JP2887617B2 - Method for producing phosphatidylcholine - Google Patents

Description

The present invention relates to a novel method for producing phosphatidylcholine.

[Conventional technology]

Lecithin contained in soybeans, egg yolks and the like has emulsifying properties and is widely used as an emulsifier for foods, cosmetics, paints, and the like. In addition, attention has been paid to the physiologically active action of lecithin, and application to functional foods, pharmaceuticals, and the like has been promoted. Furthermore, when a phospholipid containing lecithin is dispersed in water, a liposome, which is a closed vesicle having a bimolecular membrane structure, is formed, and this liposome is useful as a material for a drug delivery system or a clinical diagnostic agent.

Various attempts have been made to produce useful lecithin, that is, various methods for producing phosphatidylcholine have been studied and reported.

For example, as a method for producing phosphatidylcholine,
(1) A method of acylating glycerophosphorin choline (hereinafter abbreviated as “GPC”) with acyl imidazole (Herm
etter et al. Chem. Phys. Lipids 28 111 (1981)), (2)
A method of acylating GPC with a fatty acid anhydride using an esterification catalyst such as N, N-dimethyl-4-aminopyridine, N, N-pyrrolidinepyridine (Japanese Patent Publication No. 62-9599), (3) N, A method of acylating GPC with a fatty acid anhydride using a condensing agent such as N-dicyclohexylcarbodiimide and an esterification catalyst such as N, N-dimethyl-4-4aminopyridine (Japanese Patent Application Laid-Open No. 64-61489). Are known.

[Problems to be solved by the invention]

However, these conventionally known production methods have various problems.

For example, the method (1) cannot produce a phosphatidylcholine having a fatty acid residue other than the saturated fatty acid residue, it is difficult to control the heat of reaction, it takes a long time for the reaction, and the yield is low. In the method (2), GPC is supported on a carrier, and the GPC is contacted with a fatty acid anhydride to react.
Complicated steps are required for the operation of supporting the carrier on the carrier and the separating operation of the carrier after the reaction, and the yield is low. In the method (3), the pulverized GPC is added to the fatty acid anhydride, or the GPC is finely pulverized in the fatty acid anhydride and reacted. Since the GPC is in powder form, the contact with the fatty acid anhydride is poor. However, there is a disadvantage that the reaction is slow.

As described above, in the conventional method, when introducing an acyl group into GPC, the GPC and the raw material for introducing the acyl group do not mix with each other, so that the reaction rate becomes extremely slow, and the reaction takes a long time, Further, there is a drawback that the yield is low.

The present invention has been made in view of the above problems,
It is an object of the present invention to provide a method capable of producing phosphatidylcholine in a high yield with a simple operation and a short reaction time using C and a fatty acid anhydride.

[Means for solving the problem]

The present inventors have conducted intensive studies to solve the above problems, and as a result, by using a specific solvent in combination, GPC can be extremely finely dispersed in fatty acid anhydride, and the contact area between the two is large. The reaction progressed promptly, and a method for obtaining phosphatidylcholine in high yield was found, and the present invention was reached.

That is, the present invention is a method for producing phosphatidylcholine in which GPC and a fatty acid anhydride are dissolved in a lower alcohol, the lower alcohol is distilled off, and then an aprotic solvent and a basic catalyst are added and reacted.

GPC used in the present invention is represented by the following general formula, GPC obtained by hydrolyzing lecithin, which has been separated from soybean, egg yolk, other animal and vegetable fats and oils, and purified in accordance with a conventional method, may be mentioned.

The fatty acid anhydride used in the present invention is represented by the general formula (RCO) ₂ O, wherein R represents a saturated or unsaturated hydrocarbon group having 9 to 21 carbon atoms, or an aromatic hydrocarbon group. Fatty acid anhydrides may be used, and it is possible to use a fatty acid anhydride obtained according to a conventional method such as a method of condensing a fatty acid and a fatty acid chloride, a method of reacting a fatty acid salt with a fatty acid chloride, and a method of dehydrating a fatty acid. it can.

The fatty acids constituting the fatty acid anhydride include lauric acid, myristic acid, palmitic acid, stearic acid, arachidic acid, behenic acid, myristoleic acid, palmitoleic acid, oleic acid, elaidic acid, linoleic acid, linoleic acid, and arachidonic acid. , Eicosapentaenoic acid, docosahexaenoic acid, 10,12-octadecadienoic acid, 2,4-octadecadienoic acid, 10,12-heptadecadienoic acid, 2,4-
Nonadecadienoic acid, P-methylphenylpropionic acid,
P-vinylphenylhexanoic acid and the like are mentioned, and these fatty acid residues are used alone or in combination.

