Title: A microbial biocatalyst for preparing pharmaceutical compounds
Field of Invention
The present invention provides a process of preparing pharmaceutical compounds involving oxido reduction by means of microbial biocatalyst.
Background Information
Microbial reductions are very useful reactions in the asymmetric synthesis as they offer an alternative over the existing chemical routes by reducing the corresponding ketones with excellent selectivity. Oxidoreductases are the enzymes, which introduce chirality into the substrate (prochiral ketone) to give chiral product (alcohol). Because of this at present they compete with hydrolases as the most widely used enzyme system. They involve a hydride transfer from the cofactor (NAD(P)H or FADH) to the carbonyl center which is mediated by oxidoreductase enzyme. Depending upon the face of attack for hydride transfer on the carbonyl center, the chirality of the product alcohol is decided. If the hydride transfer occurs from the re-face, then there is formation of S-alcohol mediated by S-specific oxidoreductases and if the hydride transfer occurs from the Si-face, then there is formation of R-alcohol mediated by R-specific oxidoreductases. The inherent advantages in the microbial reductions are the possibility of obtaining upto 100% theoretical yield and above 99% enantioselectivity as commonly observed in this type of transformations.
The present invention describes a novel microbe Candida tropicalis having accession no. MTCC 5158 and deposited at IMTECH, India on 15th June, 2004 (Reference: Indian Patent Application No. 1573/DEL/2004, filed on August 23, 2004). This organism is unique in that it produces an oxidoreductase biocatalyst /enzyme, which can reduce a variety of ketonic compounds (Table 1). Since reduction of ketones is a fundamental step in the preparation of optically pure S-alcohols, which are useful intermediates in a number of pharmaceutical compounds, the microbe has considerable industrial importance.

These chiral alcohols have been prepared using several chemical and biochemical methods. Examples of chemical methods are asymmetric alkylation of aldehydes (Collomb P, Zelewsky A., Tetrahedon Asymm. 1998, 9, 3911-3917.), the asymmetric reduction of the corresponding ketones via transfer hydrogenation (Okano K,Murata K, Ikariya T, Tetrahedron Lett. 2000, 41, 9277-9280) or by using complex hydrides (Brown E, Penfornis A, Bayama J, Touet J, Tetrahedron Asymmetry, 1991, 2, 339-342; Quallich GJ, Woodall TM. Tetrahedron Lett, 1993, 34, 4145-4158).
Biochemically, chiral alcohols can be prepared the kinetic resolution of the racemic alcohol or the corresponding racemic esters by lipases (Kaneoya M, Naoyuki Y, Manabu U., US patent 4971909, Kaminska J, Gornicka I, Sikora M, Gora J, Tetrahedron Asymmetry, 1996, 7, 907-910; Orrenius C, Mattson A, Norin T, Ohrner N, Hult K, Tetrahedron Asymmetry, 1994, 5, 1363-1366), by oxidative kinetic resolution of the racemic alcohol (Fantini G, Fogagnolo M, Medici A, Pedrini P, Poli S, Gardini F, Tetrahedron Asymmetry, 1993, 4, 1607-1612) or by asymmetric reduction employing whole cells or isolated enzymes (Garrett MD, Scott RS, Sheldrake GN, Tetrahedron Asymmetry, 2002, 13, 2201-2204; Nakamura K, Takenaka K, Fujii M, Ida Y, Tetrahedron Lett., 2002, 43, 3629-3631; Bradshaw CW, Hummel W, Wong CH, J.Org. Chem., 1992, 57, 1532-1536; Uskokovic MR, Lewis RL, Partridge JJ, Despreaux DL, J. Am. Chem. Soc, 1979, 101, 6742-6744). Also, in certain cases, synthesis has been met by kinetic resolution via oxidation or deracemization of racemates by certain plant cell cultures (Takemoto M, Achiwa K, Tetrahedron Asymmetry, 1995, 6, 2925-2928).
All the chemical synthetic approaches though yielded product with good yield, even upto 100% in certain cases, as in case of Collomb et al (1998) but the give product of low enantiopurity (ee< 86%). In case of lipase catalyzed enzymatic hydrolysis of esters (Kaminska et al, 1996 and Orrenius et al, 1994), though enantiomeric excess of >95% was achieved but with low yields of merely 37-44%. In case of Kaneoya et al (us 4971909). the lipase catalysed hydrolysis of esters yielded S-alcohols with good ee but the yield was poor (as theoretically also in resolutions yield cant exceed 50) and also the reaction time was long (6-16 days). The oxidative kinetic resolution of racemic alcohol (Fantini et al, 1993) yielded a product of ee >90% (yield 70%) after 6 days of biocatalytic reaction. The asymmetric reduction of the ketone using isolated

