ethyl (3S)-3,4-dihydroxybutanoate

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

Procedure: The recombinant E. coli HB101(pNTS1G) obtained in Example 10 was inoculated in 100 ml of a 2×YT medium sterilized in a 500 ml Sakaguchi flask, and cultured with agitation at 37° C. for 13 hours. Glucose, 7.4 g, 3.2 mg of NADP, and then 4 g of ethyl 4-hydroxyacetoacetate were added to 50 ml of the resultant culture. The culture was stirred at 30° C. while being adjusted at pH 6.5 with a 5 M sodium hydroxide solution to allow for reaction for 18 hours. After the reaction, the reaction solution was subjected to extraction using ethyl acetate, the solvent was removed under decompression, and the concentrate was purified by silica gel column chromatography, to obtain 3.2 g of ethyl (S) -3,4-dihydroxybutyrate. The optical purity of the ethyl (S) -3,4-dihydroxybutyrate was analyzed as follows and found to be 100% e.e. The analysis was performed in the following manner. The sample was reacted with sodium cyanide in ethanol at room temperature to obtain 4-cyano-3-hydroxy ethyl butyrate, which was then changed to a benzoic ester using benzoyl chloride under the presence of pyridine. The optical purity of the benzoic ester was measured by the HPLC method. 1NMR(CDC13)δ(ppm): 1.30(3H,t), 2.55(2H,m), 3.18(1H,br), 3.55(1H,d), 3.68(1H,d), 4.15(1H,s), 4.20(2H,q); Column: Chiralpak AS (0.46×25 cm) manufactured by Daicel Chemical Industries, Co., Ltd.; Column temperature: 25° C.; Eluent: n-hexane/ethanol of 95/5; Flow rate: 1 ml/min.; Detection: 254 nm; Elution time: 19.6 minutes for (S), 21.3 minutes for (R). coli HB101(pNTS1G) obtained in Example 10 to allow for reaction for 18 hours After the reaction the reaction solution was subjected to extraction the solvent was removed under decompression the concentrate was purified by silica gel column chromatography

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

Procedure: The recombinant E. coli HB101(pNTS1G) obtained in Example 10 was inoculated in 100 ml of a 2×YT medium sterilized in a 500 ml Sakaguchi flask, and cultured with agitation at 37° C. for 13 hours. Glucose, 7.4 g, 3.2 mg of NADP, and then 4 g of ethyl 4-hydroxyacetoacetate were added to 50 ml of the resultant culture. The culture was stirred at 30° C. while being adjusted at pH 6.5 with a 5 M sodium hydroxide solution to allow for reaction for 18 hours. After the reaction, the reaction solution was subjected to extraction using ethyl acetate, the solvent was removed under decompression, and the concentrate was purified by silica gel column chromatography, to obtain 3.2 g of ethyl (S) -3,4-dihydroxybutyrate. The optical purity of the ethyl (S) -3,4-dihydroxybutyrate was analyzed as follows and found to be 100% e.e. The analysis was performed in the following manner. The sample was reacted with sodium cyanide in ethanol at room temperature to obtain 4-cyano-3-hydroxy ethyl butyrate, which was then changed to a benzoic ester using benzoyl chloride under the presence of pyridine. The optical purity of the benzoic ester was measured by the HPLC method. 1NMR(CDC13)δ(ppm): 1.30(3H,t), 2.55(2H,m), 3.18(1H,br), 3.55(1H,d), 3.68(1H,d), 4.15(1H,s), 4.20(2H,q); Column: Chiralpak AS (0.46×25 cm) manufactured by Daicel Chemical Industries, Co., Ltd.; Column temperature: 25° C.; Eluent: n-hexane/ethanol of 95/5; Flow rate: 1 ml/min.; Detection: 254 nm; Elution time: 19.6 minutes for (S), 21.3 minutes for (R). coli HB101(pNTS1G) obtained in Example 10 to allow for reaction for 18 hours After the reaction the reaction solution was subjected to extraction the solvent was removed under decompression the concentrate was purified by silica gel column chromatography

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

Procedure: The recombinant E. coli HB101(pNTS1G) obtained in Example 10 was inoculated in 100 ml of a 2×YT medium sterilized in a 500 ml Sakaguchi flask, and cultured with agitation at 37° C. for 13 hours. Glucose, 7.4 g, 3.2 mg of NADP, and then 4 g of ethyl 4-hydroxyacetoacetate were added to 50 ml of the resultant culture. The culture was stirred at 30° C. while being adjusted at pH 6.5 with a 5 M sodium hydroxide solution to allow for reaction for 18 hours. After the reaction, the reaction solution was subjected to extraction using ethyl acetate, the solvent was removed under decompression, and the concentrate was purified by silica gel column chromatography, to obtain 3.2 g of ethyl (S) -3,4-dihydroxybutyrate. The optical purity of the ethyl (S) -3,4-dihydroxybutyrate was analyzed as follows and found to be 100% e.e. The analysis was performed in the following manner. The sample was reacted with sodium cyanide in ethanol at room temperature to obtain 4-cyano-3-hydroxy ethyl butyrate, which was then changed to a benzoic ester using benzoyl chloride under the presence of pyridine. The optical purity of the benzoic ester was measured by the HPLC method. 1NMR(CDC13)δ(ppm): 1.30(3H,t), 2.55(2H,m), 3.18(1H,br), 3.55(1H,d), 3.68(1H,d), 4.15(1H,s), 4.20(2H,q); Column: Chiralpak AS (0.46×25 cm) manufactured by Daicel Chemical Industries, Co., Ltd.; Column temperature: 25° C.; Eluent: n-hexane/ethanol of 95/5; Flow rate: 1 ml/min.; Detection: 254 nm; Elution time: 19.6 minutes for (S), 21.3 minutes for (R). coli HB101(pNTS1G) obtained in Example 10 to allow for reaction for 18 hours After the reaction the reaction solution was subjected to extraction the solvent was removed under decompression the concentrate was purified by silica gel column chromatography

