DE10018787A1 - Lipase-catalyzed synthesis of steryl esters, useful e.g. as nutritional supplements to reduce cholesterol levels, by solventless reaction of sterol with carboxylic acid or ester - Google Patents

Abstract

Enzymatic synthesis of steryl esters (I) of carboxylic acids comprises stirring a carboxylic acid, or its ester (II) with sterol (III) in presence of lipase such that volatile by-products are removed under vacuum.

Description

The invention is characterized by the solvent-free conversion of organic Carboxylic acids or carboxylic acid esters with sterols and other steroids or their short chain esters, the reactants without solvent in the presence of lipases react in vacuo to steryl esters.

The following are naturally occurring and synthetic as sterols (sterols, steroids) Alcohols with a hydrogenated or partially hydrogenated cyclopenta [a] phenanthrene skeleton referred to, which may have other functional groups as substituents such. B. Alkyl residues, keto, hydroxy and carboxy groups. The compounds usually have 18-30 carbon atoms. The term sterols is also used below for Uses compounds that are often referred to as steroids or steroid hormones for example androstane and Pregnan derivatives.

Sterile esters are widely used as pharmaceuticals and components of cosmetics and as liquid-crystalline compounds in industry (Koshiro, A., 1987. Cholesterol ester and its manufacture using microbial lipase. Japan. Kokai Tokkyo Koho 62 296 894 [Chemical Abstracts 110: 210969p, 1989]). In recent times, fatty acid esters of phytosterols have also been called Dietary supplements are used in dietary margarines because they lower plasma cholesterol can lower (Miettinen, T., Vanhagen, H. and Wester, I., 1996. Use of stanol fatty acid ester for reducing serum cholesterol level. U.S. Patent 5,502,045; by Hellens, S., 1999. Benecol margarine enriched with stanol esters. Lipid Technology 11: 29-31; Wester, I., 2000. Cholesterol lowering effect of plant sterols. European Journal of Lipid Science and Technology, 37-44).

It is known that carboxylic acid sterylester by transfer of an acyl residue to hydroxy Groups of sterols can be made in different ways. Chemical Processes are primarily based on the transesterification of carboxylic acid esters with sterols or Reactions of carboxylic acid halides or anhydrides with hydroxyl groups of sterols (Pinter, K.G., Hamilton, J.G. and Muldrey, J.E., 1964. A method for rapid microsynthesis of radioactive cholesterol esters. Journal of Lipid Research 5: 273-274; Piretti, M.V. and Pagliuca, G., 1996. Synthesis of highly pure dolichyl and cholesteryl esters. Italian Journal of Biochemistry 45: 67-69).

Enzymatic processes for the production of carboxylic acid sterylesters go from Carboxylic acids and sterols (esterification) or carboxylic acid esters and sterols (transesterification) from, which are implemented in the presence of lipases as biocatalysts.  

Previous methods (Koshiro, A., 1987. Cholesterol ester and its manufacture using microbial lipase. Japan. Kokai Tokkyo Koho 62 296 894 [Chemical Abstracts 110: 210969p, 1989]; Kosugi, Y., Tanaka, H., Tomizuka, N., Akeboshi, K., Matsufune, Y. and Yoshikawa, S., 1989. Enzymic manufacture of sterol fatty acid esters. Japan. Kokai Tokkyo Koho 1 218 593 [Chemical Abstracts 113, 4707k, 1990]) describe the lipase-catalyzed esterification of organic carboxylic acids with hydroxy groups of sterols, e.g. B. cholesterol, in organic Solvents such as heptane, methyl tert-butyl ether, acetone and others, some in the presence of small amounts of water. Such is the lipase-catalyzed synthesis of cholesteryl docosahexaenoate known that in the presence of about 30% water to a conversion of 90% after a reaction time of 24 hours (Shimada, Y., Hirota, Y., Baba, T., Sugihara, A., Moriyama, S., Tominaga, Y. and Terai, T., 1999. Enzymatic synthesis of steryl esters of polyunsaturated fatty acids. Journal of the American Oil Chemists' Society 76: 713-716).

