Stereoselective desymmetrizations by recombinant whole cells expressing the Baeyer-Villiger monooxygenase from Xanthobacter sp. ZL5: A new biocatalyst accepting structurally demanding substrates

Article abstract of DOI:10.1002/ejoc.200700872

In this work the substrate profile and stereoselectivity of engineered whole cells overexpressing the Baeyer-Villiger monooxygenase from Xanthobacter sp. ZL5 with respect to biotransformations of prochiral substrates is characterized. This enzyme catalyzes the desymmetrization of cyclic ketones bearing different chemical features with stereoselectivity similar to that obtained with a related protein from Acinetobacter as a prototype representative of the cyclohexanone monooxygenase enzyme cluster. Moreover, this biocatalyst is able to convert sterically demanding substrates previously not transformed by other enzymes with excellent enantioselectivities. These results expand the repertoire of optically pure lactones accessible by whole-cell biotransformation processes, which are useful intermediates for the synthesis of natural and bioactive products. In addition, we observed a remarkable epoxidation reaction of a non-activated C=C bond catalyzed by this monooxygenase. Wiley-VCH Verlag GmbH & Co. KGaA, 2008.

Full text of DOI:10.1002/ejoc.200700872

FULL PAPER
DOI: 10.1002/ejoc.200700872
Stereoselective Desymmetrizations by Recombinant Whole Cells Expressing the
Baeyer–Villiger Monooxygenase from Xanthobacter sp. ZL5: A New
Biocatalyst Accepting Structurally Demanding Substrates
Daniela V. Rial,[a] Dario A. Bianchi,[a] Petra Kapitanova,[a] Alenka Lengar,[a]
Jan B. van Beilen,[b] and Marko D. Mihovilovic*[a]
Keywords: Biotransformation / Enzymes / Substrate profile / Baeyer–Villiger reaction / Epoxidation / Biocatalytic cascade
reactions / Xanthobacter sp.
In this work the substrate profile and stereoselectivity of en-
gineered whole cells overexpressing the Baeyer–Villiger
monooxygenase from Xanthobacter sp. ZL5 with respect to
biotransformations of prochiral substrates is characterized.
This enzyme catalyzes the desymmetrization of cyclic
ketones bearing different chemical features with stereoselec-
tivity similar to that obtained with a related protein from
Acinetobacter as a prototype representative of the cyclohexa-
none monooxygenase enzyme cluster. Moreover, this biocat-
alyst is able to convert sterically demanding substrates pre-
viously not transformed by other enzymes with excellent
enantioselectivities. These results expand the repertoire of
optically pure lactones accessible by whole-cell biotransfor-
mation processes, which are useful intermediates for the syn-
thesis of natural and bioactive products. In addition, we ob-
served a remarkable epoxidation reaction of a non-activated
C=C bond catalyzed by this monooxygenase.
(© Wiley-VCH Verlag GmbH & Co. KGaA, 69451 Weinheim,
Germany, 2008)
Acinetobacter sp. NCIMB 9871 (CHMO) and cyclopenta-
none monooxygenase from Comamonas sp. NCIMB 9872
(CPMO). These enzymes are prototypes of the two main
groups of cycloketone-converting Baeyer–Villiger mono-
oxygenases: CHMO- and CPMO-type clusters. The en-
zymes belonging to each group display a high primary se-
quence identity and show similar trends both in regio- and
enantiopreferences. The corresponding classification into
two groups was initially proposed by our group on the basis
of phylogenetic clustering, substrate preferences, and bio-
catalyst stereoselectivity.[8–12]
The BVMO from Xanthobacter sp. ZL5 was first cloned
in 2003,[13] and in a previous study we determined that this
new enzyme belongs to the CHMO-type branch of a phylo-
genetic tree comprising 18 BVMOs recombinantly ex-
pressed in a suitable host and with demonstrated activity
for Baeyer–Villiger oxidations. The amino acid sequences
of the CHMO from Xanthobacter sp. ZL5 and the one from
Acinetobacter show 58% identity calculated using BLAST 2
SEQUENCES,[14] a BLAST-based tool at the NCBI WWW
site (default parameters). We tested this biocatalyst both in
kinetic resolutions of several 2-substituted cyclohexanones
and in regioselective transformations of a representative set
of racemic ketones and pure terpenone precursors. We ob-
served that recombinant cells overexpressing the CHMO
from Xanthobacter sp. ZL5 behaved like the CHMO from
Acinetobacter both in regio- and enantioselectivities, again
correlating phylogenetic data with substrate preference and
Introduction
Asymmetric catalysis in general is an extremely powerful
tool to access optically pure intermediates efficiently.[1] Bi-
ocatalytic approaches offer great advantages over tradi-
tional methods since they are environmentally friendly and
very efficient. Research in the area of enzyme-mediated Ba-
eyer–Villiger oxidations has provided an outline of the great
potential of these biocatalysts with respect to their high
chemo-, regio-, and enantioselectivities.[2–6] While these
studies have in the past been limited to a comparably small
number of Baeyer–Villiger monooxygenases (BVMOs), a
constantly growing number of such enzymes is becoming
available by genome mining,[7] providing a potent platform
for utilization of this biotransformation for the oxidation of
diverse cyclic ketones to optically pure lactones. In particu-
lar, the identification of enzymes displaying enantiodiverg-
ent behavior was an important discovery, as it allows access
to both antipodal lactones in excellent purities.[6]
Examples of such BVMOs producing enantiocomple-
mentary lactones are cyclohexanone monooxygenase from
[a] Institute of Applied Synthetic Chemistry, Vienna University of
Technology,
Getreidemarkt 9/163-OC, 1060 Vienna, Austria
Fax: +43-1-58801-15499
E-mail: mmihovil@pop.tuwien.ac.at
[b] Institute of Molecular Systems Biology, ETH-Zürich,
8093 Zürich, Switzerland
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further supporting our classification into the clusters out- pectations for a CHMO-type BVMO (prototype reference:
