JPH0613538B2 - Method for producing phospholipid - Google Patents

Description

TECHNICAL FIELD The present invention relates to a method for producing a phospholipid having a definite structure from a phospholipid mixture as a raw material.

[Conventional technology]

Phospholipids can be used not only as emulsifiers, but in recent years, application to drug carriers, artificial blood, artificial cells, etc., as a base material for ribosomes has attracted attention in recent years. Various applications have been considered in the fields of medicine, pharmacy, engineering and so on. In order to meet such various demands, it is necessary to efficiently produce phospholipids having a structure according to each application.

As a method for producing a phospholipid having a well-defined structure, a method for producing by total synthesis, a method for producing an acyl group by using a glyceryl phosphate as a raw material, and a synthetic phospholipid having a well-defined structure as a raw material for forming a base moiety A method of converting to another structure by exchange is known.

[Problems to be solved by the invention]

However, the method for producing by the above-mentioned total synthesis is known for phosphatidylcholine and the like, but it has drawbacks such as long process, complicated and difficult to scale up, and separation of by-product isomers. The types of lipids are also limited.

Depending on the type of nitrogen-containing alcohol residue or polyol residue part (hereinafter referred to as a base), a method for producing an acyl group by using a glyceryl phosphate ester obtained by deacylating a phospholipid as a raw material is a glycerin part during the acylation reaction. In addition to the above, an acyl group is introduced, so that it cannot be produced by this method.

Also, only in the case of phosphatidylcholine, there is known a method for producing synthetic phosphatidylcholine by separating glycerylphosphorylcholine as a complex salt with heavy metals such as cadmium from deacylation of crude phospholipid containing phosphatidylcholine, and introducing an acyl group. However, it is difficult to remove the heavy metal remaining in a trace amount, and the use of the produced phosphatidylcholine is limited.

Furthermore, a method of converting a synthetic phospholipid having a well-defined structure as a raw material into another structure by exchanging a base moiety is as follows: phosphatidylethanolamine is produced by reacting phospholipase D with synthetic phosphatidylcholine as a raw material.
It is known to convert to phosphatidylglycerol. However, phosphatidylcholine has to be synthesized first and then converted, and loss occurs during conversion, resulting in low efficiency.

The present invention is intended to solve the above problems, and an object thereof is to propose a method for producing a phospholipid, which is a crude phospholipid as a raw material and which can efficiently produce a phospholipid having a well-defined structure in a simple process. .

[Means for solving problems]

The present invention introduces a removable protecting group into a crude phospholipid and deacylates it to the following formula (I) (In the formula, X represents a nitrogen-containing alcohol residue or a polyol residue, Y represents an easily removable protecting group, and n represents a natural number of 12 or less.), And a glyceryl phosphate derivative represented by
A method for producing a phospholipid, which comprises separating and purifying silicic acid as a separating agent, and then introducing an arbitrary acyl group to remove the protecting group.

In the present invention, a suitable protecting group (substituent) is introduced into the base of the crude phospholipid, followed by deacylation to give a glyceryl phosphate derivative, which is separated and purified by silicic acid chromatography. Then, an acyl group is introduced into each glyceryl phosphate derivative to form a phospholipid derivative, and the protecting group is eliminated to produce a phospholipid.

An important point in the present invention is to separate and purify phospholipid as a glyceryl phosphate derivative. Glyceryl phosphate, which is a deacylated phospholipid, has no method for industrially separating and purifying the mixture because of problems such as solubility. In the present invention, by introducing a protecting group into a glyceryl phosphate derivative, separation and purification by silicic acid chromatography is possible. In this case, since the number of the protective groups introduced varies depending on the type of base, the separation and purification ability is improved as compared with silicic acid chromatography in the form of phospholipid. The protecting group also plays a role of protecting an acyl group from being introduced into a base when introducing an arbitrary acyl group into the hydroxyl group of the glycerin moiety of the glyceryl phosphate derivative to form a phospholipid derivative.