Lactobacillus kefir alcohol dehydrogenase by Bradshaw et al yielded S-alcohol with 60% yield and > 97% ee. The whole cell bioreduction of 3-acetyl pyridine by Sporotrichum exile yielded optically pure S-alcohol at 60% yield (Uskokovic et al, 1979). The synthesis of optically pure pyridyl alcohols from the corresponding racemates using Cantharanthus roseus cell cultures by Takemoto et al (1995) gave enantiopure alcohols (ee about 100%) with a yield of 96%. But the reaction time in this case was 12-17 days.
All the known chemical synthetic processes provide the alcohols in good yield at a faster rate but with low enantiopurity. The lipase catalyzed resolution processes though provide alcohols of good enantiopurity but the yield is always less (<47%). The whole cell bioreductions reported so far also provide good enantiopurity but still cannot provide good yield. The plant cell culture based reduction processes suffer as they need long reaction times to carry out the reaction. They however provide good yields and ee.
This organism in the presentation invention is unique in that it produces an oxidoreductase biocatalyst /enzyme, which can reduce a variety of ketonic compounds (Table 1). It can carry out the reduction of a large number of ketones in a broad range of temperature (25-37 °C) and pH (3-10) in a very short time. The products obtained are of high enantiopurity.
Novelty aspect of the microbe
Normally, microbes cannot reduce wide variety of substrates or compounds. The microbe in the present case was isolated from soil and subjected to laboratory research and procedures to convert it into a microbe of industrial importance. The details on th e microbe isolation, culture and process of producing the microbe has been provided in our Indian Patent Application No. 1573/DEL/2004, filed on August 23, 2004.
Characteristics of the microbe which make it suitable as an industrial biocatalyst in a wide variety of reactions are:

i) Activity over a wide range of pH: The microbe was tested and found to
retain activity over a wide pH range (3-11), making it a highly versatile catalyst. Data is given in Fig.2
ii) High enantioselectivity:. Enantioselectivity refers to the ability of a catalyst to yield only one type of desirable optically active compound. In case of this particular microbe it was observed that enantioselectivity was more than 97% in most cases and even >99% in selected cases. Data is given in Table 1.
iii) Temperature: Working range of the microbe was found to be 25 to 37 °C. Data is given in Fig.3
iv) Concentration: Varies from .05% to 1% depending upon nature of substrate.. Data is given in Table 1
v) Time: Reaction time varies from 12 hours to 60 hours depending upon nature of substrate.. Data is given in Table 1
vi) Product recovery and yield: Recovery of the desired products can be carried out from the reaction mixture by solvent extraction using ethyl acetate. Yield varies from 88% to 99% depending upon substrate. Data is given in Table 1.
Objects of the Invention
The main object of the present invention provides a process of preparing pharmaceutical compounds involving oxido reduction by means of microbial biocatalyst.
Another object of the present invention provides a use of microbial biocatalyst for preparing pharmaceutical compounds involving oxido reduction.
Detailed Description of Accompanying Drawings/Figures
Figure 1 is a schematic representation of a general ketonic reduction process in which
the microbe can be utilized.
Figure 2. Effect of pH on bioreduction reaction Figure 3. Effect of pH on bioreduction reaction

