WO2008038050A2

WO2008038050A2 - Reduction of alpha-halo ketones

Info

Publication number: WO2008038050A2
Application number: PCT/GB2007/050590
Authority: WO
Inventors: Jonathan Wiffen; Brian Adger
Original assignee: Almac Sciences Limited
Priority date: 2006-09-29
Filing date: 2007-09-28
Publication date: 2008-04-03
Also published as: WO2008038050A3; GB0619240D0

Abstract

The present invention relates to a method for reducing alpha-halo ketones to alpha-halo alcohols. More specifically, the present invention is concerned with the use of isolated enzymes for reduction of alpha-halo ketones to corresponding alpha-halo alcohols. The alpha-halo alcohol is obtained in good yield from the corresponding ketone in a stereoselective manner. The reaction is conducted in the presence of an isolated enzyme or a resting cell.

Description

REDUCTION OF ALPHA-HALO KETONES

The present invention relates to a method for reducing alpha-halo ketones to alpha-halo alcohols. More specifically, the present invention is concerned with the use of isolated enzymes for reduction of alpha-halo ketones to corresponding alpha-halo alcohols.

There are numerous examples in the patent and non-patent literature of disclosures relating to the stereoselective reduction of alpha-chloroketones to the corresponding alpha-chloroalcohols. Conventional methods involve the use of inorganic reducing agents to reduce the ketone functionality. Where stereochemical control is required asymmetric catalysts and/ or asymmetric reducing agents may be used in such reactions. For example, the reaction may be carried out as a transfer hydrogenation reaction using a chiral metal catalyst and several methods using known transfer hydrogenation catalysts are reported.

More recently, workers have turned to enzymatic and fermentation processes in order to obtain enantiomerically or diastereomerically enriched products from reductions of this type. The major advantage of whole living cells relative to conventional chemical reagents is that the cells can regenerate their own co- factors. In addition, whole cells are frequently easy to produce and handle and are of relatively low cost. The drive towards increasing purity and yield, together with the need to use environmentally less damaging materials has also encouraged the use of whole living cells. However one disadvantage in using whole cells is that the volume efficiency of the reaction may be low. The reactions also frequently produce low yields. Furthermore, whilst there are examples showing good selectivity, the selectivity is not always good. In many cases, the selectivity is not sufficiently good for the intended purpose of the end products, particularly those that are going to be used in pharmaceutical or food applications. This means that further work up and purification procedures are required. There is therefore a need for a process which can provide an alpha-haloalcohol in good yield from the corresponding ketone. It is an aim to do this in a stereoselective manner. Desirably, such a reaction will have a good volume efficiency. It is also an aim of the present invention to provide a reaction in which the selectivity is strongly directed towards one or other enantiomer.

According to the present invention, there is provided a process for producing a compound of formula of I

(I)

by reduction of a compound of formula

in the presence of an isolated enzyme or a resting cell,

wherein

R¹ and R² are independently selected from the group comprising: H; Ci_-7 alkyl;

C_{2 7} alkenyl; C_{2 7} alkynyl; aryl; Ci_-7 alkylaryl; C_{2 7} alkenylaryl; het; Ci_-7 alkylhet; and ZR or ZP where Z is NH and R is Ci_-7 alkyl, C_{2 7} alkenyl, C₂.₇ alkynyl, aryl,

Ci_-7 alkylaryl, C₂.₇ alkenylaryl and P is a protecting group; wherein each of the foregoing groups may where chemically possible be optionally substituted by 1 to 4 substituents independently selected from the group comprising: Ci_-7alkyl, Ci- ₇haloalkyl, -SR⁵, -OR⁶, -NR⁶R⁶, -NO₂, SCF₃, halogen, -C(O)R⁷, -CN, and -CF₃;

