inserto CO M/J05 - Chemistry Today

Daniela V.
Rial
Marko D.
Mihovilovic
ABSTRACT
Baeyer-Villiger monooxygenases are highly promiscuous
enzymes that enable stereoselective conversions of ketones to
esters or lactones. Currently, several enzymes have been
identified and characterized for their potential as biocatalysts
capable to perform desymmetrizations, (dynamic) kinetic
resolutions and regiodivergent transformations for the production
of enantio- and regiocomplementary products. Additionally,
advanced fermentation techniques have been utilized for the upscaling
of such biooxygenations to kilogram scale using either
isolated enzymes or recombinant whole-cells.
INTRODUCTION
The oxidation of cyclic or linear ketones is a well-established
reaction in synthetic chemistry for generation of lactones or
esters, respectively. It was initially described by Adolf Baeyer and
Victor Villiger in 1899 (1). Chiral lactones are highly desired
intermediates for the synthesis of natural products and
worthy bioactive compounds. Therefore, further advances
in the field of selective Baeyer-Villiger oxidations are
greatly appreciated by the synthetic community. Certain
promising approaches using metal-assisted catalysis and
organocatalysis have been reported for chiral variants of
this transformation (2). With the increasing attention for
green chemistry methods, enzyme-mediated
biotransformations have become practical synthetic
alternatives for many different types of reactions (3-5).
During the last decades, the microbial Baeyer-Villiger
oxidation matured into a valuable tool for accessing
interesting building blocks in a highly chemo-, regio-, and
stereoselective fashion, allowing particularly useful
oxidation reactions to take place (6, 7).
In view of the ability of several microorganisms to grow on
cyclic ketones or alcohols, the metabolic pathways as well
as the catalysts were investigated. Baeyer-Villiger
monooxygenases (BVMOs) were identified as the key
enzymes involved in the oxygen insertion into a carbon-
carbon bond as crucial step. In particular within recent
years, several such biocatalysts have become available by
creating recombinant heterologous overexpression systems
based on substantial progress in molecular biology and
genome analysis (8, 9). Group I BVMOs are FADdependent
flavoenzymes (10), which display remarkable
substrate promiscuity and makes them appealing catalytic
entities. An unrooted tree showing the phylogenetic
relationships between synthetically-relevant BVMOs,
currently available as recombinant systems, is shown in
Figure 1. Names and abbreviations for the enzymes are
provided in the same graphic.
BVMOs catalyze the oxidation of a ketone at expense of
oxygen and NADPH in a way that one atom of oxygen is
inserted in the substrate and the other one is reduced to
water. The mechanism of this reaction has been a matter of
deep analysis for long time (reviewed in 7) and a model
based on kinetic studies of the catalytic performance of
PAMO was reported just recently (18). A very appealing
feature of this particular enzyme is its thermostability.
Moreover, PAMO was the first BVMO to be crystallized and
its three-dimensional structure was elucidated by X-ray
diffraction (19). This represented a great contribution in the
field and it opened additional possibilities to develop
Supplement to Chimica Oggi/CHEMISTRY TODAY Vol 26 nr 4 • Focus on Biocatalysis
Baeyer-Villiger
Monooxygenases
in asymmetric synthesis and beyond
homology models for other BVMOs. As BVMOs depend on
nicotinamid cofactors to perform their redox chemistry, recycling
of the reduction equivalent is a critical aspect in order to allow for
economic application in compound synthesis (Figure 2). This
involved dual-enzyme strategies when using purified biocatalysts
with an auxiliary substrate like formate or glucose-6-phosphate.
By applying the whole-cell concept the problem of NADPH
regeneration was simplified and further improved by the use of
recombinant expression systems for the production of large
quantities of the required enzyme (For a review see (7)).
