PROCESS FOR THE PREPARATION OF L-ALPHA-GLYCERYLPHOSPHORYLCHOLINE, L-ALPHA-GLYCERYLPHOSPHORYLETHANOLAMINE FROM CRUDE AND/OR DEOLEATED LECITHINS

Abstract: An economical and industrially applicable process for the preparation of L-α-glycerylphosphorylcholine and L-α-­ glycerylphosphorylethanolamine from soy and/or crude egg lecithin, is described. The process of the invention comprises the alcoholysis of the crude and deoleated lecithins, the subsequent formation of insoluble complexes of the mixture of glycerylphosphoryl­ choline and glycerylphosphorylethanolamine with zinc chlorides or bromides, the de-complexation with organic bases such as pyridine, collidine, lutidine and the like and the final elution on ion-exchange resins.

Description: A PROCESS FOR THE PREPARATION OF L-CC-GLYCERYLPHOSPHORYLCHO- LINE AND L-GLYCERYLPHOSPHORYLETHANOLAMINE FROM CRUDE AND/OR DEOLEATED LECITHINS The present invention refers to a process for the preparation of L-CL-glycerylphosphorylcholine and L-d-gly- cerylphosphorylethanolamine (hereinafter also named, for the sake of shorteness, with the abbreviations GFC, GFE, respectively) from crude or deoleated soy and/or egg lecithins. The processes up to now known, based on the deacylation of the corresponding pure or partially purified phosphatides, such as disclosed in patents US no. 2,864,848 and GB No. 2,058,792 and in H. Brockerhoff: J. Lipid Research Vol. 4 (1), 96 (1963); D.J. Hanahan: Biochemical Preparations 9, 55 (1962); N.H. Tattrie, C.S. McArthur: Biochemical Preparations, 6, 16 (1958); Okui et awl. : Yaka Ugaku Zasshi, 84 (12), 1206-9 (1964); E. Cubero Robles, G.F.M. Roels: Chem. Phys. Lipids 6, 31-38 (1971), suffer from such disadvantages as to limit the large-scale industrial applicability thereof. The process object of the invention, in a preferred embodiment thereof, is characterized in that a mixture of GFC + GFE, obtained by deacylation, with an alkali metal alcoholate in lower alcohols such as methanol, ethanol, isopropanol, of crude or acetone-extraction deoleated lecithins, is treated with zinc salts, preferably with the corresponding chlorides or bromides, obtaining a complex having the approximative formula I (n GFC + m GFE) ZnX2 (I) wherein: X is Cl or Br; n and m are ranging from 0 to 1 and m + n is equal to 1. The complex of formula I containing limited amounts of impurities is then treated with an organic base such as pyridine, collidine, picoline, lutidine, thereby releasing the mixture (n GFC + m GFE) purified and a complex of formula II (ZnX2 )B2 wherein X has the above mentioned meanings and B is the organic base. The obtained mixture (n GFC + m GFE) may be then separated into the single components by means of elution on resins. In this case, since the salt has been already eliminated from the complex I by precipitation and filtration of the complex II, and a mixture (n GFC + m GFE) having an high purity degree is used, very small amounts of resins are required and the process, as a whole, is very simple and economical. Deacylation of soy lecithins with sodium methylate in methanol is carried out for a time ranging from 2 to 4 hours and with an amount of alcoholate equal to about 1 mole per mole of total phosphatides (phosphati dylcholine-ethanolamine-inositole). . After filtration of the residue (which contains glycerylphosphorylinositole) the filtrate containing the (n GFC + m GFE) mixture is adjusted to pH ¯ 6 with organic or mineral acids (acetic acid, lactic, HCl, H 2S04 etc.) or by elution on an acidic resin. After concentration of the solution to small volume, the fatty acid esters are separated and removed. ZnX2 (wherein X has the above defined meanings) is then added and the complex of formula I precipitates. The use of an ethanol or methanol solution of ZnCl2 is particularly preferred. The amount of metal salt will be generally comprised from 3-6 moles per mole of nGFC + mGFE mixture. The release of the (n GFC + m GFE) mixture is then carried out by treating an aqueous