A number of other publications describe the synthesis of short-chain carboxylic acid steroid esters and of long-chain steryl esters in organic solvents, in some cases using molecular sieves in the reaction mixture to remove the water or alcohol formed (Haraldsson, GG, 1992. The application of lipases in organic In: Supplement B: The chemistry of acid derivatives, Vol. 2 [Patai, S., Ed.] pp. 1395-1473, John Wiley, Chichester; Kazlauskas, RJ and Bornscheuer, UT, 1998. Biotransformations with lipases, in: Biotechnology [Rehm H.-J., and Reed, G., Eds) 2 ^nd edition, Vol 8a: Biotransformations, pp 37-191, Wiley-VCH, Weinheim;. Riva, S. and Klibanov, AM, 1988. Enzymochemical regioselective oxidation of steroids without oxidoreductases, Journal of the American Chemical Society 110: 3291; Riva, S., Bovara, R., Ottolina, G., Secundo, F. and Carrea, G., 1989. Regioselective acylation of bile acid derivatives with Candida cylindracea lipase in anhydrous benzene. Journal of Organic Chemistry 54: 3161-3164; Faber, K. and Riva, S., 1992. Enzyme-catalyzed irreversible acyl transfer. Synthesis, 895-910).

Lipase-catalyzed esterification and transesterification reactions in vacuo to remove the volatile reaction products are already used to produce various ester compounds been used. For example, the following connections were made in this way: Alkyl esters of fatty acids (Miller, C., Austin, H., Posorske, L. and Gonzalez, J., 1988. Characteristics of an immobilized lipase for the commercial synthesis of esters. Journal of the American Oil Chemists' Society 65: 927-931), 1,3-diglycerides (Rosu, R., Yasui, M., Iwasaki, Y. and Yamane, T., 1999. Enzymatic synthesis of symmetrical 1,3-diacylglycerols by direct esterification of glycerol in solvent-free system. Journal of the American Oil Chemists' Society 76: 839-843), structured triglycerides (Han, J.J. and Yamane, T., 1999. Enhancement of both  reaction yield and rate of synthesis of structured triacylglycerol containing eicosapentaenoic acid under vacuum with water activity control. Lipids 34: 989-995) and thioesters of fatty acids (Weber, N., Klein, E. and Mukherjee, K.D., 1999. Enzymatic process for the production of Carboxylic acid thioesters. Ger. Patent Application 199 05 962 A1; Weber, N., Klein, E., Mukherjee, K.D. and Vosmann, K., 1999. Enzymatic process for the preparation of steric uniform unsaturated carboxylic acid thioester and isomeric saturated S-alkylcarboxylic acid thioester. Ger. Patent Application 199 36 549 A1).

In contrast to the known methods mentioned above, the following are enzymatic syntheses described in detail without solvents and without Molecular sieve or other desiccant carried out in the reaction mixture. Rather be the mixtures of the reactants, d. s. Carboxylic acids or carboxylic acid esters and sterols or their short-chain esters, enzyme-catalyzed in the presence of lipases and that water or short-chain alcohols, acids or esters formed in a vacuum removed the reaction mixture. About this lipase-catalyzed esterification and Transesterification reactions under vacuum for the preparation of carboxylic acid sterylesters has so far been nothing known.

Esterification: R ₁ -CO-OH + R ₂ -OH ⇄ R ₁ -CO-OR ₂ + H ₂ O

Transesterification:
R ₁ -CO-OR ₃ + R ₂ -OH ⇄ R ₁ -CO-OR ₂ + R ₃ OH
R ₁ -CO-OH + R ₂ O-CO-R ₄ ⇄ R ₁ -CO-OR ₂ + R ₄ -CO-OH
R ₁ -CO-OR ₃ + R ₂ O-CO-R ₄ ⇄ R ₁ -CO-OR ₂ + R ₄ -CO-OR ₃
where R _{1 is,} for example, alkyl radicals with 2 to 30 carbon atoms and R _{2 is} steryl radicals, while R _{3 is} preferably short-chain alkyl radicals and R ₄ is short-chain acyl radicals with 1 to 6 carbon atoms

For the production of carboxylic acid sterylesters, saturated and unsaturated are used Sterols such as B. sitostanol (stigmastanol), α-cholestan-3β-ol (dihyrocholesterol), sitosterol (β- Sitosterol), cholesterol, ergosterol and 7-dehydrocholesterol as well as sterols with other alkyl Groups such as B. lanosterol and dihydrolanosterol, or sterols with other hydroxy Groups or other functional groups, such as. B. 5α-androstane-3β, 17β-diol, 5α-Pregnan-  3β-ol-20-one, 5-Pregnen-3β-ol-20-one and cholic acid, deoxycholic acid and others Bile acids used.

