WO2007128469A1

WO2007128469A1 - Process for the preparation of enantiomerically enriched nitriles

Info

Publication number: WO2007128469A1
Application number: PCT/EP2007/003852
Authority: WO
Inventors: Daniel Mink; Jeffrey Harald Lutje Spelberg; De Erik Jan Vries
Original assignee: Dsm Ip Assets B.V.
Priority date: 2006-05-03
Filing date: 2007-05-02
Publication date: 2007-11-15

Abstract

The invention relates to a process for the preparation of an enantiomerically enriched nitrile by reacting an epihalohydrin (derivative) with Br- and CN- in the presence of an enantioselective haloalcohol dehalogenase. The process of the invention leads to enantiomerically enriched nitriles in a high yield and in a high enantiomeric excess. Preferably the haloalcohol dehalogenase used is HheC, more preferably HheC from Agrobacterium radiobacter AD1, most preferably the W249F mutant from HheC from Agrobacterium radiobacter AD1. In one preferred embodiment of the invention the epihalohydrin (derivative) is epichlorohydrin. The enantiomerically enriched nitriles obtained by the process of the invention are especially suitable as intermediates in the preparation of statins, in particular of atorvastatin or rosuvastatin.

Description

PROCESS FOR THE PREPARATION OF ENANTIOMERICALLY ENRICHED

NITRILES

The invention relates to a process for the preparation of an enantiomerically enriched nitrile of formula (2),

and/or of formula (2a)

, wherein R³ stands for Cl or Br and wherein R² stands for H or methyl, preferably for H.

Enantiomerically enriched nitriles are important building blocks in the synthesis of various active pharmaceutical ingredients, for example in the preparation of HMG-CoA reductase inhibitors, more in particular in the preparation of statines, for example lovastatin, cerivastatin, rosuvastatin, simvastatin, pravastatin, atorvastatin or fluvastatin.

Inventors have now found a process in which it is possible to prepare enantiomerically enriched nitriles of formula (2) and/or of formula (2a) in a high yield in a high enantiomeric excess (e.e.).

In a first aspect, the invention relates to a process comprising the step of reacting a mixture of enantiomers of the epoxide of formula (1 )

wherein R¹ stands for Cl or for Br, and wherein R² stands for H or for methyl, and/or a mixture of enantiomers of the corresponding ring-opened form thereof of formula (1 a)

, wherein R¹ and R² are as defined above and wherein R⁴ independently stands for Cl or Br, with CN^" and Br^" in the presence of an enantioselective haloalcohol dehalogenase to form the corresponding enantiomerically enriched compound of formula (2)

wherein R² is defined above and/or the corresponding enantiomerically enriched ring- opened form thereof of formula (2a)

wherein R³ stands for Cl or Br and wherein R² is as defined above.

Conventional kinetic resolution takes advantage of the fact that enantiomers react at different rates in the presence of a chiral catalyst or a chiral reagent. Because one enantiomer reacts much faster than the other, the product is obtained in enantiomeric excess and also the remaining substrate is then obtained in enantiomeric excess. The limitations of this conventional kinetic resolution are that (i) the other enantiomer may not be converted at all (leading to a maximum yield of the product of 50%) and/or

(ii) the other enantiomer may be converted, reducing the enantiomeric excess of the product to below the theoretically maximal obtainable enantiomeric excess If however, next to enantioselective conversion, there is a rapid equilibrium between the two enantiomers, i.e. racemization, (which equilibrium is more rapid than the enantioselective conversion), it is possible to obtain a product in an enantiomeric excess in a high yield. This phenomenon is called dynamic kinetic resolution.

The inventors have now found that in the process of the present invention, where the epoxide of formula (1) is reacted in the presence of Br^" with CN^" in the presence of an enantioselective haloalcohol dehalogenase, dynamic kinetic resolution takes place. This is surprising in view of the fact, that if Cl^" instead of Br^" is present, hardly any or even no dynamic kinetic resolution takes place. Therefore, the process of the present invention surprisingly leads to the preparation of enantiomerically enriched nitriles of formula (2) and/or of formula (2a) in a high yield in high enantiomeric excess.

