恩格列净杂质29

反应条件: temp:60.01; pressure:0.00; stir:; See reaction.notes.procedure_details.

实验步骤: To a 3-necked jacketed 3 L flask equipped with a mechanical stirrer and connected to an automatic titrater by a pH electrode and a feeding tube for addition of base, was charged H20 (1200 mL), NaCN (37.25 g) and NaH2PO4 (125 g) to bring the solution to pH 7. The water circulator was set to 40° C. After 10 minutes, halohydrin dehalogenase SEQ ID NO: 32 as cell lysate (250 mL) was added. The reaction mixture was allowed to stir for 5 minutes. Using an addition funnel, ethyl (S)-4-chloro-3-hydroxybutyrate (45 g) was slowly added over 1 hour. The pH was maintained at 7 by the automatic titrater by the addition of 10 M NaOH (27 mL) over 17 hours. Subsequently, gas chromatography of a reaction sample showed complete conversion to product. Celite (16 g) was added to the flask, which was then connected to a diaphragm pump, whose exhaust is bubbled into 5M NaOH (200 mL), to remove HCN. The mixture was heated to 60° C. under 100 mm Hg pressure. After 1 hour, a submerged air bubbler was added to the solution to aid the removal of the HCN. After 3 hours, an HCN detector indicated less than 5 ppm HCN in the off-gas. The mixture was allowed to cool to room temperature, then filtered through a celite pad. The filtrate was extracted with butyl acetate (3×800 mL) and the combined organic layers filtered through a pad of activated charcoal. The solvent was removed under vacuum by rotary evaporation to provide 28.5 g of ethyl (R)-4-cyano-3-hydroxybutyrate. The purity was 98% (w/w) by HPLC and the enantiomeric excess was >99% (by chiral GC, the S enantiomer was undetectable). As used herein, the term “enantiomeric excess” or “e.e.” refers to the absolute difference between the mole or weight fractions of major (F(+)) and minor (F(−)) enantiomers (i.e., |F(+)−F(−)|), where F(+)+F(−)=1. Percent e.e. is 100×|F(+)−F(−)|. Enantiomeric composition can be readily characterized by using the gas chromatography method described in Example 6, above, and using methods that are known in the art. To a 3-necked jacketed 3 L flask equipped with a mechanical stirrer connected to an automatic titrater by a pH electrode and a feeding tube for addition of base was set to 40° C Celite (16 g) was added to the flask, which is bubbled into 5M NaOH (200 mL) to remove HCN After 1 hour a submerged air bubbler was added to the solution the removal of the HCN After 3 hours to cool to room temperature filtered through a celite pad The filtrate was extracted with butyl acetate (3×800 mL) the combined organic layers filtered through a pad of activated charcoal The solvent was removed under vacuum by rotary evaporation

反应条件: temp:0.00; pressure:0.00; stir:; See reaction.notes.procedure_details.

实验步骤: To a mixture of ethyl (S)-4-chloro-3-hydroxybutyrate (1 93 g, 1.16 mol, optical purity:98.5% ee) and N,N-dimethylformamide (1.4 Lit.) is added sodium azide (151 g, 2.32 mol) and the mixture was stirred at 100°-110° C. for 2 hours. After cooling N,N-dimethylformamide was distilled off and to the residue was added water (1 Lit.). The mixture was extracted with ethyl acetate and the extract was dried over magnesium sulfate. By distillation off ethyl acetate under vacuo, there is obtained ethyl (S)-4-azido-3-hydroxybutyrate as a crude product (206 g). Then the crude product was dissolved in methanol (1 Lit.) and to it 5% palladium-carbon (10 g) was added. The mixture was stirred for 17 hours at room temperature under a hydrogen atmosphere. After the reaction was over, palladium-carbon was filtered off and the solvent was distilled off under vacuo. The resulting product was recrystallized from acetone-water to give (S)-4-hydroxy-2-pyrrolidone (99 g, 0.98 mol, yield:84%) as a colorless crystalline solid. Optical purity:98.3% ee, Specific rotation:[α]D22 -57.6°(c=1.00, H2O). was distilled off and to the residue was added water (1 Lit.) The mixture was extracted with ethyl acetate the extract was dried over magnesium sulfate By distillation off ethyl acetate under vacuo

反应条件: temp:30.01; pressure:0.00; stir:; See reaction.notes.procedure_details.

