Synthesis and Enantioselective Baeyer-Villiger Oxidation of Prochiral Perhydro-pyranones with Recombinant E. coli Producing Cyclohexanone Monooxygenase

Article abstract of DOI:10.1055/s-2003-42035

Recombinant whole cells of Escherichia coli overexpressing Adnetobacter sp. NCIMB 9871 cyclohexanone monooxygenase (E.C. 1.14.13.22) have been utilized for the Baeyer-Villiger oxidation of prochiral perhydro-pyranones. The spatial limitations of the enzym

Full text of DOI:10.1055/s-2003-42035

LETTER
1973
Synthesis and Enantioselective Baeyer–Villiger Oxidation of Prochiral
Perhydro-pyranones with Recombinant E. coli Producing Cyclohexanone
Monooxygenase
a
a
a
a
a
b
S
M
ynthesis and Baeyer–
a
V
illiger
O
x
r
idation
o
k
f
Prochira
l
Po
erhydro-pyranone sD . Mihovilovic,* Florian Rudroff, Wolfgang Kandioller, Birgit Grötzl, Peter Stanetty, Helmut Spreitzer
a
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
b
Vienna University, Institute of Pharmaceutical Chemistry, Vienna, Austria
Received 14 August 2003
proteome of the organism. Hence, undesired side
reactions can compromise both yield and enantiomeric
excess of the required product. To overcome this problem,
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 prochiral perhydro-pyranones. The spatial limitations
we have recently developed an E. coli based
of the enzyme’s active site have been estimated by increasing the overexpression system1
4,15
for cyclohexanone monooxy-
chain length of cis-substituents in positions 2 and 6. A diastereose- genase (CHMO) from Acinetobacter sp. NCIMB 9871
lective synthetic sequence to the required substrate ketones has been
developed utilizing high pressure hydrogenation.
16
17
(
E.C. 1.14.13.22) as the best studied BVMO so far. In
this engineered strain the production of CHMO is
significantly increased and such unwanted side reactions
are suppressed. We have demonstrated the efficiency of
this CHMO producer for the production of chiral
Key words: biocatalysis, recombinant whole-cell biotransforma-
tion, cyclohexanone monooxygenase, Baeyer–Villiger oxidation,
enantioselectivity
18
compounds1 and contributed to the active site model,
9
previously. More recently we have proven the power of
Enzyme mediated Baeyer–Villiger reactions offer an
environmentally benign entry to chiral lactones as
important building blocks in synthetic chemistry.1 To
date, a significant number of organisms has been
identified to utilize such an oxygen insertion process in
recombinant whole cell mediated Baeyer–Villiger oxida-
tions by identifying pairs of enantiodivergent BVMOs
originating from different microorganisms to allow access
–6
20–22
to both antipodal lactones.
7
–9
We demonstrated that CHMO can convert heterocyclic
cores such as 4-piperidones, perhydro-4-pyranones, and
perhydro-4-thiopyranones in addition to carbocyclic
substrates, which have been studied extensively, in high
chemoselectivity and good isolated yields.23 In this
contribution we expand these substrate acceptance studies
on prochiral heterocyclic ketones and present the first
enantioselective Baeyer–Villiger oxidation of hetero-
cyclic substrate ketones by recombinant cells. By this
strategy, it is possible to generate a set of new chiral
centers in an asymmetrization step with a theoretical yield
of 100% in contrast to kinetic resolutions, which are
their metabolic pathways. Several genes encoding such
flavin dependent Baeyer–Villiger monooxygenases (BV-
1
0–12
MOs) have been characterized.
Since most of these
biocatalysts accept a multitude of non-natural substrates,
this offers the option, to selectively overexpress the
enzymes for protein isolation purposes or direct whole-
cell mediated biotransformations.