each of R³ to R⁶ is independently selected from the group comprising: H, Ci_-7 alkyl, C₂-₇ alkenyl, C₂-₇ alkynyl, -CF₃, -CH₂F, -CHF₂, CH₂CF₃, CH₂OCi_₇ alkyl, CH₂SCi-₇ alkyl, phenyl, benzyl, and 2-phenethyl, wherein each of phenyl, benzyl, or 2-phenethyl may be optionally substituted by 1 to 3 substituents independently selected from the group comprising: d-₇alkyl, -SR⁵, -OR⁶, -NR⁶R⁶, -NO₂, SCF₃, halogen, -C(O)R⁷, -CN, and -CF₃; and / or independently any two of the R² to R¹³ groups joined to the same carbon, may together with the carbon to which they are attached form a ring of up to 6 members which contains from O to 2 heteroatoms chosen from O, S, and N the ring being optionally substituted with from 1 to 3 halo atoms;

het is selected from the group comprising: C-linked pyrrolyl, imidazolyl, triazolyl, thienyl, furyl, thiazolyl, oxazolyl, thiadiazolyl, oxadiazolyl, pyridinyl, pyrimidinyl, pyridazinyl, pyrazinyl, quinolinyl, isoquinolinyl, benzimidazolyl, quinazolinyl, phthalazinyl, benzoxazolyl and quinoxalinyl;

X is a leaving group;

m is from O to 5; and

n is from O to 5.

Preferably X is halo or sulphonate. Halo means fluoro, chloro, bromo, or iodo. More preferably X is halo. Preferably, X is chloro or bromo. Most preferably, X is chloro.

Preferably, the method uses an isolated enzyme. The invention also relates to novel compounds of Formula (I) whether produced by the process of the invention or otherwise. These compounds can most easily be produced by the process of the invention. Preferred novel compounds are those produced by the preferred embodiments described below.

In a preferred embodiment, R¹ is selected from the group comprising: Ci_-7 alkyl; Ci_-7 alkylaryl; and ZR or ZP where Z is NH and R is Ci_-7 alkyl, C_{2 7} alkenyl, C_{2 7} alkynyl, Ci_-7 alkylaryl and P is a protecting group; wherein each of the foregoing groups may where chemically possible be optionally substituted by 1 to 3 substituents independently selected from the group comprising: Ci_-7alkyl, Ci- ₇haloalkyl, -SR⁵, -OR⁶, -NR⁶R⁶, -NO₂, SCF₃, halogen, -C(O)R⁷, -CN, and -CF₃

In a further preferred embodiment, R¹ is selected from the group comprising: ZR or ZP.

In a further embodiment P is a benzyl or trityl group.

In another embodiment, R² is selected from the group comprising: Ci_-7 alkyl; aryl; Ci_-7 alkylaryl; het; and Ci_-7 alkylhet, wherein each of the foregoing groups may where chemically possible be optionally substituted by 1 to 4 substituents independently selected from the group comprising: Ci_-7alkyl, Ci-₇haloalkyl, -SR⁵, - OR⁶, -NR⁶R⁶, -NO₂, SCF₃, halogen, -C(O)R⁷, -CN, and -CF₃.

In a further embodiment, R² is selected from the group comprising: aryl and het. Most preferably R² is aryl.

Aryl includes any aromatic carbocyclic ring or ring system comprising one or more rings which may be fused, conjugated or isolated from one another and containing up to 24 carbon atoms in the ring system skeleton. Aryl thus includes systems such as phenyl, naphthyl, anthracyl, bisphenyl, phenanthryl, and indenyl. Most preferably, aryl is optionally substituted phenyl. In another embodiment R³ is selected from the group comprising: H, and optionally substituted Ci_-7 alkyl. Preferably R³ is H.

In another embodiment R⁴ is selected from the group comprising: H, and optionally substituted Ci-₇ alkyl. Preferably R⁴ is H.

In another embodiment R⁵ is selected from the group comprising: H, and optionally substituted Ci_-7 alkyl. Preferably R⁵ is H.

