Engineering of Reductive Aminases for Asymmetric Synthesis of Enantiopure Rasagiline

Abstract

Reductive aminases (RedAms) for the stereoselective amination of ketones represent an environmentally benign and economically viable alternative to transition metal–catalyzed asymmetric chemical synthesis. Here, we report two RedAms from Aspergillus calidoustus (AcRedAm) and bacteria (BaRedAm) with NADPH-dependent features. The enzymes can synthesize a set of secondary amines using a broad range of ketone and amine substrates with up to 97% conversion. To synthesize the pharmaceutical ingredient (R)-rasagiline, we engineered AcRedAm through rational design to obtain highly stereoselective mutants. The best mutant Q237A from AcRedAm could synthesize (R)-rasagiline with >99% enantiomeric excess with moderate conversion. The features of AcRedAm and BaRedAm highlight their potential for further study and expand the biocatalytic toolbox for industrial applications.

Unlocking Catalytic Diversity of a Formate Dehydrogenase: Formamide Activity for NADPH Regeneration and Amine Supply for Asymmetric Reductive Amination

“…This efficiency backfires in the form of a narrow substrate spectrum for hydride acceptors as the kinetic parameters for the regeneration of NADPH are extremely unfavorable . NADPH is involved in several processes for asymmetric synthesis of various pharmaceuticals and precursors, including ene- and ketoreductases (the latter also called alcohol dehydrogenases), transaminases, amine dehydrogenases, and imine reductases/reductive aminases among others. – This issue was addressed by using alternative regeneration systems such as a glucose dehydrogenase (GDH) with d -glucose, an alcohol dehydrogenase (ADH) with, e.g., isopropanol, and a phosphite dehydrogenase (PDH) with phosphite among others. , However, all of these systems demonstrate certain process limitations. For GDH, the synthesis of glucuronic acid requires additional pH regulation, while PDHs display a restricted range of optimal pH and temperature. , ADHs are temperature sensitive and might be problematic in reactions involving ketones, such as amine dehydrogenase or imine reductase-driven reductive aminations, , due to the reversibility of the alcohol oxidation, potentially leading to ketone reduction and alcohol side product formation.…”

Engineering of a Reductive Aminase to Enable the Synthesis of a Key Intermediate to a CDK 2/4/6 Inhibitor

“…The reactions reported in academic laboratories predominantly use carbonyl compounds at relatively low concentrations paired with simple amines, often used in large excess to drive the reactions to completion. [10][11][12][13]16,17 Both of these limitations must be overcome for a viable commercial manufacturing process. Herein we report the successful directed evolution of a RedAm to enable the synthesis of key chiral intermediate 4.…”

“…RedAms have the potential to be highly valuable catalysts for the synthesis of active pharmaceutical ingredients (APIs), but their application to manufacturing processes , is still maturing and many challenges remain. The reactions reported in academic laboratories predominantly use carbonyl compounds at relatively low concentrations paired with simple amines, often used in large excess to drive the reactions to completion. − ,, Both of these limitations must be overcome for a viable commercial manufacturing process.…”

Structure of the imine reductase from Ajellomyces dermatitidis in three crystal forms

“…These and other RedAms from fungi have been applied in a number of preparative imine reductions and reductive aminations (Mangas-Sanchez et al, 2020;Ramsden et al, 2019; Gonza ´lez-Martı ´nez et al, 2020; Zhang et al, 2021). One of them, AdRedAm from Ajellomyces dermatitidis, has been reported to be more stable than AspRedAm (Zachos et al, 2021) and thus perhaps more suitable for process applications.…”