Kinetic resolution of aliphatic acyclic β-hydroxyketones by recombinant whole-cell Baeyer-Villiger monooxygenases-Formation of enantiocomplementary regioisomeric esters

Article abstract of DOI:10.1016/j.bmcl.2009.05.014

A set of various linear aliphatic β-hydroxyketones was investigated as substrates in the enzymatic kinetic and regioselective Baeyer-Villiger oxidation catalyzed by 12 Baeyer-Villiger monooxygenases from different bacterial origin. Excellent enantioselect

Full text of DOI:10.1016/j.bmcl.2009.05.014

Bioorganic & Medicinal Chemistry Letters 19 (2009) 3739–3743
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Kinetic resolution of aliphatic acyclic b-hydroxyketones by recombinant
whole-cell Baeyer–Villiger monooxygenases—Formation of
enantiocomplementary regioisomeric esters
b,
Jessica Rehdorf a, Alenka Lengar b, Uwe T. Bornscheuer a, , Marko D. Mihovilovic
*
*
a University of Greifswald, Institute of Biochemistry, Department of Biotechnology and Enzyme Catalysis, Felix-Hausdorff-Str. 4, 17487 Greifswald, Germany
b Vienna University of Technology, Institute of Applied Synthetic Chemistry, Getreidemarkt 9/163-OC, 1060 Vienna, Austria
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 16 February 2009
Revised 4 May 2009
Accepted 6 May 2009
Available online 9 May 2009
A set of various linear aliphatic b-hydroxyketones was investigated as substrates in the enzymatic kinetic
and regioselective Baeyer–Villiger oxidation catalyzed by 12 Baeyer–Villiger monooxygenases from dif-
ferent bacterial origin. Excellent enantioselectivities (E >100) could be observed with 4-hydroxy-2-
ketones. After acyl migration, the ester undergoes hydrolysis followed by the formation of optically active
1,2-diols. Furthermore, resolution of 5-hydroxy-3-ketones gave access to the ‘abnormal’ esters, which
broadens applicability of these enzymes in organic chemistry. Additionally, it was noticed, that several
substrates were converted by different enzymes in an enantiocomplementary way and with high optical
purities.
Keywords:
Biocatalysis
Biotransformation
Biooxygenation
Asymmetric synthesis
Chiral compounds
Ó 2009 Elsevier Ltd. All rights reserved.
As a powerful methodology in synthesis to break carbon–
carbon bonds in an oxygen insertion process,1 the Baeyer–Villiger
oxidation is an appealing reaction in organic synthesis.2 The trans-
formation of ketones into the corresponding esters or lactones,
respectively, gives access to valuable intermediates in organic
chemistry on both bulk and fine chemical scale. The enzyme-med-
iated Baeyer–Villiger oxidation has received increasing attention in
recent years.1,3 Key advantages are chemo-, regio- and stereoselec-
tivity combined with utilization of molecular oxygen as primary
oxidant compared to metal-based mediated catalytic strategies.4,5
Such biocatalytic strategies offer sustainable access to chiral com-
pounds of pharmaceutical, nutritional and industrial interest.6
During the last years, the number of recombinantly available Bae-
yer–Villiger monooxygenases (BVMOs) has increased, leading to an
enzyme platform representing novel substrate specificities and
complementary properties. The exploitation of whole-cell bio-
transformations using recombinant expression hosts for such
NAD(P)H-dependent flavoenzymes7 is currently of particular pop-
ularity in both laboratory and industrial scale applications, as the
operational aspects are simple and facile, hence, enabling straight
forward utilization in synthetic chemistry.8
ketones serving as model substrates.9 We discovered that a BVMO
10
from Pseudomonas fluorescens DSM 50106 (BVMOPfl
)
oxidizes 4-
hydroxy-2-octanone-, decanone- and dodecanone with moderate
enantioselectivities, giving access to both optically active acetates
and hydroxyketones (Scheme 1). Reports on biooxygenations of
linear ketones by BVMOs are remarkably limited; only few precen-
dences for the conversion of aryl-aliphatic compounds bearing the
carbonyl group in a side chain have been published for arylketone
accepting enzymes.11,12
In this study we enlarge the number of aliphatic acyclic
b-hydroxyketones including 4-hydroxy-2-nonanone and -undeca-
none as well as the structurally more demanding compounds 5-hy-
droxy-3-nonanone and -decanone (synthesized after Kourouli
O
OPG
O
O
OPG
BVMO
O
OPG
R
+
+
R
R
R
O
O
n
n
n
n
O2
NADPH
H2O
OPG
NADP+
1
(R,S)-
(S)-2
(R)-1
(R)-4
3
6
(R,S)-4
(S)-5
1
: R = CH3
4: R = C2H5
Recently, we have reported on the first BVMO-catalyzed kinetic
resolution of aliphatic acyclic ketones with racemic 4-hydroxy-2-
1a e
- : PG = H
a
: n = 3
b
4a 4b
, : PG = H
: n = 4
c: n = 5
d: n = 6
e: n = 7
1f: n = 5; PG = COCH3
1g: n = 3; PG = COH
* Corresponding authors.
