Microbial Baeyer-Villiger oxidation of ketones by cyclohexanone and cyclopentanone monooxygenase - A computational rational for biocatalyst performance

Article abstract of DOI:10.1007/s00706-006-0468-2

Recombinant Escherichia coli overexpressing Pseudomonas sp. NCIMB 9872 cyclopentanone monooxygenase (CPMO, EC 1.14.13.16) and Acinetobacter sp. NCIMB 9871 cyclohexanone monooxygenase (CHMO, EC 1.14.13.22) have been utilized in whole-cell Baeyer-Villiger biotransformations of prochiral bicycloketones. A significant difference in substrate acceptance and stereoselectivity was observed for bicyclo[3.3.0] and bicyclo[4.3.0] substrates. A plausible mechanism of these transformations was established by means of high level DFT/B3LYP calculations suggesting an essential difference in electronic requirements for a successful enzymatic conversion, which was similarly encountered in recombinant whole-cell mediated biooxidations. Some of the lactones produced in the biocatalytic Baeyer-Villiger oxidation represent key intermediates for the synthesis of indole alkaloids. Springer-Verlag 2006.

Full text of DOI:10.1007/s00706-006-0468-2

Monatshefte f u¨ r Chemie 137, 785–794 (2006)
DOI 10.1007/s00706-006-0468-2
Microbial Baeyer-Villiger Oxidation
of Ketones by Cyclohexanone
and Cyclopentanone Monooxygenase –
A Computational Rational for Biocatalyst
Performance
ꢀ
Arjumand I. Durrani, Peter Stanetty, Georg Dazinger, and Karl Kirchner
Marko D. Mihovilovic , Bernhard M u¨ ller, Markus Spina,
Institute of Applied Synthetic Chemistry, Vienna University of Technology, Vienna, Austria
Received February 13, 2006; accepted March 3, 2006
Published online April 28, 2006 # Springer-Verlag 2006
Summary. Recombinant Escherichia coli overexpressing Pseudomonas sp. NCIMB 9872 cyclopen-
tanone monooxygenase (CPMO, EC 1.14.13.16) and Acinetobacter sp. NCIMB 9871 cyclohexanone
monooxygenase (CHMO, EC 1.14.13.22) have been utilized in whole-cell Baeyer-Villiger biotrans-
formations of prochiral bicycloketones. A significant difference in substrate acceptance and stereo-
selectivity was observed for bicyclo[3.3.0] and bicyclo[4.3.0] substrates. A plausible mechanism of
these transformations was established by means of high level DFT=B3LYP calculations suggesting an
essential difference in electronic requirements for a successful enzymatic conversion, which was simi-
larly encountered in recombinant whole-cell mediated biooxidations. Some of the lactones produced
in the biocatalytic Baeyer-Villiger oxidation represent key intermediates for the synthesis of indole
alkaloids.
Keywords. Biocatalysis; Recombinant whole-cell biotransformation; Baeyer-Villiger oxidation; DFT
calculations; Transition states.
Introduction
Based on the successful application of enzymes in laboratory scale, biocatalytic
methods have been introduced into the chemical and pharmaceutical industry due
to an increasing demand for optically pure building blocks [1]. Out of more than
300 such processes [2], the microbial Baeyer-Villiger oxidation represents a partic-
ularly useful reaction for biotransformations in asymmetric mode [3–5].
ꢀ Corresponding author. E-mail: mmihovil@pop.tuwien.ac.at