The application of BVMOs in asymmetric synthesis was hampered
for a long time by substrate and product inhibition,
complemented by possible toxicity in whole-cell
biotransformations. Recent studies have outlined strategies to
circumvent such obstacles: Particularly appealing approaches
involve the utilization of non-growing cells [20] or two-phase
fermentation protocols. Applying the SFPR (substrate feeding –
Figure 1. Phylogenetic tree of BVMOs. Full references concerning the cloning
and expression of these enzymes until early 2006 can be found in (7). Names
and Abbreviations: cyclohexanone monooxygenase from Acinetobacter
NCIMB 9871 (CHMO Acineto), cyclopentanone monooxygenase from
Comamonas NCIMB 9872 (CPMO), phenylacetone monooxygenase from
Thermobifida fusca (PAMO), cyclohexanone monooxygenase 1 from
Brevibacterium HCU (CHMO Brevi1), cyclohexanone monooxygenase 2 from
Brevibacterium HCU (CHMO Brevi2), cyclohexanone monooxygenase from
Rhodococcus Phi1 (CHMO Rhodo1), CHMO from Rhodococcus Phi2
(CHMO Rhodo2), cyclohexanone monooxygenase from Arthrobacter BP2
(CHMO Arthro), cyclohexanone monooxygenase from Brachymonas
petroleovorans (CHMO Brachy), cyclohexanone monooxygenase from
Xanthobacter sp. ZL5 (CHMO Xantho), cyclododecanone monooxygenase from
Rhodococcus ruber SCI (CDMO), 4-hydroxyacetone monooxygenase from
Pseudomonas fluorescens ACB (HAPMO). More recently cloned enzymes are:
Baeyer-Villiger monooxygenase from Mycobacterium tuberculosis H37Rv (gene
Rv3854c) (BVMO M. tuberculosis Rv3854c (EtaA)) (11), as well as Baeyer-Villiger
monooxygenase from Mycobacterium tuberculosis H37Rv (gene Rv3083)
(BVMO M. tuberculosis Rv3083) and Baeyer-Villiger monooxygenase from
Mycobacterium tuberculosis H37Rv (gene Rv3049c) (BVMO M. tuberculosis Rv3049c)
(12), cyclopentadecanone monooxygenase from Pseudomonas sp. HI-70
(CPDMO) (13), Baeyer-Villiger monooxygenase from Pseudomonas fluorescens
DSM 50106 (BVMO P. fluorescens) (14), Baeyer-Villiger monooxygenase from
Pseudomonas putida KT2440 (BVMO P. putida) (15), Baeyer-Villiger
monooxygenase from Pseudomonas veronii MEK700 (BVMO P. veronii) (16) and
cyclohexanone monooxygenase from Arthrobacter sp. L661 (17).
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product removal) concept, administration
of a resin to act as reservoir for both
substrate and product has been
successfully implemented in recombinant
whole-cell biotransformations on various
substrates and expression strains [21,
22]. This strategy enables access to kgscale
biotransformations upon further
optimization of the fermentation
parameters and equipment [23].
The following sections highlight some of
the most recent synthetic applications of
Baeyer-Villiger monooxygenases
complementing and updating previous
comprehensive reviews (6,7).
DESYMMETRIZATION REACTIONS
Stereoselectivity and enantiocomplementarity are critical aspects
for the general application of BVMOs in synthesis and were
extensively investigated by our group. Eight BVMOs - as
recombinant whole-cell systems – were challenged to catalyze the
desymmetrization of several structurally diverse prochiral ketones
(Scheme 1) (24). According to their stereopreferences and
substrate specificities, BVMOs were classified into two main
groups of cycloketone-converting enzymes: the CHMO- and the
CPMO-type BVMOs. The CHMO-type BVMOs (CHMOAcineto, CHMORhodo1, CHMORhodo2, CHMOArthro, CHMOBrachy) displayed
the same enantioselectivity as CHMOAcineto while the CPMO-type
BVMOs (CPMO and CHMOBrevi2) gave antipodal lactones. A
borderline position was adopted by CHMOBrevi1 that showed a
modified biocatalytic behaviour. These experimental results were
correlated with protein information by phylogenetic analysis. The
protein sequences clustered into two distinct groups which
matched the enantiodivergent trend observed for the conversion
of cyclic ketones and CHMOBrevi1 adopted a position between
these clusters (24).
This divergent behaviour was confirmed in subsequent reports.
The CHMO from Xanthobacter sp. ZL5 was comprehensively
studied and established as a synthetically relevant BVMO. A
phylogenetic analysis clustered this enzyme in the branch of
CHMO-type cycloketone-converting BVMOs in agreement with
the substrate preferences and stereoselectivities observed for the
enzyme as a whole-cell biocatalyst (25, 26). This biocatalyst was
the first to convert 4-methyl-4-phenyl-cyclohexanone in excellent
e.e. Moreover, as a whole-cell biocatalyst it also converted 4methyl-4-phenyl-cyclohexen-2-one
to the corresponding fully
saturated lactone in a sequential two step biotransformation
process probably taking advantage of an intracellular reductase.
