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Deracemisation of aminesRelated Patent Categories: Chemistry: Molecular Biology And Microbiology, Enzyme (e.g., Ligases (6. ), Etc.), Proenzyme; Compositions Thereof; Process For Preparing, Activating, Inhibiting, Separating, Or Purifying EnzymesDeracemisation of amines description/claimsThe Patent Description & Claims data below is from USPTO Patent Application 20070166811, Deracemisation of amines. Brief Patent Description - Full Patent Description - Patent Application Claims
[0001] The present invention relates to a method for the deracemisation or chiral inversion of chiral amines by enzymatic treatment of a mixture of enantiomers. The method employs a stereoselective enzymatic conversion and either a non-selective or partially selective chemical or enzymatic conversion, simultaneously or sequentially. The invention also provides a method for selecting a suitable enzyme, particularly a suitable amine oxidase, and for the generation of novel enzymes suitable for use in the deracemisation method. [0002] Enantiomerically pure chiral amines are valuable synthetic intermediates, particularly for the preparation of pharmaceutical target molecules. Traditionally, chiral amines have been obtained by separation methods such as diastereomeric crystallisation using a chiral acid to form a salt of one of the enantiomers, or by kinetic resolution of a racemate using an enzyme to selectively react one enantiomer allowing easier separation by physical methods such as solvent partitioning or chromatography (1). Whilst such methods can achieve high enantiomeric excess (e.e.), they can yield only a maximum of 50% of the racemic starting material as the required enantiomer. As with many chiral compounds, there is an increasing desire to develop synthetic strategies for amines that involve either asymmetric approaches or which combine resolution with racemisation of the undesired enantiomer, both of which can in principle deliver the product in 100% yield and 100% e.e. Asymmetric methods suggested to date include the use of transaminases for conversion of ketones to chiral amines (2, 3, 4). Furthermore the kinetic resolution of amines using lipases such as Burkholderia plantarii lipase (5, 6) or Candida antarctica lipase (7) has been combined with racemisation of the unreacted amine either by formation of an imine (5,6) or by transfer hydrogenation with Pd/C as the catalyst (7). [0003] An alternative approach, which has been termed deracemisation, involves the stereoinversion of one enantiomer to the other e.g. using a cyclic oxidation-reduction sequence. To date it has been shown that such a system can be applied to the preparation of L-.alpha.-amino acids by the use of an enantioselective D-amino acid oxidase in combination with a non-selective reducing agent. The original work (8) reported the stereoinversion of D- to L-alanine, albeit in low yield, using sodium borohydride as the reducing agent. The instability of sodium borohydride at pH 7 precludes its use on a practical scale, and recently we have shown that deracemisation of amino acids can be made more efficient by the use of more suitable reducing agents including sodium cyanoborohydride (13), ammonium formate with Pd/C and also borohydride complexes or amine:boranes (14). However, no-one to date has successfully applied a deracemisation method to amines. SUMMARY OF THE INVENTION [0004] The present invention provides a method for the deracemisation or chiral inversion (generally referred to herein as enantiomeric conversion) of chiral amines by treatment of a mixture of amine enantiomers with an enzyme capable of catalysing oxidation of the amine in a stereoselective manner and, subsequently or simultaneously, treating the mixture with a reducing agent. The method is applicable to mixtures of enantiomers in varying proportions, including racemic mixtures, and to conversion (epimerisation) of one single enantiomer to the other. For example the method is applicable to mixtures of R and S forms of an amine in a ratio of 1:1, 1:2, 1:5, 1:10, 2:1, 5:1, 10:1, 100:1 or other ratios. The product of the enantiomeric conversion is enriched in the desired enantiomer over the starting material i.e. the desired enantiomer is in enantiomeric excess. Preferably the product