Extensive substrate profiling of cyclopentadecanone monooxygenase as Baeyer-Villiger biocatalyst reveals novel regiodivergent oxidations

Article abstract of DOI:10.1016/j.molcatb.2011.07.003

Cyclopentadecanone monooxygenase (CPDMO) is one of the latest additions to the established library of Baeyer-Villiger monooxygenases. Desymmetrizations of substituted cyclobutanones and -hexanones as well as kinetic resolutions of racemic cycloketones are efficiently catalyzed by CPDMO. Moreover the enzyme shows unprecedented preference in regiodivergent oxidations of terpenones and the bicyclic Geissman-Waiss lactone precursor giving access to the optical antipode of retronecine and other pyrrolizidine alkaloids.

Full text of DOI:10.1016/j.molcatb.2011.07.003

Journal of Molecular Catalysis B: Enzymatic 73 (2011) 9–16
Contents lists available at ScienceDirect
Journal of Molecular Catalysis B: Enzymatic
journal homepage: www.elsevier.com/locate/molcatb
Extensive substrate profiling of cyclopentadecanone monooxygenase as
Baeyer–Villiger biocatalyst reveals novel regiodivergent oxidations
Michael J. Finka, Thomas C. Fischera, Florian Rudroffa, Hanna Dudekb, Marco W. Fraaijeb,
Marko D. Mihovilovica,∗
a Institute for Applied Synthetic Chemistry, Vienna University of Technology, Getreidemarkt 9/163, A-1090 Vienna, Austria
b Laboratory of Biochemistry, University of Groningen, Nijenborgh 4, 9747 AG Groningen, The Netherlands
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 8 April 2011
Received in revised form 25 June 2011
Accepted 4 July 2011
Available online 27 July 2011
Cyclopentadecanone monooxygenase (CPDMO) is one of the latest additions to the established library
of Baeyer–Villiger monooxygenases. Desymmetrizations of substituted cyclobutanones and -hexanones
as well as kinetic resolutions of racemic cycloketones are efficiently catalyzed by CPDMO. Moreover the
enzyme shows unprecedented preference in regiodivergent oxidations of terpenones and the bicyclic
Geissman–Waiss lactone precursor giving access to the optical antipode of retronecine and other
pyrrolizidine alkaloids.
Keywords:
Baeyer–Villiger monooxygenases
Terpenones
© 2011 Elsevier B.V. All rights reserved.
Geissman–Waiss lactone
Regioselective biotransformation
Biooxygenation
1. Introduction
monooxygenase from Acinetobacter sp. NCIMB 9871 (CHMOAcineto),
cyclopentanone monooxygenase from Comamonas sp. NCIMB 9872
The Baeyer–Villiger reaction represents a distinct approach in
synthesis of esters and lactones from carbonyl precursors [1,2]. The
possibility of de novo generation of chirality upon transformation
of prochiral cyclic ketones or the oxidative resolution of racemic
starting materials calls for discovery of suitable stereoselective cat-
alysts [3]. Although such catalytic entities have been developed
on a transition metal base displaying moderate to reasonable chi-
ral induction [4,5], Baeyer–Villiger monooxygenases (BVMOs) have
attained more attention mostly due to their superior chemo-, regio-
, and stereoselectivity [6–8] in combination with their ability to
substitute potentially dangerous peroxide reagents with cheap and
environmentally benign atmospheric oxygen [9].
Whereas the mentioned selectivities are generally mastered,
the quest in biocatalysis remains to broaden the substrate scope
and to gain access to both optical antipodes of a desired prod-
uct. This can be achieved either by genetic modifications of
already known enzymes or by discovery and isolation of new
enzymes from natural resources. The comprehensive assessment
of substrate profile, performance and selectivity of novel catalytic
entities is therefore mandatory. Within the BVMO family exam-
ples for such enzymes are the first isolated BVMO cyclohexanone
(CPMOComa) [10] and two CHMOs from Brevibacterium [11]. These
enzymes have a remarkably broad substrate profile and a strong
stereopreference producing enantiocomplementary lactones. We
have shown earlier that CHMOAcineto and CPMOComa represent pro-
totypes of two predominant subgroups of BVMOs: members of
these enzyme clusters usually have a high protein sequence iden-
tity (Fig. 1). Moreover, they generally exhibit a striking overlap in
substrate scope and enantiopreference [12]. At present, a remark-
ably broad collection of BVMOs has become available with some
notable differences in substrate acceptance. In particular, recent
contributions outlined several aryl-ketone converting enzymes
[13–15], as well as biocatalysts operating on linear precursors
[16–19].
Whenever new BVMOs are discovered, it is reasonable to put
their activity, performance, and selectivity into relation with the
defined enzyme clusters.
CPDMO was first isolated from Pseudomonas sp. strain HI-70
by Lau et al. in 2006 [20]. The authors have demonstrated that
this biocatalyst only shows a 29–50% sequence identity to any
other known BVMO. The enzyme displayed the highest catalytic
efficiency towards cyclopentadecanone, hence its name. Their
initial findings included high activity not only with other large ring
ketones (C11–C13) but also substituted and fused cyclohexanones.
In a follow-up publication by the same group [21] the enzyme’s
activity on ketosteroids was tested and its high selectivity was
∗
Corresponding author. Tel.: +43 1 588 01 154 20; fax: +43 1 588 01 154 99.
E-mail address: mmihovil@pop.tuwien.ac.at (M.D. Mihovilovic).