lined above.[15]
CHMO from Acinetobacter). Interestingly, sterically de-
Here we report on desymmetrization reactions of cyclic manding 4-tert-butylcyclohexanone (7a) was an excellent
ketones as a highly attractive method for the generation of substrate for the CHMO from Xanthobacter sp. ZL5 as a
chirality in a de novo fashion from symmetrical precur- whole-cell biocatalyst, and a preparative-scale biotransfor-
sors.[16] During screening experiments of the substrate scope mation was performed to confirm and characterize the
of this BVMO we discovered that the biocatalyst is able product. The conversion of this ketone by the CHMO iso-
to convert ketones bearing functionalized substituents with lated from Acinetobacter sp. NCIMB 9871 was first re-
enantiomeric excesses comparable to or even better than ported in 1993.[18] Even though the yield was poor (17%),
those obtained with previous reference biocatalysts. Sur- the lactones were obtained with an excellent enantiomeric
prisingly, the CHMO from Xanthobacter sp. ZL5 in particu- excess (Ͼ98%) in favor of the (–)-enantiomer.[18] Most re-
lar was able to accept and oxidize sterically demanding sub- cently, the enzyme cyclopentadecanone monooxygenase
strates with excellent enantioselectivities, offering a starting from Pseudomonas has been reported to transform this
point for the synthesis of natural products or analogues. ketone in 72% conversion and with 99% ee.[19] Another
We also observed that the recombinant whole-cell system outstanding example is illustrated by the ability of the bio-
overexpressing this enzyme is able to catalyze two further catalyst fully to convert bulky 4-phenylcyclohexanone (8a),
reactions other than Baeyer–Villiger oxidation: the epoxid- producing lactone 8b with a 98% ee in favor of the (–)-
ation of an oxo-bridged bicyclic ketone and the sequential enantiomer. This result represents the first report of a whole
two-step bioconversion of a cyclic alkenone.
cell-mediated Baeyer–Villiger biooxidation of 4-phenylcy-
clohexanone. To the best of our knowledge the only de-
scription of a Baeyer–Villiger biooxidation of this ketone
was reported in 1999, using the purified CHMO from Aci-
netobacter sp. NCIMB 9871 and different co-solvents for
the ketone.[20] The highest conversion (80%) was obtained
by solubilizing the ketone in 5% ethylene glycol, but only a
moderate enantiomeric excess (60%) was achieved.[20]
In a similar way, several 4,4-disubstituted cyclohexa-
nones 9a–12a were oxidized to completion by the Xan-
thobacter biocatalyst under standard screening conditions,
though with only moderate enantioselectivity in the case of
the 4-ethyl-4-methyl-disubstituted cyclohexanone 10a. This
may reflect the moderate energy differences for the methyl
and ethyl groups adopting equatorial and axial positions,
as proposed by us previously.[21] Surprisingly, 4-methyl-4-
phenylcyclohexanone (12a) was completely oxidized in
whole-cell mediated biotransformations with excellent en-
antiomeric excesses (95%), although the substrate is very
bulky. In a previous survey by our laboratory this ketone
was converted by none of eight recombinant whole-cell sys-
tems producing bacterial BVMOs of diverse origin.[22]
Therefore, BL21 (DE3) expressing the CHMO from Xan-
thobacter sp. ZL5 is the first biocatalyst reported to date
that is capable of accommodating and oxidizing sterically
constrained 4-methyl-4-phenylcyclohexanone.
Results and Discussion
1. Substrate Acceptance Profile of the CHMO from
Xanthobacter sp. ZL5
In this work we challenged the ability of CHMO from
Xanthobacter sp. ZL5 to catalyze desymmetrizations of pro-
chiral ketones containing diverse structural features
(Scheme 1). A large collection of mono-, bi-, and tricyclic
ketones carrying different substituents were tested as sub-
strates in screening scale experiments as described in the
Experimental Section, and results are compiled in Table 1;
reference data for prototype biocatalysts of the CHMO-
and CPMO-type clusters are also given. All experiments
were conducted with a recombinant expression strain of
E. coli BL21 (DE3) transformed with the plasmid p11X5.1
carrying the structural gene for the BVMO from Xan-
thobacter sp. ZL5.[13]
The efficient conversions and excellent enantioselectivi-
ties obtained for lactones 13b–17b are in agreement with
previous observations for CHMO-type enzymes[10,17,22,23]
Scheme 1. Typical Baeyer–Villiger biooxidation reaction.
The desymmetrization of prochiral 4-monosubstituted and indicate that at least the tested substituents in the 4-
cyclohexanones 1a–8a to lactones 1b–8b proceeded with ex- positions of 3,5-dimethylcyclohexanones impose no spatial
cellent enantiomeric excesses, except in the case of 4-hy- limitations on this BVMO.
droxycyclohexanone (2a) which was converted moderately
into racemic lactone (Table 1). As observed in previous bio- from Xanthobacter sp. to catalyze the Baeyer–Villiger oxi-
transformation studies on hydroxy-functionalized dation of heterocyclic ketones, six members of this series
To investigate the ability of the recombinant BVMO
ketones,[8,17,18] rearrangement of the initial lactone into a (18a–23a) were tested as potential substrates in whole-cell
five-membered ring system was observed. With this excep- biotransformation reactions. It has been demonstrated pre-
tion, all the monosubstituted cyclohexanones were accepted viously that the CHMO family oxidizes these ketones in
by the enzyme with excellent stereoselectivities, and the con- good yields.[24–26] As expected, all six ketones were good
figurations of the lactones were in agreement with our ex- substrates for CHMO from Xanthobacter as well. In the
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Monooxygenase Desymmetrization in Whole-Cell Systems
Table 1. Biooxidation of functionalized prochiral (hetero)cyclohexanones.
