Title of Invention

ENZYMATIC ACYLATION

Abstract

A process for the manufacture of (UZ, 13Z)-7, lO-dihydro-lO-hydroxy-retinyl acylates comprises selectively monoacylating (UZ, 13Z)-7,10-dihydro-10-hydroxy- retinol in an organic solvent in the presence of an acylating agent with a lipase, especially of the enzyme class EC 3.1.1.3, which is present in suspension. In order to improve the accessibility to the lipase and its re-use, the lipase can be immobilized on various carrier materials. The immobilization can also be effected in the presence of a cholanic salt, e.g. sodium cholate or deoxycholate, by which means the activity can be increased to some extent. The process in accordance with the invention is outstandingly suitable for the manufacture of (IIZ, 13Z)-7,10- dihydro-10-hydroxy-retinyl acetate, an important startmg materia! for the production of vitamin A acetate, and this use as well as generally the use of a (1IZ, 13Z)-7, 10-dihydro-l0-hydroxy-retinyl acylate obtained in accordance with the invention for the production of the corresponding vitamin A acylate is a further aspect of the present invention. PRICE: THIRTY RUPEES

Full Text

The present invention is concerned with a novel process for 5 the manufacture of (11Z,1 3Z)-7,10-dihydro-10-hydroxy-retinyl acylates [(2Z,4Z,7E)-carboxylic acid 3,7-dimethyl-6-hydroxy-9-[2',2',6'-trimethyl-cyclohex-6'-en-1 '-yl]nona-2,4,7-trienyl esters] of the formula 10 wherein R signifies a Ci-zs-alkyl group or a C2-23-all 20 (1 1Z,13Z)-7,10-Dihydro-10-hydroxy-retinyl acylates are starting materials for the production of the corresponding vitamin A acylates, namely by cleavage of water and simultaneous cis-trans isomerization, which can be carried out in 2 5 a manner known per se. Using the usual standard esterification methods there is obtained, for example, by acetylating (11Z,13Z)-7,10-dihydro-10-hydroxy-retinol in addition to the desired and especially important (11Z,1 3Z)-7,1 0-dihydro-l 0-hydroxy-retinyl acetate also the byproduct (11Z,1 3Z)-7,1 0-dihydro-l 0-acetoxy- 30 retinyl acetate [(2Z,4Z,7E)-acetic acid 6-acetoxy-3,7-dimethyl-9-[2',2',6'-trimethyl-cyclohex-6'-en-T-yl]nona-2,4,7-trienyl ester] of the formula 5 which occurs in varying amounts. The (11Z,13Z)-7,10-dihyclro-10-acetoxy-retinyl acetate of formula III is inert under the conditions of the catalytic dehydration and is therefore troublesome not only in the working- 10 up of (11Z,13Z)-7,10-dihydro-10-hydroxy-retinyl acetate (formula I, in which R signifies methyl), but also in the working-up of vitamin A acetate. Accordingly, a process not only for the selective monoacetylation, but also generally for the selective monoacylation of (11Z,1 3Z)-7,1 O-dihydro-10-hydroxy-retinol of 15 formula II is of great interest from the industrial point of view. The object of the present invention is to provide a process for the manufacture of (1 ^Z,13Z)-7,^0-dihydro-^0-hydroxy-retinyl acylates of formula I by the esterification of (11Z,13Z)- 20 7,10-dihydro-10-hydroxy-retinol which does not have the disadvantages of previously known procedures (e.g. undesired formation of (11Z,1 3Z)-7,10-dihydro-10-acetoxy-retinyl acetate of formula III). For this it is necessary that the enzyme-catalyzed reaction proceeds extremely selectively and in high 25 yields and that the enzyme displays the catalytic activity even in low amounts, can be readily separated and can be used again several times. In the scope of the present invention this object has been 30 achieved by carrying out the acylation of (1 ^Z,^3Z)-7,^0-dihydro-1 0-hydroxy-retinol of formula II in the presence of a lipase (enzyme class EC 3.1.1.3) and under quite specific pre-reaction and reaction conditions. 