WO2005095629A1

WO2005095629A1 - Process for the preparation of enantiomerically enriched esters and alcohols by means of azeotropically dried enzyme compositions

Info

Publication number: WO2005095629A1
Application number: PCT/EP2005/003312
Authority: WO
Inventors: Gerardus Karel Maria Verzijl
Original assignee: Dsm Ip Assets B.V.
Priority date: 2004-03-29
Filing date: 2005-03-25
Publication date: 2005-10-13

Abstract

Process for the preparation of an enantiomerically enriched ester, which comprises subjecting a mixture of enantiomers of the corresponding secondary alcohol, in the presence of an acyl donor, to an enantioselective enzymatic conversion, wherein the conversion is carried out by using a mixture containing an enantioselective acylating enzyme, which enzyme mixture is prepared by mixing an aqueous solution of the enantioselective acylating enzyme with an organic compound which forms an azeotrope with water and by subsequently azeotropically removing the water.

Description

PROCESS FOR THE PREPARATION OF ENANTIOMERICALLY ENRICHED ESTERS AND ALCOHOLS BY MEANS OF AZEOTROP CALLY DRIED ENZYME COMPOSITIONS

The invention relates to a process for the preparation of an enantiomerically enriched ester, in which a mixture of enantiomers of the corresponding secondary alcohol is subjected, in the presence of an acyl donor, to an enantioselective enzymatic conversion. Such a process is called a Kinetic Resolution 10 (KR) process. It is for example known from EP 0 321 918, in the name of Hoechst, that it is possible to resolve racemic alcohols in the presence of a vinylester into an enantiomerically enriched ester and the remaining alcohol of opposite configuration by using a lipase P or lipase FP enzyme. The lipase P and FP enzymes are commercially 15 available in free or immobilised form. It is further disclosed in EP 0 321 918 to subsequently separate the alcohol/ester mixture by extraction, crystallisation or distillation. A disadvantage of the enzymatic esterification process disclosed in EP 0 321 918 is that commercially available lipase enzymes in free or 20 fixed/immobilized form are used which are expensive and/or need to be prepared by a relatively complex method that requires, for example, additional steps for drying or immobilizing the lipase enzyme. It is therefore less desirable to use these commercially available free (as isolated dry powder) and immobilized lipase enzymes in enantioselective esterification processes at industrial scale. A further disadvantage 25 of using these commercially available free and immobilised lipase enzymes is that the enzyme activity may be inferior upon reuse and recycle in an additional esterification reaction. It is an object of the present invention to provide a process for the preparation of an enantiomerically enriched ester in relatively high yield without the 30 need of using a commercially available and expensive free (dry) or immobilised enzyme. It is a further object that the process is relatively easy to operate, commercially attractive, and thus practical in use at industrial scale. This object is achieved by a process according to the present invention wherein the enantioselective enzymatic conversion is carried out by using a 35 mixture comprising an enantioselective acylating enzyme, wherein said enzyme mixture is prepared by mixing an aqueous solution of an appropriate enantioselective acylating enzyme with an organic compound which forms an azeotrope with water, and by subsequently azeotropically removing the water. Surprisingly, relatively inexpensive aqueous acylating enzyme solutions can be directly used in a relatively simple -and thus commercially attractive- way in the process of the invention while still delivering product in an acceptable yield and % enantiomeric excess (e.e.). This process has a clear economical advantage because the step of first producing and isolating an appropriate enzyme, for example in free or in immobilised form, may be skipped. The enantioselective enzymatic conversion in the process of the present invention may be carried out in the presence of the organic compound that is used to azeotropically remove water from the aqueous solution of the enzyme. According to a preferred embodiment of the invention, the azeotropic removal of water from the aqueous solution of the enantioselective acylating enzyme is carried out in situ. In the context of the present invention, this "in situ" modification means that the reagents of the process of the invention are added to the mixture comprising the enantioselective acylating enzyme. The advantage of this preferred embodiment is that there is no need to isolate and/or purify the enzyme mixture first. The invention further relates to a Dynamic Kinetic Resolution (DKR) process. In such a DKR process, in situ racemisation of the substrate alcohol (secondary alcohol) -and not of the product ester- is achieved by applying the reaction in the presence of a racemisation catalyst. As a result, the desired, enantiomerically enriched, product ester may be obtained in more than 50% yield by this '100% yield 100% e.e.' - concept. Thus, the invention further relates to a process for the preparation of an enantiomerically enriched ester, in which a mixture of the enantiomers of the corresponding secondary alcohol is subjected, in the presence of an acyl donor, to an enantioselective enzymatic conversion in the presence of a racemisation catalyst, wherein the enantioselective conversion is carried out by using a mixture comprising an enantioselective acylating enzyme, which mixture is prepared by mixing an aqueous solution of an appropriate enantioselective acylating enzyme with an organic compound which forms an azeotrope with water, and by subsequently azeotropically removing the water. Throughout the application, the "mixture comprising the enantioselective acylating enzyme" (the mixture from which water has been azeotropically removed) is further defined as "the enzyme mixture". The original "aqueous solution of the enantioselective acylating enzyme" is further defined as "the aqueous enzyme solution". The enzyme mixture of the invention can be a homogeneous or an heterogeneous mixture. Preferably, the enzyme mixture further contains the organic compound of the present invention that was used to azeotropically remove the water of the aqueous enzyme solution (the organic compound which forms an azeotrope with water, preferably, an organic solvent). According to a preferred embodiment, the enantioselective enzymatic conversion of the invention is carried out in the presence of an inorganic heterogeneous base. Preferably, the enzyme mixture of the invention further comprises an inorganic heterogeneous base. In that case, the mixture is a heterogeneous mixture. Surprisingly, the inorganic heterogeneous base does not interfere significantly with the activity of the enantioselective acylating enzyme. Furthermore, the process of this preferred embodiment has the advantage that the heterogeneous mixture comprising the enzyme and the base (defined as the heterogeneous enzyme/base mixture) can be recycled and re-used after the reaction while maintaining acceptable enzymatic activity. The heterogeneous enzyme/base mixture may be re-used in a subsequent enantioselective enzymatic conversion. The inorganic heterogeneous base may be added at any time in the process of preparing the enzyme mixture or at any time in the process for preparing the enantiomerically enriched ester. Preferably, the base is added to or mixed with the enzyme mixture after the water has been removed from the aqueous enzymatic solution. Preferably, the enzyme mixture of the invention (after removal of the water) contains less than 5 wt.% water relative to the total wt.% of the enzyme mixture, more preferably less than 2 wt.%, even more preferred less than 1 wt.%), more preferred less than 0,5 wt.%, and most preferred substantially no water is present in the enzyme mixture of the present invention. The heterogeneous base may act as a co-precipitant of the enzyme mixture of the invention. Optionally, the base acts as a carrier for the enzyme mixture. The enzyme/base mixture, after isolation by for example filtration, can then be re-used in a subsequent enzymatic reaction cycle for the preparation of an enantiomerically enriched ester. The enzyme/base mixture can optionally also be re-used in a different esterification reaction (with different components than the previous reaction cycle). Throughout the present application the terms "(heterogeneous) enzyme/base mixture" and "enzyme-containing solid phase" are used interchangeably. When applied in the DKR process, the inorganic heterogeneous base may optionally at the same time activate the racemisation catalyst. Besides activating the racemisation catalyst, the heterogeneous base may also act as a co- precipitant of the enzyme mixture of the invention as described above. Rosu et al, J. of Biotechnology 66 (1998) 51-59 discloses a process in which Pseudomonas lipase, being first immobilized on CaCO₃, was used as a catalyst for the transesterification of docosahexaenoic acid ethyl ester with glycerol in a solvent free system. The CaCO₃ powder was added to a culture supernatant solution (i.e. lipase in an aqueous solution) of Pseudomonas sp. KWI-56 lipase, to which chilled aceton was added and the suspension was filtered, so that an immobilized enzyme preparation was formed. However, this process has the disadvantage that it cannot be applied in situ with the transesterification and therefore it would lead to a relatively more elaborated process. Further, it is not disclosed to first azeotropically remove the water from the aqueous lipase solution.

