Enzymatic cyclization of homofarnesylic acid

Abstract

The present invention relates to processes for the preparation of sclareolide and related compounds by the biocatalytic cyclization of polyunsaturated carboxylic acid compounds, in particular of homofarnesylic acid and related compounds; and to a process for the preparation of ambroxide via the biocatalytic cyclization of homofarnesylic acid to sclareolide.

1 .- 15 . (canceled) 
     
     
         16 . A process for the biocatalytic preparation of a compound of the general formula II, 
       
         
           
           
               
               
           
         
         wherein R 1 , R 2 , R 3  and R 4  independently of one another represent H or C 1 -C 4 -alkyl, 
         in stereoisomerically pure form or as a mixture of stereoisomers, 
         wherein the compound is brought into contact with a protein, which protein is capable of cyclizing a polyunsaturated carboxylic acid, in particular homofarnesylic acid. 
       
     
     
         17 . The process as claimed in  claim 16 , wherein the compound of the formula I is brought into contact with a protein which has the enzymatic activity of a squalene-hopene cyclase (SHC). 
     
     
         18 . The process as claimed in  claim 16 , wherein the substrate employed is a polyunsaturated carboxylic acid of the general formula I, 
       
         
           
           
               
               
           
         
         wherein R 1 , R 2 , R 3  and R 4  have the abovementioned meanings, 
         in particular in essentially stereoisomer-pure form. 
       
     
     
         19 . The process as per  claim 16 , wherein homofarnesylic acid, of the formula Ia, 
       
         
           
           
               
               
           
         
       
       is employed as the starting material, in particular in essentially stereoisomerically pure form, preferably in a proportion of (3E,7E)-homofarnesylic acid of at least 70 mol %, based on the total amount of homofarnesylic acid isomers present. 
     
     
         20 . The process as claimed in  claim 19 , wherein sclareolide, of the formula IIa, 
       
         
           
           
               
               
           
         
       
       is obtained in stereoisomerically pure form or as a mixture of stereoisomers. 
     
     
         21 . The process as claimed in  claim 16 , wherein the SHC is selected from among
 a) proteins comprising a polypeptide with an amino acid sequence as per SEQ ID NO: 2,   b) by deletion, insertion, substitution, addition, inversion or a combination of proteins derived as per a), comprising a polypeptide with a sequence identity of at least 45%, to the amino acid sequence as per SEQ ID NO: 2; and   c) proteins which are functionally equivalent to a) or b) and which catalyze the cyclization of homofarnesylic acid to sclareolide.   
     
     
         22 . The process as claimed in  claim 16 , wherein the biocatalytic conversion
 a) is carried out at a pH value of the reaction medium in the range from approximately 4 to 5.8; and under at least one of the following further conditions:   b) at a substrate concentration of at least 15 mM;   c) at an enzyme concentration of at least 5 mg/ml;   d) at a reaction temperature in the range from 32 to 40° C.;   e) in a sodium citrate buffer comprising 1 to 20 mM MgCl 2 ; and/or   f) at a buffer concentration of approximately 10 to 100 mM.   
     
     
         23 . The process as claimed in  claim 16 , wherein the enzymatic cyclase activity, in particular the activity of the SHC, is present in a form selected from among:
 a) a free, optionally partially or fully purified natural or recombinantly produced cyclase,   b) cyclase as per a) in immobilized form;   c) intact cells, comprising at least one cyclase;   d) cell lysates or cell homogenates of cells as per c).   
     
     
         24 . The process as claimed in  claim 16 , wherein the conversion is carried out in one-phase aqueous systems or in two-phase aqueous-organic or solid-liquid systems. 
     
     
         25 . The process as claimed in  claim 16 , wherein the conversion is carried out at a temperature in the range of 37° C., and a pH value in the range of from 5 to 5.2. 
     
     
         26 . The process as claimed in  claim 16 , wherein the SHC is isolated from a microorganism selected among  Methylococcus capsalatus, Rhodopseudomonas palustris, Bradyrhizobium japonicum, Frankia spec., Streptomyces coelicolor  and in particular  Zymomonas mobilis.    
     
     
         27 . The process as claimed in  claim 16 , wherein the SHC is isolated from an SHC-overexpressing microorganism which is selected among bacteria of the genus  Escherichia, Corynebacterium, Ralstonia, Clostridium, Pseudomonas, Bacillus, Zymomonas, Rhodobacter, Streptomyces, Burkholderia, Lactobacillus  and  Lactococcus.    
     
     
         28 . The process as claimed in  claim 16 , wherein the SHC is isolated from transgenic SHC-overexpressing bacteria of the species  Escherichia coli, Pseudomonas putida, Burkholderia glumae, Streptomyces lividans, Streptomyces coelicolor  and  Zymomonas mobilis.    
     
     
         29 . A process for the preparation of 3a,6,6,9a-tetramethyldodecahydronaphto[2,1-b]furan (ambroxide), wherein
 a) homofarnesylic acid is converted into sclareolide by a process as per  claim 16 ;   b) the product of step a) is reduced chemically to ambroxdiol, and   c) ambroxidol from step b) is cyclized chemically to ambroxide.   
     
     
         30 . The process as claimed in  claim 29 , wherein ambroxide is (−)-ambroxide ((3aR,5aS,9aS,9bR)-3a,6,6,9a-tetramethyldodecahydronaphto[2,1-b]furan [CAS 6790-58-5]).