solution of complex I with a suitable amount of a base such as pyridine, collidine, lutidine, picoline or the like, so as to precipitate complex II. The traces of metal salts in solutions may be then removed by elution on resins, obtaining thereby the pure (n GFC + m GFE) mixture, wherein n and m are as above defined. By means of subsequent elution of said mixture on a strong basic resin, separation of pure GFC and GFE is achieved. For the preparation of pure GFC only, it is more convenient to carry out the alcoholysis on the ethanol extract of the deoleated soy lecithin. In this case, the process is further made easier, in that, for instance, the complex of formula I is directly obtained from the reaction mixture (without eliminating the fatty acid esters) and moreover the formation and the isolation of the complex itself occur in an easier way. The amount of metal salt used is ranging from 1-2 moles per mole of mixture n GFC + m GFE. The following Examples further illustrate the invention without limiting it in any way. EXAMPLE 1 Preparation of deoleated soy lecithin 1 Kg of crude soy lecithin is poured into 3.5 It of acetone cooled to about +50C, under stirring. The mixture is stirred at +50C for 30', then filtered. The residue is suspended under stirring in 2 It of acetone (about +50C) then filtered, washing the residue with acetone (1 It), at room temperature. The solid is filtered and dried under vacuum (1 mm Hg) at 350C. 680 G of deoleated soy lecithin are obtained. EXAMPLE 2 Preparation of (n GFC + m GFE) ZnCl2 complex by deacy- Lation of deoleated soy lecithin in methanol The suspension of deoleated soy lecithin (3.5 g) in anhydrous methanol (10 lt) containing sodium methylate (3.5 mol = 189 g) is stirred at room temperature and in an anhydrous medium for about 3 hours. The mixture is filtered and the residue is washed with methanol (2 x 1 It), the filtrate is then neutralized (p ¯ 6) with glacial acetic acid (140 ml) and concentrated to small volume (#31 It). The (higher) fatty phase is separated, the methanol solution (lower phase) is slowly poured into a 3M zinc chloride solution in methanol (2.1 It). The obtained solution is slowly poured, in anhydrous isopropanol (7 lt) under stirring at room temperature. The mixture is filtered under anhydrous atmosphere, the residue is washed with methanol (0.5 lt), then with acetone (0.5 Lt) and dried at 700C under vacuum (1 mm Hg). 640 g of mixture (n GFC + m GFE) ZnCl2 are obtained: ZnX = 16.72% GFC% = 29.1% H2% = 2.8% Cl% = 17.65% GFE% = 22.3% The values of n and m and consequently the percentage ratios of GFC and GFE depend on the kind of the used raw material. In the tests performed, it has been noticed that n is approximately comprised from 0.45 to 0.65 and m from 0.35 to 0.55. With the same process, using crude soy lecithin (100 g), methanol (300 ml), sodium methylate (48 mmoles), 10 g of mixture (n GFC + m GFE) ZnCl 2 having similar composition to that above described, are obtained. The purity degree of the mixture (n GFC + m GFE) ZnCl2 obtained by the process described in the Example 2 may vary according to the used raw material. A high purity degree (90-95%) may be however always obtained by elution of zinc chloride (cfr. Example 6), subsequent elimination of the solvent and new formation of the complex of formula I as described in the Example 2. EXAMPLE 3 Preparation of (n GFC + m GFE) ZnCL complex by deacy- ration of ethanol extract of the deoleated soy lecithin 460 g of deoleated soy lecithin are suspended in absolute ethanol (1.2 It), the suspension is stirred at room temperature for 1 hour, then filtered washing the residue with ethanol (400 ml). The filtrate is collected with the washings, sodium methylate (89 mmol = 4.8 g), diatomaceous earth (10 g) are added and the mixture is stirred at room temperature for 2 hours. After filtration, the filtrate is neutralized (pH - 6) with glacial acetic acid (- 3.6 ml) then a 2.5M ZnCl2 solution in absolute ethanol (53 ml) is slowly added