JP2792091B2 - Method for producing 1-acyl-lysophosphatidylcholine - Google Patents

Description

The present invention relates to a method for producing 1-acyl-lysophosphatidylcholine by regiospecific deacylation using monoacid diacylphosphatidylcholine obtained by chemical synthesis as a raw material. About.

(Prior Art) Phosphatidylcholine in which arachidonic acid or eicosapentaenoic acid is bonded to the Sn-2 position of 1-acyl-lysophosphatidylcholine serves as a storage of eicosanoid precursors in a living body. It is an important substance in the pharmaceutical field.

Although 1-acyl-lysophosphatidylcholine is present in a small amount in nature, it has been conventionally prepared by the following method.

M. Kates: Lipid Research Methods, Biochemical Experimental Methods 5, 255
Page, Tokyo Chemical Dojinsha, 1975, Tokyo Harumi Okuyama, Shigeo Murase: Protein Nucleic Acid Enzyme, 32 , 267, 1987 G.H. Dehas et al .: Biochim. Biophys. Acta, 159 ,
105,1968 Enzymatic degradation method of phospholipids (JP-A-63-44893) Production method of lysolecithin (JP-A-63-225388) However, among the above-mentioned conventional preparation methods, the method of It is hydrolyzed using small and very expensive snake venom. In addition, unreacted phosphatidylcholine is observed. An improved method has been proposed in which several hundred mg of yolk phosphatidylcholine is treated with 10 mg of hub venom, but the problem of removing unreacted phosphatidylcholine in the reaction product has not been solved. The method, throughput is as small as less 1 mg, yet be used phospholipase A ₂ snake venom origin are more expensive 1-
Acyl-lysophosphatidylcholine collection is limited to 85%. Method, the egg yolk was treated with a small amount of about 1mg as a substrate, similar to the above, using phospholipase A ₂ originating expensive porcine pancreas. Method, a mixture of phospholipids or phospholipid and oil as a substrate, but is made to convert 80-90% into lysophospholipids using phospholipase A ₂ originating expensive pig pancreas, phosphatidylcholine own conversion And the stereospecificity of the lysophosphatidylcholine produced are unknown. Is a method for producing lysophosphatidylcholine by chemical synthesis, but it has a problem that a catalyst and a high boiling point solvent which are difficult to remove from the reaction product coexist.

(Problems to be Solved by the Invention) In the above-mentioned method, the amount that can be treated is small, and it is necessary to use an expensive hydrolase that is difficult to obtain, and it is difficult to obtain a pure reaction product and mass production is not possible. There was a drawback.

Therefore, at present, there is a need for a method for producing 1-acyl-lysophosphatidylcholine that can be mass-produced industrially by using an easily available and inexpensive enzyme of natural origin and that allows easy separation of the reaction product. .

The present invention hydrolyzes a monoacid diacylphosphatidylcholine obtained by chemical synthesis by using an inexpensive enzyme of natural origin having regiospecificity to produce a large amount of pure 1-acyl-lysophosphatidylcholine industrially. It seeks to provide a new way of producing.

(Means for Solving the Problems) The present invention provides a method for producing a monoacid group diacylphosphatidylcholine obtained by chemically synthesizing in the presence of an esterification catalyst (pyridine derivative or tertiary amine), followed by treating with a neutral alumina column, followed by pancreatin A method for producing 1-acyl-lysophosphatidylcholine, characterized by hydrolyzing with use of

The enzyme used in the present invention is pancreatine, which is easily available and very inexpensive, and is subjected to a hydrolysis reaction using this enzyme to produce a monoacid group diacylphosphatidylcholine.
The deacylation at the Sn-2 position can be selectively advanced, and can be converted to 1-acyl-lysophosphatidylcholine at a high conversion rate.

Hereinafter, the method for producing 1-acyl-lysophosphatidylcholine of the present invention will be described in detail.

In the hydrolysis method of the present invention, after subjecting a chemically synthesized monoacid group diacylphosphatidylcholine to an alumina column treatment, one part thereof is dissolved in 50 to 500 parts of an organic solvent,
For example, 10 to 30 parts of a calcium chloride solution, 10 to 30 parts of a sodium borate solution, and 0.5 to 2.0 parts of pancreatin are added and reacted. As this pancreatin, one derived from swine pancreas having a high lipase content is desirable.

