Ethanol → HCID 9899181, Verbindung

Temperatur

21 °C

Six Fernbach flasks, each containing 0.5 L of medium 2 as described in Example 2 were inoculated with spores of C. echinulata ATCC 8688b and incubated at 29° C. on a rotary shaker (220 rpm). After 24 hours, 5 mL of a 60 mg/mL ethanol solution of pyrrolidine-1-carboxylic acid phenylester (II) were added to each flask, and incubated for 16 more days. The bioconversion was monitored in two of the six flasks (flasks A and B) by removing samples of fermentation broth, extracting with ETOAc, and analyzing the extracts by reverse phase and chiral HPLC as described in Example 1. The results of these HPLC analyses are summarized in Table 3. The contents of all six flasks were pooled after the 16 days incubation and filtered through a triple-layer of cheesecloth to remove cells. The filtrate was then stirred with 50 g of Amberlite® XAD-16 resin (Rohm and Haas, Philadelphia, Pa.) for 4.5 hours at 21° C. The resin was collected onto filter paper and washed with 0.8 L of ETOAc. The ETOAc extract was washed with water, dried with anhydrous magnesium sulfate, and concentrated under reduced pressure to give 0.93 g of crude product. The crude product was applied to a 5 g silica Seppak® cartridge (Waters Corporation, Milford, Mass.) and eluted with mixtures of ETOAc and hexane, (2:3, 1:1) and the fractions containing the desired product were concentrated under reduced pressure to give 0.64 g (33% yield, 89% ee) of (S)-3-hydroxypyrrolidine-1-carboxylic acid phenylester (II).