JPH02101086A - Production of glycero-sn-3-phosphocholine powder - Google Patents

Abstract

PURPOSE:To obtain a large amount of the subject inexpensive safe powder, having a natural structure and useful as a raw material for phosphatidyl choline by deacylating a natural phospholipid with metallic sodium in an alcohol and then powdering the resultant product by a specific method. CONSTITUTION:A natural phospholipid is initially deacylated with metallic sodium or a sodium alcoholate in an alcohol, preferably at ambient temperature for >=3hr. The resultant deacylated product is then acidified with hydrochloric acid in methanol, then added into acetone and crystallized at -30 to -10 deg.C. The crystallized deacylated product is subsequently filtered and subsequently powdered in methanol by adding silica to afford the objective substance. Furthermore, the composition of the natural phospholipid which is the raw material is preferably >=90% phosphatidyl chlorine content and the rest is preferably lysophosphatidyl choline.

Description

DETAILED DESCRIPTION OF THE INVENTION (Industrial Application Field) The present invention relates to a method for producing glycero-3n-3-phosphorylcholine powder that maintains its natural structure directly from natural phospholipids.

(Prior art) In order to synthesize phosphatidylcholine, that is, 1,2-diacyl-3n-glycero-3-phosphorylcholine, which has the stereospecificity of natural phospholipids, glycero-5n-3-phosphorylcholine that maintains this natural structure is required. It is. This is because natural phospholipids exhibit optical activity because they have an asymmetric carbon at the second position of the glycerol skeleton. Therefore, when synthesizing an optical antipode with the same coordination as the natural one by conventional organic synthesis methods, usually substituted glycerol with the corresponding stereochemistry, such as 1,2-0-isopropylidene-D-glycerol, is D- A method is used in which mannitol is synthesized as a starting material (M, B).

Jung, T.J., Shaw, J., Am., C.
hew, Soc, 102+6304 (1980
)). The phospholipids synthesized from these starting materials are propyl-2-trimethylammonioethyl phosphate derivatives.

In addition, as a means of obtaining glycero-3n-3-phosphorylcholine that maintains its natural structure, in addition to the above-mentioned total synthesis method, in natural phospholipids, only the ester bond that connects glycero-3n-3-phosphorylcholine and fatty acid can be used. A method for specifically hydrolyzing is also known. This hydrolysis method includes an enzyme method and a chemical method. In the enzymatic method, lipid ester-degrading enzymes such as phospholipase and lipase are used.

Glycero 5n-3- with natural structure by chemical method
The following reports are known as methods for obtaining phosphorylcholine.

■H, Brockerhoff, M, York
ski, Can, J, Bi.

chem, 43.1777 (1965) ■J, S
, Chadha, Chem, Phys, Lip
ids, al 104■ Experimental Chemistry Course, Volume 3, “
Lipid Chemistry” 7.1, Diacylglycerophospholipids, Part 2
Page 61, Tokyo Kagaku Dojinsha The above methods commonly involve hydrolyzing phosphatidylcholine with tetrabutylammonium hydroxide in anhydrous ether, filtering the resulting decomposition product, and then treating it with glycero-3-phosphorylcholine using a cadmium chloride solution. - It is precipitated in crystalline form as a cadmium chloride complex.

(Problems to be Solved by the Invention) The total synthesis method for producing a propyl 2-trimethylammonioethyl phosphate derivative using a glycerol derivative as a starting material has a large number of synthesis steps, and requires a purification process for the reaction product at each step. It is complex and its final product yield is low. In addition, phosphatidylcholine synthesized using this glycero-3n-3-phosphorylcholine as a raw material,
The yield is very low based on the fatty acids used. Therefore, the cost of the reaction product is extremely high, making it unsuitable as an industrial production method.

In addition, in the total synthesis method, there is a possibility that Walden inversion, in which the steric configuration is inverted to the opposite configuration, occurs during the reaction, and there is a risk that steric coordinates that do not have a natural structure will be mixed.

Means for specifically hydrolyzing the ester bond that binds glycero-3n-3-phosphorylcholine and fatty acid in natural phospholipids with enzymes include: (1) non-specific degradation by lipase, (2) phospholipase A2 and phospholipase A, or phospholipase. There is stepwise separation by A2 and lysophospholipase, and non-specific degradation by phospholipase B.