Our approach towards the production of chiral lactones
utilizes such engineered cells. This offers the advantage to
circumvent the sometimes tedious process of protein
purification and also simplifies the required cofactor recy-
cling. BVMOs catalyze the Baeyer–Villiger oxidation by
utilizing molecular oxygen as oxidant, whereas one O
atom is incorporated into the substrate while the second is
reduced to water at the expense of a reducing cofactor. As
a consequence, this enzyme family is dependent on
NAD(P)H, which has to be recycled to facilitate an
efficient and economical biotransformation. The simplest
way to achieve this goal is the utilization of living whole
cells, in which all natural cofactor recycling systems are
24
limited to 50% yield of the required antipode. Based on
our previous results, we selected the perhydro-4-pyranone
system for a first model study, since this system gave the
best conversion rates and isolated yields when studying
unsubstituted hetero-ketones. A series of cis-2,6-dialkyl-
perhydropyrans 1 with increasing chain length for the sub-
stituents was oxidized to the corresponding lactones 2 to
investigate spatial requirements of the active site of
CHMO to accommodate such substrates (Scheme 1). The
1
3
still intact.
25–30
refinement of active site models for BVMOs
A disadvantage of using native cells is the low exclusively depends on such substrate acceptance
concentration of the required biocatalyst in the total profiles, since crystallographic data for the structure of
this enzyme family is not available, to date.
A number of methods has been reported previously to
SYNLETT 2003, No. 13, pp 1973–1976
Advanced online publication: 08.10.2003
DOI: 10.1055/s-2003-42035; Art ID: D20803ST
1
6
.1
0
.2
0
0
3
access 2,6-disubstituted g-pyrones 5 as key precursors for
the required ketones 1.3
1,32
We expanded a strategy
©
Georg Thieme Verlag Stuttgart · New York

1
974
M. D. Mihovilovic et al.
LETTER
Results of the biotransformations of substrates 1a–e with
whole-cells of BL21(DE3)(pMM4) are summarized in
Table 1. The size of substituents R had a substantial
influence on the conversion of the substrates: While small
straight chain groups such as methyl and ethyl were
oxidized in excellent yield by the whole-cells, we
observed a significantly decreased conversion for the n-
propyl ketone 1c. A straight chain with 3 carbons seems
to represent the borderline with respect to steric demands
of the substrate in the active site. Both branched
Scheme 1 Whole-cell biotransformations of prochiral perhydro-4-
pyranones.
utilizing the magnesium-diacetonedicarboxylate complex substituents (i-propyl, 1d) or longer chains (butyl, 1e)
for an array of different substituents in the 2 and 6- seem to exceed the spatial limits imposed by the enzyme.
3
3
3
positions (Scheme 2). Initial cyclization leads to the These compounds are not (or only very poor in the case of
corresponding 3,5-dicarboxylic acid esters 4 followed by 1d) substrates for CHMO in preparative whole-cell
3
4
hydrolytic decarboxylation. Diastereoselective reduc- biotransformations.
tion was accomplished by high pressure hydrogenation
Table 1 Recombinant Whole-Cell Biotransformations of 2,6-
Disubstitued Perhydro-4-pyranones
with Pd/C in MeOH. High selectivity for the direct forma-
tion of thermodynamically favored cis-products is only
ensured at 20 bar or above, while hydrogenation in a Parr-
apparatus at approximately 5bar gave mixtures of partial-
ly reduced compounds in contrast to previous reports in
Prod- Yielda
uct
eeb
20
Sub-
strate
R
[a]D
3
5
1a
1b
Me
Et
2a
2b
2c
2d
2e
80%
90%
19%
traces
>99.5% –50.6 (c 1.25, CHCl3)
>99.5%c –4.3 (c 1.32, CHCl
the literature.
It is interesting to note that in several cases ketals 6 were
formed upon high-pressure hydrogenation. These
products were only isolated as crude material and directly
3
)
1
c
n-Pr
i-Pr
Bu
98%
n.d.
