Here the synthetic utility of fluoroacetate
dehalogenase RPA1163 is explored for the production of enantiomerically
pure (R)-α-fluorocarboxylic acids and (R)-α-hydroxylcarboxylic acids via kinetic resolution
of racemic α-fluorocarboxylic acids. While wild-type (WT) RPA1163
shows high thermostability and fairly wide substrate scope, many interesting
yet poorly or moderately accepted substrates exist. In order to solve
this problem and to develop upscaled production, in silico calculations and semirational mutagenesis were employed. Residue
W185 was engineered to alanine, serine, threonine, or asparagine.
The two best mutants, W185N and W185T, showed significantly improved
performance in the reactions of these substrates, while in
silico calculations shed light on the origin of these improvements.
Finally, 10 α-fluorocarboxylic acids and 10 α-hydroxycarboxylic
acids were prepared on a gram scale via kinetic resolution enabled
by WT, W185T, or W185N. This work expands the biocatalytic toolbox
and allows a deep insight into the fluoroacetate dehalogenase catalyzed
C–F cleavage mechanism.
While
the enzymatic oxidative cleavage of C–F bonds at achiral
centers catalyzed by P450 monooxygenases has been studied extensively,
less is known about the oxidative defluorination mechanism of chiral
substrates with fluorine being at chirality centers. Here, we report
that the use of ethyl 2-fluoro-2-phenylacetate as a stereochemical
probe enabled us to discover two oxidative defluorination paths catalyzed
by P450-BM3: α-site hydroxylation and hydroxylation at the remote
phenyl group. By means of intermediate identification and capture,
chemical mimics, key intermediate derivatization, and deuterium insertion
experiments as well as QM theoretical calculations, we succeeded in
demonstrating an unusual mechanism in which these two oxidative defluorination
routes occur in a P450 monooxygenase. This work presents valuable
insights into the oxidative degradation of organofluorines and provides
a convenient method for remote C–F bond activation.
As a kind of important three-carbon building block, vinamidinium salts have been widely used in organic synthesis. In this paper the synthetic methods of vinamidinium salts are summarized, and its applications in the synthesis of aldehydes, aromatic and heterocyclic compounds in recent years are reviewed.
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