In a preferred embodiment of the invention, the invention relates to a process comprising the step of reacting a mixture of enantiomers of epichlorohydrin with CN^" and Br^" in the presence of an enantioselective haloalcohol dehalogenase to form the corresponding enantiomerically enriched oxiranyl acetonitrile and/or the corresponding enantiomerically enriched ring opened form thereof of formula (2b)

wherein R³ stands for Cl or Br. It is preferred to use epichlorohydrin (compound of formula (1) wherein R¹ stands for Cl and R² stands for H) as a starting material, since epichlorohydrin is environmentally and commercially attractive. For example, epichlorohydrin has a better solubility in water than epibromohydrin and with epichlorohydrin less grams of waste are produced than with epibromohydrin. Furthermore, epichlorohydrin is widely available at low cost.

The amount of Br^" used in the process of the present invention is in principle not critical. Usually, Br^" is used in molar equivalent amount of between 0.5 and 5 relative to the amount of epoxide of formula (1). Br^' may be added in the form of a salt or an acid, for example NaBr or HBr. The concentration of the epoxide of formula (1 ) or of the compound of formula (1a) is in principle not critical, but is preferably chosen as high as possible. For example, it is shown in the examples that it is possible to use epichlorohydrin in a concentration of 10OmM. Ways of synthesizing the compound of formula (1) and the compound of formula (1a) are known to the person skilled in the art. Furthermore, both epichlorohydrin and epibromohydrin are widely available from commercial suppliers.

The cyanide ion may, for example, be added to the reaction in the form of a cyanide salt or as a combination of HCN and optionally a base. In principle any cyanide salt may be used. Examples of cyanide salts include cyanide salts with an - A -

alkali metal as a cation (e.g., sodium cyanide, potassium cyanide, and lithium cyanide) and those with a bulky cation (e.g., tetrabutylammonium cyanide and tetrabutyl phosphonium cyanide). For commercial use, sodium cyanide or potassium cyanide is preferred. CN^' is preferably used in a molar equivalent amount of between 0.8 and 1.5 relative to the amount of epoxide of formula (1), but it is of course also possible to use more CN^'. Preferably CN^" is dosed to the reaction in more than one (small) portion.

Depending on the reaction conditions (in particular the pH of the reaction medium, the concentration of Br^" and/or of CN^"), the enantiomerically enriched nitrile may be in its ring-closed form (the compound of formula (2)) and/or in its ring- opened form (the compound of formula (2a)). As is clear to the person skilled in the art, the compound of formula (2a), wherein R³ stands for Cl can only be formed if R¹ in the compound of formula (1 a) or of the compound of formula (1 ) stands for Cl₁ or if there is another Cl^" - source present in the reaction.

In the framework of the invention with the term 'enantiomerically enriched' is meant 'having an enantiomeric excess (e.e.) of either the (R)- or (S) - enantiomer of a compound'. Preferably, the enantiomeric excess is > 80%, more preferably > 85%, even more preferably > 90%, most preferably >92%. In the framework of the invention with 'mixture of enantiomers' is meant a random mixture of (R) and (S)-enantiomers. A mixture of enantiomers therefore includes a racemic mixture of two enantiomers as well as a mixture of enantiomers wherein one or the other enantiomer is in excess.