Summary of the invention
The present invention relates to a highly efficient biocatalyst that produces oxidoreductase* capable of reducing a number of compounds. The biocatalyst is very versatile, acting over a broad range of pH and temperature conditions, making it suitable for industrial applications. More preferably the biocatalyst produces S-specific oxidoreductase for reducing large number of compounds. (* Compounds having same molecular formula but different 3 dimensional configuration are called optically active compounds or "enantiomers", because when light passes through them, its direction gets altered. Depending upon 3 dimensional arrangement, the compounds are classified as S or R. The letters do not represent any short form, but are merely internationally accepted technical terms for optically active chemical compounds)
Detailed description of the invention
The invention provides a novel process for the microbial reduction of a number of industrially important chemical compounds. The process consists of the following steps:
(a) Propagation of the culture
(b) Reduction of ketonic substrates
(c) Recovery
Thus, oxidoreductase activity of Candida tropicalis MTCC 5158 assists in the reduction of a number of ketonic substrates (mentioned in Table 1) to the corresponding optically pure S-alcohols. The conversion may be >70% in most of the cases with enantioselectivity more than 99%.

(Table Removed)
Accordingly, the main embodiment of the present invention provides a process of preparing pharmaceutical compounds involving oxido reduction by means of a microbial biocatalyst said process comprising the steps of:

a) culturing the biocatalyst in nutrient medium comprising of carbon source, nitrogen source, mineral source, for a period of 36 hrs at temperature of 30°C,
b) recovering the biocatalyst from the cultured medium of step (a) by means centrifugation and suspending them in a buffer medium having pH of 7.0,
c) incubating the isolated cells of step (b) with ketone substrates to form a reaction mixture,
d) carrying out bioreduction of reaction mixture of step (c) in the temperature range of 20°C to 35°C, for time period of 12 to 60 hrs, and
e) recovering the desired product from the reaction mixture of step (d) by conventional method solvent extraction using ethyl acetate as the solvent.
Another embodiment of the present invention relates to carbon source in step (a) is selected from group comprising of mannitol or sorbitol 0.5 to 2 %, nitrogen source in the range of peptone or yeast extract having range of 0.10% to 1 % w/v, mineral source selected from group comprising of calcium, sodium chloride, potassium chloride, zinc chloride or potassium hydrogen diphosphate in the range of 0.1 to 0.75% w/v.
Another embodiment of the present invention relates to carbon and mineral source, wherein carbon and mineral source selected are mannitol and calcium, respectively. Another embodiment of the present invention relates to ketones, wherein concentration of ketone is in the range of 0.1 to 20 g/L.
Another embodiment of the present invention relates to ketones wherein concentration of ketone is 0.5 to 10 g/L
Another embodiment of the present invention relates to pharmaceutical compounds wherein pharmaceutical compounds obtained have yield in the range of 70% to 99 %. Another embodiment of the present invention relates to biocatalyst wherein biocatalyst is a fungal microbe Candida tropicalis having accession no. MTCC 5158.

Another embodiment of the present invention relates to the microbial reduction of a number of ketones has been achieved with good yield and excellent enantiomeric excess and all the chiral products (as mentioned in Table 1) are useful chiral intermediates of immense pharmaceutical importance.
Another embodiment of the present invention relates to a use of biocatalyst fungal microbe Candida tropicalis having accession no. MTCC 5158 capable of catalyzing oxidoreduction of ketonic compounds.
Another embodiment of the present invention relates to a use wherein the microbial reduction of a number of ketones has been achieved with good yield and excellent enantiomeric excess and all the chiral products (as mentioned in Table 1) are useful chiral intermediates of immense pharmaceutical importance.
The use of microbe Candida tropicalis having accession no. MTCC 5158, as biocatalyst is very unique and unexpected finding. The present invention for the first time identifies and presents a fungal microbial biocatalyst for preparation of pharmaceutical compounds. Moreover, these pharmaceutical compounds are specifically ketone compounds. The present invention was not premised from any of the prior arts, since earlier studies have not shown identification and isolation any fungal microbe which can perform such a unexpected and surprising functions. The inventors arrived at the attributes of biocatalyst of the present invention only after they isolated and identified the microbe and undertook thorough experimental studies on microbial attributes. The present invention also derives its novelty and inventiveness by the fact, that until unless the biocatalyst was provided with specific artificial conditions (i.e. artificial environmental conditions by means of specific identified and studied medium, temperature conditions, etc.) it was not known until they were provided artificially by the inventors. Further, the very conjecture, that biocatalyst of the presentation invention is only suited for reducing only ketones and thus useful in preparing pharmaceutical compounds by reduction of ketones, is not premised from any known facts or prior arts. The inventors arrived at the present invention only after analyzing various compounds including ketones. This exercise required conscientious planning, meticulous experimentation and thorough analysis of