7
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M. D. Mihovilovic et al.
Recent advances in molecular biology enabled access to an increasing number
of novel Baeyer-Villiger monooxygenases (BVMOs) originating from various nat-
ural sources [6–8]. Nature’s diversity is additionally complemented by previous
attempts to modify the stereospecificity of such enzymes [9], ultimately generating
a toolbox of such biooxidation catalysts for the transformation of a large variety of
structurally diverse substrates into high-value chiral lactones. In this context, the
characterization of stereopreference and substrate acceptance of these enzymes is
becoming a key aspect in order to understand and control the biocatalytic process
on molecular level and to evaluate and tune their potential as biocatalysts in
organic synthesis [10].
Utilization of engineered strains overexpressing particular BVMOs in place
of wild-type organisms allows production of the required enzyme at high level
by the use of strong and highly controlled promoters and simultaneously minimiz-
ing problems with unwanted side reactions and low monooxygenase activity.
In addition, whole-cell mediated biotransformations offer a solution to obstacles
associated with cofactor regeneration, as BVMOs are NADPH-dependent flavoen-
zymes. Finally, overexpression of enzymes in suitable non-pathogenic strains such
as E.coli provides ‘‘easy-to-use’’ catalytic systems even for proteins originating
from pathogenic organisms and require a minimum of special laboratory equip-
ment and microbiological expertise. Consequently, such systems can be provided
to preparative chemists for subsequent applications in asymmetric organic synthe-
sis [11–13].
Results and Discussion
Recently, we have introduced a platform of BVMOs with enantiocomplementary
stereopreference and overlapping substrate profiles [10, 14, 15]. The most promi-
nent representatives were cycloehexanone monooxygenase (CHMO, EC 1.14.13.22
[
16]) and cyclopentanone monooxygenase (CPMO, EC 1.14.13.16 [17]), which
possess highly relaxed substrate acceptance for a variety of non-natural substrates.
However, we were puzzled by the significant difference in substrate specificity and
stereoselectivity for the biooxidation of fused bicycloketone 1b (Scheme 1) [15],
which we were studying in more detail in the current contribution. Compounds of
this type are intermediates for several indole alkaloids [18].
Substrate 1a as a representative of the bicyclo[3.3.0] scaffold was accessed via
literature methods [19] from diketone 3 (Scheme 2) [20]. Using diol 4 as entry into
Scheme 1

Microbial Baeyer-Villiger Oxidation
787
Scheme 2. (i) Ref. [19]; (ii) Ref. [15]; (iii) Ac O, pyridine, then 0.5 N HCl, 61%; (iv) Pd=C, H
2
2
81psi, THF, 77%
the bicyclo[4.3.0] series, we adapted [21] and optimized protocols [22] for tosyla-
tion, cyanation, and hydrolysis to the diacid 5 [15]. One-pot cyclization in acetic
anhydride=pyridine and concomitant decarboxylation represents a shortcut to
ketone 1b compared to the usual Dieckmann condensation route [23]. Catalytic
hydrogenation of olefin 1b afforded 1c.
Biocatalytic desymmetrizations were carried out using recombinant E.coli
under growing conditions in baffled Erlenmeyer flasks according to our recently
introduced standard protocol [24]. Protein production was induced by addition of
isopropyl-ꢀ-D-galactopyranoside and biotransformations were usually carried out
in the presence of ꢀ-cyclodextrin to improve solubility of ketones and in order to
limit possible toxicity vis- ꢀa -vis whole-cells. Results of the biooxidation are sum-
marized in Table 1.
Table 1. Biotransformation of fused bicyclo substrates with CHMO and CPMO expressing recom-
binant cells
a
Yield =%
b
e.e. =%
20
D
ꢂ1
2
ꢂ1
Ketone
X
Lactone
Strain
½ꢁꢁ =10 deg cm g
1
1
a
a
CH2
2a
CHMO
50
85
89
9
ꢂ4.7
c ¼ 0.34, CDCl
3
CH2
2a
CPMO
þ0.4
c ¼ 1.0, CHCl
3
1
1
b
b
CH¼CH
CH¼CH
2b
2b
CHMO
CPMO
33 (85)
76
ꢃ5
ꢂ0.7
c ¼ 0.36, CH Cl
2
2
>99
þ24.5
c ¼ 1.0, CHCl
ꢂ1.2
3
1
c
c
CH CH
2
2c
2c
CHMO
CPMO
21 (65)
83
ꢃ3
2
c ¼ 0.46, EtOH
þ39.1
1
CH CH
2
99
2
c ¼ 1.0, CHCl
3
a
Isolated yield after chromatographic purification; yield in parenthesis is based on consumed starting
b
material; e.e. determined by chiral phase gas chromatography; racemic reference material prepared
by m-CPBA oxidation of ketones 1a=1c

