Title of Invention	AN ENZYMATIC METHOD FOR THE PREPARATION OF AN ORGANIC ACID ESTER OF 4-T-BUTY LCYCLOHEXANOL
Abstract	The present invention relates to an enzymatic method for the preparation of an organic acid ester of 4-t-butylcyclohexanol. The process relates to an enzymatic method for the preparation of an organic acid ester of 4-t-butylcyclohexanol, where 4-t-butylcyclohexanol is used as an anchored substrate in peracetylated ß-cyclodextrin. The process steps are: reacting 4-t-butylcyclohexanol with an organic acid of carbon chain length ranging from C2 - C18, or its anhydride in a ratio of 4-t-butylcyclohexanol to organic acid ranging from 1:1 to 1:10, refluxing the above reaction mixture with 50 - 250 mol of nonpolar organic solvent at a temperature ranging from 50 - 80°C for a period of 24-120hrs, in the presence of 0.05 - 2 equivalents of peracetylated P-cyclodextrin with respect to 4-t-butylcyclohexanol concentration and porcine pancreas lipase, and removing the water formed as a byproduct of reaction and recovering the product by known methods.

Title of Invention

AN ENZYMATIC METHOD FOR THE PREPARATION OF AN ORGANIC ACID ESTER OF 4-T-BUTY LCYCLOHEXANOL

Abstract

The present invention relates to an enzymatic method for the preparation of an organic acid ester of 4-t-butylcyclohexanol. The process relates to an enzymatic method for the preparation of an organic acid ester of 4-t-butylcyclohexanol, where 4-t-butylcyclohexanol is used as an anchored substrate in peracetylated ß-cyclodextrin. The process steps are: reacting 4-t-butylcyclohexanol with an organic acid of carbon chain length ranging from C2 - C18, or its anhydride in a ratio of 4-t-butylcyclohexanol to organic acid ranging from 1:1 to 1:10, refluxing the above reaction mixture with 50 - 250 mol of nonpolar organic solvent at a temperature ranging from 50 - 80°C for a period of 24-120hrs, in the presence of 0.05 - 2 equivalents of peracetylated P-cyclodextrin with respect to 4-t-butylcyclohexanol concentration and porcine pancreas lipase, and removing the water formed as a byproduct of reaction and recovering the product by known methods.

