The invention concerns a process for the preparation of alpha-substituted carboxylic acids or alpha-substituted carboxylic acid amides, particularly alpha-tri- substituted carboxylic acids or alpha-tri-substituted carboxylic acid amides, especially alpha-disubstituted-alpha-aminoacids or alpha-disubstituted-alpha-aminoacid amides in resolved or partially resolved form. There is an interest in obtaining cheap, readily available, synthetic sources of simple chiral building blocks for use as intermediates and actives in the food, agrochemical and pharmaceutical industries. While many alpha-substituted carboxylic acids and derivatives are readily available or can be cheaply synthesised in racemic form, there is a need for industrial scale synthetic procedures for obtaining the resolved or partially resolved forms of these useful building blocks. One approach is the use of amidase enzymes to selectively hydrolyse alpha- substituted amides to give resolved or partially resolved alpha-substituted carboxylic acids. In JP-A-6188894, enzyme-catalyzed, enantioselective hydrolysis of alpha- hydroxycarboxylic acid amides is described. The process described uses Aeromonas hydrophila (FERM P-7360) and Moraxella phenylpyruvica (FERM P-7359), however a disadvantage of this process is that the yield and the optical purity (enantiomeric excess) are relatively low and the substrates are limited to alpha-hydroxycarboxylic acid amides. The development of an enzyme-catalyzed, enantioselective hydrolysis of alpha-amino acid amides based on the use of Pseudomonas putida is described in Chirality in Industry, Published by Wiley & Sons 1992, VoI 1 , Chapter 8 Page 189. However, it is well established that this process using Pseudomonas putida is only effective with substrates with an alpha-hydrogen on the alpha carbon atom. In EP0494716 the use of enzymes derived from Ochrobactrum anthropi is disclosed. These enzymes do not require the presence of alpha-hydrogen, however in our hands we found that the selectivity exhibited by the enzymes is influenced by the nature of the alpha-substituents. There is therefore a need for alternative enzymes which are not subject to such influence. According to a first aspect of the present invention there is provided a process for the preparation of an optically active carboxylic acid or an optically active carboxylic acid amide wherein a carboxylic acid amide having an alpha-carbon substituted with a heteroatom group is enantioselectively hydrolysed in the presence of an enzyme obtainable from Pseudomonas fluorescens to give an optically active carboxylic acid and a residual optically active carboxylic acid amide. The starting carboxylic acid amide having an alpha-carbon substituted with a heteroatom group is preferably a mixture of enantiomers, more preferably a racemic mixture of enantiomers. The terms optically active carboxylic acid and residual optically active carboxylic acid amide include compositions comprising an enatiomeric excess of one enantiomer in the presence of the other enantiomer. The enzyme obtainable from Pseudomonas fluorescens may be in any form capable of enantioselectively hydrolysing a carboxylic acid amide having an alpha-carbon substituted with a heteroatom group to give an optically active carboxylic acid, for example in the form of a cell-free extract, a synthetic form, an immobilised form, disintegrated cells, the enzyme is cloned and over-expressed in a suitable expression system or the enzyme is present in whole cells. The process for enantioselectively hydrolysing a carboxylic acid amide having an alpha-carbon substituted with a heteroatom group using an enzyme obtainable from Pseudomonas fluorescens can be regarded as a biotransformation process. The process is preferably carried out in a liquid medium, preferably in an aqueous medium and especially in a buffered aqueous medium. Suitable buffers may be inorganic or organic and are preferably those which control the pH of the medium in the range 5 to 10.5, more preferably in the range 7.0 to 9.0 and especially