Processes for Making (R)-Ethyl 4-Cyano-3 Hydroxybutyric Acid

Abstract

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.

What is claimed is: 
     
         1 . A method for making 4-cyano-3-hydroxybutyric acid comprising:
 (a) providing a 3-hydroxyglutaronitrile;   (b) providing a polypeptide having a nitrilase activity; wherein the polypeptide is an amino acid sequence selected from the group consisting of: SEQ ID NO:2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158, 160, 162, 164, 166, 168, 170, 172, 174, 176, 178, 180, 182, 184, 186, 188, 190, 192, 194, 196, 198, 200, 202, 204, 206, 208, 210, 212, 214, 216, 218, 220, 222, 224, 226, 228, 230, 232, 234, 236, 238, 240, 242, 244, 246, 248, 250, 252, 254, 256, 258, 260, 262, 264, 266, 268, 270, 272, 274, 276, 278, 280, 282, 284, 286, 288, 290, 292, 294, 296, 298, 300, 302, 304, 306, 308, 310, 312, 314, 316, 318, 320, 322, 324, 326, 328, 330, 332, 334, 336, 338, 340, 342, 344, 346, 348, 350, 352, 354, 356, 358, 360, 362, 364, 366, 368, 370, 372, 374, 376, 378, 380, 382, 384, or 386; and   (c) catalyzing the conversion of the 3-hydroxyglutaronitrile, or equivalent 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:
 (a) providing an epichlorohydrin;   (b) providing a polypeptide having a nitrilase activity; wherein the polypeptide is an amino acid sequence selected from the group consisting of: SEQ ID NO:2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158, 160, 162, 164, 166, 168, 170, 172, 174, 176, 178, 180, 182, 184, 186, 188, 190, 192, 194, 196, 198, 200, 202, 204, 206, 208, 210, 212, 214, 216, 218, 220, 222, 224, 226, 228, 230, 232, 234, 236, 238, 240, 242, 244, 246, 248, 250, 252, 254, 256, 258, 260, 262, 264, 266, 268, 270, 272, 274, 276, 278, 280, 282, 284, 286, 288, 290, 292, 294, 296, 298, 300, 302, 304, 306, 308, 310, 312, 314, 316, 318, 320, 322, 324, 326, 328, 330, 332, 334, 336, 338, 340, 342, 344, 346, 348, 350, 352, 354, 356, 358, 360, 362, 364, 366, 368, 370, 372, 374, 376, 378, 380, 382, 384, or 386; and   (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:
 (a) providing a 3-hydroxyglutaronitrile;   (b) providing a polypeptide having a nitrilase activity; wherein the polypeptide is an amino acid sequence selected from the group consisting of: SEQ ID NO:2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158, 160, 162, 164, 166, 168, 170, 172, 174, 176, 178, 180, 182, 184, 186, 188, 190, 192, 194, 196, 198, 200, 202, 204, 206, 208, 210, 212, 214, 216, 218, 220, 222, 224, 226, 228, 230, 232, 234, 236, 238, 240, 242, 244, 246, 248, 250, 252, 254, 256, 258, 260, 262, 264, 266, 268, 270, 272, 274, 276, 278, 280, 282, 284, 286, 288, 290, 292, 294, 296, 298, 300, 302, 304, 306, 308, 310, 312, 314, 316, 318, 320, 322, 324, 326, 328, 330, 332, 334, 336, 338, 340, 342, 344, 346, 348, 350, 352, 354, 356, 358, 360, 362, 364, 366, 368, 370, 372, 374, 376, 378, 380, 382, 384, or 386;   (c) catalyzing the conversion of the 3-hydroxyglutaronitrile, or equivalent to 4-cyano-3-hydroxybutyric acid by contacting the 3-hydroxyglutaronitrile, or equivalent, 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 is an isolated or recombinant polypeptide variant, wherein the polypeptide variant is a nitrilase having an amino acid sequence selected from the group consisting of: a sequence at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, identical to the nitrilase of  claim 1 . 
     
     
         11 . The method of  claim 1 , wherein the nitrilase is an isolated or recombinant polypeptide fragment of the nitrilase of  claim 1 , wherein the polypeptide fragment also has the nitrilase activity; 
     
     
         12 . The method of  claim 1 , wherein the nitrilase comprises one or more mutations selected from the group consisting of: residue 55 lysine, residue 55 glycine, residue 55 glutamine, residue 60 glutamic acid, residue 111 serine, residue 190, residue 190 serine, residue 190 histidine, residue 190 tyrosine, residue 190 threonine, residue 191 leucine, residue 191 valine, residue 191 methionine, residue 191 aspartic acid, residue 191 glycine, residue 191 glutamic acid, residue 191 tyrosine, residue 191 threonine, residue 199 glutamic acid, residue 199 leucine, residue 222 leucine, and any combination thereof. 
     
     
         13 . The method of  claim 1 , wherein the nitrilase is encoded by a nucleic acid sequence selected from a group consisting of: SEQ ID NO:1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125, 127, 129, 131, 133, 135, 137, 139, 141, 143, 145, 147, 149, 151, 153, 155, 157, 159, 161, 163, 165, 167, 169, 171, 173, 175, 177, 179, 181, 183, 185, 187, 189, 191, 193, 195, 197, 199, 201, 203, 205, 207, 209, 211, 213, 215, 217, 219, 221, 223, 225, 227, 229, 231, 233, 235, 237, 239, 241, 243, 245, 247, 249, 251, 253, 255, 257, 259, 261, 263, 265, 267, 269, 271, 273, 275, 277, 279, 281, 283, 285, 287, 289, 291, 293, 295, 297, 299, 301, 303, 305, 307, 309, 311, 313, 315, 317, 319, 321, 323, 325, 327, 329, 331, 333, 335, 337, 339, 341, 343, 345, 347, 349, 351, 353, 355, 357, 359, 361, 363, 365, 367, 369, 371, 373, 375, 377, 379, 381, 383 or 385. 
     
     
         14 . A method for making atorvastatin comprising:
 (a) providing a 3-hydroxyglutaronitrile;   (b) providing a polypeptide having a nitrilase activity;   (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; wherein the polypeptide is an amino acid sequence selected from the group consisting of: SEQ ID NO:2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158, 160, 162, 164, 166, 168, 170, 172, 174, 176, 178, 180, 182, 184, 186, 188, 190, 192, 194, 196, 198, 200, 202, 204, 206, 208, 210, 212, 214, 216, 218, 220, 222, 224, 226, 228, 230, 232, 234, 236, 238, 240, 242, 244, 246, 248, 250, 252, 254, 256, 258, 260, 262, 264, 266, 268, 270, 272, 274, 276, 278, 280, 282, 284, 286, 288, 290, 292, 294, 296, 298, 300, 302, 304, 306, 308, 310, 312, 314, 316, 318, 320, 322, 324, 326, 328, 330, 332, 334, 336, 338, 340, 342, 344, 346, 348, 350, 352, 354, 356, 358, 360, 362, 364, 366, 368, 370, 372, 374, 376, 378, 380, 382, 384, or 386   (d) converting the 4-cyano-3-hydroxybutyric acid to ethyl-4-cyano-3-hydroxybutyric acid; and   (e) converting the ethyl-4-cyano-3-hydroxybutyric acid to atorvastatin.