3 5 Accordingly, the present invention is concerned with a process for the manufacture of a (11Z,1 3Z)-7,10-dihydro-1 0- hydroxy-retinyl acylate of formula I given above, wherein R signifies a Ci-zs-alkyl group or a C2-23-ali The selective acylation of primary hydroxy groups in 10 addition to secondary in the presence of lipases is known from the literature. Thus, for example, according to J. Org. Chem. 5^, 2366-2369 (1990) the acylation of amphenicols, i.e. of phenyl-substituted short-chain aliphatic diols, by esterification with a trifluoroethyl acylate or a cyclic anhydride is carried out using a 1 5 lipase isolated from Pseudomonas cyclopium, with the yields being 83% and 64%, respectively, without the purity being given. The racemate resolution of 2-methyl-5-(4-methoxyphenyl)-pentane-1,3-diol by esterification with vinyl acetate in the 20 presence of immobilized lipase PS is described in Tetrahedron: Asymmetry 4, 757-760 (1993), with the yield of monoacylated product being 66% and the optical purity being 42% e.e. In Appl. Biochem. Biotechnol. H, 401-407 (1985) a series of 25 1,2- and 1,3-diols (dissolved in ethyl acylates) has been acylated with lipase from porcine pancreas in yields of According to J. Chem. Soc, Chem. Commun., 1989, 1 535-1536, 2-ethyl-hexane-l ,3-diol has been acylated with lipase 30 from porcine pancreas in 60% yield. Tetrahedron Lett. H, 3405-3408 (1990) describes the selective acylation of aliphatic l,n-diols with anhydrides in the presence of lipase from porcine pancreas, with the selectivities 3 5 being The selective acylation (98% selectivity) of 1,5-hexanediol with n-decanoic acid with lipase from Chromobacterium viscosum is described in Ind. J. Chem. 12., 30-34 (1993). All of these previously known processes have certain disadvantages: thus all of these processes yield the desired products, but the selectivity and therewith the purity and/or the yield leave much to be desired. Moreover, none of these literature references mentions a repeated use of the lipase employed, i.e. the stability of the lipase, let alone a simultaneous substrate purification. The acylation of (11Z,^3Z)-7,10-dihydro-10-hydroxy-retinol of formula II is carried out as already mentioned in an organic solvent (which is preferably almost anhydrous) in the presence of an acylating agent with a lipase which is present in suspension. Suitable lipases for the purpose of the present invention are those of the enzyme class EC 3.1.1.3, which show good activity and selectivity, especially lipase PL from Alcaligenes sp., its immobilized forms lipase PLC and lipase PLC, and lipase MY-30 from Candida cylindracea (renamed Candida rugosa) (Meito Sangyo, Tokyo, Japan), Lipozyme® IM-20 from Mucor miehei (renamed Rliizomucor mieliei) (Novo Nordisk, Bagsvaerd, Denmark), lipase CE-5 from Humicola lanuginosa and lipase G from Penicillium cyclopium (both Amano Pharmaceutical Co. Ltd., Nagoya, Japan) as well as Chirazyme® L-2 from Candida antarctica (Boehringer Mannheim GmbH, Germany; formerly Novozym® SP 435 from Novo Nordisk). Lipase PL, lipase PLC, lipase PLG, Lipozyme® IM-20 and Chirazyme® L-2 are especially preferred, with lipase PL, lipase PLC, lipase PLG and Chirazyme® L-2 being particularly preferred. Under the term "Ci-23-alkyl group" or "Cz-zs-alkenyl group containing 1 to 3 double bonds" (R) there are to be understood depending on the number of carbon atoms not only straight-chain, but also branched alkyl or alkenyl groups. Examples of C1-23- alkyl groups are methyl, ethyl, propyl, pentyl, heptyl, undecyl, pentadecyl and heptadecyl, and examples of C2-23-all 10 In a batch process the lipase, which in the commercially available form is present as a powder, granulate or small beadlets, is used in up to 20 weight % (wt.