实验步骤: The recombinant E. coil 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, 19.2 g, and 2.5 mg of NADP were added to 40 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. While stirring, a total of 16.1 g of ethyl 4-chloroacetoacetate was continuously added to the culture at a rate of about 2 g per hour. The reaction was performed 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 15.6 g of ethyl (S)-4-chloro-3-hydroxybutyrate. The optical purity of the ethyl (S)-4-chloro-3-hydroxybutyrate was analyzed by an HPLC method and found to be 100% e.e. 1H-NMR(CDC13)δ(ppm): 1.33(3H,t), 2.65(2H,d), 3.31(1H,d), 3.60(2H,d), 4.2(3H,m); Column: Chiralcel OB (0.46×25 cm) manufactured by Daicel Chemical Industries, Co., Ltd.; Column temperature: 0° C.; Eluent: n-hexane/2-propanol of 9/1; Flow rate: 0.8 ml/min.; Detection: 215 nm; Elution time: 19.2 minutes for (S), 17.0 minutes for (R). coil HB101(pNTS1G) obtained in Example 10 While stirring The reaction was performed 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

反应条件: temp:0.00; pressure:0.00; stir:; See reaction.notes.procedure_details.

实验步骤: 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

反应条件: temp:0.00; pressure:0.00; stir:; See reaction.notes.procedure_details.

实验步骤: 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

反应条件: temp:0.00; pressure:0.00; stir:; See reaction.notes.procedure_details.

实验步骤: 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

反应条件: temp:60.01; pressure:0.00; stir:; See reaction.notes.procedure_details.

实验步骤: To a 3-necked jacketed 3 L flask equipped with a mechanical stirrer and connected to an automatic titrater by a pH electrode and a feeding tube for addition of base, was charged H2O (1200 mL), NaCN (37.25 g) and NaH2PO4 (125 g) to bring the solution to pH 7. The water circulator was set to 40° C. After 10 minutes, halohydrin dehalogenase of SEQ ID NO: 32 as cell lysate (250 mL) was added. The reaction mixture was allowed to stir for 5 minutes. Using an addition funnel, ethyl (S)-4-chloro-3-hydroxybutyrate (45 g) was slowly added over 1 hour. The pH was maintained at 7 by the automatic titrater by the addition of 10 M NaOH (27 mL) over 17 hours. Subsequently, gas chromatography of a reaction sample showed complete conversion to product. Celite™ (16 g) was added to the flask, which was then connected to a diaphragm pump, whose exhaust is bubbled into 5M NaOH (200 mL), to remove HCN. The mixture was heated to 60° C. under 100 mm Hg pressure. After 1 hour, a submerged air bubbler was added to the solution to aid the removal of the HCN. After 3 hours, an HCN detector indicated less than 5 ppm HCN in the off-gas. The mixture was allowed to cool to room temperature, then filtered through a Celite™ pad. The filtrate was extracted with butyl acetate (3×800 mL) and the combined organic layers filtered through a pad of activated charcoal. The solvent was removed under vacuum by rotary evaporation to provide 28.5 g of ethyl (R)-4-cyano-3-hydroxybutyrate. The purity was 98% (w/w) by HPLC and the enantiomeric excess was >99% (by chiral GC, the S enantiomer was undetectable). As used herein, the term “enantiomeric excess” or “e.e.” refers to the absolute difference between the mole or weight fractions of major (F(+)) and minor (F(−)) enantiomers (i.e., |F(+)−F(−)|), where F(+)+F(−)=1. Percent e.e. is 100×|F(+)−F(−)|. Enantiomeric composition can be readily characterized by using the gas chromatography method described in Example 6, above, and using methods that are known in the art. To a 3-necked jacketed 3 L flask equipped with a mechanical stirrer connected to an automatic titrater by a pH electrode and a feeding tube for addition of base was set to 40° C Celite™ (16 g) was added to the flask, which is bubbled into 5M NaOH (200 mL) to remove HCN After 1 hour a submerged air bubbler was added to the solution the removal of the HCN After 3 hours to cool to room temperature filtered through a Celite™ pad The filtrate was extracted with butyl acetate (3×800 mL) the combined organic layers filtered through a pad of activated charcoal The solvent was removed under vacuum by rotary evaporation

反应条件: temp:0.00; pressure:0.00; stir:; See reaction.notes.procedure_details.

