Resolution of fused bicyclic ketones by a recombinant biocatalyst expressing the Baeyer-Villiger monooxygenase gene Rv3049c from Mycobacterium tuberculosis H37Rv

Article abstract of DOI:10.1016/j.bmcl.2006.06.072

Recombinant Escherichia coli B834 (DE3) pDB5 expressing the Rv3049c gene encoding a Baeyer-Villiger monooxygenase from Mycobacterium tuberculosis H37Rv was used for regioselective oxidations of fused bicyclic ketones. This whole-cell system represents the first recombinant Baeyer-Villiger oxidation biocatalyst that effectively resolves the racemic starting materials in this series. Within biotransformations using this organism one substrate enantiomer remains in high optical purity, while the second enantiomer is oxidized to one type of regioisomeric lactone preferably.

Full text of DOI:10.1016/j.bmcl.2006.06.072

Bioorganic & Medicinal Chemistry Letters 16 (2006) 4813–4817
Resolution of fused bicyclic ketones by a recombinant biocatalyst
expressing the Baeyer–Villiger monooxygenase gene Rv3049c
from Mycobacterium tuberculosis H37Rv
a
b
a,
*
Radka Snajdrova, Gideon Grogan and Marko D. Mihovilovic
a
Vienna University of Technology, Institute of Applied Synthetic Chemistry, Getreidemarkt 9/163, A-1060, Vienna, Austria
b
York Structural Biology Laboratory, Department of Chemistry, University of York, Heslington, York, YO10 5YW, UK
Received 6 June 2006; revised 21 June 2006; accepted 21 June 2006
Available online 12 July 2006
Abstract—Recombinant Escherichia coli B834 (DE3) pDB5 expressing the Rv3049c gene encoding a Baeyer–Villiger monooxygen-
ase from Mycobacterium tuberculosis H37Rv was used for regioselective oxidations of fused bicyclic ketones. This whole-cell system
represents the first recombinant Baeyer–Villiger oxidation biocatalyst that effectively resolves the racemic starting materials in this
series. Within biotransformations using this organism one substrate enantiomer remains in high optical purity, while the second
enantiomer is oxidized to one type of regioisomeric lactone preferably.
Ó 2006 Elsevier Ltd. All rights reserved.
Biocatalysis offers efficient and sustainable access to chi-
ral building blocks on bulk, fine, and specialty chemical
scale. It is a highly interdisciplinary and tremendously
active field at the cross roads of organic synthesis, fer-
mentation technology, microbiology, and molecular
biology. Due to the exponentially growing number of
potential biocatalysts identified by genome mining, bio-
catalysis is offering an increasing number of enzyme
platforms with novel reactivities and complementary
received increasing interest in recent years due to the
3
availability of several new enzymes.
–7
The regiodivergent biooxidation of fused bicyclic ke-
tones bearing a cyclobutanone structural motif by
BVMOs has been identified as one of the more remark-
able transformations by the enzyme class. Racemic
starting material is oxidized to two regioisomers with
either the more substituted (and, hence, more nucleo-
philic) or the less substituted carbon center undergoing
migration, consequently leading to the ‘normal’ or the
1
properties.
8
–11
In particular, biological oxygenation reactions offer
attractive options to synthetic chemists, as the corre-
‘abnormal’ lactone (Scheme 1).
A mechanistic ratio-
nale has been proposed for this behavior based on
2
12
sponding enzymes utilize molecular oxygen as oxidant.
stereoelectronic effects.
Recombinant overexpression systems represent easy-to-
use catalytic entities, which can be applied by synthetic
chemists without complicated cofactor recycling tech-
niques and elaborate enzyme purification. In addition,
biocatalysts originating from pathogenic organisms
can be studied in a benign host system for their potential
in asymmetric synthesis. Among this enzyme group,
Baeyer–Villiger monooxygenases (BVMOs) have
Consistent with our recently discovered clustering of
1
3
BVMOs into two distinct groups, we observed differ-
ent behavior by cyclohexanone (CHMO)- and cyclopen-
tanone (CPMO)-type monooxygenases, with the prior
Keywords: Baeyer–Villiger oxidation; Biocatalysis; Biotransformation;
Lactone; Monooxygenase.
*
Corresponding author. Fax: +43
mmihovil@pop.tuwien.ac.at
1
58801 15499; e-mail:
Scheme 1. Regioselective Baeyer–Villiger oxidations of fused bicyclic
ketones.
