Angewandte
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prostaglandins, for example.[19] Small-scale conversions of
rac-bicyclo[3.2.0]hept-2-en-6-one were carried out with
TmCHMO, AcCHMO, and TfPAMO. Both enantiomers of
this ketone were fully converted by both CHMOs, yielding
almost exclusively one regioisomer from each enantiomer
(Figure S6A and Table S6).[20] By contrast, TfPAMO pro-
duced all four possible lactones, proving to be far less
regioselective than CHMOs. We also used the same
BVMOs to produce enantiomerically pure sulfoxides, which
are widely used in asymmetric synthesis and are often
biologically active.[21] The prochiral compound thioanisole
was chosen as a model substrate (Scheme S3). The CHMOs
exclusively produced the (R)-sulfoxide, whereas TfPAMO
produced both enantiomers, leading to an ee of only 16% for
the (R)-sulfoxide (Figure S6B, Table S6).[18c,22]
Having established that TmCHMO is an appealing
biocatalyst based on its thermostability, solvent tolerance,
and selectivity, we performed a more in-depth character-
ization of its mechanistic properties. The reaction mechanism
of a BVMO generally involves a C4a-peroxyflavin intermedi-
ate that forms a tetrahedral Criegee intermediate through
nucleophilic attack on the substrate carbonyl carbon
(Scheme S4). Rearrangement of the Criegee intermediate
yields the ester or lactone product.[23] The spectral changes for
TmCHMO during its catalytic cycle were monitored using
a stopped-flow spectrophotometer. Anaerobic reaction of
TmCHMO with NADPH resulted in the loss of the absorb-
ance peaks at 376 nm and 440 nm, which is consistent with the
formation of the two-electron-reduced enzyme. After mixing
the reduced TmCHMO with air-saturated buffer, a rapid
increase in absorbance at 355 nm was observed (k = 37 sÀ1),
together with a small absorbance decrease at 450 nm (Fig-
ure 2A). These spectral changes are indicative of the
formation of the C4a-peroxyflavin intermediate. The absorb-
ance at 355 nm was stable for 3 s and then slowly decreased
(k = 0.01 sÀ1) owing to decay of the intermediate, which is
consistent with hydrogen peroxide elimination to form the
reoxidized enzyme (k = 0.004 sÀ1). In a second set of experi-
ments, the anaerobically reduced TmCHMO was mixed with
cyclohexanone in air-saturated buffer. The absorbance at
355 nm increased for 0.1 s and then immediately decreased,
which demonstrates the low kinetic stability of the inter-
mediate in the presence of cyclohexanone (Figure 2B and
Figure S7). The rate of formation of the peroxyflavin was not
influenced by the presence of cyclohexanone, while its decay
rate was 80-fold higher than that measured in the absence of
this ketone. Collectively, these experiments suggest that
TmCHMO functions as a typical BVMO, forming a stable
flavin peroxide that can effectively perform substrate oxy-
genation. The kinetic stability of the peroxyflavin enables the
enzyme to efficiently couple NADPH and dioxygen con-
sumption with substrate oxygenation without leakage of
hydrogen peroxide, which can be harmful in the context of
large-scale biotransformations.[23a–c]
Figure 1. Effect of temperature and acetonitrile (ACN) on the stability
of TmCHMO and AcCHMO. Activities on cyclohexanone were mea-
sured.
25 min incubation in 14% acetonitrile at 208C, TmCHMO
remained highly active (> 80%) for at least 20 h under these
conditions (Figure 1B). These thermostability and solvent-
tolerance data clearly show that TmCHMO is a substantially
more robust biocatalyst than AcCHMO.
Besides influencing stability, the reaction medium can also
modify enzyme selectivity.[15–16] The effect of cosolvents
(Table S5) was determined by using 2-butanone, the con-
version of which to two possible regioisomers is of industrial
interest (Scheme S1).[17] All reactions were stopped after 48 h
at 178C. The ratio between the products methyl propanoate
and ethyl acetate was found to be about 3:7 for both purified
TmCHMO and AcCHMO in the absence of any cosolvent
(Figure S5). The same regioisomer ratio was observed using
whole cells of Escherichia coli expressing one or the other
CHMO (not shown). Next, we inspected the effect of various
solvents at 15% concentration. The two purified CHMOs
exhibited similar results, although the yields of TmCHMO
were generally higher. The strongest effect on regioselectivity
was observed with 2-methyl-1,3-dioxolane, which led to
almost exclusive production of ethyl acetate. 1,3-Dioxane
and 1,4-dioxane had a more moderate influence, since about
40% of the total product was methyl propanoate. We also
carried out reactions with 30% methanol or ethanol. These
cosolvents had a negligible effect on enzyme regioselectivity.
However, they considerably decreased the 2-butanone con-
version yield for AcCHMO (< 4%), while that for
TmCHMO remained high (96% and 56% for methanol and
ethanol, respectively). From these results, it can be concluded
that the robustness of TmCHMO makes possible to modulate
its regioselectivity through using cosolvents.
Along the lines of the previous experiments, the potential
of TmCHMO as a regioselective biocatalyst was further
probed by using rac-bicyclo[3.2.0]hept-2-en-6-one. This com-
pound can be converted by BVMOs into four products
(Scheme S2). This reaction is widely used to study the ability
of BVMOs to carry out the kinetic resolution of racemic
compounds,[18] and it is of interest for the synthesis of
For a more in-depth understanding of TmCHMO proper-
ties, its crystal structure in complex with FAD and NADP+ in
the oxidized and reduced states were solved to a resolution of
1.22 and 1.60 ꢀ (Table S7; Figure 3A, and Figure S8). In an
attempt to rationalize the relatively high thermostability, we
2
ꢀ 2016 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Angew. Chem. Int. Ed. 2016, 55, 1 – 5
These are not the final page numbers!
Romero, Elvira