E-mail addresses: uwe.bornscheuer@uni-greifswald.de (U.T. Bornscheuer),
mmihovil@pop.tuwien.ac.at (M.D. Mihovilovic).
Scheme 1. Kinetic resolution of b-substituted linear ketones using recombinant
BVMOs.
0960-894X/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2009.05.014

3740
J. Rehdorf et al. / Bioorg. Med. Chem. Lett. 19 (2009) 3739–3743
et al.13). Furthermore, we extended the toolbox of linear-ketone
accepting BVMOs by eleven additional enzymes of various bacte-
rial origin: cyclohexanone monooxygenase (CHMOAcineto) from
Acinetobacter calcoaceticus NCIMB 987114 and seven other cyclo-
hexanone monooxygenases (CHMOArthro from Arthrobacter sp.,15
CHMOBrachy from Brachymonas sp.,16 CHMOBrevi1 and CHMOBrevi2
from Brevibacterium sp.,17 CHMORhodo1 and CHMORhodo2 from Rho-
dococcus sp.15 and CHMOXantho from Xanthobacter sp. ZL518), as well
as cyclopentanone monooxygenase (CPMO) from Comamonas sp.
verted with a slightly higher velocity than the ‘less-fitting’ enantio-
mer. Surprisingly, CHMOAcineto and BVMOPfl showed only low
activity against 1e. The longer chain is possibly too bulky for BVMO
active sites. Substrate 1e was therefore excluded from further
experiments.
We also found that in the case of a protection of the free hydro-
xyl group prior to the biotransformation (incorporation of acetyl 1f
and formyl protecting groups 1g), substrate acceptance of CHMO-
type enzymes decreased significantly. However, CPMO-type
BVMOs (CPMO and CHMOBrevi2) now display good kinetic resolu-
tions providing access to enantiocomplementary esters compared
to the above biooxygenations of the hydroxyl-substrates.
Similar results could be obtained using 5-hydroxy-3-ketones as
substrates. Ketones 4a and 4b were almost fully oxidized (c >70%),
but again CHMOBrevi1 showed no and CHMOBrevi2 only very low
conversion. Conversion of 4a by CPMO stopped at 53% suggesting
a higher enantioselectivity compared to 1a–d. Interestingly, the
NCIMB 9872,19 4-hydroxyacetophenone monooxygenase from
20
Pseudomonas putida JD1 (HAPMOPpJD1
)
and alkylketone-convert-
ing BVMO from P. putida KT244021 (BVMOPpKT2440).
BVMO-mediated oxidations were initially accomplished on
screening scale using 24-well plastic dishes in parallel format.22
On the basis of screening results, individual biotransformations
were conducted as time-course experiments with selected biocata-
lysts in order to investigate the relationship between time, conver-
sion and enantiomeric excess in detail.23 Enantioselectivity values
(E; determined by computer fitting)24 and regioisomeric excess (%
re)25 were calculated to evaluate the efficiency of kinetic resolution
and the regioselectivity of the oxygen insertion process.