Enzymes that catalytically hydrolyze the ester bonds described above are purified from microbial, animal membrane-bound enzymes and animal secreted enzymes. However, these enzymes can only be isolated or purified from these raw materials by purification methods that combine multiple analytical techniques such as ammonium sulfate fractionation, DEAE-cellulose, and electrophoresis. The yields of these enzymes obtained by analytical methods from small amounts of raw materials are extremely low, and their enzymatic activity is also low, and their potency is often lost during storage. Therefore, even if these enzyme groups are used in biochemical experiments, they are unsuitable for industrial production of glycero-3n-3-phosphorylcholine.

Glycero-5n-, which has a natural structure, is produced by specifically hydrolyzing the ester bond that binds glycero-8n-3-phosphorylcholine of natural phospholipid and fatty acid with a chemical product.
In the method of producing 3-phosphorylcholine eight times, it is necessary to react expensive tetrabutylammonium hydroxide as a hydrolyzing agent in an anhydrous nonpolar solvent. A particularly problematic step in this method is the use of cadmium to recover the hydrolyzed glycero-3n3-phosphorylcholine and crystallize it as a complex.

Cadmium, which is used to crystallize glycero-8n-3-phosphorylcholine, which has remarkable water absorption, is a heavy metal that accumulates in living organisms and causes significant toxicity. The purpose of use of various phosphatidylcholines prepared using glycero-3n3-phosphorylcholine, which has a natural structure, is as pharmaceuticals and pharmaceutical raw materials. Residual cadmium in raw materials is a serious problem. Furthermore, the method for synthesizing phosphatidylcholine using a glycero-8n-3-phosphorylcholine-cadmium chloride complex has a drawback that the yield is as low as about 50%.

As described above, in order to synthesize a large amount of phospholipids having a natural structure, it is necessary to secure a large amount of glycero-3n3-phosphoborylcholine having a natural structure at low cost. There is a desire to develop a method for producing glycero-3n-3-phosphorylcholine that satisfies the requirements of using a raw material that is safe for its intended use and that also provides a good reaction yield during the synthesis of phosphatidylcholine.

Therefore, an object of the present invention is to establish a method for producing a large amount of glycero-3n-3-phosphorylcholine having a natural structure that is inexpensive and safe.

(Means for Solving the Problems) The method for producing glycero-3n-3-phosphorylcholine powder of the present invention includes deacylating natural phospholipids with metallic sodium or sodium alcoholate in alcohol, and then
The deacylated product was acidified with hydrochloric acid in methanol, then added to acetone, crystallized and filtered at -10 to -30°C, and the crystallized deacylated product was powdered in methanol by adding silica. It is characterized by

The composition of the natural phospholipid used in the present invention is preferably such that the phosphatidylcholine content is 90% or more, with the remainder being lysophosphatidylcholine. This is because glycero-8n-3-
This is because phosphorylcholine is produced.

When natural phospholipid raw materials contain abundant phosphatidylcholine, they often coexist with sphingomyelin, which has a phosphorylcholine skeleton similar to phosphatidylcholine and lysophosphatidylcholine. Therefore, following the enrichment of phosphatidylcholine, these substances are concomitantly enriched.

However, since sphingomyelin retains aliphatic substances in the form of sphingosine residues and fatty acids bonded with acid amide, sphingomyelin is stable against the alkali treatment employed in the present invention, and is soluble in chloroform. Easily removed by chloroform treatment during deacylation. Therefore, there is no problem with the presence of sphingomyelin in the natural phospholipid raw material of the present invention, so a wide range of raw materials can be used.

However, general phospholipids that do not have a phosphorylcholine skeleton undergo hydrolysis under the deacylation conditions of the present invention, producing corresponding deacylated products, which lowers the purity of glycero-3n-3-phosphorylcholine, which is the purpose of production. Therefore, it is preferable to remove this as much as possible from the natural phospholipid raw material in the present invention.

The natural phospholipids used in the present invention are made from natural lecithins such as peanuts, soybeans, and egg yolks, and are subjected to degreasing treatment with cold acetone, solvent fractionation treatment with aqueous ethanol, complex treatment with addition of divalent metal salts, and silica column treatment. It can be obtained by combining several methods such as chromatography treatment using continuous centrifugation, liquid-liquid multistage distribution, etc.

