Processes for making [R]-ethyl 4-cyano-3 hydroxybutyric acid
View Patent ↗The invention provides novel processes for making ethyl-4-cyano-3-hydroxybutyrate, e.g., (R)-ethyl 4-cyano-3-hydroxybutyric acid, and 4-cyano-3-hydroxybutyric acid. The invention provides protocols for making and 4-cyano-3-hydroxybutyric acid and ethyl-4-cyano-3-hydroxybutyrate by whole cell processes, cell lysate processes, “one pot processes” and “multi-pot” processes using a variety of parameters.
1. A method for making 4-cyano-3-hydroxybutyric acid comprising the following steps
(a) providing a 3-hydroxyglutaronitrile;
(b) providing a polypeptide having a nitrilase activity, wherein the polypeptide is encoded by a nucleic acid at least 85% identical to SEQ ID NO:209; and
(c) catalyzing the conversion of the 3-hydroxyglutaronitrile to 4-cyano-3-hydroxybutyric acid by contacting the 3-hydroxyglutaronitrile, with the polypeptide having a nitrilase activity.
2. A method for making 4-cyano-3-hydroxybutyric acid comprising the following steps
(a) providing an epichlorohydrin;
(b) providing a polypeptide having a nitrilase activity, wherein the polypeptide is encoded by a nucleic acid at least 85% identical to SEQ ID NO:209;
(c) converting the epichlorohydrin to 3-hydroxyglutaronitrile; and
(d) catalyzing the conversion of the 3-hydroxyglutaronitrile to 4-cyano-3-hydroxybutyric acid by contacting the 3-hydroxyglutaronitrile with the polypeptide having a nitrilase activity.
3. A method for making ethyl-4-cyano-3-hydroxybutyric acid comprising the following steps
(a) providing a 3-hydroxyglutaronitrile;
(b) providing a polypeptide having a nitrilase activity, wherein the polypeptide is encoded by a nucleic acid at least 85% identical to SEQ ID NO:209;
(c) catalyzing the conversion of the 3-hydroxyglutaronitrile to 4-cyano-3-hydroxybutyric acid by contacting the 3-hydroxyglutaronitrile, with the polypeptide having a nitrilase activity; and
(d) converting the 4-cyano-3-hydroxybutyric acid to ethyl-4-cyano-3-hydroxybutyric acid.
4. The method of claim 2 , wherein the epichlorohydrin is converted to 3-hydroxyglutaronitrile by cyanide treatment.
5. The method of claim 1 further comprising conversion of the 4-cyano-3-hydroxybutyric acid to ethyl-4-cyano-3-hydroxybutyric acid.
6. The method of claim 1 wherein the reaction is a one-pot reaction.
7. The method of claim 1 wherein at least two steps of the reaction take place in sequential pots.
8. The method of claim 1 further comprising converting the 4-cyano-3-hydroxybutyric acid to its potassium salt by reaction with a potassium hydroxide in water, methanol or ethanol.
9. The method of claim 2 , wherein the preparation of hydroxyglutaronitrile from epichlorohydrin is followed by the addition of a neutralizing agent, a buffer or a diluent, and then subsequent conversion to 4-cyano-3-hydroxybutyric acid with the nitrilase.
10. The method of claim 1 , wherein the nitrilase comprises a sequence as set forth in SEQ ID NO:210 and having a mutation at residue 190, wherein alanine at residue 190 is replaced with a hydrogen-binding amino acid residue.
11. The method of claim 1 , wherein the nitrilase comprises a sequence as set forth in SEQ ID NO:210 and having a mutation at residue 190, wherein alanine at residue 190 is replaced with a hydrophobic amino acid residue.