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 (R)-ethyl-4-cyano-3-hydroxybutyrate, comprising
(a) providing an epichlorohydrin;
(b) providing a polypeptide having an amino acid sequence with at least 80% sequence identity to SEQ ID NO:210 and having nitrilase activity
(c) preparing a 3-hydroxyglutaronitrile (HGN) from the epichlorohydrin, followed by the addition of a neutralizing agent, a buffer or a diluent,
wherein the epichlorohydrin is converted to 3-hydroxyglutaronitrile (HGN) by cyanide treatment;
(d) catalyzing the conversion of the 3-hydroxyglutaronitrile (HGN) to 4-cyano-3-hydroxybutyric acid by contacting the 3-hydroxyglutaronitrile (HGN) with the polypeptide of step (b);
(e) isolating the 4-cyano-3-hydroxybutyric acid made in step (d) by crystallization using a protocol comprising:
(1) adding concentrated potassium hydroxide or calcium hydroxide to the 4-cyano-3-hydroxybutyric acid, followed by concentration of the solution and crystallization precipitation using a hot alcohol, or
(2) converting the 4-cyano-3-hydroxybutyric acid to its potassium salt by reaction with potassium hydroxide in water or an alcohol, followed by crystallization of the 4-cyano-3-hydroxybutyric acid potassium salt; or
(3) the protocol of (e)(1) or (e)(2) further comprising a second or additional recrystallization after re-suspension of the crystals formed in (e)(1) or (e)(2); and
(f) converting the crystallization precipitate to (R)-Ethyl-4-Cyano-3-Hydroxybutyrate by esterification using an acid catalyst.
2. The method of claim 1 , wherein the polypeptide of step (b) is an isolated, synthetic, or recombinant nitrilase.
3. The method of claim 1 , wherein the acid catalyst esterification of the (R)-Ethyl-4-Cyano-3-Hydroxybutyrate comprises use of a hydrochloric acid (HCl) catalyst or a sulfuric acid (H 2 SO 4 ) catalyst.
4. The method of claim 1 , wherein the reaction is a one-pot reaction.
5. The method of claim 1 , wherein at least two steps of the reaction take place in sequential pots.
6. The method of claim 1 , wherein the preparing of the 3-hydroxyglutaronitrile (HGN) from the epichlorohydrin comprises adding the epichlorohydrin to an aqueous solution of potassium cyanide (KCN) at an alkaline pH.
7. The method of claim 1 , wherein hydroxyglutaronitrile (HGN) is converted to 4-cyano-3-hydroxybutyric acid with the polypeptide of step (b) under conditions comprising:
(a) adding to an HGN-comprising pH neutral phosphate buffered solution a cell lysate comprising a recombinantly expressed polypeptide having nitrilase activity, wherein the polypeptide catalyzes the hydrolysis of the HGN to produce 4-cyano-3-hydroxybutyric acid; or,
(b) the method of (a), wherein the 4-cyano-3-hydroxybutyric acid-comprising hydrolysis solution is acidified and the 4-cyano-3-hydroxybutyric acid extracted and isolated as an oil.
8. The method of claim 1 , wherein the polypeptide of step (b) comprises the sequence as set forth in SEQ ID NO:210 and having a mutation at residue 190 from alanine to a histidine residue.
9. The method of claim 1 , wherein the polypeptide of step (b) comprises the sequence as set forth in SEQ ID NO:210 having a mutation at residue 190, wherein alanine at position 190 is replaced with an amino acid capable of receiving or donating a hydrogen bond.
10. The method of claim 1 , wherein the polypeptide of step (b) comprises the sequence as set forth in SEQ ID NO: 210 and having a mutation at residue 190, wherein alanine at position 190 is replaced with a hydrophobic amino acid.