With 'enantioselectivity' of the haloalcohol dehalogenase is meant that the haloalcohol dehalogenase preferably catalyzes the cyanation of one of the enantiomers of the epoxide of formula (1) or of the ring-opened form thereof of formula (1a). The enantioselectivity of an enzyme may be expressed in terms of E-ratio, the ratio of the specificity constants V_maχ /K_m of the two enantiomers as described in C-S. Chen, Y Fujimoto, G. Girdaukas, C. J. Sih., J. Am. Chem. Soc. 1982, 104, 7294-7299. Preferably, the haloalcohol dehalogenase has an E-ratio > 5, more preferably an E- ratio > 10, even more preferably an E-ratio > 20, most preferably an E-ratio > 50. Preferably in the process of the invention, the enantiomer of the compound of formula (2) that is in excess is the compound represented by the compound of formula (II).

wherein R² stands for methyl or H, preferably for H; or the compound represented by the corresponding ring-opened form thereof: i.e. the compound of formula (Ma)

wherein R³ stands for Cl or Br and wherein R² is as defined above. Of course, depending on for which enantiomer of the compound of formula (1) the used haloalcohol dehalogenase is enantioselective, it is also possible with the process of the invention to prepare the enantiomer represented by the compound of formula (M')

wherein R² stands for H or methyl, preferably for H, or the ring opened form of the enantiomer of formula (M'), i.e. the compound of formula (Na')

wherein R² is as defined above and wherein R³ stands for Cl or Br, in excess.

In the framework of the invention, with haloalcohol dehalogenase is meant an enzyme having haloalcohol dehalogenase activity. Haloalcohol dehalogenase activity is defined as the ability to catalyze (i) the halogenation of epoxides and/or (ii) the dehalogenation of vicinal alcohols to epoxides. Haloalcohol dehalogenases are sometimes also referred to as halohydrin dehalogenases, halohydrin epoxidases, or halohydrin hydrogen-halide lyases.

The haloalcohol dehalogenases may be used in any form. For example, the haloalcohol dehalogenase may be used - in the form of a dispersion, emulsion, a solution or in immobilized form - as crude enzyme, as a commercially available enzyme, as an enzyme further purified from a commercially available preparation, as a haloalcohol dehalogenase obtained from its source by a combination of known purification methods, in whole (optionally permeabilized and/or immobilized) cells that naturally or through genetic modification possess haloalcohol dehalogenase activity, or in a lysate of cells with such activity etc. etc. Haloalcohol dehalogenase may for example be found in bacteria, for example in Agrobacterium sp., Pseudomonas sp., Alcaligenes sp. or Corynebacterium sp. The term haloalcohol dehalogenases includes, but is not limited to, the haloalcohol dehalogenases types HheA, HheB and HheC, for example HheA from Corynebacterium sp. strain N-1074 or HheA from Arthrobacter sp. strain AD2; HheB from Corynebacterium sp. N-1074 or HheB from Mycobacterium sp. strain GP1 ; HheC from Agrobacterium radiobacter AD1 , or HheC from A. tumefaciens (see for example van Hylckama Vlieg et al., 2001 , Journal of bacteriology, vol. 183, p 5058-5066).

Preferably, in the process of the present invention a haloalcohol dehalogenase type HheC, more preferably by a HheC that has at least a 95% identity, more preferably at least a 96% identity, even more preferably at least a 97% identity, most preferably at least a 98% identity, in particular at least a 99% identity with the amino acid sequence of the HheC from Agrobacterium radiobacter AD1 , is used. The amino acid sequence of HheC from Agrobacterium radiobacter AD1 and nucleic acid sequence coding therefore are disclosed in WO01 /90397. The % identity of two amino acid sequences may for example be determined by using ClustalW version 1.82

(http://www.ebi.ac.uk/clustalw) at default settings (matrix: Gonnet 250; GAP OPEN: 10; END GAPS: 10; GAP EXTENSION: 0.05; GAP DISTANCES: 8).

