This application is divided out of Application No. 2168 M 97.
This invention relates to a dcracerrrisation process of obtaining optically pure (5>a-hydroxy esters from the corresponding racemic, (RS) - esters in the presence of a biocatalyst. More specifically, the invention is a new process by which the racemic, (RS) - esters of aryl and arylalkyl a-hydroxy acids can be completely converted (100 % conversion) into only one enantiomer - the optically pure (5>alkyl esters of aryl and arylalkyl a-hydroxy acids in >99% enantiomeric excess, ee.
Chirality is important in the context of biological activity because, in bioactive molecules where a stereogenic center is present, great differences are observed in the activities of the enantiomers. This phenomenon is seen in all bioactive substances like drugs, insecticides, herbicides, flavors and fragrances and food additives. Most naturally occurring drug molecules are chiral and exist in nature (and are marketed) as the single, active enantiomer. But in the case of synthetic chiral drugs, for many years most of them were marketed as racemates. However, the present situation reveals a definite trend towards enantiopurity in chiral synthetic drugs. Regulatory requirements (e.g. FDA, USA) call for complete information with regard to the pharmacodynamics and pharmacokinetics of all the individual isomers and the racemic mixtures. It has become exceedingly important therefore to find methods of preparing drugs or drug intermediates which are optically pure.
Optically pure a-hydroxy acid derivatives are important synthons for the synthesis of various pharmaceuticals and natural products. One of the most versatile a-hydroxy esters is 2-hydroxy-4-phenylbutanoic acid ethyl ester which is a precursor for the production of a variety of Angiotensin Converting Enzyme (ACE) inhibitors. These are compounds which are useful in

Mocking the rise in blood pressure caused by increases in vascular resistance and fluid volume due to the formation of angiotensin I from angiotensin n. These drugs have been developed and marketed as single optical isomers. In the synthesis of enalapril, (ft;-2-hydroxy-4-phenyibutanoic acid ethyl ester can be coupled with (S>alanine and men with L-proHnc to give enalapril of the required stereochemistry. It is important therefore to make the optically pure a-hydroxy ester. The ^;-a-hydroxy ester can be inverted to the ^-enantiomer (Mitsunobo O. et al, Bull. Chem. Soc. Japan, 44, 3427, 1971) and used in the same synthetic scheme.
Mandehc acid ethyl ester and its derivatives are important chiral intermediates used in the manufacture of useful bactericidal compounds (Treuner Uwe et al, US 3,943,130;1976; Oozeki et al, Jpn. Kokai Tokkyo Koho JP 06,239,742; 1994)) and resolving agents for compounds which find extensive use as pharmaceuticals (Nakamura Mhsuo et al, PCT bit Appl. WO 31,436;1995; Sohar Paul et al, Get. Offen. 2,436,686; 1975; US AppL 384,185; Jour. Org. Chem., 1983, 48, 3548; Jour. Org. Chem., 1988, 53, 5335).
Several methodologies have been developed for the synthesis of these compounds. Of these, the reduction of a-keto esters to a-hydroxy esters using microbial transformation, catalytic hydrogenation and chiral organometaffic reagents, and the chemical as well enzymatic resohition of the racemate are well documented for 2-hydroxy-4-phenylbutanoic acid and its derivatives.
Among the known microbial processes to prepare optically pure 2-hydroxy-4-phenylbutanoic acid and its derivatives, Matsuyama Akinobu et al, (PCT Int. Appl. WO 89 07,648- Japanese patent, 1989) have used the microorganism, Leuconostoc dextranicum and Myata Reiko et al (Jpn. Kokkai Tokkyo Koho JP 02,16,987, Japanese patent, 1988) have used Apiotrichum,