11. The method of claim 1 , wherein the polypeptide of step (b) comprises the sequence as set forth in SEQ ID NO:210 and having one or more mutations at amino acid residue 55 from alanine to a lysine, glycine, or glutamine; at amino acid residue 60 from isoleucine to a glutamic acid; at amino acid residue 111 from asparagine to a serine, at amino acid residue 190 from alanine to a serine, histidine, tyrosine or threonine; at amino acid residue 191 from phenylalanine to a leucine, valine, methionine, aspartic acid, glycine, glutamic acid, tyrosine or threonine; at amino acid residue 199 from methionine to a glutamic acid or leucine; or at amino acid residue 222 from aspartic acid to a leucine.
12. The method of claim 1 , wherein the polypeptide having nitrilase activity is encoded by a nucleic acid having a sequence with at least 95% sequence identity to SEQ ID NO:209.
13. The method of claim 1 , wherein the nitrilase polypeptide has a sequence with at least 90% sequence identity to SEQ ID NO:210.
14. The method of claim 13 , wherein the nitrilase polypeptide has a sequence with at least 95% sequence identity to SEQ ID NO:210.
15. The method of claim 14 , wherein the nitrilase polypeptide has a sequence with at least 98% sequence identity to SEQ ID NO:210.
16. A method for making (R)-ethyl-4-cyano-3-hydroxybutyrate comprising
(A) (a) providing an epichlorohydrin;
(b) providing a polypeptide having an amino acid sequence with at least 80% identity to SEQ ID NO:210 and having nitrilase activity;
(c) preparing a 3-hydroxyglutaronitrile (HGN) from the epichlorohydrin, followed by the addition of a neutralizing agent, a buffer or a diluent,
wherein the epichlorohydrin is converted to 3-hydroxyglutaronitrile (HGN) by cyanide treatment under pH and temperature control, and the conversion process comprises adding the epichlorohydrin to an aqueous solution of potassium cyanide (KCN) at an alkaline pH;
(d) without any purification of the HGN made in step (c), catalyzing the conversion of the 3-hydroxyglutaronitrile (HGN) to 4-cyano-3-hydroxybutyric acid by contacting the 3-hydroxyglutaronitrile (HGN) with the polypeptide of step (b) under conditions comprising:
(1) adding to an HGN-comprising pH neutral phosphate buffered solution a cell lysate comprising a recombinantly expressed polypeptide of step (b), wherein the polypeptide catalyzes the hydrolysis of the HGN to produce 4-cyano-3-hydroxybutyric acid; or,
(2) the method of (a), wherein the 4-cyano-3-hydroxybutyric acid-comprising hydrolysis solution is acidified and the 4-cyano-3-hydroxybutyric acid extracted and isolated as an oil;
(e) isolating the 4-cyano-3-hydroxybutyric acid made in step (d) by crystallization using a protocol comprising:
(1) adding concentrated potassium hydroxide or calcium hydroxide to the 4-cyano-3-hydroxybutyric acid, followed by concentration of the solution and crystallization precipitation using hot ethanol, or
(2) converting the 4-cyano-3-hydroxybutyric acid to its potassium salt by reaction with potassium hydroxide in water, methanol or ethanol, followed by crystallization of the 4-cyano-3-hydroxybutyric acid potassium salt; or
(3) a second or additional recrystallization after re-suspension of the crystals formed in (e)(1) or (e)(2); and
(f) converting the crystallization precipitate to (R)-Ethyl-4-Cyano-3-Hydroxybutyrate by esterification using an acid catalyst,
wherein the acid catalyst esterification of the (R)-Ethyl-4-Cyano-3-Hydroxybutyrate comprises use of a hydrochloric acid (HCl) catalyst or a sulfuric acid (H 2 SO 4 ) catalyst.
17. The method of claim 16 , wherein the reaction is a one-pot reaction.
18. The method of claim 16 , wherein at least two steps of the reaction take place in sequential pots.
19. The method of claim 16 , further comprising converting the 4-cyano-3-hydroxybutyric acid to its potassium salt by reaction with a potassium hydroxide in water, methanol or ethanol.
20. The method of claim 15 , wherein the nitrilase polypeptide has a sequence that has 100% sequence identity to SEQ ID NO:210, or has a sequence as set forth in SEQ ID NO:210 with a residue change comprising A190H.
