Process for preparing optically active 3-hydroxy-pyrrolidine derivatives by enzymatic hydroxylation

Abstract

A process for the preparation of optically active 3-hydroxypyrrolidine or N-substituted 3-hydroxypyrrolidines, wherein an oxygen atom is inserted stereoselectively into the corresponding pyrrolidines, respectively, by use of a bacterium having hydroxylation activity, or a prokaryotic host-organism having the gene(s) necessary for the hydroxylation, or an enzyme having hydroxylation activity derived therefrom. The bacterium may be selected from strains having alkane hydroxylases, strains degrading alkanes or mono-alicyclic compounds, or strains from the genera Pseudomonas, Mycobacterium, Corynebacterium, Nocardia, Sphingomonas, Cordona, Rhodococcus, Bacillus, Streptomyces, Sebekia and Methylococcus.

1 . A process for the preparation of optically active 3-hydroxypyrrolidine or N-substituted 3-hydroxypyrrolidine, wherein an oxygen atom is inserted stereoselectively into the corresponding non-hydroxylated pyrrolidine compound by using, as a biocatalyst, a bacterium having hydroxylation activity or an enzyme having hydroxylation activity derived from the said bacterium.  
     
     
         2 . The process of  claim 1 , wherein the bacterium is selected from the group consisting of alkane-degrading bacteria.  
     
     
         3 . The process of  claim 2 , wherein the bacterium is selected from the group consisting of bacteria degrading n-alkane containing 4 to 20 carbon atoms.  
     
     
         4 . The process of  claim 2 , wherein the bacterium is selected from the group consisting of bacteria degrading n-octane.  
     
     
         5 . The process of  claim 2 , wherein the bacterium is selected from the group consisting of bacteria degrading n-hexane.  
     
     
         6 . The process of  claim 1 , wherein the bacterium is selected from the group consisting of bacteria degrading mono-alicyclic compounds containing 4 to 20 carbon atoms.  
     
     
         7 . The process of  claim 1 , wherein the bacterium is selected from the group consisting of bacteria having alkane hydroxylase (s).  
     
     
         8 . The process of  claim 1 , wherein the bacterium is selected from the group of genera consisting of Pseudomonas, Mycobacterium, Corynebacterium, Nocardia, Sphingomonas, Gordona, Rhodococcus, Bacillus, Streptomyces, Sebekia and Methylococcus.  
     
     
         9 . The process of  claim 8 , wherein the bacterium is selected from the group consisting of  Pseudomonas oleovorans  strains, such as Pseudomonas GPo1 , Pseudomonas putida  strains, such as  Pseudomonas putida  P1, isolate HXN-200, and isolate HXN-1100.  
     
     
         10 . The process according to any one of claims  1 - 9 , wherein the biocatalyst is a recombinant bacterium having the gene(s) necessary for the hydroxylation.  
     
     
         11 . The process of  claim 10 , wherein said gene(s) necessary for the hydroxylation comprise (s) one or more of the alkane hydroxylase genes from an alkane degrading bacterium.  
     
     
         12 . The process of  claim 11 , wherein said alkane degrading bacterium is a  Pseudomonas oleovorans  strain, such as  Pseudomonas oleovorans  GPo1 (ATCC 29347).  
     
     
         13 . The process of  claim 10 , wherein the recombinant bacterium is a recombinant  Escherichia coli  strain, such as  Escherichia coli  GEc137 (pGEc47).  
     
     
         14 . The process of any one of claims  1 - 13 , wherein resting bacterial cells, growing bacterial cells, or both, are used as biocatalyst.  
     
     
         15 . The process of any one of claims  1 - 13 , wherein a crude cell extract, or a purified, or partially purified, enzyme preparation is used as biocatalyst.  
     
     
         16 . The process of any one of claims  1 - 15 , wherein the bio-catalyst is immobilized on or in a water insoluble carrier or support system.  
     
     
         17 . The process of any one of claims  1 - 16 , wherein the biocatalytic reaction is performed in aqueous medium.  
     
     
         18 . The process of any one of claims  1 - 16 , wherein the biocatalytic reaction is performed in multiphase media containing two or more of the following: a solid phase, an aqueous phase, an organic phase, and a gaseous phase.  
     
     
         19 . The process of  claim 18 , wherein an organic phase is used which comprises one or more alkanes with 5 or more C atoms, dialkyl ethers with 4 or more C atoms, or aromatic or heteroaromatic hydrocarbons, optionally with substitution.  
     
     
         20 . The process of any one of claims  1 - 19 , wherein the reaction temperature is 5-50° C., preferably 20-40° C.  
     
     
         21 . The process of any one of claims  1 - 20 , wherein the pH of the medium is 4-10, preferably 6-8.  
     
     
         22 . The process of any one of claims  1 - 21 , wherein the product is separated by column chromatography with an inorganic, organic or synthetic adsorbent used as a support.  
     
     
         23 . The process of any one of claims  1 - 21 , wherein the product is separated by means of extraction, wherein the substrate is first recovered from the reaction mixture by extraction with a less polar solvent, the remaining reaction mixture is adjusted to pH=10-12, and the product is extracted with a more polar solvent.  
     
     
         24 . The process of  claim 23 , wherein the extraction agent used is selected from the group consisting of alkanes with 5 or more C atoms, dialkyl ethers with 4 or more C atoms, chlorine-containing alkanes with 3 or fewer C atoms, alkyl aromatics with 7-10 C atoms, and carboxylic esters with 3 or more C atoms.  
     
     
         25 . The process of any one of claims  1 - 21 , wherein the product is separated by use of membrane filtration.  
     
     
         26 . The process of any one of claims  1 - 25 , wherein the optically active N-substituted 3-hydroxypyrrolidine is N-benzoyl-3-hydroxypyrrolidine.  
     
     
         27 . The process of any one of claims  1 - 25 , wherein the optically active N-substituted 3-hydroxypyrrolidine is N-benzyloxycarbonyl-3-hydroxypyrrolidine.  
     
     
         28 . The process of any one of claims  1 - 25 , wherein the optically active N-substituted 3-hydroxypyrrolidine is N-phenoxycarbonyl-3-hydroxypyrrolidine.  
     
     
         29 . The process of any one of claims  1 - 25 , wherein the optically active N-substituted 3-hydroxypyrrolidine is N-tert-butoxycarbonyl-3-hydroxypyrrolidine.  
     
     
         30 . The process of any one of claims  1 - 25 , wherein the optically active N-substituted 3-hydroxypyrrolidine is N-benzyl-3-hydroxypyrrolidine.