CHEMOENZYMATIC SYNTHESIS OF 4-AMINO-2-HYDROXY ACIDS - A COMPARISON OFMUTANT AND WILD-TYPE OXIDOREDUCTASES

We describe a new chemoenzymatic synthesis of enantiopure 4-amino-2-hy droxy acids using two biotransformations in a single-pot process in aq ueous medium. These compounds are valuable as gamma-turn mimics for in vestigations into the secondary structure of peptides. The enzyme subs trates are a series of carbobenzyloxy (CBZ)-protected 4-amino-2-keto e sters, prepared efficiently from the L-amino acids, alanine, leucine, phenylalanine, and valine. First, the alpha-amino acids were converted to the corresponding beta-amino acids in a simple five-step procedure . A further one-carbon homologation via ozonolysis of the correspondin g beta-keto cyanophosphoranes gave the required alpha-keto esters in g ood yield. The enzyme catalyzed hydrolyses of all the alpha-keto eater s to the corresponding alpha-keto acids proceeded smoothly with the li pase from Candida rugosa. Using the same reaction pot, it was found th at wild-type lactate dehydrogenases from either Bacillus stearothermop hilus (BS-LDH) or Staphylococcus epidermidis (SE-LDH) could be used to specifically reduce the ketone of the alanine-derived alpha-keto acid 2, giving the (S)- and (R)-2-hydroxy acids, respectively, in good yie lds. However, the more bulky alpha-keto acids 3, 4, and 5 (derived fro m valine, leucine, and phenylalanine) were not substrates for these en zymes. In contrast, the genetically engineered H205Q mutant of D-hydro xyisocaproate dehydrogenase proved to be an ideal catalyst for the red uction of all the alpha-keto acids 2-5, giving excellent yields of the CBZ-protected (2R,4S)-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 catalyti c activity. For example, reduction of 1 mmol of N-protected (S)-4-amin o-2-oxopentanoic acid 2 took just 4 h with the H205Q mutant giving, af ter 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.