The deacylation method using an alkaline transesterification method used in the present invention is suitable for producing glycero-5n-3-phosphorylcholine. Reaction conditions of 3 hours or more at room temperature are preferred;
The natural structure of the resulting glycero-5-phosphorylcholine is not changed.

Although deacylation is also possible by acidic transesterification using mineral acids such as sulfuric acid and hydrochloric acid, the resulting glycero-3n-3
- Concerning phosphorylcholine, a conformational change is observed in which the phosphate group of 5n-3 choline phosphate and the hydroxyl group of 5n-2 appear to be condensed within the molecule. In addition, although the alkaline catalyst is easily neutralized with acid after the reaction is completed, this reaction does not change the steric structure of glycero-5n-3-phosphorylcholine, and the resulting salt is also similar to glycero-3n-3. - It does not have any effect on the three-dimensional structure of phosphorylcholine or the reaction during synthesis of phosphatidylcholine.

The transesterification method using sodium metal and sodium alcoholate used in the present invention provides favorable conditions for separating the target product of the reaction, glycero-3n-3-phosphorylcholine, from the transesterified fatty acid derivative.

Glycero-3n-3-phosphorylcholine, which is the target of the reaction, has a hydroxyl group and a phosphoric acid group, so it is a water-soluble compound and is soluble in methanol, which is a polar solvent, but is insoluble in acetone and chloroform. Naturally, it is insoluble in nonpolar solvents. On the other hand, transesterified fatty acid derivatives are soluble in methanol, but since they are alcohol esters, they are soluble in acetone and chloroform.

That is, when a natural phospholipid is transesterified in methanol, the reaction proceeds homogeneously because the starting materials and any reaction products are soluble in methanol. By adding chloroform to the homogeneous solution after the reaction, fatty acid derivatives, unreacted phospholipid raw materials, and lipid impurities derived from the raw materials migrate into the chloroform, but the target glycero-3n3-phosphorylcholine does not migrate. Therefore, this chloroform treatment is suitable for removing reactants.

Next, can the target product of the reaction, glycero-5-phosphorylcholine, be converted into methanol? glycero-3n-
After preparing a methanol solution of 3-phosphorylcholine with hydrochloric acid, acetone is added.

As a result, methanol loses its ability to dissolve glycero-5n-3-phosphorylcholine due to the dissolved acetone,
Glycero-3n-3-phosphorylcholine is released out of solution. Since glycero-5-phosphorylcholine is insoluble in acetone, it precipitates as crystals.

In solvent fractionation treatment using chloroform or acetone,
In order to more efficiently crystallize glycero-3n-3-phosphorylcholine from methanol, -15°C to -30°C
It is preferable to hold the temperature for at least several hours, preferably at least 2 hours. Also, glycero 5n-3 with acetone
- Washing and crystallization of the phosphorylcholine crystals are desirably repeated four or more times, and preferably at least two times or more.

In the present invention, the metal sodium and sodium alcoholate such as sodium methylate and sodium ethylate used as the alkaline catalyst are used in a maximum amount of 2% by weight, preferably 0% by weight, based on the raw natural phospholipid.
．． Add 5% by weight.

In order to use the catalyst efficiently, the natural phospholipid is dissolved in methanol under stirring for 0.5 to 2 hours prior to the addition of the catalyst. A catalyst is added to a methanol solution of this natural phospholipid and allowed to react at room temperature for 3 hours or more. The progress of the reaction is terminated when phosphatidylcholine is no longer detected using thin layer chromatography. The added catalyst is hydrochloric acid, preferably 16 to 2
Neutralize to P)14.0-6.0 with 0% hydrochloric acid to inactivate. At this time, the phosphorylcholine salt of glycero-3n-3-phosphorylcholine having a quaternary amine is converted into a hydrochloride and stabilized as a quaternary amine salt. This hydrochloride has no effect on the reaction during phosphatidylcholine synthesis.

