General procedure for the synthesis of benzyl (S)-3-hydroxypiperidine-1-carboxylate from 1-N-Cbz-3-piperidone: a reaction mixture of 1 mL of potassium phosphate buffer (100 mM, pH 7.0) containing 200 mM substrate (1-N-Cbz-3-piperidone), 1 mM NAD+, 5% (v/v) 2-propanol, and 10 mg of crude enzyme READH was incubated at 50°C warming. For ChKRED20, the reaction conditions were adjusted to contain 40% (v/v) 2-propanol and the reaction temperature was 40°C. The reaction progress was monitored by TLC and the reaction was extracted with methyl tert-butyl ether (1 mL) at the termination of the reaction. The organic phase was dried with anhydrous sodium sulfate and concentrated. The conversion and enantiomeric excess were determined by chiral HPLC. The products were purified by silica gel column chromatography and structurally characterized by NMR analysis, spinometry and mass spectrometry.