Raw crude phospholipids used in the present invention include phosphatidylserine extracted from nature or commercially available,
Phosphatidyl ethanolamine, phosphatidyl N-
Methyl ethanol amine, phosphatidyl N, N-dimethyl ethanol amine, phosphatidyl N, N, N-trimethyl ethanol amine, phosphatidyl glycerol, phosphatidyl aminoacyl glycerol, phosphatidyl glycerophosphate, cardiolipin, phosphatidyl inositol, phosphatidyl inositol diphosphate, phosphatidyl inositol triphosphate, The crude phospholipid, which is a mixture of sugar phospholipids, can be used as it is without purification. Examples of such crude phospholipids include:
Examples include egg yolk lecithin, defatted soybean lecithin, phospholipid fraction extracted from yeast, crude cephalin fraction extracted from bovine brain, and the like. The crude phospholipid can be used after being pretreated by solvent fractionation or the like or purified by chromatography or the like, but it is industrially advantageous to use the crude phospholipid as it is without purification.

Since a protecting group is not introduced into phosphatidylcholine, synthetic phosphatidylcholine can be obtained through glycerylfolylcholine as it is.

The easily removable protective group is a group represented by Y in the formula (I), and examples thereof include a linear, branched or cyclic alkyl group, a linear, branched or cyclic alkyloyl group. A group selected from a straight-chain, branched-chain or cyclic alkenyl group, a straight-chain, branched-chain or cyclic alkeroyl group, and an aryl group can be used.
Examples of such protecting groups include ethers such as alkyl ether, allyl ether, benzyl ether, triarylmethyl ether, trialkylsilyl ether, tetrahydropyranyl ether which are bonded to the base moiety of the crude phospholipid; acetic acid ester, Examples thereof include groups that form esters such as formic acid esters, trifluoroacetic acid esters, and benzoic acid esters; acetals such as methylene acetal, ethylidene acetal, benzylidene acetal, and isopropylidene acetal. The selection of these groups is preferably determined by the reactivity of the functional group of the base of the phospholipid to be introduced, and the reaction method and reaction conditions are generally adopted for the reaction of sugars and amino acids. The method and conditions that are present can be adopted. However, the protective group must not be removed during the subsequent reaction of introducing any acyl group.

The method of introducing the protecting group is, for example, in the case of benzylation, dimethyl sulfoxide, N, N-dimethylformamide, tetrahydrofuran, dioxane, or the like, which is well dehydrated, is used, and sodium hydride or sodium metal is used. The benzyl ether can be formed by the formation of an alkali metal alcoholate followed by the addition of a benzyl halide to protect the hydroxyl group with a benzyl group. In the case of tetrahydropyranylation, a solvent that dissolves or disperses phospholipids such as dichloromethane, chloroform, carbon tetrachloride, and benzene is used as a solvent, and acetic acid, p-toluenesulfonic acid, methanesulfonic acid, H Using an acid such as ⁺ type ion exchange resin, 3,
By reacting with 4-dihydro-α-pyran under stirring, tetrahydropyranyl ether can be formed and the hydroxyl group can be protected. Other reactions can also be performed according to the above. The reaction temperature is -20 to 10 depending on the type of protecting group.
A range of 0 ° C is suitable, but -20 to 40 due to the stability of the substrate.
C is most preferred. The reaction time can be appropriately selected within the range of 15 minutes to several days.

A mixture of the glyceryl phosphate derivative represented by the formula (I) can be obtained by introducing a protecting group represented by Y into the raw crude phospholipid and then deacylating. As the method for deacinating, methyl esterification decomposition or saponification decomposition generally used for deacylation of phospholipids can be adopted.

In this case, deacylation of the phospholipid with a protected hydroxyl group can be carried out by alcoholysis using a quaternary alkylammonium hydroxide such as tetrabutylammonium hydroxide or an alkali metal, but a low concentration of alkali Etc. to gently hydrolyze. Examples of the solvent used in the deacylation reaction include chloroform, dichloromethane, carbon tetrachloride, ether and the like, but if necessary, a mixed solvent with a hydrophilic solvent such as methanol and ethanol can also be used. Reaction temperature is 0
The range of ~ 100 ° C is preferred, but 0 ~ 40 due to the stability of the substrate.
C is especially preferred. A suitable reaction time may be selected within the range of 15 to several days.

