The amination of racemic alcohols to produce enantiopure amines is an important green chemistry reaction for pharmaceutical manufacturing,r equiring simple and efficient solutions.H erein, we report the development of acascade biotransformation to aminate racemic alcohols.This cascade utilizes an ambidextrous alcohol dehydrogenase (ADH) to oxidize ar acemic alcohol, an enantioselective transaminase (TA) to convert the ketone intermediate to chiral amine,a nd isopropylamine to recycle PMP and NAD + cofactors via the reversed cascade reactions.T he concept was proven by using an ambidextrous CpSADH-W286A engineered from (S)-enantioselective CpSADH as the first example of evolving ambidextrous ADHs,a ne nantioselective BmTA, and isopropylamine.Abiosystem containing isopropylamine and E. coli (CpSADH-W286A/BmTA) expressing the two enzymes was developed for the amination of racemic alcohols to produce eight useful and high-value (S)-amines in 72-99 % yield and 98-99 %e e, providing with as imple and practical solution to this type of reaction.
Regioselective
hydroxylations of aromatic compounds are useful
reactions but often lack appropriate catalysts. Here a group of P450BM3
mutants (R47I/A82F/A328F, R47L/Y51F/F87V/L188P/I401P, R47I/Y51F/F87V,
R47L/Y51F/F87V/L181Q/L188P/I401P, and R47I/F87V/L188P) were developed
as unique catalysts for the p-hydroxylation of m-alkylphenols 1a–e with
high regioselectivity (91–99%) and conversion (95–99%)
to produce the corresponding useful and valuable m-alkylbenzene-1,4-diols 2a–e, respectively.
The mutated hydroxylases were developed by protein engineering of
P450BM3 monooxygenase via site-directed mutagenesis based on designed
mutations to reshape the substrate binding pocket and access channel.
Several engineered P450BM3 mutants showed good catalytic efficiency
(k
cat/K
M of
234–381 mM–1 min–1) for
the p-hydroxylations of m-alkylphenols 1a–e, respectively. Molecular docking
and simulation gave some insights into the structure-based understanding
of the enhanced regioselectivity and activity for the developed P450BM3
mutants, including the shorter distance between heme-oxygen atom and
C4-carbon (p-position) of substrates than the wild-type
enzyme in the catalytic pockets. Preparative biohydroxylations of m-alkylphenols 1a–e were
demonstrated by using E. coli cells coexpressing
individual P450BM3 mutants and glucose dehydrogenase GDH, giving high-yielding
synthesis of useful and valuable m-alkylbenzene-1,4-diols 2a–e.
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