In another embodiment R⁶ is selected from the group comprising: H, and optionally substituted Ci_-7 alkyl. Preferably R⁶ is H.

Preferably m is 1.

Preferably n is 0.

The isolated enzymes are ketone reductase enzymes and are commercially available from Biocatalytics lnc of 129 N. Hill Avenue, Suite 103, Pasadena, CA 91106 USA (web address www.biocatalytics.com).

In a particularly preferred embodiment, the alpha-halo ketone is an alpha-chloro ketone derived from phenyl alanine

which is reduced to the corresponding alpha-chloro alcohol

The amino group is protected by a suitable protecting group. Suitable protecting groups include benzyloxycarbonyl and triphenylmethyl (trityl) group. Other suitable protecting groups are known in the art.

It will be apparent to those skilled in the art that in general, sensitive functional groups may need to be protected and deprotected during synthesis of a compound of the invention. This may be achieved by conventional methods.

The present invention also relates to the use of an isolated enzyme for the reduction of an alpha-halo ketone. The use of an isolated enzyme for the reduction of an alpha-halo ketone derived from phenyl alanine is of particular interest.

The enantioselective reduction of alpha-halo ketones is of particular interest because it provides valuable intermediates that can be produced as chiral building blocks in organic synthesis. Although the bioreduction of alpha-halo ketones has been described previously, to date this has been done using whole cell systems.

A further aspect of the invention relates to entiomeric or diastereomeric enrichment of an alpha-halo ketone of formula I. This is achieved by the use of a hydrolase enzyme derived from a resting cell. We have found that the treatment of alpha-halo alcohols with hydrolase provides up to 99% enantiomeric or diastereomeric excess, the latter being the case in which a new chiral centre is created in a molecule which is already optically active.

Surprisingly, the bioreduction works on both crude and purified chloroketone. This is another advantageous feature of the KRED reductions of the present invention because conventional asymmetric reductions (including transition metal catalysed hydrogenations) need to use purified chloroketone. We have shown that crude chloroketone (81.9% purity) can be reduced using a KRED to give the (S₁R) chloroalcohol in upto 40% conversion; other KREDs can reduce this crude chloroketone to produce the (S₁S) chloroalcohol in > 96% ee with up to 50% conversion. Thus a further aspect of the invention relates to a process for the entiomeric or diastereomeric enrichment of an alpha-halo ketone as defined above in Formula I using a hydrolase enzyme derived from a resting cell. In one embodiment, the enrichment is achieved in greater than 80% ee or de, and more preferably greater than 90% ee or de.

The present invention discloses the use of isolated ketoreductase enzymes for the diastereoselective reduction of certain chloroketone substrates, as outlined elsewhere in the application. Such enzymes are typically produced by a process that starts with the fermentation of a genetically manipulated host microorganism that overexpresses the ketoreductase. Following fermentative production of the enzyme by the host, the enzyme is retained either within the cell (in soluble form or as inclusion bodies) or transported into the external medium, and is isolated by commonly practised downstream processing techniques.

For a soluble enzyme that is retained in the cell (which may be in a recombinant form or a native wild-type enzyme) it is usual to recover the cell biomass first by centrifugation, then to resuspend the cells in an appropriate buffer solution. The cells are then broken open to release the enzyme- this may be done by mechanical or chemical means, then the cell debris is removed, again by centrifugation or by addition of a flocculant followed by filtration or centrifugation.

The resulting cell-free extract may be further purified (for example by salt precipitation techniques or by chromatography), or it may be used as directly recovered in a semi-purified form. The enzyme may be concentrated at this stage, for example by ultrafiltration, or it may be processed into a powder form, for example by lyophilisation. It will be understood by those skilled in the art that this invention could be adapted to use other less pure forms of the enzyme, such as the whole-cells themselves. However, the use of isolated enzymes is preferred in accordance with the invention.