In the glyceryl phosphate derivative of formula (I) thus obtained, X is a serine group, ethanolamine group, N-methylethanolamine group, N,
N-dimethylethanolamine group, N, N, N-trimethylethanolamine group, glycerol group, aminoacylglycerol group, glycerophosphate group, glycerylphosphatidyl group, inositol group, inositol phosphate group, inositol diphosphate group, monosaccharide, disaccharide It is a mixture of sugars or oligosaccharides. The introduction of the protective group Y in this glyceryl phosphate derivative reduces the overall polarity and makes it soluble in an organic solvent, which enables separation and purification by liquid chromatography, which was impossible with conventional glyceryl phosphate. . In addition, fatty acids generated during deacylation, unreacted substances and the like can also be separated from the glyceryl phosphate derivative mixture. Phospholipids having alkyl chains such as ether bonds other than ester bonds and acid amide bonds, such as plasmalogens and sphingolipids, are difficult to separate with phospholipids, but the introduction of Y has extremely low polarity. Therefore, it can be easily separated.

The silicic acid used as a separating agent may be a crushed type or a gel type (silica gel, etc.) and has a particle size of 10-80.
Those having a diameter of 0 μm and a pore diameter of 0.25 to 10 μm are preferable.
Commercial products can also be used, for example Clarkson Chemica
Unisil manufactured by ls Co., Wakogel manufactured by Wako Pure Chemical Industries, Ltd., Me
Silica Gel 60 manufactured by rck, iatro beads manufactured by Yatron Co., Ltd. (both are trademarks), and the like are preferable in terms of separation performance and the like.
Although silicic acid, which is commercially available for high performance liquid chromatography, is more excellent in separation performance, it is disadvantageous in cost. Other products can be used if they are sufficiently refined such that the particle size is made uniform before use.

As a method of separation, a method based on a known column chromatography using the above-mentioned silicic acid can be adopted. That is, silicic acid is pretreated with a low-polarity solvent capable of adsorbing a glyceryl phosphate ester derivative, and a sample containing a glyceryl phosphate ester derivative dissolved in a similar low-polarity solvent is added to spread it onto silicic acid to dispose of it. After washing off, the glyceryl phosphate derivative is sequentially eluted with a highly polar solvent. In order to enhance the cleaning effect of washing away the unnecessary substances from the adsorbent, it is desirable to use a solvent having a high polarity so that the compound does not elute. In order to sequentially elute the glyceryl phosphate ester derivative for each type of X, it is desirable to perform operations such as sequentially flowing the highly polar solvent while gradually switching to a more polar solvent, and performing gradient elution.

As such a solvent system, for example, the following combinations can be shown. Highly polar solvent has 1 carbon
~ 10 linear, branched or cyclic aliphatic alcohols or aldehydes and the like and water or a mixture thereof and the like, which may have an ether bond, an ester bond or a double bond in the molecule. , An alkyl group,
It may have a functional group such as a cyan group or an amine group. Examples of such a high polar solvent include methanol, 3% hydrous isopropanol, acetonitrile, propyl butyrate, and a mixed system of two or more of these. The low-polarity solvent includes a linear, branched or cyclic hydrocarbon compound having 1 to 10 carbon atoms or a halogenated hydrocarbon compound having 1 to 2 carbon atoms. It may have an ether bond, or may have a functional group such as an alkyl group. Further, a mixture of these and at least one kind selected from the above-mentioned highly polar solvent group can also be used. Examples of such low polar solvents include n-hexane, isooctane, diethyl ether, diisopropyl ether, cyclohexane, benzene, xylene, chloroform, carbon tetrachloride, dichloroethane, isooctane-chloroform (3: 7), n-hexane-benzene. (4: 7), chloroform-methanol-water (65: 25: 4)
Etc.

The glyceryl phosphate derivative thus separated and purified can be made into a phospholipid derivative by introducing an arbitrary acyl group. As the method for introducing an acyl group, any method such as a method using a fatty acid chloride, a method using an acid anhydride, or a method using an acid imidazole salt can be adopted, but when an unsaturated fatty acid is to be introduced, an acid anhydride is used. The thing method is safe.

