Deracemisation of Amines

Abstract

Methods are described for the deracemisation or chiral inversion of chiral amines by enzymatic treatment, such as by a stereoselective enzymatic conversion and either a non-selective or partially selective chemical or enzymatic conversion, simultaneously or sequentially. Also described are methods for selecting a suitable enzyme, particularly a suitable amine oxidase, and for the generation of novel enzymes suitable for use in the deracemisation method.

1 . A method for the enantiomeric conversion of amines comprising treating a homochiral amine or a mixture of amine enantiomers with an enzyme capable of catalysing oxidation of the amine in a stereoselective manner and, subsequently or simultaneously, treating with a reducing agent.  
     
     
         2 . A method according to  claim 1  which is a method of epimerisation of one single amine enantiomer to the other.  
     
     
         3 . A method according to  claim 1  in which the mixture of amine enantiomers is a racemic mixture.  
     
     
         4 . A method according to  claim 1  in which the reducing agent is partially enantioselective.  
     
     
         5 . A method according to  claim 1  in which the reducing agent is a chemical reducing agent and is non-enantioselective.  
     
     
         6 . A method according to  claim 5  in which the reducing agent is selected from sodium borohydride, sodium cyanoborohydride, an amine:borane complex or a transfer hydrogenation reagent.  
     
     
         7 . A method according to  claim 6  in which the transfer hydrogenation reagent is ammonium formate with Pd/C.  
     
     
         8 . A method according to  claim 6  in which the amine:borane complex is an ammonia:borane complex.  
     
     
         9 . A method according to  claim 1  comprising an oxidation-reduction cycle in which the steps of treating with an enzyme capable of catalysing oxidation of the amine and treating with a reducing agent are performed sequentially, a plurality of times.  
     
     
         10 . A method of improving the activity, substrate specificity, or enantioselectivity of an amine oxidase enzyme by directed evolution, the method comprising mutation of the enzyme and selection of a mutant having improved activity against an homochiral substrate.  
     
     
         11 . A method of directing the evolution of an amino oxidase enzyme comprising the steps of: 
 a) mutating the enzyme to create at least one enzyme variant;    b) screening said enzyme variant for activity against a homochiral substrate; and    c) selecting one or more enzyme variants which show greater activity toward the substrate than does the originator enzyme.    
     
     
         12 . A method according to  claim 11 , further comprising repeating steps a), b) and c) one or more times, using the enzyme variant selected in step c) as the enzyme to be mutated.  
     
     
         13 . A method according to  claim 11  further comprising performing site directed mutagenesis on an enzyme selected in step c).  
     
     
         14 . A method according to  claim 11  in which the enzyme is a monoamine oxidase.  
     
     
         15 . A method according to  claim 14  in which the enzyme is a microbial monoamine oxidase.  
     
     
         16 . A method according to  claim 15  in which the enzyme is the  Aspergillus niger  monoamine oxidase, or a variant thereof.  
     
     
         17 . A method according to  claim 16  in which the enzyme is a variant of  A. niger  monoamine oxidase which differs from wild-type  A. niger  monoamine oxidase by mutation in the region of amino acids 334-350.  
     
     
         18 . A method according to  claim 14  in which the enzyme is a variant of  A. niger  monoamine oxidase which differs from wild type  A. niger  monoamine oxidase by mutation of the amino acid at position number 336.  
     
     
         19 . A method according to  claim 14  in which the enzyme is a variant of  A. niger  monoamine oxidase which differs from wild type  A. niger  monoamine oxidase by incorporation of the mutation: N336S.  
     
     
         20 .- 23 . (canceled)  
     
     
         24 . A method according to  claim 1  in which said enzyme is an enantioselective monoamine oxidase (MAO) enzyme having the amino acid sequence of SEQ ID NO:2.  
     
     
         25 . A method according to  claim 1  in which said enzyme is an enantioselective monoamine oxidase enzyme which is a variant of wild-type  A. niger  MAO, where said variant differs from wild-type  A. niger  monoamine oxidase by incorporation of the mutation: N336S.  
     
     
         26 . A method according to  claim 1  in which said enzyme is an enantioselective monoamine oxidase enzyme which is a variant of wild-type  A. niger  MAO, where said variant differs from wild-type  A. niger  monoamine oxidase by incorporation of the mutations: N336S and M348K.  
     
     
         27 . A method according to  claim 1  in which said enzyme is an enantioselective monoamine oxidase enzyme which is a variant of wild-type  A. niger  MAO, where said variant differs from wild-type  A. niger  monoamine oxidase by incorporation of the mutations: N336S and one or more of M348K, R259L and R260L.  
     
     
         28 . (canceled)