Title of Invention

AN IMPROVED PROCESS FOR THE PREPARATION OF TERPINYL ESTERS

Abstract

An enzymatic process for the preparation of terpinyl esters by reacting terpineol with carboxylic acid of chain length C2-C5 in equimolar ratio in the range 0.02-0.15M, in presence of an immobilised lipase wherein the ratio of enzyme to substrate should be minimum 10 in presence of non-polar solvent so as to keep the enzyme completely immersed in the solvent under vigorous agitating conditions up to 10h at a temperature ranging from room temperature to 60°C and recovering the ester by conventional method. The terpinyl esters obtained by this process is widely used in flavour and perfume industry.

Full Text

This invention relates to an improved process for the preparation of terpinyl esters. This invention particularly relates to an improved process for the preparation of a - terpinyl esters from a - terpineol and carboxylic acids of chain length C2-C5 using immobilised lipase from Mucor melhei and porcine pancreas. More particularly the process relates to the preparation of a - terpinyl acetate, -propionate, -butyrate, -isobutyrate, -valerate and -isovalerate esters. a - Terpinyl propionate is a commercially important flavour ester which possesses sweet - herbaceous, mildly fruity piny, refreshing odour of moderate tenacity. The ester is also being used as a modifier for Lavender and Bergamot and is very stable in alkaline media. It is used in perfumery for Pine variations, Citrus Colognes, Lavender, Sage and other herbaceous fragrances. This ester requires some specialised esterification conditions for its preparation chemically. A few chemical methods employed in the preparation of a - terpinyl propionate are listed below : REFERENCE I CONDITIONS I DRAWBACKS 1) West, Strauz and Barton 1) Esterification through Slow esterification with low : Synthetic Perfumes, their reflux of a - terpineol and yields. Chemistry and Preparation propionic acid with a neutral - London 1949, Edw. Arnold diluent, and Co. 2) Azeotropic type esterification of a - terpineol with propionic acid. 2) Militzer, Berichte der VVB Chemische Fabrik Same as above Same as above Militz, 1956. | | The following are some methodologies employed in the preparation of a -terpinyl acetate which are also applicable for the preparation of a - terpinyl propionate. 3) Tsutomo Kuwata J. Soc. Chem. Ind. Jap, 1939, No. 42 4) Boake, Roberts and Company - French patent (1290417), 1962. 5) Badische Anilin - und Soda Fabrik - French Patent (2013254), 1970. Japanese acid clay containing limonene is used as a condensing agent with equimolar mixture of d - limonene and acetic acid. Allylic terpinic chlorides treated with carboxylic acids in the presence of Cu2CI2 and amines. Purification by high vacuum distillation. Transesterification in the presence of sodium methoxide in a reaction which involves efficient fractionation. Low yield, slow reaction A procedure with many unit operations. reaction Strenuous conditions. Major drawbacks in the chemical preparation of a - terpinyl esters are that they cannot be prepared by the usual esterification procedures involving refluxing the tertiary alcohol with the acid in the presence of a strong acid catalyst, the reason being that the reaction would lead to a variety of products like a - terpinene, 1,8 cineole, p - cymene, terpin hydrate and isoterpinolene (S. Geyer, W. Zeiger and R. Mayer, Z Chem 6(4) 138 - 46 1966). Hence, the procedures for the preparation of acetates and other esters involve reaction conditions which demand a low water environment and the absence of a strong acid catalyst. Although quite a few chemical procedures for the preparation of a - terpinyl acetate are known, those for the preparation of esters of other low molecular weight acids are scanty. Most of these procedures involve rigorous fractionation and azeotropic distillations with many unit operations and the formation of other ester impurities. The workout procedures are also quite tedious and many of these are not cost - effective. Till date, no method on the enzymatic production is known for the preparation of any of the terpinyl esters mentioned above. The present invention involves an enzymatic procedure for the preparation of esters, particularly a -terpinyl propionate. The enzymatic synthesis has many advantages over chemical synthesis such as milder reaction conditions, good yield and pure products. Main objective of the present invention is to develop an enzymatic process for the preparation of a - terpinyl esters where good yields of a - terpinyl esters are obtained by the reaction of lipase preferably obtained from any source such as Mucor meihei and porcine pancreas in a non - polar solvent. New principles and advantages underlying the invention are : Esterification is achieved in a low water environment under milder reaction conditions in a non - polar solvent. The reaction, although slow, yielded a clean product without any side reaction. The method involves the use of carboxylic acid of chain length C2-C5 per se. The yields are almost