%) (wt./wt. based on (11Z,13Z)-7,10-dihydro-10-hydroxy-retinol), but the amount of lipase preferably lies between about 0.1 wt.% and about 10 wt.%, 1 5 particularly between about 1 wt.% and 5 wt.%. As organic solvents which are suitable for the purpose of the present invention there can be named: aliphatic hydrocarbons with 5-8 carbon atoms, such as hexane and heptane; alicyclic 20 hydrocarbons with 6-10 carbon atoms, such as cyclohexane, methylcyclohexane and decalin; chlorinated aliphatic hydro¬carbons, such as methylene chloride, chloroform and carbon tetrachloride; nitro-substituted aliphatic hydrocarbons, such as nitromethane; aromatic hydrocarbons, such as toluene and xylene; 25 aliphatic ethers, such as 1,2-dimethoxyethane, diethyl ether, diisopropyl ether and tert.butyl methyl ether; cyclic ethers, such as tetrahydrofuran, methylfuran and 1,4-dioxan; aliphatic esters, such as trimethyl orthoformate, ethyl acetate, butyl acetate, vinyl acetate, vinyl propionate and isopropenyl acetate; aliphatic 30 ketones, such as acetone; aliphatic nitriles, such as acetonitrile; aliphatic amines, such as triethylamine; aliphatic acetals, such as formaldehyde dimethyl acetal; and mixtures of such solvents. Preferred are hexane, cyclohexane, methylene chloride, carbon tetrachloride, toluene, diisopropyl ether, vinyl acetate, vinyl 3 5 propionate, tetrahydrofuran, methylfuran and formaldehyde dimethyl acetal, especially methylene chloride, diisopropyl ether, vinyl acetate, vinyl propionate, tetrahydrofuran, formaldehyde dimethyl acetal and mixtures of these solvents. A variety of conventional alky! acylates and alkenyl acylates, such as methyl acetate, ethyl acetate, butyl acetate, vinyl acetate, allyl acetate, isopropenyl acetate, ethyl propionate, ethyl butyrate and vinyl propionate as well as esters of long-chain fatty acids, e.g. vinyl laurate, can be used as the acylating agent. Preferably, ethyl acetate, butyl acetate or vinyl acetate, particularly vinyl acetate, is used for the acetylation. Vinyl propionate is preferred for the manufacture of the propionate (R = ethyl), and the vinyl esters of the corresponding fatty acids are also preferred for the manufacture of long-chain acylates. The amount of acylating agent used can lie between 1 molar equivalent and a several times greater excess; the acylating agent is used in excess especially when it simultaneously serves as the solvent, which can be the case with alkyl and alkenyl acylates. The concentration of (11Z,1 3Z)-7,10-dihydro-10-hydroxy-retinol is conveniently 10% to 50%, preferably 20% to 45% [expressed in weight/volume (wt./vol.)]. The solubility of the educt is controlled by the choice of solvent or solvent mixture and/or the temperature. Thus, the reaction temperature advan¬tageously lies between about lOoC and the reflux temperature of the reaction mixture, preferably between room temperature and about 900C, especially preferred between room temperature and about 600C. In the batch process the (1 lZ,13Z)-7,10-dihydro-10-hydroxy-retinol need not be in solution at the start of the reaction; rather a suspension can also be present at the start of the reaction. In order to improve the accessibility to the lipase and its re-use, it can be immobilized on various carrier materials. This immobilization can be effected covalently or non-covalently, preferably non-covalently, by simple adsorption on a suitable carrier material having a large surface. Since lipase and carrier material are insoluble in organic solvents, no measurable desorption takes place during the reaction. Suitable carrier materials are