–22.5 (c 1.0, CHCl3)
cleaved to give the required ketones 1. The overall 1d
n.d.
n.a.
sequence allowed efficient access to an array of C to C4
1
1
e
no conv. n.a.
substituted straight chain and branched perhydro-4-
pyranones 1a–e as probes to investigate the tolerance of
the enzyme’s active site for sterically demanding
a
Isolated yield after chromatographic purification.
Ee determined by chiral gas phase chromatography; racemic
b
3
6
reference material prepared by MCPBA oxidation of ketones 1.
residues.
c
Ee determined by chiral phase gas chromatography after reduction
Protein expression of CHMO in growing cultures of re- of lactone 2b with LiAlH to the corresponding diol.
4
combinant strain BL21(DE3)(pMM4) was triggered by
addition of isopropyl-b-D-thiogalactopyranoside (IPTG).
For those ketones, which can be accommodated by the
Fermentations were carried out according to our previous-
active site of CHMO (1a–c), the biooxidation generates
ly reported procedure on 100 mg scale in Erlenmeyer
two new stereogenic centers and proceeds with excellent
2
1,37
flasks
and b-cyclodextrin was added to facilitate
38
enantioselectivity. Assignment of the absolute configura-
tion is based on previous results on similar carbocyclic
systems by Taschner and coworkers.39 Assuming the
same configuration for lactones 2, which is supported by
membrane penetration. For reference purposes, racemic
lactones 2 were prepared by chemical oxidation of ke-
tones 1 with MCPBA.
Scheme 2 (i) RCOCl/Et N or (RCO) O, 32–59%; (ii) AcOH, HCl, H O, heat, 53–87%; (iii) 20 bar H , Pd/C, MeOH; (iv) 2N HCl, THF, r.t.,
3
2
2
2
35–57% (2 steps).
Synlett 2003, No. 13, 1973–1976 © Thieme Stuttgart · New York

LETTER
Synthesis and Baeyer–Villiger Oxidation of Prochiral Perhydro-pyranones
1975
the same sign of specific rotation, and considering the
change of priorities according to the Cahn–Ingold–Prelog
rules, the absolute configuration of fermentation products
(20) Mihovilovic, M. D.; Müller, B.; Kayser, M. M.; Stanetty, P.
Synlett 2002, 700.
(
(
(
21) Mihovilovic, M. D.; Müller, B.; Schulze, A.; Stanetty, P.;
Kayser, M. M. Eur. J. Org. Chem. 2003, 2243.
22) Mihovilovic, M. D.; Rudroff, F.; Müller, B.; Stanetty, P.
Bioorg. Med. Chem. Lett. 2003, 13, 1479.
23) 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.
2
a–c can be assigned as 2S,7R.40
In summary, we have developed a diastereoselective route
to substituted hetero-ketones 1 as substrates for whole-
cell biotransformations. The compounds serve as probes
to determine spatial requirements by the enzyme and we
(
24) For the first dynamic kinetic resolution mediated by Baeyer–
Villigerases see: Berezina, N.; Alphand, V.; Furstoss, R.
Tetrahedron: Asymmetry 2002, 13, 1953.
demonstrated, that straight chain substitution up to C is
3
accepted by the recombinant whole-cell expression sys-
tem for the production of chiral lactones 2.
(25) Alphand, V.; Furstoss, R. Tetrahedron: Asymmetry 1992, 3,
79.
3
(
(
(
26) Taschner, M. J.; Peddada, L.; Cyr, P.; Chen, Q. Z.; Black, D.
Acknowledgment
J. NATO ASI Ser., Ser. C 1992, 381, 347.
27) Kelly, D. R.; Knowles, C. J.; Mahdi, J. G.; Taylor, I. N.;
Wright, M. A. J. Chem. Soc., Chem. Commun. 1995, 729.
28) Kelly, D. R.; Knowles, C. J.; Mahdi, J. G.; Wright, M. A.;
Taylor, I. N.; Hibbs, D. E.; Hursthouse, M. B.; Mish’al, A.
