Title of Invention

AN IMPROVED PROCESS FOR THE PREPARATION OF ESTERS OF ORGANIC ACIDS AND ALCOHOLS

Abstract

This invention relates to an improved process for the preparation of esters of organic acids and alcohols. The process uses immobilized lipases in organic media. The reaction of primary and secondary alcohols of carbon atoms 1 to 20 with acids of carbon atoms 2 to 20 has been carried out in presence of immobilised lipase at 40-70 deg.C in presence of solvent for 12-80 hours to obtain the desired product.

Full Text

This invention relates to an improved process for the preparation of esters of organic acids and alcohols. This invention particularly relates to an improved process for the preparation of esters using immobilized lipases in organic media. The monolayer around the enzyme constituted by the microphase formed due to accumulation of the water of reaction is the scene of action for esterification (Ref: Enzyme catalyzed process in organic solvents, (1985) Zaks A and Klibanov A.M., Proc.. Natl. Acad. Sci. USA 82: 3192-3196). Dissociation of the weak acid which takes place in the aqueous interface between the organic solvent and the enzyme leads to the formation of conjugate acid which favours hydrolysis. Since esterification in organic media is affected by the accumulation of water of reaction and the conjugate acid, factors which can efficiently deal with these two have been shown to favour esterification. (Ref: The effect of water on enzyme reaction in organic media, (I988) Zaks A. and Klibanov A.M., J. Biol. Chem. 263: 8017-8021). Use of desiccants to remove the excess water formed and salts which reduce the effect of conjugate acid and which favour effective pH memory (Ref: Prapulia S.G., Divakar S. and Karanth N.G. (1996) Applied for an Indian patent) favour esterification. The major drawbacks of the existing methods have been that for a given amount of enzyme employed, a limiting concentration of substrate, water and probably H30* results in incomplete conversion leading to very low yields. Low substrate and higher enzyme concentrations, pre-treatment of the solvents employed, use of large volume of the solvent, longer duration of incubation on mechanical agitators, addition of desiccants lead to very low yields adding to the cost-intensiveness of the approach. Hence no satisfactory preparatory procedures which are cost-effective are available. There has been a report on an experimental setup which resulted in greater than 99% yield in ethyl esters of oleic, linolenic and other fatty acids where the reactions were carried out on a preparatory scale with continuous removal of water by using molecular sieves through which the solvent vapours were passed (Ref: Facile synthesis of fatty acid esters in high yields (1992) Bloomer S., Adlercreutz P. and Maftiason B. Enzyme Microb.Technol. 14: 89-97). However, the setup employed was good only for the preparation of the above mentioned ethyl esters which cannot be employed satisfactorily for the preparation of a wide variety of esters as claimed in the present proposal. Esterification has been carried out with high concentrations of enzyme and higher temperatures (which may not be conducive for the preparation of a wide variety of esters) than those employed in the present proposal. Hills et al (Ref: Eur. Pat. Appl. EP 383405, 1990, Hills G.A., Macrae A.R., and Poulina R.R., Unichema Chemie B.V.; Unilever N.V.) have reported ester preparations using lipases where the water of reaction is removed by azeotropic distillation. The ester synthesis is carried out at pressures of 20,000 Pa, with medium chain mono and dicarboxylic acids and long chain fatty acids. The procedure employed in the present proposal does not involve application of any vacuum to remove the water formed. The present invention involves a procedure whereby continuous removal of water is achieved in a setup consisting of minimum amount of lipase in minimum amount of solvent with high substrate concentrations which enables higher yields of esters to be obtained for the synthesis of a wide variety of esters. The present invention also provides carrying out the reaction at a higher temperature without denaturing the enzyme which also enablec continuous removal of water of reaction thereby facilitating higher conversions. New principles underlying and advantages of the invention are: 1. Milder reaction conditions when compared to chemical syntheses. 2. Maintenance of low water environment. 3. Low concentrations of enzyme. 4. Higher substrate and alcohol concentrations. 5. Less strenuous reaction conditions when compared to chemical synthesis. 6. Chemical activation of substrates and alcohols is not required. 7. Higher yields of products when compared to usual enzymatic synthesis as that employed in shake flasks. 