Pyruvate aldolases in chiral carbon–carbon bond formation

A procedure for the preparation of optically pure α-keto-γ-hydroxy carboxylic acids through stereospecific aldol addition catalyzed by pyruvate aldolases from the Entner–Doudoroff and the DeLey–Doudoroff glycolytic pathways is described. This highly versatile fragment serves as a precursor for a variety of commonly encountered functionalities, including β-hydroxy aldehydes and carboxylic acids, α-amino-γ-hydroxy carboxylic acids and α,γ-dihydroxy carboxylic acids. The protocol described here uses recombinant His6-tagged KDPG aldolase for the synthesis of (S)-4-hydroxy-2-keto-4-(2′-pyridyl)butyrate. A protocol for evaluating enantiomeric excess through formation of the γ-lactone of the dithioacetal followed by chiral-phase gas–liquid chromatography is also described. Enzyme expression and enzymatic synthesis can be accomplished in approximately 1 week. The enzymatic aldol addition proceeds in nearly quantitative yields with enantiomeric excesses greater than 99.7%.