Full Text	The present invention relates to an enzymatic method for the preparation of an organic acid ester of 4-t-butylcyclohexanol. The present invention particularly relates to an enzymatic method for the preparation of an organic acid ester of 4-t-butylcyclohexanol, where 4-t-butylcyclohexanol is used as an anchored substrate in peracetylated ß-cyclodextrin Esters of 4-t-butylcyclohexanol find wide applications as flavoring, fragrance and perfumery raw materials (Hooper D C, Johnson G A, Peter D and Rennie G K, Brit. UK Patent Appl. 2020 977, 1979; Moeddel, R W, Procter and Gamble Co., US Patent 4515705, 1985). They are used in soaps, detergents, creams, lotions and perfumes (Opdyke D L J, Food Cosmet. Toxicol., 1978, 16, Supl. 1, 657). They are also used as solubility reducing agents for surfactants (Kacher M L, Procter and Gamble Co., Eur. Patent Appl. 18679, 1980). 4-t-Butylcyclohexanol exists as cis and trans isomers. At room temperature, the trans/cis ratio of the alcohol is ~ 3:1 (Karger B L, Stern R L and Zannucci J F, Anal. Chem. 40 (4), 727 - 735, 1968). The esters of the cis and trans isomers exhibit different flavor notes. The trans esters have a woody odor, while the cis isomers have a stronger, intense flowery note (Podberezina N S, Kheifits L A, Aref eva L I, Klimakhina S T and Mindlin L O, Maslo - Zhir. Prom - St, 1985, 3, 24 - 5, Russian, Chem. Abstr. No. 103: 87528v). Some chemical methods are available for the preparation of the esters of 4-t-butylcyclohexanol. Reference may be made to Bollmann M and Kroning E of Bayer A G, German Patent 864 593, 1948, wherein synthesis of the acetate ester of 4-t-butylcyclohexanol is carried out by the hydrogenation of 4-/-butylphenol and subsequent acetylation. The major drawbacks of this method is that the reaction conditions are not milder and involve use of toxic solvents. The ester is sold under the trade name of Oryclon™ by Haarmann and Reimer, Germany. Reference may be made to Yokogawa A, Anezaki T, Yamasaki H and Ozawa K, Maruzen Sekiyo Giho 16, 77-87, 1871, (Japanese, Chem. Abstr. No. 80:14700s). This is a chemical method wherein cis- 4-t-butylcyclohexyl acetate is prepared from 4-t-butylphenol, where the cis isomer of the alcohol itself is first preferentially prepared by selective hydrogenation catalysts like Rhodium and Ruthenium, and this is subsequently acetylated. . The major drawbacks of this method is that the reaction conditions are not milder, involve use of toxic solvents and lead to tedious work out procedures. Reference may be made to Karger B L, Stern R L and Zannucci J F, Anal. Chem. 40(4), 727 - 735, 1968, where syntheses of esters of 4-t-butylcyclohexanol with organic acids from acetic to heptanoic acid have been carried out by chemical means. The esters were prepared by reacting 100 mmol of 4-t-butylcyclohexanol with 150 mmol of the appropriate acid chloride on a steam bath for 2h with stirring. The trans/cis ratio of acetate, propionate, n-butyrate, valerate, caproate, heptylate, isobutyrate and pivalate remained at approximately 3.5:1 in all the cases. The major drawbacks of this method are that it requires the use of acid chlorides, stringent reaction conditions and toxic solvents. Reference may be made to Pattekhan H H and Divakar S.Indian Patent, 2000, 422del2001 who have used ß-cyclodextrin for the encapsulation of 4-t-butylcycIohexanol and subsequently acetylated the alcohol in order to achieve better trans/cis ratio. The maximum trans/cis ratio of esters obtained by this method was 5.5. The major drawbacks of this method are that it does not involve milder reaction conditions and require use of toxic solvents. No alternate methods of preparation of these esters of 4-t-butylcyclohexanol other than the chemical methods described above are available. No reports exist till date on the enzymatic method of synthesis of the esters of 4-t-butylcyclohexanol. The present investigation provides an enzymatic method for the preparation of esters of 4-t-butylcyclohexanol with organic acids. The present invention therefore describes an improved enzymatic procedure for the esterification of 4-t-butylcyclohexanol, in which 4-t-butylcyclohexanol is used as a substrate anchored in peracetylated ß-cyclodextrin. Peracetylated ß-cyclodextrin has been used instead of ß-cyclodextrin as such, because the free hydroxyl groups on P-cyclodextrin can also get esterified when an esterification catalyst like lipase is present in the reaction medium along with an organic acid. Inclusion of 4-t-butylcyclohexanol inside the peracetylated ß-cyclodextrin cavity preferentially facilitates the esterification of the trans isomer of the alcohol. The cis isomer of the alcohol is stearically hindered from reacting with the incoming acyl group due to the presence of bulky acetyl groups on peracetylated P-cyclodextrin. The novelty of the present invention is that it is an alternate method to chemical synthesis, wherein 4-t-butylcyclohexanol is anchored in peracetylated ß-cyclodextrin for esterification by Upases, which not only promotes esterification under milder reaction conditions, but also appreciably alters the trans/cis ratio of the isomeric esters formed. The main object of the present invention is to provide an enzymatic method for the preparation of an organic acid ester of 4-t-butylcyclohexanol, which obviates the draw backs as described above. Yet another object of the present invention is to carry out the reaction in a water free environment, where the non-polar solvent used forms an azeotrope with water at temperatures between 50 -80°C. Still another object of the present invention is to use lipases from porcine pancreas, Rhizomucor miehei, Candia cylindracea, Pseudomonas fluorescens, wheat germ lipase and chicken liver esterase as esterification catalysts. Accordingly, the present invention provides an enzymatic method for the preparation of an organic acid ester of 4-t-butylcyclohexanol which comprises: reacting 4-t-butylcyclohexanol with an organic acid of carbon chain length ranging from Ca - C]g, or its anhydride in a ratio of 4-t-butylcyclohexanol to organic acid ranging from 1:1 to 1:10, refluxing the above reaction mixture with 50 - 250 mol of nonpolar organic solvent at a temperature ranging from 50 - 80°C for a period of 24-120 hrs, in the presence of 0.05 - 2 equivalents of peracetylated p-cyclodextrin with respect to 4-t-butylcyclohexanol concentration and porcine pancreas lipase, and removing the water formed as a byproduct of reaction and recovering the product by known methods. In another embodiment of the present invention, the ratio of 4-t-butylcyclohexanol