many of the usual, inexpensive filter aids, adsorbents, ion exchangers and chromatography materials, such as Florisil®, diatomaceous earth, bentonite, cellulose, molecular sieve, Amberlite®, Amberlyst®, silica gel or aluminium oxide and the like, as well as other inexpensive materials having large surface areas, such as sand, sintered glass or hollow fibres and 5 the like. The use of diatomaceous earth and sea sand is preferred. Alternatively, commercially available, already immobilized lipase preparations can also be used, for example the lipase preparations from Meito Sangyo and Boehringer Mannheim GmbH: 10 • Lipase PLC: Lipase PL immobilized on diatomaceous earth; • Lipase PLG: Lipase PL immobilized on granulated diatomaceous earth. • Chirazyme® L-2 (formerly Novozym® SP 435): lipase 15 from Candida antarctica, immobilized on macroporous polyacryl. If desired, the immobilization of the lipase can also be effected in the presence of a "cholanic salt" (co-immobilization), 20 by means of which the activity can in part be controlled (activator). Suitable cholanic salts are e.g. sodium chelate and sodium deoxycholate. In order to exclude the danger of chemical side-reactions 25 practically from the outset, the reaction with the lipase is conveniently carried out under an inert gas atmosphere, e.g. nitrogen or argon, and with the exclusion of light and/or in the presence of a radical scavenger, e.g. hydroquinone or 2,6-di(tert.butyl))-p-cresol. 30 In the batch process the catalyst can be filtered off after one run and re-used. The water content of the reaction solution -and thus of the lipase - inter alia plays a role with respect to the stability and activity of the lipase. The addition of a small 35 amount of water ( an even better effect. The addition of the water can also be effected periodically or in the sense of an equilibration step. For the efficient re-use of the lipase, which is extremely 5 important for the economy of the process, the purity of the educt is also decisive: the long-term stability of the lipase preparation can be improved considerably by an appropriate purification of the educt, which from the earlier stage still contains, inter alia, heavy metals. Filtration over various aids, such as diatomaceous 10 earth, silica gel, ethylenediaminetetraacetic acid (EDTA) salts and aluminium oxide, and washing with aqueous EDTA solution having a pH value of about 8 have been found to be simple and effective purification procedures, with the aid of which, inter alia, the heavy metal content of the educt can be lowered and the 1 5 efficacy of the lipase preparation improved. The process in accordance with the invention can be carried out as a repeated batch process or as a continuous process, namely using conventional types of reactor, such as, for example, 20 a solid bed reactor, cylinder reactor, filament reactor, rotary reactor, fluidized bed reactor (with a false bottom) or slurry reactor. The aforementioned use of a (11Z,^3Z)-7,^0-dihydro-^0-25 hydroxy-retinyl acylate, preferably the acetate, obtained in accordance with the invention, for the production of the corresponding vitamin A acylate or, respectively, of vitamin A acetate, represents a further aspect of the present invention 30 The following Examples for the manufacture of various (nZ,13Z)-7,10-dihydro-10-hydroxy-retinyl acylates by the enzyme-catalyzed, selective monoacylation of (11Z,1 3Z)-7,10-dihydro-10-hydroxy-retinol illustrate advantageous embodiments of the process in accordance with the invention, but do not in any 3 5 manner represent a limitation. All temperatures are given in degrees Celsius. Accordingly the present invention provides a process for the