As the arbitrary fatty acid to be introduced, a linear or branched saturated or unsaturated fatty acid having a carbon number of 30 or less can be used, and examples thereof include acetic acid, butyric acid, caprylic acid, lauric acid, stearic acid, and serotonin. Examples thereof include acids, palmitoleic acid, elaidic acid, erucic acid, nervonic acid, linoleic acid, α-cinolenic acid, γ-linolenic acid, arachidonic acid, docosahexaenoic acid, isostearic acid and phytanic acid. In principle, it is possible to introduce other than this. Depending on the purpose of these carboxylic acids,
They can be used alone or in any combination.

The reaction method for introducing an acyl group is, for example, an acid anhydride, an acid halide, or a fatty acid imidazole compound which is in an active acylation state as an acylating agent, and a hydroxyl group-protected glyceryl compound of formula (I). React with the hydroxyl group of the glyceryl moiety of the phosphate derivative. The amount of the acylating agent added is 1 to 20 equivalents, preferably 1 to the reaction substrate.
5 equivalents is good.

The catalyst used in the reaction is a basic catalyst, preferably a mild one that does not cause isomerization of phospholipids. When an acid anhydride or acid halide is used as the acylating agent, the catalyst may be pyridine or N, N-dimethyl-4-
Pyridine derivatives such as aminopyridine, N, N-dimethyl-4-amino-2-methylpyridine and 4-pyrrolidinopyrrolidine, and tertiary amines such as triethylamine and tributylamine can be used, but acid anhydrides in case of
N, N-Dimethyl-4-aminepyridine or 4-pyrrolidinopyridine, preferably pyridine in the case of an acid halide. When a fatty acid imidazole compound is used, an alkali salt of a nitrogen-containing five-membered heterocyclic compound such as imidazole sodium, triazole sodium, and benzimidazole sodium can be used, but imidazole sodium is preferable. The amount of catalyst added is 0.01 to the raw material
About 20 equivalents, preferably 0.1 to 2 equivalents are good.

Examples of the solvent include halogenated hydrocarbons such as chloroform, dichloromethane and carbon tetrachloride, aromatic hydrocarbons such as benzene and toluene, hydrocarbons such as hexane and heptane; esters such as ethyl acetate and propyl acetate; diethyl ether, tetrahydrofuran and the like. Ethers and the like can be used, and these solvents are preferably dried.

The acylation reaction can be carried out in the range of about 0 to 80 ° C, preferably 10 to 50 ° C, usually 15 minutes to several days, preferably 30 minutes.
After a lapse of from 3 minutes to 3 days, a desired phospholipid derivative having a desired acyl group can be obtained. The reaction system is not necessarily limited, but when a highly unsaturated fatty acid is used for the acyl group, it is preferably carried out under a stream of an inert gas such as nitrogen or argon.

By removing the protective group of the phospholipid derivative obtained by introducing an arbitrary acyl group as described above, a phospholipid having a definite structure can be obtained. As a method for removing the protecting group, a method usually used for breaking the bond of the protecting group, such as hydrolysis with an acid catalyst and reduction reaction, can be adopted. However, under alkaline conditions, elimination of the acyl group may occur, so it is desirable to avoid it. When a protecting group that is eliminated by reduction is used, the reaction is generally catalytic reduction with hydrogen, but a milder reduction reaction using formic acid, ammonium formate, cyclohexadiene, or the like as a hydrogen source can be used.

As the solvent, halogenated hydrocarbons such as dichloromethane and chloroform; ethers such as tetrahydrofuran and diethyl ether; aromatic hydrocarbons such as benzene and toluene can be used.
Palladium oxide, palladium-carbon, or the like can be used. The amount of catalyst added is 10 to 100 g per mol of the protective group.
Is preferred.

The reaction protects the hydroxyl group, dissolves the phospholipid derivative recombined with an arbitrary acyl group in a suitable solvent, and after adding a catalyst, in the case of catalytic reduction, hydrogen may be blown in and stirred, formic acid, ammonium formate, cyclo When a cocatalyst such as hexadiene is used, it can be obtained by adding 1 to 20 equivalents of this cocatalyst to 1 mol of the protective group in the substrate and stirring under an inert gas stream. The temperature of this reaction is -20 to 80 ℃
The temperature is preferably 0 to 40 ° C. The reaction is completed in 10 minutes to 4 hours, and as a post-treatment, the reaction product is removed by filtration.
The filtrate is thoroughly washed with water, and after drying, the solvent is removed to obtain a desired phospholipid in which any desired acyl group is recombined.