In the case of triazylglycerol, transesterification is possible by reaction with a small amount of alcohol at room temperature for 12 hours or more using an alkali metal salt such as sodium hydroxide or potassium hydroxide as a catalyst, but natural phospholipids can be transesterified using a catalyst. Even if the amount or reaction time is increased, the reaction rate is low and is unsuitable for the purpose of the present invention.

In addition, the method of deacylating natural phospholipid raw materials by saponification and decomposition at high temperature using an alkali metal salt as described above is as follows.
The solubility of the resulting fatty acid salt in water, alcohol, and acetone is close to that of glycero-3n3-phosphorylcholine, and the solubility in the deacylation solvent chloroform is insufficient, so the reaction product glycero-3n
-3-phosphorylcholine and fatty acid salt cannot be immediately separated.

Furthermore, in the saponification decomposition method, an alkali metal salt is added to the alkali metal salt in a number of moles corresponding to the number of moles of the natural phospholipid raw material, with an excess reaction rate added thereto. Therefore, a large amount of alkali metal salt remains in the reaction system. That is, during the neutralization of excess alkali at the end of the reaction, and furthermore, when removing fatty acids after conversion to fatty acids by neutralizing fatty acid salts, glycero-5n-3
- A large amount of salts such as potassium chloride and sodium sulfate are present in phosphorylcholine. Glycero-3n-3-phosphorylcholine obtained under these conditions is difficult to crystallize;
It often has a yellow-brown coloration, and the reaction yield during phosphatidylcholine synthesis is low.

In contrast, glycero-3n-3- in the present invention
The yield of phosphorylcholine is quantitative. The theoretical yield of glycero-3n3-phosphorylcholine produced from phosphatidylcholine is 34.0%.

In the present invention, the crystallized deacylated product is powdered by adding silica in methanol. From 100 g of the natural phospholipid raw material used, which is composed of 95% phosphatidylcholine and 5% zophosphatidylcholine, 32 to 37 g of glycero-3n- A silica support corresponding to the amount of 3-phosphorylcholine is obtained. Glycero-5n-3- in silica support
The phosphorylcholine content is preferably at least 50%, and the upper limit is 80%, particularly 60 to 70%, in terms of dry weight; above this content, handling and storage become difficult, and below this content, the capacity of the carrier becomes large. , is unfavorable due to the reaction volume and stirring effect during phosphatidylcholine synthesis.

The silica used in the present invention has a purity of 99.8% or more of silicic anhydride, has a specific surface area of 110 ± 20 rrr/g, and is chemically coated with methyl groups to make the surface hydrophobic.
It is preferable that the average diameter is about 16 am. Silica is chemically inert and glycero-3n-3 in methanol.
- Contains a high concentration of phosphorylcholine, about 60% or more, and becomes a powder. At this time, when silica is added in a small amount, it improves the fluidity to reduce the bulk volume, but if the amount added is increased to below the above concentration, the bulk volume increases significantly, which is not preferable.

Furthermore, by adding silica, it covers the surface of glycero-3n-3-phosphorylcholine, preventing caking and improving the fluidity of the powder.

Glycero-3n-3-phosphorylcholine obtained in the present invention has an Rf value and a color reaction by thin layer chromatography.
Molecular weight measurement by GC-MS, spectrum analysis by NMR, choline content by Reinecke salt colorimetric method, phosphorus content by molybdovanadophosphate colorimetric method, nitrogen content by Kelpur method, etc., show that the product maintains its natural structure with high purity. It was confirmed that there is.

The silica-supported glycero-3n-3-phosphorylcholine obtained in the present invention is very easy to handle as a raw material for the synthesis of phosphatidylcholine.

Using the glycero-5n-3-phosphorylcholine powder obtained in the present invention, a method of reacting with an acid chloride in the presence of hexamethylphosphoric acid triamide, and a method of reacting the powder with an acid anhydride in the presence of a basic esterification catalyst and chloroform are possible. Phosphatidylcholine can be preferably synthesized by a method of reacting with a compound. In particular, the reaction in the presence of hexamethylphosphoric triamide can be carried out in a short time and in high yield with a minimum amount of acid chloride.

In addition, silica is completely removed during phosphatidylcholine synthesis by filtration of the reaction solution with a distilled chloroform solution and centrifugation, and there is no problem as a raw material for the phosphatidylcholine reaction.

