A light-driven stereoselective biocatalytic oxidation

Article abstract of DOI:10.1002/anie.200605169

Let the sunshine in: Light can be used to drive enantioselective Baeyer-Villiger oxidations of cyclic ketones catalyzed by a flavin-dependent enzyme. Photochemical reduction of the flavin using ethylenediaminetetraacetate (EDTA) as the sacrificial electron donor closes the catalytic cycle, thus providing a means to directly regenerate reduced flavin cofactors without the need for costly nicotinamide cofactors as electron donors. (Chemical Equation Presented).

Full text of DOI:10.1002/anie.200605169

Angewandte
Chemie
DOI: 10.1002/anie.200605169
Biocatalysis
A Light-Driven Stereoselective Biocatalytic Oxidation**
Frank Hollmann, Andreas Taglieber, Frank Schulz, and Manfred T. Reetz*
During the last decades, numerous flavin-dependent mono-
oxygenases have been discovered, characterized, and studied
mechanistically.[1] They catalyze the efficient and selective
insertion of one oxygen atom from O2 into organic substrates,
as in Baeyer–Villiger (BV) reactions, aromatic hydroxyla-
tions, epoxidations, sulfoxidations, or even halogenations.[1,2]
As some of these reactions are difficult to perform selectively
using chemical catalysts,[3] this class of enzymes has gained
considerable interest as catalysts in synthetic organic chemis-
try.[2] Mechanistically, the reduced enzyme-bound flavin
(FAD or FMN) reacts with dioxygen from air to form a
flavin-peroxide species, which functions as the oxidant in the
desired oxygenation reaction.[1,2] To close the biocatalytic
Scheme 1. Comparison of the conventional regeneration of a Baeyer–
Villiger monooxygenase (BVMO, e.g. PAMO) for catalysis through
cycle, the reduced form of the flavin is then regenerated by a
nicotinamide cofactor (NADH or NADPH). As these
cofactors are expensive, a number of chemical, electrochem-
ical, and enzymatic regeneration methods have been devel-
oped to enable their use in only catalytic amounts.[4] However,
owing to the necessity of an additional catalytic system and
the lack of generality, these approaches are limited to
specialized applications. Thus, the quest for new approaches
to regeneration systems continues. Herein, we demonstrate
the utility of light as the driving force for the regeneration of
the reduced flavo-enzyme for catalysis. It is known that flavins
can be photochemically reduced using simple sacrificial
electron donors such as ethylenediaminetetraacetate
(EDTA).[5] Even though this finding opens up a fascinating
new way for the direct regeneration of reduced flavin
cofactors, thus circumventing the need for reduced nicotina-
mides as electron donors, it has never been exploited for
biocatalytic purposes.
We therefore devised a catalytic cycle in which NADPH is
replaced by light in combination with the sacrificial electron
donor EDTA, with the model reaction being the BVoxidation
of ketones. Thus, we introduce a new drastically simplified
system (Scheme 1B) compared to the traditional multicom-
ponent regeneration approach (Scheme 1A). As the flavin-
dependent enzyme, we chose a mutant of phenylacetone
monooxygenase[6] (PAMO-P3), which we had previously
engineered to catalyze the enantioselective BV reaction of
ketones such as 1a, 1b, and 3. These substrates are either not
enzymatic regeneration of NADPH (A) and the novel simplified light-
driven pathway using a flavin (B). Part (A) shows an enzyme-coupled
regeneration system employing the common glucose dehydrogenase
to catalyze the regeneration of the reduced nicotinamide cofactor. The
use of this coupled enzyme can be replaced by visible light, which
reduces the complexity of the setup by eliminating the need for a
coupled enzyme. (E-FAD: enzyme bound flavin adenine dinucleotide.)