[a] Consumption of starting material according to gas chromatography: +++ Ͼ90%, ++ 50–90%, + Ͻ50%. [b] ee values determined by
chiral-phase gas chromatography; sign of specific rotation is given in parentheses. [c] Isolated as rearranged product (see corresponding
references). [d] Reported conversion and ee for a biotransformation using isolated CHMO from Acinetobacter sp. NCIMB 9871; specific
rotation was not reported in the literature. [e] No clear baseline separation. [f] After 6 h of biotransformation; n.a.: not applicable, n.c.:
no conversion, rac.: racemic.
cases of lactones 19b and 20b the (–)-enantiomers were sion was carried out, with samples being taken at 0, 1, 6,
formed, again showing a behavior similar to that of CHMO 18, and 24 h after addition of the substrate, extracted, and
from Acinetobacter.[25,26] Like other BVMOs tested pre- analyzed by GC-MS. An evident peak corresponding to lac-
viously,[26] the CHMO from Xanthobacter displayed great tone was already identifiable after 6 h of biotransformation
chemoselectivity for the conversion of divinyl ketone 20a, and, in the course of time, most of this product was obvi-
since no evidence of epoxidation at the two double bonds ously further metabolized by the recombinant organism
was detected and only lactone was obtained. All reactions and disappeared, leaving only a small amount of lactone at
were stopped after 24 h except in the case of ketone 22a; 24 h reaction time.
this substrate had been fully consumed after 24 h, although
Prochiral 3-substituted cyclobutanones (Table 2) are
only a small amount of lactone was observed. To analyze amongst the most readily accepted substrates within the
this reaction in detail, a time-course study of the bioconver- BVMO family. Even BVMOs with narrow substrate accept-
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Table 2. Biooxidation of monosubstituted cyclobutanones.
[a] Consumption of starting material according to gas chromatography: +++ Ͼ90%, ++ 50–90%, + Ͻ50%. [b] ee values determined by
chiral-phase gas chromatography; sign of specific rotation is given in parentheses; n.a.: not applicable, n.c.: no conversion, rac.: racemic.
ance profiles such as PAMO[32,33] are able to convert this ketone in moderate yields but with very good enantio-
ketones of this class when they are substituted with aro- meric excesses in favor of the (+)-lactone 34b.[8]
matic groups. As the corresponding butyrolactones are
Different and more appealing behavior was found in the
interesting precursors for the synthesis of bioactive com- case of the carbo-bridged ketone 33a. In agreement with
pounds and natural products,[34–37] we decided to include the proposed stereodivergence between biooxidations by
a systematic investigation of representative cyclobutanones CHMO- and by CPMO-type enzymes, this ketone is con-
carrying aromatic and aliphatic substituents in position 3 verted into antipodal lactones 33b by the two enzyme
(see 24a–30a).
groups. While CPMO-type enzymes give full conversion to
As shown in Table 2, the Xanthobacter sp. ZL5 BVMO the (+)-lactone with good ees, previous members of the
converted these substrates with the same stereopreference, CHMO family only displayed fair formation of the (–)-lac-
with enantiomeric excess of lactones comparable to or even tone at best (unpublished results). So far, the BVMO from
better than those obtained with the CHMO from Acineto- Xanthobacter sp. is the first BVMO reported to catalyze the
bacter. Surprisingly, 3-isobutylcyclobutanone (29a) was ac- desymmetrization of ketone 33a in whole-cell systems, pro-
cepted by this biocatalyst, giving the corresponding (–)-lac- viding excellent access to the resultant carbo-bridged bicy-
tone 29b with a moderately good enantiomeric excess clic lactone (–)-33b. In contrast with the biooxidation of the
(76%), in contrast to the racemic product obtained with oxo-bridged ketone, no indication of epoxidation at the
CHMO from Acinetobacter but in agreement with biooxi- C=C double bond was detected. Thus, both (+)- and (–)-
dations catalyzed by other BVMOs in whole-cell systems.[37] lactones are now efficiently accessible from ketone 33a in
Table 3 summarizes the performance of the novel biocat- either CPMO- or CHMO-mediated Baeyer–Villiger reac-
alyst on polycyclic ketones. Fused bicyclic ketones 31a and tions. However, the N-substituted bridged bicyclic ketone
32a were completely biooxidized by the CHMO from Xan- 35a was not a substrate for this biocatalyst. These results
thobacter sp. with the same stereopreference as the members may indicate a limitation to the accommodation and con-
of the CHMO family tested previously.[8–10,39] The result version of bridged bicyclic ketones of higher polarity. This
obtained for lactone 32b represents a slight improvement in trend in chemoselectivity is compatible with a prior pro-
the access to the corresponding (–)-enantiomer as an inter- posal suggesting a polar constraint on the interaction be-
esting intermediate for indole alkaloids.[9,40,41,42]
tween the active site of Acinetobacter CHMO and a set of
In line with the results obtained for the biooxidation of heterocyclic cyclohexanones.[24] In principle, the same rea-
bridged oxo-ketones by the CHMO family,[8] the CHMO soning could be applied in the case of Xanthobacter sp., al-
from Xanthobacter was unable to convert the oxo-bridged though further studies should be performed to confirm this
compound 34a into the corresponding lactone, but a major hypothesis.
biotransformation product was identified as an epoxide.
Bridged tri- and tetracyclic ketones 36a, 37a, and 38a,
Only CPMO from Comamonas and CHMO 2 from Brevi- with more sterically demanding structures, were accepted
bacterium – both members of the CPMO family – convert by the BVMO from Xanthobacter sp. and were successfully
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Monooxygenase Desymmetrization in Whole-Cell Systems
Table 3. Biooxidation of polycyclic ketones.