0
960-894X/$ - see front matter Ó 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2006.06.072

4814
R. Snajdrova et al. / Bioorg. Med. Chem. Lett. 16 (2006) 4813–4817
yielding equal amounts of both oxidation products in
usually good optical purity and the latter only providing
essentially racemic normal lactones.
sors for further synthetic sequences to bioactive
1
compounds.
8
1
4
Results obtained from microbial Baeyer–Villiger oxida-
tions of racemic ketones 1–7 after 48 h of biotransfor-
mation time using recombinant whole-cells expressing
BVMOMtb5 are compiled in Table 1. Experiments were
performed in parallel format using multi-well plastic
Very recently, a ligation independent cloning (LIC)
strategy was reported to give rapid access to a family
of recombinant biocatalysts by exploiting the genome
1
5
of a sequenced species. In this study, the methodology
was applied to express BVMOs originating from Myco-
bacterium tuberculosis H37Rv—a class 3 pathogen—in
benign and easily cultured Escherichia coli. One of the
recombinant E. coli B834 (DE3) strains, expressing gene
Rv3049c from M. tuberculosis (BVMOMtb5), was found
to perform a preparative kinetic resolution of racemic
bicyclo[3.2.0]hept-2-en-6-one 1.
1
9,20
dishes.
Enantioselectivity values (E; determined by
2
1
22
computer fitting) and regioisomeric excess re were
calculated to evaluate the efficiency of kinetic resolution
and the regioselectivity of the oxygen insertion process.
Ketones 1–3 were almost fully converted to the product
lactones within 48 h. In all cases, a single substrate enan-
tiomer (‘better-fitting’ enantiomer within the active site
of enzyme) was oxidized at slightly higher velocity than
the antipodal (‘less fitting’) enantiomer. The rather low
E values indicate limited quality of the kinetic resolu-
tion, although reasonable yields of enantiopure starting
material had been recovered when the oxidation of 1
Whilst the selective formation of ‘abnormal’ lactone 1b
from ketone 1 was reported for a wild-type strain of
1
6
Cunninghamella, a recent re-investigation of this bio-
transformation indicated that it was not, in our hands,
a useful kinetic resolution process, as no enantiomerical-
1
7
15
ly enriched substrate could be recovered.
was stopped at lower conversions. In the new exam-
ples, high enantiomeric excess values of ketones (eeS)
were observed only at a high degree of conversion. Bio-
oxidations led to the formation of ‘abnormal’ lactone,
preferably, with a regioisomeric excess of around 70%.
In order to explore this interesting behavior in detail,
we focused on biotransformations by E. coli express-
ing Rv3049c using structurally similar ketones as sub-
strates. Successful resolution of such racemic ketones
would enable access to valuable optically pure precur-
The structurally more demanding ketone 4 was convert-
ed with significantly higher E value, hence providing an
Table 1. Microbial Baeyer–Villiger oxidations of fused bicyclic ketones 1–7 using recombinant whole-cells of Escherichia coli expressing
monooxygenase from Mycobacterium tuberculosis
a
c
Substrate
ee
substrate
S
(%) of
E
2
2
3
Conversion
b
(%)
re (%)
ee
N
(%) of
normal lactone
A
ee (%) of
d,e
d,e
abnormal lactone
O
>99
95
91
93
68
78
54
50
72
56 (1S,5R)
86 (1R,5S)
1
2
O
O
9
8
68
95 (1S,5S)
87 (1S,6S)
23 (n.d.)
93 (1R,5R)
19 (1S,5S)
n.a.
76 (1R,5S)
79 (1S,6R)
54 (n.d.)
9
2
76
3
O
>99
12
96
4
O
O
O
>99
7
38
86 (1S,5R)
95 (1R,5S)
>99 (1S,6R)
5
8
2
14
29
O
O
6
O
>99
>200
100
7
n.d., not determined; n.a., not applicable.
a
Enantioselectivity values (E) were determined by computer fitting of GC data to an equation derived from the theoretical expression relating the
enantiomeric excess of residual substrates (ee ) with the fractional conversion.
S
b
c
Conversion after 48 h of biotransformation determined by chiral phase chromatography.
Percent regioisomeric excess in favor of ‘abnormal’ lactone determined by chiral phase GC.
d
e
ee
N A
and ee determined by chiral phase GC; racemic reference was prepared by mCPBA oxidation of ketones 2–7.
10,11
GC data were compared to data of CHMO mediated biotransformation and assigned according to the literature.