When a protecting group that is eliminated by an acid catalyst is used,
The solvent may be any solvent that can dissolve or disperse a substrate such as chloroform, dichloromethane, benzene, tetrahydrofuran, dimethyl sulfoxide, etc. Acid catalysts include
Inorganic acids such as hydrochloric acid, sulfuric acid, nitric acid, boric acid, and acetic acid, p
-Toluenesulfonic acid, organic acid such as methanesulfonic acid,
And H ⁺ type ion exchange resins can be used. The acid catalyst may be added in an amount of 0.1 to 5 equivalents of the above acid, preferably 0.5 to 2 equivalents, relative to 1 mol of the protective group in the molecule. The reaction time can be an appropriate time from 5 minutes to several days, and the reaction temperature can be from −20 to 100 ° C., but considering the stability of the substrate, the reaction time is from 0 to 40 ° C. for 15 minutes to Four
Time reaction is preferred. After the reaction, the product is thoroughly washed with water, dried and then the solvent is removed to obtain a desired phospholipid having a recombinant acyl group.

The thus-obtained phospholipid retains the structure of the natural phospholipid as it is and has a structure in which only the acyl group is recombined. Therefore, it is used for the purpose depending on the recombined acyl group. For example, for the purpose of clarifying the chain length, the degree of unsaturation, and the bonding position of an acyl group, they are used as clear phospholipids for confirming the pharmacological effect. For the purpose of imparting functionality to the acyl group, for example, when the acyl group is a polyunsaturated fatty acid having physiological activity, it is used as a physiologically active substance, and when the acyl group is a polymerizable group,
Used as a monomer for polymerization. Further, these phospholipids are used as salts depending on the purpose.

〔The invention's effect〕

According to the present invention, a natural phospholipid mixture that is easily obtained can be used as a raw material to produce a phospholipid having a well-defined structure with higher convenience, ease, economy, purity and yield than conventional methods. Phospholipids that could not be produced can also be produced.

〔Example〕

Hereinafter, the present invention will be specifically described based on Examples and Reference Examples.

In each example,% indicates% by weight. Further, the compound was identified mainly by thin layer chromatography (TLC) analysis shown in Analysis Method 1 by comparison with a reference substance, and if necessary, mass spectrometry was performed as shown in Analysis Method 2.

Analytical method 1 TLC analysis was performed as follows.

Merck NO.5717 plate with 20-100 μg of sample
Spot to ~ 5 mm, chloroform-methanol-water (6
5: 25: 4) or chloroform-methanol-water (120: 70:
Deployed in 5). After air-drying the developing solvent, the detection reagent was sprayed. For the detection, Dittmer reagent, 50% sulfuric acid, ninhydrin reagent, and anthrone reagent were used depending on the purpose. For quantitative measurement, 50% sulfuric acid was sprayed and heated at 120 ° C for 20 minutes to develop color, which was measured with a Shimadzu high-speed thin layer chromatography scanner model CS-920.

Analysis method 2 The sample was analyzed under the following conditions using a JMS-DX303 type quality analyzer manufactured by JEOL Ltd.

Measurement conditions Ionization method: FAB method Impact gas: Xe Primary ion acceleration voltage: 6kV Filament current: 20mA Detector: Conversion Dynode Extrusion voltage: 15kv Matrix: Triethanolamine (sodium chloride added for positive ion) Data processing: JMA- DA5000 Further, with respect to a sample which has a large amount of impurities and cannot be subjected to mass spectrometry as it is, the sample was developed by TLC in the same manner as in the analysis method 1, and the extracted portion by scraping off the corresponding spot was analyzed.

Reference Example 1 Total lipids were extracted from yeast, and phospholipids were concentrated by acetone fractionation.

