The general procedure for the synthesis of tert-butyl (S)-3-hydroxybutyrate from tert-butyl acetoacetate is as follows: laboratory scale-up bioreduction of ketones 1-7 was carried out as follows. After 72 hours of fermentation, R. arrhizus mycelium was separated from the culture broth. 10% wet mycelium was suspended in 1.5 L of sterilized fresh medium in a 5 L Erlenmeyer flask, which was used as a working volume under aseptic conditions and incubated statically at room temperature for 72 hours. After fungal growth, an ethanol solution of the substrate (1 g) was added directly to the medium, followed by incubation on a rotary oscillator (100 rpm) for 8 days at room temperature. At the end of incubation, mycelium was isolated by filtration. The mycelium was washed with water and the combined aqueous phases were extracted with chloroform. The chloroform extract was washed with water and dried with Na2SO4. After removing the solvent under reduced pressure, the product alcohol was isolated, purified and characterized as described previously. The absolute configuration was determined by specific rotation sign and compared with literature data. Isolated yield: 0.81 g, [α]D26 = +28.6 (c 0.483, CHCl3), >99% ee {Literature value: [α]D26 = +32.3 (c 1.03, CHCl3), 99.0% ee}; 1H NMR (CDCl3, 200 MHz): δ 1.25 (d, 3H, CH3), 1.48 (s, 9H, CH3), 2.27-2.49 (m, 2H, CH2), 3.09 (s, 1H, OH), 4.08-4.22 (m, 1H, CHOH); 13C NMR (CDCl3, 200MHz, ppm): 22.21, 27.93, 43.79, 64.18, 80.94, 172.15; MTPA ester of the methoxy resonance, 1H NMR: δ 3.55 [major, (S)-isomer].