Candida, Torulpsis, Rhodotorula, Trichosporon, Rhodosporidium, Pichia, Klebsiella, Rhodococcus, Paracoccus, Corynebacterium and Nocardiodes to reduce 2-oxo-4-phenyl butanoic acid derivatives. Hashimoto Yoabjhiro et al, (Jpn. Kokai Tokkyo Koho JP 07,115,989, Japanese patent, 1995) have treated the racemic cip-hydroxynitriles with microorganisms chosen from Brevibacterium, Nocardia, Corynebacterium and Rhodococcus, with Gordona terrae (Eur. Pat Appl. EP 610,048, European patent, 1994) and with microorganisms belonging to Rhodococcus, Alcaligenes, Brevibacterium or Pseudomonas (Eur. Pat. AppL EP 610,049, European patent, 1994) which asymmetrically hydroryse the nitrites to give the optically pure a-hydroxycarboxyHc acids and their antipodal a-hydroxyamides.
The manufacture of optically active mandelic acid by resolution of the racemate with Pseudomonas pofycolor IFO 3918 has been done by Mori et al (Jpn. Kokai Tokkyo Koho JP 04,341,195 , Japanese patent, 1991) and by Nakayama Kyoshi et al (Jpn. Kokai Tokkyo Koho JP 06,253,890; 1993). Endo Takakazu et al have reported a process for producing optically pure mandelic acid and derivatives thereof with Rhodococcus (Eur. Pat. Appl. EP 527,553;1991).
Candida sp. has been used to manufacture, L-(+)-p-hydroxyisobutyric acid from isobutyrie acid (Kanda et al, Jpn. Kokai Tokkyo Koho JP 05,336,981, Japanese patent, 1993) by a stereospecific hydroxylation reaction. There is no such report for oc-hydroxy acids or their esters. Preparation of optically pure 1,2-diols (Hasegawa et al, Agric. Biol Chem., 1990, 54, 1819), 1,3-butanediol from the racemate and {2R, 4/?)-2,4-pentanediol from the isomeric mixture of 2,4-pentanediol has also been reported (Matsuyama et al, Biosci. Biotech.

Biochem, 1994, 58, 1148, Matsunura et a], Biotech. Lett., 1994, 16, 485). Thus, unlike resolution of the racemate and chiral reduction of the pro-chiral keto ester which are commonly used methods, there is no reported procedure whereby the racemic esters of aryl and arylalkyl oc-hydroxy acids are converted exclusively to one enantiomer, i. e. deraceinisation. It is to be noted here that this reaction is unique because the starting material, like the product, is an a-hydroxy acid ester- both are chemically identical. The difference between the starting material and the product is that while the starting ester is racemic (RS), the product is optically pure, (S). In other procedures to obtain optically pure molecules viz. asymmetric synthesis, the starting material is chemically different from the product and in resolution processes, there is always the disadvantage of the other optically pure enantiomer also being formed.
Resolution of racemic mixtures of optically active compounds has several disadvantages for preparative applications:
i) The theoretical yield of each enantiomer is limited to 50% considering the whole process. Thus the main disadvantage is that the process does not lead to a single enantiomer in 100% yield.
ii) Separation of the product is generally elaborate and may necessitate chromatography if extraction and/or distillation fails.
iii) Normally only one stereoisomer is preferred and there is little or no use for the other. To overcome this limitation, both enantiomers could be used in an enantiocorrvergent synthesis but

this requires two independent synthetic pathways to the target compound or the undesired enantiomer has to be racemised and recycled into the resolution process. It is the object of the present invention to provide a process for the complete transformation of the racemic alkyl esters of aryl and arylalkyl a-hydroxy acids into a single stereoisomer -the (5> enantiomer, (>99% ee, 100% conversion) Le. deracemisation. As opposed to resolution, deracemisation does not need recycling because only one enantiomer is formed from the racemate, in this case the (£>enantiomer. The other enantiomer (R) in the racemic mixture also gets converted to (S). Essentially, a racemic mixture gets totally converted to a single enantiomer.
The invention describes the process of converting racemic esters, i.e the (RS) esters of a-hydroxy acids of general formula, R-dtU ( CH2 )»CHOHCOOR\ I, to optically pure (5)- a-hydroxy esters by a deracemisation reaction. In the formula, I, n=0-4, R=OH, R'= alkyl, C1.3. The conversion is mediated by cells of Candida (100% conversion; ee >99%), thus maximisung the yield for a single enantiomer. For the first time alkyl esters of a-hydroxy acids can be deracemised to give only one enantiomer.
The main advantages of this invention are:
* Only one enantiomer is formed. The racemic (&S)-ester is transformed into a single enantiomer-the fS>enantiomer (100% conversion; ee>99%).
* Reaction conditions are mild. The biotransformation is done at ambient temperature wim stirring and without any elaborate inputs of heat and energy.