The biocatalyst may also exist as a whole cell-containing preparation that has been deliberately or otherwise treated to cause release of the enzymes from the cells. In some cases the use of solvents or high concentrations of the substrate itself can cause the cells to leak their contents into the surrounding medium.

Such forms of the biocatalyst are equally applicable and useful as the lyophilised enzymes that are used in this application.

The present invention will now be illustrated by the following examples presented in Tables 1 and 2 below. These show the results of various isolated enzymes on a phenylalanine derived haloketone.

General experimental procedure for enzymatic reductions

A solution of chloroketone (60mg) in dimethylsulfoxide was added to a mixture of buffer (1mL) and NADPH. A small amount of enzyme was added; the vial was sealed and stirred overnight at room temperature. Ethyl acetate (1 ml_) was added to the vial and the organic layer was separated, filtered through cotton wool and dried (MgSO₄). Samples were analysed by HPLC (Phenomenex Luna 5μ silica), eluent 2% IPA in hexane.

Tables 1 and 2 Reductase results

Experimental for enzymatic reduction of crude Boc-chloroketone

0.1 M Potassium phosphate (monobasic) buffer (19ml) containing 2mM magnesium sulphate was charged to a reactor. Boc-Chloroketone (0.5g, 81.9% purity) was added followed by isopropanol (4ml) and toluene (1 ml). NAD (20mg) and KRED-EXP-C1 E (10mg) were then added and the reaction mixture stirred at 20 -25⁰C for 74 hours. Typically a conversion of 92-98.5% and % de was in excess of 99%.

For comparative purposes, Table 3 shows the results of conventional hydrogenation reactions such as a transfer hydrogenation performed using known transfer hydrogenation catalysts and a sodium borohydride reduction.

R = BOC, Cbz

Table 3 Transfer Hydrogenation results Experimental for asymmetric transfer hydrogenations

IPA as hydrogen source: Potassium hydroxide (4 mg) and Ru(mesιtylene)(TSDPEN) catalyst

CATHy is a registered trademark of Avecia Limited and NPIL Pharmaceuticals Limited and refers to a transfer hydrogenation catalyst produced and sold by those companies.

Experimental for asymmetric transfer hydrogenations

IPA as hydrogen source: Potassium hydroxide (4 mg) and Ru(mesitylene)(TSDPEN) catalyst (4mg) was added to a solution of chloroketone (200mg) in isopropanol under nitrogen atmosphere then heated at 6O⁰C for 60hrs. Reaction was diluted with water (1OmL), extracted into ethyl acetate (2x 2OmL), dried (Na₂SO₄) and concentrated.

TEAF as hydrogen source: Triethylammonium formate (0.4 mL) was added to a solution of chloroketone (200mg) in degassed ethyl acetate (2mL) followed by addition of Ru(mesitylene)(TSDPEN) catalyst (4mg) then stirred at 3O⁰C for 60hrs. Reaction was diluted with water (1OmL), extracted into ethyl acetate (2x 2OmL), dried (Na₂SO₄) and concentrated.

In another aspect of the invention, the enantiomeric excess of the haloalcohol product is enhanced by lipase catalysed transesterification

Enhancement of enantiomeric excess by lipase catalysed transesterification

General procedure.

The enantioenriched alcohol (2.5g, 7.8mmol, typically an 85:15 to 90:10 mixture of diastereomers, but can be lower) was dissolved in methyl t-butylether (50ml) with stirring, and one equivalent of acyl donor (7.8 mmol) added. Vinyl acetate, vinyl butyrate, vinyl stearate and succinic anhydride were used as acyl donors. Lipase (0.625g, AE07, Mann-Associates, Cambridge, UK) was added and the heterogeneous mixture stirred at ambient temperature. After 48 hrs the reactions were sampled. The enzyme was removed by filtration, and the filtrate diluted one in four into hexane. The solutions were analysed by HPLC. (Phenomenex Luna 5um silica column, 15cm length, 4.6mm internal diameter, eluting with 2% isopropanol in hexane, 1 ml/min, monitoring at 254nm, retention time of major diastereomer 8.9 mins, minor diastereomer 11.4 mins). The results are tabulated below, given as % of each isomer present as found by HPLC analysis.

