3-hydroxypentanedinitrile

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

Procedure: 3-hydroxyglutaronitrile (1 g, 9 mmol) was suspended in 37.5 mL of N2 (g) sparged, room temperature, 100 mM sodium phosphate buffer at pH 7. To this 240 mM solution of HGN was added 150 mg of cell lysate normalized for protein content. As assessed by PAGE, the nitrilase content was established to be 20% of the total protein. Therefore effectively, 30 mg of nitrilase is used in this reaction. The reaction was agitated at 100 rpm. Reaction progress was monitored by TLC (1:1 EtOAc:Hexanes, Rf=0.32, nitrile; Rf=0.0, acid) and GC. After 22 h, the reaction was filtered through a 10,000 MW cutoff filter and then concentrated in vacuo. The concentrated hydrolysis solution was acidified with 1M HCl and the product was continuously extracted with MTBE (30 mL). The acid product (R)-3-hydroxy-4-cyanobutyric acid was isolated as a yellow oil (1.15 g, 98% yield). 1H NMR (d6-DMSO, 500 MHz) δ 12.32 (s, 1H), 5.52 (s, 1H), 4.10 (m, 1H), 2.70 (dd, 1H, J=16.8, 4.1 Hz), 2.61 (dd, 1H, J=16.9, 6.3 Hz), 2.44 (dd, 1H, J=15.4, 5.3 Hz), 2.37 (dd, 1H, J=15.6, 7.8 Hz). 13C NMR (DMSO, 298K, 125 MHz) δ 171.9, 118.7, 63.4, 41.2, 25.2 MS calc'd for [C5H7NO3]: 129.0. found: 130.0 [M+H+], (ESI+). ee=95% [HPLC]. Based on the isolated yield a productivity of 40 g g−1 d−1 is calculated. sparged, room temperature To this 240 mM solution of HGN was added 150 mg of cell lysate Therefore effectively, 30 mg of nitrilase is used in this reaction Reaction progress the reaction was filtered through a 10,000 MW cutoff filter concentrated in vacuo The concentrated hydrolysis solution the product was continuously extracted with MTBE (30 mL)

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

Procedure: 3-hydroxyglutaronitrile (1 g, 9 mmol) was suspended in 37.5 mL of N2 (g) sparged, room temperature, 100 mM sodium phosphate buffer at pH 7. To this 240 mM solution of HGN was added 150 mg of cell lysate normalized for protein content. As assessed by PAGE, the nitrilase content was established to be 20% of the total protein. Therefore effectively, 30 mg of nitrilase is used in this reaction. The reaction was agitated at 100 rpm. Reaction progress was monitored by TLC (1:1 EtOAc:Hexanes, Rf=0.32, nitrile; Rf=0.0, acid) and GC. After 22 h, the reaction was filtered through a 10,000 MW cutoff filter and then concentrated in vacuo. The concentrated hydrolysis solution was acidified with 1M HCl and the product was continuously extracted with MTBE (30 mL). The acid product (R)-3-hydroxy-4-cyanobutyric acid was isolated as a yellow oil (1.15 g, 98% yield). 1H NMR (d6-DMSO, 500 MHz) δ 12.32 (s, 1H), 5.52 (s, 1H), 4.10 (m, 1H), 2.70 (dd, 1H, J=16.8, 4.1 Hz), 2.61 (dd, 1H, J=16.9, 6.3 Hz), 2.44 (dd, 1H, J=15.4, 5.3 Hz), 2.37 (dd, 1H, J=15.6, 7.8 Hz). 13C NMR (DMSO, 298K, 125 MHz) δ 171.9, 118.7, 63.4, 41.2, 25.2 MS calc'd for [C5H7NO3]: 129.0. found: 130.0 [M+H+], (ESI+). ee=95% [HPLC]. Based on the isolated yield a productivity of 40 g g−1 d−1 is calculated. sparged, room temperature To this 240 mM solution of HGN was added 150 mg of cell lysate Therefore effectively, 30 mg of nitrilase is used in this reaction Reaction progress the reaction was filtered through a 10,000 MW cutoff filter concentrated in vacuo The concentrated hydrolysis solution the product was continuously extracted with MTBE (30 mL)