results. The present invention thus is not mere known use of the microbe, as the present use of the microbe was not known until the inventors decided to provide specific and selected artificial conditions to the microbe, which made the microbe perform in specific manner i.e. inventors directed microbe to perform in specific manner. Therefore, it was the share perseverance of inventors allowed the microbe to perform the unexpected and surprising functions.
The invention is now described in detail by the following examples, which are only illustrative and may not to be construed as limitations to the scope of the invention.
EXAMPLES
Example 1
Propagation of the culture of biocatalyst Candida tropicalis MTCC-5158
The first step in using the biocatalyst is propagation of the culture. The culture can be obtained in lyophilized form from the internationally recognized depository, Microbial Type Culture Collection (MTCC), Chandigarh (India), and propagated as follows:
First of all, the lyophilized cultures can be revived in nutrient broth medium containing yeast extract (0.15%, w/v), peptone (0.5%, w/v), beef extract (0.15%, w/v), sodium chloride (0.5%, w/v), glucose (l%,w/v) and acetophenone (2 mM). It is allowed to grow at 30°C at 200 rpm. Once growth is achieved, 3% (v/v) of the active inoculum is transferred to 100 ml of production medium containing yeast extract (0.65%, w/v), peptone (0.5%, w/v), beef extract (0.15%, w/v), sodium chloride (0.5%, w/v), mannitol (1%, w/v), Ca2+ (4 mM) and acetophenone (2 mM). It is allowed to grow for 36 h at 30°C at 200 rpm.
Example 2
Reduction of ketones by Candida tropicalis MTCC-5158
For the reduction of the ketones, the process is carried out as follows. The microbial cells which have already been propagated are recovered from the media after 36 hours by centrifugation. The cells are washed with a buffer such as phosphate buffer (pH 7.0, 0.2 M). Then the cell suspension is prepared in the same buffer. The amount of cells in the medium may be 150-250 g/1 of reaction mixture depending upon the

nature of the substrate. Then this cell suspension is incubated with the substrate. The concentration of the substrate in the reaction mixture varies from 0.5 - 10 g/L depending upon the nature of the substrate. The bioreduction is carried out at ambient temperature in the range of 20°C to 35°C. The time period of the reduction reaction varies from 12 to 60 hours depending upon the nature of the substrate. List of compounds (ketones) reduced by this biocatalyst, the conditions, yield and the resulting compounds (chiral alcohols) are given in Table 1. Brief description of reaction and importance of some of the resulting compounds is given below.
Example 3.
(S)- N, N-dimethyl-3-hydroxy-3-(2-thienyl)-l-propanamine (Product number 1 in Table 1) is a vital intermediate of a new antidepressant, S-Duloxetine being manufactured by Eli Lily, USA. S-Duloxetine [(S)-N-methyl-3-(l-naphthalenyloxy)-3-(2-thienyl)-l-propanamine] is regarded as the most important chiral drug desired in single enantiomeric form for the treatment of psychiatric and metabolic disorders (U.S.Patent 5,023,269). The desired pharmacological effect resides in the "S enantiomer" of Duloxetine. Hence, it is critically important for the introduction of correct enantioselectivity in the overall synthesis of Duloxetine. Existing methods of synthesis have limitations of purity and yield. Since biologically active form of Duloxetine is the "S enantiomeric form" methods, which result in, high yield of the bioactive form are highly desirable. Methods resulting in contamination of the final product with the biologically inactive "R enantiomeric form" are not desirable, because it imposes an unnecessary chemical load on the body and may have toxicological implications. Hence, it is very much desirable that more of S-form and the least R-contamination should be there. In the present method, pure S-form of the intermediate is obtained.
The key step in the enantioselective synthesis of S-Duloxetine is the enantioselective reduction of N, N-dimethyl-3-keto-3-(2-thienyl)-l-propanamine which has been tried chemically by asymmetric reductions and by certain chemo-as well as chemoenzymatic resolutions. The asymmetric reduction of N, N-dimethyl-3-keto-3-(2-thienyl)-l-propanamine has been tried using a 2:1 complex of [(2R, 3S-(+)-4-dimethylamino-l,2-diphenyl-3-methyl-2-butanol] and lithium aluminium hydride (LAH) (Deeter et al. 1990 Tetrahedron Letters.31:7101-7104) and in another method

