Scalable and Selective β‐Hydroxy‐α‐Amino Acid Synthesis Catalyzed by Promiscuous l‐Threonine Transaldolase ObiH

Abstract

Enzymes from secondary metabolic pathways possess broad potential for the selective synthesis of complex bioactive molecules. However, the practical application of these enzymes for organic synthesis is dependent on the development of efficient, economical, operationally simple, and well-characterized systems for preparative scale reactions. We sought to bridge this knowledge gap for the selective biocatalytic synthesis of β-hydroxy-α-amino acids, which are important synthetic building blocks. To achieve this goal, we demonstrated the ability of ObiH, an l-threonine transaldolase, to achieve selective milligram-scale synthesis of a diverse array of non-standard amino acids (nsAAs) using a scalable whole cell platform. We show how the initial selectivity of the catalyst is high and how the diastereomeric ratio of products decreases at high conversion due to product re-entry into the catalytic cycle. ObiH-catalyzed reactions with a variety of aromatic, aliphatic and heterocyclic aldehydes selectively generated a panel of βhydroxy-α-amino acids possessing broad functional-group diversity. Furthermore, we demonstrated that ObiH-generated β-hydroxy-α-amino acids could be modified through additional transformations to access important motifs, such as β-chloro-αamino acids and substituted α-keto acids.

Efficient Chemoenzymatic Synthesis of α-Aryl Aldehydes as Intermediates in C–C Bond Forming Biocatalytic Cascades

“…Naphthyl-2-aldehyde was previously observed to react with ObiH. 14 Here, we observed that the naphthyl-1-epoxide 15a reacted in the cascade to produce the corresponding amino acid 15b with a 25% yield. To assess whether SOI could convert epoxide functionalized aromatic heterocycles into the corresponding α-aryl aldehyde, we synthesized oxiranyl pyridine substrates from 2-, 3-, and 4-pyridine carboxaldehydes.…”

“…Previous studies by us and others have shown that ObiH has excellent stereoselectivity. 14,31,32 The S-isomer at the 2position (C α ) is formed due to the complete facial selectivity of the ObiH quinonoid nucleophile. ObiH forms the 3-(R)isomer with high, >20:1 selectivity under initial velocity conditions for most substrates tested, which matches the absolute configuration of obafluorin, the natural product produced in the biosynthetic pathway encoding ObiH.…”

“…Notably, this high stereochemical purity is maintained even as reactions proceed to high yield, which stands in contrast to the wellstudied Thr aldolases 34 and the reaction of ObiH with aryl aldehydes. 14 SOI-ObiH Forms a Promiscuous Biocatalytic Cascade. We next sought to explore the substrate scope of the SOI-ObiH cascade.…”

“…Their ability to catalyze enantioselective transformations with simple starting materials has motivated many engineering and synthetic studies. For example, aldolases have been leveraged to perform key C–C bond forming steps in biocatalytic systems that produce complex carbohydrates, , functionalized α-ketoacids, , and amino acid analogs. − Independent of the particular enzyme employed, aldol chemistry is fundamentally constrained by substrate availability. Although thousands of aromatic and aliphatic aldehydes are commercially available, simple α-aryl aldehydes are notably scarce (Figure b).…”

Investigation of β‐Substitution Activity of O‐Acetylserine Sulfhydrolase from Citrullus vulgaris

“…While there are excellent synthetic methods to access many nsAAs, [8–10] biocatalytic routes offer distinct advantages. Enzymes operate under mild conditions with no protecting groups and can be combined in cascades to build complex molecules from simple precursors in a regio‐ and stereoselective fashion [11–13] . Identifying and characterizing new classes of enzymes that can meet the demands of preparative‐scale synthesis will provide additional tools for the facile synthesis of desirable nsAAs.…”