ethyl (3R)-4-cyano-3-hydroxybutanoate

Conditions: temp:60.01; pressure:0.00; stir:; See reaction.notes.procedure_details.

Procedure: 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

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

Procedure: (R)4-cyano-3-hydroxy butyric acid ethyl ester (1.5 g, 9.54 mmol) was dissolved in iodomethane (20 ml). Silver oxide solid (3.3 g, 14.32 mmol) was added, and the reaction mixture was agitated at room temperature overnight. TLC (PE/EA=2/1) was employed to monitor the reaction. After the reaction completed, the silver oxide solid was removed by filtration, and the solvent was evaporated. Column chromatography (PE/EA=10/1) afforded colorless transparent liquid 1.0 g, yield 63.1%. the reaction the silver oxide solid was removed by filtration the solvent was evaporated

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

Procedure: (R)4-cyano-3-hydroxy butyric acid ethyl ester (1.5 g, 9.54 mmol) was dissolved in iodomethane (20 ml). Silver oxide solid (3.3 g, 14.32 mmol) was added, and the reaction mixture was agitated at room temperature overnight. TLC (PE/EA=2/1) was employed to monitor the reaction. After the reaction completed, the silver oxide solid was removed by filtration, and the solvent was evaporated. Column chromatography (PE/EA=10/1) afforded colorless transparent liquid 1.0 g, yield 63.1%. the reaction the silver oxide solid was removed by filtration the solvent was evaporated

Conditions: temp:60.01; pressure:0.00; stir:; See reaction.notes.procedure_details.

Procedure: 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

Conditions: temp:60.01; pressure:0.00; stir:; See reaction.notes.procedure_details.

Procedure: 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

Conditions: temp:60.01; pressure:0.00; stir:; See reaction.notes.procedure_details.

Procedure: 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

Conditions: temp:60.01; pressure:0.00; stir:; See reaction.notes.procedure_details.

Procedure: 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

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

Procedure: To a well stirred solution of ethyl (S)-4-chloro3-hydroxybutyrate (8.25 g, 50 mmoles) in 300 mM potassium phosphate buffer, 300 mM NaCN pH 8.0 (1 L) at 30° C. was added halohydrin dehalogenase SEQ ID NO: 14 (9 g) as a lyophilized powder. After fifty seven hours the mixture was washed with ethyl acetate (2 times 250 mL) and the combined organics dried over anhydrous sodium sulfate. The mixture was filtered and evaporated on a rotary evaporator to give essentially pure ethyl (R)-4-cyano-3-hydroxybutyrate, as determined using the gas chromatography method and elution time data described in Example 6, hereinbelow. After fifty seven hours the mixture was washed with ethyl acetate (2 times 250 mL) the combined organics dried over anhydrous sodium sulfate The mixture was filtered evaporated on a rotary evaporator

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

Procedure: To a stirred -50° C. solution of lithium diisopropylamide (100 kg of 2 M) in tetrahydrofuranheptane was added tertiary-butyl acetate (30 kg, 255 mol) followed by a rinse of 3 kg of tetrahydrofuran, and the mixture was stirred to -45° C. to -5° C. for 50 minutes. (R)-4-cyano-3-hydroxybutyric acid, ethyl ester (Step A) (10 kg, 64 mol) as a solution in 30 kg of tetrahydrofuran was then added to the previous mixture. The reaction mixture was stirred for 30 minutes at -5° C. to -30° C., and transferred to 240 L of 0° C. 2.8N aqueous hydrochloric acid solution. The aqueous layer was extracted with 50 kg of ethyl acetate, the aqueous layer was separated and reextracted with 36 kg of ethyl acetate, the extracts are combined, and concentrated in vacuo to afford crude (5R)-1,1-dimethylethyl 6-cyano-5-hydroxy-3-oxo-hexanoate which was not isolated. A small sample was purified by column chromatography on flash silica gel eluting with 1:1 hexane:ethyl acetate. a rinse of 3 kg of tetrahydrofuran The reaction mixture was stirred for 30 minutes at -5° C. to -30° C. transferred to 240 L of 0° C The aqueous layer was extracted with 50 kg of ethyl acetate the aqueous layer was separated concentrated in vacuo