With the exception of CHMOBrevi1 and HAPMOPpJD1 ketones 1a–
e were almost fully converted (c >70%) in most cases to the product
esters 2a–e within 24 h at 24 °C (data not shown) and short- and
middle-chain ketones 1a–c seem to be preferred. This is consistent
with the discovered clustering of BVMOs into two distinct groups,
the cyclohexanone- and cyclopentanone-type monooxygenases.26
CHMOAcineto and BVMOPfl showed best results with conversions be-
tween 40% and 60% and eeP >80% for 1a–d, while for the other
BVMOs it seemed that the ‘better-fitting’ enantiomer is only con-
formation of two regioisomers could be observed with BVMOPfl
.
Either the more substituted or the less substituted carbon center
undergoes migration, leading consequently to ‘normal’ esters 5a
and 5b and ‘abnormal’ esters 6a and 6b ester (Scheme 1). Such reg-
iodivergent biooxygenations have been observed in previous stud-
ies on cyclic systems.27–30 A mechanistic relationship has been
proposed for this behavior based on stereoelectronic effects.31
In order to optimize enantiomeric excesses and enantioselectiv-
ities of products 2a–d, 5a and 5b within the kinetic resolution pro-
cess, time-course experiments were performed to stop conversion
close to 50% (for optimized resolution results). For these experi-
ments selected biocatalysts were used that showed satisfactory re-
sults in pre-screening experiments as described above: CHMOAcineto
Table 1
Microbial Baeyer–Villiger oxidations of b-hydroxy-2-ketones 1a–d and b-protected ketones 1f and 1g using recombinant whole-cells of E. coli expressing BVMOs from different
bacterial origin
ca (%)
eeS (%)
eeP (%)
abs. conf.c
Ed
b
b
Substrate
BVMO
t (h)
O
OH
CHMOAcineto
CPMO
BVMOPfl
8
3
24
49
53
46
91
64
85
96
56
>99
(S)
(S)
(S)
156
7
>200
1a
1b
n
n
n = 3
O
CHMOAcineto
CHMOXantho
CPMO
20
10
2
49
47
44
48
96
84
48
89
>99
95
62
(S)
(S)
(S)
(S)
>200
103
7
OH
BVMOPfl
8
98
>200
n = 4
O
CHMOAcineto
CPMO
BVMOPfl
20
2
6
50
44
41
92
39
68
93
50
96
(S)
(S)
(S)
90
4
100
OH
OH
1c
1d
n
n
n = 5
O
CHMOAcineto
CHMOArthro
CHMORhodo2
CPMO
30
20
10
2
20
55
18
65
13
62
17
37
52
50
80
20
(S)
(S)
(S)
(S)
4
6
10
2
n = 6
O
O
CHMOAcineto
CPMO
CHMOBrevi2
24
24
20
<5
25
23
n.d.
45
41
n.d.
>99
91
n.a.
(R)
(R)
n.d.
>200
31
O
1f
n
n = 5
O
O
CHMOAcineto
CHMOXantho
CPMO
24
24
24
24
42
57
50
28
25
73
99
27
33
66
99
>99
(S)
(S)
(R)
(R)
3
10
>200
>200
O
1g
CHMOBrevi2
n
n = 3
Reactions were monitored over 48 h. Syntheses of 2a and 2c as standards for GC analysis were performed enzymatically by esterification of 1,2-diols with vinyl acetate by
immobilized Candida antarctica lipase B as described by Kirschner and Bornscheuer.9 Syntheses of 2b and 2d was accomplished as described in Ref. 2. Spectral data are shown
in Ref. 33. Syntheses of 1f and 1g were performed from b-hydroxyketones according standard procedures.
n.d. = not determined; n.a. = not applicable.
a
Conversion calculated from % eeS (enantiomeric excess of substrate) and % eeP (enantiomeric excess of product).
b
% eeS and % eeP were determined by chiral phase GC and calculated according to Chen et al.34
c
Absolute configurations of hydroxyalkyl acetates 2a–d were determined by comparison with (R)-2-hydroxydecyl acetate ((R)-2e), which was synthesized from (R)-1,2-
decandiol by using lipase-catalyzed transesterification. Since 2a–d are structurally related (homologue series) the same configuration is assumed for 2a–c.9
d
Enantioselectivity values were determined by computer fitting of GC data from % eeS and % eeP.