(Effects of the Invention) According to the present invention, glycero-5n-3-phosphorylcholine, which is made from natural phospholipid raw materials and is solid at room temperature and has a natural three-dimensional structure that is easy to handle, can be used as a particularly expensive drug.
It can be produced in large quantities at low cost and by a simple method without using enzymes or highly toxic cadmium salts.

As a result, phosphatidylcholine containing no harmful heavy metals can be produced in high yield, and the produced phosphatidylcholine has a natural three-dimensional structure. In addition, since the silica support is powdered, it has fluidity and is easy to handle, which is advantageous in terms of workability in the synthesis of phosphatidylcholine.

(Example) Hereinafter, the present invention will be specifically explained based on Examples.

Example 1 Commercially available soybean phosphatidylcholine (phosphatidylcholine 94%,
100 g (containing 3925% lysophosphatidyl) was weighed out and 0.511 methanol was added.

A magnetic cooler was placed in the flask, and the phosphatidylcholine was dissolved while stirring for 1 hour.
Sodium methylate (Wako Pure Chemical Industries, Ltd., 2
8% sodium methylate/methyl alcohol solution 1
, 8 +d) were added. The reaction mixture was stirred at room temperature for 3 hours.

Completion of the reaction was confirmed by thin layer chromatography. The developing solvent was 65/25/chloroform/methanol/water.
4 (vol/vol/vol) composition liquid was used, and coloring was performed by sulfuric acid coloring. The process was completed when the phosphatidylcholine spot with an Rf value of 0.3 disappeared on thin layer chromatography, and only the fatty acid methyl ester spot and the deacylated product spot at the origin were observed on the solvent front.

The reaction mixture was evaporated under reduced pressure in a 70°C water bath to remove the solvent, and then 0.57! of chloroform was added all at once to dissolve impurities in the raw materials and the reaction product fatty acid methyl ester. The flask was cooled to -25°C for 2 hours. The chloroform layer was removed by decantation, the deacylated residue was dissolved in 100 mp methanol, and the pl+ was adjusted to 4.0 to 6.0 with several drops of 20% hydrochloric acid. And this methanol solution II! The mixture was added dropwise to 0.5β acetone in a flask with stirring. This flask is 125
After cooling for 3 hours at <RTIgt;C,</RTI> the acetone layer was removed by decantation.

Dissolve this residue in 300 mff of methanol and
8 liquid, 20g of silica (manufactured by Degnosa, trade name Ae)
rosil R972) was added and the solvent was removed on a rotary evaporator in a 50°C water bath. The obtained solid was ground in a mortar and dried under highly reduced pressure with phosphorus pentoxide in a desiccator until the weight became constant. 55 g of white powder of silica-adsorbed glycero-5n-3-phosphorylcholine - Phosphorylcholine content: 35 g) was obtained. The yield was 93%.

The analytical values of this product were as follows.

Thin layer chromatography, mobile phase: methanol/water (1/1) Rf value = 0.4 once bottled Dragendorff reagent coloring, orange phosphorus reagent coloring:
Blue GC-MS FAB (positive dip) matrix
Triethanolamine m/e CM + H)” 258, (M+Na
) ” 28ONMR, white powder dissolved in D 20,
The solution was centrifuged to remove insoluble silica.

3.1 ppm (s, N”(CR3)3)3
．． 2-4.3 ppm (m, glycerin-H and choline-H) Choline content, Reinesoke salt colorimetric method 47.1% (theoretical value 41.3%) Phosphorus content, molybdovanadophosphate colorimetric method 11. 9% (theoretical value 12.3%) Nitrogen content, Kelpur method 5.27% (theoretical value 5.54%) Example 2 Commercially available soybean phosphatidylcholine (phosphatidylcholine 94%) was added to a 27! flask equipped with a calcium chloride tube.
, containing 5% lysophosphatidylcholine) was weighed out and 0.57! of methanol was added.

Put a magnetic cooler into the flask and dissolve the phosphatidylcholine while stirring for 1 hour.
．． Rod-shaped sodium (manufactured by Kanto Kagaku, Protective Liquid Paraffin) corresponding to 1.0 g was added. The reaction mixture was stirred at room temperature for 3 hours.