The process is applicable to a large number of carboxylic acids and also di- and polycarboxylic acids with a linear, branched-chain and cyclic structure of chain lengths C ₂ to C ₃₀ . For example, long-chain saturated and unsaturated aliphatic carboxylic acids such as lauric acid, palmitic acid, oleic acid and linoleic acid or also those with aromatic residues such as cinnamic acid and dihydrocinnamic acid are used as carboxylic acids. Branched-chain and cyclic aliphatic carboxylic acids, for example isostearic acid and cyclopentenyl fatty acids, are also used as reactants. Furthermore, aliphatic di- and polycarboxylic acids, such as. B. glutaric, cork and 1,12-dodecanedioic acid or tricarballylic acid, converted with sterols in the presence of lipases into di- and polycarboxylic acid sterylesters or their partial esters.

As carboxylic acid esters, which are used for the synthesis of carboxylic acid steryl esters, mainly esters of the above-mentioned linear, branched or cyclic saturated and unsaturated aliphatic carboxylic acids or di- and polycarboxylic acids, such as. B. esters of myristic acid, stearic acid, oleic acid, linoleic acid, isostearic acid, dihydrocinnamic acid, 1,8-octanedioic acid (suberic acid) or tricarballylic acid, with a short-chain linear or branched chain alcohol of chain length C ₁ to C ₆ . Other carboxylic acid esters, especially triacylglycerols (triglycerides) such as. As triacetin, tripropanoin, tricaproin and triolein, but also carbonic acid esters such as. B. dimethyl and diethyl carbonate and short-chain alkyl cholesteryl carbonates are used as acyl donors in the transesterification. Natural triglycerides of fats and oils, e.g. B. beef tallow or rapeseed oil, sunflower oil and soybean oil can also be used for the transesterification with sterols. Short-chain triglycerides such as. B. triacetin and tripropanoin for the preparation of carboxylic acid sterylesters (carboxylic acid steroid esters) with short (C ₂ to C ₈ ) acyl residues, which are of interest, for example, for pharmaceutical purposes:

where R _{1 is,} for example, alkyl radicals having 1 to 30 carbon atoms, R ₂ steryl radicals and R _{3 are} hydrogen atoms or short-chain acyl radicals having 1 to 6 carbon atoms

The transesterification (alcoholysis) of triglycerides with hydroxy groups of sterols is required in contrast to the esterification of carboxylic acids or transesterification of Carboxylic acid esters basically no vacuum, since no water or short-chain alcohol is formed. Nevertheless, here too the yield increases when the reaction is in vacuo is carried out.

All known lipases can be used as enzymes for the production of the carboxylic acid sterylester catalysts are used, especially lipases from microorganisms, such as. B. from Rhizopus arrhizus, Candida antarctica, Candida rugosa (= C. cylindracea), Rhizomucor miehei and Geotrichum candidum, pancreatic lipase from various animal species and lipases Plants such as B. papaya, rapeseed, castor, rice, vernonia and pineapple.

The lipase-catalyzed syntheses of carboxylic acid sterylesters can be used in different Reaction conditions are carried out, in particular by the selected lipases depend. In general, temperatures between 10 and 100 ° C are used, preferred those between 30 and 80 ° C. The molar ratios between the reactants, i.e. s. Carboxylic acids or carboxylic acid esters and sterols or their short-chain esters, and the proportions the added lipases are not subject to any restrictions. The response time is also not limited. Stir the reaction mixture and increase the amount of lipase in the Experimental approach increase the reaction rate and accelerate the formation of Carboxylic acid ester. Repeated use of those used in a reaction approach Lipase in a new approach is without significant loss of performance of the biocatalyst possible.