It will be clear to the person skilled in the art that use can also be made of mutants of naturally occurring (wild type) enzymes with haloalcohol dehalogenase activity in the process according to the invention. Mutants of wild-type enzymes can for example be made by modifying the DNA encoding the wild type enzymes using mutagenesis techniques known to the person skilled in the art (random mutagenesis, site-directed mutagenesis, directed evolution, gene shuffling, fusion proteins, for example a fusion protein of threonine aldolase and tyrosine decarboxylase; etc.) so that the DNA encodes an enzyme that differs by at least one amino acid from the wild type enzyme and by effecting the expression of the thus modified DNA in a suitable (host) cell. Mutants of the haloalcohol dehalogenase may have improved properties, for example with respect to selectivity for the substrate and/or activity and/or stability and/or solvent resistance and/or pH profile and/or temperature profile and/or enantioselectivity. Also, or alternatively, the DNA encoding the wild type enzyme may be modified in order to enhance the expression thereof. In a preferred embodiment of the invention, a mutant HheC from Agrobacterium radiobacter, more preferably from Agrobacterium radiobacter AD1 is used, more in particular the W249F mutant from HheC from Agrobacterium radiobacter, most in particular from Agrobacterium radiobacter AD1 , or a mutant HheC from a different microorganism source with an amino acid substitution at a position corresponding to the W249F position, is used. The preparation of the W249F mutant from HheC from Agrobacterium radiobacter AD1 is, for example, described in Tang LX, van Merode AEJ, Lutje Spelberg JH, Fraaije MW, Janssen DB BIOCHEMISTRY 42 (47): 14057-14065 DEC 2 2003.

Amino acid residues of amino acid sequences corresponding to position 249 of the amino acid residue in accordance with the HheC amino acid sequence from Agrobacterium radiobacter AD1 can for example be identified by performing ClustalW version 1.82 multiple sequence alignments at default settings as defined above. An amino acid residue that is placed in the same column as the amino acid residue 249 of the HheC of Agrobacterium radiobacter AD1 is an amino acid residue corresponding to that position.

The optimal temperature and pH of the process of the present invention depends on the haloalcohol dehalogenase chosen. The temperature is preferably chosen between 4 and 60°C, more preferably between 15 and 30⁰C. The pH is preferably chosen between 3 and 12, more preferably between 5 and 9. The pH may be kept constant to carry out the reaction at more or less constant pH, for example by using a buffer or automatic titration.

Suitable solvents for the process of the invention include: water, one phase mixtures of water and a water miscible organic solvent, for example alcohols miscible with water, - for example methanol- , dimethylsulfoxide, dimethylformamide, N- methylpyrrolidone, acetonitrile; or two-phase mixtures of water and a non-miscible organic solvent, for example hydrocarbons, ethers etc; or so-called ionic liquids like, for example, 1 ,3-dialkyl imidazolium salts or N-alkyl pyridinium salts of acids like hexafluorophosphoric acid, tetrafluoroboric acid, or trifluoromethane sulphonic acid, or with (CF₃SO₂^N as anionic counterpart. In case a two-phase mixture of water and a non-miscible organic solvent is used, concentrations of the epoxide of formula (1) may - depending on the choice of non-miscible organic solvent- even be higher than in case water or a one phase mixture of water and a water miscible organic solvent are used. In a special embodiment of the invention, the epoxide of formula (1 ) or the compound of formula (1a) is (by itself) the non-miscible organic solvent. Preferably, in the process of the invention a one-phase mixture of water and dimethylsulfoxide (DMSO) is used, for example water with a DMSO content between 1 and 50% v/v.

Also, it is possible to perform the process of the present invention in an emulsion system, such as macro- or micro-emulsions, bi-continuous systems comprising an organic phase, an aqueous phase and a suitable surfactant (non-ionic, cationic or anionic) and the like.

For purpose of the present invention, an emulsion system is defined as a ternary mixture of water, a surfactant and an oil phase, which may be an aliphatic alkane. Examples of aliphatic alkanes which may be used as oil phase in an emulsion include: cyclohexane, isooctane, tetradecane, hexadecane, octadecane, squalene. Surfactants can be any non-ionic, cationic or anionic surfactant, for example Triton X- 100, sodium dodecyl-sulfate, AOT, CTAB, Tween-80, Tween-20, Span-80 etc. An oil- in-water (O/W) emulsion may for instance be formed by intense mixing which leads to an increased internal surface and thus facilitates mass transfer between the phases. Especially interesting emulsions are microemulsions that are thermodynamically stable and have a domain size in the nanometer range (see for instance Clapes et a/., Chem. Eur. J. 2005, 11 , 1392-1401 and Schwuger et al., Chem. Rev. 1995, 95, 849-864.). The order of addition of CN^" and Br^" to the compound of formula (1) and the haloalcohol dehalogenase is in principle not critical. Preferably the addition of Br^" is done prior to the addition of CN^'.