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

Procedure: To a 50 gallon reactor containing a solution of 2.2 kg (44 mol) of sodium cyanide dissolved in 40 L of demineralized water was added 7 kg (33 mol) of (S)-4-bromo-3-hydroxybutyric acid, ethyl ester (Example 3) dissolved in 8 L of ethanol. The reaction mixture was stirred for 16 hours at room temperature. Ethyl acetate (65 L) was added, the mixture agitated, and the layers were allowed to separate. The lower aqueous layer was transferred to a 50 gallon vessel containing 2.5 kg of sodium chloride and 65 L of ethyl acetate, the mixture was agitated and the layers allowed to separate, and the lower aqueous layer was cut off. The organic layers were combined and concentrated in vacuo. The residue was distilled to afford 3.1 kg of the title compound; bp 110-125° C. 0.5 mm Hg; optical rotation [α]D25 =33.1° (C=1.08, chloroform); the mixture agitated to separate The lower aqueous layer was transferred to a 50 gallon vessel containing 2.5 kg of sodium chloride and 65 L of ethyl acetate the mixture was agitated to separate concentrated in vacuo The residue was distilled

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

Procedure: To a stirred -10° C. solution of 1-acetylpiperidine (127 g, 1.0 mol) in tetrahydrofuran (1.0 L) is slowly added a solution of lithium diisopropylamide in tetrahydrofuran-heptane (0.5 L of 2M) while maintaining the temperature between -40° C. to -50° C., and the mixture is stirred at -20° C. to -30° C. for 30 minutes. (R)-4-cyano-3-hydroxybutyric acid, ethyl ester (Brower, supra) (40 g, 0.25 mol) as a solution in 200 mL of tetrahydrofuran is then added to the previous mixture. The reaction mixture is stirred for 30 minutes at -25° C. to -20° C., and transferred to a 2.2N aqueous hydrochloric acid solution (1 L). The aqueous layer is extracted with 500 mL of ethyl acetate, the aqueous layer is separated and extracted with 100 mL of ethyl acetate, the extracts are combined and concentrated in vacuo to afford (R)-ε-hydroxy-α,γ-dioxo-1-piperidineheptanenitrile as an oil. while maintaining the temperature between -40° C. to -50° C. The reaction mixture is stirred for 30 minutes at -25° C. to -20° C. The aqueous layer is extracted with 500 mL of ethyl acetate the aqueous layer is separated extracted with 100 mL of ethyl acetate concentrated in vacuo

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

Procedure: To a stirred -10° C. solution of N-1,1-dimethyl-ethyl, N-phenylmethylacetamide (prepared from N-1,1-dimethylethyl, N-phenylmethyl amine, and acetyl chloride by refluxing for 2 hours in toluene) (102.5 g, 0.5 mol) in tetrahydrofuran (0.5 L) is slowly added a solution of lithium diisopropylamide in tetrahydrofuran-heptane (0.25 L of 2M) while maintaining the temperature between -40° C. to -50° C., and the mixture is stirred at -20° C. to -30° C. for 30 minutes. (R)-4-cyano-3-hydroxybutyric acid, ethyl ester (Brower, supra) (20 g, 0.125 mol) as a solution in 200 mL or tetrahydrofuran is then added to the previous mixture. The reaction mixture is stirred for 30 minutes at -25° C. to -20° C., and transferred to a 2.2N aqueous hydrochloric acid solution (0.5 L). The aqueous layer is extracted with 300 mL of ethyl acetate, the aqueous layer is separated and reextracted with 100 mL of ethyl acetate, the extracts are combined and concentrated in vacuo to afford crude (R)-6-cyano-N-(1,1-dimethylethyl)-5-hydroxy-3-oxo-N-(phenylmethyl)hexanamide as an oil. while maintaining the temperature between -40° C. to -50° C. The reaction mixture is stirred for 30 minutes at -25° C. to -20° C. The aqueous layer is extracted with 300 mL of ethyl acetate the aqueous layer is separated concentrated in vacuo