[a] Consumption of starting material according to gas chromatography: +++: Ͼ90%, ++: 50–90%, +: Ͻ50%. [b] ee values determined
by chiral-phase gas chromatography; sign of specific rotation is given in parentheses. [c] Mainly epoxide was formed; n.a. not applicable;
n.c. no conversion. [d] Diastereomeric ratio (anti/syn). [e] Reported as conversion in the literature stated.
converted into the lactones with excellent enantiomeric ex- represents the only spatial limitation for biotransformation
cesses. In these cases this allows control over four to six of polycyclic precursors by this BVMO from Xanthobacter
stereogenic centers in a single biotransformation step. in our hands.
Moreover, the location of the ketone functionalities in the
In contrast, the enzyme hardly afforded desymmetri-
bridges of these compounds did not hamper the perform- zation of endo-tricyclic ketone 41a (though with excellent
ance of the enzyme, indicating either that its structure is enantioselectivity), as was also observed for other CHMO-
flexible or that the active site is large enough to accommo- type enzymes. The conversion of 41a with the CHMO and
date such substrates. The limits in substrate acceptance for CPMO families has been studied before:[11] CPMO from
the BVMO from Xanthobacter were obviously reached in Comamonas and CHMO 2 from Brevibacterium showed
the case of bifunctional ketone 39a bearing two ester sub- good conversion rates but moderate to poor stereoselectivi-
stituents; this compound could not be biooxidized enzymat- ties, while some members of the CHMO family only pro-
ically, so far. Excellent substrate acceptance was also ob- duced traces of the corresponding (+)-lactone, but with ex-
served for the related divinyl-substituted compound 40a. A cellent selectivities. The unsaturated exo-tricyclic ketone 42a
sample of the anti-ketone contaminated with traces of the was oxidized by the BVMO from Xanthobacter sp. ZL5, re-
corresponding syn isomer was efficiently oxidized to lactone sulting in the formation of the corresponding lactone 42b
40b (anti:syn = 98:2) in high optical purity as detected by in 43% enantiomeric excess in favor of the (+)-enantiomer.
GC-MS and confirmed by NMR analysis. Ketone 40a was Ketone 42a is not converted by wild-type CHMO from Aci-
obtained from a corresponding bridged tricyclo-precursor netobacter, although two random mutants of this enzyme
by ring-opening metathesis, analogously with the previously (1-K2-F5 and 1-H7-F4) were found to display moderate
reported protocol using ethylene.[43] This compound class conversions and enantiocomplementary behavior.[44] Hence,
was also tested for substrate acceptance; however, the en- the CHMO from Xanthobacter sp. ZL5 is the first wild-type
zyme was incapable of oxidizing such structures. So far, this BVMO to show significant acceptance of exo-ketone 42a.
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The fact that the only difference between these two tricyclic CPDMO as the most effective BVMO.[19] The efficient pro-
ketones is the steric arrangements of the ring systems makes duction of lactone 8b by a whole-cell biotransformation
these substrates very appealing candidates for further stud- process is described here for the first time. Moreover, the
ies.
recombinant E. coli overexpressing CHMO from Xan-
Summarizing our substrate profiling, we were clearly able thobacter is the first biocatalyst able to produce lactone 12b.
to support our previous classification of the BVMO from The absolute configuration of lactone 7b was assigned as
Xanthobacter sp. ZL5 as belonging to the group of CHMO- in the literature.[18] For lactones 8b and 12b, assignment of
type biocatalysts. Both the substrate acceptance and the absolute configuration is still pending.
stereoselectivity compare favorably to the behavior of the
Another interesting result was encountered with the con-
CHMO from Acinetobacter as a prototype CHMO-group version of bridged bicyclic ketones (Scheme 3). The CHMO
enzyme, as opposed to the biocatalytic performance of from Xanthobacter sp. ZL5 proved to be an excellent cata-
CPMO-type proteins.
lyst for the conversion of the carbo-bridged bicyclic ketone
33a, giving the enantiopure (–)-lactone 33b.
However, the fact that the CHMO from Xantho-
bacter sp. ZL5 mainly catalyzes the epoxidation of ketone
34a to compound 34c instead of the Baeyer–Villiger oxi-
dation to lactone is an interesting starting point for further
studies. Baeyer–Villiger monooxygenases are known to cat-
alyze oxidations at other atoms apart from the classical oxi-
dations of ketones (reviewed in;[6] see also references therein
and[33]). In 2002, the first example of the epoxidation of
vinyl-phosphonates mediated by CHMO from Acineto-
bacter was reported.[46] Indeed, we have confirmed this ob-
servation in analytical-scale parallel experiments. Judging
by chiral-phase GC analysis, control experiments with
BL21 (DE3) cells not containing the expression plasmid
p11X5.1 but in the presence of IPTG and un-induced BL21
(DE3) cells transformed with the expression plasmid coding
for CHMO from Xanthobacter were unable to catalyze the
epoxidation of ketone 34a (only traces were detected) or
partially converted it to other minor side products (not
identified). In contrast, only BL21 (DE3) transformed with
the plasmid p11X5.1 and induced with IPTG catalyzed the
epoxidation reaction. Thus, we can exclude any interfering
reaction from the host and we firmly attribute this behavior
to the CHMO from Xanthobacter sp. ZL5. Future studies
with the whole-cell biocatalyst as well as the pure enzyme
are planned in order to investigate these findings more thor-
oughly.
2. Preparative-Scale Biotransformations
Preparative-scale biotransformations and purification of
products by flash column chromatography were carried out
to characterize the most representative novel products that
can be obtained with the CHMO from Xantho-
bacter sp. ZL5 as biocatalyst. The structures of pure lac-
tones were confirmed by NMR, and their optical rotations
were measured for correct assignment (see Exp. Sect. for
details). All reactions proceeded with very good yields and
high enantiomeric purities (consistent with screening ex-
periments).
Scheme 2 summarizes the selected reactions with substi-
tuted cyclohexanones. In all three cases, (–)-lactones 7b, 8b,
and 12b were formed by Xanthobacter CHMO-mediated
Baeyer–Villiger oxidation of the corresponding ketones.
