JPS61275287A - Production of synthetic phosphatidylcholine - Google Patents

Abstract

PURPOSE:To obtain simply the titled high-quality compound useful as a drug, etc. without using cadmium chloride, etc., by reacting a bivalent metallic salt such as Ca salt, Mg salt of glycerophosphorylcholine with an activated fatty acid in the presence of a catalyst in a solvent. CONSTITUTION:Ca salt, Mg salt, Cu salt, Zn salt, Mn salt, or Ni salt of glycerophosphorylcholine is reacted with an activated fatty acid in the presence of a catalyst in a solvent (preferably dichloromethane, etc.) preferably at 20-60 deg.C for 2-72hr, to give the aimed compound. An acid anhydride, a chlorine compound, or imidazole derivative of 8-24C fatty acid is preferable as the activated fatty acid.

Description

DETAILED DESCRIPTION OF THE INVENTION [Industrial Field of Application] The present invention relates to a new method for producing phosphatidylcholine without using heavy metals.

[Conventional technology]

7. Lipids exist as components of the biological membranes of cells, where they form a bilayer membrane with a bimolecular structure, and together with proteins, cholesterol, etc., they are essential for biological phenomena such as permeation of substances or selective transport. It performs functions that cannot be performed. There are mainly phospholipids such as phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, and sphingomyelin in living organisms. Among these, phosphatidylcholine is generally called lecithin and is a substance that exists in living organisms - within its molecules. This makes it possible to create vesicles called liposomes, which are characteristic of phospholipids, or thin films in which molecules are oriented on a substrate using the Langmuir-Prodgett method.

Natural phospholipids are already used industrially as natural emulsifiers, and are used in the food, cosmetics, and other fields. In addition, it is being developed into a medicine that utilizes its physiologically active action.

Recently, it has been known that phospholipids, due to their lipid characteristics, form closed endoplasmic reticulum called liposomes, which have a bilayer membrane and have a bimolecular structure in water, and various uses are being considered. For example, in the medical and pharmaceutical fields, development into artificial cells is being considered. Recently, thin films made of phospholipids are being applied to electronics, and the development of insulating films is being considered. However, natural phospholipids are currently used in both applications, and because the phospholipids are mixtures, the reproducibility of experiments or lack of strength makes it difficult to apply the original functions of phospholipids industrially. has not yet been reached. In order to be applied to these uses, there is a need for phospholipids that have a structure and strength suitable for the purpose, and various attempts have been made for this purpose. For example, in order to use phospholipids as liposomes for drug carriers, they need to be relatively unaffected by degrading enzymes in the body and to be degraded after fulfilling their function. For this purpose, polymerizable phospholipids are used as a part of the liposome to make monopolymerized liposomes, and when decomposition by enzymes is considered, total synthesis methods and semi-synthesis methods are known in which polymerizable phospholipids are used to increase the strength of the liposomes ( Beer &er et al., Jarf) v Opza American Chemical Society (JAC8) 6
1 (4), 761 (1939), Journal of Biological Chemistry (J, Biol, ch.)
230.447 (1958), Journal of the American Chemical Society (JAC8) 72942
(1950) l, the total synthesis method requires many steps and is difficult to industrialize.

A semi-synthetic method, i.e., phosphatide (Canadian Journal of Biochemistry and Endophysiology) from fatty acids and glycerophosphorylcholine (hereinafter referred to as GPC), Can, J.
) 37,953 (1959) l, (2) Using fatty acids as imidazole salts (Japanese Unexamined Patent Publication No. 1983-1991)
213, Hermetter et al.
Phys.

Lipids) 28 111 (1981)),
(3) Those using fatty acids as acid anhydrides (Regen (
Regen) Journal of the American Chemical Society (JAC8) 104791 (198
2) ) is known.

[Problem that the invention seeks to solve]

GPC, which is the raw material of the present invention, is highly hygroscopic and highly viscous at room temperature, making it difficult to handle. Therefore, in order to facilitate handling and eliminate the influence of quaternary ammonium groups, cadmium chloride was added. It has traditionally been used as Another advantage of cadmium chloride salt is that when producing purified GPC from crude GPC, purification can be achieved by recrystallizing the cadmium chloride salt. However, this purification method does not have a high yield, and furthermore, if GPC is synthesized from a raw material with high PC purity such as egg yolk lecithin, it will not have any particular industrial advantages.