R. Snajdrova et al. / Bioorg. Med. Chem. Lett. 16 (2006) 4813–4817
4815
improved kinetic resolution. Excellent ee was obtained
S
already after 68% conversion.
within the standard biotransformation times of the
screening protocol and only marginal reaction was ob-
served during the second 24 h. While tetrahydrofuran
based systems 5 and 6 gave comparable results to
carbobicyclic substrate 4, tetrahydropyran ketone 7
was only converted to the corresponding ‘abnormal’ lac-
tone; biotransformation stopped at 50% and both prod-
uct and remaining substrate were isolated in excellent
optical purity. Such a transformation is very interesting
from the point of view of possible synthetic applications,
as enantioselectivity values above 200 ensure rapid and
Hetero-bicyclic fused ketones 5–7 containing oxygen
were even better precursors for the kinetic resolution.
Such substrates did not reach complete conversion
2
3
effective processes.
In order to underscore the uniqueness of BVMOMtb5, we
compared the progress of biotransformation of ketone 7
with recombinant E. coli strains expressing CHMO
2
Acineto
4
from
Acinetobacter
and
CPMOComa
from
2
5
Comamonas. Those strains can be regarded as ‘bench-
mark’ catalysts reflecting the key features of the two fam-
ily clusters among BVMOs.
Scheme 2. Ketone (+)-7 and lactone (ꢀ)-7b as key precursors for
transformations toward natural products.
a
100
100%
8
6
4
2
0
0
0
0
7
5%
50%
25%
0
0%
0
10
20
conversion [%]
e.e.[S] e.e. [A]
30
40
50
0
1
3
6
12
24
time [h]
b
100
100%
8
6
4
2
0
0
0
0
0
75%
50%
25%
0%
0
1
3
6
9
12
24
0
20
40
60
80
100
conversion [%]
time [h]
e.e. [S]
e.e. [A]
e.e. [N]
c
100
1
00%
5%
50%
5%
8
6
4
2
0
0
0
0
7
2
0
0
%
0
20
40
60
80
100
0
1
3
6
9
conversion [%]
time [h]
abnormal
e.e. [A]
e.e. [N]
e.e. [S]
substrate
normal
Figure 1. Regioselective Baeyer–Villiger oxidations of 7 using engineered Escherichia coli expressing: (a) BVMO from Mycobacterium tuberculosis
BVMOMtb5); (b) CPMOComa from Comamonas; (c) CHMOAcineto from Acinetobacter. S, substrate; N, normal lactone; A, abnormal lactone.
(

4816
R. Snajdrova et al. / Bioorg. Med. Chem. Lett. 16 (2006) 4813–4817
Scheme 2 outlines the different behavior of these three
whole-cell biocatalysts for the biooxidation of ketone 7
over the timeframe until the end of conversion. Engi-
neered cells expressing CHMOAcineto did not resolve
family clustering hypothesis to this enzyme based on
protein sequence information and active site models.
Future synthetic applications of the recombinant
expression system are currently addressed in our
laboratory.
starting material sufficiently; highest value of ee was
S
approx. 10% at 70% conversion. Both normal and
abnormal lactone were formed from the beginning of
the biooxidation. The prior lactone was generated
predominantly, however, in low optical purity, while
abnormal lactone was obtained in excellent enantiomer-
ic excess (Fig. 1c).
Acknowledgment
Funding for this research by the Austrian Science Fund
(FWF, Project No. I19-B10) is gratefully acknowledged.
CPMOComa converted (+)-7 to one enantiomer of nor-
mal lactone, preferably. The second enantiomer (ꢀ)-7
was oxidized to the antipodal normal lactone also, how-
ever with lower velocity. Consequently, biooxidation
with this strain allows isolation of (ꢀ)-substrate in good
ee at a conversion above approx. 70% (Fig. 1b). Forma-
tion of abnormal lactone was detected only in minor
amounts (approx. 6% of total lactone quantity) in the
last stage of transformation, but with excellent ee
References and notes
1
2
. Anthonsen, T. In Applied Biocatalysis; Straathof, A. J. J.,
Adlercreutz, P., Eds.; Harwood academic publishers:
Amsterdam, 2000.
. Van Beilen, J. B.; Duetz, W. A.; Schmid, A.; Witholt, B.
Trends Biotechnol. 2003, 21, 170.