manufacture of a (1 lZ,13Z)-7,10-dihydro-10-hydroxy-retinyl acylate of the formula wherein R signifies a Ci.23-alkyi group or a C2-23-alkenyl group containing 1 to 3 double bonds, which process comprises monoacylating (11Z,13Z)-7,10-dihydro-lO-hydroxy-retinol of the formula in an organic solvent in the presence of an acylating agent selected jfrom methyl acetate, ethyl acetate, butyl acetate, vinyl acetate, allyl acetate, isopropenyl acetate, ethyl propionate, ethyl butyrate, vinyl propionate and vinyl laurate with a lipase such as herein described which is present in suspension, tlie concentration of (llZ,13Z)-7,10-dihydro-10-hydroxy-retinol in the reaction mixture before reaction being \0% to 50% (wt./vol.), the reaction temperature lying between about 10°C and the reflux temperature of the reaction mixture, and recovering the (llZ,13Z)-7,10-dihydro-10-hydrox\-retinyl acylate, in a known manner. Example 1 10.0 g (32.8 mmol) of (11Z,13Z)-7,10-clihydro-10-hydroxy-retinol and 10 mg of 2,6-di(tert.butyl)-p-cresol (BHT) were 5 dissolved in a mixture of 30 ml of toluene and 5 ml of vinyl acetate (54.1 mmol). The reaction was started by the addition of 500 mg of lipase PLC (Meito Sangyo) and the suspension was stirred gently at room temperature for 1 7 hours on a roller. The lipase was thereafter filtered off and washed with toluene, and 10 the filtrate was evaporated. After drying under a high vacuum for one day there were obtained 11.4 g (33 mmol) of (1 lZ,13Z)-7,10-dihydro-10-hydroxy-retinyl acetate in a yield of 100.6% and a purity of >99% [according to supercritical fluid chromatography (SFC) area percent]. 15 Example 2 10.0 g (32.8 mmol) of (nZ,13Z)-7,10-dihydro-10-hydroxy-retinol were taken up in a mixture of 6.8 ml of toluene and 3.2 ml 20 (34.6 mmol) of vinyl acetate. The reaction was started by the addition of 500 mg of lipase PLC (Meito Sangyo) and the reaction mixture was stirred gently at room temperature for 1 6 hours. The lipase was thereafter filtered off and washed with toluene, and the filtrate was concentrated by evaporation. After drying 2 5 under a high vacuum and stirring for one day there were obtained 11.23 g (32.4 mmol) of (1 lZ,13Z)-7,^0-dihydro-10-hydroxy-retinyl acetate in a yield of 98.8% and a purity of 97.2% (SFC area percent). 30 Example 3 20.0 g (65.7 mmol) of (11Z,13Z)-7,10-dihydro-10-hydroxy-retinol and 20 mg of BHT were dissolved in 56 ml of toluene, 56 ml of tert.butyl methyl ether (TBME) and 7.5 ml (81.2 mmol) of 3 5 vinyl acetate. The reaction was started by the addition of 1.0 g of lipase PLC (Meito Sangyo) and the suspension was stirred gently at 400 for 1 6 hours. The lipase was thereafter filtered off and washed with toluene, and the filtrate was evaporated. After drying under a high vacuum for one day there were obtained 23.1 g (66.7 mmol) of (11Z,1 3Z)-7,1 O-dihydro-1 0-hydroxy-retinyl acetate in a yield of 101.5% and a purity of >99% (SFC area percent). 5 Example 4 A) Immobilization on various carrier materials: 2.0 ml of lipase PL solution (25 mg/ml bidistilied water) were added to 10 2.5 ml of carrier material and the suspension obtained was stirred carefully at room temperature. The similarly wetted carrier was dried by gradually and cautiously increasing the vacuum (foam formation). Finally, the carrier was dried in a high vacuum for 2 days and used as described hereinafter. 15 B) Immobilization on porous glass beads: 2.0 ml of lipase PL solution (20 mg/ml bidistilied water) were added to 2.0 g of Siran Carrier Sikug 041/02/1 20/A (porous, sintered glass from Schott Glaswerke, Mainz, Germany) and the suspension was dried 20 as described above under A). 