TITLE OF THE INVENTION Enzymatic resolution of (= ! =) methyl trans-3-phenyl glycidate TECHNICAL FIELD OF THE INVENTION This invention relates to the enzymatic resolution of trans-3- phenylglycidyl esters with high enantiomeric excess using lipozyme (a class of hydrolytic enzyme) and the synthesis of novel chiral allcyl trans- phenylglycidates. The process involves transesterification of methyl trans-3-phenylglycidate with various alcohols at ambient temperature to obtain (2R, 3S) methyl trans-phenylglycidate and (2S, 3. R) alkyl trans- phenylglycidate. Study is also focussed on the effect of solvents, reaction conditions, length of the alkyl chain on the entioselectivity of the glycidate.

BACKGROUND OF THE INVENTION The esters of 3-phenylglycidic acid are lnown compounds, described in literature and widely used as synthetic intermediates.

Methyl trans-3-phenylglycidate is a useful intermediate for the synthesis of C-13 side chain (phenyl isoserine) of taxol, an anticancer drug.

Synthesis of taxol using phenyl isoserine has been reported in Product Letters, Volume 6, 147.

The presence of the taxol side chain is very important for the antitumor activity of taxol. Hence, it is very essential to develop an enantioselective synthesis route for phenylisoserine, which can be adopted for large-scale production. The convenient and economical way of synthesising phenylisoserine is from optically active trans-3- phenylglycidic esters. These chiral glycidates can be made by the resolution of the racemic mixture by chemical or enzymatic methods.

Previously W. E. Ladner and G. M. Whitesides had employed enzymatic hydrolysis in the resolution of carboxylic acid esters to chiral

epoxy alcohols : J. Am Chem. Soc. , 106,7250-7251 and US Pat. No 4, 732, 853 (1988). US Pat. No 4923810 (1990) by A. E. Walts and E. M.

Fox describes the resolution of glycidyl esters to high enantiomeric excess employing lipases to selectively hydrolyse one enantiomer of the glycidyl esters. Lipase catalysed esterifications and transesterifications in organic solvents were pioneered by Klibanov et al. (Zaks and Klibanov in Science, 224, 1249-1251 ; A. M. Klibanov in Trends in Biochemical Sciences, 1989,14, 141-144 and A. M. Klibanov in Acc. Chem. Res.

1990, 23, 114-120). Enzymatic transformations in organic solvents offer advantages like carrying out transesterifcation reactions instead of hydrolysis. Moreover, the selectivity of lipase can be enhanced by changing the organic solvent, which is sometimes called as solvent (medium) engineering. Also moisture sensitive compounds could be resolved in organic solvents. Additionally, the separation of the hydrophilic product from organic solvents is easier than from water.

Apart from the more conventional procedures of resolution, that consist of transforming the racemic mixture into diastereoisomeric mixture by interaction with an enantiomerically pure resolving agents, a particularly important group of chiral catalysts is constituted by enzymes whose use as resolving agents has only recently been recognized and developed [A. Zaks et al. in Drugs Discovery Today (1997). 2,513].

Da-Ming Gou et al in J. Org. Chem (1993), 58, 1287 had achieved the synthesis of chiral trans-3-phenylglycidate esters by lipase mediated transesterification of the racemic ester in organic solvents. Different lipases were used for this resolution and lipase from mucor miechei gave high optical purity and chemical yields. This method had some disadvantages like incubation was required and the reaction needed to be carried out at 50°C. Moreover the products were isolated using column

chromatography. Also the substrate after isolation was again subjected to re-incubation, which made the whole process difficult to be scaled up.

Da-Ming et al had also described the conversion of both the l,' resolved isomers'ofmethyl-trans-3-phenylglycidate ; to (2R, 3S)- (-)-N- benzoyl-3phenylisoserine methyl ester which is the C-13 side chain of taxol.

Tanabe in JP06/078790 reports a similar process of kinetic resolution of phenylglycidates catalysed by esterases in which alcohol (C2-Clo) are used in the transesterification reaction. Here (2R, 3S)-3- (4- methoxyphenyl) glycidate was obtained with high optical purity; however this method involved chromatographic techniques for the separation of the esters formed.

Tanabe Seiyaku Co. Limited in JP08259552A2 describes a process to obtain optically active phenylglycidate by subjecting a racemic mixture of trans-3 phenylglycidate with an alcohol in the presence of esterases enzyme. The entire process may not be cost- effective at industrial scales, as it does not utilize supported enzymes for the transesterification.