Currently, the harm caused by heavy metals to the human body has become a serious problem. In particular, cadmium is a disease that causes a disease. Synthesizing PC using cadmium chloride requires a GPC cadmium chloride salt synthesis process and a cadmium salt removal process. This poses a number of problems, including industrial difficulties and the cost of disposing of it.

The present invention is intended to solve the above-mentioned problems, and aims to provide a method for manufacturing PC that can obtain high-quality PC through simple steps without using cadmium chloride. There is.

[Means for solving problems]

The present invention relates to a PC characterized in that a calcium salt, a magnesium salt, a copper salt, a zinc salt, a manganese salt, a di-Kel salt of GPC and an activated fatty acid are reacted in a solvent in the presence of a catalyst. It is the method of production and delivery.

The PC to be manufactured in the present invention is a compound represented by the following general formula.

2C-OR GPC, which is the raw material of the present invention, serves as the skeleton of synthesized PC, and can be obtained mainly by separating and purifying natural lecithin from soybean, egg yolk, etc., or by hydrolyzing or alcohol rinsing it as it is. Natural lecithin is separated using a silica gel column, activated alumina column, etc., and eluted with a chloroform-methanol mixed solvent. GPC can be obtained from purified or crude lecithin by alcoholysis with a quaternary alkyl ammonium hydroxide such as tetrabutylammonium hydroxide or an alkali metal, but it can be obtained by gentle hydrolysis with a low concentration of alkali, etc. It's okay. The GPC thus obtained can be reacted with a divalent metal such as calcium, magnesium, copper, zinc, manganese, nickel, etc., and then reprecipitated in acetone to obtain a GPC metal salt.

As the fatty acid which is the next raw material of the present invention, natural or synthetic saturated or unsaturated fatty acids can be used, especially those having 8 carbon atoms.
~24 are preferred. Such fatty acids include myristic acid and palmitic acid.

Fatty acids derived from natural products such as stearic acid, oleic acid, linoleic acid, eicosapentaenoic acid, docosahexaenoic acid, etc., or those having a polymerizable group in molecule I such as 2.4 octadecadienoic acid H, and others such as phenyl group. There are synthetic fatty acids that have the following properties, and these can be used singly or in any combination depending on the purpose. By using a fatty acid having a polymerizable group, polymerizable PC can be produced.

In order to convert the above-mentioned fatty acids into activated fatty acids, in the case of acid anhydrides, N,N-dicyclohexylcarbonimide (hereinafter referred to as DCC), l-cyclohexyl-3-(2-morpholinoyl)carbodiimide, It is best to use a carbodiimide condensing agent such as de, N, N'-diisopropylcarbodiimide, 1-cyclohexyl-3-(4-diethylami/cyclohexyl)carbodiimide, and 1-ethyl-3-(ethylaminopropyl)carbodiimide. preferable. In addition, acid anhydrides can be obtained by reacting saturated acids with acetic anhydride or dipping chloride. In the case of a fatty acid trichloride compound, it can be obtained by reacting a fatty acid with thionyl chloride, phosphorus trichloride, phosphorus pentachloride, etc.

Fatty acid imidazoles are obtained by reacting fatty acids with N,N'-carbonyldiimidazole.

The catalyst used in the present invention is a catalyst for producing PC by reacting GPC with an activated fatty acid, and is preferably a mild catalyst that does not cause isomerization of PC. Preferred are dimethylaminopyridine (hereinafter referred to as DMAP) and pyridinopyridine (hereinafter referred to as PPY) in the case of fatty acid anhydrides, pyridine in the case of fatty acid chlorides, and imidazole alkali metal salts in the case of fatty acid imidazole compounds.

The solvent used in the reaction is preferably one that provides a high yield of PC, and dichloromethane, chloroform, etc. are suitable. By appropriately adding dimethyl sulfoxide, dimethylformamide, dimethylacetamide, N-methylpyrrolidone, hexamethylphosphoramide, sulfolane, tetramethylurea, etc. to these, GPC
The solubility of the metal salt is increased and is favorable for the reaction. It is preferable to use these solvents after dehydration.

