ethyl 4-hydroxy-3-oxobutanoate

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

Procedure: 21.24 g of the 4-benzyloxyacetoacetic ethyl ester dissolved in acetic ester (100 ml) was inserted into a 200 ml steel autoclave provided with a magnetic stirrer and was hydrogenolized in the presence of 1.05 g Pd 5 percent on coal with hydrogen with a pressure of 5 atm. After the reaction was completed, which amounted to a period of about 2 hours, and after a 2 hour post-reaction time, the catalyst was filtered out. The filtrate was evaporated on a vacuum rotation evaporator at 35° C. The residue was dried in the high vacuum. 13.4 g of 4-hydroxyacetoacetic ethyl ester in the form of a slightly yellowish oil resulted. provided with a magnetic stirrer after a 2 hour post-reaction time the catalyst was filtered out The filtrate was evaporated on a vacuum rotation evaporator at 35° C The residue was dried in the high vacuum

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

Procedure: 21.24 g of the 4-benzyloxyacetoacetic ethyl ester dissolved in acetic ester (100 ml) was inserted into a 200 ml steel autoclave provided with a magnetic stirrer and was hydrogenolized in the presence of 1.05 g Pd 5 percent on coal with hydrogen with a pressure of 5 atm. After the reaction was completed, which amounted to a period of about 2 hours, and after a 2 hour post-reaction time, the catalyst was filtered out. The filtrate was evaporated on a vacuum rotation evaporator at 35° C. The residue was dried in the high vacuum. 13.4 g of 4-hydroxyacetoacetic ethyl ester in the form of a slightly yellowish oil resulted. provided with a magnetic stirrer after a 2 hour post-reaction time the catalyst was filtered out The filtrate was evaporated on a vacuum rotation evaporator at 35° C The residue was dried in the high vacuum

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

Procedure: To a solution of 4-bromo-3-ketobutyric acid ethyl ester (2a) (21 g, 0.10 moles) in methanol dried prior to use with Molecular Sieves is added sodium formate (10.2 g, 0.15 moles), and the mixture is refluxed for 1 hour. The reaction mixture is concentrated under reduced pressure to remove the solvent. The residue is extracted with chloroform. The extract is purified by silica gel column chromatography (eluting solvent=benzene/ethyl acetate (2:1)) to give the title compound (3a). Yield: 7.34 g, 50%. the mixture is refluxed for 1 hour The reaction mixture is concentrated under reduced pressure to remove the solvent The residue is extracted with chloroform The extract is purified by silica gel column chromatography (eluting solvent=benzene/ethyl acetate (2:1))

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: 21.24 g of the 4-benzyloxyacetoacetic ethyl ester dissolved in acetic ester (100 ml) was inserted into a 200 ml steel autoclave provided with a magnetic stirrer and was hydrogenolized in the presence of 1.05 g Pd 5 percent on coal with hydrogen with a pressure of 5 atm. After the reaction was completed, which amounted to a period of about 2 hours, and after a 2 hour post-reaction time, the catalyst was filtered out. The filtrate was evaporated on a vacuum rotation evaporator at 35° C. The residue was dried in the high vacuum. 13.4 g of 4-hydroxyacetoacetic ethyl ester in the form of a slightly yellowish oil resulted. provided with a magnetic stirrer after a 2 hour post-reaction time the catalyst was filtered out The filtrate was evaporated on a vacuum rotation evaporator at 35° C The residue was dried in the high vacuum

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