to organic acid used is preferably in the ratios of 1:2 to 1:10. In still another embodiment of the present invention, the non-polar solvent used is selected from the group consisting of hexane, benzene, toluene, chloroform, acetone, ethyl methyl ketone and mixtures thereof. In yet another embodiment of the present invention, the reaction is carried out in a water free environment, where the non-polar solvent used forms an azeotrope with water and the temperature used is preferably in the range 40 - 70°C. In yet another embodiment of the present invention, the lipase used as esterification catalyst is selected from porcine pancreas, Rhizomucor miehei, Candida cylindracea, Pseudomonasfluorescens, wheat germ lipase and chicken liver esterase. In yet another embodiment of the present invention, peracetylated ß-cyclodextrin is used in various proportions as an additive in the reaction. Typically, the reaction was carried out in a flat bottom flask by taking 0.16g (0.001 mol) of 4-t-butylcyclohexanol in 50 - 250 ml of hexane: chloroform and 0.051g -0.51 g (0.0005 - 0.005 mol) acetic anhydride with 0.5035 g - 2.014 g (0.00025 - 0.001 s mol) of peracetylated ß-cyclodextrin and 100 - 800 mg of porcine pancreas lipase (Type II crude, Steapsin) of esterification activity 0.02-0.1µmol of n-butyric acid reacted per min per mg of lipase preparation. The reaction mixture was refluxed for 24 - 120h in an apparatus as per the previously filed Indian patent No. 1243/ DEL/ 99. The unreacted acid was washed with saturated bicarbonate. The organic layer was collected and dried with anhydrous sodium sulfate, the solvent was distilled off to obtain a mixture of unreacted alcohol, ester and peracetylated ß-cyclodextrin from which the product was isolated by the known conventional procedures. The reaction mixture was monitored regularly for product formation by determining the decrease in acid content as compared to the initial value, by titration against standard NaOH. The reaction was also monitored by gas chromatography using a 10% carbowax column at 100°C, with an FID detector at 250°C and injection port at 250°C with N2 as carrier gas at a flow rate of 40 ml per min. The retention times are: Cis alcohol - 13.1 min, trans alcohol - 16.3 min, cis acetate - 10.2 min, trans acetate - 12.4 min, cis butyrate - 19.0 min, trans butyrate - 24.1 min, cis valerate - 28.3 min, trans valerate -37.4 min respectively. The following examples are given by way of illustration of the present invention and therefore should not be construed to limit the scope of the present invention. Example 1 The reaction was carried out in a flat bottom flask by taking 0.39g (0.0025 mol) 4-t-butylcyclohexanol in 100 ml n-hexane and 0.22g (0.0025 mol) butyric acid and 500 mg porcine pancreas lipase. The reaction mixture was refluxed for 140h at 69°C. The insoluble enzyme was filtered off and the unreacted acid was washed with a saturated solution of sodium bicarbonate. The organic layer, after drying with anhydrous sodium sulfate was distilled off. The sample was then analyzed by Gas Chromatography as well as by titrimetry as described above. The reaction showed 46.5% (Table 1) esterification by titrimetry but no ester formation was observed by Gas Chromatography. Example 2 The reaction was carried out in a flat bottom flask by taking 0.156g (0.001 mol) 4-t-butylcyclohexanol in 100 ml of 7 : 3 hexane : chloroform and O.5lg (0.005 mol) of acetic anhydride, 1.007g (0.0005 mol, 0.5 equivalent with respect to 4-/-butylcyclohexanol) of peracetylated ß-cyclodextrin and 450 mg porcine pancreas lipase. The reaction mixture was refluxed for 72h at 56°C. The insoluble enzyme was then filtered off and the unreacted acid was washed with saturated bicarbonate; the organic solvent was distilled off after drying with anhydrous sodium sulfate to get a mixture of 4- t-butylcyclohexanol, 4-t-butylcyclohexyl acetate and peracetylated ß-cyclodextrin. About 10 ml of n-hexane or n-heptane was used to precipitate out the peracetylated P-cyclodextrin, which was then filtered off. The filtrate containing 4-t-butylcyclohexanol and its acetate ester was analyzed by Gas Chromatography using the conditions described above. Conversion yield - Total ester 86.6%, Cis isomer 29.6%, Trans isomer 70.4%, Trans/cis ratio of ester - 2.38 (Table 1). Example 3 The reaction was carried out in a flat bottom flask by taking 0.156g (0.001 mol) 4-t-butylcyclohexanol in 100 ml of 7 : 3 hexane : chloroform and 0.2805g (0.00275 mol) of acetic anhydride, 1.007g (0.0005 mol, 0.5 equivalent with respect to 4-t-butylcyclohexanol) of peracetylated p-cyclodextrin and 450 mg porcine pancreas lipase. The reaction mixture was refluxed for 120h at 56°C. The insoluble enzyme was then filtered off and the unreacted acid was washed with saturated bicarbonate; the organic solvent was distilled off after drying with anhydrous sodium sulfate to get a mixture of 4-t-butylcyclohexanol, 4-t-butylcyclohexyl acetate and peracetylated ß-cyclodextrin. About 10 ml of n-hexane or n-heptane was used to precipitate out the peracetylated P-cyclodextrin, which was then filtered off. The filtrate containing 4-t-butylcyclohexanol and its acetate ester was analyzed by Gas Chromatography using the conditions described above. Conversion yield - Total ester 68.6%, Cis isomer 28.5%, Trans isomer 71.5%, Trans/cis ratio of ester - 2.51 (Table 1). Example 4 The reaction was carried out in a flat bottom flask by taking 0.156g (0.001 mol) 4-t-butylcyclohexanol in 100 ml of 7 : 3 hexane : chloroform and 0.1657g (0.001625 mol) of acetic anhydride, 0.5035g (0.00025 mol, 0.25 equivalent with respect to 4-t-butylcyclohexanol) of peracetylated ß-cyclodextrin and 625 mg porcine pancreas lipase. The reaction mixture was refluxed for 48h at 56°C. The insoluble enzyme was then filtered off and the unreacted acid was washed with saturated bicarbonate; the organic solvent was distilled off after drying with anhydrous sodium sulfate to get a mixture of 4-t-butylcyclohexanol, 4-t-butylcyclohexyl acetate and peracetylated ß-cyclodextrin. About 10 ml of n-hexane or n-heptane was used to precipitate out the peracetylated 3-cyclodextrin, which was then filtered off. The filtrate containing 4-t-butylcyclohexanol and its acetate ester was analyzed by Gas Chromatography using the conditions described above. Conversion yield - Total ester 18.1%, Cis isomer 18.7%, Trans isomer 81.3%, Trans/cis ratio of ester - 4.35 (Table 1). Example 5 The reaction was carried out in a flat bottom flask by taking 0.156g (0.001 mol) 4-t-butylcyclohexanol in 100 ml of 7 : 3 hexane : chloroform and 