phenyl pyrrolidine-1-carboxylate

Conditions: temp:0.00; pressure:0.00; stir:; See reaction.notes.procedure_details.

Procedure: 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). incubated at 29° C. on a rotary shaker (220 rpm) were operated at 29° C. with 600 rpm agitation and 8 liter per minute aeration After 24 hours by removing samples extracting with ETOAc The results of these HPLC analyses About 9 days after substrate addition filtered through a triple-layer of cheesecloth containing about 500 g of XAD-16 resin The resin was then washed with 1 L of water eluted with 6 L of ETOAc The ETOAc eluates were washed with water dried over anhydrous magnesium sulfate concentrated under reduced pressure to give the crude product eluted with mixtures of ETOAc and hexanes, (2:3, 1·1) increasing in 10% increments to 80% ETOAc Fractions eluted with 60-80% ETOAc concentrated under reduced pressure

Conditions: temp:21.01; pressure:0.00; stir:; See reaction.notes.procedure_details.

Procedure: 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). incubated at 29° C. on a rotary shaker (220 rpm) by removing samples of fermentation broth extracting with ETOAc The results of these HPLC analyses after the 16 days incubation filtered through a triple-layer of cheesecloth to remove cells The resin was collected onto filter paper washed with 0.8 L of ETOAc The ETOAc extract was washed with water dried with anhydrous magnesium sulfate concentrated under reduced pressure to give 0.93 g of crude product eluted with mixtures of ETOAc and hexane, (2:3, 1:1) the fractions containing the desired product were concentrated under reduced pressure