Definitions For convenience, certain terms employed in the description, examples and claims are collected here. The term "inorganic heterogeneous base" is recognised in the art, and refers to a base or basic salt that forms an heterogeneous phase with other components of the mixture. The system may , for example, consist of two separate liquid phases or of a solid and a liquid phase. Preferably, the base is in solid form. Suitable examples of the inorganic heterogeneous base are salts of earth alkali metal or alkali metal compounds with ions such as carbonates, phosphates, sulphates, hydroxides, borates, oxides, hydrides, and the like as. Suitable bases are, for example, listed in Handbook of Chemistry and Physics, 82^nd edition, (Editor-in-chief: David R. Lide), CRC press LLC, 2001-2002. Preferred, in particular in the DKR process, are bases that have a sufficient basicity to activate the racemisation catalyst and -at the same time- do not interfere with the enzymatic reaction. For example, suitable bases have a pKa of higher than 6, preferably higher than 7, and more preferable higher than 8, even more preferred higher than 10. Suitable examples can be chosen from the list consisting of K₂HPO , K₃PO₄, Na₂HPO₄, Na₂B₄O₇, Na₂CO₃, K₂CO₃, NaHCO₃, KHCO₃, Ca(OH)₂, Ba(OH)₂, CaCO₃, Cs₂CO₃, Na₂B₄O₇ (dinatriumtetraborate), MgCO₃, MgO, CaH₂, KNCO (kalium cyanate), KCN (kaliumcyanide) or any other suitable heterogeneous base listed in the above-mentioned Handbook. Most preferred are CaCO₃, K₂CO₃, K₃PO₄, Na₂CO₃, NaHCO₃, KHCO₃, Ca(OH)₂, and MgO. According to the invention the water can be removed by azeotropic distillation from a mixture of an aqueous solution of an enantioselective acylating enzyme with an organic compound which forms an azeotrope with water. The "organic compound which forms an azeotrope with water" refers to any organic compound which has the property to form an azeotrope with water and which is added to azeotropically distil off the water. Preferably, in case a solvent is applied in the esterification reaction, the organic compound is the solvent of choice. A mixture of different organic compounds may be used as well. The organic compound may be the organic solvent in which the reaction is carried out, or the substrate alcohol for the enzymatic reaction or any other organic compound which is added to distil off the water, in particular the water from the aqueous enzyme solution. Preferred are organic solvents having a boiling point at atmospheric pressure of between 50 and 150°C, more preferred between 60 and 140°C, even more preferred between 70 and 130°C, and most preferred having a boiling point below 120°C. Preferred organic compounds are aromatic solvents such as, for example, o-xylene, toluene, chlorobenzene, and the like; alcohols, such as, for example, isopropylalcohol (IPA), ethanol, and the like; esters such as, for example, isopropylacetate, ethylacetate, and the like; and any other suitable solvents, such as, for example, tetrahydrofuran (THF), CH₃CN, heptane, and the like or any mixture of two or more thereof. Most preferred are toluene, isopropyl alcohol or the like. In order to minimize the thermal stress of the enantioselective acylating enzyme, the azeotropic distillation will preferably be performed at reduced pressure and at temperatures below 90°C, more preferably below 80°C, even more preferred below 50°C and most preferred at room temperature. The pressure applied is dependent on the solvent used and the temperature selected and can be easily determined by a person skilled in the art. Suitable enantioselective acylating enzymes that can be used in the process according to the invention are for example the acylating enzymes with known hydrolytic activity and a high enantioselectivity in esterification reactions according to the invention and which are also active in an organic environment. Preferably, a (trans)esterification enzyme is used, which stands for either a transesterification enzyme that can catalyse a transesterification reaction or an esterification enzyme that can catalyse an esterification reaction. A stereoselective hydrolytic enzyme suitable for use in the present invention may for example be found in one of the general classes of hydrolytic enzymes, for instance in the group of esterases, lipases, proteases, peptidases or acylases, preferably in the group of esterases or lipases. For example, enzymes with lipase or esterase activity or, when an amide is used as acyl donor, enzymes with amidase activity combined with esterase or lipase activity, or acylases such as for example amino acylase (N-acyl-L-amino acid amidohydrolase; E.G. 3.5.1.14) originating from Aspergillus melleus , for example originating from Pseudomonas, in particular Pseudomonas fluorescens, Pseudomonas fragi; Burkholderia, for example Burkholderia cepacia; Chromobacterium, in particular Chromobacterium viscosum; Bacillus, in particular Bacillus thermocatenulatus, Bacillus licheniformis; Alcaligenes, in particular Alcaligenes faecalis; Aspergillus, in particular Aspergillus niger, Candida, in particular Candida antarctica, Candida rugosa, Candida lipolytica, Candida cylindracea; Geotrichum, in particular Geotrichum candidum; Humicola, in particular Humicola lanuginosa; Penicillium, in particular Penicillium cyclopium, Penicillium roquefortii, Penicillium camembertii; Rhizomucor, in particular Rhizomucor javanicus, Rhizomucor miehei; Mucor, in particular Mucor javanicus; Rhizopus, in particular Rhizopus oryzae, Rhizopus arrhizus, Rhizopus delemar, Rhizopus niveus, Rhizopus japonicus, Rhizopus javanicus; Porcine pancreas lipase, Wheat germ lipase, Bovine pancreas lipase, Pig liver esterase. Preferably an enzyme originating from Pseudomonas cepacia, Pseudomonas sp., Burkholderia cepacia, Porcine pancreas, Rhizomucor miehei, Humicola lanuginosa, Candida rugosa or Candida antarctica or subtilisin is used. If an R-selective enzyme is used, for example from Candida antarctica, the R-ester is obtained as product. Naturally an S-selective enzyme will lead to the S-ester. Subtilisin is an example of an S-selective enzyme. Such enzymes can be obtained via generally known technologies. Many enzymes are produced on a technical scale and are commercially available. The enzyme source for preparing the enzymatic mixture of the present invention is not limited by purity etc. and can be both a crude enzyme solution or a purified enzyme, but it can also consist of, optionally permeabilised cells that have the desired activity, or of a homogenate of cells with such an activity. The enzyme can also be used in a chemically modified form. Within the framework of the invention it is of course also possible to use an enzyme originating from a genetically modified microorganism. Suitable commercially available enzyme solutions in water are for example those containing Candida Antarctica Lipase B (CALB) in water. Preferred aqueous enzyme solutions are Novozym^®525 L (Liquid) and Novozym^®525 F (Frozen), both obtainable from Novozymes. The amount of enzyme relative to the water of the aqueous enzyme solution is preferably more than 3 wt.%, more preferably more than 5 wt.%, even more preferred more than 10 wt.%, and most preferred more than 20 wt.%. The amount of enzyme relative tot the water of the aqueous solution is preferably less than 98%, more preferred less than 90%, even more preferred less than 80% and more preferred less than 70%. Preferably, the aqueous enzyme solution is as concentrated as possible. Stabilizing agents are optionally present in the aqueous enzyme solution. Besides the aqueous enzyme solution, enzymes with other activities or in other forms, such as enzymes in immobilised form, may additionally be present as well. An additional advantage of the present invention is that acylating enzymes that are not suitable for immobilisation due to instability effects (not being stable for a long time), and thus are not commercially available in immobilised form can now advantageously be used in the process of the present invention (while maintaining acceptable activity). Thus, the present invention further provides the application of acylating enzymes that are not available in immobilised form. Acyl donors that can be used in the process of the present invention are the well known acyl donors as for instance described in Enzyme Catalysis in Organic Synthesis, A comprehensive Handbook, Second, Completely Revised and Enlarged Edition. (Editors: K. Drauz and H. Waldmann), Vol II, 2002, 472, 544, Wiley- VHS, and references cited herein and by U.T. Bornscheuer and R.J. Kazlauskas in the handbook Hydrolases in Organic Synthesis - Regio- and Stereoselective