accepted by the wild-type enzyme or they are oxidized with
poor stereoselectivity.[7] In that study, we showed that the
kinetic resolution of 1a and 1b catalyzed by mutant P3 is
highly R-selective (> 97% ee of products 2a and 2b) and that
in the case of rac-3 a 1:1 mixture of regioisomers (À)-4 (92%
ee) and (À)-5 (99% ee) is formed.[7]
Upon attempting the BV reaction of rac-1a in the
presence of EDTA, FAD, a white light bulb, and catalytic
amounts of the mutant monooxygenase P3, but in the absence
of NADP+, no conversion of 1a occurred. However, this
finding can be explained by the fact that the nicotinamide
cofactor stays bound to a Baeyer–Villiger monooxygenase
during the active catalytic cycle, suggesting a pivotal role of
the bound nicotinamide cofactor in sustaining a catalytically
active conformation of the enzyme.[1h,8]
We therefore repeated the reaction, this time in the
presence of catalytic amounts of NADP+. Gratifyingly, this
protocol was successful, giving almost the maximum theoret-
ical conversion of 50% in the kinetic resolution of ketone 1a
to lactone (R)-2a with excellent enantioselectivity (48%
conversion, 97% ee). The optical purity of the product is
essentially identical to that obtained from previous experi-
ments utilizing whole cells[7a] or an in vitro system based on
conventional cofactor regeneration.[7b] Apparently, the inher-
ent stereoselectivity of PAMO-P3 is not impaired by the
unnatural regeneration conditions (Table 1), suggesting a
non-altered mechanism of the actual oxidation reaction.
Moreover the decomposition products of EDTA have no
detrimental effect on the course of the reaction (Figure 1).
[*] Dr. F. Hollmann, A. Taglieber, F. Schulz, Prof. Dr. M. T. Reetz
Max-Planck-Institut für Kohlenforschung
Kaiser-Wilhelm-Platz 1, 45470 Mülheim/Ruhr (Germany)
Fax: (+49)208-306-2985
E-mail: reetz@mpi-muelheim.mpg.de
[**] This work was supported by the Fonds der Chemischen Industrie
and the Deutsche Forschungsgemeinschaft (RE 359/13-1). We
thank S. Ruthe and F. Kohler for GC analyses.
Supporting information for this article is available on the WWW
under http://www.angewandte.org or from the author.
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Table 1: Stereoselective Baeyer–Villiger reactions.[a]
apparent necessity for non-enzyme-bound FAD is in accord
with previous findings, suggesting a catalytic role of free flavin
in the reduction of the enzyme-bound cofactor, which is
otherwise inefficient (see Supporting Information).[5b]
In the case of ketone 1b, the reaction was likewise
successful, delivering essentially enantiomerically pure lac-
tone (R)-2b (97% ee) in 30% yield. Finally, the BV reaction
of rac-3 afforded as expected a 1:1 mixture of (À)-4 (92% ee)
and (À)-5 (95% ee) in 90% total yield. Thus, all of these light-
driven BV oxidations show essentially the same stereo- and
regioselectivity as the corresponding reaction using the
mutant P3 with the native NADPH as reductant.[7]
Substrate
Conversion [%]
ee [%]
Inspired by the encouraging results obtained using an
artificial source of light, we decided to utilize the most
abundant source of energy available, namely sunlight. This
“green” regeneration system provided results comparable to
those obtained using a light bulb. For example, in the case of
ketone 1a two hours of bright sunshine in central Europe
induced the expected stereoselective transformation, with a
conversion of 20% reached under such conditions. As the
weather turned cloudy, the conversion began to level off
(Figure 1). Compared to the use of a light bulb, the rates were
similar, whereas the conversion was limited as a result of the
sudden weather change.
1a
1b
3
48 (ꢀ50)
30 (40)
93 (95)
97 (96)
97 (99)
92 (4), ꢁ95 (5)
(92 (4), >99 (5))
[a] General conditions for light-driven reactions: 10 mm PAMO P3,
25 mm EDTA, 100 mm FAD, 250 mm NADP+, 1 or 2 mm substrate, 50 mm
Tris (pH 7.4); T=308C, 100 W Osram white light bulb. Values given in
parentheses refer to data obtained in the conventional NADPH-based
process[7] and are included here for comparison.
Note that under the current conditions, the catalytic
performance of PAMO is significantly lower than in cases
where
a
conventional regeneration system is used
(Table 2).[7b]
Table 2: Performance of the light-driven BVMO regeneration in com-
parison with a native regeneration system and an electrochemical system
of comparable simplicity.