0.682g (0.00775 mol) of butyric acid, 0.5035g (0.00025 mol, 0.25 equivalent with respect to 4-t-butylcyclohexanol) of peracetylated p-cyclodextrin and 625 mg porcine pancreas lipase. The reaction mixture was refluxed for 48h at 56°C. The insoluble enzyme was then filtered off and the unreacted acid was washed with saturated bicarbonate; the organic solvent was distilled off after drying with anhydrous sodium sulfate to get a mixture of 4-f-butylcyclohexanol, 4-t-butylcyclohexyl butyrate and peracetylated ß-cyclodextrin. About 10 ml of n-hexane or n-heptane was used to precipitate out the peracetylated P-cyclodextrin, which was then filtered off. The filtrate containing 4-t-butylcyclohexanol and its butyrate ester was analyzed by Gas Chromatography using the conditions described above. Conversion yield - Total ester 8.1%, Cis isomer 8.5%, Trans isomer 91.5%, Trans/cis ratio of ester - 10.8 (Table 1). Example 6 The reaction was carried out in a flat bottom flask by taking 0.39g (0.0025 mol) 4-t-butylcyclohexanol in 100 ml of 3 : 1 hexane : ethyl methyl ketone and 0.2805g (0.00275 mol) of acetic anhydride, 0.5035g (0.00025 mol, 0.25 equivalent with respect to 4-t-butylcyclohexanol) of peracetylated ß-cyclodextrin and 250 mg porcine pancreas lipase. The reaction mixture was refluxed for 63 h at 69°C. The insoluble enzyme was then filtered off and the unreacted acid was washed with saturated bicarbonate; the organic solvent was distilled off after drying with anhydrous sodium sulfate to get a mixture of 4-t-butylcyclohexanol, 4-t-butylcyclohexyl acetate and peracetylated ß-cyclodextrin. About 10 ml of n-hexane or n-heptane was used to precipitate out the peracetylated ß-cyclodextrin, which was then filtered off. The filtrate containing 4-t-butylcyclohexanol and its acetate ester was analyzed by Gas Chromatography using the conditions described above. Conversion yield - Total ester 44.7%, Cis isomer 28.1%, Trans isomer 71.9%, Trans/cis ratio of ester - 2.56 (Table 1). Example 7 The reaction was carried out in a flat bottom flask by taking 0.39g (0.0025 mol) 4-t-butylcyclohexanol in 100 ml of 3 : 2 hexane : toluene and 1.275g (0.0125 mol) of n-valeric acid, 1.007g (0.0005 mol, 0.5 equivalent with respect to 4-t-butylcyclohexanol) of peracetylated ß-cyclodextrin and 250 mg porcine pancreas lipase. The reaction mixture was refluxed for 90h at 69°C. The insoluble enzyme was then filtered off and the unreacted acid was washed with saturated bicarbonate; the organic solvent was distilled off after drying with anhydrous sodium sulfate to get a mixture of 4-t-butylcyclohexanol, 4-t-butylcyclohexyl valerate and peracetylated ß-cyclodextrin. About 10 ml of n-hexane or n-heptane was used to precipitate out the peracetylated p-cyclodextrin, which was then filtered off. The filtrate containing 4-t-butylcyclohexanol and its valerate ester was analyzed by Gas Chromatography using the conditions described above. Conversion yield - Total ester 8.3%, Cis isomer 25.6%, Trans isomer 74.3%, Trans/cis ratio of ester -2.9 (Table 1). Example 8 The reaction was carried out in a flat bottom flask by taking 0.156g (0.001 mol) 4-t-butylcyclohexanol in 100 ml of 7 : 3 hexane : chloroform and 0.285g (0.00275 mol) of acetic anhydride, 2.014g (0.001 mol, 1 equivalent with respect to 4-t-butylcyclohexanol) of peracetylated p-cyclodextrin and 450 mg porcine pancreas lipase. The reaction mixture was refluxed for 72h at 56°C. The insoluble enzyme was then filtered off and the unreacted acid was washed with saturated bicarbonate; the organic solvent was distilled off after drying with anhydrous sodium sulfate to get a mixture of 4-t-butylcyclohexanol, 4-t-butyl cyclohexyl acetate and peracetylated ß-cyclodextrin. About 10 ml of n-hexane or n-heptane was used to precipitate out the peracetylated P-cyclodextrin, which was then filtered off. The filtrate containing 4-t-butylcyclohexanol and its acetate ester was analyzed by Gas Chromatography using the conditions described above. Conversion yield - Total ester 36.5%, Cis isomer 21.9%, Trans isomer 78.1%, Trans/cis ratio of ester - 3.58 (Table 1). Table 1 (Table Removed) The main advantages of the invention are 1. The maximum yield of acetate ester of 86.6% was obtained in presence of 0.5 equivalent (with respect to 4-t-butylcyclohexanol) of peracetylated ß-cyclodextrin and using porcine pancreas lipase. 2. The maximum trans/cis ratio of 4-t-butyl cyclohexyl acetate obtained at a ratio of 1: 0.1 4-t-butylcyclohexanol: peracetylated p-cyclodextrin was 4.35. 3. The reaction was effected under milder reaction conditions. 4. The workout procedures was less tedious and also led to less pollution than the chemical method. 5. The reaction involved the use of non-toxic solvents. We Claim: 1. An enzymatic method for the preparation of an organic acid ester of 4-t- butylcyclohexanol which comprises: reacting 4-t-butylcyclohexanol with an organic acid of carbon chain length ranging from C2 - C18, or its anhydride in a ratio of 4-t- butylcyclohexanol to organic acid ranging from 1:1 to 1:10, refluxing the above reaction mixture with 50 - 250 mol of nonpolar organic solvent at a temperature ranging from 50 - 80°C for a period of 24-120 hrs, in the presence of 0.05 - 2 equivalents of peracetylated ß-cyclodextrin with respect to 4-t-butylcyclohexanol concentration and porcine pancreas lipase, and removing the water formed as a byproduct of reaction and recovering the product by known methods. 2. An enzymatic method as claimed in claim 1, wherein, the ratio of 4-t-butylcyclohexanol to organic acid used is preferably in the ratios of 1:2 to 1:10. 3. An enzymatic method as claimed in claims 1 and 2, wherein, non-polar solvent used is selected from the group consisting of hexane, benzene, toluene, chloroform, acetone, ethyl methyl ketone and mixtures thereof. 4. An enzymatic method as claimed in claims 1-3, wherein, the reaction is carried out in a water free environment, where the non-polar solvent used forms an azeotrope with water and the temperature used is preferably in the range 40 - 70°C. 5. An enzymatic method as claimed in claims 1 - 4, wherein, the lipase used as esterification catalyst is selected from porcine pancreas, Rhizomucor miehei, Candida cylindracea, Pseudomonas Fluorescens, wheat germ lipase and chicken liver esterase. 6. An enzymatic method as claimed in claims 1 - 5, wherein, peracetylated P-cyclodextrin is used in various proportions as an additive in the reaction. 7. An enzymatic method for the preparation of an organic acid ester of 4-t- butycyclohexanol substantially as herein described with references and examples.