7
88
M. D. Mihovilovic et al.
We observed an interesting trend for both enzymes: while CHMO converted the
bicyclo[3.3.0] ketone 1a readily and in good stereoselectivity, CPMO gave 2a in
poor optical purity with opposite specific rotation. In the case of bicyclo[4.3.0]
substrates 1b=1c also enantiocomplementary biooxidations were observed, how-
ever, now CHMO displayed poor stereopreference, while CPMO provided lactones
2b=2c in excellent selectivity. The general behavior with respect to stereocomple-
mentary oxidations was in agreement with our recently introduced model of two
clusters for BVMO enzymes [10].
A most intriguing aspect was the very sluggish biotransformation of ketones
1b=1c by CHMO, which may be attributed to spatial limitations within the active
site of the enzyme. On the other hand, this enzyme had been demonstrated to
accommodate also sterically demanding substrates [25], which prompted us to
closer investigate the electronic situation in intermediates of the Baeyer-Villiger
process.
5
For this purpose the transformation of ketones 1a (A in the model) and 1c
A in the model) to the corresponding lactones 2a (C in the model) and 2c (C in
6
5
6
(
the model) was studied by means of high level DFT=B3LYP calculations. The
energy profiles for these conversions utilizing H O as oxidizing agent (represent-
2
2
ing a simple approximation for peroxy-FAD) are shown in Figs. 1 and 2.
A remarkable difference in the two transformations was observed for the initial
6
attack by the peroxy-species at the ketone carbonyl center. In the case of A , the
5
energy barrier to be overcome is about 42 kJ=mol higher than for A and is
approaching values for the transformation of aromatic ketones (Fig. 3). It has been
established in early mechanistic studies, that aryl substituents have a retarding
effect on the rate-determining step of Criegee intermediate formation [26]. It is
5
Fig. 1. Energy profile (in kJ=mol) for the transformation of ketone A to lactone C (distances in A)
5
˚

Microbial Baeyer-Villiger Oxidation
789
6
6
˚
Fig. 2. Energy profile (in kJ=mol) for the transformation of ketone A to lactone C (distances in A)
Fig. 3. Energy profile (in kJ=mol) for the transformation of 2,3-dihydroinden-1-one A to 3,4-dihy-
˚
drochromen-2-one C (distances in A)

7
90
M. D. Mihovilovic et al.
5
interesting to note that the five-membered cyclopentanone moiety both in A and
6
A exhibits a significant ring distortion which is changed during the conversion to
5
6
intermediate B but maintained in the case of B . Such structural differences of
intermediates might be interpreted to be responsible for the complementary stereo-
chemistry imposed by the two enzymes. However, more elaborate calculations
involving the 3-dimensional structure of the proteins are required to further inves-
tigate this hypothesis.
Despite these changes in geometry the thermodynamics is very similar in both
reactions being slightly endothermic (1.3 and 9.6 kJ=mol). On the other hand, the
activation energy for the first step differs markedly suggesting that electronic rather
than steric effects may be responsible for the different kinetic behavior. It may be
speculated that this reflects a disturbance of the ꢂ-conjugation within the cyclo-
pentanone ring.
Previously, CPMO has been demonstrated to convert several indanones to the
corresponding lactones [27], while CHMO seems not active enough to facilitate
this transformation. In a single case study, kinetic resolution of an ꢁ,ꢀ-unsaturated
cyclopentanone has also been reported for CPMO [28]. The above presented data
can also be interpreted in such a way. Again, CHMO requires sufficiently activated
ketone substrates, in order to develop its biocatalytic potential. In contrast,
CPMO is able to also attack less reactive carbonyl centers as in aromatic or
ꢁ
,ꢀ-unsaturated ketones.
Conclusions
Based on the computational model, substantially different electronic requirements
within the substrate ketones seem relevant in CHMO and CPMO mediated Baeyer-
Villiger biooxidations. While this was already indicated by selective acceptance of
aromatic and unsaturated substrates, in this work we have also found a class of
aliphatic ketones, where a similar effect seems to predominate. Molecular model-
ing of the Baeyer-Villiger oxidation of ketones 1a and 1c indicates a substantial
difference in activation energy of the initial nucleophilic attack of a peroxo-species,
which was not expected in such a magnitude. However, based on the theoretical
study, the highly different efficiency of the two enzymes can be better rationalized.
Currently, our model only includes a non-chiral environment and is limited to
explain electronic differences for the enzymatic transformation. We are currently
trying to develop a more elaborate system, which should ultimately incorporate
sterical aspects based on the first 3-dimentional structure of a BVMO reported
recently [29].
Experimental
All chemicals and microbial growth media were purchased from commercial suppliers. All solvents
were distilled prior to use. Flash column chromatography was performed on silica gel 60 from
Merck (40–63ꢃm). Melting points were determined using a Kofler-type Leica Galen III micro hot
stage microscope. NMR-spectra were recorded from CDCl or DMSO-d solutions on a Bruker AC
3
6
2
00 (200MHz) or Bruker Advance UltraShield 400 (400MHz) spectrometer and chemical shifts
are reported in ppm using TMS as internal standard. Enantiomeric excess was determined via GC

