200012-06-2

Upstream product

• 72-19-5 L-threonine 
• 555-16-8 4-nitrobenzaldehdye 
• 56-40-6 glycine 
• 5872-74-2 (2RS,3RS)-2-amino-3-hydroxy-3-(4-nitro-phenyl)-propionic acid 
• 19185-86-5 (2S,3R)-2-acetylamino-3-hydroxy-3-(4-nitro-phenyl)-propionic acid methyl ester 

Relevant academic research and scientific papers

Threonine aldolases-an emerging tool for organic synthesis

In a systematic study, 21 ring-substituted benzaldehydes were reacted with glycine under catalysis with a l-threonine aldolase (lTA) from Pseudomonas putida and a d-threonine aldolase (dTA) from Alcaligenes xylosoxidans to form the corresponding β-hydroxy-α-amino acids 1-18. dTA proved to be highly selective with ee's >99% (d) and de's up to 99% (syn). Two thiamphenicol precursors were synthesized utilizing dTA on a preparative scale. lTA-catalyzed reactions led to ee's >99% (l) but low to moderate de's (20-50%, syn).

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

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 β-hydroxy-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-α-amino acids and substituted α-keto acids.

Characteristics of l-threonine transaldolase for asymmetric synthesis of β-hydroxy-α-amino acids

l-Threonine transaldolase (LTTA) is a putative serine hydroxymethyltransferase (SHMT) that can catalyze the trans-aldehyde reaction of l-threonine and aldehyde to produce l-threo-β-hydroxy-α-amino acids with excellent stereoselectivity. In the present study, an l-threonine transaldolase from Pseudomonas sp. (PsLTTA) was mined and expressed in Escherichia coli BL21 (DE3). A substrate spectrum assay indicated that PsLTTA only consumed l-threonine as the donor substrate and could accept a wide range of aromatic aldehydes as acceptor substrates. Among these substrates, PsLTTA could catalyze p-methylsulfonyl benzaldehyde and l-threonine to produce l-threo-p-methylsulfonylphenylserine with a high conversion rate (74.4%) and a high de value (79.9%). The conversion and stereoselectivity of PsLTTA were found to be dramatically influenced by the concentration of the whole cell, the co-solvent and the reaction temperature. Through conditional optimization, l-threo-p-methylsulfonylphenylserine was obtained with 67.1% conversion and a near-perfect de value (94.5%), the highest stereoselectivity for an l-threo-β-hydroxy-α-amino acid so far reported by enzymatic synthesis. Finally, synthesis of l-threo-p-methylsulfonylphenylserine at a 100 mL scale by whole-cell biocatalysis was conducted. This is the first systematic report of l-threonine transaldolase as a robust biocatalyst for preparation of β-hydroxy-α-amino acids, which can provide new insights for β-hydroxy-α-amino acids synthesis.

Improving and Inverting Cβ-Stereoselectivity of Threonine Aldolase via Substrate-Binding-Guided Mutagenesis and a Stepwise Visual Screening

Threonine aldolase (TA)-catalyzed aldol condensation is a powerful tool for C-C bond formation under mild conditions, but the low Cβ-stereoselectivity has hampered its wide application. A stepwise visual screening method was developed to measure the activity and stereoselectivity of threonine aldolase-catalyzed aldol condensation by employing a stereoselective phenylserine dehydratase, enabling direct selection of mutants with higher Cβ-stereoselectivity. Mutants of l-PsTA from Pseudomonas sp. with improved or inverted stereoselectivity toward aromatic aldehydes were obtained by simultaneously mutating amino acid residues which interact with the amino and hydroxyl groups of the substrate and screening the resulting mutant libraries with this method. The mutation and enzyme-substrate docking studies provided some insights into the regulation of the Cβ-stereoselectivity by the enzyme-substrate interactions. This study offers a tool and useful guidance for further engineering of TAs to address the Cβ-stereoselectivity problem.

ENGINEERED POLYPEPTIDES AND THEIR APPLICATIONS IN SYNTHESIS OF BETA-HYDROXY-ALPHA-AMINO ACIDS

Provided are engineered polypeptides that are useful for the asymmetric synthesis of β-hydroxy-α-amino acids under industrial-relevant conditions. The engineered polypeptides disclosed are developed through directed evolution based on the ability of catalytic synthesis of (2S, 3R) -2-amino-3-hydroxy-3- (4-nitrophenyl) propanoic acid. Also provided are polynucleotides encoding the engineered polypeptides, host cells capable of expressing engineered polypeptides, and methods of producing β-hydroxy-α-amino acids using engineered polypeptides. Compared to other processes of preparation, the use of the engineered polypeptides for the preparation of β-hydroxy-α-amino acids results in high purity of the desired stereoisomers, mild reaction conditions, low pollution and low energy consumption. It has good industrial application prospects.

Engineered L-serine hydroxymethyltransferase from streptococcus thermophilus for the synthesis of α,α-dialkyl-α-amino acids

α,α-Disubstituted a-amino acids are central to biotechnological and biomedical chemical processes for their own sake and as substructures of biologically active molecules for diverse biomedical applications. Structurally, these compounds contain a quaternary stereocenter, which is particularly challenging for stereoselective synthesis. The pyridoxal-5′-phosphate (PLP)-dependent l -serine hydroxymethyltransferase from Streptococcus thermophilus (SHMTSth; EC 2.1.2.1) was engineered to achieve the stereoselective synthesis of a broad structural variety of α,α-dialkyl-α-amino acids. This was accomplished by the formation of quaternary stereocenters through aldol addition of the amino acids D-Ala and D-Ser to a wide acceptor scope catalyzed by the minimalist SHMTSth Y55T variant overcoming the limitation of the native enzyme for Gly. The SHMTSth Y55T variant tolerates aromatic and aliphatic aldehydes as well as hydroxy- and nitrogen-containing aldehydes as acceptors.