8. Enhancement of reaction rate when compared to reported lipase mediated esterification processes and hence higher productivities 9. Cost-effective procedure. 10. Applicability to a wide .gamut of esterification reactions. 11. Employment of inexpensive desiccants. 12. Use of relatively cheap low-boiling solvents which can be recycled. 13. Reusability of enzyme. 14. Clean product. Accordingly, the present invention provides an improved process for the preparation of esters of organic acids and alcohols which comprises: a) refluxing a reaction mixture containing acid and alcohol with carbon atoms 2 to 20 and 1 to 20, respectively, in a low boiling solvent with boiling point in the range of 40-80°C characterised in that at a temperature of 40-70°C in the presence of immobilized lipase for a duration of 12 - 80 hours in the apparatus as shown in the drawing of accompanying the specification ; b) removing water by using conventional desiccants in a manner as herein described, c) recovering and purifying the ester formed by known solvent extraction methods. In some other cases the solvent was evaporated to obtain a mixture of the alcohol, the acid and the compound. The apparatus employed is shown in figure 1. The operation comprises; heating the reaction mixture consisting of the enzyme, acid and alcohol in a round bottomed flask along with the solvent (A), a Soxhlet extractor containing the suitable desiccant (B) to which is attached in condenser (C), whereby the solvent vapour which rises to the condenser carrying along with it water of reaction (zoetrope) is condensed back into the Soxhlet extractor containing the desiccant which removes the water of reaction thereby rendering the reaction mixture anhydrous favoring esterification. The reaction can be depicted as follows: R1-OH + R2-COOH --> R,-O-CO-R2 + H2O R1:C1-C20 ALCOHOL R2:C1-C19ACID In an embodiment of the present invention, the acid employed is one consisting of carbon atoms 2 - 20. In an another embodiment of the present invention the alcohol employed is one containing 1 - 20 carbon atoms. In yet another embodiment of the present invention, the enzyme used is such as immobilized lipases from Mucor miehei, Candida cylindracea, Pseudomonas fluorescens. Wheat germ and Porcine pancreas. In still another embodiment of the present invention, the solvent employed » is a low - boiling solvent in the boiling range 40-70°C, such as diethyl ether, diisopropyl ether, dichloromethane, petroleum ether (40-60°C fraction), chloroform, hexane, pentane, petroleum ether (60-80°C fraction). In a further embodiment of the present invention the desiccant employed in the path of the evaporated solvent is such as anhydrous Na2S04, MgSO4, molecular sieves, nonionic polymeric adsorbents and silica gel. Commercially available above mentioned acids and alcohols were used as such in case of solids and distilled once in case of liquids. Immobilized lipase preparation, Lipozyme IM-20 (Mucor miehei lipase) was procured from M/s. Novo Nordisk, Denmark and porcine pancreas lipase from Sigma chemicals, St. Louis, Mo, USA was used. The specific activities of the lipases were determined to be 9.945 and 2.0395 ^imoles/ min/ mg protein respectively. The activity was assayed by hydrolysis of tributyrin substrate .(Comparison of lipases by different assays, (1992), Vorderwulbecke T., Kieslich K. and Erdmann H. Enzyme Microb. Technol. 14: 631-639). Estimation of protein content was by Lowry's method (Protein estimation method using Folin-Ciocalteu reagent, (1951), Lowry O.H., Josebourgh N., Farr A.L. and Randall R.J., J. Biol. Chem. 193: 265-269). The reactions were monitored as follows: A back titration method was employed whereby a 1 ml aliquot withdrawn at regular intervals of time was treated with excess 0.1 N NaOH from which the acid content in the reaction mixture was determined by titrating the excess of NaOH against 0.1 N oxalic acid. The characterization of the product esters was done by GC analysis using a Carbowax column 20M (10%) of 6 m length with the column operating at 100°C and injection and detection ports maintained at 200°C and 250°C respectively. The characterization of the product esters was also done by recording 1H NMR spectra, on a Broker DRX 500 NMR instrument operating at 20°C. The samples were dissolved in CDCI3-DMSO-d6 mixture and the signals were referenced to TMS. The process of the invention is described in detail in the examples given below which are illustrative only and should not be construed to limit the scope of the invention. Example 1 To 0.05 mole a-terpineol and 0.05 mole propionic acid taken in 200 ml petroleum ether at 60°C is added 1 g of immobilized Mucor miehei lipase. This was taken in a two necked round bottomed flask fitted with a Dean-Stark apparatus with a thimble filled with