Enantioselective Synthesis of Pharmaceutically Active γ-Aminobutyric Acids Using a Tailor-Made Artificial Michaelase in One-Pot Cascade Reactions

Abstract

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.

In Situ Acetaldehyde Synthesis for Carboligation Reactions

“…We initially focused on the 4‐OT catalyzed Michael‐type addition of 5 to 6 yielding 7 , an important precursor for the γ‐aminobutyric acid analogue phenibut . As a catalyst, the previously engineered 4‐OT L8Y/M45Y/F50A variant was selected . Combination of 4‐OT L8Y/M45Y/F50A and 6 with in situ synthesized 5 (via both routes I and II) afforded R ‐ 7 in high enantiopurity ( er up to 98:2) and good to excellent isolated yields (up to 96 % compared to 6 ; Table ).…”

“…4‐OT is unique in that it can use 5 as substrate for C−C bond‐forming Michael‐type additions to nitroolefin acceptors and for aldol condensations with benzaldehydes . Previously, we have demonstrated that 4‐OT can be combined with other biocatalysts and chemocatalysts to convert 6 and 8 into GABA analogues using two complementary one‐pot cascade reactions . However, both routes rely on 5 as a substrate.…”

Tuning Enzyme Activity for Nonaqueous Solvents: Engineering an Enantioselective “Michaelase” for Catalysis in High Concentrations of Ethanol

“…We next investigated if we could use the information from the solvent‐tolerance mutability landscape to engineer a previously constructed highly enantioselective 4‐OT variant, L8F/M45Y/F50A (4‐OT FYA), to function in high concentrations of ethanol. As single mutants at “hotspot” position Ala33 generally exhibited higher “Michaelase” activity than those at “hotspot” position Ser30, we focused our mutagenesis strategy on position Ala33 .…”

“…[a] Assay conditions: 3 m m 4 b , 100 m m 3 , 73 μ m 4‐OT, 20 m m NaH 2 PO 4 (pH 7.3), 0.3 mL reaction volume. [b] Determined by GC using a chiral stationary phase; the absolute configuration was determined by literature comparison . [c] Reaction progress was monitored by following the depletion in absorbance at 249 nm.…”

“…For instance, the 4‐OT catalyzed Michael‐type addition of acetaldehyde ( 3 ) to nitroalkenes 4 a and 4 b yields γ‐nitroaldehydes 5 a and 5 b , important precursors for the γ‐aminobutyric acid analogues phenibut ( R ‐ 6 a ) and pregabalin ( S ‐ 6 b ), respectively (Scheme B) . Hence, several enzyme engineering studies have been performed to improve the activity and enantioselectivity of 4‐OT for this reaction …”