K.; Roberts, S. M.; Wan, P. W. H.; Grogan, G.; Willets, A.
J. J. Chem. Soc., Perkin Trans. 1 1995, 2057.
29) Ottolina, G.; Pasta, P.; Carrea, G.; Colonna, S.; Dallavalle,
S.; Holland, H. L. Tetrahedron: Asymmetry 1995, 6, 1375.
30) Ottolina, G.; Carrea, G.; Colonna, S.; Rückmann, A.
Tetrahedron: Asymmetry 1996, 7, 1123.
This project was funded by the City of Vienna, Hochschuljubi-
läumsstiftung (grant no. H-40/98). Support by Baxter Immuno
Austria and Novartis Donation & Sponsoring is gratefully acknow-
ledged. We thank Dr. Erwin Rosenberg (Vienna University of
Technology) for his assistance in the determination of enantiomeric
purity.
(
(
References
(
1) Mihovilovic, M. D.; Müller, B.; Stanetty, P. Eur. J. Org.
Chem. 2002, 3711.
(31) Yates, P.; Hand, E. S.; Singh, P.; Roy, S. K.; Still, I. W. J. J.
Org. Chem. 1969, 34, 4046.
(32) Sear, R. P.; Frenkel, D. J. Chem. Phys. 1996, 105, 10632.
(33) Yamoto, M.; Kusunoki, Y. Chem. Pharm. Bull. 1981, 29,
1214.
(
2) Flitsch, S.; Grogan, G. In Enzyme Catalysis in Organic
Synthesis; Drauz, K.; Waldmann, H., Eds.; Wiley-VCH:
Weinheim, 2002, 1202–1245.
(
(
3) (a) Kelly, D. R. Chim. Oggi 2000, 18, 33. (b) Kelly, D. R.
Chim. Oggi 2000, 18, 52.
4) Roberts, S. M.; Wan, P. W. H. J. Mol. Catal. B: Enzym.
(34) Feist, F. Justus Liebigs Ann. Chem. 1890, 257, 253.
(35) Sato, M.; Kuroda, H.; Kaneko, C.; Furuya, T. J. Chem. Soc.
Chem. Commun. 1994, 687.
(36) Typical procedure for the high pressure hydrogenation:
Precursor 5 dissolved in anhyd MeOH was hydrogenated
with Pd/C (10%, 300 mg) in a Büchi steel autoclave under
1998, 4, 111.
(
(
(
5) Willetts, A. Trends Biotechnol. 1997, 15, 55.
6) Walsh, C. T.; Chen, Y.-C. J. Angew. Chem. 1988, 100, 342.
7) Donoghue, N. A.; Trudgill, P. W. Eur. J. Biochem. 1975, 60,
H atmosphere (20 bar) for 2 d. The solution was filtered
2
®
1.
through a bed of Celite and MeOH was evaporated. In the
(8) Iwaki, H.; Hasegawa, Y.; Teraoka, M.; Tokuyama, T.;
case of partial ketal formation (6), the crude material was
treated with a 5:1 mixture of THF and 0.1 N HCl at r.t.
overnight. The solution was washed with NaHCO3,
extracted with CH Cl , dried over Na SO , filtered, and
Bergeron, H.; Lau, P. C. K. Appl. Environ. Microbiol. 1999,
65, 5158.
(
9) Cheng, Q.; Thomas, S. M.; Kostichka, K.; Valentine, J. R.;
2
2
2
4
Nagarajan, V. J. Bacteriol. 2000, 182, 4744.
concentrated in vacuo. Pure 1 was obtained after Kugelrohr
distillation or flash column chromatography.
cis-Tetrahydro-2,6-dimethyl-4H-pyran-4-one (1a): 42%
(
(
(
(
10) Chen, Y.-C. J.; Peoples, O. P.; Walsh, C. T. J. Bacteriol.