anhydrous Na2S04. The solvent was continuously refluxed and stirred for a period of 16 h. The product workout was by filtering the immobilized enzyme, washing the reaction mixture with aqueous saturated NaHCO3 to remove unreacted propionic acid and distilling off the solvent and α-terpineol to obtain α-terpinyl propionate which was further purified by distillation. Estimation of the product was by titration method. Yield of the ester obtained in the reaction mixture was 62%. Example 2 To 0.05 mole a-terpineol and 0.025 mole acetic anhydride taken in 200 ml petroleum ether at 60°C is added 0.5 g of immobilized Mucor miehei lipase. This was taken in a two necked round bottomed flask fitted with a Soxhlet apparatus with a cotton plug filled with anhydrous Na2SO4. The solvent was continuously refluxed and stirred for a period of 43 h. The product work out was by filtering the immobilized enzyme, washing the reaction mixture with aqueous saturated NaHC03 to remove unreacted acetic anhydride and distilling off the solvent and a-terpineol to obtain α-terpinyl acetate which was further purified by distillation. Estimation of the product was by the titration method. Yield of the ester obtained in the reaction mixture was 54%. Example 3 To 0.05 mole a-terpineol and 0.05 mole butyric acid taken in 200mL petroleum ether at 60°C is added 0.5 g immobilized Mucor miehei lipase. This was taken in a two necked round bottomed flask fitted with a Soxhlet apparatus filled with anhydrous Na2S04. The solvent was continuously refluxed and stirred for a period of 40 h. The product work out was by filtering the immobilized enzyme, washing the reaction mixture with aqueous saturated NaHCO3 to remove unreacted butyric acid and distilling off the solvent and α-terpineol to obtain α-terpinyl butyrate which was further purified by distillation. Estimation of the product was by the titration method. Yield of the ester obtained in the reaction mixture was 33 %. Example 4 To 0.025 mole a-terpineol and 0.025 mole isobutyric acid taken in 200 ml chloroform at 60°C is added 0.5 g immobilized Mucor miehei lipase This was taken in a two necked round bottomed flask fitted with a Soxhlet apparatus filled with anhydrous Na2SO4.and nonionic polymeric adsorbent. The solvent was continuously refluxed and stirred for a period of 31.5 h. The product work out was by filtering the immobilized enzyme, washing the reaction mixture with aqueous saturated NaHCO3 to remove unreacted isobutyric acid and distilling off the solvent and α-terpineol to obtain α -terpinyl isobutyrate which was further purified by distillation. Estimation of the product was by titration method. Yield of the ester obtained in the reaction mixture was 32 %. Example 5 To 0.025 mole isoamyl alcohol and 0.025 mole propionic acid taken in 200 ml petroleum ether at 60°C is added 0.5 g immobilized Mucor miehei lipase. This was taken in a two necked round bottomed flask fitted with a Soxhlet apparatus filled with anhydrous Na2S04. The solvent was continuously refluxed and stirred for a period of 69 h. The product work out was by filtering the immobilized enzyme, washing the reaction mixture- with aqueous saturated NaHC03 to remove unreacted propionic acid and distilling off the solvent and isoamyl alcohol to obtain isoamyl propionate which was further purified by distillation. Estimation of the product was by the titration method. Yield of the. ester obtained in the reaction mixture was 39 %. Example 6 To 0.025 mole isopropyl alcohol and 0.025 mole isovaleric. acid taken in 200 ml petroleum ether at 60°C is added 0.5 g immobilized Mucor miehei lipase. This was taken in a two necked round bottomed flask fitted with a Soxhlet apparatus filled with anhydrous Na2S04. The solvent was continuously refluxed and stirred for a period of 48 h. The product work out was by filtering the immobilized enzyme, washing the reaction mixture with aqueous saturated NaHC03 to remove unreacted isovaleric acid and distilling off the solvent and isopropyl alcohol to obtain isopropyl isovalerate which was furthe; purified by distillation. Estimation of the product was by the titration method Yield of the ester obtained in the reaction mixture was 23.07%. Example 7 To 0.025 mole ethyl lactate and 0.025 mole palmitic acid taken in 200 ml chloroform at 60°C added 0.5 g immobilized Mucor miehei lipase. This was taken in a two necked round bottomed flask fitted with a Soxhlet apparatus filled with nonionic polymeric adsorbent. The solvent was continuously refluxed and stirred for a period of 20 h. The product work out was by filtering the immobilized enzyme and distilling off the solvent to obtain'a mixture of unreacted ethyl lactate and palmitic acid and palmitoy! lactic acid. Estimation of the product was by the titration method. Yield of the ester obtained