Biochemical and Structural Characterization of an (R)‐Selective Transaminase in the Asymmetric Synthesis of Chiral Hydroxy Amines

Abstract

An (R)‐selective transaminase RbTA with excellent stereoselectivity (>99% ee) in the asymmetric amination of hydroxy ketones was identified. Biochemical characterization showed that RbTA exhibited the highest activity toward 4‐hydroxy‐2‐butanone among reported enzymes, and that it has broad substrate specificity, including for aliphatic, aromatic, and alicyclic ketones. Crystallization of RbTA were performed, as were molecular docking and mutagenesis studies. Residue Tyr125 plays a key role in substrate recognition by forming a hydrogen bond with hydroxy ketone. The applicability of the enzyme was determined in preparative‐scale synthesis of (R)‐3‐amino‐1‐butanol, demonstrating the potential of RbTA as a green biocatalyst for production of value‐added chiral hydroxy amines. This study provides an efficient tool for enzymatic synthesis of chiral hydroxy amines, as well as structural insight into substrate recognition by transaminases in the asymmetric amination of hydroxy ketones.

Biocatalytic Reductive Amination by Native Amine Dehydrogenases to Access Short Chiral Alkyl Amines and Amino Alcohols

“…CfusAmDH and MsmeAmDH harbor a less narrow substrate scope, as RbTA can convert C4-C7 ketones, but furnish the opposite (S)-enantiomer, thus widening the biocatalytic solutions for accessing such chiral building blocks. Obtained here in non-optimal condition in a 96-well plate with 48%-66% yield with excellent (S)-stereoselectivity (ee > 99%), the opposite enantiomer (S)-2f ((S)-ABOL) could be synthesized with MsmeAmDH or CfusAmDH without requiring high loading of IPA as it was done by Li et al with 1 M IPA for 100 mM substrate 1f and 30 g L −1 of whole cells in 60 h (Li et al, 2021). Again, Y125 in RbTA was postulated to play a key role in substrate hydroxy ketone recognition by forming a hydrogen bond with the hydroxyl functionality according to docking results and loss of activity with the mutation Y125A, thus supporting our hypothesis of key role of P11 in our case.…”

Redesigning an (R)-Selective Transaminase for the Efficient Synthesis of PharmaceuticalN-Heterocyclic Amines

“…Structure-Guided Design of RbTA Variants. RbTA can convert a series of aliphatic hydroxy ketones to chiral amines with high stereoselectivity 12 but shows low catalytic activity toward N-heterocyclic and aromatic ketones. Among these compounds, N-Boc-3-piperidone (7a) was selected as a research candidate because it is the most important synthon for the synthesis of DPP-4 inhibitor drugs.…”

“…However, RbTA shows a low sequence identity with reported TAs and has a unique activesite pocket. 12 No previous report has focused on modifying the activity of RbTA toward bulky substrates. The Y125 residue is a newly discovered site for regulating catalytic activity toward N-heterocyclic ketones, and several variants with enhanced efficacy were designed by modifying this site.…”

“…140A, RbTA, which exhibited wide substrate acceptance toward aromatic and alicyclic ketones but showed poor activity toward 3-amino-1-Boc-piperidone. 12 Asymmetric synthesis using (R)-selective TAs can convert cheap prochiral ketones to the corresponding (R)-enantiomer product, which can overcome the yield limitation of kinetic resolution. 13 However, most reported TAs are (S)-selective, and naturally occurring (R)-TAs suffer from low catalytic activity, which severely limits their application in the industrial production of pharmaceuticals.…”

Engineering of Reductive Aminases for Asymmetric Synthesis of Enantiopure Rasagiline

“…Chiral amines are valuable building blocks of numerous natural products, active pharmaceuticals, and other high-value chemicals ( Afanasyev et al, 2019 ; Newman and Cragg, 2020 ; Guo et al, 2021 ; Li et al, 2021 ; Nguyen and Kou, 2021 ). Constituting approximately 40% of new drugs approved by the FDA in recent years, chiral amines constituted a market value of USD 14 billion in 2020 ( de Gonzalo and Lavandera, 2021 ).…”