8.0 ml aliquots of a 2:2:1 mixture of hexane, tert.butyl methyl ether (TBME) and vinyl acetate containing 1.0 g of (11Z,1 3Z)-7,1 O-dihydro-10-hydroxy-retinol were added to the 25 dried lipase preparations obtained as described above and the reaction solution was stirred gently at room temperature on a roller. 25 ^il samples were removed after 6 hours and 27 hours for HPLC analysis. The results are compiled in Table 1 herein¬after in which A stands for (11Z,1 3Z)-7,1 O-dihydro-1 0-hydroxy- 30 retinol and B stands for (11Z,1 3Z)-7,10-dihydro-l 0-hydroxy-retinyl acetate. Example 5 8.0 ml of a solution of 1.0 g of (nZ,13Z)-7,10-dihydro-10-5 hydroxy-retinol in a 2:2:1 mixture of hexane, TBME and vinyl acetate were treated with 30 mg of lipase PLC or 30 mg of lipase PLC and the suspension was stirred gently on a roller under argon at room temperature in the dark. After 24 hours the residual content of (11Z,1 3Z)-7,10-dihydro-10-hydroxy-retinol 10 was less than 1% (HPLC). When 51 mg of lipase PLC or 51 mg of lipase PLC were used, (11Z,1 3Z)-7,10-dihydro-10-hydroxy-retinol was no longer detectable after 24 hours. Example 6 15 25 mg of lipase PL and 0.5 mg of chelate salt (sodium chelate, sodium deoxycholate) were dissolved in 2.0 ml of bidistilled water and added to 2.5 g of DICALITE® Speedex or sea sand suspended in 6 ml of bidistilled water. The suspension was 20 dried cautiously as in Example 4 and, when necessary, the dried material was pulverized. The lipase preparations produced were tested with 8 ml of (1 1Z,1 3Z)-7,10-dihydro-10-hydroxy-retinol solution as described 25 in Example 5. HPLC analysis was carried out after 24 hours in each case. The results are compiled in Example 2 hereinafter in which A stands for (11Z,1 3Z)-7,1 0-dihydro-lO-hydroxy-retinol. Example 7 5 50.0 g (164.2 mmol) of (112,1 3Z)-7,10-dihydro-10-hydroxy-retinol were dissolved in a mixture of 140 mi of hexane, 140 ml of TBME, 70 ml of vinyl acetate and 50 mg of BHT. 2.5 g of lipase PLC were added and the suspension was stirred gently on a 10 roller at room temperature under argon and in the dark for 24 hours. Then, the lipase catalyst was filtered off, washed with diethyl ether and dried for re-use. The filtrate was then concentrated by evaporation together with the wash solution and thereafter concentrated by evaporation firstly with two 300 ml 15 portions of hexane and then with 300 ml of pentane in order in this manner to remove azeotropically traces of acetic acid formed. After drying in a high vacuum at 35° for about 1 6 hours there were obtained 56.73 g (163.7 mmol, 99%) of (11Z,13Z)-7,^ 0-dihydro-l 0-hydroxy-retinyl acetate as a pale yellowish oil. 20 Analysis: >99% purity (SFC area percent); confirmed by 250-MHz-iH-NMR (CDCI3), EI-MS (m/e 346), IR (film) and micro¬analysis: 2 5 Calc: C 76.26%; H 9.89%; Found: C 76.07%; H 9.73%. Example 8 8.0 ml aliquots of a solution of 1.0 g of (nZ,13Z)-7,10-dihydro-10-hydroxy-retinol and 1 mg of BHT in a 2:2:1 mixture of 5 hexane, TBME and vinyl acetate were each added to 50 mg of lipase PLC, which had already previously been used four times (as a single batch) under the same conditions and had been washed between the individual batches merely with a 1:1 mixture of hexane and TBME. Various aqueous solutions were then added to 10 the individual samples, and the reaction suspensions were stirred gently on a roller for 1 7 hours and then analyzed by HPLC. The results are compiled in the Table 3 hereinafter in which B stands for (11 Z, 1 3Z)-7,1 0-dihydro-l 0-hydroxy-retinyl acetate. 