The compound of formula (II) or the compound of formula (Ma) may be used in the preparation of statins, such as for example rosuvastatin or atorvastatin in a manner known per se. Typically the compound of formula (II) and/or of formula (Ma) is converted by hydrolysis and subsequent esterification into the corresponding carboxylic acid ester of formula (3)

(3) wherein R³ stands for a C₁-C₃ alkyl, preferably methyl or ethyl in a manner known per se. The compound of formula (3) may be subsequently converted into either rosuvastatin in a manner known per se or into atorvastatin, for example as described by A. Kleemann, J. Engel; pharmaceutical substances, synthesis, patents, applications 4th edition, 2001 Georg Thieme Verlag, p. 146-150.

Therefore, in another aspect, the invention relates to a process for the preparation of an active pharmaceutical ingredient, in particular of a statin comprising the process according to the invention. In other words, the invention relates to the use of the process of the invention in the preparation of an active pharmaceutical ingredient, in particular of a HMG-CoA reductase inhibitor, more in particular a statin, for example lovastatin, cerivastatin, rosuvastatin, simvastatin, pravastatin, atorvastatin or fluvastatin.

The invention will now be illustrated by way of the following examples, without however being limited thereto.

Examples

Example 1 : Preparation of Haloalcohol dehalogenases

HheC from Agrobacterium radiobacter AD1 was cloned, expressed and purified according to van Hylckama Vlieg et al., 2001 , Journal of bacteriology, vol. 183, p 5058-5066. The HheC mutant (W249F) from Agrobacterium radiobacter AD1 was cloned according to Tang, L. et al. 2003, Biochemistry 42:14057-14065 (Steady- state kinetics and tryptophan fluorescence properties of halohydrin dehalogenase from Agrobacterium radiobacter. Roles of W139 and W249 in the active site and halide- induced conformational change). Expression and purification thereof was done according to Tang, L. et al. 2005, Biochemistry 44(17): 6609-6618. HheB from

Mycobacterium sp. Strain GP1 was cloned, expressed and purified according to Vlieg et al. 2001 , Journal of Bacteriology 182(17): 5058-5066.

Example 2: Demonstration of in situ racemization To prove the dynamic kinetic resolution concept to enantiomerically enriched (S)- Oxiranyl-acetonitrile, 5 mM of epichlorohydrin (A) or 5mM epibromohydrin (B) was treated with the haloalcohol dehalogenases HheB, HheC or the mutant haloalcohol dehalogenase HheC(W249F) (300-500 μl of purified enzyme in the concentration range of between 2-5 mg/ml) in the presence of 10 mM NaCN and 10 mM NaCI (in the reaction with epichlorohydrin) or 1OmM NaBr (in the reaction with epibromohydrin), respectively. Enzyme incubations were performed in 30 ml bottles at 22 ⁰C and the pH (7.5) was controlled by a 1 M Tris SO₄ buffer. At regular time intervals 1 ml samples were withdrawn from the reaction mixtures. The sample was extracted with diethylether after which the ether layers were analyzed by Chiral GC on a 30 m Chiraldex G-TA column at 130 ⁰C. Results of this example are given in Table 1 below.

Table !

Initial rate in μmol.min^' .mg^" protein

² Estimation based on initial e.e. value of the formed product ³ Estimation based on the e.e. value of remaining substrate

As can be seen from table 1 , the E-value of the HheC(W249F) mutant is the highest and the lowest for HheB (for both epichlorohydrin and epibromohydrin). Hence with the HheC(W249F) mutant the highest e.e. for the target product can be obtained.

