A mixture of 300 mg of 6-benzyl-6-aza-spiro[2.5]octan-4-one hydrochloride (1.19 mmol, 1 eq.), 1.5 ml of 2-propanol and 28 ml of 30 mM aq. TRIS-HCl buffer (pH 8.1) was heated to 35° C. The pH was re-adjusted to 8.0. The reaction was started by adding □-NAD (1 mg; free acid; Roche Diagnostics Cat. No. 10 004 626) and ketoreductase KRED-NADH-117 (29.3 mg; Codexis [ex. Biocatalytics]). The suspension was stirred at 35° C. keeping the pH constant at 8.0 by the controlled addition (pH-stat) of 1.0 M aq. sodium hydroxide solution. After roughly 80 area % conversion and 1 d, further 2-propanol (0.3 ml), β-NAD (3 mg; free acid; Roche Diagnostics Cat. No. 10 004 626), ketoreductase KRED-NADH-117 (30 mg; Codexis [ex. Biocatalytics]) and magnesium chloride (12.7 mg) were added. After 4 d, 98.5 area % conversion and 5.9 ml consumption of 1.0 M aq. sodium hydroxide solution the reaction mixture was stopped by the addition of NaCl (9 g), EtOAc (30 ml) and filter aid (1 g Dicalite Speedex). The mixture was stirred 30 min. and filtered. The filtrate was extracted 3 times with 30 ml EtOAc. The combined organic phases were dried over sodium sulfate, filtered and concentrated under reduced pressure to provide the crude product in over 99.9% e.e. Purification by flash chromatography provided the title compound as a colorless oil.
