The
enzyme 4-oxalocrotonate tautomerase (4-OT) exploits an N-terminal
proline as main catalytic residue to facilitate several promiscuous
C–C bond-forming reactions via enzyme-bound enamine intermediates.
Here we show that the active site of this enzyme can give rise to
further synthetically useful catalytic promiscuity. Specifically,
the F50A mutant of 4-OT was found to efficiently promote asymmetric
Michael additions of nitromethane to various α,β-unsaturated
aldehydes to give γ-nitroaldehydes, important precursors to
biologically active γ-aminobutyric acids. High conversions,
high enantiocontrol, and good isolated product yields were achieved.
The reactions likely proceed via iminium ion intermediates formed
between the catalytic Pro-1 residue and the α,β-unsaturated
aldehydes. In addition, a cascade of three 4-OT(F50A)-catalyzed reactions
followed by an enzymatic oxidation step enables assembly of γ-nitrocarboxylic
acids from three simple building blocks in one pot. Our results bridge
organo- and biocatalysis, and they emphasize the potential of enzyme
promiscuity for the preparation of important chiral synthons.
Enzymes catalyzing asymmetric carboligation reactions typically show very high substrate specificity for their nucleophilic donor substrate components. Structure-guided engineering of the thermostable transketolase from Geobacillus stearothermophilus by directed in vitro evolution yielded new enzyme variants that are able to utilize pyruvate and higher aliphatic homologues as nucleophilic components for acyl transfer instead of the natural polyhydroxylated ketose phosphates or hydroxypyruvate. The single mutant H102T proved the best hit toward 3-methyl-2-oxobutyrate as donor, while the double variant H102L/H474S showed highest catalytic efficiency toward pyruvate as donor. The latter variant was able to complement the auxotrophic deficiency of Escherichia coli cells arising from a deletion of the dxs gene, which encodes for activity of the first committed step into the terpenoid biosynthesis, offering the chance to employ a growth selection test for further enzyme optimization.
Directed evolution of the thermostable transketolase from Geobacillus stearothermophilus based on a pH-based colorimetric screening of smart libraries yielded several mutants with up to 16-fold higher activity for aliphatic aldehydes and high enantioselectivity (>95% ee) in the asymmetric carboligation step.
Chiral
γ-aminobutyric acid (GABA) analogues represent abundantly
prescribed drugs, which are broadly applied as anticonvulsants, as
antidepressants, and for the treatment of neuropathic pain. Here we
report a one-pot two-step biocatalytic cascade route for synthesis
of the pharmaceutically relevant enantiomers of γ-nitrobutyric
acids, starting from simple precursors (acetaldehyde and nitroalkenes),
using a tailor-made highly enantioselective artificial “Michaelase”
(4-oxalocrotonate tautomerase mutant L8Y/M45Y/F50A), an aldehyde dehydrogenase
with a broad non-natural substrate scope, and a cofactor recycling
system. We also report a three-step chemoenzymatic cascade route for
the efficient chemical reduction of enzymatically prepared γ-nitrobutyric
acids into GABA analogues in one pot, achieving high enantiopurity
(e.r. up to 99:1) and high overall yields (up to 70%). This chemoenzymatic
methodology offers a step-economic alternative route to important
pharmaceutically active GABA analogues, and highlights the exciting
opportunities available for combining chemocatalysts, natural enzymes,
and designed artificial biocatalysts in multistep syntheses.
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