Full Text

The present invention relates to an enzymatic method for the preparation of an organic acid ester of 4-t-butylcyclohexanol. The present invention particularly relates to an enzymatic method for the preparation of an organic acid ester of 4-t-butylcyclohexanol, where 4-t-butylcyclohexanol is used as an anchored substrate in peracetylated ß-cyclodextrin
Esters of 4-t-butylcyclohexanol find wide applications as flavoring, fragrance and perfumery raw materials (Hooper D C, Johnson G A, Peter D and Rennie G K, Brit. UK Patent Appl. 2020 977, 1979; Moeddel, R W, Procter and Gamble Co., US Patent 4515705, 1985). They are used in soaps, detergents, creams, lotions and perfumes (Opdyke D L J, Food Cosmet. Toxicol., 1978, 16, Supl. 1, 657). They are also used as solubility reducing agents for surfactants (Kacher M L, Procter and Gamble Co., Eur. Patent Appl. 18679, 1980). 4-t-Butylcyclohexanol exists as cis and trans isomers. At room temperature, the trans/cis ratio of the alcohol is ~ 3:1 (Karger B L, Stern R L and Zannucci J F, Anal. Chem. 40 (4), 727 - 735, 1968). The esters of the cis and trans isomers exhibit different flavor notes. The trans esters have a woody odor, while the cis isomers have a stronger, intense flowery note (Podberezina N S, Kheifits L A, Aref eva L I, Klimakhina S T and Mindlin L O, Maslo - Zhir. Prom - St, 1985, 3, 24 - 5, Russian, Chem. Abstr. No. 103: 87528v).
Some chemical methods are available for the preparation of the esters of 4-t-butylcyclohexanol. Reference may be made to Bollmann M and Kroning E of Bayer A G, German Patent 864 593, 1948, wherein synthesis of the acetate ester of 4-t-butylcyclohexanol is carried out by the hydrogenation of 4-/-butylphenol and subsequent acetylation. The major drawbacks of this method is that the reaction conditions are not

milder and involve use of toxic solvents. The ester is sold under the trade name of Oryclon™ by Haarmann and Reimer, Germany.
Reference may be made to Yokogawa A, Anezaki T, Yamasaki H and Ozawa K, Maruzen Sekiyo Giho 16, 77-87, 1871, (Japanese, Chem. Abstr. No. 80:14700s). This is a chemical method wherein cis- 4-t-butylcyclohexyl acetate is prepared from 4-t-butylphenol, where the cis isomer of the alcohol itself is first preferentially prepared by selective hydrogenation catalysts like Rhodium and Ruthenium, and this is subsequently acetylated. . The major drawbacks of this method is that the reaction conditions are not milder, involve use of toxic solvents and lead to tedious work out procedures. Reference may be made to Karger B L, Stern R L and Zannucci J F, Anal. Chem. 40(4), 727 - 735, 1968, where syntheses of esters of 4-t-butylcyclohexanol with organic acids from acetic to heptanoic acid have been carried out by chemical means. The esters were prepared by reacting 100 mmol of 4-t-butylcyclohexanol with 150 mmol of the appropriate acid chloride on a steam bath for 2h with stirring. The trans/cis ratio of acetate, propionate, n-butyrate, valerate, caproate, heptylate, isobutyrate and pivalate remained at approximately 3.5:1 in all the cases. The major drawbacks of this method are that it requires the use of acid chlorides, stringent reaction conditions and toxic solvents.
Reference may be made to Pattekhan H H and Divakar S.Indian Patent, 2000, 422del2001 who have used ß-cyclodextrin for the encapsulation of 4-t-butylcycIohexanol and subsequently acetylated the alcohol in order to achieve better trans/cis ratio. The maximum trans/cis ratio of esters obtained by this method was 5.5. The major drawbacks

of this method are that it does not involve milder reaction conditions and require use of toxic solvents.
No alternate methods of preparation of these esters of 4-t-butylcyclohexanol other than the chemical methods described above are available. No reports exist till date on the enzymatic method of synthesis of the esters of 4-t-butylcyclohexanol. The present investigation provides an enzymatic method for the preparation of esters of 4-t-butylcyclohexanol with organic acids.
The present invention therefore describes an improved enzymatic procedure for the esterification of 4-t-butylcyclohexanol, in which 4-t-butylcyclohexanol is used as a substrate anchored in peracetylated ß-cyclodextrin. Peracetylated ß-cyclodextrin has been used instead of ß-cyclodextrin as such, because the free hydroxyl groups on P-cyclodextrin can also get esterified when an esterification catalyst like lipase is present in the reaction medium along with an organic acid. Inclusion of 4-t-butylcyclohexanol inside the peracetylated ß-cyclodextrin cavity preferentially facilitates the esterification of the trans isomer of the alcohol. The cis isomer of the alcohol is stearically hindered from reacting with the incoming acyl group due to the presence of bulky acetyl groups on peracetylated P-cyclodextrin.
The novelty of the present invention is that it is an alternate method to chemical synthesis, wherein 4-t-butylcyclohexanol is anchored in peracetylated ß-cyclodextrin for esterification by Upases, which not only promotes esterification under milder reaction conditions, but also appreciably alters the trans/cis ratio of the isomeric esters formed.