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

Procedure: The recombinant E. coli HB101(pNTS1G) obtained in Example 10 was inoculated in 100 ml of a 2×YT medium sterilized in a 500 ml Sakaguchi flask, and cultured with agitation at 37° C. for 13 hours. Glucose, 7.4 g, 3.2 mg of NADP, and then 4 g of ethyl 4-hydroxyacetoacetate were added to 50 ml of the resultant culture. The culture was stirred at 30° C. while being adjusted at pH 6.5 with a 5 M sodium hydroxide solution to allow for reaction for 18 hours. After the reaction, the reaction solution was subjected to extraction using ethyl acetate, the solvent was removed under decompression, and the concentrate was purified by silica gel column chromatography, to obtain 3.2 g of ethyl (S) -3,4-dihydroxybutyrate. The optical purity of the ethyl (S) -3,4-dihydroxybutyrate was analyzed as follows and found to be 100% e.e. The analysis was performed in the following manner. The sample was reacted with sodium cyanide in ethanol at room temperature to obtain 4-cyano-3-hydroxy ethyl butyrate, which was then changed to a benzoic ester using benzoyl chloride under the presence of pyridine. The optical purity of the benzoic ester was measured by the HPLC method. 1NMR(CDCl3) δ (ppm): 1.30(3H,t), 2.55(2H,m), 3.18(1H,br), 3.55(1H,d), 3.68(1H,d), 4.15(1H,s), 4.20(2H,q); Column: Chiralpak AS (0.46×25 cm) manufactured by Daicel Chemical Industries, Co., Ltd.; Column temperature: 25° C.; Eluent: n-hexane/ethanol of 95/5; Flow rate: 1 ml/min.; Detection: 254 nm; Elution time: 19.6 minutes for (S), 21.3 minutes for (R). coli HB101(pNTS1G) obtained in Example 10 to allow for reaction for 18 hours After the reaction the reaction solution was subjected to extraction the solvent was removed under decompression the concentrate was purified by silica gel column chromatography

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

Procedure: To (S)-ethyl 3,4-dihydroxybutanoate (1.40 g, 9.45 mmol) in methylene chloride (20 ml) were added triethylamine (1.15 g, 11.36 mmol) and DMAP (17 mg, 0.139 mmol) and the solution was cooled in ice bath. Trityl chloride (2.90 g, 10.4 mol) in methylene chloride (15 ml) was dropped to the solution under stirring and then stirred at room temperature over night. The reaction mixture was washed with saturated ammonium chloride and then saturated brine, dried on magnesium sulfate, filtered and condensed in vacuo. The residue was subjected to silica gel chromatography to give (S)-ethyl 3-hydroxy-4-trityloxybutanoate (1.11 g, yield 31%). the solution was cooled in ice bath stirred at room temperature over night The reaction mixture was washed with saturated ammonium chloride saturated brine, dried on magnesium sulfate filtered condensed in vacuo

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

Procedure: The recombinant E. coli HB101(pNTS1G) obtained in Example 10 was inoculated in 100 ml of a 2×YT medium sterilized in a 500 ml Sakaguchi flask, and cultured with agitation at 37° C. for 13 hours. Glucose, 7.4 g, 3.2 mg of NADP, and then 4 g of ethyl 4-hydroxyacetoacetate were added to 50 ml of the resultant culture. The culture was stirred at 30° C. while being adjusted at pH 6.5 with a 5 M sodium hydroxide solution to allow for reaction for 18 hours. After the reaction, the reaction solution was subjected to extraction using ethyl acetate, the solvent was removed under decompression, and the concentrate was purified by silica gel column chromatography, to obtain 3.2 g of ethyl (S) -3,4-dihydroxybutyrate. The optical purity of the ethyl (S) -3,4-dihydroxybutyrate was analyzed as follows and found to be 100% e.e. The analysis was performed in the following manner. The sample was reacted with sodium cyanide in ethanol at room temperature to obtain 4-cyano-3-hydroxy ethyl butyrate, which was then changed to a benzoic ester using benzoyl chloride under the presence of pyridine. The optical purity of the benzoic ester was measured by the HPLC method. 1NMR(CDC13)δ(ppm): 1.30(3H,t), 2.55(2H,m), 3.18(1H,br), 3.55(1H,d), 3.68(1H,d), 4.15(1H,s), 4.20(2H,q); Column: Chiralpak AS (0.46×25 cm) manufactured by Daicel Chemical Industries, Co., Ltd.; Column temperature: 25° C.; Eluent: n-hexane/ethanol of 95/5; Flow rate: 1 ml/min.; Detection: 254 nm; Elution time: 19.6 minutes for (S), 21.3 minutes for (R). coli HB101(pNTS1G) obtained in Example 10 to allow for reaction for 18 hours After the reaction the reaction solution was subjected to extraction the solvent was removed under decompression the concentrate was purified by silica gel column chromatography