The method for producing PC includes G consisting of the above-mentioned metal salt with a valence of 2.
A PC metal salt and an activated fatty acid such as a fatty acid anhydride, a fatty acid chloride, or a fatty acid imidazole are mixed in a dry solvent such as dichloromethane in the presence of a catalyst with 20 to 6 (I'
The reaction is carried out with gentle stirring for 2 to 72 hours.

For example, when using a fatty acid anhydride, an acid anhydride synthesized in advance may be used, but the reaction can also be carried out by the following method. That is, a carbodiimide condensing agent such as DCC is used in an amount equal to or more than the fatty acid, and a GPC metal salt, a fatty acid, a carbodiimide condensing agent, and a catalyst such as DMAP are simultaneously charged and reacted in a dehydrated solvent such as dichloromethane. The reaction at this time is schematically shown below.

(RCO)20-1-GPC metal salt + [:DMAP]
→ 17−−−−−−−− +++ +++
+++++lThis reaction utilizes the new knowledge that when a condensing agent such as KDCC is present in the reaction system, the fatty acid does not react with a catalyst such as DMAP, but reacts with the condensing agent and becomes an acid anhydride. converting a fatty acid into an acid anhydride and reacting it with GPC to produce PC,
The by-produced free fatty acid is again converted into an acid anhydride by DCC and used for the reaction.

In this way, the fatty acids liberated by the reaction are always reacted with an excess of condensing agent to form rIR anhydride, and when this is reacted with GPC, the fatty acid is converted to anhydride and used as cidal. In addition to being able to reduce the amount of fatty acids, the expensive catalyst does not produce free fat bonds and salts, and is effective with less tK.

The required amount of the condensing agent is equal to or more than the mole of the fatty acid (even if added in large excess, the yield of PC will not be lowered, but an amount up to twice the number of moles of the fatty acid is appropriate). The agent may be added at appropriate times during the course of the reaction (or the necessary amount may be added in advance at the start of the reaction).

After the reaction is completed, filtration, concentration, and purification are performed to obtain purified *pc.

By carefully selecting fatty acids as raw materials, PCs with various structures and strengths are manufactured depending on the intended use.

〔Effect of the invention〕

According to the present invention, by mixing and reacting the GPC metal salt and the activated fatty acid in a low pressure solvent in the presence of a catalyst, there is no need to use heavy metals such as cadmium salts.
There is no need to worry about heavy metal contamination, and there is no need for a waste liquid treatment process. Therefore, it is possible to manufacture high-quality PC with high yield using simple steps and equipment. The PC obtained in this manner is highly safe and can be effectively used for pharmaceuticals and the like.

〔Example〕

The present invention will be specifically explained below based on Reference Examples and Examples.

Reference Example-1 Commercially available egg yolk lecithin 1001 was dissolved in 800 d of benzene, and 100 P of tetrabutylammonium hydroxide 10 timemethanol solution was added while stirring. Stirring was continued for 30 minutes after the precipitation began, and then the mixture was kept quiet for 30 minutes. The crude GPC' which settled at the bottom was collected by decantation and washed with benzene. Then, the crude GPC was poured into acetone 1 cooled to -20'C to obtain a precipitate. The acetone reprecipitation was repeated again in the same manner to recover GPo 33 P free of impurities such as esters. This 1tGPC
Dissolve in methanol, remove insoluble matter by centrifugation,
A GPC methanol solution was obtained.

Reference example-2 Palmitic acid 2 Q J' (0,078 mol) and DC
C8,0/ (0,04 mo/I/) was reacted in dehydrated chloroform at 5°C for 115 hours. The precipitated dicyclohexylurea was filtered off, and the filtrate was concentrated to obtain crude palmitic anhydride. This crude palmitic anhydride was separated on a silica gel column using chloroform as an eluent to obtain 13.67' (0,028 mol) of pure palmitic anhydride.

