tert-butyl (5S)-6-chloro-5-hydroxy-3-oxohexanoate

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

Procedure: Separately, 240 mg (6 mmol equivalent) of sodium hydride (60% in mineral oil) was washed with hexane and, then, 6 ml of tetrahydrofuran was added. Then, at 5° C., 1.71 g (18.0 mmol) of magnesium chloride, 1.74 g (15.0 mmol) of tert-butyl acetate and 1.0 g (6 mmol) of ethyl (3S)-4-chloro-3-hydroxybutyrate were added and the mixture was stirred for 30 minutes. To this mixture, the lithium diisopropylamide solution prepared above was added dropwise over 10 minutes at the same temperature and the reaction mixture was further stirred at an elevated temperature of 25° C. for 3 hours. above was added dropwise over 10 minutes at the same temperature the reaction mixture was further stirred at an elevated temperature of 25° C. for 3 hours

Conditions: temp:30.01; pressure:0.00; stir:; See reaction.notes.procedure_details.

Procedure: Large-sized test tubes were charged with 7 mL of said medium B and, after sterilization, inoculated with the bacteria shown in Table 2, respectively. Then, aerobic shake culture was carried out at 30° C. for 1 day. From the resulting culture broth, the cells were harvested by centrifugation and suspended in 0.5 mL of 50 mM phosphate buffer (pH 6.5) containing 0.5% of tert-butyl (5S)-6-chloro-5-hydroxy-3-oxohexanoate and 1.5% of glucose. The suspension was placed in a 10 mL test tube equipped with a stopper and the reaction was carried out under shaking at 30° C. for 20 hours. After completion of the reaction, the reaction mixture was extracted with 0.5 mL of ethyl acetate and the ethyl acetate phase was analyzed by high performance liquid chromatography (column: Nakalai-Tesque's Cosmocil 5CN-R (4.6 mm×250 mm), eluent: 1 mM phosphoric acid/H2O:acetonitrile=5:1, flow rate: 0.7 mL/min., detection: at 210 nm, column temperature: 30° C.) for reaction rate and for the diastereomer ratio of the product tert-butyl (3R,5S)-6-chloro-3,5-dihydroxyhexanoate. The results are shown in Table 2. Large-sized test tubes were charged with 7 mL of said medium B The suspension was placed in a 10 mL test tube equipped with a stopper After completion of the reaction the reaction mixture was extracted with 0.5 mL of ethyl acetate the ethyl acetate phase was analyzed by high performance liquid chromatography (column: Nakalai-Tesque's Cosmocil 5CN-R (4.6 mm×250 mm), eluent: 1 mM phosphoric acid/H2O:acetonitrile=5:1, flow rate: 0.7 mL/min., detection: at 210 nm, column temperature: 30° C.) for reaction rate and for the diastereomer ratio of the product tert-butyl (3R,5S)-6-chloro-3,5-dihydroxyhexanoate

Conditions: temp:30.01; pressure:0.00; stir:; See reaction.notes.procedure_details.

Procedure: Large-sized test tubes were charged with 7 mL of said medium B and, after sterilization, inoculated with the bacteria shown in Table 2, respectively. Then, aerobic shake culture was carried out at 30° C. for 1 day. From the resulting culture broth, the cells were harvested by centrifugation and suspended in 0.5 mL of 50 mM phosphate buffer (pH 6.5) containing 0.5% of tert-butyl (5S)-6-chloro-5-hydroxy-3-oxohexanoate and 1.5% of glucose. The suspension was placed in a 10 mL test tube equipped with a stopper and the reaction was carried out under shaking at 30° C. for 20 hours. After completion of the reaction, the reaction mixture was extracted with 0.5 mL of ethyl acetate and the ethyl acetate phase was analyzed by high performance liquid chromatography (column: Nakalai-Tesque's Cosmocil 5CN-R (4.6 mm×250 mm), eluent: 1 mM phosphoric acid/H2O:acetonitrile=5:1, flow rate: 0.7 mL/min., detection: at 210 nm, column temperature: 30° C.) for reaction rate and for the diastereomer ratio of the product tert-butyl (3R,5S)-6-chloro-3,5-dihydroxyhexanoate. The results are shown in Table 2. Large-sized test tubes were charged with 7 mL of said medium B The suspension was placed in a 10 mL test tube equipped with a stopper After completion of the reaction the reaction mixture was extracted with 0.5 mL of ethyl acetate the ethyl acetate phase was analyzed by high performance liquid chromatography (column: Nakalai-Tesque's Cosmocil 5CN-R (4.6 mm×250 mm), eluent: 1 mM phosphoric acid/H2O:acetonitrile=5:1, flow rate: 0.7 mL/min., detection: at 210 nm, column temperature: 30° C.) for reaction rate and for the diastereomer ratio of the product tert-butyl (3R,5S)-6-chloro-3,5-dihydroxyhexanoate