15 Table 3 Example 9 20 A. 1 kg of (11Z,13Z)-7,10-dihydro-l 0-hydroxy-retinol was dissolved in 8 I of ethyl acetate and washed at room temperature with two 3 I portions of a 50 mmolar EDTA solution having a pH value of 8.0. The organic phase was thereafter dried over anhydrous sodium sulphate and concentrated by evaporation, 25 and the residue was dried in a high vacuum. B. 50 ml aliquots of (11Z,1 3Z)-7,10-dihydro-l 0-hydroxy-retinol were dissolved in 400 ml of ethyl acetate and in each case filtered over 5 g of basic aluminium oxide, DICALITE 30 Speedex® or silica gel. The filtrate was concentrated by evaporation and dried in a high vacuum. C. 3.5 g of the (HZ,! 3Z)-7,^0-clihydro-^0-hydroxy-retinol samples purified according to A. or B. were dissolved in 25 ml of a 2:2:1 mixture of toluene, TBME and vinyl acetate. Three 8.0 ml samples were removed from each solution (triple 5 experiment) and were each treated with 2.5 mg of lipase PLC. The reaction suspension was stirred gently on a roller at room temperature for 24 hours and subsequently analyzed by SFC. The results are compiled in Table 4 hereinafter in which A and B stand for (nZ,13Z)-7,10-dihydro-10-hydroxy-retinol and (11Z,13Z)-10 7,1 O-dihydro-1 0-hydroxy-retinyl acetate, respectively. Table 4 1 5 Example 10 5.0 g of lipase PLC were placed in a 2 I chromatography column having a glass frit. Then, 100.0 g of (11Z,1 3Z)-7,10-dihydro-10-hydroxy-retinol, 280 ml of hexane, 280 ml of TBME, 20 140 ml of vinyl acetate, which optionally contained 500 ]x\ of a 0.1M ammonium bicarbonate solution or of a 1% ammonium hydroxide solution, and 100 mg of BHT were added in this sequence and the column was stirred gently by rotation (motorized stirrer), with the column having the outlet inclined 2 5 upwards in order that the frit was not in contact with the solution. After incubation for 17 hours at room temperature the reaction solution was drained off and analyzed by SFC. The residual lipase preparation was washed firstly three times with 100 ml of a 1:1 mixture of hexane and TBME each time (for about 1 5 minutes per washing) and thereafter with 50 ml of hexane, and then again used for a new cycle. The results are compiled in Table 5 hereinafter in which A and B stand for (11Z,1 3Z)-7,10-dihydro-10-hydroxy-retinol and (11Z,1 3Z)-7,^0-dihydro-^0- hydroxy-retinyl acetate, respectively. Table 5 10 Example 11 (11Z,1 3Z)-7,10-Dihydro-10-hydroxy-retinol was purified as described in Example 9 A and thereafter used in a repeated batch process as described in Example 10, but using 3.0 g of lipase PLC, 15 90.0 g of (11Z,13Z)-7,10-dihydro-10-hydroxy-retinol, 250 ml of toluene, 250 ml of TBME and 125 ml of vinyl acetate, which contained 450 ul of 1% ammonium hydroxide solution. The reaction time was 23 hours. Between the cycles the lipase preparation was washed twice with TOO ml of a 1:1 mixture of 20 toluene and TBME, which contained 1%o (vol./vol.) of a 1% ammonium hydroxide solution, and after every 4 cycles was left to stand for 3 days at room temperature in toluene. The results are presented in the following graph: WE CLAIM: 1. A process for the manufacture of a (1IZ, 13Z)-7, lO-dihydro-lO-hydroxv-retinyl acylate of the formula wherein R signifies a Ci.23-alk>'l group or a C^-is-alkenyl group containing 1 to 3 double bonds, which process comprises monoacylating (11Z,13Z)-7,10-dihydro-lO-hydrox^-retinol of the formula in an organic solvent in the presence of an acylating agent selected from methyl acetate, ethyl acetate, butyl acetate, vinyl acetate, allyl acetate, isopropenyl acetate, ethyl propionate, ethyl butyrate, vinyl propionate and vinyl laurate with a lipase such as herein described which is present in suspension, the concentration of (1 lZ,13Z)-7,10-dihydro-10-hydroxy-retinol in the reaction mixture before reaction being 10% to 50% (wt./vol.). the reaction temperature lying between about lO'C and the retlux temperature of the reaction mixture, and recovering the (llZ,13Z)-7,10-dihydro-10-hydroxy-retinyl acylate, in a known manner. 