Enantioselective kinetic resolution of 3-phenyl-2-ketones using Baeyer-Villiger monooxygenases

Article abstract of DOI:10.1016/j.tetasy.2007.03.025

The enantioselective kinetic resolution of two 3-phenyl-2-ketones using four different Baeyer-Villiger monooxygenases (BVMO) expressed recombinantly in Escherichia coli was studied. The highest enantioselectivity (E = 82) was achieved for 3-phenyl-2-butanone using a BVMO originating from Pseudomonas fluorescens. A BVMO from Pseudomonas putida showed an opposite (R)-enantiopreference and E = 12.

Full text of DOI:10.1016/j.tetasy.2007.03.025

Tetrahedron: Asymmetry 18 (2007) 892–895
Enantioselective kinetic resolution of 3-phenyl-2-ketones using
Baeyer–Villiger monooxygenases
a
a
a
a
Kristian Geitner, Anett Kirschner, Jessica Rehdorf, Marlen Schmidt,
b
a,
*
Marko D. Mihovilovic and Uwe T. Bornscheuer
a
Department of Biotechnology and Enzyme Catalysis, Institute of Biochemistry, Greifswald University, Felix-Hausdorff-Str. 4,
D-17487 Greifswald, Germany
Institute of Applied Synthetic Chemistry, Vienna University of Technology, Getreidemarkt 9/163-OC, A-1060 Vienna, Austria
b
Received 13 March 2007; accepted 26 March 2007
Available online 25 April 2007
Abstract—The enantioselective kinetic resolution of two 3-phenyl-2-ketones using four different Baeyer–Villiger monooxygenases
(
2
BVMO) expressed recombinantly in Escherichia coli was studied. The highest enantioselectivity (E = 82) was achieved for 3-phenyl-
-butanone using a BVMO originating from Pseudomonas fluorescens. A BVMO from Pseudomonas putida showed an opposite (R)-enan-
tiopreference and E = 12.
Ó 2007 Elsevier Ltd. All rights reserved.
1. Introduction
2. Results and discussion
Baeyer–Villiger monooxygenases (BVMOs) belong to the
class of oxidoreductases and convert aliphatic, aryl-
aliphatic and cyclic ketones into esters and lactones, respec-
Kinetic resolutions of the two 3-phenyl-2-ketones 1a and 1b
(Scheme 1), which were synthesized according to the litera-
1
9
ture, were carried out using resting cells of recombinant
E. coli expressing four different BVMOs: cyclohexanone
monooxygenase from Acinetobacter calcoaceticus NCIMB
1
–5
tively, using molecular oxygen.
Thus, they mimic the
6
chemical Baeyer–Villiger oxidation, which is usually per-
acid-catalyzed and proceeds by a two-step process as ini-
2
0
9871 (CHMO ), cyclopentanone monooxygenase from
7
21
tially proposed by Criegee. Since it is becoming more
Comamonas sp. NCIMB 9872 (CPMO ), a BVMO
and more important to perform Baeyer–Villiger oxidations
from P. fluorescens DSM 50106 (PF-BVMO) and a BVMO
from Pseudomonas putida KT2440 (PP-BVMO).
8
in an enantioselective manner, BVMOs represent a valu-
9
able alternative to metal-based chiral catalysts. Until
now, stereoselective Baeyer–Villiger oxidations using
enzymes were described for prochiral or racemic mono-
BVMO
O2
H O
2
O
R
1
0–15
R
+
R
NADPH NADP
and bicyclic ketones
as well as racemic arylaliphatic
O
3
,16
ketones.
Recently, we have shown that aliphatic acyclic
+
ketones such as 4-hydroxy-2-ketones could be enantioselec-
tively (E ꢀ 50) converted by a BVMO from Pseudomonas
fluorescens DSM 50106, which was recombinantly
O
O
(
R,S)-1
(R
)-1
(S
)-2
1
7,18
expressed in Escherichia coli.
a: R = CH3
b: R = C H
2
5
Herein we report, the kinetic resolution of two 3-phenyl-2-
ketones using four different BVMOs (Scheme 1).
Scheme 1. Kinetic resolution of two 3-phenyl-2-ketones by Baeyer–
Villiger monooxygenase-catalyzed oxidation.