Biotransformations, 1999, Wiley-VCH, chapter 4.2.3., for instance carboxylic acid esters, amides or anhydrides. For example, four major classes of suitable acyl donors are: a) non-activated esters of carboxylic acids (so-called reversible acyl donors), and activated esters of carboxylic acids, such as, b) alkenyl esters (so-called irreversible acyl donors), c) phenol esters or d) oxi e esters of general structure R¹R²C=N-OAcyl. Examples of suitable acyl donors are esters of C C₂₀ carboxylic acids, preferably isopropyl acetate, isopropenyl acetate, isobutyl acetate, vinyl acetate, ethyl acetate, isopropyl laureate, isopropenyl laureate, isopropyl butyrate, vinyl butyrate, methyl phenyl acetate, or other readily available esters of carboxylic acids and C C₇ alcohols, preferably C_rC alcohols. Most preferably non-activated carboxylic acid esters are used, in particular esters of saturated alcohols. Preferably an acyldonor is chosen such that the acyldonor itself is (relatively) not volatile under the reaction conditions while its acyl donor residue is volatile, and such that any side reactions with the substrate (for example oxidation of the substrate) is prevented as much as possible under the reaction conditions. In the process of the present invention, an enantiomerically enriched ester is formed and an acyl donor residue is obtained. Preferably, the acyl donor residue is irreversibly removed from the phase in which the enantioselective conversion is carried out. The acyl donor residue is preferably removed from the reaction mixture on a continuous basis, for example by preferentially transferring the acyl donor residue to another phase relative to the acyl donor and the other reaction components. This can be achieved by physical and by chemical methods, or by a combination thereof. Examples of physical methods by which the acyl donor residue can irreversibly be removed from the phase in which the enzymatic reaction occurs, are selective crystallisation, extraction, complexing to an insoluble complex, absorption or adsorption; or by such a choice of the acyl donor that the acyl donor residue is sufficiently volatile relative to the reaction mixture, or is converted in situ into another compound that is sufficiently volatile relative to the reaction mixture to remove the acyl donor residue irreversibly from the reaction mixture; examples of the latter are the application of isopropyl acetate as acyl donor resulting in volatile isopropyl alcohol as acyl donor residue, and the application of isopropenyl acetate as acyl donor, resulting, via isopropenyl alcohol, in volatile acetone as acyl donor residue. In order to remove the acyl donor residue use can be made of a reduced pressure, depending on the boiling point of the reaction mixture. The pressure (at a given temperature) is preferably chosen in such a way that the mixture refluxes or is close to refluxing. In addition it is known to one skilled in the art that the boiling point of a mixture can be lowered by making an azeotropic composition of the mixture. Examples of chemical methods of removal are covalent bonding or chemical or enzymatic derivatization. The concentration at which the reaction is carried out is not particularly critical. The reaction can be carried out without a solvent. For practical reasons, for instance when solid or highly viscous reactants or reaction products are involved, a solvent may be used. The reaction can suitably be carried out at higher concentrations, for example at a substrate concentration higher than 0.4 M, in particular higher than 0.8 M. Preferably, the substrate (eventually substrate mixture) concentration is higher than 1M, for instance higher than 2M. Secondary alcohols that can be used as substrate (substrate alcohol) in the process of the present invention can be any of the well known secondary alcohols of industrial relevance. According to the present invention, a mixture of the enantiomers of the substrate alcohol is used. A racemic mixture but also non-racemic mixtures of the enantiomers of the substrate alcohol can be used. Even the opposite enantiomer of the desired product alcohol can be used as substrate alcohol. The amount of the opposite enantiomer is preferably less than 95% relative tot the total amount of substrate alcohol, more preferably less than 90%, even more preferred less than 80%, more preferred less than 60%, and most preferred a racemic mixture is used. The choice of the secondary alcohol depends on the desired product and on whether the secondary alcohol can be converted by the acyl donor with acceptable enantioselectivity. Also, mixtures of different secondary alcohols may be used. A secondary alcohol can be defined by the following formula (1),

(1)

wherein R¹ ≠ R² and R¹ or R² represents independently an alkyl group with for instance 1-20 C-atoms, preferably 1-6 C-atoms, an alkenyl group with for instance 2-20 C- atoms, preferably 2-6 C-atoms, or an aryl or heteroaryl group optionally containing one or more O or N atoms, with for instance 4-20 C-atoms, preferably 5-10 C-atoms; The alkyl, alkenyl and aryl groups of R¹ or R² may contain any substituents that are inert in the reaction system. Suitable substituents are, for example, alkyl groups, aryl groups, alkoxy groups, alkenyl groups, optionally substituted a ine groups which are insusceptible for the enzymatic reaction, halogens, nitrile, nitro, acyl, carboxyl, carbamoyl or sulphonate groups which may contain for instance 0-10 C-atoms. The secondary alcohol can, if desired, be formed beforehand from the corresponding ketone in a separate step (that principally does not need to be stereoselective at all) with the aid of a reducing ancillary reagent. For example, the reduction can be a hydrogenation reaction or a transfer hydrogenation reaction catalysed by a hydrogenation catalyst or a trans hydrogenation catalyst respectively, further defined as a (transfer)hydrogenation catalyst. Preferably, when applied to the DKR process, the (transfer)hydrogenation catalyst is the racemisation catalyst, using a cheap hydrogen source. A preferred hydrogen source is a volatile alcohol or hydrogen (non stereoselective hydrogenation or transfer hydrogenation). The substrate alcohol can optionally be formed in situ from the corresponding ketone with the aid of a reducing ancillary reagent. This gives the freedom of choice to employ substrate ketone or substrate alcohol or mixtures of both as substrate. The choice can depend on the availability and the simplicity of the synthesis of the substrate alcohol. If the alcohol is formed in situ from the ketone, a hydrogen source is also added as ancillary reagent. As ancillary reagent , preferably, a secondary alcohol is added to the reaction mixture that promotes the conversion of the ketone to the substrate alcohol (e.g. by setting up a redox equilibrium) and that is not converted by the enantioselective acylating enzyme. The ancillary reagent is preferably chosen so that it is not also removed from the reaction mixture by the same irreversible removal method by which the acyl donor residue is removed and that this ancillary reagent is not acylated by the enantioselective acylating enzyme, and that it has sufficient reduction potential, relative to the substrate ketone, for the creation of a redox equilibrium. Reducing agents other than alcohols can of course also be used as ancillary reagents. One skilled in the art can simply determine by experimental means which compounds are suitable for use as ancillary reagents in his reaction system. In the DKR process according to the present invention the enantioselective enzymatic conversion is carried out in the presence of a racemisation catalyst. Examples of suitable racemisation catalysts for the racemisation of the substrate alcohol are redox catalysts as, for example, applied in hydrogenations and in transfer hydrogenations. The racemisation catalyst and the enantioselective acylating enzyme are preferably chosen so that they are mutually compatible, which means that they do not or minimally deactivate each other. The expert can establish by experimental means which combination of acylating enzyme and racemisation catalyst is suitable for his specific system. Preferred combinations of acylating enzyme mixture and racemisation catalyst are combinations of lipase enzymes with a racemisation catalyst obtained by complexing of a [RuCI₂(η⁶-cymene)]₂ transition metal compound with (R,S)-2-amino-2-phenyl-propionamide as ligand under basic conditions. Either one or both of the enantioselective acylating enzyme mixture and the racemisation catalyst can be used in heterogenised form. Examples of racemisation catalysts to be chosen are catalysts on the basis of a transition metal compound. Transition metal compounds are described for example in Comprehensive Organometallic Chemistry 'The synthesis, Reactions and Structures of Organometallic Compounds' Volumes 1 - 9, Editor: Sir Geoffrey Wilkinson, FRS, deputy editor: F. Gordon A. Stone, FRS, Executive editor Edward W. Abel, preferably volumes 4, 5, 6 and 8 and in Comprehensive Organometallic Chemistry 'A review of the literature 1982 - 1994', Editor-in-chief: Edward W. Abel, Geoffrey Wilkinson, F. Gordon A. Stone, preferably volume 4 (Scandium, Yttrium, Lanthanides and Actinides, and Titanium Group), volume 7 (Iron, Ruthenium, and Osmium), volume 8 (Cobalt, Rhodium, and Iridium), volume 9 (Nickel, Palladium, and Platinum), volume 11 (Main-group Metal Organometallics in Organic Synthesis) and volume 12 (Transition Metal Organometallics in Organic Synthesis). As transition metal compound, use is preferably made of a transition metal compound of the general formula (2): M_nX_pS_qL_r (2)

