Application of Threonine Aldolases for the Asymmetric Synthesis of α‐Quaternary α‐Amino Acids

Abstract

We report the synthesis of diverse β‐hydroxy‐α,α‐dialkyl‐α‐amino acids with perfect stereoselectivity for the α‐quaternary center through the action of l‐ and d‐specific threonine aldolases. A wide variety of aliphatic and aromatic aldehydes were accepted by the enzymes and conversions up to >80 % were obtained. In the case of d‐selective threonine aldolase from Pseudomonas sp., generally higher diastereoselectivities were observed. The applicability of the protocol was demonstrated by performing enzymatic reactions on preparative scale. Using the d‐threonine aldolase from Pseudomonas sp., (2R,3S)‐2‐amino‐3‐(2‐fluorophenyl)‐3‐hydroxy‐2‐methylpropanoic acid was generated in preparative amounts in one step with a diastereomeric ratio >100 favoring the syn‐product. A Birch‐type reduction enabled the reductive removal of the β‐hydroxy group from (2S)‐2‐amino‐3‐hydroxy‐2‐methyl‐3‐phenylpropanoic acid to generate enantiopure l‐α‐methyl‐phenylalanine via a two‐step chemo‐enzymatic transformation.

Comparison of L-Threonine Aldolase Variants in the Aldol and Retro-Aldol Reactions

“…LTAs accept a wide range of aliphatic and aromatic aldehydes as an electrophilic substrate, whereas glycine is accepted as the only nucleophile donor by most of the known LTAs (Steinreiber et al, 2007a,b; Franz and Stewart, 2014). LTAaj is a unique enzyme with broad donor specificity, accepting other small-sized D-amino acids as donors (Fesko et al, 2010; Blesl et al, 2018). The aldol additions of benzaldehyde ( 1a ) to amino acid donors [glycine ( 2a ), D-alanine ( 2b ), D-serine ( 2c ), and D-aminobutanoic acid ( 2d )] to produce L-β-phenylserine ( 3a ) and L-α-alkyl-β-phenylserine 3b-d , respectively, were used as model reactions in this study to compare the properties of the LTAaj mutant variants (Figure 4).…”

Biocatalysis: A Pharma Perspective

“…A recent example of using glycine oxidase to deaminate the excess glycine to glyoxylate at the end of the reaction offers a novel approach to facilitate product recovery . Another drawback to the use of these enzymes has been the exclusive specificity for the donor amino acid to be glycine: recently protein research has extended this specificity to include additional amino acids such as alanine, thereby increasing the opportunities for application of these enzymes, particularly to the synthesis of the challenging 2‐methyl‐substituted amino acids …”

Building Up Quaternary Stereocenters Through Biocatalyzed Direct Insertion of Carbon Nucleophiles on Ketones

“…Later, the electrophile tolerability of D‐threonine aldolase from Pseudomonas sp. (D‐TA) and L‐ allo ‐threonine aldolase from Aeromonas jandaei (L‐TA) has been further investigated, using a number of substituted benzaldehydes, etherocyclic and aliphatic aldehydes . On the other hand, the specificity towards the nucleophile proved to be strict and more sterically demanding or electronically different candidates as compared to D‐alanine, D‐serine and D‐cysteine have not been accepted by the enzymes.…”

“…The removal of the β‐hydroxy group from (2 S )‐2‐amino‐3‐hydroxy‐2‐methyl‐3‐phenylpropanoic acid by a Birch reduction has given access to enantipure L‐α‐methylphenyl alanine (Scheme ).…”