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

Procedure: A 5-L mini-jar fermenter containing 3 L of medium A was inoculated with Candida magnoliae IFO0705and incubated at 30° C. with 0.5 vvm aeration and stirring at 500 rpm for 24 hours. After completion of cultivation, 30 g of tert-butyl (5S)-6-chloro-5-hydroxy-3-oxohexanoate (synthesized by the process described in Example 1) and 60 g of glucose were added and the reaction was carried out, with the pH maintained at 6.5 with sodium hydroxide, for 18 hours. After completion of the reaction, the cells were removed centrifugally and the supernatant was extracted twice using 1.5 L of ethyl acetate each. The organic phase was separated and dehydrated over anhydrous sodium sulfate and the solvent was distilled off by heating under reduced pressure to recover 24 g of tert-butyl (3R,5S)-6-chloro-3,5-dihydroxyhexanoate as a solid. As analyzed by the method described in Example 4, the diastereomer ratio of this product was (3R,5S)/(3S,5S)=100/0. A 5-L mini-jar fermenter containing 3 L of medium incubated at 30° C. with 0.5 vvm aeration were added After completion of the reaction the cells were removed centrifugally the supernatant was extracted twice The organic phase was separated the solvent was distilled off by heating under reduced pressure to recover 24 g of tert-butyl (3R,5S)-6-chloro-3,5-dihydroxyhexanoate as a solid

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

Procedure: A 5-L mini-jar fermenter containing 3 L of medium A was inoculated with Candida magnoliae IFO0705and incubated at 30° C. with 0.5 vvm aeration and stirring at 500 rpm for 24 hours. After completion of cultivation, 30 g of tert-butyl (5S)-6-chloro-5-hydroxy-3-oxohexanoate (synthesized by the process described in Example 1) and 60 g of glucose were added and the reaction was carried out, with the pH maintained at 6.5 with sodium hydroxide, for 18 hours. After completion of the reaction, the cells were removed centrifugally and the supernatant was extracted twice using 1.5 L of ethyl acetate each. The organic phase was separated and dehydrated over anhydrous sodium sulfate and the solvent was distilled off by heating under reduced pressure to recover 24 g of tert-butyl (3R,5S)-6-chloro-3,5-dihydroxyhexanoate as a solid. As analyzed by the method described in Example 4, the diastereomer ratio of this product was (3R,5S)/(3S,5S)=100/0. A 5-L mini-jar fermenter containing 3 L of medium incubated at 30° C. with 0.5 vvm aeration were added After completion of the reaction the cells were removed centrifugally the supernatant was extracted twice The organic phase was separated the solvent was distilled off by heating under reduced pressure to recover 24 g of tert-butyl (3R,5S)-6-chloro-3,5-dihydroxyhexanoate as a solid

Conditions: temp:30.01; pressure:0.00; stir:; See reaction.notes.procedure_details.

Procedure: Sakaguchi flasks of 500 mL capacity were respectively charged with 50 mL of said medium A and, after sterilization, inoculated with the microbial strains indicated in Table 1, respectively. Aerobic shake culture was then carried out at 30° C. for 2 days. From each of the culture broths, the cells were harvested by centrifugation and suspended in 25 mL of 50 mM phosphate buffer (pH 6.5) containing 1% of tert-butyl (5S)-6-chloro-5-hydroxy-3-oxohexanoate (synthesized by the process described in Example 1) and 2% of glucose. The suspension was put in a 500 mL Sakaguchi flask and a reaction was conducted under shaking at 30° C. for 20 hours. After completion of the reaction, the reaction mixture was extracted twice with one volume of ethyl acetate each and the ethyl acetate phase was analyzed by high performance liquid chromatography (column: Nakalai-Tesque, Cosmocil 5CN-R (4.6 mm×250 mm), eluent: 1 mM phosphoric acid/H2O:acetonitrile=5:1, flow rate 0.7 mL/min., detection: at 210 nm, column temperature: 30° C.) for reaction rate and for the diastereomer ratio of the product tert-butyl (3R,5S)-6-chloro-3,5-dihydroxyhexanoate. The results are shown in Table 1. Sakaguchi flasks of 500 mL capacity were respectively charged with 50 mL of said medium A synthesized by the process After completion of the reaction the reaction mixture was extracted twice with one volume of ethyl acetate each and the ethyl acetate phase was analyzed by high performance liquid chromatography (column: Nakalai-Tesque, Cosmocil 5CN-R (4.6 mm×250 mm), eluent: 1 mM phosphoric acid/H2O:acetonitrile=5:1, flow rate 0.7 mL/min., detection: at 210 nm, column temperature: 30° C.) for reaction rate and for the diastereomer ratio of the product tert-butyl (3R,5S)-6-chloro-3,5-dihydroxyhexanoate

