JPS61275286A - Production of phosphatidylcholine - Google Patents

Abstract

PURPOSE:To obtain the high-quality substance useful in the field of drug without using a heavy metal such as cadmium and a solvent and causing problems of heavy metal contamination, treatment of waste water, etc., by reacting glycerophosphorylcholine with an activated fatty acid in the presence of a catalyst in the absence of a solvent. CONSTITUTION:Glycerophophorylcholine, is blended with an activated fatty acid (especially preferably 8-24C natural or synthetic saturated or unsaturated fatty acid anhydride, chloride, or imidazole derivative) as raw materials in the absence of a solvent, preferably uniformly stirred by a homogenizer into a creamy state, reacted at room temperature -60 deg.C for 2-72hr while allowing it to stand, to give the aimed compound. For example, when a fatty acid anhydride is used as the raw material, dimethylaminopyridine, pyrrolidinopyridine, etc., are preferably used as a catalyst.

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]

Phospholipids exist as components of 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 and selective transport. It performs functions that cannot be performed. There are mainly phospholipids such as phos-7-acytidylcholine (hereinafter referred to as PC), phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, and sphingomyelin in living organisms. Among these, PC is generally called lecithin and is the most abundant in living organisms. There are many of them, and their importance is recognized. A common feature of these phospholipids is that they have a hydrophilic group and a hydrophobic group within their molecules, which allows them to form vesicles called liposomes, which are characteristic of phospholipids, or to form vesicles called liposomes on a substrate.
Blodgett's method allows the creation of thin films with oriented molecules.

Natural phospholipids are already used industrially as natural emulsifiers.
While it is used in the food and cosmetics industries, it is also being developed into medicine that takes advantage of its physiologically active effects.

Recently, it has been known that phospholipids form closed endoplasmic reticulum called liposomes, which have a bilayer membrane and have a bimolecular structure in water, and various uses for this are being considered. For example, medicine
In the pharmaceutical field, it is being considered for use in drug carriers, artificial blood, etc., and in the engineering field, it is being considered for use in artificial blood cells. Recently, thin films made from phospholipids are being applied to electronics, and the development of insulating films is being considered.

However, natural phospholipids are currently used for both purposes, but because phospholipids are mixtures, reproducibility is poor and their strength is insufficient, making it difficult to apply the original functions of phospholipids industrially. not present. In order to be applied to these uses, phospholipids having a structure and strength depending on the purpose are desired, and various attempts have been made to round them out. For example, in order to use phospholipids as liposomes for drug carriers, they must be unaffected by degrading enzymes in the body and must be decomposed after fulfilling their function. For rounding, attempts have been made to use phospholipids with ether bonds that are less susceptible to decomposition by heavy enzymes as part of the liposomes. Furthermore, when considering the use as a thin film, polymerizable phospholipids are said to be preferable for rounding to increase its strength.

Total synthesis method and semi-synthesis method have been known as methods for synthesizing PC (Baer et al., Journal of the American Chemical Society (JAC8)),
4), 761 (1939), Journal Opiological Chemistry (J, Biol, Chem
) 230.447 (1958), Journal of American Chemical Science (JAC8) 72°9
42 (1950)), the total synthesis method requires a large number of steps and is difficult to industrialize. The semi-synthetic method is glycerophosphorylcholine (
This is a method of synthesizing PC from (hereinafter referred to as GPC) and active fatty acids], and heavy metal salts such as cadmium salts are used as GPC. Activated fatty acids include (1) a method of converting fatty acids into acid chlorides (Canadian Journal Opbiochemistry Endophysiology (Ca)
n, J.

Biochem, Phymiol, 37.953
(1959)), (2) Method of using fatty acids as imidazole salts (JP-A-51-91213, H@rmetter et al., Chemistry Endophysics Opriviz (Cern, Phys, Li)
pida 1981, and (g, xtt)), and (3
) A method using fatty acids as acid anhydrides (Regen
Gen) et al., Journal of the American Chemical Society (JAC8) (1982).

791) is known.