21. The method of claim 16 , wherein the recombinant nitrilase polypeptide is encoded by a nucleic acid having a sequence with at least 90% sequence identity to SEQ ID NO:209.
22. The method of claim 21 , wherein the recombinant nitrilase polypeptide is encoded by a nucleic acid having a sequence with at least 95% sequence identity to SEQ ID NO:209.
23. The method of claim 22 , wherein the recombinant nitrilase polypeptide is encoded by a nucleic acid having a sequence with at least 98% sequence identity to SEQ ID NO:209.
24. The method of claim 23 , wherein the recombinant nitrilase polypeptide is encoded by a nucleic acid having a sequence with at least 99% sequence identity to SEQ ID NO:209.
25. The method of claim 24 , wherein the recombinant nitrilase polypeptide is encoded by a nucleic acid having 100% sequence identity to SEQ ID NO:209, or has a sequence as set forth in SEQ ID NO:209 but encoding a polypeptide with an amino acid residue change A190H.
26. The method of claim 16 , wherein the recombinant nitrilase polypeptide has a sequence with at least 90% sequence identity to SEQ ID NO:210.
27. The method of claim 1 , wherein the alcohol comprises a methanol or an ethanol.
28. The method of claim 1 , wherein the 4-cyano-3-hydroxybutyric acid generated in step (d) has a chiral purity of at least 90% ee.
29. A method for making 4-cyano-3-hydroxybutyric acid comprising
(A) (a) providing an epichlorohydrin;
(b) providing a polypeptide having an amino acid sequence with at least 80% identity to SEQ ID NO:210 and having nitrilase activity;
(c) preparing a 3-hydroxyglutaronitrile (HGN) from the epichlorohydrin, followed by the addition of a neutralizing agent, a buffer or a diluent,
wherein the epichlorohydrin is converted to 3-hydroxyglutaronitrile (HGN) by cyanide treatment under pH and temperature control, and the conversion process comprises adding the epichlorohydrin to an aqueous solution of potassium cyanide (KCN) at an alkaline pH;
(d) without any purification of the HGN made in step (c), catalyzing the conversion of the 3-hydroxyglutaronitrile (HGN) to 4-cyano-3-hydroxybutyric acid by contacting the 3-hydroxyglutaronitrile (HGN) with the polypeptide of step (b) under conditions comprising:
(1) adding to an HGN-comprising pH neutral phosphate buffered solution a cell lysate comprising a recombinantly expressed polypeptide having nitrilase activity, wherein the polypeptide catalyzes the hydrolysis of the HGN to produce 4-cyano-3-hydroxybutyric acid; or,
(2) the method of (a), wherein the 4-cyano-3-hydroxybutyric acid-comprising hydrolysis solution is acidified and the 4-cyano-3-hydroxybutyric acid extracted and isolated as an oil.
30. The method of claim 29 , further comprising isolating the 4-cyano-3-hydroxybutyric acid made in step (d) by crystallization using a protocol comprising:
(1) adding concentrated potassium hydroxide or calcium hydroxide to the 4-cyano-3-hydroxybutyric acid, followed by concentration of the solution and crystallization precipitation using hot ethanol, or
(2) converting the 4-cyano-3-hydroxybutyric acid to its potassium salt by reaction with potassium hydroxide in water, methanol or ethanol, followed by crystallization of the 4-cyano-3-hydroxybutyric acid potassium salt; or
(3) a second or additional recrystallization after re-suspension of the crystals formed in (e)(1) or (e)(2).
31. The method of claim 30 , further comprising converting the crystallization precipitate to an ethyl-4-cyano-3-hydroxybutyrate by esterification using an acid catalyst.
32. The method of claim 31 , wherein the acid catalyst esterification of the ethyl-4-cyano-3-hydroxybutyrate comprises use of a hydrochloric acid (HCl) catalyst or a sulfuric acid (H 2 SO 4 ) catalyst.