US patent no. 6187936 B I discloses the process for the enzymatic kinetic resolution of 3-phenylglycidates by transesterification with aminoalcohols. In this process, optically active (2R, 3S) methyl trans 3- phenyl glycidates were obtained and the separation of the other amino ester formed during the above transesterification was achieved by acidification. However this process does not mention about the optical and chemical yield of the amino esters thus formed. The lower alcohols used in the process may not facilitate the easy separation of the esters formed, possibly making it difficult to determine the chemical and optical purity of the isomers.

In recent years the research is focussed on the use of immobilized enzymes for various transformations. The inherent advantages of immobilized enzyme is the enhanced stability and its more resistance towards denaturation.

Hence most of the reported processes for resolution of 3- phenylglycidates are either not cost-effective or industrially impractical as they do not utilize supported enzymes or require higher than ambient reaction temperatures or require chromatographic methods or render difficult analytical interpretation.

The object of the present invention is therefore to provide a novel process for enzymatic resolution of trans-3-phenylglycidyl esters with high enantiomeric excess.

It is a further object of the invention to provide such enzymatic resolution process that can be practised cost-effectively at commercial scale.

It is yet an object of the invention to obtain novel optically active trans-3-phenylglycidic esters that can be employed for the synthesis of Taxol.

Summary of the invention To achieve the said objects, the present invention comprises of a method for preparing (2S, 3R) alkyl trans-3-phenylglycidate or (2R, 3S) methyl traw1s-3-phenylglycidate comprising: (i) enzymatic resolution by trans-esterification of methyl trans-3-phenylglycidate with enantiomeric excess using lipozyme in the presence of a transesterification agent and an organic solvent to obtain (2R, 3S) methyl trans-3- phenylglycidate and (2S, 3R) alkyl trans-3-phenylglycidate and

(ii) isolating (2S, 3R) alkyl trans-3-phenylglycidate from (2R, 3S) methyl trans-3-phenylglycidate.

The reaction is carried out at ambient temperature. The transesterification agent is an alcohol selected from butanol, pentanol, hexanol, octanol and decanol, preferably octanol.

The organic solvent is selected from toluene, tetrahydrofuran (THF) and chloroform and more preferably toluene.

The amount of lipozyme is about 5-15 % by weight with respect to the substrate, preferably about 15 %.

In step (ii), (2S, 3R) alkyl trans-3-phenylglycidate is isolated from (2R, 3S) methyl trans-3-phenylglycidate by vacuum distillation.

The methanol released from the trans-esterification reaction is removed under vacuum.

The molar ratio of methyl tra7ls-3-phenylglycidate to trans- esterification agent is 1.0 This invention also includes novel (2S, 3 R) alkyl trans- phenylglycidates of the formula : where R = phenyl, hexyl, octyl and decyl Detailed Description of the invention The present invention relates to a method for preparing chiral trans-3-phenylglycidate esters with high enantiomeric excess, by enzymatic transesterifcation. Ph ° Lipozyme 0 COOCH3 Ph* 0 ROH ROH Ph'COOR COOMB COOMe (2R, 3S) (2S, 3R)

More specifically the invention pertains to a method for producing (2R, 3S) methyl trans-3-phenylglycidate and (2S, 3R) alkyl trans-3- phenylglycidate (alkyl = pentyl, hexyl, octyl, decyl) with high enantioselectivity. The reaction was carried out in different solvents such as chloroform, THF and toluene. All these reactions were performed at 25-30°C temperature. The quantity of enzyme used in the initial study was about 15 % by weight with respect to the substrate. Octanol was used as the transesterification agent. The reaction time was kept constant and the progress of the reaction was monitored by chiral HPLC.

Table 1 S. No Solvent Reaction Conversion (2S, 3R)-octyl- trans-3- time (%) phenyl glycidate (h) ee (%) 1. Toluene 23 23.09 90 2 THF 23 22 86. 32 3 ; Chloroform 23 21 84. 33 Among the three different solvents used for the resolution, toluene gave better results as shown in'Table 1.

As the inhibitory nature of methanol is well known for enzymatic reactions it was thought worthwhile to remove methanol by applying vacuum. Methanol removal could not only enhance the reaction rate but also improve the enantioselectivity (Table-2). For this study toluene was chosen as the solvent.