J. Rehdorf et al. / Bioorg. Med. Chem. Lett. 19 (2009) 3739–3743
3741
hydrolysis
HO
A 100%
O
O
OH
n
acyl migration
hydrolysis
HO
BVMO
n
80%
60%
40%
20%
0%
O
O
OH
n
n
OH
(S)-2a-c
O
(R,S)-1a-c
(S)-8a-c
(S)-7a-c
Scheme 2. Kinetic resolution of 1a–c followed by direct ester hydrolysis or via acyl
migration and ester bond cleavage releasing optically active 1,2-diols 8a–c.
0
2
4
6
8
24
48
time [h]
and CPMO as two ‘benchmark’ catalysts, reflecting the key features
of the two family clusters among BVMOs, CHMOXantho, CHMORhodo2
,
1a
2a
8a
CHMOBrachy, CHMOArthro, CHMOBrevi1 and BVMOPfl. Results obtained
from biotransformations with 1a–d are compiled in Table 1.
All investigated enzymes oxidized the (S)-enantiomer of 1a–d,
whereas biotransformations of 1a–c with CHMOAcineto and BVMOPfl
lead to very high enantioselectivities. Even after 48 h the (R)-
hydroxyketone is not converted at all and reaction rate is stopped
at 50%. In comparison, CPMO oxidized ketones very fast (less than
3 h for 50% conversion), but showed nearly no selectivity. The
highest E-value of 7 could be achieved for 2a and 2b. Investigating
the effect of chain-length indicated that enantioselectivity de-
creases (compare CHMOAcineto in Table 1) from 1a with eight car-
bon atoms to 1d with eleven carbon atoms. Tentatively, the
chain-length and therefore the increase in hydrophobicity are cri-
teria for selectivity.
Interestingly, kinetic resolution of 4-hydroxy-2-ketones can be
used to generate two different chemical species, which addition-
ally differ in their configuration. Besides the residual optically ac-
tive hydroxyketones (R)-1a–d, optically active hydroxyalkyl
esters (S)-2a–d are formed, which in case of 2a, 2b and 2c undergo
acyl migration to form optically pure acetates of 1,2-diols 7a–c
(Scheme 2).
The equilibrium between 2a–c and 7a–c is shifted towards the
production of the acylated 1,2-diol after 24 h reaction time fol-
lowed by hydrolysis of the ester bond yielding in enantiomerically
pure 1,2-diols 8a–c. Since acyl migration proceeds without any
conformational changes, all 1,2-diols possess the same configura-
tion as the hydroxyalkyl acetates. The maximum amount of gener-
ated 1,2-diol was 55% after 48 h using 1c as starting material and
BVMOPfl as biocatalyst (compare Fig. 1).
Indeed, this feature makes this biotransformation especially
useful and complementary to bioreductions by highly regio- and
stereospecific ketoreductases.35,36 The alternative kinetic resolu-
tion of 1,2-diols using lipases or esterases proceeds so far with
low selectivity.37–39
100%
B
80%
60%
40%
20%
0%
0
2
4
6
8
24
time [h]
1b
2b
8b
100%
80%
60%
40%
20%
0%
C
0
2
4
6
8
24
48
time [h]
1c
2c
8c
Figure 1. Enzymatic formation of 1,2-diols 8a–c via kinetic resolution of 4-
hydroxy-2-ketones 1a–c at 24 °C after 24 and 48 h with (A) 1a and CHMO from A.
calcoaceticus NCIMB 9871, (B) 1b an CHMO from A. calcoaceticus NCIMB 9871 and
(C) 1c and BVMO from P. fluorescens DSM 50106, both enzymes expressed in E. coli.