at a pH of 8.0. The buffer is preferably inorganic, more preferably a phosphate buffer type, typically an alkali metal phosphate, especially sodium/potassium phosphate buffer mixture. An especially preferred buffer is 0.1 M sodium/potassium phosphate prepared from potassium dihydrogenphosphate and sodium hydroxide. The pH of the process may optionally be maintained at the desired pH by an intermittent feed of a base, preferably an inorganic base, more preferably an alkali metal hydroxide such as dilute aqueous sodium or potassium hydroxide. The process is preferably performed at a temperature from O0C to 100°C, more preferably at from 2O0C to 450C and especially at from 3O0C to 4O0C. When the process has proceeded for a suitable period, as judged by the rate of accumulation of the optically active carboxylic acid product and reduction in concentration of starting material using a technique such as HPLC, the process may be terminated by any convenient means, for example by removing the micro-organism or enzyme by centrifugation or filtration and/or by cooling the reaction mass to a temperature of less than 50C or by separating the reactants from the micro-organism or enzyme by extraction. The process may take a few hours or many days, e.g. 1 hr to 1 week. The optically active carboxylic acid and the residual carboxylic acid amide may be isolated by any convenient means, for example by solvent extraction, preferably using a halocarbon solvent (e.g. CH2CI2), an aromatic solvent (e.g. toluene), or more preferably an ester (e.g. ethyl acetate) or an ether (e.g. tert-butyl methyl ether). Preferably, the optically active carboxylic acid and the residual carboxylic acid amide are separated during isolation. As a result of the enantioselective hydrolysis, the residual carboxylic acid amide is optically active. In a preferred isolation process, the reaction mixture is made alkaline by addition of base and then residual optically active carboxylic acid amide is isolated by solvent extraction. The residual optically active carboxylic acid amide product that is isolated by solvent extraction may be used without further purification or may be purified, for example by recrystallisation or chromatography, for example by preparative HPLC or thin layer chromatography (TLC) using silica gel and a liquid eluent, e.g. an ether, an alkane or a mixture thereof. The residual optically active carboxylic acid amide product may be hydrolysed to give a carboxylic acid. By controlling the hydrolysis conditions, the carboxylic acid obtained in entiomeric excess from the residual optically active carboxylic acid amide retains optical activity and is of opposite rotation to the optically active carboxylic acid produced directly by enantioselective hydrolysis. After extraction of the residual optically active carboxylic acid amide, the pH of the alkaline aqueous phase is then adjusted to be less alkaline, preferably to near neutral or to be acidic, by addition of acid and then the optically active carboxylic acid produced by the enzyme process is isolated by solvent extraction or crystallisation. The optically active carboxylic acid product of the process may be further purified, for example by recrystallisation or chromatography, for example by preparative HPLC or thin layer chromatography (TLC) using silica gel and a liquid eluent, e.g. an ether, an alkane or a mixture thereof. Optionally, the optically active carboxylic acid may be reacted with an amine or aminating agent to give a carboxylic acid amide. By controlling the amination conditions, the carboxylic amide obtained in enantiomeric excess by amination of the optically active carboxylic acid retains optical activity and is of opposite rotation to the residual optically active carboxylic acid amide. Preferably the carboxylic acid amide that is enantioselectively hydrolysed in the presence of an enzyme obtainable from Pseudomonas fluorescens to give an optically active carboxylic acid or an optically active carboxylic acid amide, is a carboxylic acid amide of formula (1 ):