Lactones were usually isolated in good yields and with ex-
cellent enantiomeric excesses (Scheme 2). The biocatalytic
formation of 7b in synthetically useful quantities is particu-
larly noteworthy, as only moderate conversion (72%) of the
corresponding substrate had previously been reported, for
Nevertheless, we consider our findings to represent a
very exceptional behavior for a BVMO. In the previously
reported case of a BVMO-mediated epoxidation, a Michael
acceptor-type alkene was oxygenated. This points to a nu-
cleophilic reaction, as can be expected according to the gen-
erally accepted biocatalytic reaction cycle via an FAD-per-
oxyanion intermediate.[47] However, the C=C double bond
in 34a represents an electron-rich functionality, and an elec-
trophilic oxygenation seems more likely to explain the for-
mation of epoxide 34c. Such a process is more similar to
Scheme 2. Desymmetrizations of 4-substituted cyclohexanones.
Scheme 3. CHMO Xanthobacter sp. ZL5-mediated conversion of bridged ketones.
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Monooxygenase Desymmetrization in Whole-Cell Systems
the BVMO-mediated oxidation of heteroatoms and, to the ductase” would catalyze the reduction of the double bond,
best of our knowledge, so far unprecedented at carbon cen- and then, in a second step, this saturated ketone would be
ters.
oxidized by the BVMO.[22] However, in this context the
The capacity of the enzyme to accept large substrates presence of such reduced intermediates has never been dem-
is illustrated by the excellent isolated yield for lactone 40b onstrated in situ for the biotransformation of a cyclic alk-
(Scheme 4). When we challenged nine BVMOs in screening enone. To address this question, cyclohexenone 43a was
experiments, poor isolated yields at best were observed in used as a model substrate for the reaction with the CHMO
the CHMO family, while CPMO-type enzymes did not from Xanthobacter sp. ZL5 as a whole-cell biocatalyst. Con-
transform the substrate at all. Undoubtedly, the CHMO trol experiments were carried out with BL21 (DE3) cells
from Xanthobacter sp. gave the highest isolated yield.
not containing the expression plasmid. Here, a clear kinetic
resolution of racemic unsaturated ketone 43a was observed,
yielding a mixture of prochiral 12a and (almost) optically
pure 43a. In parallel experiments, BL21 (DE3) cells trans-
formed with plasmid p11X5.1, in which protein expression
was induced with IPTG, partially converted the α,β-unsatu-
rated ketone 43a into fully saturated lactone 12b with the
same enantioselectivity as achieved in the biooxidation
starting from saturated ketone 12a. Interestingly, it was pos-
sible to detect a small amount of saturated ketone 12a by
chiral-phase GC and GC-MS, confirming the sequential
biotransformation process proposed previously (Scheme 5).
When a similar experiment was conducted with trans-
formed BL21 (DE3) cells containing the expression plasmid
p11X5.1 but without addition of IPTG, we nevertheless ob-
served formation of chiral lactone 12b; small amounts of
the intermediate 12a of the redox cascade were found ac-
cording to chiral-phase GC, and optically pure 43a re-
mained as result of the kinetic resolution catalyzed by the
reductase. We attribute these findings to basal transcription
from the promoter in the absence of inducer, especially after
long fermentation periods.
Accordingly, no unsaturated lactone was detected in the
fermentation medium. This finding has two major implica-
tions: on one hand, it confirms our previous hypothesis of
a biocatalytic redox cascade reaction. On the other hand, it
emphasizes the biotransformation of 4-methyl-4-phenylcy-
clohex-2-enone (43a) and its corresponding saturated form,
a ketone until now exclusively described as substrate for
CHMO Xanthobacter sp. As enone 43a is also a precursor
in the chemical synthesis of 12a, this redox cascade bio-
transformation reaction represents an appealing short-cut
to access optically pure 12b, underscoring the efficiency of
this recombinant whole-cell approach with respect to sys-
tem sustainability.
Scheme 4. Example of the conversion of structurally-demanding
ketones.
3. Sequential Two-Step Biotransformation Reaction
To characterize the biocatalytic behavior of this recombi-
nant whole-cell system further, we studied the oxidation of
the α,β-unsaturated cyclohexenone 43a (Scheme 5). Few ex-
amples describing acceptance and oxidation of enones by
Baeyer–Villiger monooxygenases have been reported. In an
early contribution, it was shown that wild-type whole cells
of Pseudomonas NCIMB 10007 transformed α,β-unsatu-
rated cyclohexenones into mixtures of saturated and unsat-
urated ketones and lactones.[48] In 1996, Bes et al. reported
the partial conversion of 5-hexylcyclopent-2-enone into the
unsaturated lactone by CPMO from Comamonas, both as a
whole-cell system and as pure enzyme.[49] Another BVMO
able to accept unsaturated aryl ketones is HAPMO from
Pseudomonas fluorescens ACB.[50] The chemoselective natu-
res of biocatalysts give access to lactones otherwise almost
impossible to obtain by classical chemical Baeyer–Villiger
oxidation, given that the oxidation of this kind of ketones
with mCPBA yields mainly epoxide products instead of lac-
tones.
Conclusions
In this work we have confirmed our previous classifica-
tion of the BVMO from Xanthobacter sp. ZL5 as a CHMO-
type enzyme according to its substrate selectivity and pref-
Scheme 5. Sequential two-step biotransformation of a 4-substituted
cyclohexenone.
The biotransformation of a set of cyclic alkenones was erence.[15] We have challenged the CHMO from Xan-
recently studied for recombinant E. coli-based expression thobacter sp. ZL5 as a whole-cell biocatalyst with a wide
systems for CHMO from Acinetobacter, CHMO 2 from variety of prochiral ketones containing diverse functional
Brevibacterium, and CPMO from Comamonas.[22] On the substituents in desymmetrization reactions. During this ex-
basis of parallel experiments with and without induction of tensive profiling, conversions were in most cases complete
the BVMO, our group proposed that two sequential enzy- or greater than 90%, and lactones were obtained with very
matic reactions took place. Firstly, a (so far unknown) “re- good to excellent enantiomeric excesses. The biocatalyst be-
Eur. J. Org. Chem. 2008, 1203–1213
© 2008 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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1209

M. D. Mihovilovic et al.
FULL PAPER
from recombinant Escherichia coli DH5α host cells by use of the
Wizard Plus SV Minipreps DNA Purification System (Promega).