988, 170, 781.
1
11) Iwaki, H.; Hasegawa, Y.; Lau, P. C. K.; Wang, S.; Kayser,
M. M. Appl. Environ. Microbiol. 2002, 68, 5681.
12) Brzostowicz, P. C.; Gibson, K. L.; Thomas, S. M.; Blasko,
M. S.; Rouviere, P. E. J. Bacteriol. 2000, 182, 4241.
13) Seelbach, K.; Riebel, B.; Hummel, W.; Kula, M.-R.;
Tishkov, V. I.; Egorov, H. M.; Wandrey, C.; Kragl, U.
Tetrahedron Lett. 1996, 37, 1377.
yield, colorless liquid, bp 50 °C/12mbar (Kugelrohr),
1
H NMR (200 MHz, CDCl ): d = 1.35 (d, J = 6 Hz, 6 H),
3
2.10–2.45 (m, 4 H), 3.61–3.80 (m, 2 H),
1
3
C NMR(50 MHz, CDCl ): d = 21.9 (q), 48.8 (t), 72.9 (d),
3
207.2 (s).
cis-Tetrahydro-2,6-diethyl-4H-pyran-4-one (1b): 57%
yield, colorless liquid, bp 81–83 °C/11mbar (Kugelrohr).
1
(
14) Gang, C.; Kayser, M. M.; Mihovilovic, M. D.; Mrstik, M. E.;
Martinez, C. A.; Stewart, J. D. New J. Chem. 1999, 8, 827.
15) For another recombinant overexpression system for CHMO
applied in whole-cell biocatalysis see: Doig, S. D.;
O’Sullivan, L. M.; Patel, S.; Ward, J. M.; Woodley, J. M.
Enzyme Microb. Technol. 2001, 28, 265.
H NMR (200 MHz, CDCl ): d = 1.00 (t, J = 7 Hz, 6 H),
3
1.43–1.81 (m, 4 H), 2.11–2.45 (m, 4 H), 3.39–3.55 (m, 2 H).
1
3
(
C NMR (50 MHz, CDCl ): d = 9.6 (q), 29.3 (t), 47.5 (t),
3
78.2 (d), 207.7 (s).
cis-Tetrahydro-2,6-dipropyl-4H-pyran-4-one (1c): 56%
yield, beige oil.
1
(
16) Donoghue, N. A.; Norris, D. B.; Trudgill, P. W. Eur. J.
Biochem. 1976, 63, 175.
H NMR (200 MHz, CDCl ): d = 0.90 (t, J = 6 Hz, 6 H),
3
1.30–1.80 (m, 8 H), 2.15–2.40 (m, 4 H), 3.60–3.75 (m, 2 H).
1
3
(
(
17) Stewart, J. D. Curr. Org. Chem. 1998, 2, 195.
18) Mihovilovic, M. D.; Müller, B.; Kayser, M. M.; Stewart, J.
D.; Stanetty, P. Synlett 2002, 703.
C NMR (50M Hz, CDCl ): d = 14.3 (q), 19.0 (t), 38.9 (t),
3
48.4 (t), 77.1 (d), 207.9 (s).
cis-Tetrahydro-2,6-bis-(1-methylethyl)-4H-pyran-4-one
(1d): 71% yield, colorless oil, bp: 90–95 °C/0.1 mbar
(
19) Mihovilovic, M. D.; Chen, G.; Wang, S.; Kyte, B.; Rochon,
F.; Kayser, M. M.; Stewart, J. D. J. Org. Chem. 2001, 66,
(Kugelrohr).
1
733.