The main object of the present invention is to provide an enzymatic method for the preparation of an organic acid ester of 4-t-butylcyclohexanol, which obviates the draw backs as described above.
Yet another object of the present invention is to carry out the reaction in a water free environment, where the non-polar solvent used forms an azeotrope with water at temperatures between 50 -80°C.
Still another object of the present invention is to use lipases from porcine pancreas, Rhizomucor miehei, Candia cylindracea, Pseudomonas fluorescens, wheat germ lipase and chicken liver esterase as esterification catalysts.
Accordingly, the present invention provides an enzymatic method for the preparation of an organic acid ester of 4-t-butylcyclohexanol which comprises: reacting 4-t-butylcyclohexanol with an organic acid of carbon chain length ranging from Ca - C]g, or its anhydride in a ratio of 4-t-butylcyclohexanol to organic acid ranging from 1:1 to 1:10, refluxing the above reaction mixture with 50 - 250 mol of nonpolar organic solvent at a temperature ranging from 50 - 80°C for a period of 24-120 hrs, in the presence of 0.05 - 2 equivalents of peracetylated p-cyclodextrin with respect to 4-t-butylcyclohexanol concentration and porcine pancreas lipase, and removing the water formed as a byproduct of reaction and recovering the product by known methods.
In another embodiment of the present invention, the ratio of 4-t-butylcyclohexanol to organic acid used is preferably in the ratios of 1:2 to 1:10.
In still another embodiment of the present invention, the non-polar solvent used is selected from the group consisting of hexane, benzene, toluene, chloroform, acetone, ethyl methyl ketone and mixtures thereof.

In yet another embodiment of the present invention, the reaction is carried out in a water free environment, where the non-polar solvent used forms an azeotrope with water and the temperature used is preferably in the range 40 - 70°C.
In yet another embodiment of the present invention, the lipase used as esterification catalyst is selected from porcine pancreas, Rhizomucor miehei, Candida cylindracea, Pseudomonasfluorescens, wheat germ lipase and chicken liver esterase.
In yet another embodiment of the present invention, peracetylated ß-cyclodextrin is used in various proportions as an additive in the reaction.
Typically, the reaction was carried out in a flat bottom flask by taking 0.16g (0.001 mol) of 4-t-butylcyclohexanol in 50 - 250 ml of hexane: chloroform and 0.051g -0.51 g (0.0005 - 0.005 mol) acetic anhydride with 0.5035 g - 2.014 g (0.00025 - 0.001
s
mol) of peracetylated ß-cyclodextrin and 100 - 800 mg of porcine pancreas lipase (Type II crude, Steapsin) of esterification activity 0.02-0.1µmol of n-butyric acid reacted per min per mg of lipase preparation. The reaction mixture was refluxed for 24 - 120h in an apparatus as per the previously filed Indian patent No. 1243/ DEL/ 99. The unreacted acid was washed with saturated bicarbonate. The organic layer was collected and dried with anhydrous sodium sulfate, the solvent was distilled off to obtain a mixture of unreacted alcohol, ester and peracetylated ß-cyclodextrin from which the product was isolated by the known conventional procedures. The reaction mixture was monitored regularly for product formation by determining the decrease in acid content as compared to the initial value, by titration against standard NaOH.
The reaction was also monitored by gas chromatography using a 10% carbowax column at 100°C, with an FID detector at 250°C and injection port at 250°C with N2 as

carrier gas at a flow rate of 40 ml per min. The retention times are: Cis alcohol - 13.1 min, trans alcohol - 16.3 min, cis acetate - 10.2 min, trans acetate - 12.4 min, cis butyrate - 19.0 min, trans butyrate - 24.1 min, cis valerate - 28.3 min, trans valerate -37.4 min respectively.
The following examples are given by way of illustration of the present invention and therefore should not be construed to limit the scope of the present invention.
Example 1
The reaction was carried out in a flat bottom flask by taking 0.39g (0.0025 mol) 4-t-butylcyclohexanol in 100 ml n-hexane and 0.22g (0.0025 mol) butyric acid and 500 mg porcine pancreas lipase. The reaction mixture was refluxed for 140h at 69°C. The insoluble enzyme was filtered off and the unreacted acid was washed with a saturated solution of sodium bicarbonate. The organic layer, after drying with anhydrous sodium sulfate was distilled off. The sample was then analyzed by Gas Chromatography as well as by titrimetry as described above. The reaction showed 46.5% (Table 1) esterification by titrimetry but no ester formation was observed by Gas Chromatography.
Example 2
The reaction was carried out in a flat bottom flask by taking 0.156g (0.001 mol) 4-t-butylcyclohexanol in 100 ml of 7 : 3 hexane : chloroform and O.5lg (0.005 mol) of acetic anhydride, 1.007g (0.0005 mol, 0.5 equivalent with respect to 4-/-butylcyclohexanol) of peracetylated ß-cyclodextrin and 450 mg porcine pancreas lipase. The reaction mixture was refluxed for 72h at 56°C. The insoluble enzyme was then filtered off and the unreacted acid was washed with saturated bicarbonate; the organic solvent was distilled off after drying with anhydrous sodium sulfate to get a mixture of 4-