Prior to activity assays, E. coli BL21(DE3) cells were chemically
transformed with this plasmid by standard procedures.[53]
haved similarly to CHMO from Acinetobacter with all sub-
strates reported in this work, with respect both to accept-
ance and to enantioselectivity.
While a great number of ketones were accepted and suc-
cessfully converted, six substrates gave access to highly
interesting enantiopure lactones containing sterically hin-
dered structural motifs. These were comprehensively char-
acterized after preparative biotransformations mediated by
CHMO from Xanthobacter sp. ZL5. The products are of
great interest as key intermediates for the first-time synthe-
sis of natural products by BVMO-mediated biooxidations.
A particularly promising example is the conversion of the
carbo-bridged ketone 33a in light of the great synthetic ver-
satility of this scaffold, as previously outlined by us.[51] An-
other rare and attractive feature of this enzyme is the ability
to catalyze the epoxidation of ketone 34a. Whether this en-
zyme will accept substrates other than ketones will be ex-
plored in future investigations. Further expression studies
will be also carried out to address the problem of the basal
activity encountered for this system.
Typical Procedure for Screening in Multiwell Plates: An overnight
preculture of E. coli BL21 (DE3) transformed with the plasmid
p11X5.1 was prepared by inoculating a single colony of the strain
into LB medium (10 mL) supplemented with ampicillin
(200 µgmL–1) and grown at 30 °C. Cultivation of recombinant bac-
teria for the expression of the CHMO from Xanthobacter sp. ZL5
was carried out in baffled Erlenmeyer flasks containing LB me-
dium supplemented with ampicillin (200 µgmL–1) and inoculated
with the overnight-grown preculture (1% v/v). The culture was
shaken at 120 rpm and at 30 °C until it reached an OD590 of about
0.7. At this point, IPTG was added to a final concentration of
0.1 m, and the culture was mixed well and distributed into 24-
well plate dishes. Aliquots of 1 mL were loaded in each well, the
appropriate ketone substrate (0.3–0.5 mg, as a solution in dioxane
or ethanol) was added, and the plate was shaken at 24 °C and at
120 rpm. After 24 h of reaction, samples (700 µL) were centrifuged,
and the supernatant was extracted with ethyl acetate or methylene
dichloride (700 µL) containing methyl benzoate (1 mM) as internal
standard. The organic phase was dried with anhydrous Na2SO4 and
analyzed by chiral-phase GC. Assignments were performed accord-
ing to the literature, with comparison of results with data from
CHMO Acinetobacter sp. NCIMB 9871 and/or results from other
BVMO-mediated biotransformations as indicated in Table 1,
Table 2, and Table 3. If available, experimental data from biotrans-
formations catalyzed by CHMO-type and CPMO-type enzymes
have been provided in Tables 1, 2, and 3 as references.
In conclusion, this versatile biocatalyst greatly expands
the repertoire of optically pure lactones that can be ob-
tained by whole-cell Baeyer–Villiger biooxidations. Its po-
tential for applications in key chemoenzymatic steps in the
synthesis of natural products and bioactive compounds is
presently being addressed by our group.
Preparative-Scale Biotransformations, Product Isolation, and Char-
acterization: Luria Bertani [LB: peptone (1% w/v), yeast extract
(0.5% w/v), and NaCl (1% w/v)] or Terrific Broth [TB: tryptone
(1.2% w/v), yeast extract (2.4% w/v), glycerol (0.4% v/v), KH2PO4
(17 m), and K2HPO4 (72 m)] media supplemented with ampicil-
lin (200 µgmL–1) were used for bacterial biotransformations. Pre-
cultures of E. coli BL21 (DE3) transformed with plasmid p11X5.1
were prepared in LB/ampicillin starting from an isolated colony of
fresh transformants and grown overnight at 30 °C with gentle shak-
ing (120 rpm). As a general procedure, fresh medium containing
ampicillin (200 µgmL–1) in baffled Erlenmeyer flasks was inocu-
lated with an overnight-grown preculture of the bacteria (1% v/v).
Antifoam was used if required. Cultures were shaken at 120 rpm
at 30 °C, and protein expression was induced with IPTG at the
appropriate OD590. Biotransformations were initiated upon ad-
dition of the corresponding substrate and β-cyclodextrin. Reaction
mixtures were incubated at 120 rpm and at 24 °C until completion
or until no further conversion was observed. This time-lapse was
different for each substrate, but as a general rule biotransforma-
tions lasted between 14 and 20 h. Cells were then removed by cen-
trifugation, and the supernatant was extracted repeatedly with ethyl
acetate or methylene dichloride (if necessary after saturation with
sodium chloride). The combined organic layers were dried with an-
hydrous Na2SO4, filtered, and concentrated. The crude products
were purified by standard flash column chromatography.
Experimental Section
Abbreviations: BVMO = Baeyer–Villiger monooxygenase, CHMO
=
cyclohexanone monooxygenase, CPMO
= cyclopentanone
monooxygenase, PAMO phenylacetone monooxygenase,
=
HAPMO = 4-hydroxyacetophenone monooxygenase, CPDMO =
cyclopentadecanone monooxygenase.
General: Chemicals and components of bacterial growth media
were purchased from commercial suppliers. Substrates were either
commercially available or synthesized in our laboratory (see corre-
sponding references in Tables 1, 2, and 3). Solvents were distilled
before use. Whenever possible, m-chloroperbenzoic acid was used
to oxidize ketones to lactones in order to obtain racemic stan-
dards.[52]
Chiral-phase gas chromatography was performed with a Thermo-
Finnigan Trace GC 2000 or a Focus GC instrument with a
BGB 173 or a BGB 175 column, 30 mϫ0.25 mm ID, 0.25 µm film.