Oxidation of 4a and 4b showed different results (summarized in
Table 2). In this case, enantioselectivity among utilized BVMOs is
opposite compared to 1a–d. While for CHMOAcineto and BVMOPfl
Table 2
Microbial Baeyer–Villiger oxidations of b-hydroxy-3-ketones 4a and 4b using recombinant whole-cells of E. coli expressing BVMOs from different bacterial origin
ca (%)
eeS (%)
eeP (%)
Ec
red (%)
b
b
Substrate
BVMO
t (h)
O
OH
OH
CHMOAcineto
CHMOXantho
CHMOBrachy
CPMO
10
10
8
24
10
46
53
58
50
32
75
81
>99
>99
25
88
72
70
96
53
35
15
40
>200
4
—
—
—
—
64
4a
4b
n
n
n = 3
BVMOPfl
O
CHMOAcineto
CHMOXantho
CHMOBrachy
CPMO
8
8
6
10
24
40
44
42
48
55
46
61
57
81
>99
69
78
75
87
82
8
—
—
—
—
40
14
12
35
74
n = 4
BVMOPfl
Reactions were monitored over 48 h. For syntheses of product standards see Ref. 32. Spectral data are shown in Ref. 33.
a
Conversion calculated from % eeS (enantiomeric excess of substrate) and % eeP (enantiomeric excess of product).
b
% eeS and % eeP were determined by chiral phase GC and calculated according to Chen et al.34
c
Enantioselectivity values were determined by computer fitting of GC data from % eeS and % eeP.
Percent regioisomeric excess in favor of ‘abnormal’ ester determined by chiral phase GC.
d

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J. Rehdorf et al. / Bioorg. Med. Chem. Lett. 19 (2009) 3739–3743
100%
80%
60%
40%
20%
0%
100%
80%
60%
40%
20%
0%
0
2
4
6
8
10
24
0
2
4
6
8
10
24
time [h]
time [h]
4a
normal ester
abnormal ester
4b
normal ester
abnormal ester
Figure 2. Regioselective Baeyer–Villiger oxidation of 5-hydroxy-3-ketones 4a and 4b using engineered E. coli cells expressing BVMO from P. fluorescens DSM 50106.
4. Gusso, A.; Baccin, C.; Pinna, F.; Strukul, G. Organometallics 1994, 13, 3442.
5. Strukul, G.; Varagnolo, A.; Pinna, F. J. Mol. Catal. A: Chem. 1997, 117, 413.
6. Kawamoto, M.; Utsukihara, T.; Abe, C.; Sato, M.; Saito, M.; Koshimura, M.; Kato,
E-values are between 4 and 40, CPMO now showed a very high and
even reverse selectivity for 4a (E >200). Thus, implementing
CHMOBrachy and CPMO for kinetic resolution of 4a gives access to
both enantiomers of 5a in high optically purity. This fact makes
this reaction a powerful and interesting tool in organic chemistry.
Furthermore, it could be observed that the location of the keto
group in the molecule influences regioselectivity of the enzymes.
While the methyl ester (‘abnormal’ product) was formed only up
to 10% (data not shown) for 4-hydroxy-2-ketones this fact changed
using 5-hydroxy-3-ketones. BVMOPfl even generated 64% or 40% in
favor of the ‘abnormal’ ester in case of 4a and 4b, respectively
(Fig. 2). This is the only option to synthesize ‘abnormal’ esters
enzymatically to our knowledge until now. This observation
underscores the powerful capabilities of BVMOs and not only
broadens their synthetic applicability, but also provides a synthet-
ically useful alternative to already established chemical reactions
in organic chemistry.
N.; Horiuchi, A. C. Biotechnol. Lett. 2008, 30, 1655.
7. van Berkel, W. J. H.; Kamerbeek, N. W.; Fraaije, M. W. J. Biotechnol. 2006, 124,
670.
8. Mihovilovic, M. D.; Rudroff, F.; Müller, B.; Stanetty, P. Bioorg. Med. Chem. Lett.
2003, 13, 1479.
9. Kirschner, A.; Bornscheuer, U. T. Angew. Chem., Int. Ed. 2006, 45, 7004.
10. Kirschner, A.; Altenbuchner, J.; Bornscheuer, U. T. Appl. Microbiol. Biotechnol.
2007, 73, 1065.
11. Geitner, K.; Kirschner, A.; Rehdorf, J.; Schmidt, M.; Mihovilovic, M. D.;
Bornscheuer, U. T. Tetrahedron: Asymmetry 2007, 18, 892.
12. Rodríguez, C.; de Gonzalo, G.; Fraaije, M. W.; Gotor, V. Tetrahedron: Asymmetry
2007, 18, 1338.