Pressures between 900 mbar and 0.1 mbar can be used for the reaction; Pressures between 200 mbar and 10 mbar are usually maintained.

The carboxylic acid steryl esters can be purified in different ways. To the reaction is carried out with the enzyme catalyst by centrifugation, filtration or extraction an organic, preferably apolar solvent is separated from the reaction mixture. The reaction mixture is either used directly or packed on a short column a sorbent, preferably silica gel. By elution with apolar and less polar, aprotic organic solvents, e.g. B. iso-hexane / diethyl ether mixtures, the carboxylic acid sterylesters are then converted from unreacted carboxylic acids or Carboxylic acid esters and sterols separated.

By reacting stoichiometric amounts of carboxylic acids or carboxylic acid esters and Sterols can also be converted into high purity carbonic acid esters with high purity (<90%) win without complex cleaning of the reaction products.  

Various exemplary embodiments are given below for the production of Carboxylic acid ester, which is carried out in heated stirred vessels under vacuum:

example 1

Sterol: Sitostanol 41.7 mg (0.1 mmol)
Carboxylic acid: oleic acid 84.7 mg (0.3 mmol)
Lipase: Candida rugosa lipase, immobilized, 50 mg
Temperature: 40 ° C
Pressure: 50 mbar
Duration: 4 hours
Work-up: After the reaction, the enzyme catalyst is separated from the reaction mixture by extraction twice with 3 ml of diethyl ether and subsequent centrifugation. The solvent is evaporated, the reaction mixture is dissolved in 1 mL iso-hexane / diethyl ether (9: 1, v / v) and adsorbed on a short column (20 × 1 cm ID) with silica gel in iso-hexane. Elution with 20 mL iso-hexane / diethyl ether (95: 5, v / v) gives 60.5 mg of oleic acid sitostanyl ester. Subsequent elution with 20 mL iso-hexane / diethyl ether (1: 1, v / v), excess oleic acid and unreacted sitostanol are recovered.
Analysis: The analysis of the product is carried out by GC as described in Example 2.
Conversion (GC): 95% oleic acid sitostanyl ester (based on sitostanol)
Purity (GC): <95%
Melting range: 44-45 ° C

Example 2 Analysis of the carboxylic acid sterylester Thin layer chromatography on silica gel layers

Portions of the reaction mixtures, dissolved in iso-hexane / diethyl ether, are placed on silica gel Thin-layer boards (0.3 mm layer thickness) applied in a dot or line. The plates are developed in a mixture of iso-hexane / diethyl ether (95: 5, v / v) and the different compounds of the reaction mixture separated. By spraying the plates with an aqueous sulfuric acid solution and then heating or by steaming with  The different compounds are then made visible to iodine. The identification of the individual connection classes are made by comparison with known standards. The following Rf values are given for the different classes of compounds in the reaction mixture Found use of the above eluent: carboxylic acid steryl ester 0.6-0.7; Fatty acid- methyl ester 0.4-0.5; Triglycerides 0.3-0.35; Sterols 0.1-0.15; free fatty acids and others Carboxylic acids <0.1. Silica gel thin-layer plates on which reaction mixtures with 5a- Pregnan-3β-ol-20-one and 5-Pregnen-3β-ol-20-one-propionates and with dicarboxylic acid steryl esters or carbonic acid sterylesters are separated, developed in a mixture of iso-hexane / diethyl ether (80:20, v / v); Rf values: 5α-Pregnan-3β-ol-20-one and 5-Pregnen- 3β-ol-20-one-propionate 0.20-0.25; Tripropionin 0.15-0.20; Dicarboxylic acid methyl cholesteryl ester 0.45-0.55; Dicarboxylic acid dicholesteryl ester 0.70-0.80; Dimethyl dicarboxylic acid 0.30-0.40; Dicarboxylic acids <0.1; Ethyl cholesteryl carbonate 0.60-0.65; Oleyl cholesteryl carbonate 0.75-0.80.