For GC-MS analysis a GC Top 8000/MS Voyager (quadrupole,
EI+) instrument equipped with a standard BGB5 capillary column,
30 mϫ0.32 mm ID was employed. Combustion analysis was car-
ried out in the Microanalytic Laboratory, University of Vienna.
NMR spectra were recorded from CDCl3 solutions on a Bruker
AC 200 (200 MHz) or a Bruker Avance UltraShield 400 (400 MHz)
spectrometer, and chemical shifts are reported in ppm from Me4Si
as internal standard. Peak assignment was based on correlation
experiments. Specific rotations ([α]D) were determined with a Per-
Particular biotransformation procedures and physical properties of
1
products are described in detail below. H NMR, 13C NMR, m/z
and combustion analysis data are provided only for compounds
not reported in the literature previously.
kin–Elmer Polarimeter 241 by the following equation: [α]D
100ϫα/(cϫl); c [g/100 mL], l [dm].
=
(–)-(5S)-5-tert-Butyloxepan-2-one (7b): Fresh LB-ampicillin me-
dium (125 mL) was inoculated with an overnight preculture of the
corresponding recombinant E. coli strain (1% v/v) in a baffled Er-
Strains and Plasmid: The plasmid p11X5.1[13] encoding the cyclo-
hexanone monooxygenase from Xanthobacter sp. ZL5 was isolated
1210
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© 2008 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Eur. J. Org. Chem. 2008, 1203–1213

Monooxygenase Desymmetrization in Whole-Cell Systems
lenmeyer flask. The culture was incubated at 120 rpm and at 30 °C
on an orbital shaker for 3–4 h. After the system had reached an
optical density OD590 = 0.6–0.7, IPTG stock solution was added
to provide a final concentration of 0.134 m. Substrate 7a (55 mg,
0.357 mmol) was added neat along with β-cyclodextrin (1 equiv.).
The culture was incubated at 24 °C for 20 h. The biomass was re-
moved by centrifugation, saturated with sodium chloride, and ex-
tracted repeatedly with ethyl acetate. The combined organic layers
were dried with anhydrous Na2SO4 and filtered, and the solvent
was removed in vacuo.
TB-ampicillin containing a drop of antifoam (200 mL) was used to
grow the cells at 30 °C until an optical density of 11 at 590 nm was
reached. Then, IPTG (0.1 m) was added to initiate the induction
of protein expression. After about 30 min at 24 °C, ketone 33a
(50 mg, 0.409 mmol; as a solution in dioxane) and β-cyclodextrin
(1 equiv.) were added to start the reaction. Biotransformation was
performed at 24 °C and 120 rpm over 14 h. After this period, cells
were removed by centrifugation and the supernatant was extracted
repeatedly with methylene dichloride. The combined organic layers
were dried with anhydrous Na2SO4, filtered, and concentrated.
The crude product was purified by flash column chromatography
(silica gel, PE/EtOAc, 8:2), and (–)-(5S)-7b was obtained as color-
less crystals (50 mg, 0.294 mmol, 82%), m.p. 76–78 °C (lit.
The crude product was purified by flash column chromatography
(silica gel, stepwise gradient system of PE/EtOAc from 100% PE
to 100% EtOAc), and lactone 33b was obtained as colorless crys-
tals (40 mg, 0.289 mmol, 71%), m.p. 50–52 °C. [α]2D1 = –83.3
(Ͼ99% ee, c = 0.51, CHCl3). 1H NMR (200 MHz, CDCl3): δ =
1.64 (d, J = 11.5 Hz, 1 H), 2.20–2.45 (m, 1 H), 2.65–2.87 (m, 2 H),
2.89–3.07 (m, 1 H), 4.00–4.24 (m, 2 H), 5.83 (dd, J = 2.9, 5.7 Hz,
1 H), 6.10 (dd, J = 2.9, 5.7 Hz, 1 H) ppm. 13C NMR (50 MHz,
CDCl3): δ = 36.7 (d), 42.6 (d), 44.4 (t), 44.7 (t), 70.7 (t), 131.0 (d),
137.0 (d), 174.1 (s) ppm. m/z: 138 (100) [M]+, 109 (62), 79 (45), 66
(100). C8H10O2 (138.17): calcd. C 69.55, H 7.30; found C 69.29, H
7.25.
58.5 °C[54]). [α]2D0 = –38.5 (99% ee, c = 0.75, CHCl3) (lit. [α]2D0
=
–34.9 (98% ee, c = 0.78, CHCl3[18]). Spectral properties were in ac-
cordance with previous literature reports.[55,19]
(–)-5-Phenyloxepan-2-one (8b): Fresh LB-ampicillin medium
(125 mL) was inoculated with an overnight preculture of the corre-
sponding recombinant E. coli strain (1% v/v) in a baffled Erlen-
meyer flask. The culture was incubated at 120 rpm and at 30 °C on
an orbital shaker for 3–4 h. After an optical density OD590 = 0.6–
0.7 had been reached, IPTG stock solution was added to provide
3,9-Dioxatricyclo[3.3.1.02,4]nonan-7-one (34c): Fresh LB-ampicillin
medium (125 mL) was inoculated with an overnight preculture of
E. coli BL21 (DE3) transformed with p11X5.1 (1% v/v) in a baffled
Erlenmeyer flask. A drop of antifoam was added to avoid the for-
mation of foam. The culture was incubated at 120 rpm and at 30 °C
on an orbital shaker until the optical density had reached 0.8 at
590 nm and IPTG was provided to give a final concentration of
0.1 m. After an initial induction period of 1 h, ketone 34a (50 mg,
0.403 mmol; as a solution in ethanol) and β-cyclodextrin (1 equiv.)
were added to start the reaction. Biotransformation was carried
out over 24 h at 24 °C. After this period, cells were removed by
centrifugation, and the supernatant was extracted repeatedly with
methylene dichloride. The combined organic layers were dried with
anhydrous Na2SO4, filtered, and concentrated.
a
final concentration of 0.134 m. Compound 8a (55 mg,
0.316 mmol) was added neat, along with β-cyclodextrin (1 equiv.).