13. Kourouli, T.; Kefalas, P.; Ragoussis, N.; Ragoussis, V. J. Org. Chem. 2002, 67,
4615.
14. Chen, Y.-C. J.; Peoples, O. P.; Walsh, C. T. J. Bacteriol. 1988, 170, 781.
15. Brzostowicz, P. C.; Walters, D. M.; Thomas, S. M.; Nagarajan, V.; Rouvière, P. E.
Appl. Environ. Microbiol. 2003, 69, 334.
16. Bramucci, M. G.; Brzostowicz, P. C.; Kostichka, K.; Nagarajan, V.; Rouvière, P. E.,
Thomas, S. M. 2003 Genes encoding Baeyer–Villiger monooxygenases. P. I.
Appl. WO/2003/020890; 2003, 138, 233997.
Based on our observations, aliphatic open-chain b-hydroxyke-
tones are good substrates for most of the BVMOs recombinantly
available so far, especially for those previously described as cyclok-
etone converting enzymes. The possibility to synthesize enantio-
pure 1,2-diols enzymatically is noteworthy, since these
compounds are of special interest for example, in organic industry
for the synthesis of polyesters but also in medical treatment as
antimicrobial agents.40 Furthermore, 1,2-diols are predominantly
synthesized chemically, so far; thus, kinetic resolution of 4-hydro-
xy-2-ketones using BVMOs can accomplish a considerable contri-
bution for environmental protection.
The fact that some BVMOs are capable to generate the ‘abnor-
mal’ Baeyer–Villiger product with high enantioselectivity offers
new possibilities for the synthesis of natural products. Together
with the enantiocomplementary conversion of ketones by different
enzymes it displays the potential of the natural diversity to provide
suites of catalysts for chemical operations. Currently, further
studies on the regioselectivity of BVMOs are in progress in our
laboratories.
17. Brzostowicz, P. C.; Gibson, K. a. L.; Thomas, S. M.; Blasko, M. S.; Rouvière, P. E. J.
Bacteriol. 2000, 18215, 4241.
18. van Beilen, J. B.; Mourlane, F.; Seeger, M. A.; Kovac, J.; Li, Z.; Smits, T. H. M.;
Fritsche, U.; Witholt, B. Environ. Microbiol. 2003, 5, 174.
19. Iwaki, H.; Hasegawa, Y.; Wang, S.; Kayser, M. M.; Lau, P. C. K. Appl. Environ.
Microbiol. 2002, 68, 5671.
20. Rehdorf, J.; Zimmer, C. L.; Bornscheuer, U. T. Appl. Environ. Microbiol. 2009, 75,
3106.
21. Rehdorf, J.; Kirschner, A.; Bornscheuer, U. T. Biotechnol. Lett. 2007, 29,
1393.
22. Typical procedure for screening experiments—Precultures were inoculated with a
single colony from a plate and incubated at 37 °C overnight (in case of CHMO
from Xanthobacter sp. at 30 °C) in an orbital shaker in a baffled Erlenmeyer
flask. LBamp (LBamp/chl for HAPMO from P. putida JD1) was inoculated with 1% of
the overnight preculture and incubated at 37 or 30 °C, respectively, until
OD600nm reached 0.5–0.7. Then, protein expression was induced with either
IPTG (0.1 mM final concentration) or in case of BVMO from P. putida KT2440
with L-rhamnose (0.2% final concentration). Protein expression was performed
at 24 °C for 2 h. Screening was performed in 24-well plastic plates (Greiner
BioOne company). 1 mL bacterial culture was transfered into each well (max.
volume of 2 mL) and substrate was added (2.5 mM final concentration).
Biotransformations were carried out at 24 °C and analyzed after 24 h.
Therefore, samples were extracted with ethyl acetate supplemented with
1.2 mM of an internal standard (benzoic acid methylester) and dried over
sodium sulfate.
23. Typical procedure for time-course experiments—Biotransformations were
performed as described for screening experiments, but here samples were
taken at certain time intervals (2, 4, 6, 8, 10, 24, 30 and 48 h), extracted and
analyzed via GC.
