General procedure for the synthesis of (R)-(-)-1-benzyl-3-hydroxypiperidine using 1-benzyl-3-piperidone as starting material: a reaction mixture was prepared in 1 mL of potassium phosphate buffer (100 mM, pH 7.0) containing 200 mM 1-benzyl-3-piperidone, 1 mM NAD+, 5% (v/v) 2-propanol, and 10 mg of the crude enzyme READH. The reaction mixture 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 TLC and the reaction was terminated by extraction with 1 mL of methyl tert-butyl ether. 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.