where: n is 1 ,2,3,4....; p, q and r each independently represent 0,1 ,2,3,4...;

M is a transition metal, for example a metal of group 7,8,9 or 10 of the periodic system according to the new IUPAC version as shown in the table printed in the cover of the Handbook of Chemistry and Physics, 70th edition, CRC press, 1989-1990, or a lanthanide or a mixture thereof, in particular iron, cobalt, nickel, rhenium, ruthenium, rhodium, iridium, osmium, palladium, platinum or samarium, or a mixture thereof. In the racemisation use is preferably made of palladium, ruthenium, iridium or rhodium, most preferably ruthenium or iridium; X is an anion such as e.g. hydride, halogenide, carboxylate, alkoxy, hydroxy or tetrafluoroborate;

S is a so-called spectator ligand, a neutral ligand that is difficult to exchange, for example an aromatic compound, an olefin or a diene. Examples of aromatic compounds are: benzene, toluene, xylene, cumene, cymene, naphthalene, anisole, chlorobenzene, indene, cyclopentadienyl derivatives, tetraphenyl cyclopentadienone, dihydroindene, tetrahydronaphthalene, gallic acid, benzoic acid and phenylglycine. It is also possible for the aromatic compound to be covalently bonded to the ligand. Examples of dienes are norbornadiene, 1 ,5-cyclooctadiene and 1 ,5-hexadiene. L is a neutral ligand that is relatively easy to exchange with other ligands and is for example a co-ordinating solvent, in particular acetonitrile, dimethyl sulphoxide (DMSO), methanol, water, tetrahydrofuran, dimethyl formamide, pyridine, N-methylpyrrolidinone, or a tertiary amine, for example, triethylamine. Optionally, X can act as L, when X at the same time forms an intramolecular interaction by μ-complexation in, for example, dimer-, trimer- or polymer complexes. Examples of suitable transition metal compounds are:

[RuCI₂(η⁶-benzene)]₂, [RuCI₂(η^δ-cymene)]₂, [RuCI₂(η⁶-mesitylene)]₂, [RuCI₂(η⁶- hexamethylbenzene)]₂, [RuCI₂(η⁶-1 ,2,3,4-tetramethylbenzene)]₂, [RuCI₂(η⁶-1 ,3,5- triethylbenzene)]₂, [RuCI₂(η⁶-1 ,3,5-triϊspropylbenzene)]₂, [RuCI₂(η⁶- tetramethylthiophene)]₂, [RuCI₂(η⁶-methoxybenzene)]₂, [RuBr₂(η⁶-benzene)]₂, [Rul₂(η⁶- benzeen)]₂, trans-RuCI₂(DMSO)₄, RuCl₂(PPh₃)₃, Ru₃(CO)₁₂, Ru(CO)₃(η⁴-Ph₄C₄CO), [Ru₂(CO)₄(μ-H)(C₄Ph₄COHOCC4Ph₄)], [lr(COD)₂CI], [lr(CO)₂CI]_n, [lrCl(CO)₃]_n, [lr(Acac)(COD)], [lr(NBD)CI₂]₂, [lr(COD)(C₆H₆)]⁺BF₄ ^",

(CF₃C(O)CHCOCF₃)-[lr(COE)₂]⁺, [lr(CH₃CN)₄]⁺BF₄-, [lrCl₂C_p*]_2l [lrCI₂C_p]₂, [Rh(C₆H₁₀Cϊ]₂ (where C₆Hι₀ = hexa-1 ,5-di-ene), [RhCI₂C_p*]₂, [RhCI₂C_p]₂, [Rh(COD)CI]₂, CoCI₂. If necessary the transition metal compound is converted to a transition metal complex by for example exchanging the neutral ligand L with another ligand L\ whereby the transition metal compound changes into M_nX_pS_qL_r.iL'i, or complexing the transition metal compound with a ligand U, wherein i represents 0, 1, 2, ...r. The catalyst on the basis of the transition metal compound and the ligand can be added in the form of separate components of which one is the transition metal compound and the other is the ligand L', or as a complex that contains the transition metal compound and the ligand L'. Suitable racemisation catalysts are obtained for example by complexing the transition metal compound with for example a primary or secondary amine, alcohol, diol, amino alcohol, diamine, mono-acylated diamine, mono-acylated amino alcohol, mono-tosylated diamine, mono-tosylated amino alcohol, amino acid, amino acid amide, amino-thioether, phosphine, bisphosphine, aminophosphine, preferably an aminoalcohol, monoacylated diamine, monotosylated diamine, amino acid, amino acid amide, amino thioether or an aminophosphine. Examples of ligands are described in EP-A-916637 and in Tetrahedron: Asymmetry 10 (1999) 2045-2061 , complexing not necessarily taking place with the optically active ligand, but optionally with the racemate corresponding to the optically active ligands described. The ligands are preferably used in quantities that vary between 0.5 and 8 equivalents relative to the metal, in particular between 1 and 3 equivalents. In the case of a bidentate ligand use is preferably made of 0.3-8, in particular 0.5-3 equivalents. An example of a particularly good class of ligands is the class of amino acid amides of the formula (3).