comparable to those obtained by chemical methods. Rigorous fractionations or azeotropic type esterification methods are not required. Unit operations are less with an easy workout procedure. Use of toxic catalysts is also avoided. Accordingly, the present invention provides an enzymatic process for the preparation of terpinyl esters which comprises reacting terpineol with carboxylic acid of chain length C2-C5 in equimolar ratio in the range 0.02-0.15M, in presence of a lipase wherein the ratio of enzyme to substrate should be minimum 10 in presence of non-polar solvent so as to keep the enzyme completely immersed in the solvent under vigorous agitating conditions up to 1 0h at a temperature ranging from room temperature to 60°C and recovering the ester by conventional methods such as herein described. In an embodiment of the present invention, the terpineol employed is ot a, p, Y - terpineols. In another embodiment of the present invention, the carboxylic acid employed is of chain length C2-C5 acetic, propionic, butyric, isobutyric, valeric and isovaleric acid, their anhydride derivatives and mixtures thereof. In yet another embodiment of the present invention, the lipase used is stieh as immobilized lipase from Mucor meihei, Candida cylindracea, Pseudomonas fluorescens, wheat germ, porcine pancreas and chicken liver esterase. In further embodiment of the present invention, the agitation is carried out at 100-250 rpm range. In still another embodiment of the present invention, the non-polar solvent employed is sueh-as heptane, hexane, isooctane, dichloromethane, chloroform, cyclohexane and diisopropyl ether or mixtures thereof. Immobilised Mucor meihei lipase (Lipozyme IM - 20 immobilised on duolite weak anion exchange resin) was obtained from Novo Nordisk, Denmark. Immobilised porcine pancreatic lipase from Sigma Chemical Company, USA, was used. Terpineol, carboxylic acid and non-polar solvent were from commercial sources (Analytical grade) and were distilled once before use. The reactions were monitored as follows : Aliquots of the reaction mixture (0.5 ml) were withdrawn at regular intervals of time to which 10 mL of 0.01 N NaOH was added. This was titrated against 0.01 N oxalic acid with phenolphthalein as indicator. The titre value was compared with the volume of oxalic acid consumed by 10mL of NaOH. Initially, (at 0 hour incubation with enzyme), the difference in titre values between the reaction mixture with added NaOH, and blank (NaOH alone) corresponds to the volume of NaOH consumed by propionic acid. An increase in the volume of oxalic acid consumed by the reaction mixture on titration as compared to the initial value indicates that the concentration of propionic acid has depleted implying that the reaction has proceeded to yield the ester. While the titration with NaOH showed the consumption of propionic acid in the reaction, Gas Chromatographic analysis showed the formation of the products. A Carbowax 20M column (6m) was used with the column maintained at 100°C and the injection and detection port temperatures being maintained at 200°C and 240°C respectively. GC retention times : a - Terpineol: 18.6 min.; a - Terpinyl propionate : 7.9 min.; Propionic acid : 16.6 min. The process of the invention is described in detail in the examples below which are illustrative only and should not be construed to limit the scope of the invention. EXAMPLE 1 To 10mL of n - heptane is added 0.0306M a - terpineol, 0.031 M propionic acid and immobilised lipase from Mucor meihei at an E/S ratio of 148.4 and the reaction mixture was agitated at 150 rpm, 37°C and 72h. The product workout was by filtering the enzyme and removing the unreacted acid by shaking with a saturated solution of NaHCO3, the organic supernatant was evaporated to get a mixture of a - terpinyl propionate and a - terpineol. The yield of a - terpinyl propionate was found to be 76.9 % (Table 1). EXAMPLE 2 To 10mL of n - heptane is added 0.0907M a - terpineol, 0.09M propionic acid and immobilised lipase from Mucor meihei at an E/S ratio of 152.2 and the reaction mixture was agitated at 150 rpm, 37°C and 72h. The product workout was by filtering the enzyme and removing the unreacted acid by shaking with a saturated solution of NaHCO3 , the organic supernatant was evaporated to get a mixture of a - terpinyl propionate and a - terpineol. The yield of a - terpinyl propionate was found to be 48.4 % (Table 1). EXAMPLE 3 To 10mL of n - heptane is added 0.061 M a - terpineol, 0.066M propionic acid and immobilised lipase from Mucor meihei at an E/S ratio of 145.5 and the reaction mixture was agitated at 150 rpm, 37°C and 72h. The product workout was by filtering the enzyme and removing the unreacted acid by shaking with a saturated solution of NaHCO3, the organic supernatant was evaporated to get a mixture of a - terpinyl propionate and a - terpineol. The yield of a - terpinyl propionate was found to be 43.3 % (Table 1). TABLE 1: Esterification of a - terpineol with immobilised lipase from Mucor meihei (Table Removed) a Alcohol and acid concentration = 0.09M b