实验步骤: In a 50 mL-volumetric autoclave were added deaerated ethanol (10 mL), and thereto were added cobalt acetate tetrahydrate (250 mg, 1 mmol) and 10% Pd/C (10 mg). After covering the autoclave with a cap, carbon monoxide (1 MPa) and hydrogen gas (1 MPa) were introduced therein, successively and the mixture was reacted at 80° C. for 3 hours. After 3 hours the mixture was cooled to room temperature, and the mixed gas was emitted to give a solution containing dicobaltoctacarbonyl complex. Thereto were added 4-aminopyridine (47 mg, 0.5 mmol) and (S)-epichlorohydrin (1.8 g, 20 mmol, >99% ee). After covering the autoclave with a cap, carbon monoxide (1 MPa) was introduced therein and the mixture was reacted at 35° C. for 28 hours. After temperature of the mixture was made room temperature, the solvent was removed in vacuo. The residue was subjected to Kugelrohr distillation to give (S)-4-chloro-3- hydroxybutanoic acid ethyl ester (3.1 g, 92%, >99% ee) as a colorless oil. were introduced successively and the mixture was reacted at 80° C. for 3 hours to give a solution containing dicobaltoctacarbonyl complex was introduced the mixture was reacted at 35° C. for 28 hours was made room temperature the solvent was removed in vacuo The residue was subjected to Kugelrohr distillation

反应条件: temp:0.00; pressure:0.00; stir:; See reaction.notes.procedure_details.

实验步骤: In a 50 mL-volumetric autoclave were added deaerated ethanol (10 mL), and thereto were added cobalt acetate tetrahydrate (250 mg, 1 mmol) and 10% Pd/C (10 mg). After covering the autoclave with a cap, carbon monoxide (1 MPa) and hydrogen gas (1 MPa) were introduced therein, successively and the mixture was reacted at 80° C. for 3 hours. After 3 hours the mixture was cooled to room temperature, and the mixed gas was emitted to give a solution containing dicobaltoctacarbonyl complex. Thereto were added 4-aminopyridine (47 mg, 0.5 mmol) and (S)-epichlorohydrin (1.8 g, 20 mmol, >99% ee). After covering the autoclave with a cap, carbon monoxide (1 MPa) was introduced therein and the mixture was reacted at 35° C. for 28 hours. After temperature of the mixture was made room temperature, the solvent was removed in vacuo. The residue was subjected to Kugelrohr distillation to give (S)-4-chloro-3- hydroxybutanoic acid ethyl ester (3.1 g, 92%, >99% ee) as a colorless oil. were introduced successively and the mixture was reacted at 80° C. for 3 hours to give a solution containing dicobaltoctacarbonyl complex was introduced the mixture was reacted at 35° C. for 28 hours was made room temperature the solvent was removed in vacuo The residue was subjected to Kugelrohr distillation

反应条件: temp:60.01; pressure:0.00; stir:; See reaction.notes.procedure_details.

实验步骤: To a 3-necked jacketed 3L flask equipped with a mechanical stirrer and connected to an automatic titrater by a pH electrode and a feeding tube for addition of base, was charged H2O (1200 mL), NaCN (37.25 g) and NaH2PO4 (125 g) to bring the solution to pH 7. The water circulator was set to 40° C. After 10 minutes, halohydrin dehalogenase of SEQ ID NO: 32 as cell lysate (250 mL) was added. The reaction mixture was allowed to stir for 5 minutes. Using an addition funnel, ethyl (S)-4-chloro-3-hydroxybutyrate (45 g) was slowly added over 1 hour. The pH was maintained at 7 by the automatic titrater by the addition of 10 M NaOH (27 mL) over 17 hours. Subsequently, gas chromatography of a reaction sample showed complete conversion to product. Celite™ (16 g) was added to the flask, which was then connected to a diaphragm pump, whose exhaust is bubbled into 5M NaOH (200 mL), to remove HCN. The mixture was heated to 60° C. under 100 mm Hg pressure. After 1 hour, a submerged air bubbler was added to the solution to aid the removal of the HCN. After 3 hours, an HCN detector indicated less than 5 ppm HCN in the off-gas. The mixture was allowed to cool to room temperature, then filtered through a Celite™ pad. The filtrate was extracted with butyl acetate (3×800 mL) and the combined organic layers filtered through a pad of activated charcoal. The solvent was removed under vacuum by rotary evaporation to provide 28.5 g of ethyl (R)-4-cyano-3-hydroxybutyrate. The purity was 98% (w/w) by HPLC and the enantiomeric excess was >99% (by chiral GC, the S enantiomer was undetectable). As used herein, the term “enantiomeric excess” or “e.e.” refers to the absolute difference between the mole or weight fractions of major (F(+)) and minor (F(−)) enantiomers (i.e., |F(+)−F(−)|), where F(+)+F(−)=1. Percent e.e. is 100×|F(+)−F(−)|. Enantiomeric composition can be readily characterized by using the gas chromatography method described in Example 6, above, and using methods that are known in the art. connected to an automatic titrater by a pH electrode and a feeding tube for addition of base was set to 40° C Celite™ (16 g) was added to the flask, which is bubbled into 5M NaOH (200 mL) to remove HCN After 1 hour a submerged air bubbler was added to the solution the removal of the HCN After 3 hours to cool to room temperature filtered through a Celite™ pad The filtrate was extracted with butyl acetate (3×800 mL) the combined organic layers filtered through a pad of activated charcoal The solvent was removed under vacuum by rotary evaporation

反应条件: temp:0.00; pressure:0.00; stir:; See reaction.notes.procedure_details.