wherein R1 and R2 each independently are hydrogen or a substituent group; and R3 is an optionally substituted heteroatom group; provided that R1, R2 and R3 are different. Substituent groups which may be represented by R1 and R2 include optionally substituted hydrocarbyl groups, perhalogenated hydrocarbyl groups, optionally substituted heterocyclic groups, or optionally R1 & R2 may be linked to form an unsymmetrical ring optionally comprising one or more heteroatoms. Hydrocarbyl groups which may be represented by R1 and R2 independently include alkyl, alkenyl and aryl groups, and any combination thereof, such as aralkyl and alkaryl, for example benzyl groups. Alkyl groups which may be represented by R1 and R2 include linear and branched alkyl groups comprising up to 20 carbon atoms, particularly from 1 to 7 carbon atoms and preferably from 1 to 5 carbon atoms. When the alkyl groups are branched, the groups often comprising up to 10 branch chain carbon atoms, preferably up to 4 branch chain atoms. In certain embodiments, the alkyl group may be cyclic, commonly comprising from 3 to 10 carbon atoms in the largest ring and optionally featuring one or more bridging rings. Examples of alkyl groups which may be represented by R1and R2 include methyl, ethyl, propyl, 2-propyl, butyl, 2-butyl, t-butyl and cyclohexyl groups. Alkenyl groups which may be represented by R1 and R2 include C2-2o> and preferably C2-6 alkenyl groups. One or more carbon - carbon double bonds may be present. The alkenyl group may carry one or more substituents, particularly phenyl substituents. Examples of alkenyl groups include vinyl, styryl and indenyl groups. Aryl groups which may be represented by R1 and R2 may contain 1 ring or 2 or more fused rings which may include cycloalkyl, aryl or heterocyclic rings. Examples of aryl groups which may be represented by R1 and R2 include phenyl, tolyl, fluorophenyl, chlorophenyl, bromophenyl, trifluoromethylphenyl, anisyl, naphthyl and ferrocenyl groups. Perhalogenated hydrocarbyl groups which may be represented by R1and R2 independently include perhalogenated alkyl and aryl groups, and any combination thereof, such as aralkyl and alkaryl groups. Examples of perhalogenated alkyl groups which may be represented by R1and R2 include -CF3 and -C2F5. Heterocyclic groups which may be represented by R1 and R2 independently include aromatic, saturated and partially unsaturated ring systems and may constitute 1 ring or 2 or more fused rings which may include cycloalkyl, aryl or heterocyclic rings. The heterocyclic group will contain at least one heterocyclic ring, the largest of which will commonly comprise from 3 to 7 ring atoms in which at least one atom is carbon and at least one atom is any of N, O, S or P. Examples of heterocyclic groups which may be represented by R1 and R2 include pyridyl, pyrimidyl, pyrrolyl, thiophenyl, furanyl, indolyl, quinolyl, isoquinolyl, imidazoyl and triazoyl groups. When any of R1 and R2 is a substituted hydrocarbyl or heterocyclic group, the substituent(s) should be such so as not to adversely affect the rate or stereoselectivity of any of the reaction steps or the overall process. Optional substituents include halogen, cyano, nitro, hydroxy, amino, thiol, acyl, hydrocarbyl, heterocyclyl, hydrocarbyloxy, mono or di-hydrocarbylamino, hydrocarbylthio, thioesters, esters, carbamates, carbonates, amides, sulphonyl and sulphonamido groups wherein the hydrocarbyl groups are as defined for R1 above. One or more substituents may be present. Examples of R1 and R2 groups having more than one substituent present include -CF3 and -C2F5. Optionally substituted heteroatom groups which may be represented by R3 include heteroatoms, for example halogens, and functional groups based on heteroatoms, preferably oxygen, nitrogen, sulphur, boron and phosphorous for example hydroxy, hydrocarbyloxy, amino, hydrocarbylamino, dihydrocarbylamino, thiol, and hydrocarbylthio groups. Heteroatoms which may be represented by R3 include fluorine, chlorine, bromine, iodine. Preferably, R3 is an optionally substituted heteroatom group, more preferably an optionally substituted oxygen or nitrogen group. When a carboxylic acid amide having an alpha-carbon substituted with a heteroatom group of Formula (1) is enantioselectively hydrolysed in the presence of an enzyme obtainable from Pseudomonas fluorescens, the optically active carboxylic acid obtained is an optically active carboxylic acid of formula (4):