1381-1177/$ – see front matter © 2011 Elsevier B.V. All rights reserved.
doi:10.1016/j.molcatb.2011.07.003

10
M.J. Fink et al. / Journal of Molecular Catalysis B: Enzymatic 73 (2011) 9–16
Fig. 1. Phylogenetic tree of BVMOs (not completely shown) visualizing the CHMO and CPMO clusters as well as the CPDMO branch (CHMO: cyclohexanone monooxygenase,
CPMO: cyclopentanone monooxygenase, CDMO: cyclododecanone monooxygenase, HAPMO: p-hydroxyacetophenone monooxygenase, PAMO: phenylacetone monooxyge-
nase, STMO: steroid monooxygenase, MO: monooxygenase).
confirmed. The unusual position of CPDMO in the phylogenetic
tree of BVMOs and these intriguing first results initiated our
in-depth profiling of this new biocatalyst.
with 1.0 mL EtOAc (supplemented with 1 mM methyl benzoate as
internal standard) after centrifugal separation of the cell mass.
General procedure for preparative fermentations: LBamp fermenta-
tion medium was inoculated, induced, and charged with additives
as described above. The substrate was then added directly to the
shakeflask as approx. 10% (w/v) solution in 1,4-dioxane. Incubation
at 24 ◦C was carried out until the desired degree of conversion was
determined via GC control. The aqueous solution was then cen-
trifuged, the supernatant was extracted with EtOAc or Et2O and
concentrated. The crude compounds were purified by chromatog-
raphy on silica using LP/Et2O or LP/EtOAc mixtures.
(−)-2-Allylcycloheptanone (23a) and (+)-8-allyloxocan-2-one
(23b): The fermentation was carried out with rac. 23a (100 mg,
657 ␮mol) according to the general procedure. Purification on 15 g
silica gel, LP/Et2O 10:1 gave compounds 23a (34 mg, 68%) and 23b
(33 mg, 60%) as colorless oils. (−)-23a 1H NMR (CDCl3, 200 MHz) ı:
1.20–1.87 (m, 10H), 1.98–2.12 (m, 1H), 2.35–2.62 (m, 2H), 4.96–5.05
(m, 2H), 5.61–5.82 (m, 1H); 13C NMR (CDCl3, 50 MHz) ı: 24.3 (t),
28.7 (t), 29.5 (t), 30.6 (t), 36.3 (t), 43.1 (t), 51.6 (d), 116.5 (t), 136.3
(d), 215.6 (s); ˛2D5 = −43.4 (c = 0.68, CHCl3); ee (GC) = 85%. (+)-23b
1H NMR (CDCl3, 200 MHz) ı: 1.32–1.98 (m, 8H), 2.22–2.60 (m, 4H),
4.53–4.66 (m, 1H), 5.06–5.16 (m, 2H), 5.81 (ddt, 1H, J1 = 17.08 Hz,
J2 = 10.11 Hz, J3 = 6.97 Hz); 13C NMR (CDCl3, 50 MHz) ı: 23.7 (t), 26.2
(t), 28.7 (t), 32.3 (t), 36.7 (t), 39.8 (t), 78.0 (d), 117.7 (t), 133.6 (d),
176.5 (s); ˛2D5 = +28.3 (c = 0.66, CHCl3); ee (GC) = 93%.
2. Experimental
Unless noted otherwise, all reagents were purchased from com-
mercial suppliers and used without further purification. DCM and
toluene intended for water-free reactions were desiccated over
Al2O3 columns. All other solvents were distilled prior to use.
Ketones for substrate acceptance screening were either purchased
from commercial suppliers or synthesized according to litera-
ture known protocols. Column chromatography was performed
on a Büchi Sepacore Flash System using silica gel 60 and dis-
tilled solvents. General conversion determination was conducted
via GC using an achiral capillary column (BGB5, 30 m × 0.25 mm
ID, 0.25 ␮m film). Enantiomeric excess values were measured on
modified cyclodextrin GC columns BGB 175 (30 m × 0.25 mm ID,
0.25 ␮m film) or BGB 173 (30 m × 0.25 mm ID, 0.25 ␮m film). Spe-
cific rotation was measured on an Anton Paar MCP500 polarimeter.
LB medium was used without pH correction; ampicillin concen-
tration was set to 200 mg/L (LBamp). For expression of CPDMO
a codon-optimized synthetic gene was cloned into the recently
developed pCRE2 expression vector [22].
Experimental procedure for substrate acceptance screening: A baf-
fled Erlenmeyer flask was charged with LBamp medium (10 mL),
inoculated with a bacterial single colony from an Agar plate and
incubated at 37 ◦C in an orbital shaker o/n. The fermentation
medium (LBamp) was then inoculated with 2% (v/v) of the precul-
ture and incubated for ca. 1–2 h under the same conditions until an
optical density of 0.2–0.6 was reached. l-Arabinose (0.02%, w/v) and
␤-cyclodextrin (4 mM) were added, the mixture was thouroughly
mixed and split in 1.0 mL aliquots into 24-well plates. Substrates
were added as 0.8 M solutions in 1,4-dioxane to a final concen-
tration of 4 mM. The plates were sealed with adhesive film and
incubated at 24 ◦C in an orbital shaker for up to 24 h. Analytical sam-
ples were prepared by extraction of 0.5 mL of fermentation broth
(−)-6-Methyloxepan-2-one (27b) and (−)-4-methyloxepan-2-one
(27c): The fermentation was carried out with rac. 27a (180 mg,
1.60 mmol) according to the general procedure. Purification on 45 g
silica gel, LP/Et2O 3:1 gave an inseparable mixture of compounds
27b and 27c (ratio 50:50 as determined by GC) as colorless oil
(160 mg, 78%). 1H NMR and 13C NMR spectra were in accordance
with published values [23]; 27b ee (GC) = 76%; 27c ee (GC) = 74%.