under stirring. The mixture is filtered under anhydrous atmosphere, the residue is washed with ethanol (100 ml) then with acetone (2 x 100 ml) and then dried under vacuum (1 mm Hg) at 700C. 31.2 G of (n GFC + m GFE) ZnCl are obtai 2 ned. ++ Zn = 16.5X GFC% = 50.1% Cl = 17.8% GFE% = 10.1X Working on the ethanol extract from different kinds of deoleated soy lecithin, it has been noticed that n is approximately comprised from 0.75 to 0.85 and m from 0.25 to 0.15. EXAMPLE 4 Preparation of (n GFC + m GFE) ZnCl2 complex by deacy- lation of soy lecithin in ethanol The suspension of deoleated soy lecithin (600 g) in anhydrous ethanol (1.8 lt), containing sodium methylate (0.6 moles = 32.4 g), diatomaceous earth (10 g) is stirred at room temperature for 3 hours. 20 g of decolorating charcoal are added and the mixture is then filtered, washing the residue with ethanol (2 x 200 ml). The washings are combined with the filtrate, neutralized (pH > 6) with glacial acetic acid, then a 2.5M zinc chloride solution in ethanol (24 ml) is added. The mixture is filtered in anhydrous atmosphere, the precipitate is washed with ethanol (50 ml), then with acetone (2 x 50 ml) and finally dried under vacuum (1 mm Hg) at 600C. 20 G of (n GFC + m GFE) ZnCl mixture are obtained. The composition of the mixture 2 is similar to that described in the Example 3. The methods of the previous Examples may also be applied to the crude egg lecithin; since it is completely soluble also after hydrolysis in methanol or ethanol, it is more conveniently preferred, nevertheless, to operate in concentrated solution according to the method of the following Example. EXAMPLE 5 Preparation of (n GFC + m GFE) ZnCl2 complex by deacy- lation of crude egg lecithin A solution of commercial egg lecithin (100 g) in anhydrous methanol (100 ml) containing sodium methylate (0.045 moles = 2.42 g) is left to stand at room temperature for about 2 hours. The methanol phase (lower) is separated from the (higher) fatty phase. The methanol solution is neutralized with glacial acetic acid, then it is slowly poured under stirring intoc a 3M zinc chloride solution in methanol (110 ml). The obtained solution is added with isopropanol (350 ml) under stirring. The mixture is filtered under anhydrous atmosphere, the residue is washed with methanol (50 ml) and subsequently with acetone (2 x 50 ml), then it is dried under vacuum (1 mm Hg) at 700C obtaining 28 g of (n GFC + m GFE) ZnCl 2 mixture. ++ Zn = 16.1% GFC% = 52.3% Cl = 17.6% GFE% = 11.2% The values of n and m depend, also in this case, on the used raw material. In the tests carried out on different kinds of lecithins it has been found that the GFC percentages in the mixture are approximately ranging from 52 to 70%, the GFE percentages from 15 to 20%. EXAMPLE 6 Preparation of (n GFC + m GFE) mixture Pyridine (18 ml) is added dropwise at +50C, under stirring, to the solution of (n GFC + m GFE) ZnCl 2 (31.2 g) (cfr. Example 3) in water (90 ml). The mixture is left to stand for 4 hours, then filtered, washing the residue (ZnCl2 (Py)2) with cold (+50C) water (2 x 15 ml). The aqueous solution is concentrated at reduced pressure, till disappearance of the residual pyridine, then it is filtered and the aqueous solution is first eluted on IR 120 H resin (40 ml: h C 10 cm, # 2.5 cm) and subsequently on IR 45 OH (80 ml: h '= 18 cm, Z # 2.5 cm). Water is removed under reduced pressure, obtaining 23.5 g of (n GFC + m GFE) mixture. GFC% = 66.83% GFE% = 13.85% H2 0% = 18.1% EXAMPLE 7 Preparation of GFC The aqueous solution of (n GFC + m GFE) mixture of Example 6 (23 g in 50 ml of water) is eluted on IR 401 OH resin (115 ml: h # 20 cm, AS ¯ 3 cm). The resin is washed with water (300 ml) and the mixture is then eluted on IR C50H (40 ml: h 1818 cm, # # 2 cm). Water is eliminated removed reduced pressure obtaining 16.9 g of GFC (cont. H20 = 12.3%). Assay (with HClO4 in acetic acid) = 99.6%. By crystallization from ethanol/ethyl ether, a very hygro scopic solid, having the following