The pH during the reaction is preferably 5 to 9, and more preferably 6 to 9.
-8 are suitable, and the added sodium borate solution is
It is preferable to add 0.01 to 1 M calcium chloride solution prepared by adjusting calcium chloride to a concentration of 2 to 200 mM to 0.1 M Tris / HCl buffer. Reaction is 10 ~
The reaction is carried out at 80 ° C., preferably at 20 to 50 ° C., and it is preferable to carry out high-speed stirring during the reaction and to carry out the reaction for 3 to 24 hours.

The chemically synthesized monoacid group dialphosphatidylcholine used in the present invention is, for example, an anhydride or a halide of a specific fatty acid with respect to 1 mol of glycerophosphocholine.
5 to 6 mol of an aprotic highly polar solvent such as dimethyl sulfoxide, hexamethoxyphosphoramide in the presence of a pyridine derivative or an esterification catalyst selected from tertiary amines such as dimesylaminopyridine and triisopropylamine And the like. 1 obtained from these phosphatidylcholines
The fatty acid species of the acyl-lysophosphatidylcholine can be arbitrarily selected by selecting the molecular species of the anhydride or halide of the fatty acid used in this reaction. The fatty acid is preferably a fatty acid having 12 to 24 carbon atoms, and may be a saturated or unsaturated fatty acid.

In the present invention, an alumina column treatment is performed in order to improve the decomposition rate of chemically synthesized monoacid group diacylphosphatidylcholine as a raw material by pancreatin. Commercially available alumina includes drying, thin-layer chromatography, column chromatography, decomposition of yeast cells and microorganisms, and a special activated gel having a high adsorption capacity. Are preferred. Alumina for column chromatography includes acidic, basic, and neutral types of active gels.However, phospholipids generally show weak bonds to bases, and those subjected to acidic treatment are considered to cause chemical changes in solutes. Alumina is preferred. Since the activity of alumina changes depending on the water content and its retention capacity decreases, it is sufficiently dried before use to reduce the water content of alumina to less than 3% and adjusted to Brockmann activity I. It is more preferable to use after that.

Commercial products that can be used as alumina for column chromatography include alumina manufactured by Sigma Chemical Co., activity I (pfs) WN-3 type, neutral; aluminum oxide 90 manufactured by ICN Biomedicals, active type, and Properties, activity I; aluminum oxide 90 manufactured by E. Merck AG, active type, neutral, activity I; aluminum oxide manufactured by Aldrich Chemical Company, active type, neutral, activity I; Jansen・ Simika aluminum oxide, activated, neutral;
And the like.

The alumina column treatment is preferably performed as follows.
First, 20 to 50 parts by weight of neutral alumina per 1 weight of synthetic phosphatidylcholine is suspended in chloroform, filled in a column, and sufficiently conditioned with chloroform to remove fine alumina fragments to prepare an alumina column. Next, the synthetic phosphatidylcholine is eluted with chloroform as a chloroform solution and then with chloroform and then with a mixed solution of chloroform / methanol. By this method, phosphatidylcholine can be recovered in a very short time with a yield of 80 to 95%. When the thus obtained synthetic phosphatidylcholine treated with an alumina column and synthetic phosphatidylcholine not treated with alumina are hydrolyzed with a fixed amount of pancreatin, the yield of 1-acyl-lysophosphatidylcholine of the treated product is higher than that of the untreated product. The rate improved by about 20%.

Pancreatin used in the present invention can be, for example, of pancreatic origin and contains many enzymes such as amylase, trypsin, lipase, ribonuclease and protease. Commercially available pancreatin is classified according to its constituent enzymes, amylase, protease and lipase activities, measured according to the American Pharmaceutical Prescription List and the United States Pharmacopeia. The pancreatin used in the present invention is, in particular, a titer equivalent × 1, a titer equivalent × 3, a titer equivalent × 4, and a titer equivalent × 8 according to the results of measuring the activity of amylase in the United States Pharmacopeia. And those classified by class are preferred. In addition, pancreatin, which is a digestive enzyme preparation for pharmaceutical use, is not suitable because dextrin, lactose, sugar and the like are added to a diluent and the titer is lowered.

Commercial products that can be used as pancreatin include Sigma
Pancreatin derived from pig pancreas manufactured by Chemical Company (Activity 1 × USPspecification, Activity 3 × US
P.specification, Activity 4 × USPspecification, Ac
tivity 8 × USPspecification), pancreatin derived from pig pancreas (Activity 4 × JP) manufactured by Tokyo Kasei, pancreatin manufactured by Kanto Chemical, pancreatin for chemicals manufactured by Ishizu Pharmaceutical, pancreatin manufactured by Junsei Chemical, and the like.