Gas chromatography

Portions of the reaction mixtures are suspended in diethyl ether and the Lipase catalysts by centrifugation and subsequent filtration through a syringe filter (Pore size 0.35 µm) separated. Free carboxylic acids in the reaction mixture are by Derivatization with diazomethane converted into the corresponding methyl ester. The resulting Mixture of carboxylic acid methyl esters, unreacted sterols and carboxylic acid steryl esters are analyzed by gas chromatography. The separation on a 15 m × 0.25 mm i. D. Quadrex 400-1HT capillary column (hydrogen as a carrier gas with a linear flow speed of 25-35 cm / sec; Temperature program: 160 ° C [2 min isothermal] followed by a linear temperature increase from 160 to 180 ° C at 5 ° C / min, then at 20 ° C / min to 410 ° C [10 min isothermal]) provides for the different compounds of the reaction mixtures for example the following retention times (Rt): methyl laurate (0.8 min); Myristic acid methyl ester (0.9 min); Ethyl dihydrocinnamic acid (1.0 min); Methyl stearate (1.9 min); Oleic acid methyl ester (1.8 min), linoleic acid methyl ester (1.7 min); Dimethyl coric acid (Dimethyl 1,8-octanedicarboxylate, 0.9 min); Dimethyl 1,12-dodecanedicarboxylate (1.0 min); 9-octadecenedicarboxylic acid dimethyl ester (3.1 min); Tripropanoin (0.8 min); Tributyrin (1.1 min); Cholesterol (8.9 min); Sitosterol (9.7 min); Stigmaster (9.2 min); Ergosterol (8.9 min); 7-dehydrocholesterol (8.8 min); Lanosterol (9.5 min); Dihydrolanosterol (9.3 min); α-cholestan-3β-ol (dihydrocholesterol) (8.8 min); Sitostanol (9.6 min); Epicoprostanol (8.6 min); 5α-Pregnan-3β-ol-20-one (5.6 min); 5-Pregnen-3β-ol-20-one (5.7 min); Butyric acid cholesteryl ester (10.0 min); Cholesteryl laurate (12.5 min); Myristic acid  cholesteryl ester (12.9 min); Cholesteryl stearic acid (13.8 min); Oleic acid cholesteryl ester (13.9 min); Oleic acid cholestanyl ester (14.0 min); Oleic acid sitostanyl ester (14.3 min); Linoleic acid sitostanyl ester (14.3 min); Oleic acid ergosterylester (14.0 min); Oleic acid 7-dehydrocholesteryl ester (13.9 min); Oleic acid lanosteryl ester (14.2 min); Oleic acid dihydrolanosteryl ester (14.1 min); 5α-Pregnan-3β-ol-20-one-propionate (7.7 min); 5-pregnen-3β-ol-20-one-propionate (7.7 min); Ethyl cholesteryl carbonate (10.0 min); Korkäure-methylcholesteryldiester (1,8- Octanedicarboxylic acid methylcholesteryl diester, 12.0 min); 1,12-dodecanedicarboxylic acid methyl cholesteryl diester (12.8 min); 9-octadecen-1,18-dicarboxylic acid methylcholesteryl diester (14.1 min).

Reversed-Phase High Performance Liquid Chromatography (RP-HPLC)

The conversion of the lipase-catalyzed transesterification of ethyl cholesteryl carbonate with oleyl alcohol is determined by RP-HPLC. Portions of the reaction mixture are suspended in acetone and the lipase catalyst is separated off by centrifugation and subsequent filtration through a syringe filter (pore size 0.35 μm). The resulting solution, consisting of ethyl cholesteryl carbonate, oleyl alcohol and oleyl cholesteryl carbonate, is analyzed using HPLC on a C ₁₈ reverse phase column. A Rheodyne Injector with 20 µL sample loop is used for sample application. The separation takes place on a 250 × 4 mm Hibar RP-18 column, packed with 10 µm LiChrospher 100 (Merck AG, Darmstadt), with acetonitrile / acetone (25: 75, v / v) as eluent at a flow rate of 1 mL / min. The detection is carried out with a light scattering detector. The following retention times are measured under these conditions: oleyl alcohol (3.8 min); Ethyl cholesteryl carbonate (7.3 min); Oleyl cholesteryl carbonate (22.1 min).