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

Procedure: Separately, 240 mg (6 mmol equivalent) of sodium hydride (60% in mineral oil) was washed with hexane and, then, 6 ml of tetrahydrofuran was added. Then, at 5° C., 1.71 g (18.0 mmol) of magnesium chloride, 1.74 g (15.0 mmol) of tert-butyl acetate and 1.0 g (6 mmol) of ethyl(3S)-4-chloro-3-hydroxybutyrate were added and the mixture was stirred for 30 minutes. To this mixture, the lithium diisopropylamide solution prepared above was added dropwise over 10 minutes at the same temperature and the reaction mixture was further stirred at an elevated temperature of 25° C. for 3 hours. above was added dropwise over 10 minutes at the same temperature the reaction mixture was further stirred at an elevated temperature of 25° C. for 3 hours

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

Procedure: Separately, 240 mg (6 mmol equivalent) of sodium hydride (60% in mineral oil) was washed with hexane and, then, 6 ml of tetrahydrofuran was added. Then, at 5° C., 1.71 g (18.0 mmol) of magnesium chloride, 1.74 g (15.0 mmol) of tert-butyl acetate and 1.0 g (6 mmol) of ethyl(3S)-4-chloro-3-hydroxybutyrate were added and the mixture was stirred for 30 minutes. To this mixture, the lithium diisopropylamide solution prepared above was added dropwise over 10 minutes at the same temperature and the reaction mixture was further stirred at an elevated temperature of 25° C. for 3 hours. above was added dropwise over 10 minutes at the same temperature the reaction mixture was further stirred at an elevated temperature of 25° C. for 3 hours

Conditions: temp:30.01; pressure:0.00; stir:; See reaction.notes.procedure_details.

Procedure: Large-sized test tubes were charged with 7 mL of said medium B and, after sterilization, inoculated with the bacteria shown in Table 2, respectively. Then, aerobic shake culture was carried out at 30° C. for 1 day. From the resulting culture broth, the cells were harvested by centrifugation and suspended in 0.5 mL of 50 mM phosphate buffer (pH 6.5) containing 0.5% of tert-butyl(5S)-6-chloro-5-hydroxy-3-oxohexanoate and 1.5% of glucose. The suspension was placed in a 10 mL test tube equipped with a stopper and the reaction was carried out under shaking at 30° C. for 20 hours. After completion of the reaction, the reaction mixture was extracted with 0.5 mL of ethyl acetate and the ethyl acetate phase was analyzed by high performance liquid chromatography (column: Nakalai-Tesque's Cosmocil 5CN-R (4.6 mm×250 mm), eluent: 1 mM phosphoric acid/H2O:acetonitrile=5:1, flow rate: 0.7 mL/min., detection: at 210 nm, column temperature: 30° C.) for reaction rate and for the diastereomer ratio of the product tert-butyl(3R,5S)-6-chloro-3,5-dihydroxyhexanoate. The results are shown in Table 2. Large-sized test tubes were charged with 7 mL of said medium B The suspension was placed in a 10 mL test tube equipped with a stopper After completion of the reaction the reaction mixture was extracted with 0.5 mL of ethyl acetate the ethyl acetate phase was analyzed by high performance liquid chromatography (column: Nakalai-Tesque's Cosmocil 5CN-R (4.6 mm×250 mm), eluent: 1 mM phosphoric acid/H2O:acetonitrile=5:1, flow rate: 0.7 mL/min., detection: at 210 nm, column temperature: 30° C.) for reaction rate and for the diastereomer ratio of the product tert-butyl(3R,5S)-6-chloro-3,5-dihydroxyhexanoate

Conditions: temp:30.01; pressure:0.00; stir:; See reaction.notes.procedure_details.

Procedure: A 5-L mini-jar fermenter containing 3 L of medium A was inoculated with Candida magnoliae IFO0705 and incubated at 30° C. with 0.5 vvm aeration and stirring at 500 rpm for 24 hours. After completion of cultivation, 30 g of tert-butyl(5S)-6-chloro-5-hydroxy-3-oxohexanoate (synthesized by the process described in Example 1) and 60 g of glucose were added and the reaction was carried out, with the pH maintained at 6.5 with sodium hydroxide, for 18 hours. After completion of the reaction, the cells were removed centrifugally and the supernatant was extracted twice using 1.5 L of ethyl acetate each. The organic phase was separated and dehydrated over anhydrous sodium sulfate and the solvent was distilled off by heating under reduced pressure to recover 24 g of tert-butyl(3R,5S)-6-chloro-3,5-dihydroxyhexanoate as a solid. As analyzed by the method described in Example 4, the diastereomer ratio of this product was (3R,5S)/(3S,5S)=100/0. 1H-NMR (CDCl3, 400 MHz/ppm): 1.47 (9H, s), 1.62-1.78 (2H, m), 2.43 (2H, d, J=6.4. Hz), 3.51-3.58 (2H, m), 3.75 (1H, bs), 3.84 (1H, bs), 4.07-4.13 (1H, m), 4.23-4.28 (1H, m) A 5-L mini-jar fermenter containing 3 L of medium were added After completion of the reaction the cells were removed centrifugally the supernatant was extracted twice The organic phase was separated the solvent was distilled off by heating under reduced pressure to recover 24 g of tert-butyl(3R,5S)-6-chloro-3,5-dihydroxyhexanoate as a solid