According to the present invention, a reaction product obtained by hydrolyzing synthetic monoacid group diacylphosphatidylsylcholine with pancreatin can be easily separated into 1-acyl-lysophosphatidylcholine and fatty acids by operations such as decantation and acetone fractionation. I can do it.

The present invention (Effect of the Invention), without using very expensive phospholipase A _2, using a very inexpensive pancreatin industrially available, pure 1-acyl synthetic phosphatidylcholine - lysophosphatidylcholine a large amount Can be manufactured. Therefore, 1-acyl-lysophosphatidylcholine having various biological activities in vivo can be easily obtained. The 1-acyl-lysophosphatidylcholine obtained according to the present invention can be widely used in the field of pharmaceuticals and cosmetics as a raw material of mixed acid diacylphosphatidylcholine having a polyunsaturated fatty acid incorporated at the Sn-2 position.

(Examples) Hereinafter, the present invention will be specifically described based on examples.
In each case,% is based on weight.

Reference Example 1 Alumina column treatment Neutral activated aluminum oxide for column chromatography (activity I, 70 to 29) manufactured by ICN Biomedicals
0 mesh ASTM, particle size 0.05-0.02mm, pH 7.5, flow density 0.
9 g / ml, the density 3.97, pore diameter 58 Å, surface area 155m ² / g) 20g
Was suspended in chloroform in a 2φ × 7 cm glass column and filled (filling volume: 22 cm ³ , filling height: 7 cm).
1 g of synthetic phosphatidylcholine passed through 300 ml of a mixed solution of 0 ml and chloroform / methanol (3/1) was applied to an alumina column as a 3 ml chloroform solution.
l, then chloroform / methanol (3 / 1-5 / 1, v / v)
Elute phosphatidylcholine by passing 200 ml of the mixture,
The eluate was distilled off to obtain phosphatidylcholine treated with an alumina column.

Reference Example 2 Confirmation of Regiospecificity of Pancreatin Sn-1-oleoyl-2-docosahexaenoylphosphatidylcholine of chemically synthesized product treated with alumina column
1.2 g was dissolved in 150 ml of a mixed solution of ether / ethanol (95/5), and 1 g of pancreatin derived from swine pancreas (Pharmacopoeia of the United States, titer equivalent × 1, Sigma) was suspended.
Mortris / hydrochloric acid / mmol calcium chloride solution (pH 7.
4) Add 15ml and 15ml of 0.1M sodium borate solution float,
The mixture was left at room temperature for several hours with rapid stirring. After the elapse of time, a part of the reaction solution was ligated and dried, and a chloroform / methanol (2/1, v / v) extract was spotted on thin layer chromatography (TLC) to give chloroform / methanol / water (65%). / 25/4, v / v / v) The mixture was developed as a developing solvent. After the development, the free fatty acid section and the lysophosphatidylcholine section were scraped off, respectively, and fatty acid methyl was prepared by a methanol sulfate method, followed by gas chromatography analysis. As a result, 85% of docosahexaenoic acid was detected from the free fatty acid section, and 80% of oleic acid was detected from the lysophosphatidylcholine section.

After the lapse of time, most of the remaining ether in the reaction solution was removed by decantation, 150 ml of fresh ether was added and stirred.After removing ether by decantation, 100 ml of acetone was added and stirred, and 100 ml of new acetone was further added. After additional decantation, the crude product was dried and concentrated. This concentrate was added to chloroform / methanol (1/1 /
1, v / v) dissolved in 100 ml of a mixed solution, 1 g of a filter aid (Dikalite / Perlite, manufactured by Dikalite Orient) was added to the solution, and the mixture was stirred, filtered, and the mother liquor was evaporated to dryness. 840 mg of a yellow oil were obtained.

 The analysis values of the yellow oil were as follows.

TLC developing solution Chloroform / Methanol / Water 65/25/4 (v / v / v) Rf value: 0.12 Color former 2 ', 7'-dichlorofluorescein Reagent: orange Jittmer-Lester reagent: blue TLC-FID (Eiyatro Scanning method) Developing solution Same as TLC Lysophosphatidylcholine 97%, Unknown 3% FAB-MS (Pos.) TEA [M + H] ⁺ : 522 [Oleoyl lysophosphatidylcholine + H] ⁺ 522 (Neg.) TEA [MH]: 281 [C ₁₇ H ₃₃ COO ⁻ ], ie oleic acid _- 281.