As can also be seen from table 1 , racemization proceeds better in the reaction with epibromohydrin and Br^" than in the reaction with epichlorohydrin and Cl^' (from ++ to +++ for HheB, from n.d. (not detectable) to +/- for HheC and from +/- to + for the HheC(W249F) mutant. If the racemization of the epihalohydhn occurs faster than the epoxide ring opening by CN^" (dynamic kinetic resolution) one would expect that the e.e. of the desired product is about equal to the theoretical maximal e.e. of the desired product.

For example, in the reaction with HheC(W249F) with CN^' and epibromohydrin in the presence of NaBr₁ the desired (S)-Oxiranyl-acetonitrile product (i.e. the compound of formula (II) and/or the ring-opened form thereof: the compound of formula (Ma)) was obtained in 92% enantiomeric excess and >90% yield. The E-value of the HheC(W249F) mutant is 23, which corresponds to a theoretical maximal e.e. of the desired product of 92%. This e.e. was in fact obtained and hence a very good dynamic kinetic resolution occurs with the process of the present invention.

In other words, the reaction of epibromohydrin with CN^" in the presence of an enantioselective haloalcohol dehalogenase^" leads to the enantiomerically enriched nitrile in a high e.e. and in a high yield.

Example 3: Demonstration of Dynamic Kinetic Resolution of epichlorohvdrin

In an alternative experimental setup, in a 1 ml GC vial, 3 μM 90% pure HheC(W249F) was incubated at pH 7.5 (controlled by a 200 mM TrJs-SO₄ buffer) and 22°C with 5 mM epichlorohydrin and 10 mM NaCN in the presence of 10 and 25 mM NaBr. A comparative example was done in exactly the same way, except that 10 mM NaCI was used instead of NaBr.

At regular time intervals 25 microliter samples were withdrawn from the reaction mixtures, and dissolved in 1.5 ml diethyl ether. Finally, the samples were analyzed by Chiral GC on a 30 m Chiraldex G-TA column at 13O⁰C. The results of this example are given in tables 2 and 3 below.

Table 2: Results of the dynamic kinetic resolution of epichlorohydrin (A) with HheC(W249F) in the presence of NaBr and NaCI.

1O mM 25 mM 1O mM

NaBr NaBr NaCI

Initial activity¹ 1.4 U 10 U 3.6 U

Final e.e. of (S) oxiranyl

86% 91% not formed acetonitrile

Final e.e. of 4-chloro-3-

91% (± 3%) 91% (± 3%) < 80%² hydroxy-butyronitrile

¹ Initial rate in μmol.min^"1.mg^"1 protein

² At 50% conversion

Table 3: Time course of enantiomeric excess of the remaining epichlorohydrin in the different reaction mixtures with HheC(W249F).

In the reaction with 1OmM NaCI, the enantiomeric excess of the remaining epichlorohydrin steadily increased in time (see table 3, and finally to >99 % at 62 % conversion). In the case of complete racemization, the e.e. of the remaining epichlorohydrin would be 0. An increase of the e.e. of the remaining epichlorohydrin is an indication that racemization is not optimal or even absent. Therefore, the increase in e.e. of the remaining epichlorohydrin in the reaction with 1OmM NaCI indicates that (almost) no racemization occurred when NaCI was present. Furthermore, also the e.e. of the formed 4-chloro-3-hydroxy-butyronitrile was lower than the theoretical maximal value of 90% (an e.e. of 80% at 50% conversion see table 2). Hence the results show that the reaction of epichlorohydrin in the rpesence of NaCI is predominantly a kinetic resolution and not a dynamic kinetic resolution. Addition of 10 mM NaBr led to a reduced increase in enantiomeric excess of the remaining epichlorohydrin (to 71 % at 86 % conversion) as compared to the reaction with 1OmMNaCI pointing to the occurrence of racemization of the remaining epichlorohydrin substrate. In this case, the desired product (S)-Oxiranyl- acetonitrile was formed in an enantiomeric excess of 86%, which is higher than the e.e. obtained in the reaction with 1OmM NaCI.