实验步骤: To a 100 mL vessel connected to an automatic titrater by a pH electrode and a feeding tube for addition of base was charged a solution of glucose (12. g) in water (30 mL). To this solution was charged ketoreductase SEQ ID NO: 42 (100 mg); 50 mg GDH SEQ ID NO: 66 and NADP (6.25 mg). Butyl acetate (10 ml) was then charged. Ethyl 4-chloroacetoacetate (10 g) was then charged via syringe pump as follows: 1 mL was charged rapidly and the remainder was then charged at a rate of 1 mL/hr). The pH was maintained at 7 by the automatic titrater by the addition of 4M NaOH over 18 hours hrs. The stirring was stopped and the phases allowed to separate. The organic layer included some emulsion. The organic layer, including some emulsion, was separated and washed with 10 mL of water. The combined aqueous layers were extracted twice with 20 mL of butyl acetate. The organic extracts were combined and rotary evaporated under vacuum to remove water. Additional butyl acetate was added during the evaporation to help remove the water. When the water was removed the butyl acetate solution was decanted from solids in the flask. Evaporation of the solvent under vacuum then gave 8.85 g of ethyl (S)-4-chloro-3-hydroxybutyrate (87.4% yield) of very good purity. To a 100 mL vessel connected to an automatic titrater by a pH electrode and a feeding tube for addition of base To this solution was charged ketoreductase SEQ ID NO 1 mL was charged rapidly the remainder was then charged at a rate of 1 mL/hr) to separate was separated washed with 10 mL of water The combined aqueous layers were extracted twice with 20 mL of butyl acetate rotary evaporated under vacuum to remove water Additional butyl acetate was added during the evaporation to help remove the water When the water was removed the butyl acetate solution was decanted from solids in the flask Evaporation of the solvent under vacuum

反应条件: temp:60.01; pressure:0.00; stir:; See reaction.notes.procedure_details.

实验步骤: To a 3-necked jacketed 3 L flask equipped with a mechanical stirrer and connected to an automatic titrater by a pH electrode and a feeding tube for addition of base, was charged H2O (1200 mL), NaCN (37.25 g) and NaH2PO4 (125 g) to bring the solution to pH 7. The water circulator was set to 40° C. After 10 minutes, halohydrin dehalogenase SEQ ID NO: 32 as cell lysate (250 mL) was added. The reaction mixture was allowed to stir for 5 minutes. Using an addition funnel, ethyl (S)-4-chloro-3-hydroxybutyrate (45 g of the material from Example 7) was slowly added over 1 hour. The pH was maintained at 7 by the automatic titrater by the addition of 10 M NaOH (27 mL) over 17 hrs. Subsequently, gas chromatography of a reaction sample showed complete conversion to product. Celite (16 g) was added to the flask, which was then connected to a diaphragm, whose exhaust is bubbled into 5M NaOH (200 mL), to remove HCN. The mixture was heated to 60° C. under 100 mm Hg pressure. After 1 hour a submerged air bubbler was added to the solution to aid the removal of the HCN. After 3 hours, an HCN detector indicated less than 5 ppm HCN in the off-gas. The mixture was allowed to cool to room temperature, then filtered through a celite pad. The filtrate was extracted with butyl acetate (3×800 mL) and the combined organic layers filtered through a pad of activated charcoal. The solvent was removed under vacuum by rotary evaporation to provide 28.5 g of ethyl (R)-4-cyano-3-hydroxybutyrate. The purity was 98% (w/w) by HPLC and the enantiomeric excess was >99% (by chiral GC, the S enantiomer was undetectable). To a 3-necked jacketed 3 L flask equipped with a mechanical stirrer connected to an automatic titrater by a pH electrode and a feeding tube for addition of base was set to 40° C Celite (16 g) was added to the flask, which is bubbled into 5M NaOH (200 mL) to remove HCN After 1 hour a submerged air bubbler was added to the solution the removal of the HCN After 3 hours to cool to room temperature filtered through a celite pad The filtrate was extracted with butyl acetate (3×800 mL) the combined organic layers filtered through a pad of activated charcoal The solvent was removed under vacuum by rotary evaporation