10.1002/anie.202009733
Angewandte Chemie International Edition
RESEARCH ARTICLE
The authors declare no conflict of interest.
Conclusion
Keywords: Alcohol amination • Alcohol dehydrogenase •
Biocatalysis • Cascade biotransformation • Directed evolution
A simple and efficient cascade reaction system for the conversion
of racemic alcohols to enantiopure amines was developed, with a
novel concept of using an ambidextrous ADH and an
enantioselective TA as the only two ezymes for the target reaction
and isopropylamine as the only one co-substrate for the
regeneration of the two co-factors PMP and NAD+ via reversed
cascade reactions. The novel concept was successfully proven
by utilizing a newly engineered ambidextrous ADH (CpSADH-
W286A) for the oxidation of a racemic alcohol, an enantioselective
transaminase (BmTA) for the amination of the ketone
intermediate to produce chiral amine product, and isopropylamine
for co-factors regeneration. It has also been achieved by using a
biosystem containing the cells of recombinant E. coli (CpSADH-
W286A/BmTA) expressing the ADH and the TA, together with
isopropylamine. The use of biosystem containing E. coli
(CpSADH-W286A/BmTA) cells allows for the production of eight
useful and high-value (S)-amines 3a-c, 3e-g, 3i-j in 72-99% yield
and 98-99% ee from the corresponding easily available racemic
alcohols, respectively. This biocatalytic system is the simplest one
for ADH-TA catalyzed amination of racemic alcohols, enables
high-yielding production of the corresponding chiral amines with
high ee in a sustainable manner, and provides with a useful
solution to this challenging green chemistry reaction in
pharmaceutical synthesis. Two new ambidextrous ADHs
(CpSADH-W286A and CpSADH-W286G) were successfully
evolved from the highly (S)-enantioselective CpSADH, being able
to oxidize both enantiomers of racemic alcohols 1a-k to give the
corresponding ketones 2a-k with high conversions. To the best of
our knowledge, this represents the first example of engineering
an ambidextrous ADH via directed evolution. The success was
accomplished by mutating a large amino acid (tryptophan) in the
binding pocket into a smaller one (alanine or glycine), thus
widening the binding pocket of the enzyme. This could be a
general principle for the future engineering of ambidextrous
enzymes to accept both enantiomers of substrates. CpSADH-
W286A and CpSADH-W286G show unique substrate specificity,
being the only available ADHs for the high-yielding oxidation of
racemic benzylic alcohols and also capable of oxidizing other
types of racemic alcohols with high conversion. This significantly
expands the substrate scope of the biooxidation of racemic
alcohols and opens new possibility of using ambidextrous alcohol
oxidation enzymes for cascade reactions in green chemical
synthesis.
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Acknowledgements
This research was supported by GlaxoSmithKline (GSK) and
Singapore Economic Development Board (EDB) through a Green
and Sustainable Manufacturing (GSM) grant (project no. 279-000-
506-592). We are grateful to Gheorghe-Doru Roiban (GSK) and
Cyril Boudet (GSK) for the useful scientific discussion.
Conflict of interest
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