1 kg of commercially available baker's yeast was mixed with chloroform-methanol (1:
1) Extraction was performed 3 times in total of 4, 2, 1. The extracts were collected, 0.2 M aqueous potassium chloride solution was added to separate the layers, and the chloroform layer was dried under reduced pressure to give 16.8 g of total lipid.

This was dispersed in 500 ml of cold acetone, stirred at 5 ° C. for 30 minutes, and the resulting precipitate was collected by centrifugation. Do the same
This was repeated twice with 200 ml and 100 ml of acetone, and 2.7 g of the obtained precipitate was used as a crude yeast phospholipid.

Its composition was 28% neutral lipid, 34% phosphatidylinositol, 18% phosphatidyl acid, 9% phosphatidylcholine and 8% phosphatidylethanolamine.

Reference Example 2 M. Lees' method from cow brain [in "Methods in Enxymology" (S.
P. Colowick and NOKaplaned.), Vo1.3, pp328, Achademi
C Press, New York (1957)] was used to extract crude cephalin.

300 g of fresh bovine brain meninges, devascularized, obtained at a local slaughterhouse were homogenized in 1.2 acetone and extracted. The filtration residue was extracted once again with 1.2 acetone and the filtration residue was extracted with 1.2 ethanol. Further, the filtration residue was extracted twice with 1.2 petroleum ether in the same manner, and the extracts were collected and dried under reduced pressure to obtain 3.9 g of a crude sephaline fraction.

The composition of this product is phosphatidyl ethanoalamine 31.
%, Phosphatidylcholine 23%, phosphatidylinositol 14%, sphingomyelin 12%, phosphatidylserine 11%, and phosphatidic acid 7%.

Example 1 Commercially available defatted soybean lecithin (neutral lipid 6%, phosphatidylcholine 26%, phosphatidylethanolamine 19%, phosphatidic acid 16%, phosphatidylinositol 15)
%) 5 g dissolved in 500 ml benzene, methanesulfonic acid
0.32 g and 3.5 ml of ethyl vinyl ether were added, and the mixture was stirred at room temperature overnight. The reaction mixture was washed with 5% NaHCO ₃ aqueous solution (500 ml), dried over Na ₂ SO ₄ , and concentrated under reduced pressure.

The obtained reaction product was dissolved in 300 ml of chloroform, and 35 ml of a 1M NaOH methanol solution was added dropwise with stirring at 30 ° C. to carry out alkali hydrolysis to deacylate. Methanol and water were added to the reaction mixture, and the method of Folch et al. [J. Folch, et.al., J. Biol.
Chem. 226 , 497 (1957)]. The chloroform layer was removed, dehydrated with Na ₂ SO ₄ , dried under reduced pressure and dissolved in about 5 ml of n-hexane.

Unisil column made by Clarkson Chemicals Co. (diameter 2.2 cm,
It was filled to a height of 20 cm) and pretreated with n-hexane. The above reaction product was spread, and n-hexane-chloroform (1:
1) After washing the column with 150 ml, 300 ml of n-hexane-methanol (1: 9) was poured to collect 100 ml of the latter half. Further, 150 ml of methanol was poured to collect 100 ml of the latter half.

The n-hexane-methanol (1: 9) elution fraction and the methanol elution fraction were each dehydrated with Na ₂ SO ₄ , dried under reduced pressure, and subjected to mass spectrometry to find that n-hexane-methanol (1: 9). It was presumed that the ethoxyethyl group was introduced into glycerylphosphorylinositol in the elution fraction, and the presumed ethoxyethyl group was introduced into glycerylphosphorylethanolamine in the methanol elution fraction.

To the n-hexane-methanol (1: 9) fraction eluate, linoleic acid was made to act as an anhydride in the presence of dimethylaminopyridine using dicyclohexylcarbodiimide as a catalyst, and introduced. The reaction mixture was evaporated to dryness under reduced pressure, dissolved in chloroform-methanol-acetic acid (4: 2: 1) and stirred,
An appropriate amount of water was added to separate the layers, and the chloroform layer was collected and dried under reduced pressure. TLC analysis of the reaction product from which the protecting group had been removed revealed spots where the Rf values coincided with those of phosphatidylinositol. TLC separation and mass spectrometry confirmed that it was dilinoleylphosphatidylinositol.