wherein R¹ and R⁴ each independently represent H or a substituted or unsubstituted alkyl or phenyl group with for instance 1-9 C-atoms; R² and R³ each independently represent H or a substituted or unsubstituted alkyl group with for instance 1-9 C-atoms, or R¹ and R²form a ring together with the N and C atom to which they are bound. In most cases, activation of catalysts, for example catalysts obtained by complexing of the transition metal compound and the ligand, can be effected for example by treating the transition metal compound or the complex of the transition metal compound and the ligand in a separate step with a base, for example KOH, KOtBu, and subsequently isolating it by removing the base, or by activating the transition metal compound or the complex of the transition metal compound and the ligand in situ, when the acylation/racemisation takes place, with a mild base, for example a heterogeneous base (as listed above), in particular KHCO₃ or K₂ CO₃, or a homogeneous base, in particular an organic amine, for example triethylamine. It is also possible to activate the transition metal compound with the aid of a reducing agent, for example H₂, formic acid and salts thereof, Zn and NaBH₄. According to a preferred embodiment of the present invention, a heterogeneous base is used to activate the racemisation catalyst, which base may at the same time act as co-precipitant of the enzyme mixture as defined above. The quantities of racemisation catalyst and enantioselective acylating enzyme to be used are not particularly critical and are for example less than 10 mole%, preferably less than 5 mole%, more preferred less than 3 mole% and even more preferred less than 1 mole%, calculated relative to the substrate. The optimum quantities of both catalysts are linked to each other; the quantity of enantioselective acylating enzyme is preferably adapted so that the overall reaction continues to proceed efficiently, that is to say, that the racemisation reaction does not proceed much slower than the acylation reaction and thus the e.e. of the remaining substrate does not become too high. The optimum ratio between racemisation catalyst and enantioselective acylating enzyme for a given reaction/catalyst system can simply be established by experimental means. A particularly preferred example of a racemisation catalyst according to the invention is the one obtained by complexing of a [RuCI₂(η⁶-cymene)]₂ transition metal compound and (RS)-2-amino-2-phenyl-propionamide ligand (or (RS)-α-methyl phenyl glycine amide) and activating in the presence of a mild inorganic heterogeneous base as defined above. The DKR process according to the invention can be carried out in the presence of a supporting ketone (e.g. added for accelerating the racemisation reaction). The supporting ketone is preferably chosen in such a way that it corresponds to the alcohol that is used as substrate, or it can be another ketone that is preferably chosen in such a way that it is not also removed from the reaction mixture by the same irreversible method of removal used to remove the acyl donor residue, and that its corresponding alcohol is not acylated by the acylating enzyme. One skilled in the art can simply establish by experimental means which ketones are the most suitable for use in his specific reaction system. When carried out in the presence of an inorganic heterogeneous base, the reaction mixture obtained in the KR or DKR process of the present invention, can be used in a work-up procedure. The reaction mixture may comprise the enantiomerically enriched ester, the acyl donor residue, and the mixture of the enantioselective enzyme and inorganic heterogeneous base. The reaction mixture may further contain a residue of the enantiomerically enriched substrate alcohol, and optionally a solvent. When the DKR process is applied in the presence of the heterogeneous base, the reaction mixture further comprises the racemisation catalyst and optionally a ketone. The reaction mixture from the KR/DKR process of the invention, when carried out in the presence of an inorganic heterogeneous base, is heterogeneous. The solid phase may be separated from the reaction mixture by techniques commonly used in the art, for example by filtration. The seperated solid phase comprises at least the enzyme mixture and the base (also defined as enzyme/base mixture). This enzyme-containing solid phase can be used as such in a subsequent enzymatic reaction. Surprisingly, the recycled enzyme-containing solid phase still contains sufficient enzymatic activity to be re-used in an esterification reaction. The enzyme-containing solid phase may optionally be re-used in a different esterification reaction (with different components than the previous reaction). The product ester obtained may subsequently be isolated from the mother liquor using common practice isolation techniques, depending on the nature of the ester, for instance by extraction, destination, chromatography or crystallization. If the product is isolated by crystallization further enantiomeric enrichment may be obtained. If desired, the mother liquor (which may contain the alcohol, ester and/or ketone involved in the reaction) may be recycled to the non-stereoselective reduction of the substrate ketone or to the enzymatic resolution. Normally, before recycling, the solids will be removed from the mother liquor and, according to common practice, a purge will be built in in order to prevent built up of impurities. If desired, the ester in the mother liquor will first be saponified. With the process according to the invention an enantiomerically enriched ester can be obtained with enantiomeric excess (e.e.) higher than 85%, preferably higher than 90%, more preferred higher than 95%, even more preferred higher than 98%, and most preferred higher than 99%, optionally after recrystallization. When re-using the enzyme-containing solid phase of the invention for a subsequent esterification reaction (second cycle), an enantiomerically enriched ester can be obtained with a yield of at least 20%, preferably at least 30%, more preferably at least 40%, even more preferred at least 50% and with similar enantiomeric excess (e.e.) as during the first cycle; after the third cycle, a yield of at least 15%, more preferably at least 25%, even more preferred at least 40% can be obtained with an enantiomeric excess (e.e.) higher than 90%, preferably higher than 95%, more preferably higher than 98%, optionally after recrystallization. When re-using the enzyme-containing solid phase according to the invention for a subsequent esterification reaction (second, third or any subsequent cycle), fresh acylating enzyme mixture may be nonetheless added when this would be desired to increase the possibly diminished enzyme activity. The amounts of fresh enzyme mixture to be added can be easily determined by a person skilled in the art. The enantiomerically enriched ester obtained can subsequently be used as such. If the corresponding enantiomerically enriched alcohol is the desired product, the enantiomerically enriched ester is subsequently converted by a known procedure into the corresponding enantiomerically enriched alcohol. This can for example be effected by means of a conversion catalysed by an acid, base or enzyme. When an enantioselective acylating enzyme is used the enantiomeric excess of the product alcohol can be improved compared to the enantiomerically enriched ester obtained. The same acylating enzyme as used for the enantioselective esterification according to the invention can very suitably be used for the conversion of the enantiomerically enriched ester into the enantiomerically enriched alcohol. When the ultimate goal is the preparation of the alcohol, the acyl donor can be freely chosen in such a way that the physical or chemical properties of the acyl donor and the acyl donor residue are optimal for the irreversible removal of the acyl donor residue and the treatment of the reaction mixture. With the process according to the invention enantiomerically enriched alcohols can be obtained with an enantiomeric excess (e.e.) higher than 85%, preferably higher than 90%, more preferably higher than 95%, even more preferred higher than 98%, most preferred higher than 99%, optionally after recrystallization and/or hydrolysis with the aid of an enantioselective acylating enzyme. The alcohols thus obtained form commonly used building blocks in the preparation of for example liquid crystals, agrochemicals and pharmaceutical products, for example of secondary aliphatic alcohols or aryl alcohols, for example of 1- aryl-ethanols, -propanols, -butanols or -pentanols. In the literature applications are known of for example 1-(4-methoxyphenyl)-2-propenyl-1-ol in J. Heterocycl. Chem (1977), 14 (5), 717-23; 1-(4-methoxyphenyl)-1 -propanol in the preparation of liquid crystals and pharmaceuticals (JP-A-01000068); 1-(4-fluorophenyl)-1-ethanol in the preparation of antiarrhytmic agents (d-Sotalol; Org. Process Res. Dec. (1997), 1(2), 176-178); 1-(2-chlorophenyl)-1-butanol in EP-A-314003; 1-(2,6-difluorophenyl)-1- ethanol in the preparation of antiepileptics (EP-A-248414); 1-(3,5-difluorophenyl)-1- pentanol in the preparation of liquid crystals (WO-A-8902425); 1-(3,4-difluorophenyl)-1- propanol in the preparation of means for the treatment of prostatic hyperplasia and prostatitis (WO-A-9948530); 1-(2-trifluoromethyl-phenyl)-1-ethanol in the preparation of a tocolytic oxytocine receptor antagonist (US-A-5726172); 1-(3-trifluoromethyl-phenyl)- 1-ethanol in the preparation of a fungicide (JP-A-10245889) ; 1-(3,5-bis(trifluoromethyl)-phenyl)-1-ethanol in the preparation of tachykinin receptor antagonist (US-A-5750549); 1-(2-fluor-5-nitro-phenyl)-1-ethanol in the preparation of a herbicide (DE-A-4237920); 1-(3-chloro-4,5-dimethoxy-phenyl)-1-ethanol in inhibitors of plasmogen activator inhibitor- 1 (WO-A-9736864); 1-methyl-3-(4-acetylphenyI)-1- propanol and 1-methyl-2-(4-acetylphenyl)-1-ethanol in the preparation of liquid crystals (EP-A-360622/JP-A-03236347); 4-(1-hydroxyethyl)-benzonitrile in the preparation of nicotine amides as PDE4 D isoenzyme inhibitors (WO-A-9845268); 1 -naphthalene- 1- ethanol in the separation of enantiomers via HPLC with chiral stationary phase on the basis of polysaccharides (WO-A-9627615); 1-naphthalene-1 -propanol as an example of a product in the asymmetrically catalysed dialkyl zinc addition to aldehydes (Chem. Lett. (1983), (6), 841-2); 1-(1,3-benzodioxol-5-yl)-1-butanol in the preparation of a proteinase 3 inhibitor in the treatment of leukemia (US-A-8508056). The invention also relates to the preparation of an enantiomerically enriched alcohol from the enantiomerically enriched ester obtained.