wherein R1 and R2 each independently are hydrogen or a substituent group; and R3 is an optionally substituted heteroatom group; provided that R1, R2 and R3 are different such that * is a chiral centre. Thus, a preferred reaction scheme is illustrated as follows:-

mixture of enantiomers optically active carboxylic acid residual optically active (ee of 1st enantiomer) carboxylic acid amide (ee of 2nd enantiomer)

amination hydrolysis

R1 R1 2 I ( ,2 I O 2 I .0 R2 ^3' NH, O t H

(ee of 1 st enantiomer) (ee of 2nd enantiomer) Most preferably the carboxylic acid amide is an alpha-aminocarboxylic acid amide of formula (2):

wherein R4 and R5 each independently are hydrogen or a substituent group; and R6 is NH2; provided that R4, R5 and R6 are different. Substituent groups which may be represented by R4 and R5 are as described above for R1. Typically, R4 and R5 may be selected from the substituent groups which are analogous to those commonly found in naturally occurring amino acids. For example, -CH3, -CH(CH3)2, -CH2CH(CH3)2, -CH2OH, -CH(OH)CH3, -CH2CO2H, -CH2CH2CO2H, -CH2SH, -CH2Ph, -(CHz)4NH2, -(CH2)3NHC(=NH)NH2, -CH2imidazoyl and -CH2indolyl. However, R4 and R5 may also be selected from substituents not found in naturally occurring amino acids, for example simple aryl and aralkyl groups such as phenyl, naphthyl or substituted benzyl groups, other alkyl groups such as -CH2CH3 and -C(CH3)3, halogenated alkyl groups such as -CF3, and thio substituted alkyl groups such as -CH2SRa where Ra is an optionally substituted hydrocarbyl, for example tert-butyl or benzyl group, or other protecting group. Preferably both R4 and R5 are substituent groups. When an alpha-aminocarboxylic acid amide of formula (2) is enantioselectively hydrolysed in the presence of an enzyme obtainable from Pseudomonas fluorescens, the optically active alpha-aminocarboxylic acid obtained is an optically active alpha- aminocarboxylic acid of formula (5):

wherein R4 and R5 each independently are hydrogen or a substituent group; and R6 is NH2; provided that R4, R5 and R6 are different such that * is a chiral centre. In a highly preferred aspect of the present invention, the carboxylic acid amide is an alpha-aminocarboxylic acid amide of formula (3): R7

R9 NH* wherein R7 is an alkyl group, preferably C1-6alkyl and most preferably methyl. R8 is a substituted alkyl group comprising an optionally substituted heteroatom group; and R9 is NH2. Alkyl groups which may be represented by R7 are as described above for R1. Substituted alkyl groups which may be represented by R8 include alkyl groups, as described above for R1, but substituted with an optionally substituted heteroatom group, as described above for R3. Preferably, the optionally substituted heteroatom group is a functional group based on sulphur, for example a thiol or a hydrocarbylthio group. Preferably, R8 is a substituted C1-6alkyl group comprising an optionally substituted hydrocarbylthio group. More preferably, R8 is a C1-6alkyl group substituted with an optionally substituted tert-butylthio or optionally substituted benzylthio group. Most preferably, R8 is -CH2SCH2Ph group. When an alpha-aminocarboxylic acid amide of formula (3) is enantioselectively hydrolysed in the presence of an enzyme obtainable from Pseudomonas fluorescens, the optically active alpha-aminocarboxylic acid obtained is an optically active alpha- aminocarboxylic acid of formula (6):

R9 OH wherein R7 is an alkyl group, preferably C1-6alkyl and most preferably methyl. R8 is a substituted alkyl group comprising an optionally substituted heteroatom group; R9 is NH2; and * is a chiral centre. The invention is further illustrated by the following Examples.

Pseudomonas fluorescens AL45 (NCIMB 41223) was deposited on 8 June 2004 with NCIMB Ltd, Ferguson Building, Craibstone Estate, Bucksburn, Aberdeen AB21 9YA (formerly of 23 St Machar Drive, Aberdeen, Scotland, AB24 3RY).