Alcohol and acid concentration = 0.06M Unless otherwise stated, both alcohol and acid concentration = 0.03M 0 Refers to the slope value obtained from a plot of % esterification versus time in h EXAMPLE 4 To 10mL of n - heptane is added 0.0307M a - terpineol, 0.031 M propionic acid and immobilised lipase from porcine pancreas at an E/S ratio of 58.96 and the reaction mixture was agitated at 150 rpm, 37°C and 72h. The product workout was by filtering the enzyme and removing the unreacted acid by shaking with a saturated solution of NaHCO3, the organic supernatant was evaporated to get a mixture of a - terpinyl propionate and a - terpineol. The yield of a - terpinyl propionate was found to be 28.8%. EXAMPLE 5 To 10mL of n - heptane is added 0.033M a - terpineol, 0.036M propionic acid and immobilised lipase from porcine pancreas at an E/S ratio of 10.3 and the reaction mixture was agitated at 150 rpm, 37°C and 72h. The product workout was by filtering the enzyme and removing the unreacted acid by shaking with a saturated solution of NaHCO3, the organic supernatant was evaporated to get a mixture of a - terpinyl propionate and a - terpineol. The yield of a - terpinyl propionate was found to be 23.2%. EXAMPLE 6 To 10mL of n - heptane is added 0.061 M a - terpineol, 0.062M propionic acid and immobilised lipase from porcine pancreas at an E/S ratio of 35.05 and the reaction mixture was agitated at 150 rpm, 37°C and 72h. The product workout was by filtering the enzyme and removing the unreacted acid by shaking with a saturated solution of NaHCOa • the organic supernatant was evaporated to get a mixture of a - terpinyl propionate and a - terpineol. The yield of a - terpinyl propionate was found to be 19.3%. EXAMPLE 7 To 10mL of n - heptane is added 0.0905M a - terpineol, 0.0456M acetic anhydride and immobilised lipase from Mucor meihei at an E/S ratio of 148.7 and the reaction mixture was agitated at 150 rpm, 37°C and 74h. The product workout was by filtering the enzyme and removing the unreacted acid by shaking with a saturated solution of NaHCO3, the organic supernatant was evaporated to get a mixture of a - terpinyl acetate and a - terpineol. The yield of a - terpinyl acetate was found to be 62.4%. EXAMPLE 8 To 10mL of n - heptane is added 0.091 M a - terpineol, 0.0452M acetic anhydride and immobilised lipase from Mucor meihei at an E/S ratio of 100.1 and the reaction mixture was agitated at 150 rpm, 37°C and 74h. The product workout was by filtering the enzyme and removing the unreacted acid by shaking with a saturated solution of NaHCO3 , the organic supernatant was evaporated to get a mixture of a - terpinyl acetate and a - terpineol. The yield of a - terpinyl acetate was found to be 64.0%. The main advantages of the present invention are : 1) Esterification is achieved in a low water environment under milder reaction conditions in a non - polar solvent. 2) The reaction yielded a clean product without any side reaction 3) The method involves the use of propionic acid per se, and not propionic anhydride. 4) Rigorous fractionation methods are not required. 5) Unit operations are less with an easy workout procedure. 6) The use of toxic catalysts is avoided making the process eco-friendly and economical. 7) Recovery and reuse of enzymes results in a cost - effective procedure. We claim: 1) An enzymatic process for the preparation of terpinyl esters which comprises reacting terpineoi with carboxyiic acid of chain length C2-C5 in equimolar ratio in the range 0.02-0.15M, in presence of an immobilized lipase wherein the ratio of enzyme to substrate should be minimum 10 in presence of non-polar solvent so as to keep the enzyme completely immersed in the solvent under vigorous agitating conditions up to 10h at a temperature ranging from room temperature to 60°C and recovering the ester by conventional methods such as herein described. 2) An enzymatic process as claimed in claim 1 wherein the terpineoi employed is a, (3, 7 - terpineols. 3) An enzymatic process as claimed in claims 1 and 2 wherein the carboxyiic acid employed is of chain length C2-C5 acetic, propionic, butyric, isobutyric, valeric and isovaleric acid. 4) An enzymatic process as claimed in claims 1 to 3 wherein the immobilized lipase used is from Mucor meihei, Candida cylindracea, Pseudomonas fluorescens, wheat germ, porcine pancreas and chicken liver esterase. 5) An enzymatic process as claimed in claims 1 to 4 wherein the agitation is carried out at 100-250 rpm range. 6) An enzymatic process as claimed in claims 1 to 5 wherein the non-polar solvent employed is heptane, hexane, isooctane, dichloromethane, chloroform, cyclohexane and diisopropyl ether or mixtures thereof. 7) An enzymatic process for the preparation of terpinyl esters, substantially as herein described with reference to the examples.

Documents:

2381-del-1998-abstract.pdf

2381-del-1998-claims.pdf

2381-del-1998-correspondence-others.pdf

2381-del-1998-correspondence-po.pdf

2381-del-1998-description (complete).pdf

2381-del-1998-form-1.pdf

2381-del-1998-form-19.pdf

2381-del-1998-form-2.pdf