The entry to enantiocomplementary lactones in high optical
purities provides great platforms
for the synthesis of natural products
(Scheme 3). Several indole
alkaloids can be synthesized from
a fused bicyclic precursor (27-29).
Antipodal lactones of various fused
cyclopentanones can be obtained
by CHMO- and CPMO- mediated
biooxidation of the corresponding
starting ketones (26, 30, 31).
Using recombinant whole-cells
overexpressing CHMOXantho it was
possible for the first time to access
the sterically demanding carbabridged
bicyclic lactones (26). A
chiral oxo-bridged bicyclic lactone
obtained by stereoselective Baeyer-
Villiger biooxidation using
recombinant whole-cells
overexpressing CPMO was used as
Figure 2. Enzymatic regeneration of NADPH
coupled to BVMO biooxidation. Group I
BVMOs catalyze the oxidation of cyclic
ketones to lactones in a NADPH-dependent
manner. Fusion proteins between a NADPHrecycling
enzyme and BVMOs represent selfsufficient
systems for Baeyer-Villiger
biooxidations. See text for details.
Scheme 1. Desymmetrizations of prochiral cyclic ketones
pivotal intermediate for the formal
synthesis of several natural products
containing a tetrahydrofuran core such
as carba-nucleosides like (+)showdomycin,
(+)-trans-kumausyne, and
goniofufurone analogs (32). A combined
photochemo-enzymatic route to the
bicyclo[4.2.0]octane ring system was
also reported recently (33).
The above library of complementary
BVMOs was also applied to the
desymmetrization of a series of βsubstituted
cyclobutanones. Access to
antipodal lactones was gained using
CHMO- and CPMO-type enzymes while
CHMO Brevi1 showed again a particular
behaviour (26, 34). Chiral β-substituted-γ-butyrolactones are key
intermediates in the synthesis of pharmaceutical products like
baclofen, several lignans, and modified amino acids.
BVMOs are usually favouring the Baeyer-Villiger pathway even in
the presence of other oxidizable heteroatoms incorporated into
the substrate structures (35, 36). However, in the absence of a
carbonyl functionality as primary target, also sulfoxidations and
N-oxide formations (7 and references therein) were observed
with PAMO and HAPMO as particularly useful enzymes (37, 38,
39). The asymmetric oxidation of several aromatic sulfides to
sulfoxides proceeded in good enantioselectivities in most of the
cases. Moreover, racemic sulfites were converted to sulfates in a
kinetic resolution process in moderate to good selectivities for
both chiral sulfites and sulfates (40). Other substrates were
accepted as well, broadening the synthetic applicability of
BVMOs, especially considering the high stability of PAMO. A
patent describing the use of a BVMO for the synthesis of
modafinil, a psychostimulant agent, underscores BVMOs as
sulfoxidation agents (41, 42). Epoxidation of vinyl-phosphonates
was observed for the CHMO Acineto by a nucleophilic reaction in
which a Michael-acceptor type alkene is oxygenated (43). More
recently, also epoxidation at a non-activated alkene was reported
to be catalyzed by CHMO Xantho via a process in which an
electrophilic reaction presumably explains the formation of the
epoxide (26).
REGIOSELECTIVE BIOTRANSFORMATIONS
AND KINETIC RESOLUTIONS
Regioselectivity is another key aspect for synthetic applications of
BVMOs. Scheme 2 shows the four possible products obtainable
by biooxidation of racemic bicyclo[3.2.0]hept-2-en-6-one. This
racemic ketone can be converted into four lactones: two
regioisomeric lactones (“normal” and “abnormal” based on the
classical migratory preference of
the higher substituted carbon
center) and two enantiomers of
each. A regiodivergent trend for
the Baeyer-Villiger biooxidation of
such substrates was established
already in early work and the
present enzyme collection enables
access to specific isomers: CHMOtype
enzymes and CHMOBrevi1 gave a nearly equimolar mixture of
“normal” and “abnormal” lactones
in excellent enantiomeric excess
while the CPMO family yielded
mainly a racemic mixture of the
“normal” lactone (44). This
behaviour is complemented by the
kinetic resolution of racemic
precursors to yield predominantly
“abnormal” optically active
Supplement to Chimica Oggi/CHEMISTRY TODAY Vol 26 nr 4 • Focus on Biocatalysis

lactones using a BVMO from
Mycobacterium tuberculosis H37Rv
(45, 46). All biooxidation products
represent highly valuable
intermediates for various marine
pheromones and bioactive compounds such as prostaglandins.
