General procedure for the synthesis of N-CBZ-3-(R)-hydroxypyrrolidine using N-Cbz-3-pyrrolidone as starting material: a reaction mixture of 1 mL of potassium phosphate buffer (100 mM, pH 7.0) containing 200 mM of substrate, 1 mM NAD+, 5% (v/v) 2-propanol, and 10 mg of crude enzyme READH was warmed at 50 °C. For ChKRED20, the reaction conditions were adjusted to 40% (v/v) 2-propanol and 40°C reaction temperature. The reaction progress was monitored by thin layer chromatography (TLC) and the reaction was terminated by the addition of 1 mL of methyl tert-butyl ether extraction. The organic phase was dried with anhydrous sodium sulfate and concentrated. The conversion and enantiomeric excess were determined by chiral high performance liquid chromatography (HPLC). The products were purified by silica gel column chromatography and structurally characterized by nuclear magnetic resonance (NMR) analysis, spinometry and mass spectrometry (MS).