In the same way, the turnover of the lipase-catalyzed transesterification of 1.8 Octanedicarboxylic acid dimethyl ester with cholesterol determined by RP-HPLC. The separation takes place on a 250 × 4 mm Hibar RP-18 column, packed with 10 µm LiChrospher 100 (Merck AG, Darmstadt), at a flow rate of 1 mL / min. Elution begins with acetonitrile / acetone (25: 75, v / v) for 15 min, then there is a linear increase in the acetone concentration within from 5 min to a ratio of acetonitrile / acetone (1: 9, v / v), which for a further 30 min is maintained. The detection is carried out with a light scattering detector. The following Retention times are measured under these conditions: 1,8-octanedicarboxylic acid dimethyl ester (2.1 min); Cholesterol (13.1 min); 1,8-octanedicarboxylic acid methyl cholesteryl diester (7.1 min); 1,8-octanedicarboxylic acid dicholesteryl diester (36.1 min).  

Example 3

Sterol: cholesterol 38.7 mg (0.1 mmol)
Carboxylic acid ester: methyl stearate 89.6 mg (0.3 mmol)
Lipase: Rhizomucor miehei lipase, immobilized (Lipozyme®) 50 mg
Temperature: 80 ° C
Pressure: 30 mbar
Duration: 48 hours

The product is worked up as in Example 1, the analysis by GC as described in Example 2.
Turnover (GC): 78% stearic acid cholesteryl ester (based on cholesterol)
Purity (GC): 98%
Melting range: 83-84 ° C

Example 4

Sterol: ergosterol 39.7 mg (0.1 mmol)
Carboxylic acid: oleic acid 84.7 mg (0.3 mmol)
Lipase: Candida rugosa lipase, immobilized, 50 mg
Temperature: 40 ° C
Pressure: 30 mbar
Duration: 24 hours

The product is worked up as in Example 1, analysis by GC as described in Example 2.
Turnover (GC): 88% oleic acid ergosterylester (based on ergosterol)
Purity (GC): 95%
Melting range: 76-77 ° C

Example 5

Sterol: 7-dehydrocholesterol 38.5 mg (0.1 mmol)
Carboxylic acid: oleic acid 84.7 mg (0.3 mmol)
Lipase: Candida rugosa lipase, immobilized, 50 mg
Temperature: 40 ° C
Pressure: 30 mbar
Duration: 24 hours

The product is worked up as in Example 1, the analysis by GC as described in Example 2.
Sales (GC): 85% oleic acid 7-dehydrocholesteryl ester (based on 7-dehydrocholesterol)
Purity (GC): 94%
Melting range: 69-70 ° C

Example 6

Sterol: Lanosterin ^a)

42.7 mg (0.1 mmol)
Carboxylic acid ester: oleic acid methyl ester 178.0 mg (0.6 mmol)
Lipase: Rhizomucor miehei lipase, immobilized (Lipozyme®) 100 mg
Temperature: 80 ° C
Pressure: 20 mbar
Duration: 120 hours

The product is worked up as in Example 1, the analysis by GC as described in Example 2.
Conversion (GC): 17% oleic acid lanosterylester + oleic acid dihydrolanosterylester (based on lanosterol ^a) )
Purity (GC): 90% oleic acid lanosterylester + oleic acid dihydrolanosterylester ^a)

^a) The proportion of dihydrolanosterol or dihydrolanosterylester is about 30%.