Enzyme
Mediator
Souce of
reducing
equivalents
TOF[a]
TON[b]
[hÀ1
]
Enzyme
Mediator
PAMO[c]
StyA[9b]
FAD
EDTA, hn
cathode
2-propanol
10
104
394
96
26
9471
9.6
0.2
400
FAD
PAMO[7b]
NADPH[d]
Figure 1. Time-resolved conversion of the light-driven BV reaction of
rac-1a. Conditions for sunlight-driven reactions: 10 mm PAMO P3,
25 mm EDTA, 100 mm FAD, 250 mm NADP+, 1 mm substrate, 50 mm
Tris (pH 7.4). Reaction vessels were placed on aluminum foil and
exposed to sunlight. For comparison, the reaction employing a lamp
as light source is also shown.
[a] TOF: catalyst turnover frequency (initial rate). [b] TON: total turnover
number. [c] This study. [d] NADPH regeneration by a coupled enzyme.[7b]
Several reasons may account for this reduced perfor-
mance of the novel light-driven approach. Decoupling of the
regeneration from the enzymatic oxidation reaction to result
in formation of H2O2 can in principle severely impair enzyme
activity and stability. Therefore, we investigated the influence
of H2O2 on PAMO activity (Figure 2). PAMO proved to be
remarkably resistant to elevated concentrations of H2O2.
Thus, any inactivation of the enzyme caused by H2O2 accounts
only partially for the reduced performance. Therefore, we
suspect slow electron-transfer kinetics to be rate-limiting
under the present conditions, which is also in accord with the
linear course of the reaction (Figure 1). Further experiments
are underway to elucidate this hypothesis.
Control experiments corroborated the mechanistic postu-
late of Scheme 1B, including reactions in the absence of
either one of the components shown in Scheme 1B (i.e. light,
EDTA, FAD, or PAMO), with each experiment showing no
conversion of 1a. It is therefore clear that the tris(hydroxy-
methyl)aminomethane (Tris) base used as buffer is not the
source of electrons under these reaction conditions. More-
over, a putative flavo-H2O2-shunt pathway[4d] was excluded, as
in the presence of H2O2, FAD, and PAMO no BV oxidation
was found to occur (see below). Control experiments also
showed that FAD can be replaced by riboflavin or FMN,
which proves that no significant exchange of reduced FAD
with protein-bound cofactor occurs. In further experiments,
we found no evidence for the photoreduction of NADP+. The
In summary, we have devised and implemented exper-
imentally for the first time a catalytic scheme for a light-
driven flavin-dependent enzymatic reaction. Using light as a
source of energy and EDTA as a reservoir of electrons,
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Tris-HCl (pH 7.4) was incubated under aerobic conditions at 308C in
a water bath exposed to the light of a 100-W OSRAM white light bulb
(CLASSIC A, CLAS A CL 100, 230 V, E27/ES). The light was filtered
through 1 cm water and approximately 0.5 cm DURAN glass. The
approximate distance between the light source and the reaction
vessels was 6 cm. The reaction mixture was extracted with ethyl
acetate (275 mL; containing 40 mm n-hexadecane GC standard for 2-
phenyl cyclohexanone) and analyzed by GC and GC/MS using
authentic standards. For all experiments described here, PAMO-P3
was produced and purified by affinity chromatography as described
previously[7a] to allow for accurate quantification of the results.
However, the reaction works as well using crude enzyme as obtained
after bacterial lysis.[7b]
Received: December 21, 2006
Revised: January 29, 2007
Published online: March 13, 2007
Keywords: asymmetric catalysis · cofactors · enzyme catalysis ·
.
Figure 2. Tolerance of hydrogen peroxide by PAMO-P3. Preparations of
PAMO P3 were incubated in the presence of various concentrations of
H2O2 for 20 h. Subsequently, residual activities were determined using
a NADPH-depletion assay.[7] The graph shows the residual activities
relative to a sample incubated in the absence of H2O2.
kinetic resolution · photochemistry
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other oxidation and reduction reactions.
Experimental Section
A typical light-driven reaction was set up as follows: A mixture (final
reaction volume 250 mL) containing 10 mm PAMO-P3, 25 mm EDTA,
100 mm FAD, 250 mm NADP+, 1 mm or 2m m substrate, and 50 mm
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