When the eluate of the methanol fraction was treated with myristic acid chloride and the protective group was eliminated in the same manner, analysis was conducted, and production of dimyristoylphosphatidylethanolamine was observed. Purification by silica gel column chromatography gave 324 mg of dimyristoylphosphatidylethanolamine.

Example 2 2.5 g of yeast crude phospholipid obtained in Reference Example 1 was dissolved in 250 ml of benzene-dimethyl sulfoxide (1: 1), and a carbanion solution of sodium hydride-dimethyl sulfoxide was prepared in advance under a nitrogen stream. (Sodium hydride 1.
65 g of 2 g in 72 ml of dimethyl sulfoxide under nitrogen pressure flow.
62.5 ml of a dark green solution obtained by stirring at 70 ° C. was added dropwise with stirring. After stirring for 3 hours, cool with benzyl chloride 20
ml was added dropwise, and the mixture was further stirred for 6 hours. It was extracted with 450 ml of benzene, washed 3 times with water of 1 and concentrated under reduced pressure.

It was deacylated and washed with water in the same manner as in Example 1. The chloroform layer was dried, dried under reduced pressure, and dissolved in 3 ml of benzene.

Column packed with Merck Silica Gel 60 and pretreated with isooctane-benzene (9: 1) (diameter 2 cm, height 18 c
The test fee was spread to m). Column is isooctane
After washing with 200 ml of acetone (3: 2), it was eluted with 200 ml of isooctane-acetone (2: 8). The isooctane-acetone (2: 8) fraction was concentrated to obtain 205 mg of glyceryl phosphoryl inositol benzyl compound.

This is dissolved in 200 ml of dichloromethane and 0.1 ml of pyridine,
Add 20 ml of 2% myristate chloride chloroform solution,
The mixture was stirred at room temperature for 5 hours, washed with 200 ml of water three times, and concentrated under reduced pressure. The reaction product dissolved in 10 ml of chloroform was spread on a silica gel column (diameter 1.5 cm, height 20 cm) pretreated with chloroform-methanol-water (65: 25: 4) and eluted with the same solvent to remove unreacted products. Removed. Fractions containing the desired reaction product were collected and dried under reduced pressure.

This was dissolved in 200 ml of benzene, 0.63 g of 10% palladium-charcoal was added, and the mixture was stirred at 42 ° C under a hydrogen stream. Palladium-charcoal was separated by filtration, concentrated under reduced pressure, and dissolved in 10 ml of chloroform. Chloroform-methanol-water (65: 25: 4) pre-treated silica gel column (diameter 1.5 cm, height 20 cm) was spread and eluted with the same solvent as yeast standard phosphatidylinositol and Rf value on TLC. Fractions from which the desired reaction product giving almost identical spots were eluted and dried under reduced pressure to obtain 219 mg of a solid substance.

It was confirmed by mass spectrometry that this was dimyristoylphosphatidylinositol.

Example 3 3.9 g of the crude bovine brain cephalin fraction obtained in Reference Example 2 was dissolved in 380 ml of chloroform, 1 g of p-toluenesulfonic acid and 4.5 ml of isopropyl ketone were added, and the mixture was stirred for 353 hours. The product was washed in the same manner as in Example 1, and once concentrated under reduced pressure, it was deacylated as a chloroform solution.

The sample was spread on a Jatro Inatiabeads 6RS-80100 column (diameter 2.2 cm, height 33 cm) pretreated with chloroform, and the column was washed with 240 ml of chloroform-menolol (2: 1), followed by chloroform-methanol. (3: 2)
130 ml, chloroform-methanol (3: 7) 200 ml, and chloroform-methanol-water (65: 25: 4) 300 ml were sequentially eluted.

Chloroform-methanol (3: 2) fraction with isopropylidene group introduced into glycerylphosphorylinositol, chloroform-methanol (3: 7) fraction with isopropylidene group introduced into glycerylphosphorylserine But chloroform-methanol-water
In the (65: 25: 4) fraction, glycerylphosphorylethanolamine with isopropylidene group introduced was eluted.