24. Faber, K.; Hönig, H., and Kleewein, A. Free shareware programs (‘Selectivity-
1.0’) for calculation of the enantiomeric ratio. Available on the authors website:
<http://borgc182.kfunigraz.ac.at./>.
25. Percent regioisomeric excess (% re) is defined as the percentage of the major
regioisomer (both enantiomers) minus the percentage of the minor
regioisomer (both enantiomers).
Acknowledgements
We thank the German Federal Foundation of Environment
(Deutsche Bundesstiftung Umwelt, DBU) for a stipend to Jessica
Rehdorf. Part of this research was funded by the Austrian Science
Fund FWF (P19845). Furthermore, we thank Dr. Pierre Rouviere
(DuPont USA) for providing six recombinantly expressed BVMOs.
26. Mihovilovic, M. D.; Rudroff, F.; Grötzl, B.; Kapitan, P.; Snajdrova, R.; Rydz, J.
Angew. Chem., Int. Ed. 2005, 44, 3609.
27. Snajdrova, R.; Grogan, G.; Mihovilovic, M. D. Bioorg. Med. Chem. Lett. 2006, 16,
4813.
References and notes
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3. Kamerbeek, N. M.; Janssen, D. B.; van Berkel, W. J. H.; Fraaije, M. W. Adv. Synth.
Catal. 2003, 345, 667.
28. Mihovilovic, M. D.; Kapitan, P.; Kapitanova, P. ChemSusChem 2008, 1, 143.
29. Cernuchová, P.; Mihovilovic, M. D. Org. Biomol. Chem. 2007, 5, 1715.
30. Kyte, B. G.; Rouvière, P. E.; Cheng, Q.; Stewart, J. D. J. Org. Chem. 2004,
69, 12.

J. Rehdorf et al. / Bioorg. Med. Chem. Lett. 19 (2009) 3739–3743
3743
31. Kelly, D. R.; Knowles, C. J.; Mahdi, J. G.; Taylor, I. N.; Wright, M. A. Chem. Soc.,
Chem. Commun. 1995, 12, 729.
CDCl3) 9.4 (q), 14.1 (q), 23.1 (t), 27.2 (t), 27.6 (t), 32.2 (t), 70.9 (d), 71.2 (t), 171.4
(s). Physical and spectral data of 6a: colorless liquid (23 mg, 46%); specific
32. Preparative scale biotransformations and enzymatic synthesis of 2b, 2d, 5a, 5b, 6a
and 6b—Baffled Erlenmeyer flask with LBamp (500 mL) was inoculated with an
overnight bacterial culture, IPTG was added at OD 0.6 (0.1 mM final
concentration) together with substrate (50 mg). Flasks were shaken at 24 °C
for 24 h, then biomass was removed by centrifugation, the aqueous phase was
saturated with sodium chloride and extracted five times with ethyl acetate.
Organic layers were combined, dried over sodium sulfate and concentrated.
Esters were purified by silica gel chromatography.
rotation of sample obtained in enzyme mediated kinetic resolution: ½a D20
ꢀ
=
-18.2 (c 0.43, CHCl3; ee = 93%); dH (200 MHz, CDCl3) 0.91 (t, J = 11 Hz, 3H), 1.24
(t, J = 7.2 Hz, 3H), 1.31–1.46 (m, 6H), 2.32–2.56 (m, 2H), 3.89–4.0 (m, 1H), 4.12–
4.23 (q, J = 7.4 Hz, 2H); dC (50 MHz, CDCl3) 12.9 (q), 13.2 (q), 21.6 (t), 26.6 (t),
35.2 (t), 40.3 (t), 59.7 (d), 67.0 (t), 172.1 (s). Physical and spectral data of 5b:
colorless liquid (17 mg, 34%); specific rotation of sample obtained in enzyme
mediated kinetic resolution: ½a D20
ꢀ
= +2.2 (c 0.74, CHCl3; ee = 93%); dH (200 MHz,
CDCl3) J = 0.91 (t, 6 Hz, 3H), 1.15 (t, J = 7.6 Hz, 3H), 1.24–1.45 (m, 8H), 2.35 (q,
J = 5.4 Hz, 2H), 3.8–3.92 (m, 1H), 3.91–4.19 (m, 2H); dC (50 MHz, CDCl3) 9.4 (q),
14.1 (q), 22.7 (t), 24.8 (t), 27.6 (t), 32.1 (t), 32.5 (t), 70.8 (d), 71.2 (t), 174,1 (s).
