The
direct asymmetric reductive amination of ketones using ammonia
as the sole amino donor is a growing field of research in both chemocatalysis
and biocatalysis. Recent research has focused on the enzyme engineering
of amino acid dehydrogenases (to obtain amine dehydrogenases), and
this technology promises to be a potentially exploitable route for
chiral amine synthesis. However, the use of these enzymes in industrial
biocatalysis has not yet been demonstrated with substrate loadings
above 80 mM, because of the enzymes’ generally low turnover
numbers (k
cat < 0.1 s–1) and variable stability under reaction conditions. In this work,
a newly engineered amine dehydrogenase from a phenylalanine dehydrogenase
(PheDH) from Caldalkalibacillus thermarum was recruited and compared against an existing amine dehydrogenase
(AmDH) from Bacillus badius for both
kinetic and thermostability parameters, with the former exhibiting
an increased thermostability (melting temperature, T
m) of 83.5 °C, compared to 56.5 °C for the latter.
The recruited enzyme was further used in the reductive amination of
up to 400 mM of phenoxy-2-propanone (c = 96%, ee
(R) < 99%) in a biphasic reaction system utilizing
a lyophilized whole-cell preparation. Finally, we performed computational
docking simulations to rationalize the generally lower turnover numbers
of AmDHs, compared to their PheDH counterparts.
Biocatalysis is an effective tool
to access chiral molecules that
are otherwise hard to synthesize or purify. Time-efficient processes
are needed to develop enzymes that adequately perform the desired
chemistry. We evaluated machine-directed evolution as an enzyme engineering
strategy using a moderately stereoselective imine reductase as the
model system. We compared machine-directed evolution approaches to
deep mutational scanning (DMS) and error-prone PCR. Within one cycle,
it was found that machine-directed evolution yielded a library of
high-activity mutants with a dramatically shifted activity distribution
compared to that of traditional directed evolution. Structure-guided
analysis revealed that linear additivity might provide a simple explanation
for the effectiveness of machine-directed evolution. The most active
and selective enzyme mutant, which was identified through DMS and
error-prone PCR, was used for the gram-scale synthesis of the H4 receptor
antagonist ZPL389 with full conversion, > 99% ee (R), and a 72% yield.
This account focuses on the application
of ω-transaminases,
lyases, and oxidases for the preparation of amines considering mainly
work from our own lab. Examples are given to access α-chiral
primary amines from the corresponding ketones as well as terminal
amines from primary alcohols via a two-step biocascade. 2,6-Disubstituted
piperidines, as examples for secondary amines, are prepared by biocatalytical
regioselective asymmetric monoamination of designated diketones followed
by spontaneous ring closure and a subsequent diastereoselective reduction
step. Optically pure tert-amines such as berbines
and N-methyl benzylisoquinolines are obtained by
kinetic resolution via an enantioselective aerobic oxidative C–C
bond formation.
Research highlights► Novel C–C bond formations from lyases, oxidoreductases and transferases reviewed. ► Highlights from lyases are the Stetter reaction, and the synthesis of N-heterocyclases and the first intermolecular Diels-Alderase. ► The highlight from oxidoreductases is the aerobic oxidative C–C coupling.
Prochiral bicyclic diketones were transformed to a single diastereomer of 3‐substituted cyclohexylamine derivatives via three consecutive biocatalytic steps. The two chiral centres were set up by a CC hydrolase (6‐oxocamphor hydrolase) in the first step and by an ω‐transaminase in the last step. The esterification of the intermediate keto acid was catalysed by a lipase in the second step if possible. For two substrates the CC hydrolytic step as well as the esterification could be run simultaneously in a one‐pot cascade in an organic solvent. In one example, the reaction mixture of the first two steps could be directly subjected to bio‐amination in an organic solvent without the need to change the reaction medium. Depending on the choice of the ω‐transaminase employed and the substrate the cis‐ as well as the trans‐diastereomers could be obtained in optically pure forms.
Although CC bond hydrolases are distributed widely in Nature, they has as yet have received only limited attention in the area of biocatalysis compared to their counterpart the C‐heteroatom hydrolases, such as lipases and proteases. However, the substrate range of CC hydrolases, and their non‐dependence on cofactors, suggest that these enzymes may have considerable potential for applications in synthesis. In addition, hydrolases such as the β‐diketone hydrolase from Rhodococcus (OCH) are known, that catalyse the formation of interesting chiral intermediates. Further enzymes, such as kynureninase and a meta‐cleavage product hydrolase (MhpC), are able to catalyse carbon‐carbon bond formation, suggesting wider applications in biocatalysis than previously envisaged. In this review, the distribution, catalytic characteristics and applications of CC hydrolases are described, with a view to assessing their potentialfor use in biocatalytic processes in the future.
Background: Recent methodology development in directed evolution of stereoselective enzymes has shown that various mutagenesis strategies based on saturation mutagenesis at sites lining the binding pocket enable the generation of small and smart mutant libraries requiring minimal screening.
Methods:In this endeavor, limonene epoxide hydrolase (LEH) has served as an experimental platform, the hydrolytic desymmetrization of cyclohexene oxide being the model reaction with formation of (R,R)-and (S,S)-cyclohexane-1,2-diol. This system has now been employed for testing reduced amino acid alphabets based on the Hecht concept of binary patterning, with and without additional hydrophobic amino acids.
Results and Conclusions:It turns out that in binary pattern based saturation mutagenesis as applied to LEH, polar amino acids are seldom introduced. When applying binary patterning in combination with additional hydrophobic amino acids as building blocks in iterative saturation mutagenesis, excellent LEH variants were evolved for the production of both (R,R)-and (S,S)-diols (80-97 % ee), but again the introduction of polar amino acids occurs rarely. Docking computations explain the source of enhanced and inverted stereoselectivity. Some of the best variants are also excellent catalysts in the hydrolytic desymmetrization of other meso-epoxides, although both enantiomeric diols are not always accessible.
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