Enzymatic processes for the preparation of (±)-2-(difluoromethyl)-1-(alkoxycarbonyl)-cyclopropanecarboxylic acid and (±)-2-(vinyl)-1-(alkoxycarbonyl)-cyclopropanecarboxylic acid

Abstract

Disclosed are methods of synthesizing racemic 2-(difluoromethyl)-1-(alkoxycarbonyl)-cyclopropanecarboxylic acids and 2-(vinyl)-1-(alkoxycarbonyl)-cyclopropanecarboxylic acids and their salts, such as the dicyclohexylamine salt. Also disclosed are methods for preparing enantioenriched (1R,2R)-1-((tert-butoxycarbonyl)amino)-2-(difluoromethyl)cyclopropane-1-carboxylic acid and esters of the same. These compounds are useful intermediates in the synthesis of viral protease inhibitors.

1 - 29 . (canceled) 
     
     
         30 . A method according to reaction Scheme A: 
       
         
           
           
               
               
           
         
         wherein 
         R 1  is (C 1 -C 6 )alkyl; 
         R 2  is —CF 2 H; and 
         the first enzyme is a mutant of BsteE esterase; 
         further comprising the step according to reaction Scheme B: 
       
       
         
           
           
               
               
           
         
         wherein: 
         the second enzyme is selected from the group consisting of Alcalase® 2.4 L, Esperase® 8.0 L, and Savinase® 16.0 L. 
       
     
     
         31 . The method of  claim 30 , wherein the second enzyme is Alcalase® 2.4 L. 
     
     
         32 . The method of  claim 30 , wherein the loading of the second enzyme is about 50 wt % to about 200 wt % as compared to the starting material in Scheme B. 
     
     
         33 . The method of  claim 30 , wherein the second solvent comprises an aqueous buffer. 
     
     
         34 . The method of  claim 33 , wherein second solvent comprises a sodium bicarbonate/sodium carbonate buffer solution. 
     
     
         35 . The method of  claim 33 , wherein the second solvent further comprises an organic solvent such as acetone. 
     
     
         36 - 38 . (canceled) 
     
     
         39 . A polypeptide, comprising
 an amino acid sequence having at least two amino acid substitutions relative to SEQ ID NO: 2, wherein the amino acid sequence comprises a first mutation of T25H, and a second mutation of L92H.   
     
     
         40 . The polypeptide of  claim 39 , comprising a third mutation selected from the group consisting of C115S, C115A, K121R, K121Y, K121H, K121D, K121L, K121T, K121A, K121N, E123T, M126F, M126V, M126I, M126Q, M126N, K164S, L166N, L166M, L166I, L166T, L166A, I170N, I170Q, I170V, I170T, I170A, D172N, M194A, M194L, I195G, I195A, I195V, and K215N. 
     
     
         41 . The polypeptide of  claim 40 , comprising a third mutation selected from the group consisting of K121R, K121Y, K121H, K121D, K121L, M126F, M126V, M126I, M126Q, M194L, M194A, L166N, L166M, I170N, and I170Q. 
     
     
         42 . The polypeptide of  claim 41 , wherein the third mutation is selected from the group consisting of K121R, K121Y, K121H, K121D, K121L, M126V, M126I, M126Q, M194L, and L166M. 
     
     
         43 . The polypeptide of  claim 42 , wherein the third mutation is selected from the group consisting of K121Y, K121H, K121D, K121L, M126V, M126I, M126Q, and L166M. 
     
     
         44 . The polypeptide of  claim 42 , wherein the third mutation is selected from the group consisting of K121Y, K121H, K121D, and K121L. 
     
     
         45 . A method according to reaction Scheme B: 
       
         
           
           
               
               
           
         
         wherein 
         R 1  is (C 1 -C 6 )alkyl; 
         R 2  is —CF 2 H; and 
         the second enzyme is selected from the group consisting of Alcalase® 2.4 L, Esperase® 8.0 L, and Savinase® 16.0 L. 
       
     
     
         46 . The method of  claim 45 , wherein the second enzyme is Alcalase® 2.4 L. 
     
     
         47 . The method of  claim 45 , wherein the loading of the second enzyme is about 50 wt % to about 200 wt % as compared to the starting material in Scheme B. 
     
     
         48 . The method of  claim 45 , wherein the second solvent comprises an aqueous buffer. 
     
     
         49 . The method of  claim 48 , wherein second solvent comprises a sodium bicarbonate/sodium carbonate buffer solution. 
     
     
         50 . The method of  claim 48 , wherein the second solvent further comprises an organic solvent such as acetone. 
     
     
         51 - 53 . (canceled)