Example 7

Sterol: Sitostanol 41.7 mg (0.1 mmol)
Carboxylic acid ester: oleic acid methyl ester 89.0 mg (0.3 mmol)
Lipase: Rhizomucor miehei lipase, immobilized (Lipozyme®) 50 mg
Temperature: 80 ° C
Pressure: 40 mbar
Duration: 24 hours

The product is worked up as in Example 1, the analysis by GC as described in Example 2.
Conversion (GC): 95% oleic acid sitostanyl ester (based on sitostanol)

Example 8

Sterol: Sitostanol 41.7 mg (0.1 mmol)
Carboxylic acid ester: trioleoylglycerol (triolein) 132.8 mg (0.3 mequival to sn-1,3 esterified oleic acid residues; 0.15 mmol)
Lipase: Candida rugosa lipase, immobilized, 50 mg
Temperature: 40 ° C
Pressure: 20 mbar
Duration: 8 hours

The product is worked up as in Example 1, the analysis by GC as described in Example 2.
Sales (GC): 89% oleic acid sitostanyl ester (based on sitostanol)

Example 9

Sterol: Sitostanol 41.7 mg (0.1 mmol)
Carboxylic acid ester: trioleoylglycerol (triolein) 132.8 mg (0.3 mequival to sn-1,3 esterified oleic acid residues; 0.15 mmol)
Lipase: Papaya latex lipase 50 mg
Temperature: 80 ° C
Pressure: 20 mbar
Duration: 48 hours

The product is worked up as in Example 1, the analysis by GC as described in Example 2.
Turnover (GC): 60% oleic acid sitostanyl ester (based on sitostanol)

Example 10

Sterol: 5-Pregnen-3β-ol-20-one 31.6 mg (0.1 mmol)
Carboxylic acid ester: tripropionin 651 mg (2.5 mmol)
Lipase: Candida rugosa lipase, immobilized, 100 mg
Temperature: 40 ° C
Pressure: 20 mbar
Duration: 24 hours

The product is worked up by extracting the reaction mixture as iso-hexane Solution with a methanol / water mixture and subsequent column chromatography:

Purification of the 5-Pregnen-3β-ol-20-one-propionate

The reaction mixture of the lipase-catalyzed synthesis of 5-pregnen-3β-ol-20-one propionate is extracted twice with diethyl ether and the lipase catalyst is passed through Centrifugation separated. The solvent is distilled off, the residue in 5 mL iso- Dissolved hexane and the majority of the tripropanoin by extracting three times with 3 mL each Methanol / water (95: 5, v / v) removed. The iso-hexane phases are then over dried anhydrous sodium sulfate. The pre-cleaned product mixture is on a Silica gel column (20 × 1 cm) separated as described in Example 1.

The analysis is carried out by GC as described in Example 2.
Conversion (GC): 78% 5-Pregnen-3β-ol-20-one-propionate (based on 5-Pregnen-3β-ol-20-one)
Purity (GC): 93%
Melting range: 122-123 ° C

Example 11

Sterol: cholesterol 386.7 mg (1 mmol)
Carboxylic acid: oleic acid 282.5 mg (1 mmol)
Lipase: Candida rugosa lipase, immobilized, 500 mg
Temperature: 40 ° C
Pressure: 50 mbar
Duration: 48 hours

The product is worked up by filtration or centrifugation in the heat without further purification steps, and analysis by GC as described in Example 2.
Sales (GC): 93% oleic acid cholesteryl ester (based on cholesterol)
Purity (GC): 93%
Melting range: 48-50 ° C

Example 12

Sterol: Sitostanol 41.7 mg (0.1 mmol)
Carboxylic acid ester: sunflower oil ^b)

132.8 mg (0.15 mmol; 0.3 mequival based on sn-1,3 esterified fatty acid residues)
Lipase: Candida rugosa lipase, immobilized, 50 mg
Temperature: 40 ° C
Pressure: 30 mbar
Duration: 24 hours

The product is worked up as in Example 1, the analysis by GC as described in Example 2.
Conversion (GC): 99% fatty acid sitostanyl ester ^b) (based on sitostanol)
Purity (GC): <95%

^b) esterified fatty acid residues of sunflower oil: 8.5% palmitic acid, 3.9% stearic acid, 24.0% oleic acid and 63.4% linoleic acid residues

Example 13

Sterol: ethyl cholesteryl carbonate 45.9 mg (0.1 mmol)
Alcohol: oleyl alcohol 80.6 mg (0.3 mmol)
Lipase: Rhizomucor miehei lipase, immobilized (Lipozyme®) 50 mg
Temperature: 80 ° C
Pressure: 40 mbar
Duration: 48 hours