* Work up is simple. The product is obtained after concentration of the organic extract.
* Purification of the product is not required Since only one enantiomer is fonned which is extracted into the organic layer during work up, no further purification of the product is needed.
♦Continuous production of the product is possible using immobilised cells. Algmated cells can be charged into and with continous feeding of the racemic ester into the reactor, the optically pure product, (5>hydroxy ester obtained can be siphoned out.
In a typical experiment carried out by us, the cell pellet was obtained from Candida sp. grown in Yeast Malt Broth (YMB). The medium was sterilsed and then innoculated with stock culture. After 48h, the cells were harvested by centrifugation and finally suspended in sterile distilled water. The appropriate racemic ester was added to a known quantity of the cell pellet suspended in phosphate buffer (O.S-l.OM, pH 5-7). The racemic ester is dissolved in just sufficient organic solvent, preferrably ethanol, to enhance its soulubility, before adding it to the cell suspension. The ratio of the S and R enantiomers formed when other solvents are used to dissolve the racemic ester are shown in the table below. The cell suspension with the racemic ester was men incubated with the cells (10:1 by weight) at ambient temperature, 200-400 rpm. The reaction mixture was incubated for l-12h after which it was extracted with ether, concentrated and analysed to give the (S)-enantiomer (100% conversion; >99% ee, where ee denotes enantiomeric excess).


The invention describes a process for converting racemic esters, ie the (AS) esters of a-hydroxy acids dissolved in sufficient ethanol to enhance the solubility in aqueous medium, of general formula, R-QH* (CH2)„CHOHCOOR', I, where n=0-4, R=OH, R'= alkyl, Cvit to optically pure (3>a-hydroxy esters in phosphate buffer (0.5M-1.0M; pH 5-7) at ambient temperature with stirring between 200-400 rpm over a period of lh to 12h by a deracemisation reaction (100% conversion; >99%ee) mediated by free or immobilised cells of Candida sp.; the cell pellet obtained in a known manner keeping the ratio of the weight of racemic ester to the weight of the cells as 10:1; and the product isolated by separation of the cells by filtration followed by extraction with a suitable organic solvent like ether.
The process is further illustrated by the following examples which are provided by way of illustration only and therefore should not be construed to limit the scope of the invention.
Example 1 Preparation of (gV2-hvdroxv-4-phenylbutanoic acid ethyl ester Candida cells were grown in the YMB Medium for 48h after which cells were harvested by centrifugation and suspended in phosphate buffer (0.5-1.0M, pH 5-7); (0.18-0.29mg/ml). 2-

hydroxy-4-phenyIbutanoic acid ethyl ester was added to the cell suspension to get a final concentration of 3mg/ml. The reaction mixture was incubated at ambient temperature in a shake-flask, 200-400 rpm for 5h after which it was extracted with ether, concentrated and anah/sed to yield fS>2-hydroxy-4-pheny!-butanoic acid ethyl ester in >99%ee ; 100% conversion.
When the (i?S>2-hydroxy-4-phenylbutanoic acid methyl ester was used, the product was (5)-2-hydroxy-4-phenylbutanoic acid methyl ester.
Example 2 Preparation of ffi-mandelic acid ethvl ester Candida cells were grown in the YMB Medium for 48h after which cells were harvested by centrifugation and suspended in phosphate buffer (0.5-1.0M, pH 5-7); (0.18-0.29mg/ml). Mandelic acid ethyl ester was added to the ceD suspension to get a final concentration of 3mg/ml. The reaction mixture was incubated at ambient temperature in a shake-flask, 200-400 rpm for lOh after which it was extracted with ether, concentrated and analysed to yield (S>2-mandelic acid ethyl ester in >99%ee; 100% conversion.
When the (ftS)-mandelic acid methyl ester was used, the product was (5)- mandelic acid methyl ester.
Example 3
Preparation of ^S)-j?-hydroxv-mandeHc acid ethvl ester
Candida cells were grown in the YMB Medium for 48h after which cells were harvested by
centrifugation and suspended in phosphate buffer (0.5-1.0M, pH 5-7); (0.18-0.29mg/ml). p-
hydroxy mandelic acid ethyl ester was added to the cell suspension to get a final concentration