with BH3 in the presence of oxazaborolidine (Wheeler et.al.1995 J Am Comp Radiopharm.36.213-223.1995). Both methods yield product with a moderate enantiomeric excess of 80-88%, which needs to be increased to more than 99%.
Apart from this, some methods are based on resolution processes. One of them involves resolution of 3-(methylamino)-l-(2-thienyl)-propan-l-ol using S-Mandelic acid as a resolving agent and 2-butanol containing 2-equimolar amounts of water as a solvent (Sakai et al. 2003 Tetrahedron Asymmetry.14.1631-1636) and resolution of 3-(dimethylamino)-l-(2-thienyl) propan-1-ol via distereomeric salt formation (U.S.Patent 5,362,886).
Chemical synthesis methods have the limitation that they yield bioactive product having moderate enantiomeric excess i.e. 80-88%, which is not biologically desirable. However, product yield by the chemical synthesis route is good i.e. about 80%. Another route for the synthesis of the compound is by use of enzymatic methods, involving lipase enzymes. Enzymatic resolution of 3-chloro-l-(2-thienyl)-l-propanol employing Candida antartica lipase has been described (Anthonsen et al.2000 Chirality.12 26-29). Resolution of 3-hydroxy-3-(2-thienyl)-propanenitrile has also been carried out using Pseudomonas cepacia lipase (Kamal et al. 2003 Tetrahedron Letters.44: 4783-4787). In case of enzymatic resolution of 3-chloro-l-(2-thienyl)-l-propanol employing Candida antartica lipase by Anthonsen et al (2000) the yield was 35% and enantiomeric excess 97% while resolution of 3-hydroxy-3-(2-thienyl)-propanenitrile using Pseudomonas cepacia lipase done by Kamal et al (2003) yielded 42% product of >99% enantiomeric excess.
Thus, enzymatic methods though offering the advantage of giving an enantiomeric excess >97-99%, suffer from the limitation that maximum theoretical yield possible through this route is only 50%, while yields obtained practically are still lower, only 35-42%. The present process thus offers distinct advantages in terms of purity and yield of the product. Disadvantages associated with existing processes viz. low purity and low yield have been overcome. Utilizing the present process, not only is the product of high purity (>99% enantiomeric excess) but also the yield is quite high (>80-84%).