(4R,7S)-7-Isopropyl-4-methyloxepan-2-one (29b): The fermenta-
tion was carried out with (−)-menthone 29a (100 mg, 648 ␮mol)
according to the general procedure. Purification on 20 g silica gel,
LP/Et2O 3:1 gave compound 29b as a colorless oil (72 mg, 65%).
1H NMR (CDCl3, 200 MHz) ı: 0.94 (d, 3H, J = 6.8 Hz), 0.96 (d, 3H,

M.J. Fink et al. / Journal of Molecular Catalysis B: Enzymatic 73 (2011) 9–16
11
J = 6.8 Hz), 1.02 (d, 3H, J = 6.6 Hz), 1.10–1.99 (m, 6H), 2.38–2.60 (m,
2H), 4.02 (dd, 1H, J1 = 9.0 Hz, J2 = 4.4 Hz); 13C NMR (CDCl3, 50 MHz)
ı: 17.1 (q), 18.4 (q), 24.0 (q), 30.5 (d), 31.0 (t), 33.4 (d), 37.5 (t), 42.6
(t), 84.8 (d), 175.1 (s); ˛2D0 = −19.7 (c = 0.72, CHCl3); ee (GC) = >99%.
Ethyl (4,4-diethoxybutyl)carbamate (33): Amine 32 (5.64 g,
35 mmol) and NEt3 (9.8 mL, 70 mmol, 2.0 equiv) were dissolved in
dry DCM (100 mL) and cooled to 0 ◦C under an argon atmosphere.
Then ethyl chloroformate (10.0 mL, 70 mmol, 2.0 equiv) was added
quickly causing local reflux and clouding. The mixture was kept
in the ice/water bath and stirred o/n. After 19 h the reaction com-
posite was washed with saturated NH4Cl solution (2× 50 mL) and
brine, then dried over Na2SO4 and concentrated under reduced
pressure yielding carbamate 33 as a pale yellow liquid (9.40 g, 90%).
1H NMR (CDCl3, 200 MHz) ı: 1.06–1.33 (m, 9H), 1.51–1.72 (m, 4H),
3.13–3.23 (m, 2H), 3.40–3.71 (m, 4H), 4.03–4.20 (m, 2H), 4.47 (t,
1H, J = 5.2 Hz), 4.79 (br s, 1H).
Ethyl 2-hydroxypyrrolidine-1-carboxylate (34): Carbamate 33
(9.00 g, 36.6 mmol) was dissolved in THF (65 mL) before aqueous
HCl (ca. 4%, w/v, 65 mL) was added. The mixture was stirred at
rt. After 10 min complete conversion was determined via TLC. The
solution was extracted with Et2O (3× 150 mL), the pooled organic
extracts were then washed with 10% K2CO3 solution (100 mL) and
brine, dried over Na2SO4 and concentrated. Heterocycle 34 was
obtained as colorless oil in >95% purity according to GC–MS as a
mixture of isomers (5.80 g, quant.).
(2× 200 mL) and the pooled organic layers were dried over Na2SO4
and concentrated, yielding the crude product as a brown liquid.
Column chromatography on 70 g silica gel eluting with LP/EtOAc
1:1 gave pure 26a as a mixture of rotamers (615 mg, 54%). 1H
NMR (CDCl3, 200 MHz) ı: 1.22 (t, 3H, J = 7.1 Hz), 1.82–2.03 (m,
1H), 2.12–2.24 (m, 1H), 2.76–2.86 (br m, 1H), 3.30–3.39 (br m,
2H), 3.75–3.85 (br m, 2H), 4.12 (q, 2H, J = 7.1 Hz), 4.53 (br s, 1H);
13C NMR (CDCl3, 50 MHz) ı: 14.8 (q), 25.3/26.0 (t), 46.1/46.5 (t),
48.2/48.8 (d), 53.7/53.8 (t), 61.4 (t), 63.7/64.8 (d), 155.0 (s), 210.0
(s).