The industrial applicability of the asymmetric bioconversion of
racemic bicyclo[3.2.0]hept-2-en-6-one by CHMO Acineto was
demonstrated when the reaction was successfully scaled up to
kilogram level using both fed-batch conditions (47) as well as a
resin-based in situ substrate feeding and product removal
technology, glycerol feed control and improved oxygenation
conditions (48). In 2006 and 2008 two further optimized
procedures were reported using recombinant E. coli cells
overexpressing the CHMO Acineto as biocatalysts, the SFPR concept,
an optimal provision of oxygen and a fine control of the
bioprocess to reach high productivity levels (49, 50).
The BVMO mediated regiodivergent oxidation of terpenones is
another impressive example on how the active site of the enzyme
can affect the migratory preference by imposing a particular
geometry and, hence, favouring only one stereoelectronic
arrangement to produce a single product. Utilizing the
regiodivergent trends displayed by the presently available
BVMOs, it was possible to access all regio- and enantiomeric
lactones of trans- and cis-dihydrocarvone and carvomenthone
using pure enantiomers of the ketones as starting materials (51).
Similar observations were made within the biooxygenation of 3substituted
cyclopentanones and -hexanones providing access to
“proximal” and “distal” lactones in high optical purities (52, 53).
The conventional kinetic resolution of a racemic substrate enables
the stereoselective oxidation of one enantiomer of the substrate
leaving the antipodal ketone intact in enantiopure form with a
maximum yield of 50% for each optically enriched product.
Several synthetically relevant substrates derived from cycloketones
were studied in recent years (for a comprehensive summary see
7) and the biotransformation products were successfully used in
the chiral synthesis of pharmaceutical products and pheromones.
A significant contribution in this area of enzyme mediated
Baeyer-Villiger oxidations was the
development of a dynamic kinetic
resolution process (Scheme 3) in which
the substrate was racemized at elevated
pH within a whole-cell biotransformation
achieving 86% yield of lactone in 96 %
e.e. (54). The initial approach was
subsequently further refined by employing
an anionic exchanger resin as racemizing
agent at neutral pH (55).
While previously neglected, linear ketones received increasing
attention in very recent times. Kinetic resolution of racemic
aliphatic ketones was reported for the BVMO from Pseudomonas
fluorescens DSM 50106 (56). The oxygenation of 4-hydroxy-2ketones
to give hydroxyalkyl acetates is particularly interesting
and represents a synthetic alternative to the dioxygenation of
terminal alkenes. The remaining hydroxyketones can also be
isolated in optically pure form. Recently, two other BVMOs from
Pseudomonas sp. were cloned and were shown to prefer aliphatic
acyclic ketones as substrates expanding the synthetic applicability
of BVMOs (15, 16).
ADVANCES IN BAEYER-VILLIGER
MONOOXYGENASES DESIGN
While an increasing diversity of BVMOs has become available
over recent years (and this process is not completed yet due to a
progressing number of genomes being sequenced), wild-type
enzymes can have limitations with respect to substrate scope,
stereospecificity, catalytic efficacy, or stability. Consequently,
modifications to tune the biocatalytic performance of such
enzymes may be required. The creation of such “designer
Scheme 2. Regiodivergent biooxidation of racemic
bicyclo[3.2.0]hept-2-en-6-one
Scheme 3. Dynamic kinetic resolution of
2-substituted cycloketones
Supplement to Chimica Oggi/CHEMISTRY TODAY Vol 26 nr 4 • Focus on Biocatalysis
enzymes” requires a detailed
understanding of the catalytic cycle
on molecular level. Presently, this is
still an ongoing process with
contributions from structure
determination, kinetics, and substrate profiling. Undoubtedly, very
valuable information is provided by the identification of “hotspots”
that affect the catalytic performance of the enzymes.