Conditions: temp:30.01; pressure:0.00; stir:; See reaction.notes.procedure_details.

Procedure: Sakaguchi flasks of 500 mL capacity were respectively charged with 50 mL of said medium A and, after sterilization, inoculated with the microbial strains indicated in Table 1, respectively. Aerobic shake culture was then carried out at 30° C. for 2 days. From each of the culture broths, the cells were harvested by centrifugation and suspended in 25 mL of 50 mM phosphate buffer (pH 6.5) containing 1% of tert-butyl(5S)-6-chloro-5-hydroxy-3-oxohexanoate (synthesized by the process described in Example 1) and 2% of glucose. The suspension was put in a 500 mL Sakaguchi flask and a reaction was conducted under shaking at 30° C. for 20 hours. After completion of the reaction, the reaction mixture was extracted twice with one volume of ethyl acetate each and the ethyl acetate phase was analyzed by high performance liquid chromatography (column: Nakalai-Tesque, Cosmocil 5CN-R (4.6 mm×250 mm), eluent: 1 mM phosphoric acid/H2O:acetonitrile=5:1, flow rate 0.7 mL/min., detection: at 210 nm, column temperature: 30° C.) for reaction rate and for the diastereomer ratio of the product tert-butyl(3R,5S)-6-chloro-3,5-dihydroxyhexanoate. The results are shown in Table 1. Sakaguchi flasks of 500 mL capacity were respectively charged with 50 mL of said medium A synthesized by the process After completion of the reaction the reaction mixture was extracted twice with one volume of ethyl acetate each and the ethyl acetate phase was analyzed by high performance liquid chromatography (column: Nakalai-Tesque, Cosmocil 5CN-R (4.6 mm×250 mm), eluent: 1 mM phosphoric acid/H2O:acetonitrile=5:1, flow rate 0.7 mL/min., detection: at 210 nm, column temperature: 30° C.) for reaction rate and for the diastereomer ratio of the product tert-butyl(3R,5S)-6-chloro-3,5-dihydroxyhexanoate

Conditions: temp:30.01; pressure:0.00; stir:; See reaction.notes.procedure_details.

Procedure: Sakaguchi flasks of 500 mL capacity were respectively charged with 50 mL of said medium A and, after sterilization, inoculated with the microbial strains indicated in Table 1, respectively. Aerobic shake culture was then carried out at 30° C. for 2 days. From each of the culture broths, the cells were harvested by centrifugation and suspended in 25 mL of 50 mM phosphate buffer (pH 6.5) containing 1% of tert-butyl(5S)-6-chloro-5-hydroxy-3-oxohexanoate (synthesized by the process described in Example 1) and 2% of glucose. The suspension was put in a 500 mL Sakaguchi flask and a reaction was conducted under shaking at 30° C. for 20 hours. After completion of the reaction, the reaction mixture was extracted twice with one volume of ethyl acetate each and the ethyl acetate phase was analyzed by high performance liquid chromatography (column: Nakalai-Tesque, Cosmocil 5CN-R (4.6 mm×250 mm), eluent: 1 mM phosphoric acid/H2O:acetonitrile=5:1, flow rate 0.7 mL/min., detection: at 210 nm, column temperature: 30° C.) for reaction rate and for the diastereomer ratio of the product tert-butyl(3R,5S)-6-chloro-3,5-dihydroxyhexanoate. The results are shown in Table 1. Sakaguchi flasks of 500 mL capacity were respectively charged with 50 mL of said medium A synthesized by the process After completion of the reaction the reaction mixture was extracted twice with one volume of ethyl acetate each and the ethyl acetate phase was analyzed by high performance liquid chromatography (column: Nakalai-Tesque, Cosmocil 5CN-R (4.6 mm×250 mm), eluent: 1 mM phosphoric acid/H2O:acetonitrile=5:1, flow rate 0.7 mL/min., detection: at 210 nm, column temperature: 30° C.) for reaction rate and for the diastereomer ratio of the product tert-butyl(3R,5S)-6-chloro-3,5-dihydroxyhexanoate