Ethanol → HCID 9899181, 화합물

Three 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 about 24 hours, the contents of the three flasks were combined and used to inoculate two fermentors, each containing 8 L of medium 2. Fermentors (Fermentors A and B) were operated at 29° C. with 600 rpm agitation and 8 liter per minute aeration. The pH was controlled between 6 and 7. After 24 hours, the bioconversion was started by adding 4.8 g of substrate (I), dissolved in 20 mL ethanol, to each fermentor. The bioconversion was followed by removing samples, extracting with ETOAc, and analyzing by reverse phase and chiral HPLC as described in Example 1. The results of these HPLC analyses are summarized in Table 4. About 9 days after substrate addition, the contents of the fermentors were pooled and filtered through a triple-layer of cheesecloth. The filtrate was passed through a column containing about 500 g of XAD-16 resin. The resin was then washed with 1 L of water and eluted with 6 L of ETOAc. The ETOAc eluates were washed with water, dried over anhydrous magnesium sulfate, and concentrated under reduced pressure to give the crude product. The crude product was applied to a silica flash cartridge (Biotage Herts SGI3 7NW ENGLAND) and eluted with mixtures of ETOAc and hexanes, (2:3, 1·1) beginning with 10% ETOAc, and increasing in 10% increments to 80% ETOAc. Fractions eluted with 60-80% ETOAc were combined and concentrated under reduced pressure to give 2.9 g (28% yield, 88% ee) of (S)-3-hydroxypyrrolidine-1-carboxylic acid phenylester (II).