Examples

The invention will be elucidated on the basis of the examples, without however being limited by them. Examples are illustrated by following model reaction:

3a Acyl = acetyl 3b Acyl = butyryl

Step 1 : transferhydrogenation using a hydrogen source, for example, isopropanol and a ruthenium catalyst

Step 2, option 1: removal of water from Novozym^® 525 L and subsequent DKR of 2

Step 2, option 2: DKR of 2 applying a mixture of Novozym^® 525 L (isolated from Step 2, option 1) and heterogeneous base

Yields and enantiomeric excess (e.e.) are determined by GC-analvsis:

Sample: ~20 μL of clear reaction mixture was diluted in CH₂CI₂ (1 mL) and injected on WCOT Fused Silica 25 m x 0.25 mm coating CP Chirasil-dex CB DF=0.25 column.

Program: isothermal at 120°C

R_t: acetophenone (1): 2.6 min, (S)-l-phenyl-ethyl acetate ((S)-3a): 3.5 min and (R)-1- phenyl-ethyl acetate ((R)-3a): 3.9 min.

(R)-l-phenylethanol ((R)-2): 5.4 min, (S)-l-phenylethanol ((S)-2): 5.7 min, (S)-1- phenyl-ethyl butyrate ((S)-3b): 7.5 min and (R)-l-phenyl-ethyl butyrate ((R)-3b): 7.8 min. The result of the process according to the present invention is an enantiomeric balance (e.b.) higher than 0. The enantiomeric balance is defined by the following formula wherein (R) and (S)-substrate and (R) and (S)-product are expressed in moles:

e.b. = (R)-substrate + (RVproduct - (SVsubstrate - (S)-product . (R)-substrate + (S)-substrate + (R)-product + (S)-product

Example I (Step 2, option 1)

A 100 mL three-neck round bottom flask equipped with thermometer, distillation unit and magnetic stirring bar was charged with isopropanol (5 mL) and Novozym^® 525 L (150μL). Under continuous stirring of the obtained solution, toluene (24 mL) was added to the enzyme solution. The pressure was slowly adjusted to 250 mbar at 70°C in order to remove water by azeotropic distillation with toluene and isopropanol. When distillation was finished the obtained enzyme/toluene mixture was ready to use in DKR. For DKR purpose, (RS)-l-phenylethanol (2) (4.39 g, 36 mmol), isopropenyl acetate (7.2 g, 72 mmol), [RuCI₂(η⁶-cymene)]₂ (11 mg, 0.018 mmol) and (RS)-2-amino-2-phenyl-propionamide (7.1 mg, 0.043 mmol) were added sequentially to the enzyme solution. The reaction mixture was degassed by 5 cycles of vacuum and dry nitrogen purge. The racemisation catalyst was activated by the addition of K₂CO₃ (3.6 g) as a heterogeneous base. The reaction mixture was stirred at 70°C for 24 h accompanied by continuous distillation of acetone at approximately 240 mbar to give (R)-l-phenyl-ethyl acetate ((R)-3a) in 85 % yield and 99 % e.e..

Result: a DKR process is applied and K₂CO₃ is used as mild heterogeneous base, both to activate the racemisation catalyst and to form a K₂CO₃/ Novozym^® 525 L heterogeneous enzyme mixture that may be recycled and re-used. Suprisingly, the desired product (R)-l-phenyl-ethyl acetate ((R)-3a) is obtained in acceptable yield and high e.e. without the need of using a commercially available and expensive immobilised enzyme. Example II

Cycle 1 :

Transferhydrogenation of 1 (stepD: In a 100 mL three-neck round bottom flask equipped with a thermometer, distillation unit and magnetic stirring bar, acetophenone (1) (4.32 g, 36 mmol), [RuCI₂(η⁶-cymene)]₂ (11 mg, 0.018 mmol), (RS)-2-amino-2-phenyl- propionamide (7.1 mg, 0.043 mmol) were dissolved in isopropanol (20 mL). After degassing the homogeneous solution with dry nitrogen, the transferhydrogenation catalyst was activated by K₂CO₃ (15.2 mg, 0.11 mmol) and the solution was stirred at 70°C for 1 hour. Then, isopropanol was completely distilled at slight reduced pressure over a period of 1 hour to furnish 2 in 99 % yield. The ruthenium catalyst remains in 2 as a homogeneous mixture and can be employed as racemisation catalyst in the following DKR-reaction.

DKR of 2 (Step2, option 1): Under stirring of the obtained homogeneous mixture of 2, Novozym^®

525 L (140 μL) and toluene (10 ml) were added sequentially. The reaction mixture was degassed by 5 cycles of vacuum and dry N₂ purge. Water was azeotropically removed by distillation of toluene. For this purpose, the reaction pressure was slowly reduced to 10 mbar at 70°C. When finishing the distillation, the reaction was continued by DKR of 2. The residue containing the enzyme, ruthenium transferhydrogenation catalyst (from now, it will be applied as racemisation catalyst) and 2 is dissolved in toluene (24 mL). Then, isopropenyl acetate (7,2 g, 72 mmol) and K₂CO₃ (1.79 g, 13 mmol) was added to the toluene solution and the heterogeneous reaction mixture was degassed by 5 cycles of vacuum and dry nitrogen purge. The reaction mixture was stirred at 70°C for 24 h accompanied by continuous distillation of acetone at approximately 240 mbar to give (R)-l-phenyl-ethyl acetate ((R)-3a) in 87 % yield and 99 % e.e. The reaction mixture was filtrated over a glass-filter and the residue was washed with 3 portions of toluene. The obtained wet cake of K₂CO₃ and enzyme-containing solid phase was stored as such and re-used in a second DKR cycle.