comprises a substantially pure single enantiomer. Thus, in preferred embodiments the enantiomeric conversion process of the invention is employed to convert a mixture of amine enantiomers into a composition consisting essentially of a single enantiomer, or is employed to convert one substantially pure amine enantiomer into the other, again in enantiomerically pure form. [0005] The reducing agent may be partially stereoselective or non-stereoselective and may be a chemical reducing agent. Alternatively the reduction may be enzymatically catalysed. If a chemical reducing agent is to be employed, this may advantageously be selected from sodium borohydride, sodium cyanoborohydride, amine:borane complexes or a transfer hydrogenation reagent such as ammonium formate with Pd/C. If the stereoselective oxidation and non-stereoselective (or partially selective) reduction are performed sequentially, in an oxidation-reduction cycle, the cycle may be performed a plurality of times until the desired enantiomeric excess is achieved. [0006] The enzyme capable of catalysing oxidation of the amine in a stereoselective manner may be a monoamine oxidase (MAO), particularly a microbial monoamine oxidase, but any amine oxidase enzyme may be employed. One MAO which may advantageously be employed in the method of the present invention is the Aspergillus niger monoamine oxidase or a variant thereof, for example a variant in which the enzyme differs from wild-type A. niger MAO by incorporation of one or more mutations, especially in the region of amino acids 25-265 and 334-350, particularly prefere4d are enzymes having a mutation at one or more of amino acids 259, 260, 336 and 348, more particularly the mutation N336S or the double mutation N336S, M348K. [0007] The present invention also provides a method of directing the evolution of an originator enzyme by: a) mutating the originator 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 homochiral substrate than does the originator enzyme. Optionally steps a), b) and c) may be repeated, using the enzyme variant selected in step c) as an originator enzyme. At appropriate stages, the enzyme variant(s) may be assayed against the opposite enantiomer of the substrate, or against a mixture of the substrate enantiomers, to confirm enantioselectivity. In particular embodiments, the originator enzyme is an oxidase which shows activity against amines, for example an amine oxidase, especially a monoamine oxidase. The substrate may be any chiral amine which can be oxidised to an imine, including cyclic secondary amines, for example amines of Formula I: In which: a) R is H or C.sub.1-4alkyl; R1 and R2 are independently selected from substituted or unsubstituted C.sub.1-10alkyl, C.sub.1-10alkenyl, C.sub.1-10cycloalkyl, C.sub.1-10heterocycle, C.sub.1-10aryl, C.sub.1-10heteroaryl, C.sub.1-4alkyl-aryl, C.sub.1-4alkyl-heteroaryl, C.sub.1-4alkyl-C.sub.1-6cycloalkyl and C.sub.1-4alkyl-C.sub.1-6 heterocycle; or b) R is H or C.sub.1-4alkyl, R1 and R2 together form a substituted or unsubstituted C.sub.1-10cycloalkyl ring system or C.sub.1-10aryl ring containing one or more heteroatoms; or c) R and R1 together form a substituted or unsubstituted C.sub.1-10cycloalkyl or C.sub.1-10aryl ring system which may contain one or more heteroatoms and R2 is defined as in a) above. [0008] As used herein, the terms "halo" or "halogen" refer to fluorine, chlorine, bromine and iodine. [0009] As used herein, the term "alkyl" refers to a straight or branched hydrocarbon chain containing the specified number of carbon atoms. For example, C.sub.1-C.sub.3alkyl means a straight or branched hydrocarbon chain containing at least 1 and at most 3 carbon atoms. Examples of alkyl as used herein include, but are not limited to; methyl, ethyl, n-propyl, i-propyl. [0010] As used herein, the term "cycloalkyl" refers to a fully saturated hydrocarbon ring containing the specified number of carbon atoms. The term "cycloalkyl" encompasses single and bicyclic ring structures. Examples of cycloalkyl as used herein include, but are not limited to cyclohexyl, cyclopropyl. [0011] As used herein, the term "aryl" refers to an unsaturated which may be saturated or unsaturated hydrocarbon ring containing the specified number of carbon atoms. The term "aryl" encompasses single and bicyclic