Microbial Baeyer-Villiger Oxidation
791
using a BGB 175 column (30 mꢄ0.25 mm ID, 0.25 ꢃm film) on a HP 6890 Series chromatograph.
20
Specific rotation ½ꢁꢁ was determined using a Perkin Elmer Polarimeter 241 by the following
D
2
D
0
ꢀ
ꢀ
3
equation: ½ꢁꢁ ¼ 100 ꢁ=(c l); c=g=100 cm , l=dm.
1
,3,3a,4,7,7a-Hexahydro-2H-inden-2-one (1b)
3
Diacid 5 (8.25 g, 41.6 mmol [15]) was dissolved in 80cm Ac O and 6.4 cm pyridine were added. The
3
2
mixture became dark brown upon refluxing for 62h. The mixture was evaporated in vacuo to remove
3
pyridine. After addition of 55cm 0.5 N HCl the mixture was refluxed for 1.5 h, cooled to rt, and
treated with saturated K CO solution until no further CO evolution was observed. The aqueous layer
2
3
2
3
3
was extracted with 3ꢄ100 cm Et O. The combined organic layers were washed with 2ꢄ80cm 2 N
2
HCl, saturated NaHCO solution, brine, dried (Na SO ), and evaporated. The remaining yellow-orange
3
2
4
ꢅ
oil was purified by Kugelrohr distillation to give 3.44 g 1b (61%) as colorless oil. Bp 105–108 C=
5mbar; spectral properties agreed to Ref. [23].
1
cis-Octahydro-2H-inden-2-one (1c)
3
Ketone 1b (1.00 g, 5.87mmol) and 0.08g 10% Pd=C were suspended in 15cm dry THF. The mixture
+
was hydrogenated with H (81 psi) using a Parr-apparatus for 24h. After filtration (Celite -bed) and
2
evaporation, the slightly yellowish oil was purified by flash column chromatography (20 g SiO2,
LP:EtOAc¼ 15:1) to give 0.78 g 1c (77%) as colorless oil with spectral properties in agreement with
Ref. [22].
General Procedure for m-CPBA Oxidation (GP I)
The corresponding ketone was dissolved in dry CH Cl (10% solution), m-CPBA (1.1–1.3 equiv, 50%
2
2
chemical grade) was added, and the mixture was stirred overnight at rt until TLC indicated complete
conversion. A white precipitate was formed. Excess triethylamine was added and the mixture was
stirred for 0.5 h. Then the reaction was hydrolyzed upon addition of H O and extracted with CH Cl .
2
2
2
The combined organic layers were washed with 2 N HCl, saturated NaHCO solution, dried (Na SO ),
3
2
4
and the solvent was removed in vacuo.
General Procedure for Whole-Cell Biooxidation (GP II)
3
Fresh LB-ampicillin medium (250 cm ; LB medium: 1% Bacto-Peptone, 0.5% Bacto-Yeast Extract,
3
3
1
% NaCl in deion H O, supplemented by 200ꢃg=cm ampicillin) was inoculated with a 2.5 cm
2
aliquot of an overnight preculture of the corresponding expression strain for CHMO or CPMO in a
3
000 cm baffled Erlenmeyer flask. The culture was shaken at 120 rpm at 37 C until it reached an
ꢅ
1
optical density at 600 nm (OD600) between 0.2 and 0.4, then isopropylthio-ꢀ-D-galactoside (IPTG) was
added to a final concentration of 0.025 mM. The substrate was added neat and 1 equiv ꢀ-cyclodextrin
was supplemented if required. The culture was shaken at 150rpm at rt. Conversion was monitored by
GC and reached completion between 18 and 36h. Then, the biomass was separated by centrifugation
+
(3500 rpm, 10 min) and the supernatant was filtered through a pad of Celite . The clear solution was
extracted with EtOAc, the combined organic layers were dried (Na SO ), and evaporated. The residue
2
4
was purified by column chromatography.
cis-Hexahydrocyclopenta[c]pyran-3(1H)-one (2a)
Conversion of 1a (0.50 g, 4.00 mmol) with m-CPBA according to GP I gave 0.28g racemic 2a (50%) as
colorless liquid after flash column chromatography (LP:EtOAc¼ 2:1).
Biotransformation of 0.53g 1a (4.27 mmol) according to GP II with CHMO expressing recombi-
nant E.coli gave 0.30g (ꢂ)-2a (50%) after flash column chromatography (LP:EtOAc¼ 2:1) as color-
less liquid with physical properties according to Table 1.
Biotransformation of 106 mg 1a (0.84 mmol) according to GP II with CPMO expressing recom-
binant E.coli gave 106 mg (þ)-2a (89%) after flash column chromatography (LP:EtOAc¼ 2:1) as
colorless liquid with physical properties according to Table 1.