t-butylcyclohexanol, 4-t-butylcyclohexyl acetate and peracetylated ß-cyclodextrin. About 10 ml of n-hexane or n-heptane was used to precipitate out the peracetylated P-cyclodextrin, which was then filtered off. The filtrate containing 4-t-butylcyclohexanol and its acetate ester was analyzed by Gas Chromatography using the conditions described above. Conversion yield - Total ester 86.6%, Cis isomer 29.6%, Trans isomer 70.4%, Trans/cis ratio of ester - 2.38 (Table 1).
Example 3
The reaction was carried out in a flat bottom flask by taking 0.156g (0.001 mol) 4-t-butylcyclohexanol in 100 ml of 7 : 3 hexane : chloroform and 0.2805g (0.00275 mol) of acetic anhydride, 1.007g (0.0005 mol, 0.5 equivalent with respect to 4-t-butylcyclohexanol) of peracetylated p-cyclodextrin and 450 mg porcine pancreas lipase. The reaction mixture was refluxed for 120h at 56°C. The insoluble enzyme was then filtered off and the unreacted acid was washed with saturated bicarbonate; the organic solvent was distilled off after drying with anhydrous sodium sulfate to get a mixture of 4-t-butylcyclohexanol, 4-t-butylcyclohexyl acetate and peracetylated ß-cyclodextrin. About 10 ml of n-hexane or n-heptane was used to precipitate out the peracetylated P-cyclodextrin, which was then filtered off. The filtrate containing 4-t-butylcyclohexanol and its acetate ester was analyzed by Gas Chromatography using the conditions described above. Conversion yield - Total ester 68.6%, Cis isomer 28.5%, Trans isomer 71.5%, Trans/cis ratio of ester - 2.51 (Table 1).
Example 4
The reaction was carried out in a flat bottom flask by taking 0.156g (0.001 mol) 4-t-butylcyclohexanol in 100 ml of 7 : 3 hexane : chloroform and 0.1657g (0.001625

mol) of acetic anhydride, 0.5035g (0.00025 mol, 0.25 equivalent with respect to 4-t-butylcyclohexanol) of peracetylated ß-cyclodextrin and 625 mg porcine pancreas lipase. The reaction mixture was refluxed for 48h at 56°C. The insoluble enzyme was then filtered off and the unreacted acid was washed with saturated bicarbonate; the organic solvent was distilled off after drying with anhydrous sodium sulfate to get a mixture of 4-t-butylcyclohexanol, 4-t-butylcyclohexyl acetate and peracetylated ß-cyclodextrin. About 10 ml of n-hexane or n-heptane was used to precipitate out the peracetylated 3-cyclodextrin, which was then filtered off. The filtrate containing 4-t-butylcyclohexanol and its acetate ester was analyzed by Gas Chromatography using the conditions described above. Conversion yield - Total ester 18.1%, Cis isomer 18.7%, Trans isomer 81.3%, Trans/cis ratio of ester - 4.35 (Table 1).
Example 5
The reaction was carried out in a flat bottom flask by taking 0.156g (0.001 mol) 4-t-butylcyclohexanol in 100 ml of 7 : 3 hexane : chloroform and 0.682g (0.00775 mol) of butyric acid, 0.5035g (0.00025 mol, 0.25 equivalent with respect to 4-t-butylcyclohexanol) of peracetylated p-cyclodextrin and 625 mg porcine pancreas lipase. The reaction mixture was refluxed for 48h at 56°C. The insoluble enzyme was then filtered off and the unreacted acid was washed with saturated bicarbonate; the organic solvent was distilled off after drying with anhydrous sodium sulfate to get a mixture of 4-f-butylcyclohexanol, 4-t-butylcyclohexyl butyrate and peracetylated ß-cyclodextrin. About 10 ml of n-hexane or n-heptane was used to precipitate out the peracetylated P-cyclodextrin, which was then filtered off. The filtrate containing 4-t-butylcyclohexanol and its butyrate ester was analyzed by Gas Chromatography using the conditions

described above. Conversion yield - Total ester 8.1%, Cis isomer 8.5%, Trans isomer 91.5%, Trans/cis ratio of ester - 10.8 (Table 1).
Example 6
The reaction was carried out in a flat bottom flask by taking 0.39g (0.0025 mol) 4-t-butylcyclohexanol in 100 ml of 3 : 1 hexane : ethyl methyl ketone and 0.2805g (0.00275 mol) of acetic anhydride, 0.5035g (0.00025 mol, 0.25 equivalent with respect to 4-t-butylcyclohexanol) of peracetylated ß-cyclodextrin and 250 mg porcine pancreas lipase. The reaction mixture was refluxed for 63 h at 69°C. The insoluble enzyme was then filtered off and the unreacted acid was washed with saturated bicarbonate; the organic solvent was distilled off after drying with anhydrous sodium sulfate to get a mixture of 4-t-butylcyclohexanol, 4-t-butylcyclohexyl acetate and peracetylated ß-cyclodextrin. About 10 ml of n-hexane or n-heptane was used to precipitate out the peracetylated ß-cyclodextrin, which was then filtered off. The filtrate containing 4-t-butylcyclohexanol and its acetate ester was analyzed by Gas Chromatography using the conditions described above. Conversion yield - Total ester 44.7%, Cis isomer 28.1%, Trans isomer 71.9%, Trans/cis ratio of ester - 2.56 (Table 1).
Example 7
The reaction was carried out in a flat bottom flask by taking 0.39g (0.0025 mol) 4-t-butylcyclohexanol in 100 ml of 3 : 2 hexane : toluene and 1.275g (0.0125 mol) of n-valeric acid, 1.007g (0.0005 mol, 0.5 equivalent with respect to 4-t-butylcyclohexanol) of peracetylated ß-cyclodextrin and 250 mg porcine pancreas lipase. The reaction mixture was refluxed for 90h at 69°C. The insoluble enzyme was then filtered off and the unreacted acid was washed with saturated bicarbonate; the organic solvent was distilled