Completion of the reaction was confirmed by thin layer chromatography. The developing solvent was 65/25/chloroform/methanol/water.
4 (vol/vol/vol) composition liquid was used, and coloring was performed by sulfuric acid coloring. Rf on thin layer chromatography
(!) The process was completed when the phosphatidylcholine spot of 0.35 disappeared and only the fatty acid methyl ester spot and the deacylated product spot at the origin were observed on the solvent front.

The reaction mixture was evaporated under reduced pressure in a 70°C water bath to remove the solvent, and then 0.57! of chloroform was added all at once to dissolve impurities in the raw materials and the reaction product fatty acid methyl ester. The flask was cooled to -20°C for 2 hours. The chloroform layer was removed by decantation, and the deacylated residue was dissolved in 100 ml of methanol, and the pH was adjusted to 4.0 to 6.0 with several drops of 20% hydrochloric acid. Then, this methanol solution was placed in a 0.57!II flask. was added dropwise to acetone with stirring. This flask is heated to -20℃.
After cooling for 3 hours, the acetone layer was removed by decantation.

This residue was dissolved in 300+++# methanol, and 20 g of silica (manufactured by Degusosa, trade name ^e) was added to this solution.
rosil R972) was added and the solvent was removed on a rotary evaporator in a 50°C water bath. The obtained solid was ground in a mortar and dried under high vacuum with phosphorus pentoxide in a desiccator until the weight became constant. 52 g of white powder of silica-adsorbed glycero-3n'-3-phosphorylcholine 5n3-phosphorylcholine content: 33 g) was obtained. The yield was 88%.

The analytical values of this product were the same as in Example 1.

The effectiveness of silica-adsorbed glycero-5n-3-phosphorylcholine as a raw material for phosphatidylcholine synthesis is shown in the following reference example.

Reference example 1 5n of silica-adsorbed glycero in a 29ml eggplant-shaped flask
-3-phosphorylcholine 1613mg (glycero-5
Contains 1g of n3-phosphorylcholine: 3.9 mmol)
and suspended by adding chloroform IQml.

After stirring this suspension for 30 minutes with a magnetic stirrer, 4.0 g (9.1 mmol) of myristic acid anhydride and 1.11 g (9.1 mmol) of dimethylaminopyridine were added, and the mixture was stirred at room temperature for 5 hours. . After the reaction is complete,
The mixture was poured into dry acetone 300++1 and the precipitate formed was isolated by centrifugation. The precipitate was dissolved in chloroform, the silica was filtered off, and the solvent was distilled off under reduced pressure. 2.0 g of 1,2-simyristoylphosphatidylcholine was obtained, with a yield of 75.9%.

As a result of analyzing this substance by FAB-MS, m/e 678
(M + H)+ was confirmed.

Reference example 2 Silica-adsorbed glycero 5n-3 in a 20- eggplant-shaped flask
-Phosphorylcholine 1613mg (glycero-3n3
-phosphorylcholine (containing 1 g; 3.9 mmol) was added, and hexamethylphosphoric acid triamide 10#+2 was added and suspended. After stirring this suspension in a magnetic cooler for 30 minutes, 4.6 g of rill oil chloride was added.
(15.3 mmol) was added and stirred on an oil bath at 60°C for 3 hours. After the reaction was completed, the mixture was poured into 150 bottles of ice water and extracted with hexane. After drying the hexane layer with sodium sulfate, the residue was packed with silica gel.
Place it on a φX 50cm0 column and use a chloroform-methanol gradient? When separated, 2.31 g of 1
．． There was 2-silyloylphosphatidylcholine. As a result of analyzing this substance by FAB-MS, m/e
782 (M + H)+ is confirmed.

[Brief explanation of the drawing]

FIG. 1 shows glycero Sn-3- obtained in Examples.
This is a diagram showing the results of analysis of phosphorylcholine using the positive method of First Atom Bombard Ionization Mass Chromatography.
80) is recognized.

Claims (1)

[Claims] After deacylating natural phospholipids with sodium metal or sodium alcoholate in alcohol, the deacylated product is acidified with hydrochloric acid in methanol, and then added to acetone and crystallized and filtered at -10 to -30 °C. A method for producing glycero-Sn-3-phosphorylcholine powder, which further comprises powdering the crystallized deacylated product by adding silica in methanol.