Example 1 58.3 J' of barium chloride was added to the GPC methanol solution obtained by the method of Reference Example 1, and after reaction, a GPC barium salt was obtained by reprecipitation with acetone. This GPC barium salt 9.37', (0.02 mol), barmitic acid 8.5
F (0,03 mol), DCClo, 3.7 (0,0
5 mol), DMAP 0.61? (0,005 mol) was reacted in total chloroform at room temperature for 48 hours. The reaction solution was filtered, and the filtrate was passed through a column of ion exchange resin (Amberlite 200C, manufactured by Rohm and Haas) to remove DMAP. After concentrating the effluent, it was purified with a silica gel column using chloroform/methanol/water = 65/25/4 as an eluent to obtain civalmitic acid P.
C6,2P was obtained.

Note that PC was identified by comparing the spot position of thin layer a-mography and the NMR (nuclear magnetic resonance) absorption spectrum with a standard substance.

Example-2 GPC calcium salt 7°37' obtained according to Example-1
(0.02 mol), palmitic acid 8.5 P C0,
03 mol), DCCI 0.3 P (0.05 mol), and DMA P 0.617 (0.00 mol) were reacted in chloroform at room temperature for 48 hours. Thereafter, treatment was carried out in the same manner as in Example-1, and civalmitic acid PC6,4
I got 7'.

Example-3 9 GPC magnesium salt 7, O obtained according to Example-1
J' (0.02 mol), oleic acid 7.9 F (0
,03 mol), DCClo,37'(0,05 mol),
DMA P 0.71A (0,006 mol) was reacted in chloroform at room temperature for 48 hours. Hereafter, Example-1
was treated in the same manner as Pc6. Got OJ'.

Example-4 GPC manganese salt 8.77 (
0.02 mol), palmitic acid 8.57' (0.03
mol), DCC10,27' (0.05 mol), DM
AP 0.7 Jl' (0,006 solu) was reacted in chloroform at room temperature for 48 hours. The following is an example-
The treatment was carried out in the same manner as in 3 to obtain cibalmitic acid PC6,I P.

Example-5 GPC nickel metal salt 7, 71 obtained according to Example-1
(0.02 mol), stearic acid 8.77 (0,
006 mol), DCCIo, 37' (0.05 mol),
DMAPo, 61? (0,005 mol) was reacted in dichloromethane at room temperature for 48 hours. Thereafter, the same treatment as in Example-1 was carried out to obtain distearic acid PC6,3.

Example-6 Same GPC magnesium salt as used in Example-3 7.0
Palmitic acid anhydride obtained in 7 and Reference Example-2 12.87
'(0,0235 mol) and DMAP2.2P C0,
00,176 mol) was reacted in dichloromethane 50P at room temperature for 48 hours. Thereafter, treatment was carried out in the same manner as in Example-1 to obtain cybalmitic acid PC2,96J'.

Example 7 The same GPC nickel salt used in Example 5 and hypopalmitic acid chloride were reacted with pyridine as a catalyst. That is, 7.71 J' of GPC nickel salt was dispersed in 50 J' of dichloromethane in an ice bath, and 6,5 J' (0,0235 mol) of palmitic acid chloride was dissolved in 25 J' of dichloromethane, which was slowly added dropwise.

Then pyridine 1.4 J' ff-dichloromethane 2
5J' was dissolved and added dropwise to the reaction mixture for 030 minutes at room temperature.
Allowed time to react. The reaction solution was concentrated and poured into acetone 20011j at -20°C to obtain a precipitate. The rest is an example.
Purify using a silica gel column in the same manner as in 1 to obtain cibalmitic acid PC? , I got a friend on the 14th floor.

Claims (3)

[Claims] (1) A method for producing phosphatidylcholine, which comprises reacting a calcium salt, magnesium salt, copper salt, zinc salt, manganese salt, or nickel salt of glycerophosphorylcholine with an activated fatty acid in a solvent in the presence of a catalyst. . (2) The production method according to claim 1, wherein the activated fatty acid is an anhydride, a chloride, or an imidazolate of a natural or synthetic saturated or unsaturated fatty acid having 8 to 24 carbon atoms. (3) The production method according to claim 2, wherein the fatty acid anhydride is synthesized using a fatty acid and a carbodiimide condensing agent during the production of phosphatidylcholine.