Example 4.
(S)-l-(3-pyridyl) ethanol (Product number 3 in Table 1) is a key intermediate of akuamidine and heteroyohimidine, pharmacologically important alkaloids (Uskokovic MR, Lewis RL, Partridge JJ, Despreaux CW. J.Am.Chem.Soc, 1979, 6742-6743). (S)-l-(2-pyridyl) ethanol (Product number 2 in Table 1) and (S)-l-(4-pyridyl) ethanol (Product number 4 in Table 1) are useful as dopants, (atoms deliberately added to the substrate to change its electronic properties) which give spiral structures for liquid crystal molecules in liquid crystal compositions (Kaneoya M, Yoshida N, Uchida M. USPTO 4971909). Apart from this, chiral 1-pyridylethanols are useful chiral auxiliary. There has been considerable interest in the synthetic applications of chiral 1-pyridylethanols. Examples include chiral ligands in the zinc-catalysed asymmetric alkylation of aldehydes (Ishizaki M, Hoshino O. Chem. Lett. 1994, 1337-1340) and involvement in asymmetric hydroboration reactions (Quallich GJ, Woodall TM. Tetrahedron Lett. 1993, 34, 4145-4158). Also pyridylalcohols have proved to be an efficient catalyst in the asymmetric addition of dialkyl zinc to benzaldehyde (Collomb P, Zelewsky A., Tetrahedon Asymm. 1998, 9, 3911-3917).
These alcohols have been prepared using the chemical and biochemical methods. Examples of chemical methods are asymmetric alkylation of aldehydes (Collomb P, Zelewsky A., Tetrahedon Asymm. 1998, 9, 3911-3917.), the asymmetric reduction of the corresponding ketones via transfer hydrogenation (Okano K,Murata K, Ikariya T, Tetrahedron Lett. 2000, 41, 9277-9280) or by using complex hydrides (Brown E, Penfornis A, Bayama J, Touet J, Tetrahedron Asymmetry, 1991, 2, 339-342; Quallich GJ, Woodall TM. Tetrahedron Lett, 1993, 34, 4145-4158).
Biochemically, these alcohols can be prepared the kinetic resolution of the racemic alcohol or the corresponding racemic esters by lipases (Kaneoya M, Naoyuki Y, Manabu U., US patent 4971909, Kaminska J, Gornicka I, Sikora M, Gora J, Tetrahedron Asymmetry, 1996, 7, 907-910; Orrenius C, Mattson A, Norin T, Ohrner N, Hult K, Tetrahedron Asymmetry, 1994, 5, 1363-1366), by oxidative kinetic resolution of the racemic alcohol (Fantini G, Fogagnolo M, Medici A, Pedrini P, Poli S, Gardini F, Tetrahedron Asymmetry, 1993, 4, 1607-1612) or by asymmetric reduction employing whole cells or isolated enzymes (Garrett MD, Scott RS, Sheldrake GN, Tetrahedron Asymmetry, 2002, 13, 2201-2204; Nakamura K,

Takenaka K, Fujii M, Ida Y, Tetrahedron Lett., 2002, 43, 3629-3631; Bradshaw CW, Hummel W, Wong CH, J.Org. Chem., 1992, 57, 1532-1536; Uskokovic MR, Lewis RL, Partridge JJ, Despreaux DL, J. Am. Chem. Soc., 1979, 101, 6742-6744). Also, in certain cases, its synthesis has been met by kinetic resolution via oxidation or deracemization of racemates by certain plant cell cultures (Takemoto M, Achiwa K, Tetrahedron Asymmetry, 1995, 6, 2925-2928).
All the chemical synthetic approaches though yielded product with good yield, even upto 100% in certain cases, as in case of Collomb et al (1998) but the give product of low enantiopurity (ee< 86%). In case of lipase catalyzed enzymatic hydrolysis of esters (Kaminska et al, 1996 and Orrenius et al, 1994), though enantiomeric excess of >95% was achieved but with low yields of merely 37-44%. In case of Kaneoya et al (US 4971909) the lipase catalysed hydrolysis of esters yielded S-alcohols with good ee but the yield was poor (as theoretically also in resolutions yield cant exceed 50) and also the reaction time was long (6-16 days) . The oxidative kinetic resolution of racemic alcohol (Fantini et al 1993) yielded a product of ee >90% (yield 70%) after 6 days of biocatalytic reaction. The asymmetric reduction of the ketone using isolated Lactobacillus kefir alcohol dehydrogenase by Bradshaw et al yielded S-alcohol with 60% yield and > 97% ee. The whole cell bioreduction of 3-acetyl pyridine by Sporotrichum exile yielded optically pure S-alcohol at 60% yield (Uskokovic et al 1979). The synthesis of optically pure pyridyl alcohols from the corresponding racemates using Cantharanthus roseus cell cultures by Takemoto et al (1995) gave enantiopure alcohols (ee about 100%) with a yield of 96%. But the reaction time in this case was 12-17 days. All the known chemical synthetic processes provide the alcohols in good yield at a faster rate but with low enantiopurity. The lipase catalyzed resolution processes though provide alcohols of good enantiopurity but the yield is always less (<47%). The whole cell bioreductions reported so far also provide good enantiopurity but still cannot provide good yield. The plant cell culture based reduction processes suffer as they need long reaction times to carry out the reaction. They however provide good yields and ee.
The present invention makes use of whole resting cells of a novel yeast strain Candida tropicalis to carry out the bioreduction of 2-, 3- and 4-acetyl pyridine to produce of (S)-l-(2-pyridyl) ethanol, (S)-l-(3-pyridyl) ethanol and (S)-l-(4-pyridyl)