7
92
M. D. Mihovilovic et al.
1
H NMR (CDCl ): ꢄ ¼ 1.20–2.08 (m, 6H), 2.25–2.69 (m, 4H), 4.00, 4.37 (2dd, J ¼ 7, 20 Hz,
3
1
3
2
1
H) ppm; C NMR (CDCl ): ꢄ ¼ 25.0 (t), 28.9 (t), 34.2 (t), 34.3 (t), 34.4 (d), 36.3 (d), 67.4 (t),
3
73.4 (s) ppm.
1
,4,4a,5,8,8a-Hexahydro-3H-2-benzopyran-3-one (2b)
Biotransformation of 106 mg 1b (0.78 mmol) according to GP II with CHMO expressing recombinant
E.coli followed by flash column chromatography (LP:EtOAc¼ 10:1) resulted in recovery of 38 mg 1b
(
36%) and afforded 40mg (4aS,8aS)-(ꢂ)-2b (33%; 85% based on consumed and unrecovered starting
material) as colorless oil with physical properties according to Table 1.
Biotransformation of 100 mg 1b (0.73 mmol) according to GP II with CPMO expressing
recombinant E.coli gave 91 mg (4aR,8aR)-(þ)-2b (76%) after flash column chromatography
(
LP:EtOAc¼ 10:1) as colorless oil with physical properties according to Table 1.
1
H NMR (CDCl ): ꢄ ¼ 1.80–2.13 (m, 2H), 2.16–2.44 (m, 4H), 2.50–2.60 (m, 2H), 4.22–4.40
3
1
3
(
(
m, 2H), 5.67 (bs, 2H) ppm; C NMR (CDCl ): ꢄ ¼ 24.0 (t), 28.4 (t), 28.5 (d), 29.6 (d), 33.7 (t), 72.1
3
t), 124.1 (d), 124.6 (d), 170.6 (s) ppm.
cis-Octahydro-3H-2-benzopyran-3-one (2c, C H O )
9 14 2
Ketone 1c (200mg, 1.44mmol) was oxidized according to GP I to give 109mg 2c (49%) as colorless
oil after flash column chromatography (LP:EtOAc¼ 20:1).
Biooxidation of 106 mg 1c (0.70 mmol) according to GP II with CHMO expressing recombinant
E.coli followed by flash column chromatography (LP:EtOAc¼ 10:1) resulted in recovery of 40 mg 1c
(
40%) and afforded 23 mg (4aS,8aS)-(ꢂ)-2c (21%; 65% based on consumed and unrecovered starting
material) as colorless oil with physical properties according to Table 1.
Biotransformation of 106 mg 1c (0.70 mmol) according to GP II with CPMO expressing
recombinant E.coli gave 100 mg (4aR,8aR)-(þ)-2c (83%) after flash column chromatography
(
LP:EtOAc¼ 10:1) as colorless oil with physical properties according to Table 1.
1
H NMR (CDCl ): ꢄ ¼ 1.22–1.63 (m, 8H), 1.83–2.01 (m, 1H), 2.05–2.25 (m, 1H), 2.40–2.55
3
1
3
(
(
m, 2H), 4.25 (d, 2H, J ¼ 8 Hz) ppm; C NMR (CDCl ): ꢄ ¼ 21.5 (t), 23.3 (t), 24.6 (t), 28.6 (t), 31.0
3
d), 32.7 (d), 32.8 (t), 72.4 (t), 171.1 (s) ppm.
Computational Details
All calculations were performed using the Gaussian98 software package on the Silicon Graphics
Origin 2000 of Vienna University of Technology [30]. The geometry and energy of the model
compounds and the transition states were optimized at the B3LYP level [31]. For C, O, and H atoms
the 6–31gꢀꢀ basis set was employed [32]. A vibrational analysis was performed to confirm that the
structures of the model compounds have no imaginary frequency. Transition state optimizations were
performed with the Synchronous Transit-Guided Quasi-Newton Method (STQN) developed by
Schlegel et al. [33]. Frequency calculations were performed to confirm the nature of the stationary
points, yielding one imaginary frequency for the transition states and none for the minima. The
vibrational eigenvectors corresponding to the reaction coordinate (with imaginary frequency) of all
transition states were visually checked to confirm the connectivity of transition states with the reactants
and the products. All geometries were optimized without symmetry constraints.
Acknowledgements
This project was funded by the Austrian Science Fund (FWF, project nos. P16373 and P16600-N11).
Continuous support of our research activities in the field of biooxidations by COST Action
D25 (Applied Biocatalysis, work group Biooxidations) is acknowledged. The authors thank
Prof. M.M. Kayser (University of New Brunswick) for the generous donation of the CPMO
expression system.

Microbial Baeyer-Villiger Oxidation
793
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