Preparation of β-hydroxy-α-amino Acid Using Recombinant d-Threonine Aldolase

The chiral β-hydroxy-α-amino acid, (2R,3S)-2-amino-3-hydroxy-3-(pyridin-4-yl)-propanoic acid, is a key intermediate in the synthesis of the API (2R,3S)-2-amino-3-hydroxy-3-(pyridin-4-yl)-1-(pyrrolidin-1-yl)propan-1-one, a developmental drug candidate. Two d-threonine aldolase enzymes were identified to catalyze the aldol addition of glycine and pyridine 4-carboxaldehyde for the synthesis of the β-hydroxy-α-amino acid. The two d-threonine aldolase enzymes have similar properties. Efficient recombinant E. coli fermentation processes were developed for producing the enzymes. The stabilities of the enzymes were significantly improved by addition of divalent cations. An unexpected and beneficial finding was that the β-hydroxy-α-amino acid aldol addition product directly crystallized out from the reaction mixture in high purity and high diastereo- and enantioselectivity, contributing also to high yield and allowing easy isolation, processing, and downstream utilization. The temperature, pH, and amounts of reactants and enzyme were optimized to minimize reaction time and enzyme and raw material usage and maximize amino acid formation. Efficient d-threonine aldolase-catalyzed synthesis and recovery of the β-hydroxy-α-amino acid at the 100 L scale was demonstrated leading to a highly efficient and environmentally friendly process for the production of the API.