A second transferhydrogenation/DKR-run of 1 was performed (cycle 2) using the obtained K₂CO₃/enzyme batch from cycle 1.

Cycle 2: Transferhvdrogenation of 1 (stepD:

Transferhydrogenation of 1 has been performed according to the procedure described in cycle 1 giving 2 in 99 % yield.

DKR of 2 (Step2. option 2): To the obtained homogeneous mixture of 2 was added sequentially, toluene (24 mL), isopropenyl acetate (7,2 g, 72 mmol) and the K₂CO₃/ Novozym^® 525 L wet cake collected from previous transferhydrogenation/DKR cycle. The reaction mixture was degassed by 5 cycles of vacuum and dry N₂ purge. The DKR of 2 was performed by stirring the heterogeneous reaction mixture at 70°C accompanying by continuous distillation of acetone at approximately 240 mbar. Continuation of the DKR for 20 hours at given conditions furnished (R)-l-phenyl-ethyl acetate ((R)-3a) in 81 % yield and 99 % e.e.. The reaction mixture was filtrated over a glass-filter and washed with 3 portions of toluene. The obtained wet cake of K₂CO₃ and enzyme was stored as such and re-used in a third cycle. A third transferhydrogenation/DKR-run of 1 was performed (cycle 3) using the obtained K₂CO₃/enzyme batch from cycle 2.

Cycle 3:

Transferhydrogenation of 1 (stepD:

Transferhydrogenation of 1 has been performed according to the procedure described in cycle 1 giving 2 in 98 % yield.

DKR of 2 fStep2. option 2):

DKR of 2 has been performed following the procedure described in cycle 2 to furnish (R)-l-phenyl-ethyl acetate ((R)-3a) in 70 % yield and 98 % e.e..

In a fourth cycle it is demonstrated that the slightly declined lipase activity of the K₂CO₃/enzyme mixture obtained from previous run could be upgraded by the addition of fresh lipase according to procedure step 2 option 1.

Cycle 4:

Transferhydrogenation of 1 (stepl): Transferhydrogenation of 1 has been performed according to the procedure described in cycle 1 giving 2 in 97 % yield.

DKR of 2 (Step2. option 1):

Under stirring of the obtained homogeneous mixture of 2, fresh enzyme Novozym^® 525 L (70 μL) and toluene (5 ml) were added sequentially. The reaction mixture was degassed by 5 cycles of vacuum and dry N₂ purge. Water was azeotropically removed by distillation of toluene. For this purpose, the reaction pressure was slowly reduced to 10 mbar. When finishing the distillation, the reaction was continued by DKR of 2. The residue containing the enzyme, ruthenium catalyst and 2 is dissolved in toluene (24 mL). Then, isopropenyl acetate (7,2 g, 72 mmol) and the K₂CO₃/ Novozym^® 525 L wet cake collected from previous transferhydrogenation/DKR cycle was added to the toluene solution and the heterogeneous reaction mixture was degassed by 5 cycles of vacuum and dry nitrogen purge. The reaction mixture was stirred at 70°C for 21 h accompanied by continuous distillation of acetone at approximately 240 mbar to give (R)-l-phenyl-ethyl acetate ((R)-3a) in 72 % yield and 98 % e.e.. The reaction mixture was filtrated over a glass- filter and the residue was washed with 3 portions of toluene. The obtained wet cake of K₂CO₃ and enzyme was stored as such and re-used in a next DKR cycle.

Cycle 5: In this cycle, the procedure of cycle 4 was repeated using K₂CO₃/

Novozym^® 525 L wet cake collected from previous transferhydrogenation/DKR cycle giving (R)-l-phenyl-ethyl acetate ((R)-3a) in 77 % yield and 99 % e.e..

Result: it is clear from Example II that the K₂CO₃ /Novozym enzyme heterogeneous mixture (base/enzyme batch) can be recycled and re-used in subsequent reaction cycles to prepare the enantiomerically enriched ester (R)-1- phenyl-ethyl acetate ((R)-3a) in acceptable yield and high enantiomeric excess. After several cycles, the addition of a small amount of fresh enzyme may help to reach an acceptable level of activity. Example III

DKR of 2 (Step2, option 1):

In a 100 mL three-neck round bottom flask equipped with thermometer, distillation unit and magenetic stirring bar, Novozym^® 525 L (400 μL) was dissolved in (RS)-l-phenylethanol (2) (4.39 g, 36 mmol) and isopropanol (5 mL). Under continuous stirring of the obtained homogeneous solution, pressure was slowly adjusted to 10 mbar at 70°C in order to remove water by azeotropic distillation with isopropanol. When distillation was finished the obtained enzyme solution in 2 was dissolved in toluene (24 mL). A small fraction of toluene was distilled at reduced pressure and 70°C in order to remove residual amounts of isopropanol. Then, isopropyl butyrate (9.36 g, 72 mmol), [RuCI₂(η⁶-cymene)]₂ (22 mg, 0.036 mmol) and (RS)-2- amino-2-phenyl-propionamide (14.2 mg, 0.086 mmol) were added sequentially to the reaction mixture. The reaction mixture was degassed by 5 cycles of vacuum and dry nitrogen purge. The racemisation catalyst was activated by the addition of K₂CO₃ (3.6 g) as a heterogeneous base. The reaction mixture was stirred at 70°C for 24 hours accompanied by continuous distillation of isopropanol at approximately 190 mbar to give (R)-l-phenyl-ethyl butyrate ((R)-3b).

The reaction mixture was filtrated over a glass-filter and washed with 3 portions of toluene. The obtained wet cake of K₂CO₃ and enzyme was stored as such and re-used in a next DKR-cycle.

Cycle 2:

DKR of 2 tStep2, option 2):

A 100 mL three-neck round bottom flask equipped with thermometer, distillation unit and magenetic stirring bar was charged with toluene (24 mL), (RS)-1- phenylethanol (2) (4.39 g, 36 mmol), isopropyl butyrate (9.36 g, 72 mmol), [RuCI₂(η⁶~ cymene)]₂ (22 mg, 0.036 mmol), (RS)-2~amino-2-phenyl-propionamide (14.2 mg, 0.086 mmol) and the K₂CO / Novozym^® 525 L wet cake collected from previous DKR run. After degassing the reaction mixture by 5 cycles of vacuum and dry nitrogen purge, the reaction mixture was stirred at 70°C for 23 h accompanied by continuous distillation of isopropanol at approximately 190 mbar to give (R)-l-phenyl-ethyl butyrate ((R)-3b) in 97 % yield and >99 % e.e.. The reaction mixture was filtrated over a glass-filter and washed with 3 portions of toluene. The obtained wet cake of K₂CO₃ and enzyme was stored as such and re-used in a next DKR-cycle.

Cycle 3:

DKR of 2 (Step2, option 2):

Cycle 2 was repeated using K₂CO₃/ Novozym^® 525 L wet cake collected from previous DKR cycle to furnish (R)-l-phenyl-ethyl butyrate ((R)-3b) in 82 % yield and >99 % e.e..

Result: A different acyl donor isopropyl butyrate is used in this example III. It is clear from Example III that the K₂CO₃ /Novozym enzyme heterogeneous mixture (enzyme/base mixture or enzyme-containing solid phase) can be recycled and re-used in subsequent reaction cycles to prepare the enantiomerically enriched ester (R)-l-phenyl-ethyl butyrate ((R)-3a) in acceptable yield and high enantiomeric excess.