The culture was incubated at 24 °C for 20 h. The biomass was re-
moved by centrifugation, saturated with sodium chloride, and ex-
tracted repeatedly with ethyl acetate. The combined organic layers
were dried with anhydrous Na2SO4 and filtered, and the solvent
was removed in vacuo.
The crude product was purified by standard flash column
chromatography (silica gel, PE/EtOAc, 8:2), and 8b was obtained
as colorless crystals (53 mg, 0.279 mmol, 88%), m.p. 90–92 °C (lit.
100–101 °C[56]). [α]2D0 = –63.5 (98% ee, c = 2.41, CHCl3). Spectral
properties were in accordance with previous literature reports.[57]
(–)-5-Methyl-5-phenyloxepan-2-one (12b): Fresh LB-ampicillin me-
dium (230 mL) was inoculated with an overnight preculture of
E. coli BL21 (DE3) transformed with p11X5.1 (1% v/v) in a baffled
Erlenmeyer flask. Antifoam was added to prevent the formation of
foam. The culture was incubated at 120 rpm and at 30 °C on an
orbital shaker until an optical density of 0.7 at 590 nm was reached.
Then, IPTG was added to provide a final concentration of 0.1 m
and, after an initial induction period of about 1 h, ketone 12a
(100 mg, 0.532 mmol, solution in dioxane) and β-cyclodextrin
(0.1 equiv.) were further added to start the reaction. Biotransfor-
mation was carried out over 15 h at 24 °C. After this period, cells
were removed by centrifugation and the supernatant was repeatedly
extracted with ethyl acetate after saturation with sodium chloride.
The combined organic layers were dried with anhydrous Na2SO4,
filtered, and concentrated.
The crude product was purified by flash column chromatography
(silica gel, stepwise gradient system of PE/EtOAc from 100% PE
to 100% EtOAc), and epoxide 34c was obtained as a colorless solid
(33 mg, 0.235 mmol, 58%), m.p. 85–87 °C (pentane) {lit. 97–
98.5 °C [ethyl acetate,[58] 93 °C (CCl4)[59]]}. Spectral properties were
in accordance with previous literature reports;[58,59] m/z: 140 (72)
[M]+, 111 (8), 98 (72), 69 (100).
2,4-Divinyl-6-oxabicyclo[3.2.2]nonan-7-one (40b): Fresh LB-ampi-
cillin medium (250 mL) was inoculated with an overnight precul-
ture of the corresponding recombinant E. coli strain (1% v/v) in a
baffled Erlenmeyer flask. The culture was incubated at 120 rpm
and at 30 °C on an orbital shaker. After 3 h, the culture had
reached an optical density of 0.6 at 590 nm, and IPTG was added
to give a final concentration of 0.168 m. Substrate 40a (70 mg,
0.400 mmol) was added, together with β-cyclodextrin (1 equiv.).
The culture was incubated at 24 °C for 20 h. The biomass was re-
moved by centrifugation; the supernatant was saturated with so-
dium chloride, and extracted repeatedly with ethyl acetate. The
combined organic layers were dried with anhydrous Na2SO4 and
filtered, and the solvent was removed.
The crude material was purified by flash column chromatography
(silica gel, stepwise gradient system of PE/EtOAc from 100% PE
to 100% EtOAc), and (–)-12b was obtained as colorless crystals
(65 mg, 0.318 mmol, 60%), m.p. 94–96 °C. [α]2D1 = –23.2 (95% ee, c
= 4.13, CHCl3). 1H NMR (200 MHz, CDCl3): δ = 1.30 (s, 3 H),
1.75–2.05 (m, 2 H), 2.30–2.80 (m, 4 H), 4.05–4.35 (m, 2 H), 7.20–
7.45 (m, 5 H) ppm. 13C NMR (50 MHz, CDCl3): δ = 30.6 (t), 31.9
(q), 33.9 (t), 40.3 (t), 40.4 (s), 65.3 (t), 125.9 (d), 126.4 (d), 128.9
(d), 145.6 (s), 175.9 (s) ppm. 1H NMR and 13C NMR results repre-
sent a correction to previously reported data.[22] m/z: 207 (2) [M]+,
121 (100), 93 (60), 65 (68).
The crude material was purified by flash column chromatography
(silica gel, PE/EtOAc, 8:1), and lactone 40b was obtained as a col-
orless oil (70 mg, 0.360 mmol, 92%, diastereomeric ratio 98:2
anti/syn). [α]2D6 = +35.5 (c = 1.91, CHCl3). 1H NMR (400 MHz,
CDCl3, 25 °C): δ = 1.28–1.32 (m, 1 H), 1.72–1.77 (m, 1 H), 1.79–
1.81 (m, 1 H), 1.91–1.96 (m, 1 H), 2.07–2.14 (m, 2 H), 2.25–2.30
(–)-3-Oxabicyclo[4.2.1]non-7-en-4-one (33b): To improve the per-
formance of CHMO from Xanthobacter sp. ZL5 on ketone 33a,
Eur. J. Org. Chem. 2008, 1203–1213
© 2008 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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1211

M. D. Mihovilovic et al.
FULL PAPER
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(m, 1 H), 2.32–2.38 (m, 1 H), 2.77 (d, J = 5.8 Hz, 1 H), 4.56 (d, J
= 5.9 Hz, 1 H), 4.95–5.07 (m, 4 H), 5.77–5.91 (m, 2 H) ppm. 13C
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