*
Corresponding author. Tel.: +49 3834 86 4367; fax: +49 3834 86
0066; e-mail: uwe.bornscheuer@uni-greifswald.de
After expression of the recombinant enzymes, cells were
harvested by centrifugation, washed once with phosphate
8
0957-4166/$ - see front matter Ó 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetasy.2007.03.025

K. Geitner et al. / Tetrahedron: Asymmetry 18 (2007) 892–895
893
buffer and used directly afterwards for biocatalysis reac-
tions on an analytical scale. Cofactor recycling was ensured
by employing the whole cell E. coli and glucose was added
to each reaction vial. Therefore, the addition of cofactor
NADPH was not necessary. For efficient uptake of the
substrates 1a and 1b by the cells, b-cyclodextrin had to
to ethyl changed the enantiopreference of CPMO from (S)-
to (R)-selectivity, while CHMO remained (S)-selective.
Using resting cells of E. coli JM109 pGro7 pJOE4072.6,
biocatalysis with 1a was also performed in preparative
scale (1 mmol substrate), this time using shake flasks and
only 0.5 equiv of b-cyclodextrin. Previous experiments with
different cyclodextrin concentrations revealed that conver-
sions only marginally decreased when reducing the amount
of cyclodextrin to 0.5 equiv (data not shown). After 6 h at
30 °C, GC analysis showed 46% conversion. The enantio-
meric excess of substrate and product was 80% ee and
94% ee, respectively, corresponding to an E = 82. For bet-
ter separation of the ketone and ester during work-up, 2a
was enzymatically hydrolyzed to the corresponding alcohol
using Candida antarctica lipase A (CAL-A). CAL-A was
identified to be non-selective and therefore could be used
without affecting the enantiomeric excess of the ester. Thus,
1-phenylethanol 3a could be isolated in 35% yield and 93%
ee. GC analysis of the substrate fraction revealed that a
small amount of ester 2a was still present. Interestingly,
using an esterase from P. fluorescens instead of CAL-A,
the ee-value of 2a could be increased up to 99% since this
enzyme preferentially hydrolyzes the (R)-ester (data not
shown).
2
2,23
be included.
Reaction progress was monitored by GC
analyses using a chiral column.
3
-Phenyl-2-butanone 1a was converted by all four enzymes
to the corresponding 1-phenylethyl acetate 2a, while PF-
BVMO and PP-BVMO did not oxidize 3-phenyl-2-penta-
none 1b to 1-phenylpropyl acetate 2b (Tables 1 and 2).
The enzymes exhibited different activities and enantioselec-
tivities towards 1a, for example, CPMO showed the highest
activity but only low enantioselectivity (E < 3). On the
other hand, PF-BVMO exhibited significantly less activity
but at the same time was the most selective, yielding the
highest E-value (analytical scale: E = 43, preparative scale:
E = 82). This value substantially exceeds the selectivity
1
6
(
E ꢀ 7) estimated for a BVMO from Thermobifida fusca.
Interestingly, PP-BVMO preferentially converted the (R)-
enantiomer of 1a (E = 12), while the three other BVMOs
were (S)-selective.
Table 1. Whole cell biocatalysis reactions (analytical scale) with different
BVMOs expressed in recombinant E. coli for ketone 1a at 30 °C (CHMO:
cyclohexanone monooxygenase from Acinetobacter calcoaceticus NCIMB
3. Conclusions
9871; CPMO: cyclopentanone monooxygenase from Comamonas sp.
NCIMB 9872; PF-BVMO: BVMO from P. fluorescens DSM 50106; PP-
BVMO: BVMO from P. putida KT2440)
Herein, we have shown that arylaliphatic ketones were
accepted as substrates by different Baeyer–Villiger mono-
oxygenases. Only the BVMO from P. fluorescens DSM
50106 gave satisfactory enantioselectivity in the kinetic res-
olution of 3-phenyl-2-butanone yielding the corresponding
a
a
Enzyme
Time (h) Conversion (%)
Enantiomeric
excess
E
%
ee
S
% ee
P
(
S)-acetate with 93% ee. On a preparative-scale reaction the
CHMO
CPMO
PF-BVMO
PP-BVMO
8
2
8
2
41
93
46
50
24
2.6
8.3 (S) <3
6.0 (S) <3
corresponding chiral alcohol 1-phenylethanol was obtained
in 35% yield and 93% ee. Interestingly, one enzyme showed
opposite enantiopreference towards 1a and the increase of
the alkyl chain from methyl to ethyl gave no conversion
using the BVMOs originating from Pseudomonas sp. but
inverted the enantiopreference of CPMO.