ethanol with good conversion (>99%) and excellent enantioselectivity (ee>99%). Moreover this microbial process can tolerate high substrate concentration (10 g/L) and occurs at a very fast rate (reaction time 12 hours).
Example 5.
(S)-l-(l'-napthyl) ethanol (Product number 5 in Table 1) is reported to be an important intermediate in the production of keto phosphonate, which is suitable synthon for the lactone moiety of hydroxy methyl glutaryl co-enzyme A reductase inhibitors (Thiesen, P. and Heathcock, C. 1988, J Org Chem. 53: 2374-2378.).
The present microbial process makes use of whole resting cells of a novel yeast strain Candida tropicalis to carry out the reduction of 1-acetonapthone to produce (S)-l-(l'-napthyl) ethanol with good conversion (>85%) and excellent enantioselectivity (ee>99%). Another advantage of the process is that it occurs at a very fast rate (reaction time 12 hours).
Example 6
Other Products viz. (S)-l-(2-thienyl) ethanol, (S)-l-(2-furyl) ethanol, (S)-l-(2-pyrroyl) ethanol, (S)-l-phenyl ethanol, (S)-2-fluoro-l- phenyl ethanol, (S)-3-fluoro-l-phenyl ethanol, (S)-4-fluoro-l- phenyl ethanol, (S)-4-chloro-l-phenyl ethanol, (S)-4-bromo-1 -phenyl ethanol, (S)-4-iodo-l-phenyl ethanol, (S)-4-acetoxy-l-phenyl ethanol, (S)-4-hydroxy-l-phenyl ethanol (Products no. 6-17 in Table 1) are useful chiral auxiliaries and fine chemicals being synthesized by chemical industries using chemical catalysts which are expensive, environmentally toxic and give products of low chiral purity. The present microbial process employing the above said microbe gives product of very high enantiopurity in all the cases and also the process is environment friendly.
Example 7.
Recovery of the product
After the reaction is completed, the cells are separated out from the reaction mixture by centrifugation (10,000 rpm, 10 min). Then supernatant is extracted with ethyl acetate and the solvent is evaporated under reduced pressure. The product is obtained as a viscous liquid/oily mass.