Chloroform-methanol-water (65:25) was used to cause caprylic acid to act in the chloroform-methanol (3: 2) elution fraction and isostearic acid to act in the chloroform-methanol (3: 7) elution fraction, respectively. : 4) Arachidonic acid was introduced as the acid anhydride into the eluted fraction.

The isopropylidene group was eliminated and the product was purified by silica gel column chromatography to obtain 97 mg of dioctanoylphosphatidylinositol, 353 mg of diisostearoylphosphatidylethanolamine, and 112 mg of diarachidoylphosphatidylserine.

Example 4 Commercial egg yolk lecithin (phosphatidylcholine 64%, phosphatidylethanolamine 19%, phosphatidic acid 7)
%, Sphingomyelin 5%, lysophosphatidylcholine 3%) 10 g were dissolved in carbon tetrachloride 500 ml, and p-toluenesulfonic acid 0.75 g and 3,4-dihydro-α-pyran 5.2 g
Was added, and the mixture was stirred at room temperature for 4 hours. After washing in the same manner as in Example 1, the solution was concentrated under reduced pressure and deacylated by alkaline hydrolysis in chloroform. The alkali was neutralized with ethyl formate, dried under reduced pressure to dissolve it in 50 ml of diisopropyl ether, and a sample was prepared.

200 Wako Gel C300 manufactured by Wako Pure Chemical Industries, Ltd. pretreated with diisopropyl ether-acetonitrile (8: 2)
g The above sample was added to a packed column and processed in batch mode. The gel was washed 3 times with the same solvent 2, then extracted 3 times with diisopropyl ether-acetonitrile (2: 8) 2 and 5 times with 10% hydrous methanol 2.

TLC analysis revealed that the diisopropyl ether-acetonitrile (2: 8) fraction was eluted with a tetrahydropyranyl group introduced into glycerylphosphorylethanolamine, and the 10% hydrous methanol fraction was eluted with glycerylphosphorylcholine. It was

Phytanic acid was introduced as a fatty acid chloride into the elution fraction of diisopropyl ether-acetonitrile (2: 8) to remove the tetrahydropyranyl group and purified by column chromatography to obtain diphytanoylphosphatidylethanolamine 1.08. got g.

Palmitic acid was introduced as a fatty acid chloride into the 10% hydrous methanol elution fraction, and purified by column chromatography to obtain dipalmitoylphosphatidylcholine.
4.15 g was obtained.

Claims (6)

[Claims] 1. A crude phospholipid having a removable protective group introduced therein,
Following deacylation, the following formula (I) (In the formula, X represents a nitrogen-containing alcohol residue or a polyol residue, Y represents an easily removable protecting group, and n represents a natural number of 12 or less.), And a glyceryl phosphate derivative represented by
A method for producing a phospholipid, which comprises separating and purifying silicic acid as a separating agent, and then introducing an arbitrary acyl group to eliminate the protecting group. 2. X is serine group, ethanolamine group, N-
Methylethanolamine group, N, N-dimethylethanolamine group, N, N, N-trimethylethanolamine group, glycerol group, aminoacylglycerol group, glycerophosphate group, glycerylphosphatidyl group, inositol group,
The production method according to claim 1, which comprises at least one selected from an inositol phosphate group, an inositol diphosphate group, a monosaccharide, a disaccharide and an oligosaccharide. 3. The method according to claim 1 or 2, wherein Y is a linear, branched or cyclic alkyl group, an alkyloyl group, an alkenyl group, an alkeroyl group or an aryl group. 4. The production according to any one of claims 1 to 3, wherein the acyl group to be introduced is a linear, branched or cyclic saturated or unsaturated fatty acid having 30 or less carbon atoms. Method. 5. Silicic acid as a separating agent has a particle size of 10 to 800 μm.
The manufacturing method according to any one of claims 1 to 4, wherein m and pore diameter are 0.25 to 10 µm. 6. A glyceryl phosphate derivative is adsorbed on a separating agent in the presence of a solvent of low polarity, the separating agent is washed, and then the glyceryl phosphate derivative is eluted with a solvent of high polarity. Item 6. The manufacturing method according to any one of Items 5 to 5.