(3aS,6aS)-Ethyl
b]pyrrole-4(5H)-carboxylate
2-oxotetrahydro-2H-furo[3,2-
(26b) and (3aR,6aS)-ethyl
4-oxohexahydro-1H-furo[3,4-b]pyrrole-1-carboxylate (26c): The
fermentation was carried out with rac. 26a (100 mg, 546 ␮mol)
according to the general procedure. Purification on 25 g silica gel
eluting with LP/EtOAc 1:1 gave compounds 26b (34 mg, 63%) and
26c (51 mg, 93%) as yellow oils. 26b 1H NMR (CDCl3, 400 MHz) ı:
1.25 (t, 3H, J = 7.1 Hz, H-4ꢀ), 1.95–2.10 (m, 1H, H-3), 2.31 (dd, 1H,
J1 = 14.2 Hz, J2 = 6.0 Hz, H-3), 2.71–2.89 (m, 2H, H-6), 3.33–3.45 (m,
1H, H-2), 3.79 (dt, 1H, J1 = 39.6 Hz, J2 = 9.8 Hz, H-2), 4.13 (q, 2H,
J = 7.0 Hz, H-3ꢀ), 4.41–4.52 (m, 1H, H-6a), 5.02–5.12 (m, 1H, H-3a);
13C NMR (CDCl3, 100 MHz) ı: 14.7/14.8 (q, C-4ꢀ), 30.3/30.8 (t, C-3),
35.8/36.6 (t, C-6), 44.2/44.5 (t, C-2), 57.8/58.4 (d, C-6a), 61.6/61.7 (t,
C-3ꢀ), 83.1/84.1 (d, C-3a), 154.2/154.7 (s, C-1ꢀ), 175.4/175.8 (s, C-5);
25
D
˛
= +132.7 (c = 0.50, CHCl3); Lit. +144.1 (c = 0.52, CHCl3) [25];
Ethyl
7,7-dichloro-6-oxo-2-azabicyclo[3.2.0]heptane-2-
ee (GC) = 98%; HR-MS: calc. for [M+H] 200.0917; found 200.0917
(ꢀ = 0.00 ppm). 26c 1H NMR (CDCl3, 400 MHz) ı: 1.25 (t, 3H,
J = 7.1 Hz, H-4ꢀ), 2.06–2.23 (m, 1H, H-3), 2.31–2.41 (m, 1H, H-3),
3.17–3.30 (m, 2H, H-2/H-3a*), 3.74 (dt, 1H, J1 = 40.2 Hz, J2 = 9.8 Hz,
H-2), 4.08–4.24 (m, 2H, H-3ꢀ), 4.39 (dd, 1H, J1 = 10.6 Hz, J2 = 4.5 Hz,
H-6), 4.45–4.60 (m, 2H, H-6/H-6a*); 13C NMR (CDCl3, 100 MHz) ı:
14.7/14.8 (q, C-4ꢀ), 27.1/27.6 (t, C-3), 44.0/44.8 (d, C-3a), 45.3/45.6
(t, C-2), 57.8/58.4 (d, C-6a), 61.6/61.7 (t, C-3ꢀ), 72.7/73.3 (t, C-6),
154.0/154.8 (s, C-1ꢀ), 177.8/178.1 (s, C-4); ˛2D5 = −165.1 (c = 0.42,
CHCl3) ee (GC) = 89%; HR-MS: calc. for [M+H] 200.0917; found
200.0922 (ꢀ = 2.50 ppm).
carboxylate (35): Hydroxypyrrolidine 34 (3.00 g, 18.85 mmol)
was dissolved in toluene and refluxed using a Dean-Stark trap
o/n under an Ar atmosphere. GC–MS reaction control after 17 h
showed selective formation of the desired ethyl 2,3-dihydro-1H-
pyrrole-1-carboxylate intermediate but incomplete conversion of
starting material. After 29 h the reaction had progressed further,
but starting material could still be detected. The Dean-Stark
trap was then replaced by a reflux condenser and triethylamine
(7.88 mL, 56.54 mmol, 3.0 equiv) was added at reflux temperature
in one shot via syringe. Then dichloroacetyl chloride (5.44 mL,
56.54 mmol, 3.0 equiv) was quickly added with submersed syringe
tip. The pale yellow solution turned brown immediately under
heavy reflux and formation of ammonium chloride. The oil bath
temperature was reduced from 135 to 120 ◦C. TLC reaction control
after 17 h showed both the alcohol 34 as well as the intermediate
elimination product to be fully consumed and indicated formation
of two new products, confirmed by GC–MS as chlorine-containing
molecules. The reaction mixture was cooled to rt, washed with
H2O (300 mL), 2 N HCl (2× 150 mL), saturated NaHCO3 solution
(3× 150 mL) and brine. The organic phase was dried over Na2SO4
and concentrated under reduced pressure. Careful column chro-
matography on 500 g silica gel under gradient elution from 10:1 to
1:1 LP/EtOAc yielded the desired dichloroketone 35 as a mixture
of rotamers as brown oil (1.75 g, 37%). 1H NMR (CDCl3, 200 MHz)
ı: 1.93–1.34 (m, 6H), 1.93–2.16 (m, 2H), 2.29 (dd, 2H, J1 = 13.3 Hz,
J2 = 6.7 Hz), 3.23–3.41 (m, 2H), 3.94 (q, 2H, J = 7.3 Hz), 4.09–4.35 (m,
6H), 4.81 (d, 1H, J = 7.3 Hz), 4.93 (d, 1H, 7.4 Hz); 13C NMR (CDCl3,
50 MHz) ı: 14.3/14.6 (q), 26.2/27.0 (t), 46.4/46.7 (t), 60.0/61.1 (d),
62.0 (t), 64.8/65.2 (d), 88.3 (s), 154.5 (s), 196.6 (s).
3. Results and discussion
In order to generate a complete picture of the catalyst perfor-
mance, CPDMO was used to oxidize a large library of substituted
cyclobutanones, cyclohexanones, fused and bridged bi- and tri-
cyclic ketones. The experiments were conducted on analytical scale
as described in Section 2. For novel transformations the fermen-
tations were scaled up to 100 mg preparative scale. Results are
referenced to published values obtained from fermentations with
enzymes from the CHMO- and/or CPMO-cluster. The substrates are
thematically grouped into desymmetrizations, kinetic resolutions
and regiodivergent oxidations. CPDMO appears to be a CHMO-type
enzyme except for the regiodivergent oxidations as compiled in
Section 3.3, where the main potential for novel biotransformations
of this enzyme was identified.