Directed Evolution of BVMOs
The stereoselectivity of CHMO Acineto towards 4hydroxycyclohexanone
was evolutionary improved by errorprone
PCR (57). An increase in substrate promiscuity and even
the formation of enantiocomplementary products were observed
within 1-2 rounds of mutagenesis (58). Mutations L143F and
F432S were identified to have a drastic effect on
enantioselectivity. A semirational approach (restricted CASTing
using NDT degeneracy) was applied to CPMO in order to
improve its enantioselectivity (59). The approach focused on two
regions in the neighbourhood of positions 156 and 450 of
CPMO (homolog to positions 143 and 432 in CHMO Acineto).
Some of the mutants obtained showed enhanced
enantioselectivity for 4-methylcyclohexanone and 4acetoxycyclohexanone.
Rational Re-design of BVMOs
In order to expand the substrate range of PAMO while retaining its
thermostability, mutations were designed by comparing the
structure of PAMO with a homology model of CHMO Acineto. Hence,
PAMO was turned into a phenylcyclohexanone monooxygenase
by deleting major parts of a bulge found in the arginineinteracting
loop near the active site (60). To further expand the
substrate acceptance profile of PAMO, a rational re-design of the
enzyme was carried out by site-directed mutagenesis based on the
modelled structure of CPMO, a related BVMO (41 % identity in
sequence), although substrate profiles are very different (39). The
mutation of M446 of PAMO to G effectively broadened the
substrate profile to some prochiral cyclic ketones, sulfides and
amines not accepted by wild type PAMO.
Even more interestingly, it increased the
enantioselectivity especially for the
oxidation of sulfides. This mutant also
converted indole to indigo blue in a
reaction described for the first time for a
BVMO. Very recently, three fusion proteins
composed of phosphite dehydrogenase
(PTDH) for NADPH regeneration and
NADPH-dependent BVMOs (PTDH-PAMO,
PTDH-CHMO Acineto and PTDH-CPMO) were constructed and
successfully tested in desymmetrization reactions and
regiodivergent oxygenations using whole-cells systems, crude cell
extracts and purified enzymes (61). The aim of this prove-ofconcept
study was the creation of self-sufficient BVMOs capable to
recycle NADPH by covalent linkage of a suitable additional
domain. It was interesting to note, that the addition of a completely
novel domain had only very minor effects on the biocatalytic
behaviour of the fusion enzymes. As a major improvement, the
bifunctional enzymes can operate on very low concentrations of
NADPH and utilize a cheap and convenient auxiliary substrate for
cofactor regeneration (Figure 2). This novel concept may represent
a very promising advance in NADPH-dependent flavoenzyme
mediated-biocatalysis aiming at the application of BVMOs in
industrial processes.
CONCLUSIONS AND PERSPECTIVES
Nowadays, a highly complementary platform of BVMOs for
various reactions such as desymmetrizations, regiodivergent
oxygenations, and (dynamic) kinetic resolutions is available to
operate on functionally and structurally diverse substrates
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(cycloketones, aromatic ketones, linear ketones). It is possible to
access both enantiomers of a lactone in an optically pure form
and good synthetic yields using purified enzymes or recombinant
whole-cell systems. Kinetic resolution and regiodivergent
biooxidation of racemic ketones give access to lactones
impossible to obtain by conventional chemical methods. Although
versatile, BVMOs are extremely chemoselective. Therefore,
ketones carrying functionalities incompatible with chemical
oxidation can also be used as starting materials. Scale-up of
BVMO mediated biotransformations has been demonstrated to
pilot-plant capacities. However, it is still a developing field and
several aspects (enzyme stability, improved efficiency) have to be
addressed and solved in a satisfactory manner. Present day
molecular biology provides the required tools and additional
progress may be expected in the immediate future to ultimately
establish enzyme mediated Baeyer-Villiger oxidation as an
attractive process in asymmetric organic synthesis from lab-scale
to industrial production.
ACKNOWLEDGEMENTS
The authors thank the Austrian Science Fund FWF for the
continuous support of our research in BVMO mediated
biooxygenations (presently funded within project no. P18945).
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Readers interested in a complete list of references are kindly invited to write to the
author at mmihovil@pop.tuwien.ac.at
DANIELA V. RIAL, MARKO D. MIHOVILOVIC*
*Corresponding author
Vienna University of Technology, Institute of Applied
Synthetic Chemistry
Getreidemarkt 9/163-OC, A-1060, Vienna
Austria