As the lower alcohol used in the present invention, methyl alcohol, ethyl alcohol, propyl alcohol,
Isopropyl alcohol, butyl alcohol, sec-butyl alcohol, tert-butyl alcohol, amyl alcohol, isoamyl alcohol, hexyl alcohol,
Heptyl alcohol and the like, methyl alcohol,
Ethyl alcohol is preferred.

The aprotic solvent used in the present invention is preferably an aprotic solvent capable of dissolving or dispersing the fatty acid anhydride and the basic catalyst, for example, n-pentane, n-hexane, isohexane, n-heptane, n-
Octane, isooctane, petroleum ether, cyclohexane, benzene, toluene, xylene, methylene chloride, chloroform, carbon tetrachloride, ethylene chloride, trichloroethane, chlorobenzene, dimethyl sulfoxide, dimethylformamide, dimethylacetamide, n-methylpyrrolidone, hexamethylphosphonamide , Methyl ethyl ketone and the like, and these can be used alone or in combination of two or more.

As the basic catalyst used in the present invention, N, N-dimethyl-4-aminopyridine, N, N-dimethyl-4-amino-2-methylpyridine, N, N-diethyl-4-aminopyridine, N, N-dibutyl-4-amino-3-ethylpyridine, 4-pyrrolidinopyridine, 4-pyrrolidino-2-
Methylpyridine, 4-pyrrolidino-3-butylpyridine, pyridine, triethylamine, tributylamine,
Examples thereof include diisopropylethylamine, trioctylamine, tripropylamine, and triisopropylamine, and these can be used alone or as a mixture.

To produce phosphatidylcholine according to the present invention,
First, 1 mol of GPC and 1 to 2 mol of fatty acid anhydride are dissolved in a lower alcohol. The used amount of the lower alcohol may be any amount that can dissolve the GPC and the fatty acid anhydride, and is preferably 1 to 5 times the weight of the total amount of both. In the above-mentioned dissolving operation, the order of addition of each raw material may be any order, and the stirring power required for dissolving may be small, and an ordinary stirrer can be used.

When GPC and fatty acid anhydride are dissolved in the lower alcohol and become uniform, the lower alcohol is then distilled off. Distillation of the lower alcohol is carried out by heating, but it can be carried out under reduced pressure or by heating under reduced pressure. In the case of heating, it is preferable to carry out the heating at a temperature of 100 ° C. or lower in order to prevent deterioration of GPC and fatty acid anhydride. Even if lower alcohol is distilled off here, GP
Neither C nor fatty acid anhydride is aggregated, and a solid or liquid mixture is obtained at 30 ° C. in which either one is finely dispersed in the other.

Next, an aprotic solvent is added to the above mixture. When the aprotic solvent is added, the fatty acid anhydride is dissolved in the aprotic solvent, and a dispersion is obtained in which GPC is dispersed in the form of fine particles of 50 μm or less, mainly 20 μm or less. The amount of the aprotic solvent used here is preferably at least 3 times the weight of the total amount of GPC and fatty acid anhydride.

An acylation reaction is performed by adding a basic catalyst to the dispersion. The addition amount of the basic catalyst is preferably 1 to 5% by weight based on GPC. The acylation reaction is carried out under a flow of an inert gas such as nitrogen gas, helium gas, argon gas or the like while stirring.
Perform at 70 ° C. for 2-12 hours. As the reaction proceeds, the dispersion changes from a cloudy state to a transparent state, and the reaction is completed in 4 to 12 hours.

After completion of the reaction, the reaction solution was concentrated, washed with acetone to remove the basic catalyst, the acetone-insoluble portion was dissolved in a methanol / chloroform solvent, and separated and purified using a silica gel column to obtain high-purity phosphatidylcholine. Obtainable.

The resulting phosphatidylcholine has the following general formula (Wherein, R is the same as the fatty acid residue of the fatty acid anhydride used as a reaction raw material), and has an emulsifier for food, cosmetics, paint, etc., a drug carrier as a liposome in pharmaceuticals, artificial blood, It can be applied to artificial cells and the like, an insulating film using a phospholipid as a thin film, a chemical sensor having an artificial phospholipid film adsorbed on a filter, and the like.

Hereinafter, the present invention will be described in more detail with reference to Examples.
The present invention is not limited to these without departing from the gist thereof.