[Problem that the invention seeks to solve]

In the conventional semi-synthetic method, GP, which is the starting material for synthesis,
Because C is extremely hygroscopic and highly viscous at room temperature, it is difficult to handle, so it has been used as a crystalline cadmium chloride salt that is easy to handle by reacting with GPCK cadmium chloride.

Currently, the harm caused by heavy metals to the human body is a serious problem, and cadmium is said to be the cause of Itai-tai disease, so it is not desirable to use heavy metals such as cadmium chloride. Also GPC
When PC is synthesized using cadmium chloride salt, GPC
Since this method requires a cadmium chloride salt synthesis step and a cadmium salt removal step, it is not only industrially difficult, but also requires a great deal of cost to dispose of.

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

[Means for Solving the Problems] The present invention is a method for producing PC, which is characterized in that GPC and activated fatty acids are mixed and reacted in the presence of a catalyst without a solvent.

The PC to be manufactured in the present invention has the following general formula (1
) is a compound represented by

GPC, which is the raw material of the present invention, serves as the skeleton of synthesized PC, and can be obtained mainly by hydrolysis or alcoholysis of natural lecithin from soybean or egg yolk after separation and purification, or as it is. .

Natural lecithin is separated using a silica gel column, activated alumina column, etc., and eluted with a chloroform/methanol mixed solvent. To obtain GPC from purified or crude lecithin, a quaternary alkali such as tetrabutylammonium hydroxide can be used.Natural or synthetic saturated or unsaturated fatty acids can be used as the next raw material fatty acids of the present invention, especially those having 8 to 8 carbon atoms. 24 is preferred. Such fatty acids include naturally occurring fatty acids such as myristic acid, palmitic acid, stearic acid, oleic acid, linoleic acid, eicosapentaenoic acid, and docosahexaenoic acid, or polymerizable fatty acids within the molecule such as 2.4-octadecadienoic acid. Synthetic fatty acids with groups such as those with 7-2 groups, and synthetic fatty acids with other groups such as 722 groups can be used alone or in any combination depending on the purpose. Polymerizable PC can be produced by using a fatty acid having a polymerizable group.

In order to convert the fatty acid into an activated fatty acid, in the case of an acid anhydride, N,N'-dicyclohexyl (hereinafter referred to as DCC), l-cyclohexyl-3-(2-morpholinoethyl) Carbodiimide, N,N'-diisopropylcarbodiimide, 1-cyclohexyl-3-(4-
diethylaminocyclohexyl)carbodiimide, 1-
It is preferable to convert the fatty acid into an acid anhydride using a carbodiimide condensing agent such as ethyl-3-(diethylaminoglobil)carbodiimide. In the case of this slightly saturated acid, an acid anhydride can be obtained by reacting it with acetic anhydride and then reacting it with its acid chloride.

In the case of fatty acid chlorides, they can be obtained by reacting fatty acids with thionyl chloride, phosphorus trichloride, phosphorus pentachloride, etc. Fatty acid imidazolates convert fatty acids into N,N'-
Obtained by reaction with 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 examples include dimethylaminopyridine (hereinafter referred to as DMAP) and pyrrolidinopyridine (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 imidazolates.

The method for manufacturing PC is to mix GPC, activated fatty acids such as fatty acid anhydrides, fatty acid chlorides, or fatty acid imidazole, and a catalyst, stir uniformly with a homogenizer to form a cream, and then heat the mixture at room temperature to 60°C for 2 to 30 minutes. This is carried out by allowing the reaction to stand for 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 mixed with a fatty acid, GPC, a fatty acid, a carbodiimide condensing agent, and a catalyst are simultaneously charged and reacted. The reaction at this time is schematically shown below (If)
It is shown by the formula.

SodDMAP' This reaction occurs when a condensing agent such as DCC is present in the reaction system.
It utilizes the fact that fatty acids do not react with catalysts such as DMAP, but react with condensing agents to form acid anhydrides.
) In the formula, a fatty acid is converted into an acid anhydride by DCC, then reacted with GPC to generate PC, and the free fatty acid produced as a by-product is again converted into an acid anhydride by DCC and used for the reaction.

In this way, if the fatty acids liberated by the reaction are always reacted with an excess of condensing agent to form acid anhydrides, and this is then reacted with GPC, the fatty acids are recycled into acid anhydrides. In addition to reducing the amount of fatty acids,
Expensive catalysts also do not produce free fatty acids and salts, and are effective even in small amounts.