Table 2 S. No Reaction Condition Conversion (2S, 3R)-octyl-trans- 3- time (h) (%) phenylglycidate ee (%) 1. 26 Without methanol 23.0 93 removal With methanol 2 26 42.0 93.21 removal

It was observed that the transesterification rate and the conversion (%) were increased when methanol was stripped off from the reaction medium.

The amount of enzyme required for the reaction was studied and optimised as shown in Table 3. The resolution of (methyl trans-3- phenylglycidate was carried out by varying the enzyme quantity like 15 %, 10 % and 5 % with respect to the substrate with octanol as a transesterification agent and toluene as the solvent.

Table 3 S. No Enzyme Reaction Conversion wu (%) Time (h) (%) 1.15 26 43 2 10 26 41.7 3 5 26 41

The table clearly indicates that only 5 % of enzyme was necessary for the resolution.

Various alcohols were used for the transesterification studies. The results are summarised in Table 4 Table-4

S. No Alcohol Reaction time Conversion (2S, 3R)-alkyl-trans- (h) (%) 3-phenylglycidate ee (%) 1. Butanol 4 14 83.3 2 Pentanol 32 37.23 85.4 3 Hexanol 26 37.39 94.0 4 Octanol 26 42 93.6 5 Decanol 31 41.55 93.6 The results indicates that transesterification with lower alcohol proceeded with low enantioselectivity than the higher analogs.

General method used for resolution is described as follows. The results described here relate primarily to the resolution of methyl trans- phenylglycidate, the invention may be applied to other racemic phenylglycidates also. The enzymatic transesterifications were carried out with octanol at 25-30°C temperature in toluene as the solvent with constant removal of methanol under vacuum. (2R, 3S) -methyl trans- phenylglycidate was separated from (2S, 3R) -alkyl trans-phenylglycidate by vacuum distillation.

In order to enhance the optical purity of (2R, 3S) -methyl trans- phenylglycidate the lipase catalyzed transesterification with octanol was allowed to reach close to 50 % conversion. Once close to 50 % conversion was achieved, the reaction was quenched by filtering off the enzyme; the methyl isomer thus isolated showed 92 % ee as shown in table 5.

Table-5

S.No Alcohol Reaction time Conversion (2R, 3S)-methyl-trans- (h) (%) 3-phenylglycidate ee(%0 1 Octanol 62 49.5 92 The following Examples further illustrate the invention, it being understood that the invention is not intended to be limited by the details disclosed therein.

Example: 1 : General procedure for the transesterification of racemic methyl trans-3-phenyl glycidate with octanol in toluene Racemic methyl trans-3-phenylglycidate (10 g) was taken in toluene (6v). 0.5 g of lipozyme and ocatanol (1 equiv.) were added and stirred at 25-30 °C. Methanol fonned in the reaction was removed by applying vacuum to the reaction mixture. The progress of the reaction was monitored by chiral HPLC. After 26 h nearly 43% of (2S, 3R)-octyl- trans-3-phenyl glycidate isomer was formed with the chiral purity of > 90%. The enzyme was filtered and the solvent was concentrated. The (2R, 3S) methyl tra7s-3-phenylglycidyl was separated from (2S, 3R) octyl trans-3-phenylglycidate by high vacuum distillation.

The transesterification with various alcohols were also carried out by the above method.

(2R, 3S) Methyl trans-3-phenylglycidate: Colourless liquid, [a] 25 D-52.0 [c 1, chloroform] Chiral purity: 85. 33 %, IR: 3034, 2954, 1752, 1440, 1292,1209. Mass: m/z, 178, 121, 91, 77,51 Base

peak: 121, 1H NMR : # 4. 1 (1H, d, J=1. 8 Hz), 3.55 (1H, d, J=1. 88 Hz), 3. 8 (3H, m), 1.8 (2H, m), 1.36 (4H, m), 7.32 (5H, m).

(2S, 3R) Octyl trans-3-phenylglycidate: Yellow liquid, [a] 25 D +96.0 [c 1, chloroform], Chiral purity: 94.46 %, IR: 3019,2926, 2928, 1744, 1217, Mass: m/z 276,219, 164,146, 136,118, 107, Base peak: 106. 90, 1H NMR : # 3. 5 (1H, d, J=1. 69 Hz), 4.08 (1H, d, J=1.69 Hz), 4.2 (2H, m), 1.7 (2H, m), 1.3 (IOH, m), 0.882 (3H, t), 7.3 (5H, m).

