. 2001, 16, 101.
(30) Riva, R.; Banfi, L.; Danieli, B.; Guanti, G.; Lesma, G.;
Palmisano, G. J. Chem. Soc., Chem. Commun. 1987, 299.
(31) Hieble, J. P.; Nichols, A. J.; Langer, S. Z.; Ruffolo, R. R. Jr.
In Principles of Pharmacology; Munson, P. L., Ed.;
Chapman and Hall: New York, 1996, 135.
(32) Szántay, C.; Honzy, K. Chem. Heterocycl. Compd. 1994, 25
(Suppl.), 161.
(33) Danieli, B.; Lesma, G.; Mauro, M.; Palmisano, G.;
Passerella, D. Tetrahedron 1994, 50, 8837.
(34) Johns, S. R.; Lamberton, J. A.; Occolowitz, J. L. J. Chem.
Soc., Chem. Commun. 1967, 229.
DH5a(pCMP201) in a baffled Erlenmeyer flask. The culture was
incubated at 120 rpm at 37 °C on an orbital shaker for 2 h, then
IPTG was added to a final concentration of 0.025 mM. The sub-
strate 1 (3–5 mM) was added neat along with -cyclodextrin (1
equiv). The culture was incubated at r.t. for 48 h. The biomass was
removed by centrifugation (3500 rpm, 10 min), the aq layer was
passed through a bed of Celite®, and the product was isolated by re-
peated extraction with EtOAc. The combined organic layers were
dried over sodium sulfate and concentrated. Lactones 241 were pu-
rified by flash column chromatography.
Acknowledgement
This project was funded by the Oesterreichische Nationalbank
(grant no. JF-7619). Support by Baxter Immuno Austria and Novar-
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.
(35) Krawczyk, A. R.; Jones, J. B. J. Org. Chem. 1989, 54, 1795.
(36) Mundy, B. P.; Theodore, J. J. J. Am. Chem. Soc. 1980, 102,
2005.
(37) Aube, J.; Gosh, S.; Tanol, M. J. Am. Chem. Soc. 1994, 116,
9009.
(38) Barret, A. G. M.; Boys, M. L.; Boehm, T. L. J. Org. Chem.
1996, 61, 685.
(39) Bar, R. Trends Biotechnol. 1989, 7, 2.
References
(1) Krow, G. R. Org. React. 1993, 43, 251.
(2) Walsh, C. T.; Chen, Y.-C. J. Angew. Chem. 1988, 100, 342.
(3) Willetts, A. Trends Biotechnol. 1997, 15, 55.
(4) Roberts, S. M. J. Mol. Catal. B: Enzym. 1998, 4, 111.
(5) Stewart, J. D. Curr. Org. Chem. 1998, 2, 195.
(6) (a) Kelly, D. R. Chim. Oggi 2000, 18, 33. (b) Kelly, D. R.
Chim. Oggi 2000, 18, 52.
(7) Bolm, C.; Schlingloff, G.; Weickhardt, K. Angew. Chem.,
Int. Ed. Engl. 1994, 33, 1848.
(8) Gusso, A.; Baccin, C.; Pinna, F.; Strukul, G.
Organometallics 1994, 13, 3442.
(9) Bolm, C.; Schlingloff, G. J. Chem. Soc., Chem. Commun.
1995, 1247.
(10) Bolm, C.; Schlingloff, G.; Bienewald, F. J. Mol. Catal. A:
Chemical 1997, 117, 347.
(11) Bolm, C.; Luong, T. K. K.; Schlingloff, G. Synlett 1998,
1151.
(12) Strukul, G.; Varagnolo, A.; Pinna, F. J. Mol. Catal. A:
Chemical 1997, 117, 413.
(40) Miyafuji, A.; Ito, K.; Katsuki, T. Heterocycles 2000, 52,
261.
(41) Physical and spectroscopic data of lactones 2:
(4aR,8aR)-1,4,4a,5,8,8a-Hexahydro-3H-2-benzopyran-3-
one(2a). Colorless oil; [ ]D20 = +24.5 (c 1.0, CHCl3); ee
>99% (chiral phase GC); 1H NMR (200 MHz, CDCl3):
=
1.80–2.13 (m, 2 H), 2.16–2.44 (m, 4 H), 2.50–2.60 (m, 2 H),
4.22–4.40 (m, 2 H), 5.67 (br s, 2 H); 13C NMR (50 MHz,
CDCl3): = 24.0 (t), 28.4 (t), 28.5 (d), 29.6 (d), 33.7 (t), 72.1
(t), 124.1 (d), 124.6 (d), 170.6 (s).
(4aR,8aR)-Octahydro-3H-2-benzopyran-3-one(2b).
Colorless oil; [ ]D20 = +39.1 (c 1.0, CHCl3); ee = 99%
(chiral phase GC); 1H NMR (200 MHz, CDCl3): = 1.22–
1.63 (m, 8 H), 1.83–2.01 (m, 1 H), 2.05–2.25 (m, 1 H), 2.40–
2.55 (m, 2 H), 4.25 (d, 2 H, J = 8 Hz); 13C NMR (50 MHz,
CDCl3): = 21.5 (t), 23.3 (t), 24.6 (t), 28.6 (t), 31.0 (d), 32.7
(d), 32.8 (t), 72.4 (t), 171.1 (s).
(13) Bolm, C.; Beckmann, O.; Kühn, T.; Palazzi, C.; Adam, W.;
Rao, P. B.; Saha-Möller, C. R. Tetrahedron: Asymmetry
2001, 12, 2441.
(14) Uchida, T.; Katsuki, T. Tetrahedron Lett. 2001, 42, 6911.
(15) Vogel, M.; Scharz-Linek, U. Bioorg. Chem. 1999, 102.
Synlett 2002, No. 5, 700–702 ISSN 0936-5214 © Thieme Stuttgart · New York