3. Mihovilovic, M. D.; Rudroff, F.; Gr o¨ tzl, B. Curr. Org.
Chem. 2004, 8, 1057.
(
ee = >99%).
A
4
5
6
7
. Kamerbeek, N. M.; Janssen, D. B.; van Berkel, W. J. H.;
Fraaije, M. W. Adv. Synth. Catal. 2003, 345, 667.
. Mihovilovic, M. D.; M u¨ ller, B.; Stanetty, P. Eur. J. Org.
Chem. 2002, 3711.
BVMOMtb5 displays excellent kinetic resolution with for-
mation of abnormal lactone in high optical purity from
the very beginning of the biotransformation. The reaction
. Roberts, S. M.; Wan, P. W. H. J. Mol. Catal. B: Enzym.
stops at 50% conversion, and at this stage ee of remaining
S
1
. Luna, A.; Gutierrez, M. C.; Furstoss, R.; Alphand, V.
998, 4, 111.
(+)-7 is also >99% (Fig. 1a). It is noteworthy that this
whole-cell system provides access to the antipodal ketone
compared to CPMOComa mediated transformations.
Tetrahedron: Asymmetry 2005, 16, 2521.
8. Carnell, A. J.; Roberts, S. M.; Sik, V.; Willetts, A. J.
J. Chem. Soc., Chem. Commun. 1990, 1438.
A preparative scale biotransformation using 7 as
starting material and BVMOMtb5 as biocatalyst was
performed to confirm assignment of absolute stereo-
9. Carnell, A. J.; Roberts, S. M.; Sik, V.; Willetts, A. J.
J. Chem. Soc., Perkin Trans. 1 1991, 2385.
0. Alphand, V.; Furstoss, R. J. Org. Chem. 1992, 57, 1306.
1
2
6
11. Petit, F.; Furstoss, R. Tetrahedron: Asymmetry 1993, 4,
341.
12. Kelly, D. R.; Knowles, C. J.; Mahdi, J. G.; Taylor, I. N.;
chemistry from GC based screening experiments. Data
for optical rotation were compared to the literature val-
1
1
1
ues.
(
After purification of products, we obtained
1R,6S)-(+)-2-oxabicyclo[4.2.0]octan-7-one (+)-7 and
Wright, M. A. J. Chem. Soc., Chem. Commun. 1995, 729.
3. Mihovilovic, M. D.; Rudroff, F.; Gr o¨ tzl, B.; Kapitan, P.;
1
(
>
1S,6R)-(ꢀ)-3-oxabicyclo[3.3.0]octan-2-one (ꢀ)-7b in
Snajdrova, R.; Rydz, J.; Mach, R. Angew. Chem. 2005,
1
2
7
99% ee and in good yields.
17, 3675.
1
4. Mihovilovic, M. D.; Kapitan, P. Tetrahedron Lett. 2004,
45, 2751.
15. Bonsor, D.; Butz, S. F.; Solomons, J.; Grant, S.; Fairlamb,
Based on our observations, biooxidations using
BVMOMtb5 display general, novel, and very valuable
properties for the kinetic resolution of racemic ketone
substrates for the first time in enzyme mediated
Baeyer–Villiger oxidations. We consider this new
whole-cell system as superior biocatalyst compared to
previous catalytic entities for such transformations
based on the simplicity to grow and handle recombinant
E. coli together with the high control of biocatalyst pro-
duction by simultaneously minimizing possible side
reactions in living cells. The above results open up
new possible pathways toward synthesis of natural
products. Ketone (+)-7 represents a key intermediate
for preparation of potentially important prostanoid syn-
I. J. S.; Fogg, M. J.; Grogan, G. Org. Biomol. Chem. 2006,
4
, 1252.
6. Lebreton, J.; Alphand, V.; Furstoss, R. Tetrahedron Lett.
996, 37, 1011.
1
1
1
1
7. Fairlamb, I. J. S.; Grant, S.; Grogan, G.; Maddrell, D. A.;
Nicols, J. C. Org. Biomol. Chem. 2004, 2, 1831.
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McCague, R.; Olivo, H. F.; Roberts, S. M.; Willets, A.
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19. Mihovilovic, M. D.; Snajdrova, R.; Winninger, A.; Rudr-
off, F. Synlett 2005, 2751.