3.1. Desymmetrization reactions
Ethyl 6-oxo-2-azabicyclo[3.2.0]heptane-2-carboxylate (26a):
Cu/Zn couple was prepared as described by Krepski and Hassner
[24]. Dichloroketone 35 (1.57 g, 6.23 mmol) was dissolved in
MeOH (150 mL) saturated with NH4Cl (approx. 40 g/L). The mix-
ture was set under Ar atmosphere before freshly prepared Cu/Zn
couple (2.00 g, 31.10 mmol, 5.0 equiv) was added slowly at rt. The
resulting suspension was stirred at rt for 17 h. Full conversion
was determined by TLC. The mixture was filtered through a pad
of Celite® and washed with MeOH (200 mL). The solvent was
evaporated and the residue was partitioned between EtOAc and
H2O (200 mL each). The aqueous phase was extracted with EtOAc
A series of 20 symmetrical ketones was screened in desym-
metrization biooxygenations (Table 1, Scheme 1). 3-Substituted
cyclobutanones (substrates 1a–4a) were all converted by CPDMO,
usually showing full conversion to the desired lactones with high
enantiomeric excesses. The optical purity of product 1b was even
slightly higher than previously published values (91 vs. 88% ee).
Only 4-chlorophenyl compound 3a showed poor performance as
well as a rather low selectivity of 25%. The enantiomeric preference
in this series goes in line with representatives of the CHMO-type
enzyme cluster.

12
M.J. Fink et al. / Journal of Molecular Catalysis B: Enzymatic 73 (2011) 9–16
Table 1
Desymmetrizations of substituted cyclobutanones, cyclohexanones and bi-/tricyclic ketones (substrates 1–20).
Substrate
R, Rꢀ, X
Compound No.
CPDMO
% Conv.a
Reference biotransformation
% eeb
% Conv.a
% eeb
Biocatalyst
Reference
[26]
+++
+
+++
n.c.
+++
++
88 (−)
31 (−)
95 (−)
n.a.
85 (+)
44 (+)
99 (−)
40 (−)
CHMOXantho
CPMOComa
CHMOXantho
CPMOComa
CHMOAcineto
CPMOComa
CHMOXantho
CPMOComa
+++
+++
++
91 (−)
82 (−)
25 (+)
80 (−)
Bn
1
2
3
4
[26]
[26]
[26]
3,4,5-triMeO-Bn
4-Cl-Ph
+++
++
3,4-(OCH2O)-Bn
+++
+++
++
+++
n.a.
+++
n.a.
98 (−)
64 (+)
98 (−)
n.a.
CHMOXantho
CPMOComa
CHMOXantho
CPMOComa
CHMOXantho
CPMOComa
[26]
[26]
[26]
CO2Et
Ph
+++
+++
+++
>99 (−)
99 (−)
5
6
7
99 (−)
>99 (−)
tBu
n.a.
R = Me
Rꢀ = Me
R = Me
Rꢀ = Ph
+++
+
+++
n.c.
n.a.
n.a.
CHMOXantho
CPMOComa
CHMOXantho
CPMOComa
[26]
[26]
n.a.
n.a.
+++
n.c.
8
9
95 (−)
n.a.
X = CH2
R = Me
X = O
R = Me
X = O
R = H
X = S
R = H
X = NMe
R = H
+++
++
+++
n.c.
+++
++
+++
n.a.
+++
n.a.
+++
n.a.
97 (−)
99 (+)
>99 (−)
n.a.
n.a.
n.a.
n.a.
n.a.
n.a.
n.a.
CHMOXantho
CPMOComa
CHMOXantho
CPMOComa
CHMOXantho
CPMOComa
CHMOXantho
CPMOComa
CHMOXantho
CPMOComa
CHMOXantho
CPMOComa
[26]
[26]
[26]
[26]
[26]
[26]
+++
+
99 (−)
>99 (−)
n.a.
10
11
12
13
14
15
n.c.
n.c.
n.c.
n.c.
n.a.
n.a.
X = NCO2Me
R = H
n.a.
n.a.
n.a.
+++
++
88 (−)
CHMOXantho
CPMOComa
[26]
++
>99 (−)
16
>99 (+)
>99 (−)
n.a.
n.a.
CHMOXantho
CPMOComa
CHMOXantho
CPMOComa
[26]
[26]
n.c.
n.c.
n.a.
n.a.
X = CH2
X = O
++
17
18
n.c.c
++
95 (+)
n.c.
n.c.
+++
+
n.a.
n.a.
CHMOXantho
CPMOComa
CHMOXantho
CPMOComa
[26]
[26]
n.c.
+++
n.a.
CO2Me
19
20
97 (−)
–(CH2)3–
64 (−)
91 (+)
n.a.: not available, n.c.: no conversion, rac.: racemic.
a
Relative conversion (Conv.) of starting material determined by chiral phase GC after 24 h: +++ > 90%, ++ 50–90%, + < 50%.
Enantiomeric excess values determined by chiral phase GC; sign of optical rotation or absolute configuration is given in parentheses and assigned on the basis of reference
b
biotransformations.
c
Mainly epoxide was formed.