in the reaction mixture was 44.44%. Example 8 To 0.05 mole lactic acid taken in 200 mL chloroform at 60°C is added 0.5 g Mucor miehei lipase. This was taken in a two necked round bottomed flask fitted with a Soxhlet apparatus filled with anhydrous Na2S04. The solvent was continuously refluxed and stirred for a period of 60 h. The product work out was by filtering the immobilized enzyme and distilling off the solvent to obtain a mixture of lactic acid oligomer and lactic acid. Estimation of product was by the titration method. Yield of the ester obtained in the reaction mixture was 48%. Example 9 To 0.025 mole lactic acid and 0.025 mole palmitic acid taken in 200 ml chloroform at 60°C is added 0.5 g of porcine pancreas lipase. This was taken in a two necked round bottomed flask fitted with a Soxhlet apparatus filled with anhydrous Na2S04. The solvent was continuously refluxed and stirred for a period of 112 h. The product work out was by filtering the immobilized enzyme and distilling off the solvent to obtain a mixture of unreacted lactic acid and palmitic acid and palmitoyl lactic acid. Estimation of the product was by the titration method. Yield of the ester obtained in the reaction mixture was 72%. Example 10 To 0.055 mole lactic acid and 0.025 mole palmitic acid taken in 200 ml chloroform at 60°C is added 0.5 g of porcine pancreas lipase. This was taken in a two necked round bottomed flask fitted with a Soxhlet apparatus filled with anhydrous Na2S04. The solvent was continuously refluxed and stirred for a period of 40 h. The product work out was by filtering the immobilized enzyme and distilling off the solvent to obtain a mixture of unreacted acids and palmitoyl lactic acid. Estimation of the product was by the titration method. Yield of the ester obtained in the reaction mixture was 76%. The main advantages of the invention are 1. Milder reaction conditions. 2. Maintenance of low water environment. 3. Lesser concentrations of enzyme. 4. Higher substrate and alcohol concentrations. 5. Less strenuous reaction conditions when compared to chemical synthesis. 6. Chemical activation of substrates and alcohols is not required. 7. Higher yields of products when compared to usual enzymatic synthesis as that employed in shake flasks. 8. Enhancement of reaction rate when compared to other lipase mediated esterification processes. 9. Cost effective procedure. 10. Applicability to a wide gamut of esterification reactions. I1. Employment of inexpensive dessicants. 12. Use of relatively cheap low-boiling solvents which can be recycled. 13. Reusability of enzyme. 14. Clean product. We Claim: 1. An improved process for the preparation of esters of organic acids and alcohols which comprises: a) refluxing a reaction mixture containing acid and alcohol with carbon atoms 2 to 20 and 1 to 20, respectively, in a low boiling solvent with boiling point in the range of 40-80°C characterised in that at a temperature of 40-70°C in the presence of immobilized lipase for a duration of 12 - 80 hours in the apparatus as shown in the drawing of accompanying the specification ; b) removing water by using conventional desiccants in a manner as herein described, c) recovering and purifying the ester formed by known solvent extraction methods. 2. An improved process as claimed in claim 1 wherein the acid used is selected from acetic, butyric, propionic, valeric, isovaleric, octanoic, decanoic, oleic, myristic, lauric, palmitic, stearic acids consisting of carbon atoms 2 - 20. 3. An improved process as claimed in claims 1 and 2 wherein the alcohol used is selected from methyl, ethyl, propyl, butyl, amyl, lauryl, palmityl, stearyl alcohols and hydroxy acids such as lactic, malic, citric acids containing 1-20 carbon atoms. 4. An improved process as claimed in claims 1 to 3 wherein the immobilized lipases is from Mucor miehei, Candida cylindracea, Pseudomonas fluorescens, Wheat germ and Porcine pancreas. 5. An improved process as claimed in claims 1 to 4 wherein the solvent of a low- boiling solvent selected from diethyl ester, diisopropyl ether, dichloromethane, petroleum ether 40-60°C fraction, chloroform, hexane, pentane and petroleum ether 60-80°C fraction. 6. An improved process as claimed in claims 1 to 5 wherein the desiccant employed is anhydrous Na2SO4, MgSO4, conventional molecular sieves, nonionic polymeric adsorbents and silica gel. 7. An improved process for the preparation of esters organic acids and alcohols substantially as herein described with references and examples.

Documents:

1244-del-1999-abstract.pdf

1244-del-1999-claims.pdf

1244-del-1999-correspondence-others.pdf

1244-del-1999-correspondence-po.pdf

1244-del-1999-description (complete).pdf

1244-del-1999-drawings.pdf

1244-del-1999-form-1.pdf

1244-del-1999-form-19.pdf

1244-del-1999-form-2.pdf