90
75
72
90 (S)
71 (R)
43
12
a
Calculated according to Chen et al.
Table 2. Whole cell biocatalysis reactions (analytical scale) with different
BVMOs expressed in recombinant E. coli for ketone 1b at 30 °C. For
abbreviations, see Table 1
a
b
4. Experimental
Enzyme
Time (h) Conversion (%)
Enantiomeric
excess
E
4.1. General
%
ee
S
% ee
P
c
CHMO
CPMO
4
2
36
44
n.d.
n.d.
73 (S) 4.5
48 (R) 4.0
All chemicals were purchased from Sigma–Aldrich (Tauf-
kirchen, Germany), Fisher Scientific (Schwerte, Germany),
VWR (Darmstadt, Germany), ABCR (Karlsruhe, Ger-
many) and Roth GmbH (Karlsruhe, Germany) unless
c
PF-BVMO
PP-BVMO
No conversion
No conversion
a
1
13
Calculated by the following equation: (peak areas of product enantio-
mers)/(peak areas of substrate enantiomers + peak areas of product
enantiomers).
Calculated according to Chen et al.
n.d.—not determined due to unsatisfactory separation of enantiomers by
GC analysis.
otherwise specified. H and
C NMR-spectra were
recorded on a 300 MHz or 600 MHz (Bruker) instrument.
b
c
24
4.2. Synthesis of the starting material
3
-Phenyl-2-butanone 1a and 3-phenyl-2-pentanone 1b were
1
9
synthesized according to the literature by conversion of
the corresponding carboxylic acids with methyllithium.
Synthesis of 1-phenylpropyl acetate 2b was carried out by
acetylation of 1-phenylpropanol with acetylchloride.
Structural identity was confirmed by NMR spectroscopy.
A similar pattern was found for 1b, for which CHMO and
CPMO again exhibited only low enantioselectivity, but the
E-values were slightly higher compared to 1a (E ꢀ 4; Table
2
5
2
). Increasing the length of the alkyl side chain from methyl

894
K. Geitner et al. / Tetrahedron: Asymmetry 18 (2007) 892–895
4
.3. BVMO-production
were added. The reaction mixture was incubated at 30 °C
and 220 rpm. After four hours another 2 mL sterile glucose
solution was added. The reaction was stopped after 6 h and
extracted five times with 50 mL ethyl acetate. The com-
bined organic phase was dried over anhydrous sodium sul-
fate and solvent was removed in vacuo.
Cyclohexanone monooxygenase (CHMO) from Acineto-
bacter calcoaceticus NCIMB 9871: E. coli BL21 (DE3)
pKJE7 pMM4 was grown in 200 mL LBcm+amp at
2
6
3
0 °C to an optical density of 0.5. Then, 0.5 mg/mL L-arab-
27
inose for induction of chaperone coexpression
and
0
.1 mM IPTG for induction of CHMO expression were
For better separation of ketone 1a and ester 2a, the prod-
uct mixture was hydrolyzed by Candida antarctica lipase A
(CAL-A, Chirazyme L-5) in 60 mL of a 5:1 mixture of
sodium phosphate buffer and hexane yielding unreacted
added. After further growth for 4 h at 30 °C, cells were har-
vested by centrifugation (10 min, 4400g, 4 °C) and washed
once with sterile phosphate buffer (50 mM, pH 7.5).