The transesterification with various alcohols were also carried out by the above method and the (2S, 3R) alkyl trans-3-phenylglycidates were isolated and characterized.

(2S, 3R) Pentyl trans-3-phenylglycidate : Pale yellow liquid, [a] 25D + 114. 0 [c 1, chloroform] Chiral purity : 87. 13 %, IR: 3019, 2926, 2928, 1744, 1217, Mass : m/z 236,179, 164, 149, 131, 107,90, 66,56.

Base peak: 106. 9, 1H NMR : 6 4.205 (1H, d, J=1. 7 Hz), 3.52 (1H, d, J=1. 7ho), 4.4 (2H, m), 1. 8 (2H, m), 1.36 (4H, in), 0. 98 (3H, t), 7.32 (5H, m).

(2S, 3R) Hexyl-trans-3-phenylglycidate: Pale yellow liquid [α] 25 D +109.6 [c 1, chloroform], Chiral purity: 95. 81 %, IR : 3066, 3035, 2957, 2931, 2859,1749, 1460, 1289, 1195 cm~l, Mass : m/z, 248, 191, 164, 149, 131, 107, 105,90, 65,55, Base peak : 106. 85,'H NMR : 6 4.11 (1H, d, J=1.7 Hz), 3.5 (IH. d, J=1.7 Hz), 4.2 (2H, m), 1.7 (2H, m), 1.34 (6H, m), 0.94 (3H, t), 7.32 (5H, m).

(2S, 3R)-Decyl trans-3-phenylglycidate : Yellow liquid, [a] 25 D +91.6 [c 1, chloroform], Chiral purity: 93.12 %, IR: 3066,3035, 2926, 2855, 1751,1459, 1289, 1195cm', Mass: m/z, 304, 247, 164, 146, 131, 107,90, 77,57, Base peak: 107,'H NMR : # 4.11 (1H, d, J=1.7 Hz),

3. 53 (1H, d, J=1. 7 Hz), 4.24 (2H, m), 1.75 (2H, m), 1.29 (14H, m), 0.9 (3H, t), 7.31 (5H, m).

Example 2: Procedure for enhancing the optical purity of (2R, 3S) methyl trans-3-phenyl glycidate by transesterification of racemic methyl tfafzs-3-phenyl glycidate with octanol in toluene Racemic methyl trans-3-phenylglycidate (10 g) was taken in toluene (6v). 0.5 g of lipozyme and ocatanol (1 equiv. ) were added and stirred at 25-30°C. Methanol formed in the reaction was removed by applying vacuum to the reaction mixture. The progress of the reaction was monitored by chiral HPLC. When this lipase catalyzed transesterification reached about 50 % conversion in 62h, the reaction was quenched by filtering off the enzyme and the (2R, 3S) methyl trans- 3-phenylglycidate was separated from (2S, 3R) octyl trans-3- phenylglycidate by high vacuum distillation and both the esters were characterized. (2R, 3S) methyl trans-3-phenylglycidate thus isolated showed 92 % ee.

(2R, 3S) Methyl trans-3-phenylglycidate: Colourless liquid, [α]25D - 122. 5 [c 1, chloroform] Chiral purity : 96.14 %, IR : 3034, 2954, 1752,1440, 1292, 1209. Mass : m/z, 178, 121, 91,77, 51 Base peak : 121,'H NMR : 8 4.1 (1H, d, J=1. 8 Hz), 3.55 (1H, d, J=1. 88 Hz), 3. 8 (3H, m), 1. 8 (2H, m), 1.36 (4H, m), 7.32 (5H, m).

(2S, 3R) Octyl trans-3-phenylglycidate : Yellow liquid, [a] 25 D +66.7 [c 1, chloroform], Chiral purity: 85.22%, IR: 3019,2926, 2928, 1744, 1217, Mass: m/z 276, 219,164, 146,136, 118, 107, Base peak: 106. 90, 1H NMR : 8 3.5 (1H, d, J=1. 69 Hz), 4. 08 (1H, d, J=1. 69 Hz), 4.2 (2H, m), 1. 7 (2H, m), 1.3 (10H, m), 0.882 (3H, t), 7.3 (5H, m).