Physical and spectral data of 6b: colorless liquid (25 mg, 50%); specific rotation
33. Physical and spectral data of 4a: Obtained from 3-oxoheptanoate and n-
valeraldehyde as a colorless liquid (685 mg, 84%); dH (300 MHz, CDCl3) 0.90 (t,
J = 6.9 Hz, 3H), 1.07 (t, J = 6.6 Hz, 3H), 1.23–1.55 (m, 6H), 2.42–2.58 (m, 2H),
2.59 (q, J = 3 Hz, 2H), 4.0–4.07 (m, 1H); dC (50 Hz, CDCl3) 7.5 (q), 14.0 (q), 22.6
(t), 27.6 (t), 36.1 (t), 36.8 (t), 48.6 (t), 67.7 (d), 212.8 (s). Physical and spectral
data of 4b: obtained from 3-oxoheptanoate and capronaldehyde as a colorless
liquid (220 mg, 60%); specific rotation of sample obtained in enzyme mediated
of sample obtained in enzyme mediated kinetic resolution: ½a D20
= ꢁ20.5 (c
ꢀ
0.69, CHCl3; ee >99%); dH (200 MHz, CDCl3) 0.88 (t, J = 6.4 Hz, 3H), 1.27 (t,
J = 7.2 Hz, 3H), 1.31–1.43 (m, 8H), 2.4–2.48 (m, 2H), 3.95–4.03 (m, 1H), 4.06–
4.19 (m, 2H). dC (50 MHz, CDCl3) 14.0 (q), 14.2 (q), 21.0 (t), 22.6 (t), 25.1 (t),
31.7 (t), 36.5 (t), 41.3 (t), 60.7 (d), 173.1 (s).
kinetic resolution: ½a D20
= ꢁ34.8 (c 0.34, CHCl3; ee = 93%); dH (300 MHz, CDCl3)
ꢀ
0.89 (t, J = 7 Hz, 3H), 1.06 (t, J = 7.4 Hz, 3H), 1.25–1.52 (m, 8H), 2.48–2.67 (m,
2H), 2.55 (q, J = 4 Hz, 2H), 4.0–4.07 (m, 1H); dC (50 MHz, CDCl3) 7.9 (q), 14.1 (q),
22.7 (t), 24.8 (t), 31.4 (t), 32.1 (t), 36.2 (t), 48.6 (t), 67.6 (d), 211.6 (s). Physical
and spectral data of 2b: colorless liquid (15 mg, 30%); dH (200 MHz, CDCl3) 0.85
(t, J = 8 Hz, 3H), 1.29-1.45 (m, 8H), 2.09 (s, 3H), 3.83-3.95 (m, 1H), 3.98-4.1 (m,
2H); dC (50 MHz, CDCl3) 14.1 (q), 21.0 (q), 22.7 (t), 25.1 (t), 31.9 (t), 33.4 (t),
68.9 (d), 70.0 (t), 171.4 (s). Physical and spectral data of 2d: colorless liquid
(20 mg, 40%); dH (200 MHz, CDCl3) 0.86 (t, J = 9 Hz, 3H), 1.26–1.49 (m, 12H), 2.1
(s, 3H), 3.81–3.90 (m, 1H), 4.0–4.18 (m, 2H); dC (50 MHz, CDCl3) 14.3 (q), 21.1
(q), 22.8 (t), 25.5 (t), 29.3 (t), 29.7 (t), 31.9 (t), 33.5 (t), 68.9 (d), 70.1 (t), 171.4
(s). Physical and spectral data of 5a: colorless liquid (24 mg, 48%); dH
(200 MHz, CDCl3) 0.9 (t, J = 10 Hz, 3H), 1.14 (t, J = 8 Hz, 3H), 1.25–1.44 (m,
6H), 2.29 (q, 18 Hz, 2H), 3.74–3.87 (m, 1H), 4.0–4.36 (m, 2H); dC (50 MHz,
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