The main advantages of the present invention are:
1. Limitations of existing chemical and enzymatic methods regarding low purity and low yield have been overcome.
2. Present method offers advantages in that the oxidoreductase mediated bioreduction of a number of ketonic substrates (listed in table 1) is always advantageous over synthetic chemical route, since the reaction can be performed at ambient temperature and pressure, resulting in lowering of production costs.
3. The yield and enantioselectivity of the product is also very high.
References
1. Collomb P, Zelewsky A. (1998, Tetrahedon Asymm, vol. 9, page 3911-3917)
2. Okano et al (2000, Tetrahedron Lett vol. 41, page 9277-9280)
3. Brown et al (1991, Tetrahedron Asymmetry, vol. 2, page 339-342)
4. Quallich et al (1993, Tetrahedron Lett, vol. 34, page 4145-4158);.
5. Kaneoya M, Naoyuki Y, Manabu U. (US patent 4971909)
6. Kaminska et al (1996, Tetrahedron Asymmetry, vol. 7, page 907-910)
7. Orrenius et al (1994, Tetrahedron Asymmetry, vol. 5, page 1363-1366)
8. Fantini et al (1993, Tetrahedron Asymmetry, vol. 4, page 1607-1612
9. Garrett et al (2002, Tetrahedron Asymmetry, vol. 13, page 2201 -2204)
10. Nakamura et al (2002, Tetrahedron Lett, vol. 43, page 3629-3631)
11. Bradshaw et al (1992, J.Org. Chem, vol. 57, page 1532-1536)
12. Uskokovic et al (1979, J. Am. Chem. Soc, vol. 101, page 6742-6744)
13. Takemoto et al (1995, Tetrahedron Asymmetry, vol. 6, page 2925-2928).
14. Deeter et al (1990, Tetrahedron Letters, vol..31, page 7101-7104 )
15. Wheeler et al (1995, J Am Comp Radiopharm, vol.36, page.213-223)
16. U.S.Patent 5,362,886
17. Anthonsen et al (2000, Chirality, vol. 12, page 26-29)
18. Kamal et al (2003, Tetrahedron Letters, vol. .44, page 4783-4787)
19. Uskokovic et al (1979, J.Am.Chem.Soc , page 6742-6743)
20. Kaneoya et al USPTO 4971909
21. Ishizaki M, Hoshino (1994, O. Chem. Lett, 1337-1340)
22. Quallich GJ, Woodall TM (1993, Tetrahedron Lett., vol. 34, page 4145-4158 )
23. Collomb P, Zelewsky A. (1998, Tetrahedon Asymm., vol. 9, page 3911-3917)
24. Okano K, Murata K, Ikariya T (2000, Tetrahedron Lett vol. 41, page 9277-9280)
25. Brown et al (1991, Tetrahedron Asymmetry, vol. 2, page 339-342)
26. Quallich GJ, Woodall TM. T (1993, Tetrahedron Lett., vol. 34, page 4145-4158)
27. Kaneoya et al US patent 4971909,
28. Kaminska et al(1996, Tetrahedron Asymmetry, vol. 7, page 907-910)
29. Orrenius et al (1994, Tetrahedron Asymmetry, vol. 5, page 1363-1366)
30. Fantini et al 1993, Tetrahedron Asymmetry, vol. 4, page 1607-1612)
31. Garrett MD, Scott RS, Sheldrake GN, 2002, Tetrahedron Asymmetry, vol. 13, page 2201-2204)
32. Nakamura et al (2002, Tetrahedron Lett. vol. 43, page 3629-3631)
33. Bradshaw et al (1992, J.Org. Chem. vol. 57, page 1532-1536)
34. Uskokovic et al (1979, J. Am. Chem. Soc, vol. 101, page 6742-6744)
35. Takemoto M, Achiwa K (1995, Tetrahedron Asymmetry, vol. 6, page 2925-2928)

We claim:
1. A process of preparing optically pure S-alcohols such as herein described,
involving oxido-reduction of compounds by means of a microbial catalyst,
wherein the microbial catalyst is the cells of yeast Candida tropicalis having
accession no. MTCC 5158, the said process involves the oxido-reduction of
ketonic substrates such as herein described and comprises the steps of:
a) culturing the cells of Candida tropicalis having accession no. MTCC 5158 in nutrient medium comprising of carbon source, nitrogen source, mineral source, for a period of 36 hours at temperature of 30°C,
b) recovering the cells of Candida tropicalis having accession no. MTCC 5158 from the cultured medium of step (a) by means of centrifugation and suspending them in a buffer medium having pH of 7.0,
c) incubating the cells of step (b) with ketonic substrates to form a reaction mixture,
d) carrying out bio-reduction of reaction mixture of step (c) in the temperature range of 20°C to 35°C, for time period of 12 to 60 hrs, and
e) recovering the S-alcohols from the reaction mixture of step (d) by solvent extraction using ethyl acetate as the solvent.

2. A process as claimed in claim 1, wherein the nutrient medium in step (a) comprises of the carbon source selected from a group comprising of mannitol or sorbitol, nitrogen source is peptone in the range of 0.5 to 2 % or yeast extract in range of 0.10% to 1 % w/v, mineral source selected from group comprising of calcium, sodium chloride, potassium chloride, zinc chloride or potassium hydrogen diphosphate is in the range of 0.1 to 0.75 % w/v.
3. A process as claimed in claim 2, wherein the preferred carbon and mineral source are mannitol and calcium, respectively.
4. A process as claimed in claim 1, wherein concentration of ketonic substrates is in the range of 0.I to 20glL

5. A process as claimed in claim 4, wherein concentration of ketonic substrates is 0.5 to 10 g/L
6. A process as claimed in claim 1, wherein the product S-alcohols obtained has yield in the range of 70% to 99 %.
7. A process as claimed in claim 1, wherein the obtained S-alcohols have optical purity of >97%.