From the above results, it was found that pancreatin derived from swine pancreas efficiently hydrolyzes the fatty acid at the Sn-2 position of synthetic phosphatidylcholine to produce 1-acyl-lysophosphatidylcholine.

Example 1 1.0 g of chemically synthesized dipalmitoyl phosphatidylcholine treated with an alumina column was prepared. Next, Reference Example 2
The hydrolysis reaction was carried out under the same conditions as described above. The reaction solution over time was mixed with chloroform / methanol / water (1/2 / 0.8, v / v / v) 10
Extract with 0 ml, add 100 ml of chloroform and distilled water
100 ml was added, and the separated chloroform layer was separated. After evaporating the chloroform layer to dryness, add 4 ml of 50 ml of cold acetone.
The precipitate was washed, and it was confirmed that there was no free fatty acid in the acetone washing solution. The yield was 0.62 g and the properties were white powder.

 The analysis values of the white powder were as follows.

TLC Same conditions as in Reference Example 2. Spots were observed at Rf values: 0.12 and 0.52, and the color development for the color former was the same.

TLC-FID (Eatroscan method) Developing solution Same as TLC Lysophosphatidylcholine 92%, Phosphatidylcholine 8% Based on the results above, the hydrolysis of synthetic phosphatidylcholine by pancreatin of pig pancreas origin is 90% or more for alumina column-treated products. It turned out to be.

The above composition was dissolved in chloroform (3 ml), and the solution was subjected to silica column chromatography (2φ × 7 cm). The solution was passed from a chloroform alone system while increasing the methanol content, and chloroform / methanol (4/1 to 1/1) was passed. 1, v /
v) Lysophosphatidylcholine eluted with the mixture was fractionated. The yield was 520 mg.

The FAB-MS analysis values of this lysophosphatidylcholine were as follows.

(Pos.) TEA [M + H] ⁺ : 496 [palmitoyl lysophosphatidylcholine + H] ⁺ 196 (Neg.) TEA [MH]: 255 [C ₁₅ H ₃₁ COO ⁻ ], that is, palmitic acid ^- 255 Example 2 Alumina column 1.0 g of the treated synthetic synthetic product dilinoleyl phosphatidylcholine was prepared. Next, a hydrolysis reaction was performed under the same conditions as in Reference Example 2. According to Reference Example 2, the aged reaction solution was subjected to solvent separation with ether and acetone and treated with a filter aid to obtain 801 mg of a yellow oil.

 The analysis values of the yellow oil were as follows.

The same conditions as in the TLC Example Rf values: Color development was observed in spots of 0.13 and 0.54,
The spot at 0.85 was significantly weaker in color.

TLC-FID (yatroscan method) Developing solution Same as TLC 74% lysophosphatidylcholine, 3% phosphatidylcholine 3% fatty acid 24% Lysophosphatidylcholine was fractionated from the reaction composition by silica gel column chromatography as in Example 2, yield Was 576 mg.

The FAB-MS analysis values of this lysophosphatidylcholine were as follows.

(Pos.) TEA [M + H] ⁺ : 520 [Linoleyl lysophosphatidylcholine + H] ⁺ 520 (Neg.) TEA [MH]: 279 [C ₁₇ H ₃₁ COO ⁻ ], ie linoleic acid ^- 279 Comparative example Chemical synthesis 1.0 g of the thus-treated dipalmitoyl phosphatidylcholine untreated with alumina was prepared. Next, the same operation was performed according to Example 1. The yield was 0.75 g and the properties were white powder.

 The analysis values of the white powder were as follows.

TLC Same conditions as in Reference Example 2. Spots were observed at Rf values: 0.10 and 0.48, and the color development for the color former was the same.

TLC-FID (Eatroscan method) Developing solution Same as TLC Lysophosphatidylcholine 45%, Phosphatidylcholine 55% Based on the above results, hydrolysis of synthetic phosphatidylcholine by pancreatin derived from swine pancreas was degraded without alumina column treatment. The rate was found to decrease.

──────────────────────────────────────────────────続 き Continued on the front page (58) Field surveyed (Int.Cl. ⁶ , DB name) C07F 9/10 CA (STN)

Claims (2)

(57) [Claims] 1. A method comprising subjecting a monoacid group diacylphosphatidylcholine obtained by chemical synthesis in the presence of an esterification catalyst (pyridine derivative or tertiary amine) to a neutral alumina column treatment, followed by hydrolysis using pancreatine. A method for producing 1-acyl-lysophosphatidylcholine, which is a feature. 2. The method according to claim 1, wherein the neutral alumina is adjusted to Brockmann activity I.