off after drying with anhydrous sodium sulfate to get a mixture of 4-t-butylcyclohexanol, 4-t-butylcyclohexyl valerate and peracetylated ß-cyclodextrin. About 10 ml of n-hexane or n-heptane was used to precipitate out the peracetylated p-cyclodextrin, which was then filtered off. The filtrate containing 4-t-butylcyclohexanol and its valerate ester was analyzed by Gas Chromatography using the conditions described above. Conversion yield - Total ester 8.3%, Cis isomer 25.6%, Trans isomer 74.3%, Trans/cis ratio of ester -2.9 (Table 1).
Example 8
The reaction was carried out in a flat bottom flask by taking 0.156g (0.001 mol) 4-t-butylcyclohexanol in 100 ml of 7 : 3 hexane : chloroform and 0.285g (0.00275 mol) of acetic anhydride, 2.014g (0.001 mol, 1 equivalent with respect to 4-t-butylcyclohexanol) of peracetylated p-cyclodextrin and 450 mg porcine pancreas lipase. The reaction mixture was refluxed for 72h at 56°C. The insoluble enzyme was then filtered off and the unreacted acid was washed with saturated bicarbonate; the organic solvent was distilled off after drying with anhydrous sodium sulfate to get a mixture of 4-t-butylcyclohexanol, 4-t-butyl cyclohexyl acetate and peracetylated ß-cyclodextrin. About 10 ml of n-hexane or n-heptane was used to precipitate out the peracetylated P-cyclodextrin, which was then filtered off. The filtrate containing 4-t-butylcyclohexanol and its acetate ester was analyzed by Gas Chromatography using the conditions described above. Conversion yield - Total ester 36.5%, Cis isomer 21.9%, Trans isomer 78.1%, Trans/cis ratio of ester - 3.58 (Table 1).

Table 1

(Table Removed)
The main advantages of the invention are
1. The maximum yield of acetate ester of 86.6% was obtained in presence of 0.5
equivalent (with respect to 4-t-butylcyclohexanol) of peracetylated ß-cyclodextrin
and using porcine pancreas lipase.
2. The maximum trans/cis ratio of 4-t-butyl cyclohexyl acetate obtained at a ratio of 1:
0.1 4-t-butylcyclohexanol: peracetylated p-cyclodextrin was 4.35.
3. The reaction was effected under milder reaction conditions.
4. The workout procedures was less tedious and also led to less pollution than the
chemical method.
5. The reaction involved the use of non-toxic solvents.

We Claim:
1. An enzymatic method for the preparation of an organic acid ester of 4-t-
butylcyclohexanol which comprises: reacting 4-t-butylcyclohexanol with an organic
acid of carbon chain length ranging from C2 - C18, or its anhydride in a ratio of 4-t-
butylcyclohexanol to organic acid ranging from 1:1 to 1:10, refluxing the above reaction
mixture with 50 - 250 mol of nonpolar organic solvent at a temperature ranging from 50
- 80°C for a period of 24-120 hrs, in the presence of 0.05 - 2 equivalents of
peracetylated ß-cyclodextrin with respect to 4-t-butylcyclohexanol concentration and
porcine pancreas lipase, and removing the water formed as a byproduct of reaction and
recovering the product by known methods.
2. An enzymatic method as claimed in claim 1, wherein, the ratio of 4-t-butylcyclohexanol
to organic acid used is preferably in the ratios of 1:2 to 1:10.
3. An enzymatic method as claimed in claims 1 and 2, wherein, non-polar solvent used is
selected from the group consisting of hexane, benzene, toluene, chloroform, acetone,
ethyl methyl ketone and mixtures thereof.
4. An enzymatic method as claimed in claims 1-3, wherein, the reaction is carried out in a
water free environment, where the non-polar solvent used forms an azeotrope with water
and the temperature used is preferably in the range 40 - 70°C.
5. An enzymatic method as claimed in claims 1 - 4, wherein, the lipase used as
esterification catalyst is selected from porcine pancreas, Rhizomucor miehei, Candida
cylindracea, Pseudomonas Fluorescens, wheat germ lipase and chicken liver esterase.
6. An enzymatic method as claimed in claims 1 - 5, wherein, peracetylated P-cyclodextrin
is used in various proportions as an additive in the reaction.
7. An enzymatic method for the preparation of an organic acid ester of 4-t-
butycyclohexanol substantially as herein described with references and examples.

Documents:

1205-del-2000-abstract.pdf

1205-del-2000-claims.pdf

1205-del-2000-correspondence-others.pdf

1205-del-2000-correspondence-po.pdf

1205-del-2000-description (complete).pdf

1205-del-2000-form-1.pdf

1205-del-2000-form-19.pdf

1205-del-2000-form-2.pdf

1205-del-2000-form-3.pdf

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Patent Number

242188

Indian Patent Application Number

1205/DEL/2000

PG Journal Number

34/2010

Publication Date

20-Aug-2010

Grant Date

18-Aug-2010

Date of Filing

26-Dec-2000

Name of Patentee

COUNCIL OF SCIENTIFIC AND INDUSTRIAL RESEARCH

Applicant Address

RAFI MARG, NEW DELHI-110001, INDIA.

Inventors:

#	Inventor's Name	Inventor's Address
1	SOUNDAR DIVAKAR	CENTRAL FOOD TECHNOLOGICAL RESEARCH INSTITUTE, MYSORE-570013.
2	PRAMILA RAO	CENTRAL FOOD TECHNOLOGICAL RESEARCH INSTITUTE, MYSORE-570013.

PCT International Classification Number

C07C 29/145

PCT International Application Number

N/A

PCT International Filing date

PCT Conventions:

#	PCT Application Number	Date of Convention	Priority Country
1			NA