1a and 1-phenylethanol 3a. After 24 h, the reaction mixture
Cyclopentanone monooxygenase (CPMO) from Coma-
monas sp. NCIMB 9872: expression of CPMO in E. coli
DH5a pCMO206 was performed in 200 mL LBamp. Cells
were grown at 37 °C to an optical density of 0.5 where
CPMO expression was induced by the addition of
was extracted four times with 30 mL of ethyl acetate. After
drying over anhydrous sodium sulfate and evaporation of
the organic solvent, the product was purified by column
chromatography (hexane/ethyl acetate 5:1). Compound
3a could be isolated in 35% yield (43 mg, 0.35 mmol).
2
8
0
.1 mM IPTG. After further growth for 6 h at 25 °C, cells
were harvested by centrifugation (10 min, 4400g, 4 °C) and
washed once with sterile phosphate buffer (50 mM,
pH 7.5).
4.6. Chiral GC analysis
GC analyses on a chiral stationary phase were carried out
on a Shimadzu GC-14A gas chromatograph with a chiral
b-cyclodextrin column (Hydrodex -b-3P, Macherey-
Nagel, D u¨ ren, Germany). Injection and detection tempera-
ture were set to 220 °C. Absolute configurations were
determined by comparison of the retention times with
those of optically active standards of (R)-1a and (S)-2b
(see Table 3).
BVMO from P. fluorescens DSM 50106 (PF-BVMO):
Ò
1
8
E. coli JM109 pGro7 pJOE4072.6 was grown in 200 mL
LBcm+amp containing 0.5 mg/mL L-arabinose at 30 °C. At
OD = 0.6, enzyme expression was induced by addition of
L-rhamnose (0.2% (w/v) final concentration) and cells were
further incubated at 30 °C for 4 h. Afterwards, cells were
harvested by centrifugation (10 min, 4400g, 4 °C) and
washed once with sterile phosphate buffer (50 mM,
pH 7.5).
Table 3. GC analysis using a chiral column
Compound
T
Column (°C)
a
Retention times (min)
BVMO from P. putida KT2440 (PP-BVMO): expression of
PP-BVMO in E. coli JM109 pGro7 pBVMO2440 was per-
formed as described for PF-BVMO.
c
1a
2a
100
100
100
90
90
7.1/7.6
6.7/9.7
a
a
d
c
3
1
2
a
b
b
10.8/12.1
b
c
35.6/36.1
After expression of the recombinant BVMOs, the resting
cells were used for biocatalysis reactions.
b
d
34.6/48.7
a
b
c
Column pressure: 125 kPa.
Column pressure: 65 kPa.
Elution order of enantiomers is (R) before (S).
Elution order of enantiomers is (S) before (R).
4
.4. Biocatalysis reactions on an analytical scale
d
Recombinant E. coli cells carrying the BVMOs were resus-
pended in a sodium phosphate buffer (50 mM, pH 7.5) to a
final OD of around 50. Aliquots (1 mL) of these cell
suspensions were mixed in 2 mL Eppendorf (Hamburg,
Germany) reaction vials with 15 lmol substrate, 15 lmol
b-cyclodextrin and 10 lL of a sterile 1 M glucose solution.
Acknowledgements
Anett Kirschner thanks the Fonds der Chemischen Indust-
rie (Frankfurt, Germany) and the Studienstiftung des
Deutschen Volkes (Bonn, Germany) for stipends. We also
thank Anette Riebel and Torge Vorhaben for their support
during their practical courses.
The vials were closed with air permeable caps (LidBac
,
Eppendorf) and incubated in a thermoshaker (Eppendorf)
at 1400 rpm. After certain time intervals, samples (300 lL)
were taken, extracted twice with ethyl acetate and dried
over anhydrous sodium sulfate. Excess solvent was
removed under nitrogen and samples were analyzed via
GC.
References
4
.5. Biocatalysis reaction on a preparative scale
1
2
3
. Walsh, C. T.; Chen, Y. C. J. Angew. Chem., Int. Ed. Engl.
1
988, 27, 333–343.
Recombinant E. coli cells containing the expressed PF-
BVMO were resuspended in sodium phosphate buffer
. Mihovilovic, M. D.; M u¨ ller, B.; Stanetty, P. Eur. J. Org.
Chem. 2002, 3711–3730.