ring structures. Examples of cycloalkyl as used herein include, but are not limited to phenyl, naphthyl. [0012] Where cycloalkyl or aryl ring systems contain one or more heteroatoms, these are selected from N, S or O, preferably N. Thus, the terms "heterocyclic" and "heteroaryl" refer to cycloalkyl and aryl groups, respectively, which contain up to three heteroatoms selected from N, S or O, preferably N. [0013] Where one or more of R, R1 and R2, or a ring formed therebetween, are substituted, one to three substituents may be present and are selected from halogen, hydroxy, C.sub.1-3alkyl, C.sub.1-3alkenyl, C.sub.1-3alkoxy, nitro, nitrile and CONH.sub.2. [0014] Conveniently, the target substrate can be used for evolution of enzymes with improved activity and enantioselectivity for that particular substrate. [0015] The invention therefore provides a method of improving the catalytic activity, and optionally also the enantioselectivity, of an amine oxidase, especially a monoamine oxidase, by directed evolution comprising mutation of the enzyme and selection of a mutant having improved activity against a homochiral substrate. The use of an enzyme variant, selected by such a method, in a method for the deracemisation or epimerisation of amines is also provided. [0016] The wild-type amino acid sequence of A. niger MAO is set out in SEQ. ID No. 1. In one embodiment the invention provides a variant of the A. niger MAO in which the enzyme differs from wild-type A. niger MAO in the region of amino acids 334-350, particularly amino acid 336 and/or amino acid 348, more particularly by incorporation of the mutation N336S or the double mutation N336S, M348K. The invention thus provides a variant of the A. niger MAO having the amino acid sequence set out in SEQ. ID No. 2 and, in a further embodiment, a variant of the A. niger MAO having the amino acid sequence set out in SEQ. ID No. 3. [0017] Throughout the specification and the claims which follow, unless the context requires otherwise, the word `comprise`, and variations such as `comprises` and `comprising`, will be understood to imply the inclusion of a stated integer or step or group of integers but not to the exclusion of any other integer or step or group of integers or steps. [0018] A chiral compound will have two or more enantiomers which are stereochemically dissimilar. A composition which contains more than one enantiomer of a chiral compound is referred to as a "racemic mixture" if it contains the enantiomers in equal or substantially equal amounts. By contrast a composition is "homochiral", "enantiomerically pure" or a "substantially pure single enantiomer" if it contains a single enantiomer, substantially free of the corresponding enantiomer, or consists essentially of one enantiomer in the absence of the other. By `substantially free` is meant no more than about 5% w/w of the corresponding enantiomer, particularly no more than about 3% w/w, and more particularly less than about 1% w/w is present. "Enantioselective" and "stereoselective" are used herein interchangeably and refer to the tendency of a reaction to favour one enantiomer of a chiral compound over the other. "Partially stereoselective" (or "partially enantioselective"), "non-enantioselective" etc. shall be understood accordingly. [0019] The invention will now be described in more detail, with reference to the accompanying drawings and sequence listings, in which: [0020] FIG. 1 shows the reaction scheme for the oxidation-reduction cycle which results in deracemisation; [0021] FIG. 2 shows diagrammatically the assay used to detect enzymes having the desired enantioselective amine oxidase activity; [0022] FIG. 3 shows the results of 27 enzymes selected from the detection assay of FIG. 2, assayed against L-AMBA and D-AMBA; [0023] FIG. 4 shows the substrate specificity of the N336S, M348K mutant A. niger monoamine oxidase; Continue reading about Deracemisation of amines... Full patent description for Deracemisation of amines Brief Patent Description - Full Patent Description - Patent Application Claims Click on the above for other options relating to this Deracemisation of amines patent application. ###  How KEYWORD MONITOR works... a FREE service from FreshPatents1. Sign up (takes 30 seconds). 2. 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