A 0.1 M potassium phosphate buffer, 2.0 mM in magnesium chloride, was prepared by combining potassium dihydrogenphosphate (9.86 g, 72.4 mmol), potassium hydrogenphosphate (22.2 g, 127 mmol) and magnesium chloride hexahydrate (0.812 g, 4.0 mmol) in water (2 L); the pH of the resulting solution was 7.05. To this phosphate buffer (1.8 L) was added nicotinamide adenine dinucleotide phosphate, disodium salt trihydrate (1.9 g, 2.4 mmol) and ketoreductase enzyme (Codexis, KRED-P1-E05) (8 g), and the mixture was stirred for 45 minutes at 22° C. to dissolve the ketoreductase. A solution of tert-butyl [(2S)-4-chloro-3-oxobutan-2-yl]carbamate (C43) (50.0 g, 226 mmol) in 2-propanol (200 mL) was added, and the resulting suspension was stirred for 46 hours at 30° C., under a nitrogen flow (10 mL/minute) from a sparge inlet containing 2-propanol and water (1:1, 300 mL). At this point, tert-butyl methyl ether (1 L) was added to the reaction mixture, which was swirled for 20 minutes. The resulting emulsion was filtered through diatomaceous earth (200 g), and the filter cake was broken and washed with tert-butyl methyl ether (3×400 mL). The combined organic layers from the filtrates were dried with sodium sulfate (625 g), filtered, and concentrated in vacuo to afford the crude product as a red oil (50 g). This material was mixed with ethyl acetate (80 mL) and treated with decolorizing carbon (5 g) over 10 minutes with gentle heating. After filtration through Celite, the solution was concentrated in vacuo and mixed with warm hexanes (40 mL) under stirring. After 18 hours, the resulting solid was collected via filtration and rinsed with pentane, providing the product as a white powder (14.14 g). The mother liquor was concentrated under reduced pressure to yield an oil (32 g), which was crystallized in the same way using warm hexanes (30 mL) to obtain additional product as a white powder (13.65 g). Total yield: 27.79 g, 124 mmol, 55%. 1H NMR (400 MHz, CDCl3) δ 4.77 (br s, 1H), 3.77-3.87 (m, 1H), 3.69-3.76 (m, 1H), 3.65 (dd, half of ABX pattern, J=11.1, 3.9 Hz, 1H), 3.53 (dd, half of ABX pattern, J=11.3, 8.0 Hz, 1H), 3.00 (br s, 1H), 1.45 (s, 9H), 1.26 (d, J=6.8 Hz, 3H).