Example IV

DKR of 2 (Step2, option 1):

In a 100 mL three-neck round bottom flask equipped with thermometer, distillation unit and magenetic stirring bar, Novozym^® 525 L (60 μL) was dissolved in (RS)-l-phenylethanol (2) (976 mg, 8 mmol) and isopropanol (5 mL). The pressure was slowly reduced to vacuum at 70°C in order to remove water by azeotropic distillation with isopropanol. When distillation was finished, toluene (10 mL), isopropyl butyrate (2.08 g, 16 mmol), acetophenone 1 (96 mg, 0.8 mmol) and [Ru₂(CO)₄(μ-H) (C₄Ph₄COHOCC₄Ph₄)] (so-called Shvo-catalyst) (43 mg, 0.04 mmol) were added sequentially to the enzyme solution. The reaction mixture was degassed by 5 cycles of vacuum and dry nitrogen purge. The reaction mixture was stirred at 70°C for 16 h accompanied by continuous distillation of isopropanol at approximately 190 mbar to give (R)-l-phenyl-ethyl butyrate ((R)-3b) in 54 % yield and >99 % e.e.and (S)~ 2 in 46 % yield and 46 % e.e.. The enantiomeric balance (e.b.) was calculated to be 33 %. Result: a DKR process is applied by using a different racemisation catalyst and without using an inorganic heterogeneous base. The enzyme mixture of the invention is thus obtained in a relative simple and commercially attractive way (after azeotropical removal of the water from the aqueous Novozym^® 525 L mixture) while still resulting in acceptable yield and enantiomeric balance. Example V A 50 mL three-neck round bottom flask equipped with distillation unit and magnetic stirring bar was charged with isopropanol (5 mL) and Novozym® 525 L (150μL). Under continuous stirring, toluene (15 mL) was added to the obtained enzyme suspension in isopropanol. The suspension changed to an almost clear solution. The pressure was slowly adjusted to 250 mbar at 70°C in order to remove water by azeotropic distillation with toluene and isopropanol. When distillation was finished the obtained enzyme/toluene mixture was cooled to room temperature. For KR purpose, (RS)-l-phenylethanol (2) (2.20 g, 18 mmol) isopropenyl acetate (3.6 g, 36 mmol) and MgO as base were added sequentially to the enzyme solution. The reaction mixture was stirred at conditions given in table furnishing a mixture of (R)-l-phenyl-ethyl acetate ((R)-3a) and (S)-l-phenylethanol ((S)-2a).

Table 1

Exp. T t MgO (S)-2a (S)-2a (R)-3a (R)-3a (°C) (h) (mmol) (%) e.e (%) (%) e.e. (%) 1 20 20 0 50 > 99 50 > 99 2 70 2 0 56 77 44 > 99 3 70 1 15 50 > 99 50 > 99

Example VI

Kinetic resolution of RSI -1-cvclohexvlethanol (4)

(R)-5 (S)-4 A 50 mL three-neck round bottom flask equipped with distillation unit and magnetic stirring bar was charged with isopropanol (5 mL) and Novozym® 525 L (150μL). Under continuous stirring, toluene (15 mL) was added to the obtained enzyme suspension in isopropanol. The suspension changed to an almost clear solution. The pressure was slowly adjusted to 250 mbar at 70°C. Water was removed azeotropically by concomitant distillation of toluene and isopropanol. For KR purpose, (RS)-1- cyclohexylethanol (2) (2.30 g, 18 mmol), isopropenyl acetate (3.6 g, 36 mmol) and base (13 mmol) were added sequentially to the enzyme solution. The reaction mixture was stirred for 1 night at 70°C furnishing a mixture of (R)-l-cyclohexylethyl acetate ((R)-5) and (S)-l-cyclohexyllethanol ((S)-4). Samples were analysed using chiral G.C. according to method described before. For the e.e. determination of the remaining alcohol, 4 was converted to the butyric ester by treatment of sample with butyric anhydride in the presence of triethylamine and dimethylaminopyridine (DMAP). After derivatisation, DMAP and triethylamine were removed by extraction with 2 N HCI aqueous solution.

Table

Exp. Base (S)-4 (S)-4 (R)-5 (R)-5 (%) e.e (%) (%) e.e. (%) 1 75 29 25 87 2 Na₂B₄O₇ 69 44 31 98 3 NaHCO₃ 60 64 40 98 4 Ca(OH)₂ 50 > 99 50 98 5 MgO 50 > 99 50 98

Example VII

Resolution (RS)-l-phenvlethanol using subtilisine

A 50 mL three-neck round bottom flask equipped with distillation unit and magnetic stirring bar was charged with isopropanol (10 mL). The addition of subtilisine (0.5 mL) to isopropanol was accompanied by partial precipitation of the enzyme. At 40°C, 5 ml isopropanol was removed in vacuo (azeotropic distillation). Under continuous stirring, toluene (15 mL) was added to the obtained enzyme suspension in isopropanol. The suspension changed to an almost clear solution. In order to perform further removal of residual amounts of isopropanol, a second fraction (4 ml) was distilled from the reaction mixture at 40°C (azeotropic distillation). Then, the enzyme mixture was cooled to room temperature and (RS)-l-phenylethanol (2) (2.20 g, 18 mmol) and vinyl butyrate (2.05 g, 18 mmol) was added sequentially to the enzyme solution. The pressure was slowly adjusted to 100 mbar at room temperature for 1 hour and then the temperature was raised to 40°C for 1 night to furnish a mixture of (S)-1- phenyl-ethyl butyrate ((S)-3b) in 27 % yield and 85 % e.e., and (R)-l-phenylethanol (2) in 73 % yield and 31 % e.e.

Claims

1. Process for the preparation of an enantiomerically enriched ester, which comprises subjecting a mixture of enantiomers of the corresponding secondary alcohol and an acyl donor to an enantioselective enzymatic conversion in the presence of a mixture containing an enantioselective acylating enzyme, which enzyme mixture is prepared by mixing an aqueous solution of the enantioselective acylating enzyme with an organic compound which forms an azeotrope with water and by subsequently azeotropically removing the water.

2. Process according to claim 1 , wherein the enantioselective enzymatic conversion is carried out in the presence of said organic compound.

3. Process according to any one of claims 1-2, wherein the enantioselective conversion is carried out in the presence of a racemisation catalyst. 4. Process according to any one of claims 1-3, in which the enantioselective acylating enzyme has lipase or esterase activity.

5. Process according to any one of claims 1-4, wherein the azeotropic distillation is carried out at a temperature of below 80°C.

6. Process according to any one of claims 1-5, in which the mixture containing the enantioselective acylating enzyme contains less than 5 wt.% water relative to the total wt.% of the enzyme mixture.

7. Process according to any one of claims 1-6, wherein the conversion is carried out in the presence of an inorganic heterogeneous base.

8. Process according to claim 7, wherein a solid phase comprising the inorganic heterogeneous base and the mixture containing the enantioselective acylating enzyme is separated from the reaction mixture.

9. Process according to claim 8, wherein the separated solid phase is re-used in a subsequent enantioselective enzymatic conversion.

10. Process according to any one of claims 1-9, wherein the enantioselective enzymatic conversion takes place in the presence of a ketone.

11. Process according to any one of claims 1-10, wherein the secondary alcohol is formed in situ from the corresponding ketone in the presence of a hydrogen source.

12. Process according to any one of claims 1-11 , wherein a mixture of the secondary alcohol and the corresponding ketone is used as the substrate.

3. Process according to any one of claims 1-12, in which the enantiomerically enriched ester obtained is subsequently converted into the corresponding enantiomerically enriched alcohol.