2
0. Typical procedure for bacterial cultivation. Fresh LBamp
CHMOAcineto, CPMOComa) or LBkan (BVMOMtb5) was
(
2
8
inoculated with 1% of an overnight preculture of the
appropriate recombinant E. coli strain in a baffled
Erlenmayer flask. The culture was incubated at 120 rpm
at 37 °C on an orbital shaker until the culture reached the
OD of 0.5.
thons, while lactone (ꢀ)-7b is envisioned to become an
interesting precursor for natural products bearing a
tetrahydropyran structural core as the central feature
2
9
(
Scheme 2).
6
00
Typical procedure for screening experiments in multi-well
dishes. Each well was charged with LBamp/kan grown
bacterial culture (2 mL); IPTG was added (final concentra-
tion of 0.025 mM) together with substrate (1 mg) and
BVMOMtb5 displays
behavior to previously reported Baeyer–Villiger mono-
oxygenases. We are presently trying to extend our
a
very different biocatalytic

R. Snajdrova et al. / Bioorg. Med. Chem. Lett. 16 (2006) 4813–4817
4817
b-cyclodextrin (1 equiv). Theplate was shaken atrtand after
certain time periods (1, 3, 6, 9, 12, 24, and 48 h) samples were
analyzed by chiral phase GC measurement (after extraction
with EtOAc supplemented with internal standard).
1. Faber, K.; H o¨ nig, H.; Kleewein, A. Free shareware
programs (’Selectivity-1.0’) for calculation of the Enantio-
meric Ratio, available from the author’s website: <http://
borgc185.kfunigraz.ac.at./>.
2. Percent regioisomeric excess (% re) is defined as the
percentage of the major regioisomer (both enantiomers)
minus the percentage of the minor regioisomer (both
enantiomers).
(100 mg) and b-cyclodextrin (1 equiv). The flask was
shaken at rt until the conversion was complete (24 h). The
biomass was removed by centrifugation; the aqueous
phase was repeatedly extracted with dichloromethane. The
combined organic layers were dried over sodium sulfate
and concentrated. Ketone 7 and lactone 7b were purified
by flash column chromatography.
2
2
27. Physical and spectral data of (+)-7. Yellow oil; yield 64%
(32 mg); d
2.12 (m, 1H), 2.74 (d, J = 17 Hz, 1H), 3.17–3.38 (m, 3H),
3.81–3.87 (m, 1H), 4.45 (t, J = 6 Hz, 1H). d (50 MHz,
CDCl ) 18.7 (t), 22.1 (t), 53.4 (t), 57.1 (d), 64.0 (t), 65.1 (t),
H 3
(200 MHz, CDCl ) 1.46–1.64 (m, 3H), 2.04–
C
3
2
0
2
3. Faber, K. In Biotransformations in Organic Chemistry;
Springer: Berlin, Heidelberg, 2004; p 41.
4. Mihovilovic, M. D.; Chen, G.; Wang, S.; Kyte, B.;
Rochon, F.; Kayser, M. M.; Stewart, J. D. J. Org. Chem.
207.5 (s). ½aꢁ +15.27 (c 0.81, CHCl
3
). Physical and
spectral data of (ꢀ)-7b. Colorless oil; yield 71% (40 mg);
D
2
d
H
(200 MHz, CDCl
1H), 2.26–2.32 (m, 1H), 2.55–2.60 (m, 1H), 3.30–3.43 (m,
1H), 3.87–3.93 (m, 1H), 4.24–4.31 (m, 3H). d (50 MHz,
CDCl ) 20.0 (t), 22.0 (t), 39.9 (d), 66.0 (t), 72.4 (t), 73.5 (d),
3
) 1.48–1.62 (m, 2H), 1.74–1.93 (m,
2
001, 66, 733.
C
2
5. Iwaki, H.; Hasegawa, Y.; Wang, S.; Kayser, M. M.; Lau,
P. C. K. Appl. Environ. Microbiol. 2002, 68, 5671.
6. Typical procedure for biotransformation on preparative
scale. Baffled Erlenmayer flask was charged with LBkan
grown bacterial culture (250 mL). IPTG was added (final
concentration of 0.025 mM) together with substrate
3
2
0
176.8 (s). ½aꢁ ꢀ24.50 (c 0.82, CHCl
3
).
D
2
28. Finch, H.; Mjalli, A. M. M.; Montana, J. G.; Roberts, S.
M.; Taylor, R. J. K. Tetrahedron 1990, 46, 4925.
29. Kelly, M. J.; Newton, R. F.; Roberts, S. M.; Whitehead,
J. F. J. Chem. Soc., Perkin. Trans. 1 1990, 2352.