〔Example〕

Example 1 A 300 ml flask equipped with a stirrer was charged with 10 g (0.0388 mol) of L-α-glycerophosphatidylcholine, 21.2 g (0.0427 mol) of palmitic anhydride and 30 g of methanol, and heated to 40 ° C. to dissolve. After dissolution, methanol was distilled off at 70 ° C. under reduced pressure. After the distillation, the pressure was further reduced at the same temperature for 5 hours, followed by drying. After drying, dehydrated and dried chloroform 10
0 ml was added and stirred gently to give a cloudy solution.
Then 4.7 g of 4-dimethylaminopyridine (0.04
27 mol) and stirred at 50 ° C under nitrogen gas flow.
For 5 hours. After completion of the reaction, chloroform was distilled off under reduced pressure at 40 ° C, and then acetone 100m
l, and the mixture was stirred for 1 hour. The generated crystals were separated by filtration and dried to obtain 29.5 g of crystals. The obtained crystals were dissolved in 60 ml of a solvent of chloroform / methanol = 75/25 and adsorbed on a silica gel column.
Elute with 65/25/4 eluent, concentrate the eluent,
28.5 g of white crystals were obtained. As a result of the analysis,
L-β, γ-dipalmitoyl-α-phosphatidylcholine, and the yield was 95%.

The results of IR, NMR, and TLC analysis of the obtained crystals were compared well with those of L-β, γ-dipalmitoyl-α-phosphatidylcholine standard reagent (purity 99%) manufactured by Sigma.

Example 2 A 500 ml flask equipped with a stirrer was charged with 20 g (0.0776 mol) of L-α-glycerophosphorylcholine, 44.2 g (0.0802 mol) of stearic anhydride and 100 g of methanol.
The mixture was heated to 0 ° C. for dissolution, and methanol was distilled off under slow stirring in the same manner as in Example 1. The residue was dissolved by adding 200 ml of dehydrated and dried chloroform and 50 ml of dimethyl sulfoxide. 18.9 g (0.155 mol) of N, N-dimethyl-4-aminopyridine was added to the obtained solution, and an acylation reaction was carried out at room temperature for 8 hours under a nitrogen gas stream. After completion of the reaction, desolvation, acetone purification, and silica gel column (solvent chloroform / methanol = 75/25, eluent
Purification with chloroform / methanol / water = 65/28/5) gave 55.8 g (98% yield) of white crystals of L-β, γ-distearoyl-α-phosphatidylcholine.

Example 3 A 300 ml flask with a stirrer was charged with 10 g (0.0388 mol) of L-α-glycerophosphorylcholine, 25.8 g (0.044 mol) of eicosapentaenoic anhydride and 100 g of methanol, and heated to 25 ° C. to dissolve. 25 ° C under reduced pressure with slow stirring
Then, methanol was distilled off, followed by drying under reduced pressure at 20 ° C. for 5 hours. The residue was dissolved by adding 150 ml of dehydrated and dried chloroform. To the resulting solution was added 9.4 g (0.0854 mol) of 4-dimethylaminopyridine, and under a stream of nitrogen gas,
The reaction was performed at room temperature for 12 hours. After completion of the reaction, Example 1
Solvent removal, acetone purification, silica gel column (solvent chloroform / methanol = 75/25, eluent
Purification with chloroform / methanol / water = 65/25/4) yielded 28 g of white crystals of L-β, γ-diicosapentaenoylphosphatidylcholine (93% yield).

Comparative Example 1 In a 300 ml flask equipped with a stirrer, 10 g of L-α-glycerophosphorylcholine (0.0388
Mol), 21.2 g (0.0427 mol) of palmitic anhydride, 150 ml of dehydrated and dried chloroform, and 9.5 g (0.0776 mol) of 4-dimethylaminopyridine, and under a nitrogen gas stream,
The reaction was carried out at 50 ° C. for 10 hours with rapid stirring. After completion of the reaction, desolvation, acetone purification, and silica gel column purification were carried out in the same manner as in Example 1 to obtain L-β,
13.5 g of white crystals of γ-dipalmitoyl-α-phosphatidylcholine were obtained. The yield was 45%.

〔The invention's effect〕

As described above, the present invention relates to a method for producing phosphatidylcholine by reacting GPC with a fatty acid anhydride,
After the reaction raw materials are dissolved in lower alcohol and uniformly mixed, the lower alcohol is distilled off, and the reaction is performed by adding an aprotic solvent and a basic catalyst. Since GPC is dispersed in a very fine state in the solution, it is possible to carry out a reaction with a high reaction rate in a short time as compared with the conventional method, and since no carrier is used, the reaction after Purification becomes easier,
In addition, highly pure phosphatidylcholine can be obtained in a high yield. In addition, the method of the present invention has effects such as excellent economical efficiency without the need for complicated steps, large scale, manufacturing equipment, etc., because the operation of dispersing GPC is simple.

Claims (1)

(57) [Claims] 1. A method for producing phosphatidylcholine, comprising dissolving glycerophosphorylcholine and a fatty acid anhydride in a lower alcohol, distilling the lower alcohol, and then reacting by adding an aprotic solvent and a basic catalyst. Law.