2. The process according to claim 1, wherein the said lipase is lipase PL from Alcaligenes sp., lipase PLC, lipade PLG, lipase MY-30 from Candida cylindracea, (Candida rugosa), Lipozyme® IM-20 from Mucor miehei (Rhizomucor meihei), lipase CE-5 from Humicola lanuginosa or lipase G from Penicillium cyclopium, preferably lipase PL, lipase PLC, lipase PLG or lipozyme(g) IM-20; particularly lipase PL, lipase PLC or lipase PLG. 3. The process according to claim 1, wherein the said lipase is Chirazyme® L-2 from Candida antarctica. 4. The process according to any one of claims 1 to 3, wherein R in formula 1 signifies methyl, whereby (llZ,13Z)-7,10-dihydro-10-hydrox\-retinol of formula II is monoacetviated. 5. The process according to any one of claims 1 to 4, wherein the amount of the said lipase is up to 20% wt.% based on (11Z,13Z)-7,10-dihydro-10-hydroxy-retino preferably between 0.1 wt.% and 10 wt.% particularly between about 1 wt.% and 5wt.%. 6. The process according to any one of claims 1 to 5, wherein the said lipase is present in immobilized form. 7. The process according to claim 6. wherein the said lipase has been immobilized in the presence of a cholanic salt, preferably of sodium chelate or sodium deoxycholate. 8. The process according to any one of claims 1 to 7, wherein the reaction is carried out under an inert gas atmosphere and with the exclusion of light and/or in the presence of a radical scavenger. 9. The process according to any one of claims 1 to 8, wherein an aliphatic hydrocarbon with 5-8 carbon atoms, a cyclic hydrocarbon with 6-10 carbon atoms, a chlorinated aliphatic hydrocarbon, a nitro-substituted aliphatic hydrocarbon, an aromatic hydrocarbon, an aliphatic ether, a cyclic ether, an aliphatic ester, an aliphatic ketone, an aliphatic nitrile, an aliphatic amine, an aliphatic acetal or a mixture of such solvents is used as the solvent. 10. The process according to claim 9, wherein n-hexane, cyclohexane, methylene chloride, carbon tetrachloride, toluene, diisopropyl ether, tetrahydrofuran, methylfuran, vinyl acetate, vinyl propionate or fomialdehyde dimethyl acetal, preferably methylene chloride, diisopionate ether, tetrahydrofuran, vinyl acetate, vinyl propionate, tetrahydrofuran, methylfuran or fonnaldehyde dimethyl acetal, or a mixture of such solvents is used. 11. The process according to any one of claims 1 to 10, wherein the concentration of (1 lZ,13Z)-7,I0-dihydro-10-hydroxy-retinol is 20% to 40% (wt./vol.). 12. The process according to any one of claims 1 to 11, wherein the reaction temperature lies between room temperature and about 90C, particularly between room temperature and 60°C. 13. The process according to any one of claims 1 to 12, wherein the reaction solution contains a small amount ( 14. The process according to any one of claims 1 to 13, wherein the (llZ,13Z)-7,10-dihydro-10-hydroxy-retinol is filtered over a filter aid, preferably diatomaceous earth, an ethylenediaminetetraacetic acid salt, silica gel or aluminium oxide, or washed with aqueous ethylenediaminetetraacetic acid solution having a pH value of about 8 in order to remove harmful impurities prior to use. 15. The process according to any one of claims 1 to 14, wherein the process is carried out as a repeated batch process or as a continuous process. 16. A process for the manufacture of a (1IZ, 13Z)-7,10-dihydro-lO- hydroxy-retinyl acylate, substantially as herein above described and exemplified.

Documents:

0579-mas-1997 abstract.pdf

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