The process of the invention has utility in preparing intermediate compounds which in turn can be used in the preparation of a number of pharmaceutically active compounds as well as other materials. Examples of actives which can be prepared from intermediates produced using the present invention include amprenavir, atazanavir and fosamprenavir.

In the case of amprenavir, the invention finds utility in the second of the following sequence of steps from compound XVIII to XIX in the process to produce amprenavir:

BOCHN ..C!

XVi^ XVII XCK

In the case of fosamprenavir, the invention finds utility in the step from compound XX to XXI in the scheme below:

o O

B o cH N N₂ BocHN .Cl

HCI

XIX XX

QH BocHN BocHN . ,P.

XXI IV The skilled man will appreciate that adaptation of the methods herein described and/or adaptation of methods known in the art could be applied to the processes of the present invention.

For example, the skilled person will be immediately familiar with standard textbooks such as "Comprehensive Organic Transformations - A Guide to Functional Group Transformations", RC Larock, Wiley-VCH (1999 or later editions), "March's Advanced Organic Chemistry - Reactions, Mechanisms and Structure", MB Smith, J. March, Wiley, (5th edition or later) "Advanced Organic Chemistry, Part B, Reactions and Synthesis", FA Carey, RJ Sundberg, Kluwer Academic/Plenum Publications, (2001 or later editions), "Organic Synthesis - The Disconnection Approach", S Warren (Wiley), (1982 or later editions), "Designing Organic Syntheses" S Warren (Wiley) (1983 or later editions), "Guidebook To Organic Synthesis" RK Mackie and DM Smith (Longman) (1982 or later editions), etc., and the references therein as a guide.

It is to be understood that the synthetic transformation methods mentioned herein are exemplary only and they may be carried out in various different sequences in order that the desired compounds can be efficiently assembled. The skilled chemist will exercise his judgement and skill as to the most efficient sequence of reactions for synthesis of a given target compound and will employ protecting groups as necessary. This will depend inter alia on factors such as the nature of other functional groups present in a particular substrate. Clearly, the type of chemistry involved will influence the choice of reagent that is used in the said synthetic steps, the need, and type, of protecting groups that are employed, and the sequence for accomplishing the protection / deprotection steps. These and other reaction parameters will be evident to the skilled person by reference to standard textbooks and to the examples provided herein.

Sensitive functional groups may need to be protected and deprotected during synthesis of a compound of the invention. This may be achieved by conventional methods, for example as described in "Protective Groups in Organic Synthesis" by TW Greene and PGM Wuts, John Wiley & Sons lnc (1999), and references therein.

The important features of the invention can be summarised as follows:

1. A process for reducing an alpha-halo ketone in the presence of an isolated enzyme or resting cell.

The isolated enzyme is capable of catalyzing the reduction of a ketone. Preferably, the isolated enzyme is a ketoreductase.

The process can be conducted in the presence of a reducing agent. The reducing agent may be NADPH or NADP.

The ketoreductase may be an NADPH dependent ketoreductase. The ketoreductase may also be an NADH dependent ketoreductase.

2. Improvements in methodology for bioreduction using isolated enzyme or whole cells of a prochiral ketone of typical formula

Where X = leaving group e.g. sulfonate, halogen preferably Cl or Br P = protecting group, preferably carbamate. R = alkyl, substituted alkyl, preferably -CH₂Ph. The alkyl group may have from 1 to 10 carbon atoms and may be optionally substituted by one or more groups such as phenyl.

3. Purity and quality enhancement by hydrolase resolution of the resulting alcohol product as shown by schemes below. P is a protecting group and X is a halo group. R is an alkyl group which may have from 1 to 10 carbon atoms and which may be optionally substituted by one or more groups such as phenyl. Suitable types of hydrolase which can be used include lipase, protease and aminoacylase.

