methyl (3S)-4-chloro-3-hydroxybutanoate

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, 7.2 g, 3.2 mg of NADP, and then 4 g of methyl 4-chloroacetoacetate 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 24 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 3.85 g of methyl (S)-4-chloro-3-hydroxybutyrate. The optical purity of the methyl (S)-4-chloro-3-hydroxybutyrate was analyzed as follows and found to be 100% e.e. That is, the sample was converted to a carbamate using phenyl isocyanate under the presence of pyridine and measuring the optical purity of the carbamate by an HPLC method. 1H-NMR(CDC13)δ(ppm): 2.65(2H,m), 3.20(1H,br), 3.63(2H,m), 3.73(3H,s), 4.28(1H,m); Column: Chiralcel OJ (0.46×25 cm) manufactured by Daicel Chemical Industries, Co., Ltd.; Column temperature: 25° C.; Eluent: n-hexane/2-propanol of 8/2; Flow rate: 1 ml/min.; Detection: 254 nm; Elution time: 19.2 minutes for (S), 22.6 minutes for (R). coli HB101(pNTS1G) obtained in Example 10 to allow for reaction for 24 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: In a 50 mL-volumetric autoclave were added deaerated methanol (5 mL) and t-butanol (3 mL), and thereto were added 4-aminopyridine (47 mg, 0.5 mmol) and (S)-epichlorohydrin (1.9 g, 20 mmol, >99% ee). Then to the mixture was added crystalline dicobaltoctacarbonyl complex (171 mg, 0.5 mmol). After covering the autoclave with a cap, carbon monoxide (1 MPa) was introduced therein and the mixture was reacted at 40° C. for 5 hours. After cooling to room temperature, the solvent was removed in vacuo. The residue was subjected to Kugelrohr distillation to give (S)-4-chloro-3-hydroxybutanoic acid methyl ester (2.8 g, 92%, >99% ee) as a colorless oil. was introduced the mixture was reacted at 40° C. for 5 hours the solvent was removed in vacuo The residue was subjected to Kugelrohr distillation

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

Procedure: In a 50 mL-volumetric autoclave were added deaerated methanol (5 mL) and t-butanol (3 mL), and thereto were added 4-aminopyridine (47 mg, 0.5 mmol) and (S)-epichlorohydrin (1.9 g, 20 mmol, >99% ee). Then to the mixture was added crystalline dicobaltoctacarbonyl complex (171 mg, 0.5 mmol). After covering the autoclave with a cap, carbon monoxide (1 MPa) was introduced therein and the mixture was reacted at 40° C. for 5 hours. After cooling to room temperature, the solvent was removed in vacuo. The residue was subjected to Kugelrohr distillation to give (S)-4-chloro-3-hydroxybutanoic acid methyl ester (2.8 g, 92%, >99% ee) as a colorless oil. was introduced the mixture was reacted at 40° C. for 5 hours the solvent was removed in vacuo The residue was subjected to Kugelrohr distillation