Example 1 Preparative Scale Resolution S-Benzyl-protected-α-methyl cysteine amide by Pseudomonas fluorescens AL45 (NCIMB 41223) grown on lactamide

Growth of Microorganism Ps fluorescens (NCIMB 41223) was grown in a 10 litre fermenter in 5 litres of mineral salts medium pH 7.2 supplemented with yeast extract (2g/litre) and lactamide (2.5g/litre) and maintained at 28°C. The fermenter was aerated at 5 litres/minute and stirred at 400 rpm for 24 hours. Cells were harvested by centrifugation and the recovered cells were washed by resuspension in 10OmM phosphate buffer pH 7.2 and recentrifuged, the recovered cells were stored at 40C overnight prior to use in the biotransformation. Biotransformation Conditions The cell pellet was resuspended in 1 litre of phosphate buffer (10OmM, pH7.2) and to this was added δgrams of racemic amino amide and the stirred mixture incubated at 28°C and samples removed periodically for analysis. After 8.5 hours quantitative HPLC indicated that the reaction had reached 50% hydrolysis, this was supported by chiral analysis ((R) amino amide 96% ee, (S)-amino acid 97.5% ee) eeS/ees + eeP = 49.6% conversion.

Analytical Methods Amino amide chiral Chiralcel OD 250 x 4.6 mm 90% isohexane plus 0.1% diethylamine : 10% ethanol 1 ml/min Detection 210nm Retention times - (R)-amino amide 15.3 minutes, (S)-amino amide 18.6 minutes

Amino acid chiral Astec Chirobiotic T 250 x 4.6mm 80% aqueous ethanol 1 ml/min Detection 210nm Retention times - (R) - amino acid 6.8 minutes, (S)-amino acid 8.2 minutes

Quantitative Jones Genesis C18 150 x 4.6 mm 85% water plus 0.1 % TFA: 15% acetonitrile 1 ml/minute Detection 210 nm Retention times - amino amide 7.9 minutes, amino acid 12.1 minutes

Reaction Work-Up Cells were removed from the biotransformation broth by centrifugation and the supernatant (approximately 1000ml) adjusted to pH10.5 using 20% sodium carbonate (final volume approximately 1200ml). The alkaline supernatant was extracted with 750ml of tert-butyl methyl ether. The aqueous layer was decanted and the organic layer plus an emulsion layer was mixed with approximately 250ml of acetone which resulted in the formation of a precipitate. The acetone/tBME mixture was dried with anhydrous sodium sulfate and the solvent evaporated to yield a straw-coloured oil. The aqueous layer from the initial extraction was re-extracted with a further 750ml of tBME, on addition of acetone (as above) an aqueous layer (approximately 50ml) separated and was decanted and combined with the other aqueous fraction. The combined tBME fractions were dried with anhydrous sodium sulfate and evaporated to dryness to yield an off-white crystalline solid, amino amide (1.7g, 68% yield, >99% ee). The aqueous phase was adjusted to pH 6.8 using 2 molar HCI and concentrated by freeze drying to approximately 300ml which on thawing produced a white slurry. The crystalline solid was recovered by filtration and dried over night at 370C to yield 1.8g of fine white crystals. HPLC analysis confirmed that the crystals were amino acid (chiral purity >99% ee (S), chemical purity 98% by HPLC, isolated yield 72%).

Example 2 Preparative scale resolution of racemic 2-amino-3-(benzylthio)-2- methylpropanamide using Pseudomonas fluorescens AL45 (NCIMB 41223) grown on glucose

Stock cultures of Pseudomonas fluorescens AL45 (NCIMB 41223) were grown for 48 hours on CLED agar (Oxoid) at 28°C and stored at 40C until required. A fermenter inoculum was developed by inoculating a colony from a stock plate into a 1 litre flask containing 200ml of mineral salts medium supplemented with yeast extract (2g/litre) and glucose (5g/litre) and growing at 28°C for 24 hours. This culture was used to inoculate a fermenter containing 8 litres of a mineral salts medium, pH 7.2, supplemented with yeast extract (2 g/litre) and glucose (20 g/litre). The culture was stirred at 600 rpm, aerated at 8 litres/minute and maintained at 28°C. Antifoam (polypropylene glycol) was added throughout growth at a rate of 0.5ml/hour. Additional glucose was added at intervals to maintain growth and the cells were harvested by centrifugation after 23 hours growth. The cell pellet was washed by resuspension in 8 litres of 10OmM phosphate buffer, pH7.2 and recovered by centrifugation. The wet cell mass was dispensed into vials in 25ml portions and stored at -20°C until required.

