Chemoenzymatic Synthesis of 4-Amino-2-hydroxy Acids: A Comparison of Mutant and Wild-Type Oxidoreductases

We describe a new chemoenzymatic synthesis of enantiopure 4-amino-2-hydroxy acids using two biotransformations in a single-pot process in aqueous medium. These compounds are valuable as γ-turn mimics for investigations into the secondary structure of peptides. The enzyme substrates are a series of carbobenzyloxy (CBZ)-protected 4-amino-2-keto esters, prepared efficiently from the l -amino acids, alanine, leucine, phenylalanine, and valine. First, the α-amino acids were converted to the corresponding β-amino acids in a simple five-step procedure. A further one-carbon homologation via ozonolysis of the corresponding β-keto cyanophosphoranes gave the required α-keto esters in good yield. The enzyme catalyzed hydrolyses of all the α-keto esters to the corresponding α-keto acids proceeded smoothly with the lipase from Candida rugosa . Using the same reaction pot, it was found that wild-type lactate dehydrogenases from either Bacillus stearothermophilus ( BS -LDH) or Staphylococcus epidermidis ( SE -LDH) could be used to specifically reduce the ketone of the alanine-derived α-keto acid 2, giving the ( S )- and ( R )-2-hydroxy acids, respectively, in good yields. However, the more bulky α-keto acids 3, 4, and 5 (derived from valine, leucine, and phenylalanine) were not substrates for these enzymes. In contrast, the genetically engineered H205Q mutant of d -hydroxyisocaproate dehydrogenase proved to be an ideal catalyst for the reduction of all the α-keto acids 2 − 5, giving excellent yields of the CBZ-protected (2 R,4 S )-4-amino-2-hydroxy acids as single diastereomers. This genetically engineered oxidoreductase has great potential value in synthesis due to its broad substrate specificity and high catalytic activity. For example, reduction of 1 mmol of N-protected ( S )-4-amino-2-oxopentanoic acid 2 took just 4 h with the H205Q mutant giving, after esterification, the ( R )-2-alcohol 25 in 85% yield, whereas with SE -LDH the reaction required 4 days to give a 67% yield of 25 .