The product is worked up as in Example 1, the analysis by RP-HPLC as described in Example 2.
Conversion (RP-HPLC): 19% oleyl cholesteryl carbonate (based on ethyl cholesteryl carbonate)
Purity (RP-HPLC): <95%
Melting range: 32-33 ° C

Example 14

Sterol: cholesterol 38.7 mg (0.1 mmol)
Carboxylic acid ester: dihydrocinnamic acid ethyl ester 178.2 mg (1.0 mmol)
Lipase: Rhizomucor miehei lipase, immobilized (Lipozyme®) 100 mg
Temperature: 80 ° C
Pressure: 50 mbar
Duration: 96 hours

The product is worked up as in Example 1, the analysis by GC as described in Example 2.
Turnover (GC): 70% dihydrocinnamic acid cholesteryl ester (based on cholesterol)
Purity (GC): 90%
Melting range: 111-113 ° C

Example 15

Sterol: Sitostanol 41.7 mg (0.1 mmol)
Carboxylic acid: oleic acid 84.7 mg (0.3 mmol)
Lipase: Candida rugosa lipase, immobilized, 50 mg
Temperature: 40 ° C
Pressure: 50 mbar
Duration: 4 hours

Deacidification

The product is purified by removing the free fatty acid from the reaction mixture ("deacidifying"): portions of excess free oleic acid are removed from the reaction mixture by deacidifying with dilute aqueous sodium carbonate solution. For this purpose, the reaction mixture of the lipase-catalyzed synthesis of oleic acid sitostanyl ester is extracted twice with diethyl ether and the lipase catalyst is separated off by centrifugation. Free oleic acid is removed from the ether solution by extraction three times with 3 ml of 2% sodium carbonate solution and subsequent washing with water. The ether phase is dried over sodium sulfate and an aliquot is analyzed by gas chromatography as described in Example 2.
Conversion (GC): 95% oleic acid sitostanyl ester (based on sitostanol)
Purity (GC): 94% oleic acid sitostanyl ester (contains 5% sitostanol and <1% oleic acid)

Example 16

Sterol: cholesterol 77.3 mg (0.2 mmol)
Carboxylic acid ester: 1,8-octanedioic acid dimethylester 1214 mg (1.2 mmol)
Lipase: Rhizomucor miehei lipase, immobilized (Lipozyme®) 200 mg
Temperature: 80 ° C
Pressure: 50 mbar
Duration: 72 hours

The product is worked up as described in Example 1, but the 1,8-octanedioic acid dicholesteryl diester is mixed with 20 ml of iso-hexane / diethyl ether (9: 1, v / v) and the 1,8-octanedioic acid methylcholesteryl diester with 20 ml Iso-hexane / diethyl ether (4: 1, v / v) eluted. The analysis is carried out by GC and RP-HPLC as described in Example 2.
Yield (isolated): 60% 1,8-octanedioic acid methylcholesteryl diester + 5% 1,8-octanedioic acid dicholesteryl diester (based on cholesterol)
Purity (GC, RP-HPLC): <95% 1,8-octanedioic acid methylcholesteryl diester <95% 1,8-octanedioic acid dicholesteryl diester)
Melting range: 83-84 ° C 1,8-octanedioic acid methylcholesteryl diester 188-191 ° C 1,8-octanedioic acid dicholesteryl diester

Claims (6)

1. Starting enzymatic process for the synthesis of organic carboxylic acid sterylesters of organic carboxylic acids or carboxylic acid esters and sterols with stirring in Presence of lipases, whereby volatile by-products are removed in vacuo become. 2. The method according to claim 1, wherein the carboxylic acid by a di- or polycarboxylic acid is replaced. 3. The method according to claim 1, wherein the carboxylic acid ester by a di- or Polycarboxylic ester is replaced. 4. The method according to claim 1, wherein the carboxylic acid ester is a triacylglycerol (triglyceride) or the carboxylic acid polyester of another polyol. 5. The method according to claim 1, wherein the sterols have further functional groups. 6. The method according to claims 1 to 5, in which steryl esters of short-chain carboxylic acids instead of sterols.