characteristics, is obtained: LRID - 2.89 (c = 10% in H2 0) D PU = 12.15% calc. for C8H20NO6P found P% = 12.04% -N% = 5.58% calc. for C8H20NO6P found N% = 5.45% m.p. = 132-1340C. EXAMPLE 8 Preearation of GFE After the separation of GFC (cfr. Example 7), IR 401 OH resin is washed with a 1% aqueous solution of acetic acid (100 ml) and, thereafter, with a 5% aqueous solution of acetic acid. The solvent is eliminated from the second solution at reduced pressure obtaining 4 g of GFE (H20% = 15.2X). A sample precipitated from water/ethanol and allowed to crystallize showed the analysis: [&alpha;]]D = -2.8 (c = 6.5%) m.p. = 84-860C. Using (n GFC + m GFE) ZnCl2 complexes containing an higher GFE percent and following the procedures of Examples 6, 7, 8, GFE is obviously recovered in larger amount. For instance, from 91 g of mixture (n GFC + m GFE) ZnCl2 + (cfr. Example 2) using IR 120 H , IR 45 OH (180 ml), IR 401 OH (350 ml), 22.6 g of GFE (H20 = 16.1X) are obtained. Using the process described in the Example 6, from 35 g of complex (n GFC + m GFE) ZnCl 2 (cfr. Example 5) 22 g of mixture (n GFC + m GFE) (h20% = 18.2X) are obtained. GFC% = 81.5% GFE% = 16.3X.

Claims: CLAIMS¯f r tbe Contractin9 stateS. AT, BE, CH, DE, FR, GB, GR, IT, LI, LU, NL, SE, SP 1. A process for the preparation of L-Q-glycerylpho- sphorylcholine and L-a-glycerylphosphorylethanolamine comprising: a) deacylation of crude or deoleated soy or egg lecithin, in methanol or ethanol with an alkali metal alcoholate; b) complexation of mixture L-O(-glycerylphosphorylcholine and L-a-glycerylphosphorylethanolamine with zinc hali des, insoluble in lower alcohols C1-C 4, to give comple xes approximatively of formula I (n CFC + m GFE) ZnX2 (I) wherein: X is Cl or Br; n and m are ranging from 0 to 1 and m + n is equal to 1; c) releasing of the salt of complex I by treatment with a base selected from the group comprising pyridine, col lidine, lutidine or picoline; d) purification and separation of the mixture of gly cerylphosphorylcholine and glycerylphosphorylethanola mine by means of ion-exchange resins 2. A process according to claim 1, characterized in that methanol or ethanol ZnCl solutions are used as com 2 plexing agents and that the salt is subsequently released by treating aaueous solutions of the complex of formula I with pyridine. 3. A process for the preparation of L--glycerylpho- sphorylcholine and L-a-glycerylphosphorylethanolamine from deoleated soy lecithin, characterized in that soy lecithin or its ethanolic extract are treated in ethanol with sodium methylate or ethylate and that, after filtration and acidification, the mixture of deacylated phospholipids is treated according to steps b), c) and d) of claim 1. 4. A process according to any one of the previous claims, characterized in that crude soy lecithin is used as the starting material. 5. A process according to any one of the previous claims, characterized in that crude egg lecithin is used as the starting material. 6. A process for the preparation of L-O(-glycerylpho- sphorylcholine by alcoholysis in methanol/ethanol of lecithins with an alkali metal alcoholate and subsequent purification with ion-exchange resins, characterized in that pure GFC is obtained by eluting with water the GFC + GFE mixture on a strongly basic resin. 7. A process for the preparation of L-t-glycerylpho- sphorylethanolamine, characterized in that pure GFE is recovered from the GFC + GFE mixture after separation of the GFC according to claim 6, by elution with a diluted aqueous solution of acetic acid. 8. A process according to claim 6 or 7, characterized in that crude egg lecithin is used as the starting mate rial 9. A process for the splitting of GFC and GFE complex with metal compounds characterized in that organic bases such as pyridine, collidine, lutidine or picoline are used.

Inventors: TRONCONI, GIOVANNI
Applicants: ISTITUTO CHEMIOTERAPICO ITALIANO FINE CHEMICALS S.P.A.
Application year: 1986
Main classification: C07F9
IPC codes: C07F9