Substituted cyclohexanones 5a–9a provide a more diverse pic-
ture of the substrate scope of CPDMO. It was reported before that
the model CHMO from Acinetobacter performs rather poorly on
bulky 4-phenyl- and 4-tert-butylcyclohexanone, however, these
precursors are readily transformed by CHMOXantho with excel-
lent selectivity. The allegedly large active site of CPDMO can
accommodate those sterically demanding compounds, but not 4-
methyl-4-phenyl substituted substrate 9a in contrast to reference
enzymes. Nevertheless, the achieved optical purities are 99% or
greater and align with the CHMO cluster preference. This also holds
true for 3,5-dimethyl substrates 10a and 11a, although conver-
sion of the oxa-heterocycle was rather low. Generally, CPDMO does
not accept heteroatom-containing 6-rings. This could be substan-
tiated with substrates 12a–15a where only starting material was
recovered after 24 h.
Scheme 1. Desymmetrization of prochiral cycloketones with BVMOs.
verted (substrate 20a) with moderate ee. Summarizing this section,
it can be concluded that, although CPDMO’s active site accepts sub-
strates as large as cyclopentadecanone and smaller rings bearing
bulky substituents, it is not as flexible as CHMOXantho. Enantiose-
lectivities though are in the same region as other well-established
BVMOs.
The enantioselectivity on fused ring system 16a could be
improved from 88% as obtained with CHMOXantho to >99%, although
with lower conversion. Only one other polycyclic system was con-

M.J. Fink et al. / Journal of Molecular Catalysis B: Enzymatic 73 (2011) 9–16
13
Table 2
Kinetic resolutions of 2-substituted six and seven-membered cycloketones (substrates 21–24).
Substrate
R
Compound No.
CPDMO
% Conv.a
Reference biotransformation
% eeS,
% eeP
Eb
% Conv.a
% eeS,
Eb
Biocatalyst
Reference
a
a
% eeP
Me
Ph
21
22
57
97 (+),
82 (−)
rac.
41
24
48
29 (+),
62 (−)
>99 (+),
76 (−)
6
CHMOXantho
CHMOXantho
[30]
[30]
100
n.a.
>200
allyl
Bn
23
24
n.c.
n.c.
n.a.
n.a.
n.a.
n.a.
53
20
85 (−),
93 (+)
13,
74
3
CHMOAcineto
CHMOXantho
This work
This work
45
n.a.: not available, n.c.: no conversion, rac.: racemic.
a
Relative conversion (Conv.) of starting material and enantiomeric excess values determined by chiral phase GC; sign of optical rotation or absolute configuration is given
in parentheses and assigned on the basis of reference biotransformations; eeS (substrate) in italics, eeP (product) in normal font.
b
Enantiomeric ratio E was calculated using the Selectivity software developed by Faber.
3.2. Kinetic resolutions
After dealing with desymmetrizations of various cyclic ketones
we were interested in the performance of CPDMO in kinetic reso-
lutions of racemic ketones (21a–24a).
Enantiomeric excess values are a function of conversion in
kinetic resolution reactions, thus progress of these biotransforma-
tions was monitored over time in order to determine the 50% mark
where a compound optimum of selectivity and yield is reached. For
individual purposes where only enantiomerically pure ketone or
lactone is desired, it may be necessary to shift the point of stopping
the reaction: for maximal optical purity of the starting material the
conversion should significantly exceed 50%. For highly enantioen-
riched product collection on the other hand it is advisable to stop
at ca. 40%. In order to assess the stereoselectivity of an enzyme for
a particular substrate, the enantiomeric ratio E was introduced as
it is independent from conversion [27].
Scheme 2. Schematic view of a kinetic resolution of 2-substituted cyclic ketones
catalyzed by BVMOs.
Scheme 3. Regiodivergent Baeyer–Villiger oxidations of fused bicyclic cyclobu-
Usually consumption of one enantiomer is completed within
8 h for normal ring sizes when employing recombinant whole-cell
based CPDMO fermentations. Medium and larger rings may take up
to 24 h to reach completion. Enantiomeric ratio E was calculated by
fitting the experimental data to a mathematical equation using the
Selectivity software developed by Faber et al. [28]. All values are
compiled in Table 2.
tanones.
3.3. Regiodivergent bio-oxidations
In general, fused bicyclic cyclobutanones are good substrates
for BVMOs due to highly favored alleviation of ring strain by inser-
tion of an oxygen atom. Moreover, they highlight another level
of selective Baeyer–Villiger oxidation: regiodivergent transforma-
tions (Scheme 3). It was found that the mechanism of the reaction
allows for prediction of product formation not only based upon
nucleophilicity of carbonyl-adjacent carbon atoms but also via
stereoelectronic requirements of the rearrangement [31]. For a suc-
cessful migration the migrating bond must adopt a conformation
antiperiplanar to the bond between the two oxygens of the peroxy-
reagent and anti to one of the two non-bonding electron pairs of
the carbonyl oxygen. Only when these prerequisites are met, the
oxygen insertion can take place (Scheme 4). Based on these two
effects, formation of the “normal” lactone as product of the nucle-
ophilicity driven rearrangement as well as the “abnormal” lactone
It has been reported that CPDMO catalyzes kinetic resolutions
of five and six-membered cycloketones (Scheme 2) [20]. While
substituted cyclopentanones are fully converted to racemic lac-
tones, it was possible to selectively oxidize only the (−)-enantiomer
of 2-methylcyclohexanone 21a to the normal lactone in 82% ee
(E = 41). The only other BVMO known to surpass this is a cyclodo-
decanone monooxygenase (CDMO) from Rhodococcus SC1 (E > 200)
[29]. The best result from the CHMO-cluster so far was obtained
with CHMOXantho producing 21b in 62% ee (E = 6). When the methyl
substituent is replaced with the more sterically demanding and
electronically different phenyl residue (substrate 22a), no selec-
tivity at all could be observed and only racemic lactone 22b
was isolated. To our surprise 2-substituted cycloheptanones 23a
and 24a were not accepted by CPDMO. For reference reasons
these substrates were screened with CHMOAcineto and CHMOXantho
and for the allyl compound 23a a preparative fermentation was
carried out (see Section 2). Due to poor selectivity and con-
version substrate 24a and product 24b were not isolated and
characterized.