Use of a higher NaBr concentration (25mM) led to an even more reduced increase in enantiomeric excess of the remaining epichlorohydrin (25% throughout the reaction), indicating an even better racemization than with 1OmM NaBr. Furthermore, also the e.e. of the desired product (S)-Oxiranyl-acetonitrile (91 %, see table 2) was higher than in the reaction with 10 mM NaBr, also indicating that racemization with 25mM NaBr proceeded better than with 1OmM NaBr. Therefore, these results show that in the reaction of epichlorohydrin in the presence of Br^' a dynamic kinetic resolution occurs. To sum up, the presence of Br^" in the reaction of epichlorohydrin and

CN^" with an enantioselective haloalcohol dehalogenase leads to the desired product (in this case (S)-Oxiranyl-acetonitrile and/or one of the corresponding ring-opened forms thereof) in a high e.e. in a high yield.

Example 4: Dynamic Kinetic Resolution of epichlorohydrin at high substrate concentration

To a GC vial containing a solution of 112 mM epichlorohydrin, 28 mM NaBr and 12.4 μM haloalcohol dehalogenase HheC(W249F) (90% pure) in 1 M Tris- SO₄ buffer of pH 7.3, NaCN was added in 4 equal portions from a 3M stock solution to a final concentration of 136 mM. These portions were added to the reaction mixture 40, 90, 190 and 310 minutes after the start of the reaction. The reaction was carried out at 22 ⁰C. Samples (25 microliter) were withdrawn from the reaction mixtures at regular time intervals and dissolved in 1.5 ml diethyl ether, after which the samples were analyzed by Chiral GC on a 30 m Chiraldex G-TA column at 130⁰C. In this example, the desired product (S)-Oxiranyl-acetonitrile (i.e. the compound of formula (II) and/or the ring-opened form thereof: the compound of formula (Ma)) was obtained in 90% enantiomeric excess at 90 % conversion based on the remaining substrate. This is close to the maximum e.e. in case of an optimal dynamic kinetic resolution by an enzyme with an E-value of 23 (which is 92% as mentioned in example 2). This result proves that the dynamic kinetic resolution concept is also feasible at high epichlorohydrin concentrations.

Claims

1. Process comprising the step of reacting a mixture of enantiomers of the epoxide of formula (1 )

wherein R¹ stands for Cl or for Br, and wherein R² stands for H or for methyl, and/or a mixture of enantiomers of the corresponding ring-opened form thereof of formula (1a)

, wherein R¹ and R² are as defined above and wherein R⁴ independently stands for Cl or Br, with CN^' and Br^' in the presence of an enantioselective haloalcohol dehalogenase to form the corresponding enantiomerically enriched compound of formula (2)

wherein R² is defined above and/or the corresponding enantiomerically enriched ring-opened form thereof of formula (2a)

wherein R³ stands for Cl or Br and wherein R² is as defined above. 2. Process according to claim 1 , wherein R¹ stands for Cl and wherein R² stands for H.

Process according to claim 1 or claim 2, wherein the compound of formula (2) has an excess of the enantiomer of formula (II)

wherein R² is as defined above and/or wherein the compound of formula (2a) has an excess of the enantiomer of formula (Ua)

wherein R³ and R² are as defined above.

4. Process according to any one of claims 1-3, wherein the haloalcohol dehalogenase is HheC from Agrobacterium radiobacter, preferably from

Agrobacterium radiobacter AD 1.

5. Process according to any one of claims 1-3, wherein the haloalcohol dehalogenase is the W249F mutant from HheC from Agrobacterium radiobacter, preferably from Agrobacterium radiobacter AD1 6. Process for the preparation of an active pharmaceutical ingredient comprising the process according to any one of claims 1-5.

7. Use of a process according to any one of claims 1-5 in the preparation of an active pharmaceutical ingredient, preferably of a statin, in particular of atorvastatin or rosuvastatin