The Schemes above are only indicative and any stereoisomer can be isolated as the alcohol or as the acylated product.

4. Compounds of formula

where

R¹ is selected from the group comprising: H; Ci_-7 alkyl; C_{2 7} alkenyl; C_{2 7} alkynyl; aryl; Ci_-7 alkylaryl; C_{2 7} alkenylaryl; het; and Ci_-7 alkylhet;

R² is selected from the group comprising: H; Ci_-7 alkyl; C_{2 7} alkenyl; C₂.₇ alkynyl; aryl; Ci_-7 alkylaryl; C₂.₇ alkenylaryl; het; and Ci_-7 alkylhet; wherein each of the foregoing groups defined for R¹ and R² may where chemically possible be optionally substituted by 1 to 4 substituents independently selected from the group comprising: Ci_-7alkyl, Ci-₇haloalkyl, -SR⁵, - OR⁶, -NR⁶R⁶, -NO₂, SCF₃, halogen, -C(O)R⁷, -CN, and -CF₃; and P is a protecting group.

Preferably, R¹ is optionally substituted Ci_-7 alkyl, and more prefereably it is Me.

Preferably ,R² is optionally substituted Ci_-7 alkyl; aryl; or Ci_-7 alkylaryl, and more preferably it is Ci_-7 alkylaryl. Most preferably it is benzyl.

P is preferably a benzyl or BOC group.

Compounds of particular interest are compounds of formula:

4. Catalytic transfer hydrogenation of compounds in paragraph 1 above using Ru(mesitylene) (S,S) or (R₁R)TSDPEN complex.

Claims

1 A process for producing a compound of formula of I

by reduction of a compound of formula

in the presence of an isolated enzyme or a resting cell,

wherein

R¹ and R² are independently selected from the group comprising: H; Ci_-7 alkyl; C₂-7 alkenyl; C2-7 alkynyl; aryl; Ci_-7 alkylaryl; C₂-₇ alkenylaryl; het; Ci_-7 alkylhet; and ZR or ZP where Z is NH and R is Ci_-7 alkyl, C_{2 7} alkenyl, C_{2 7} alkynyl, aryl, Ci_-7 alkylaryl, C_{2 7} alkenylaryl and P is a protecting group; wherein each of the foregoing groups may where chemically possible be optionally substituted by 1 to 4 substituents independently selected from the group comprising: Ci_-7alkyl, Ci- ₇haloalkyl, -SR⁵, -OR⁶, -NR⁶R⁶, -NO₂, SCF₃, halogen, -C(O)R⁷, -CN, and -CF₃;

each of R³ to R⁶ is independently selected from the group comprising: H, Ci_-7 alkyl, C₂-₇ alkenyl, C₂-₇ alkynyl, -CF₃, -CH₂F, -CHF₂, CH₂CF₃, CH₂OC_L7 alkyl, CH₂SCi-₇ alkyl, phenyl, benzyl, and 2-phenethyl, wherein each of phenyl, benzyl, or 2-phenethyl may be optionally substituted by 1 to 3 substituents independently selected from the group comprising: d-₇alkyl, -SR⁵, -OR⁶, -NR⁶R⁶, -NO₂, SCF₃, halogen, -C(O)R⁷, -CN, and -CF₃; and / or independently any two of the R² to R¹³ groups joined to the same carbon, may together with the carbon to which they are attached form a ring of up to 6 members which contains from 0 to 2 heteroatoms chosen from O, S, and N the ring being optionally substituted with from 1 to 3 halo atoms;

X is a leaving group;

m is from 0 to 5; and

n is from 0 to 5.

