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) 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.

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-ce68bc9b8b1343708d1a0c229c5a8f0f

Source dataset

uspto-grants-2011_04

DOI

10.6084/m9.figshare.5104873.v1

Patent number

US07932064B2