Hydrochloric Acid → (R)-4-cyano-3-hydroxybutyric acid

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.

1. customsparged, room temperature
2. additionTo this 240 mM solution of HGN was added 150 mg of cell lysate
3. customTherefore effectively, 30 mg of nitrilase is used in this reaction
4. customReaction progress
5. filtrationthe reaction was filtered through a 10,000 MW cutoff
6. filtrationfilter
7. concentrationconcentrated in vacuo
8. customThe concentrated hydrolysis solution
9. extractionthe product was continuously extracted with MTBE (30 mL)

ORD record

ord-742035f652cc4f7baf9b7de7a28ca5a4

Source dataset

uspto-grants-2013_02

DOI

10.6084/m9.figshare.5104873.v1

Patent number

US08372608B2