Into 9 litres of sodium/potassium phosphate (10OmM, pH 7.2) was added 90 grams of racemic 2-amino-3-(benzylthio)-2-methylpropanamide. To this was added 65 grams of cell paste and the reaction was gently stirred at room temperature (approximately 200C). Samples were removed at intervals and analysed by reverse phase HPLC as described in Example 1. After 26 hours reaction reverse phase HPLC indicated that 51 % hydrolysis of the starting material had occurred, the enantiomeric purity of the residual amide was checked using the HPLC method described in Example 1 and found to be greater than 98% enantiomeric excess. The reaction mixture was centrifuged to remove the cells and then the pH of the cell-free reaction mixture was adjusted to 9.5 with 48% sodium hydroxide. Sodium sulfate was added to the reaction mixture to a final concentration of 50 g/litre and the solution extracted twice with 4.5 litres of ethyl acetate. The organic extracts were combined and dried with anhydrous sodium sulfate, filtered to remove solids and then the solvent was removed under reduced pressure to yield a white solid. The solid was recovered and dried under high vacuum for 48 hours to yield 43.24 grams of (R)- amide, 98.7% enantiomeric excess. Example 3 Effect of pH on hydrolysis of racemic 2-amino-3-(benzylthio)-2-methylpropanamide by Pseudomonas fluorescens AL45 (NCIMB 41223).

Racemic 2-amino-3-(benzylthio)-2-methylpropanamide was dissolved in buffer to give a final concentration of 10 grams/litre. The buffers used were sodium phosphate (pH 6, 7, 8), tris (hydroxymethyl)aminomethane hydrochloride (pH 9), and sodium carbonate/sodium bicarbonate pH 10. Cell paste prepared as described in Example 2 was resuspended in the appropriate 10OmM buffer to a concentration of 1gram dry cell weight per litre and the rate of reaction monitored by measuring the disappearance of the starting material.

Comparative Example 1 Resolution of racemic 2-amino-3-(benzylthio)-2-methylpropanamide using Ochrobactrum anthropi grown on lactamide

Ochrobactrum anthropi was isolated from soil by growth at 28 degrees C on a minimal salts medium supplemented with yeast extract (0.2 grams/litre) and 2- hydroxypropanamide (2.5grams/litre). The isolate was identified as O. anthropi on the basis of API 20NE test kit (Bio Merieux). This strain of O. anthropi has been deposited with NCIMB Ltd, Ferguson Building, Craibstone Estate, Bucksburn, Aberdeen AB21 9YA and assigned the code NCIMB 41225.

Ochrobactrum anthropi strain R39 was grown for 24 hours at 280C in a 1 litre flask containing 200ml of mineral salts medium pH 7.2 supplemented with yeast extract (2g/litre) and racemic 2-hydroxy propanamide (2.5g/litre). Cells were recovered by centrifugation and washed in 10OmM phosphate buffer pH7.2.

To a vial containing 10OmM phosphate buffer pH 7.2 (5 ml) was added 44mg of cells and 25 mg of racemic 2-amino-3-(benzylthio)-2-methylpropanamide and the mixture stirred at 28°C, samples were removed at intervals for analysis. After 6.5 hours the extent of hydrolysis of the starting material had reached 50% and the enantiomeric excess of the residual starting material was 56% (S).

Conclusion - Pseudomonas fluorescens shows opposite selectivity to Ochrobactrum anthropi. Pseudomonas fluorescens also shows enhanced selectivity in the hydrolysis of 2-amino-3-(benzylthio)-2-methylpropanamide as evidenced by the higher ee of the residual amide.