Although CPDMO proficiently catalyzes the difficult kinetic res-
olution of 2-methylcyclohexanone 21a, it did not emerge as a
suitable biocatalyst for this reaction type, in general.
Scheme 4. Left: attack of the flavin adenine dinucleotide (FAD) peroxo species
and formation of the Criegee intermediate; right: required conformation for the
Baeyer–Villiger rearrangement (Rstat = static residue, Rmig = migrating residue).

14
M.J. Fink et al. / Journal of Molecular Catalysis B: Enzymatic 73 (2011) 9–16
Scheme 5. Comparison of CHMO-catalyzed kinetic resolution and CPDMO-catalyzed regiodivergent oxidation of 26a to Geissman–Waiss lactone 26b and the role of 26b in
the total synthesis of the necine bases (+)-retronecine and (−)-turneforcidine.
as result of the rearrangement governed by stereoelectronic effects
may be expected.
important intermediate in the synthesis of (+)-retronecine [35] and
other pyrrolizidine alkaloids [36]. By switching between the two
catalysts, they were able to facilitate a preferential formation of
normal lactone 26b (R = Bn) with (R,R)-configuration or of abnor-
mal 26c (R = Bn) with (R,S)-configuration. Both enzymes catalyzed
the oxidation in a kinetic resolution fashion, leaving behind the
(S,S)-enantiomer of the ketone precursor (Scheme 5). In contrast
to this, CPDMO provided access to the antipodal Geissman–Waiss
lactone (S,S)-26b (R = Et) as well as the abnormal product (R,S)-
26c (R = Et) in a perfect 50:50 mixture via regiodivergent oxidation
in line with carbocyclic analogues of this compound class. A
preparative fermentation on 100 mg scale yielded (S,S)-26b in
63% yield (98% ee) and (R,S)-26c in 93% yield (89% ee). This in
turn means that through the different behavior of CPDMO the
non-natural enantiomer of retronecine and other necine bases
are accessible via this chiral intermediate (Scheme 5). Moreover,
in a detailed screen using 10 BVMOs of various microbial ori-
While the quite fast auto-oxidation of these starting materials
with atmospheric oxygen leads to only one pair of racemic enan-
tiomers, BVMOs are able to stabilize the Criegee intermediates in a
way that two optically pure regioisomers can be obtained in many
cases. It must be stressed that this reaction type is not a kinetic reso-
lution as both enantiomers are converted to different products with
comparable rates. In contrast to stereoselective desymmetrizations
or kinetic resolutions of racemic ketones which were also con-
ducted under metal-assisted catalysis with a wider substrate scope,
such asymmetric and regiodivergent chemical Baeyer–Villiger oxi-
dations are only reported on compound 25a [3]. Consequently, they
represent a unique feature of BVMOs.
Two examples were chosen to demonstrate the ability of CPDMO
to catalyze this reaction type (Table 3). Commercially available
bicyclic ketone 25a is transformed to a 51:49 mixture of regioi-
someric lactones with high selectivity (86 and 85% ee). Again, the
enzyme groups with the CHMO-type stereopreference domain.
Previously, different behaviors were identified for CPMO-type bio-
catalysts converting both antipodal substrates into racemic normal
lactones [32] as well as for a BVMO from Mycobacterium tuberculosis
giving predominant access to optically enriched abnormal lactones
[33].
gin (CHMOAcineto, CHMOXantho, CHMOBrevi1, CHMOBrevi2, CHMOBrachy
,
CHMOArthro, CHMORhodo1, CHMORhodo2, CPMOComa, CPDMO) it was
revealed, that only CPDMO performed this reaction with ideal
regioselectivity (unpublished results; for further information on
the strains used see [6] and references cited therein).
Ketone 26a was synthesized according to the protocol for the
benzyl carbamate published by Alphand et al. [34]. Since a detailed
experimental procedure was not reported there, full experimental
description was disclosed in this work (Scheme 6).
3-Methylcyclohexanone 27a is another model substrate for the
demonstration of CPDMO’s regioselectivity. Here the difference
in nucleophilicity between the carbonyl substituents is less pro-
nounced as in 2-substituted cycloketones and a mixture of proximal
An exception to this clustering of CPDMO and CHMO was
observed in the Baeyer–Villiger oxidation of N-heterocyclic bicyclic
ketone 26a.
In 2005 Alphand et al. published BVMO-mediated access
to a protected Geissman–Waiss lactone 26b (R = Bn) by two
different CHMOs from Acinetobacter sp. [34]. This compound is an
Scheme 6. Synthetic route to rac. 26a, the ketone precursor for the N-protected Geissman–Waiss lactone 26b (a: ClCO2Et, NEt3, DCM; b: HCl, THF; c: toluene, Dean-Stark
trap; d: Cl2HCOCl, NEt3, toluene; e: Cu/Zn couple, NH4Cl, MeOH).

