ethyl 4-iodo-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, 0.5 g, 3.2 mg of NADP, and then 0.5 g of ethyl 4-iodoacetoacetate 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 72 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 900 mg of ethyl (S)-4-iodo-3-hydroxybutyrate. The optical purity of the ethyl (S)-4-iodo-3-hydroxybutyrate was analyzed as follows and found to be 91.6% e.e. That is, the sample was heated together with sodium cyanide in dimethyl sulfoxide to obtain ethyl 4-cyano-3-hydroxybutyrate, 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 an HPLC method. NMR(CDC13)δ(ppm): 1.28(3H,t), 2.65(2H,d), 3.31(3H,m), 4.00(1H,m), 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 72 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:0.01; pressure:0.00; stir:; See reaction.notes.procedure_details.

Procedure: Sodium iodide (228 g) was dissolved in acetone (135 mL) and cooled to 0° C. 4-chloro-3-oxobutanoic acid, ethyl ester (5.0 g) was added to the sodium iodide solution and the mixture was stirred at 0° C. for 20 minutes It was diluted with pentane (135 mL) and water (135 mL). The reaction mixture was diluted with ethyl acetate and washed with water, brine and dried over anhydrous sodium sulfate to give an intermediate (4-Iodo-3-oxobutanoic acid, ethyl ester; 6.2 g yield). washed with water, brine dried over anhydrous sodium sulfate to give

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, 0.5 g, 3.2 mg of NADP, and then 0.5 g of ethyl 4-iodoacetoacetate 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 72 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 900 mg of ethyl (S)-4-iodo-3-hydroxybutyrate. The optical purity of the ethyl (S)-4-iodo-3-hydroxybutyrate was analyzed as follows and found to be 91.6% e.e. That is, the sample was heated together with sodium cyanide in dimethyl sulfoxide to obtain ethyl 4-cyano-3-hydroxybutyrate, 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 an HPLC method. NMR(CDC13)δ(ppm): 1.28(3H,t), 2.65(2H,d), 3.31(3H,m), 4.00(1H,m), 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 72 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, 0.5 g, 3.2 mg of NADP, and then 0.5 g of ethyl 4-iodoacetoacetate 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 72 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 900 mg of ethyl (S)-4-iodo-3-hydroxybutyrate. The optical purity of the ethyl (S)-4-iodo-3-hydroxybutyrate was analyzed as follows and found to be 91.6% e.e. That is, the sample was heated together with sodium cyanide in dimethyl sulfoxide to obtain ethyl 4-cyano-3-hydroxybutyrate, 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 an HPLC method. NMR(CDC13)δ(ppm): 1.28(3H,t), 2.65(2H,d), 3.31(3H,m), 4.00(1H,m), 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 72 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:0.01; pressure:0.00; stir:; See reaction.notes.procedure_details.

Procedure: Sodium iodide (228 g) was dissolved in acetone (135 mL) and cooled to 0° C. 4-chloro-3-oxobutanoic acid, ethyl ester (5.0 g) was added to the sodium iodide solution and the mixture was stirred at 0° C. for 20 minutes. It was diluted with pentane (135 mL) and water (135 mL). The reaction mixture was diluted with ethyl acetate and washed with water, brine and dried over anhydrous sodium sulfate to give an intermediate (4-Iodo-3-oxobutanoic acid, ethyl ester; 6.2 g yield). washed with water, brine dried over anhydrous sodium sulfate to give

Conditions: temp:0.01; pressure:0.00; stir:; See reaction.notes.procedure_details.

Procedure: Sodium iodide (228 g) was dissolved in acetone (135 mL) and cooled to 0° C. 4-chloro-3-oxobutanoic acid, ethyl ester (5.0 g) was added to the sodium iodide solution and the mixture was stirred at 0° C. for 20 minutes. It was diluted with pentane (135 mL) and water (135 mL). The reaction mixture was diluted with ethyl acetate and washed with water, brine and dried over anhydrous sodium sulfate to give an intermediate (4-Iodo-3-oxobutanoic acid, ethyl ester; 6.2 g yield). washed with water, brine dried over anhydrous sodium sulfate to give

Conditions: temp:0.01; pressure:0.00; stir:; See reaction.notes.procedure_details.

Procedure: Sodium iodide (228 g) was dissolved in acetone (135 mL) and cooled to 0° C. 4-chloro-3-oxobutanoic acid, ethyl ester (5.0 g) was added to the sodium iodide solution and the mixture was stirred at 0° C. for 20 minutes. It was diluted with pentane (135 mL) and water (135 mL). The reaction mixture was diluted with ethyl acetate and washed with water, brine and dried over anhydrous sodium sulfate to give an intermediate (4-Iodo-3-oxobutanoic acid, ethyl ester; 6.2 g yield). washed with water, brine dried over anhydrous sodium sulfate to give

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, 0.5 g, 3.2 mg of NADP, and then 0.5 g of ethyl 4-iodoacetoacetate 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 72 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 900 mg of ethyl (S)-4-iodo-3-hydroxybutyrate. The optical purity of the ethyl (S)-4-iodo-3-hydroxybutyrate was analyzed as follows and found to be 91.6% e.e. That is, the sample was heated together with sodium cyanide in dimethyl sulfoxide to obtain ethyl 4-cyano-3-hydroxybutyrate, 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 an HPLC method. NMR(CDCl3) δ (ppm): 1.28(3H,t), 2.65(2H,d), 3.31(3H,m), 4.00(1H,m), 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). (pNTS1G) obtained in Example 10 to allow for reaction for 72 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:0.01; pressure:0.00; stir:; See reaction.notes.procedure_details.

Procedure: Sodium iodide (228 g) was dissolved in acetone (135 mL) and cooled to 0° C. 4-chloro-3-oxobutanoic acid, ethyl ester (5.0 g) was added to the sodium iodide solution and the mixture was stirred at 0° C. for 20 minutes It was diluted with pentane (135 mL) and water (135 mL). The reaction mixture was diluted with ethyl acetate and washed with water, brine and dried over anhydrous sodium sulfate to give an intermediate (4-Iodo-3-oxobutanoic acid, ethyl ester; 6.2 g yield). washed with water, brine dried over anhydrous sodium sulfate to give

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, 0.5 g, 3.2 mg of NADP, and then 0.5 g of ethyl 4-iodoacetoacetate 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 72 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 900 mg of ethyl (S)-4-iodo-3-hydroxybutyrate. The optical purity of the ethyl (S)-4-iodo-3-hydroxybutyrate was analyzed as follows and found to be 91.6% e.e. That is, the sample was heated together with sodium cyanide in dimethyl sulfoxide to obtain ethyl 4-cyano-3-hydroxybutyrate, 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 an HPLC method. NMR(CDC13)δ(ppm): 1.28(3H,t), 2.65(2H,d), 3.31(3H,m), 4.00(1H,m), 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 72 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