of 3mg/ml. The reaction mixture was incubated at ambient temperature in a shake-flask, 200-400 rpm for lOh after which it was extracted with ether, concentrated and analysed to yield (S>/7-hydroxy mandclic acid ethyl ester in >99%ee ; 100% conversion. When the (KS>/;-hydroxy mandclic acid methyl ester was used, the product was (5>^-hydroxy mandclic acid methyl ester.
Example 4 Preparation of immobilised cells of Candida Immobilisation of Candida cells grown in YMB medium for 48h, was done by making a slurry of the cells in sodium alginate to get a final concentration of 15% cells (dry wt.) and 1.6% sodium alginate. The slurry was added to 3% CaCk solution. The beads formed were stored overnight at 4°C for curing and used after washing with distilled water.
Example 5 Effect of organic solvents on the deracemisation reaction To study the effects of organic solvents on the reaction, the 2-hydroxy-4-phenylbutanoic acid ethyl ester was dissolved in ~1 ml of organic solvent and added to the aqueous cell suspension following which the standard assay was done. The solvents studied were dimethyl formamide pMF), dimethyl sulfoxide (DMSO), terahydrofuran (THF), acetone, hexane, chloroform, methanol and ethanol. DMF, DMSO, acetone and ethanol showed an ee of 95-99% wim 100% conversion of the (RS)- 2-hydroxy-4-phenylbutanoic acid ethyl ester to fS>2-hydroxy-4-phenylbutanoic acid ethyl ester and (ft$-mandelic acid ethyl ester to fS>mandelic acid ethyl ester.

Preparation of ^-2-hvdroxv-4-phenvlbutanoic acid ethvl ester using immobilised cells Candida cells entrapped in calcium alginate (57g) were suspended in 300ml of phosphate buffer (0.5-1.0M, pH 3-7) to which was added 853mg of (^>2^droxy-4-phenyIbutanoic acid ethyl ester. The flask was kept shaking on a rotary shaker for 6h at 2S°C. The contents were then filtered and extracted with ether. (S)- 2-hydroxy-4-phenyIbutanoic acid ethyl ester was formed, >99% ee, 100% conversion.

We claim:
1. A process for converting racemic esters, i.e the (RS) esters of a-hydroxy acids dissolved in
sufficient ethanol to enhance the solubility in aqueous medium, of general formula, R-QH*
(CH^CHOHCOOR', I, where n=0-4, R=OH, R'= alky], C1-5, to optically pure (S)-a-hydroxy
esters in phosphate buffer (0.5M-1.0M; pH 5-7) at ambient temperature with stirring between
200-400 rpm over a period of lh to 12h by a deracemisation reaction (100% conversion;
>99%ee) mediated by free or immobilised cells of Candida sp.; the cell pellet obtained in a
known manner keeping the ratio of the weight of racemic ester to the weight of the cells as
10:1; and the product isolated by separation of the cells by filtration followed by extraction
with a suitable organic solvent like ether.

In the formula I, where n=2, R is hydrogen and R' is preferrabry ethyl or methyl and where n=0, R is either hydrogen or p-hydroxy and R' is preferrabry ethyl or methyl.
2. A process as claimed in claim 1 where the ethyl and methyl esters of (5)-2-hydroxy-4-
phenylbutanoic acid were prepared.

3. A process as claimed in claim 1 where the ethyl and methyl esters of (S)-mandelic
acid were prepared.
4. A process as claimed in claim 1 where the ethyl and methyl esters of (S)-p-hydroxy-
mandclic acid were prepared.