H NMR (200 MHz, CDCl ): d = 0.90, 0.95 (2 × d, J = 6 Hz,
3
Synlett 2003, No. 13, 1973–1976 © Thieme Stuttgart · New York

1
976
M. D. Mihovilovic et al.
LETTER
2
3
× 6 H), 1.75 (oct, J = 6 Hz, 2 H), 2.10–2.45 (m, 4 H), 3.20–
(40) Physical and spectroscopic data of lactones 2:
.30 (m, 2 H).
cis-2,7-Dimethyl-1,4-dioxepan-5-one (2a): colorless oil.
13
1
C NMR (50 MHz, CDCl ): d = 18.2 (t), 33.4 (d), 45.3 (t),
H NMR (400 MHz, CDCl ): d = 1.18 (d, J = 6 Hz, 3 H), 1.29
3
3
81.7 (d), 208.9 (s).
(d, J = 6 Hz, 3 H), 2.67 (dd, J = 14 Hz, ca 1 Hz, 1 H), 2.92
(dd, J = 14 Hz, 5 Hz, 1 H), 3.79–3.99 (m, 2 H), 4.02 (dd, J =
cis-Tetrahydro-2,6-dibutyl-4H-pyran-4-one (1e): 35%
yield, beige oil.
H NMR (200 MHz, CDCl ): d = 0.90–1.00 (m, 6 H), 1.20–
13 Hz, ca 1 Hz, 1 H), 4.20 (dd, J = 14 Hz, 6 Hz, 1 H).
1
13
C NMR (100 MHz, CDCl ): d = 18.6 (q), 23.5 (q), 45.6 (t),
3
3
1
.80 (m, 12 H), 2.10–2.40 (m, 4 H), 3.40–3.55 (m, 2 H).
C NMR (50 MHz, CDCl ): d = 14.4 (q), 22.9 (t), 27.9 (t),
71.0 (d), 74.4 (t). 75.5 (d), 173.8 (s).
13
cis-2,7-Diethyl-1,4-dioxepan-5-one (2b): colorless oil.
3
1
36.5 (t), 48.8 (t), 77.4 (d), 208.4 (s).
H NMR (400 MHz, CDCl ): d = 0.99 (t, J = 9 Hz, 3 H), 1.00
3
(
37) CH Cl was required to achieve efficient extraction of
(t, J = 9 Hz, 3 H), 1.40–1.75 (m, 4 H), 2.69 (dd, J = 16 Hz,
ca 1 Hz, 1 H), 2.90 (dd, J = 16 Hz, 10 Hz, 1 H), 3.50–3.67
(m, 2 H), 4.08 (dd, J = 13 Hz, ca 1 Hz, 1 H), 4.21 (dd, J = 13
Hz, 6 Hz, 1 H).
2
2
lactones 2a–c from the fermentation broth.
38) Bar, R. Trends Biotechnol. 1989, 7, 2.
39) Taschner, M. J.; Black, D. J. J. Am. Chem. Soc. 1988, 110,
(
(
1
3
6892.
C NMR (100 MHz, CDCl ): d = 9.8 (q), 9.9 (q), 25.5 (t),
3
3
0.0 (t), 43.8 (t), 73.4 (t), 75.5 (d), 80.3 (d), 173.6 (s).
cis-2,7-Dipropyl-1,4-dioxepan-5-one (2c): beige colored
oil.
1
H NMR (400 MHz, CDCl ): d = 0.90 (t, J = 7 Hz, 6 H),
3
1
.20–1.60 (m, 8 H), 2.65 (d, J = 13 Hz, 1 H), 2.90 (dd, J = 13
Hz, 8 Hz, 1 H), 3.55–3.70 (m, 4 H), 4.05 (d, J = 13 Hz, 1 H)
4
.25 (dd, J = 13 Hz, 8 Hz, 1 H).
Figure 1
1
3
C NMR (100 MHz, CDCl ): d = 13.6 (q), 18.6 (t), 34.2 (t),
3
38.9 (t), 44.2 (t), 73.7 (t), 73.9 (d), 78.7 (d), 173.5 (s).
Synlett 2003, No. 13, 1973–1976 © Thieme Stuttgart · New York