The required amount of the condensing agent may be at least equimolar to the fatty acid, and 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 suitable. The condensing agent may be added at appropriate times during the course of the reaction, or may be added in the required amount in advance at the start of the reaction.

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

By selecting the fatty acid as the raw material, PC can be manufactured with various structures and strengths depending on the application.

〔Effect of the invention〕

According to the present invention, by mixing and reacting GPC and activated fatty acids without a solvent in the presence of a catalyst, there is no need to use heavy metals such as cadmium salts and solvents, so there is no need to worry about heavy metal contamination. Furthermore, there is no need for waste liquid treatment or solvent recovery steps. Therefore, high-yield, high-quality PC can be manufactured using simple steps and equipment.

〔Example〕

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

Reference Example 1 100 F of egg yolk lecithin was dissolved in 800 ml of benzene, and 1001 F of tetrabutylammonium hydroxide 10 timeethanol solution was added while stirring. Stirring was continued for 30 minutes after precipitation started, and then the mixture was kept quiet for 30 minutes. Crude GPC precipitated at the bottom was collected with Deka/Cheeseon and washed with benzene. Then, the crude GPC was poured into acetone 11 cooled to -20°C to obtain a precipitate. The acetone reprecipitation was repeated in the same manner, and crude GPC containing no impurities such as esters was recovered. This crude GPC was dissolved in methanol, and insoluble matter was removed by centrifugation.

Evaporate the supernatant, 33? APC of 100% was obtained.

'Reference example-2 Palmitic acid 20F (0,078 mol) and DCC8, O
f (0.04 mol) in dry chloroform at 5°C for 1
The reaction was allowed to proceed for 5 hours. The precipitated dicyclohexyl flare 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 13.6 F (0,0287 l) of pure palmitic anhydride.

The above reaction formula is shown below.

DCC Example-1 l! obtained by the method of Reference Example-1! [CpctosP, palmitic acid anhydride 401 and DIAP19F obtained by the method of Reference Example-2 were mixed well, and mixed with a homogenizer for 50 minutes.
The mixture was stirred for a minute and left to react at room temperature for 48 hours. After the reaction, methanol was added, and the solution was passed through a column of ion exchange resin Amberlite 200C (trademark of Rohm and Haas) to remove DMAP. The effluent was concentrated and passed through a silica gel column using chloro μform/methanol/
By purifying with water = 65/25/4, phosphatidylcholine cybalmitate (DPPC) 15,5
I got an F.

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

Example-2 One GPC10r obtained by the method of Reference Example-1, palmitic acid 602, DCC50S' and DMAP19f were mixed well, stirred for 5 minutes with a homogenizer, and heated to 48% at room temperature.
The reaction was allowed to stand for a period of time. Methanol was added to this, the solution was passed through an Amberlite 200C column, and DMAP
was excluded. Below, DPPC1 was purified in the same manner as in Example-1.
I got 6,Of.゛Example-3 GPC5F obtained in the same manner as in Reference Example-1, stearic acid chloride 151F, and pyridine 3.22 were mixed well, stirred with a homogenizer for 5 minutes, and left to react at room temperature for 2 hours. Ta. The reaction mixture was diluted with acetone at 20°C.
00- to obtain a precipitate. Filter this precipitate,
Purified using a silica gel column in the same manner as Example-1,
8.51 of phosphatidylcholine distearate was obtained.

Example-4 1iGpcxor obtained by the method of Reference Example-1, oleic acid 60? , DC050F and DMAP19? Mix well, stir with a homogenizer for 5 minutes, and stir at room temperature for 48 minutes.
The reaction was allowed to stand for a period of time. After the reaction, phosphatidylcholine dioleate 15.8F was obtained in the same manner as in Example-1.

Claims (3)

[Claims] (1) A method for producing phosphatidylcholine, which comprises mixing and reacting glycerophosphorylcholine and activated fatty acid in the presence of a catalyst without a solvent. (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 in an amount equal to or more than the same molar amount as the fatty acid during the synthesis of phosphatidylcholine.