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

Procedure: 3-hydroxyglutaronitrile (1 g, 9 mmol) was suspended in 37.5 mL of N2 (g) sparged, room temperature, 100 mM sodium phosphate buffer at pH 7. To this 240 mM solution of HGN was added 150 mg of cell lysate normalized for protein content. As assessed by PAGE, the nitrilase content was established to be 20% of the total protein. Therefore effectively, 30 mg of nitrilase is used in this reaction. The reaction was agitated at 100 rpm. Reaction progress was monitored by TLC (1:1 EtOAc:Hexanes, Rf=0.32, nitrile; Rf=0.0, acid) and GC. After 22 h, the reaction was filtered through a 10,000 MW cutoff filter and then concentrated in vacuo. The concentrated hydrolysis solution was acidified with 1M HCl and the product was continuously extracted with MTBE (30 mL). The acid product (R)-3-hydroxy-4-cyanobutyric acid was isolated as a yellow oil (1.15 g, 98% yield). 1H NMR (d6-DMSO, 500 MHz) δ12.32 (s, 1H), 5.52 (s, 1H), 4.10 (m, 1H), 2.70 (dd, 1H, J=16.8, 4.1 Hz), 2.61 (dd, 1H, J=16.9, 6.3 Hz), 2.44 (dd, 1H, J=15.4, 5.3 Hz), 2.37 (dd, 1H, J=15.6, 7.8 Hz). 13C NMR (DMSO, 298K, 125 MHz) δ171.9, 118.7, 63.4, 41.2, 25.2 MS calc'd for [C5H7NO3]: 129.0, found: 130.0 [M+H+], (ESI+). ee=95% [HPLC]. Based on the isolated yield a productivity of 40 g g−1 d−1 is calculated. sparged, room temperature To this 240 mM solution of HGN was added 150 mg of cell lysate Therefore effectively, 30 mg of nitrilase is used in this reaction Reaction progress the reaction was filtered through a 10,000 MW cutoff filter concentrated in vacuo The concentrated hydrolysis solution the product was continuously extracted with MTBE (30 mL)

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

Procedure: Into a tetrahydrofuran solution, 0.7 g of sodium hydride was added. After cooling to 0° C., 3 g of 3-hydroxyglutaronitrile dissolved in a tetrahydrofuran solvent was slowly dropped. After completing the dropping, dimethyl sulfate was slowly added dropwise and stirred at 0° C. for 15 minutes. After stirring at room temperature for 6 hours, the reactant was extracted using water and dichloromethane and distilled under a reduced pressure to obtain 3-methoxy glutaronitrile (yield 82%). was slowly dropped was slowly added dropwise After stirring at room temperature for 6 hours the reactant was extracted distilled under a reduced pressure

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

Procedure: 3-hydroxyglutaronitrile (1 g, 9 mmol) was suspended in 37.5 mL of N2 (g) sparged, room temperature, 100 mM sodium phosphate buffer at pH 7. To this 240 mM solution of HGN was added 150 mg of cell lysate normalized for protein content. As assessed by PAGE, the nitrilase content was established to be 20% of the total protein. Therefore effectively, 30 mg of nitrilase is used in this reaction. The reaction was agitated at 100 rpm. Reaction progress was monitored by TLC (1:1 EtOAc:Hexanes, Rf=0.32, nitrile; Rf=0.0, acid) and GC. After 22 h, the reaction was filtered through a 10,000 MW cutoff filter and then concentrated in vacuo. The concentrated hydrolysis solution was acidified with 1M HCl and the product was continuously extracted with MTBE (30 mL). The acid product (R)-3-hydroxy-4-cyanobutyric acid was isolated as a yellow oil (1.15 g, 98% yield). 1H NMR (d6-DMSO, 500 MHz) δ 12.32 (s, 1H), 5.52 (s, 1H), 4.10 (m, 1H), 2.70 (dd, 1H, J=16.8, 4.1 Hz), 2.61 (dd, 1H, J=16.9, 6.3 Hz), 2.44 (dd, 1H, J=15.4, 5.3 Hz), 2.37 (dd, 1H, J=15.6, 7.8 Hz). 13C NMR (DMSO, 298K, 125 MHz) δ 171.9, 118.7, 63.4, 41.2, 25.2 MS calc'd for [C5H7NO3]: 129.0. found: 130.0 [M+H+], (ESI+). ee=95% [HPLC]. Based on the isolated yield a productivity of 40 g g−1 d−1 is calculated. sparged, room temperature To this 240 mM solution of HGN was added 150 mg of cell lysate Therefore effectively, 30 mg of nitrilase is used in this reaction Reaction progress the reaction was filtered through a 10,000 MW cutoff filter concentrated in vacuo The concentrated hydrolysis solution the product was continuously extracted with MTBE (30 mL)