. Kamerbeek, N. M.; Olsthoorn, J. J.; Fraaije, M. W.; Janssen,
D. B. Appl. Environ. Microbiol. 2003, 69, 419–426.
(
50 mM, pH 7.5) to a final OD of around 20. To 100 mL
of this suspension 1 mmol of substrate 1a, 0.5 mmol b-
cyclodextrin and 2 mL of a sterile 1 M glucose solution
4. Mihovilovic, M. D. Curr. Org. Chem. 2006, 10, 1265–1287.

K. Geitner et al. / Tetrahedron: Asymmetry 18 (2007) 892–895
895
5
. Kamerbeek, N. M.; Janssen, D. B.; van Berkel, W. J. H.;
Fraaije, M. W. Adv. Synth. Catal. 2003, 345, 667–678.
. Baeyer, A.; Villiger, V. Chem. Ber. 1899, 32, 3625–3633.
. Criegee, R. Justus Liebigs Ann. Chem. 1948, 560, 127–141.
. Mihovilovic, M. D.; Rudroff, F.; Gr o¨ tzl, B. Curr. Org. Chem.
17. Kirschner, A.; Bornscheuer, U. T. Angew. Chem., Int. Ed.
2006, 45, 7004–7006.
18. Kirschner, A.; Altenbuchner, J.; Bornscheuer, U. T. Appl.
Microbiol. Biotechnol. 2007, 73, 1065–1072.
19. Rubottom, G. M.; Kim, C.-W. J. Org. Chem. 1983, 48, 1550–
1552.
6
7
8
2
004, 8, 1057–1069.
9
. Strukul, G. Angew. Chem., Int. Ed. 1998, 37, 1199–1209.
20. Donoghue, N. A.; Norris, D. B.; Trudgill, P. W. Eur. J.
Biochem. 1976, 63, 175–192.
1
0. Mihovilovic, M. D.; Rudroff, F.; Gr o¨ tzl, B.; Kapitan, P.;
Snajdrova, R.; Rydz, J.; Mach, R. Angew. Chem., Int. Ed.
21. Griffin, M.; Trudgill, P. W. Eur. J. Biochem. 1976, 63, 199–
209.
2
005, 44, 3609–3613.
1
1
1
1
1
1
1. Taschner, M. J.; Black, D. J.; Chen, Q. Z. Tetrahedron:
Asymmetry 1993, 4, 1387–1390.
2. Taschner, M. J.; Peddada, L. J. Chem. Soc., Chem. Commun.
22. Hedges, A. R. Chem. Rev. 1998, 98, 2035–2044.
23. Saenger, W. Angew. Chem., Int. Ed. 1980, 19, 344–362.
24. Chen, C.-S.; Fujimoto, Y.; Girdaukas, G.; Sih, C. J. J. Am.
Chem. Soc. 1982, 104, 7294–7299.
25. Krebsf a¨ nger, N.; Zocher, F.; Altenbuchner, J.; Bornscheuer,
U. T. Enzyme Microb. Technol. 1998, 22, 641–646.
26. Chen, G.; Kayser, M. M.; Mihovilovic, M. D.; Mrstik, M. E.;
Martinez, C. A.; Stewart, J. D. New J. Chem. 1999, 23, 827–
832.
27. Lee, D. H.; Kim, M. D.; Lee, W. H.; Kweon, D. H.; Seo,
J. H. Appl. Microbiol. Biotechnol. 2004, 63, 549–552.
28. Iwaki, H.; Hasegawa, Y.; Wang, S.; Kayser, M. M.; Lau,
P. C. K. Appl. Environ. Microbiol. 2002, 68, 5671–5684.
1
992, 1384–1385.
3. Taschner, M. J.; Black, D. J. J. Am. Chem. Soc. 1988, 110,
892–6893.
6
4. Carnell, A. J.; Roberts, S. M.; Sik, V.; Willetts, A. J. J. Chem.
Soc., Perkin Trans. 1 1991, 2385–2389.
5. Alphand, V.; Furstoss, R. J. Org. Chem. 1992, 57, 1306–
1
309.
6. Fraaije, M. W.; Wu, J.; Heuts, D. P. H. M.; van Hellemond,
E. W.; Spelberg, J. H. L.; Janssen, D. B. Appl. Microbiol.
Biotechnol. 2005, 66, 393–400.