2. A process as claimed in claim 1 wherein, X is halo or sulphonate.

3. A process as claimed in claim 2, wherein, X is halo.

4. A process as claimed in any preceding claim, wherein the method uses an isolated enzyme.

5. A process as claimed in claim 4, wherein the isolated enzyme is a ketone reductase enzyme.

6. A process as claimed in any preceding claim, wherein R¹ is selected from the group comprising: Ci_-7 alkyl; Ci_-7 alkylaryl; and ZR or ZP where Z is NH and R is Ci_-7 alkyl, C_{2 7} alkenyl, C_{2 7} alkynyl, Ci_-7 alkylaryl and P is a protecting group; wherein each of the foregoing groups may where chemically possible be optionally substituted by 1 to 3 substituents independently selected from the group comprising: d-₇alkyl, Ci_-7haloalkyl, -SR⁵, -OR⁶, -NR⁶R⁶, -NO₂, SCF₃, halogen, -C(O)R⁷, -CN, and -CF₃ Preferably, R¹ is selected from the group comprising: ZR or ZP.

7. A process as claimed in any preceding claim, wherein P is a benzyl or trityl group.

8. A process as claimed in any preceding claim, wherein R² is selected from the group comprising: Ci_-7 alkyl; aryl; Ci_-7 alkylaryl; het; and Ci_-7 alkylhet, wherein each of the foregoing groups may where chemically possible be optionally substituted by 1 to 4 substituents independently selected from the group comprising: Ci_-7alkyl, Ci_-7haloalkyl, -SR⁵, -OR⁶, -NR⁶R⁶, -NO₂, SCF₃, halogen, - C(O)R⁷, -CN, and -CF₃. Preferably, R² is selected from the group comprising: aryl and het.

9. A process as claimed in any preceding claim, wherein R³ is selected from the group comprising: H, and optionally substituted Ci_-7 alkyl. Preferably R³ is H.

10. A process as claimed in any preceding claim, wherein R⁴ is selected from the group comprising: H, and optionally substituted Ci_-7 alkyl. Preferably R⁴ is H.

11. A process as claimed in any preceding claim, wherein R⁵ is selected from the group comprising: H, and optionally substituted Ci_-7 alkyl. Preferably R⁵ is H.

12. A process as claimed in any preceding claim, wherein R⁶ is selected from the group comprising: H, and optionally substituted Ci_-7 alkyl. Preferably R⁶ is H.

13. A process as claimed in any preceding claim, wherein m is 1.

14. A process as claimed in any preceding claim, wherein n is 0.

15. A compound of formula

wherein R¹ is selected from the group comprising: H; Ci_-7 alkyl; C₂.₇ alkenyl; C₂.₇ alkynyl; aryl; Ci_-7 alkylaryl; C_{2 7} alkenylaryl; het; and Ci_-7 alkylhet;

R² is selected from the group comprising: H; Ci_-7 alkyl; C₂.₇ alkenyl; C₂.₇ alkynyl; aryl; Ci_-7 alkylaryl; C_{2 7} alkenylaryl; het; and Ci_-7 alkylhet; wherein each of the foregoing groups defined for R¹ and R² may where chemically possible be optionally substituted by 1 to 4 substituents independently selected from the group comprising: Ci_-7alkyl, Ci-₇haloalkyl, -SR⁵,

OR⁶, -NR⁶R⁶, -NO₂, SCF₃, halogen, -C(O)R⁷, -CN, and -CF₃; and

P is a protecting group.

16. A compound as claimed in claim 15, wherein R¹ is optionally substituted Ci_-7 alkyl. More prefereably it is Me.

17. A compound as claimed in claim 15 or 16, wherein R² is optionally substituted Ci_-7 alkyl; aryl; or Ci_-7 alkylaryl. More preferably it is Ci_-7 alkylaryl. Most preferably it is benzyl.

18. A compound as claimed in any of claims 15, 16, or 17 wherein P is a benzyl or BOC group.

19. A compound of formula:

20. Thus a further aspect of the invention relates to a process for the entiomeric or diastereomeric enrichment of an alpha-halo ketone as defined above in Formula I using a hydrolase enzyme derived from a resting cell.