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, 7.2 g, 3.2 mg of NADP, and then 4 g of methyl 4-chloroacetoacetate 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 24 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 3.85 g of methyl (S)-4-chloro-3-hydroxybutyrate. The optical purity of the methyl (S)-4-chloro-3-hydroxybutyrate was analyzed as follows and found to be 100% e.e. That is, the sample was converted to a carbamate using phenyl isocyanate under the presence of pyridine and measuring the optical purity of the carbamate by an HPLC method. 1H-NMR(CDC13)δ(ppm): 2.65(2H,m), 3.20(1H,br), 3.63(2H,m), 3.73(3H,s), 4.28(1H,m); Column: Chiralcel OJ (0.46×25 cm) manufactured by Daicel Chemical Industries, Co., Ltd.; Column temperature: 25° C.; Eluent: n-hexane/2-propanol of 8/2; Flow rate: 1 ml/min.; Detection: 254 nm; Elution time: 19.2 minutes for (S), 22.6 minutes for (R). coli HB101(pNTS1G) obtained in Example 10 to allow for reaction for 24 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, 7.2 g, 3.2 mg of NADP, and then 4 g of methyl 4-chloroacetoacetate 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 24 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 3.85 g of methyl (S)-4-chloro-3-hydroxybutyrate. The optical purity of the methyl (S)-4-chloro-3-hydroxybutyrate was analyzed as follows and found to be 100% e.e. That is, the sample was converted to a carbamate using phenyl isocyanate under the presence of pyridine and measuring the optical purity of the carbamate by an HPLC method. 1H-NMR(CDC13)δ(ppm): 2.65(2H,m), 3.20(1H,br), 3.63(2H,m), 3.73(3H,s), 4.28(1H,m); Column: Chiralcel OJ (0.46×25 cm) manufactured by Daicel Chemical Industries, Co., Ltd.; Column temperature: 25° C.; Eluent: n-hexane/2-propanol of 8/2; Flow rate: 1 ml/min.; Detection: 254 nm; Elution time: 19.2 minutes for (S), 22.6 minutes for (R). coli HB101(pNTS1G) obtained in Example 10 to allow for reaction for 24 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, 7.2 g, 3.2 mg of NADP, and then 4 g of methyl 4-chloroacetoacetate 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 24 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 3.85 g of methyl (S)-4-chloro-3-hydroxybutyrate. The optical purity of the methyl (S)-4-chloro-3-hydroxybutyrate was analyzed as follows and found to be 100% e.e. That is, the sample was converted to a carbamate using phenyl isocyanate under the presence of pyridine and measuring the optical purity of the carbamate by an HPLC method. 1H-NMR(CDCl3) δ (ppm): 2.65(2H,m), 3.20(1H,br), 3.63(2H,m), 3.73(3H,s), 4.28(1H,m); Column: Chiralcel OJ (0.46×25 cm) manufactured by Daicel Chemical Industries, Co., Ltd.; Column temperature: 25° C.; Eluent: n-hexane/2-propanol of 8/2; Flow rate: 1 ml/min.; Detection: 254 nm; Elution time: 19.2 minutes for (S), 22.6 minutes for (R). (pNTS1G) obtained in Example 10 to allow for reaction for 24 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: In a 50 mL-volumetric autoclave were added deaerated methanol (5 mL) and t-butanol (3 mL), and thereto were added 4-aminopyridine (47 mg, 0.5 mmol) and (S)-epichlorohydrin (1.9 g, 20 mmol, >99% ee). Then to the mixture was added crystalline dicobaltoctacarbonyl complex (171 mg, 0.5 mmol). After covering the autoclave with a cap, carbon monoxide (1 MPa) was introduced therein and the mixture was reacted at 40° C. for 5 hours. After cooling to room temperature, the solvent was removed in vacuo. The residue was subjected to Kugelrohr distillation to give (S)-4-chloro-3-hydroxybutanoic acid methyl ester (2.8 g, 92%, >99% ee) as a colorless oil. was introduced the mixture was reacted at 40° C. for 5 hours the solvent was removed in vacuo The residue was subjected to Kugelrohr distillation

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, 7.2 g, 3.2 mg of NADP, and then 4 g of methyl 4-chloroacetoacetate 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 24 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 3.85 g of methyl (S)-4-chloro-3-hydroxybutyrate. The optical purity of the methyl (S)-4-chloro-3-hydroxybutyrate was analyzed as follows and found to be 100% e.e. That is, the sample was converted to a carbamate using phenyl isocyanate under the presence of pyridine and measuring the optical purity of the carbamate by an HPLC method. 1H-NMR(CDC13)δ(ppm): 2.65(2H,m), 3.20(1H,br), 3.63(2H,m), 3.73(3H,s), 4.28(1H,m); Column: Chiralcel OJ (0.46×25 cm) manufactured by Daicel Chemical Industries, Co., Ltd.; Column temperature: 25° C.; Eluent: n-hexane/2-propanol of 8/2; Flow rate: 1 ml/min.; Detection: 254 nm; Elution time: 19.2 minutes for (S), 22.6 minutes for (R). coli HB101(pNTS1G) obtained in Example 10 to allow for reaction for 24 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