M.J. Fink et al. / Journal of Molecular Catalysis B: Enzymatic 73 (2011) 9–16
15
Table 3
Regiodivergent bio-oxidations of racemic (substrates 25–27) and optically pure ketones (substrates 28–31).
Substrate
Compound No.
CPDMO
Reference biotransformation
% Conv.a/ratiob
% eec
% Conv.a/ratiob
% eec
Biocatalyst
CHMOXantho
Reference
[26]
+++
51:49
86 (−),
85 (−)
+++
52:48
91 (−),
25
>99 (−)
d
+++
50:50
98 (+),
89 (−)
+
96 (−),
93 (−)d
CHMOAcineto
[34]
26
27
78:22d
+++
50:50
76 (−),
74 (−)
+++/50:50
+++/100:0
95 (−), 94 (−)
CHMOAcineto
CPMOComa
This work
[30]
rac.
28
+++
100:0
+++
100:0
CHMOAcineto
>99 (+)e
>99 (−)e
>99 (−)e
>99 (+)e
(+)-Menthone
29
+++
100:0
n.c.
n.c.
n.a.
n.a.
CHMOXantho
CHMOAcineto
[30]
(−)-Menthone
30
+++
0:100
+++
0:100
CHMOXantho
>99 (−)e
[30]
(+)-trans-Dihydrocarvone
31
+++
100:0
+++
100:0
CHMOXantho
>99 (+)e
>99 (+)e
[30]
(−)-trans-Dihydrocarvone
n.a.: not available, n.c.: no conversion, rac.: racemic.
a
Relative conversion (Conv.) of starting material determined by chiral phase GC after 24 h: +++ > 90%, ++ 50–90%, + < 50%.
Ratio of regioisomers (normal:abnormal).
Enantiomeric excess values determined by chiral phase GC; sign of optical rotation or absolute configuration is given in parentheses and assigned on the basis of reference
b
c
biotransformations; eenormal in italics, eeabnormal in normal font.
d
The reference biotransformation proceeded in a kinetic resolution fashion (see text).
Starting material was optically pure; no epimerization observed.
e
and distal lactones is to be expected (Scheme 7). Studies with
isolated enantiomers of 27a have shown that BVMOs from both
the CHMO and the CPMO cluster behave highly regio- and stere-
oselectively [23]. For comparison fermentations were repeated
slower (4 h vs. 20 h) and that our results deviate from the reported
values (ratio distal/proximal 3:1, both 96% ee, 62% yield) [20].
The last substrate class in this series consists of four terpenones
(substrates 28a–31a). The biocatalytic Baeyer–Villiger oxidation
of terpenones yields both the expected normal migration prod-
ucts b as well as the abnormal lactones c. As was shown by our
group in 2007 [37] enzymes from the CHMO cluster usually perform
these reactions as enantiodivergent transformations. These start-
ing materials are commercially available in their diastereomerically
and enantiomerically pure forms, hence, the fermentations lead
to a single product when employing optically pure precursors.
In this case, formation of either normal or abnormal lactone
depends on the absolute configuration of the starting material
(Scheme 8).
with racemic starting material using CHMOAcineto and CPMOComa
.
In a 200 mg preparative biotransformation CPDMO produced both
proximal and distal lactones 27b and 27c in good enantiomeric
purities (ee27b 76%, ee27c 74%) as a clean 50:50 mixture in 78%
yield (isomers are inseparable by preparative chromatography).
This once again complies with the CHMO-cluster, as CPMO-type
enzymes usually produce proximal lactones in racemic form only.
It is noteworthy that the transformation by CPDMO is significantly
CPDMO follows this trend among BVMOs, as could be shown
with the completely selective oxidation of trans-dihydrocarvones
30a and 31a to abnormal lactone 30c and normal lactone 31b.
Unexpectedly, an unprecedented biotransformation was found
when menthone was tested: (−)-menthone 29a was accepted by a
BVMO for the first time and was converted to normal lactone 29b,
confirmed by a preparative biotransformation on 100 mg scale (65%
yield, >99% ee, 100:0 29b:29c). It is intriguing that CPDMO does
Scheme 7. Regiodivergent oxidation of ␤-substituted cycloketones to proximal and
distal lactones.

16
M.J. Fink et al. / Journal of Molecular Catalysis B: Enzymatic 73 (2011) 9–16
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In this study we have reported on the substrate scope and per-
formance of cyclopentadecanone monooxygenase as a whole-cell
biocatalyst overexpressed in Escherichia coli. It was clearly shown
that CPDMO performs well as a biocatalyst in the Baeyer–Villiger
oxidation of commercially available and synthesized cycloketones
serving as model substrates and intermediates in natural com-
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with members of the CHMO cluster, it shows a similar substrate
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achieved and the (S,S)-Geissman–Waiss lactone 26b was obtained
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and (−)-turneforcidine. Consequently, CPDMO enables a number
of interesting novel biooxygenations to complement the already
established portfolio of highly selective transformations by this
enzyme class. The particular behavior of the enzyme in regiodiver-
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relative to the previously identified enzyme clusters based on sub-
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Acknowledgement
Funding for this work is generously provided by the OxyGreen
project (FP7 EU Grant No. 212281).
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