JP3127571B2 - Method for producing lysophosphatidylcholine - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to a method for producing lysophosphatidylcholine directly from glycerophosphocholine.

[0002]

2. Description of the Related Art Lysophosphatidylcholine, also called lysolecithin, is a generic term for compounds in which one acyl group of lecithin has been eliminated, and is present in most animals and plants.
Generally, it is a structural phospholipid that forms a cell membrane like lecithin, but it is known that it also has a physiological effect such as causing a strong hemolytic action on the erythrocyte membrane. As a method for obtaining lysophosphatidylcholine, the following methods are already known.

(1) A small amount of lysophosphatidylcholine contained in natural lecithin and crude phospholipid is separated by solvent,
Separate by column chromatography or the like. ｛For example, Biochemistry Experiment Course, Volume 3, Lipid Chemistry P256-260 (edited by The Biochemical Society of Japan, Tokyo Chemical Dojin), JP-A 1-233290｝

[0004] (2) a natural or synthetic lecithin Hosuhoripa - decompose selectively an acyl group by Ze A ₂ process to obtain a lysophosphatidylcholine. {For example, Biochemistry Experiment Course, Vol. 3, Lipid Chemistry, P264-265 (edited by The Biochemical Society of Japan, Tokyo Kagaku Dojin), JP-A-63-279753, JP-A-2-49593, JP-A-62-262998}

(3) Glycerophosphocholine is reacted with a fatty acid anhydride or acyl chloride in the presence of a base catalyst to obtain lysophosphatidylcholine. (For example, JP-A-1-11088)

Further, (4) a method of producing phosphatidylcholine (lecithin) by reacting glycerophosphocholine with a polymer-supported imidazole resin acylated with a fatty acid is known (JP-A-1-29468).
2).

[0007]

However, among the above-mentioned methods for producing lysophosphatidylcholine, the method (1) comprises:
The content of lysophosphatidylcholine in the commercially available lecithin as a raw material is low and it is not economical to separate and purify. Furthermore, a large amount of solvent and a long time are required for fractionation. The resulting lysophosphatidylcholine acyl chains are not unique because they are of natural origin.

[0008] In the method (2), the enzyme is expensive, easily deactivated, and is difficult to reuse. Furthermore, the supply of the enzyme raw material is limited due to southern snake venom and bee venom, and the enzyme itself is very poisonous, which is dangerous for operation and may be mixed into the product.

The method (3) produces a large amount of lecithin,
Not only is the yield of lysophosphatidylcholine low, but also the selectivity for the bond position of the acyl group of the lysophosphatidylcholine produced is low. In addition, since a highly polar and non-volatile reaction solvent is used to improve the yield and selectivity of lysophosphatidylcholine, purification becomes difficult.

The method (4) is an excellent method for producing phosphatidylcholine, but it is difficult to produce lysophosphatidylcholine.

The present invention has been made to solve the above-mentioned various problems, and has a single acyl composition without using a large amount of solvents, enzymes, special devices, etc., and has regiospecificity. An object of the present invention is to provide a method capable of efficiently producing lysophosphatidylcholine in large quantities.

[0012]

According to the present invention, there is provided a polymer-supported and immobilized azabicyclo catalyst in which a crosslinked resin is bonded with an azabicyclo compound represented by the formula [I] as a side chain and supported and fixed. A process for producing lysophosphatidylcholine, which comprises reacting glycerophosphocholine with acylimidazole.

[0013]

Embedded image (Wherein, X represents a phenylene group, an alkylene group or a divalent group containing these groups, Y represents CH or N 2, a, b,
c represents an integer from 1 to 15. )

Lysophosphatidylcholine, which is the object of the present invention, retains its natural three-dimensional structure and is position-specific.
A compound called acyl-2-lyso-sn-3-phosphatidylcholine.

Glycerophosphocholine (GPC), which is a raw material of the present invention, serves as a skeleton of lysophosphatidylcholine to be produced. Naturally, natural lecithin such as soybean and egg yolk is separated and purified, and then hydrolyzed or subjected to alcoholysis. Obtainable. For separation of natural lecithin, use a silica gel column, activated alumina column, etc., and elute with a chloroform / methanol mixed solvent. GPC can be obtained from the separated lecithin by alcoholysis with a quaternary alkylammonium hydroxide such as tetrabutylammonium hydroxide or an alkali metal. Is also good.

Purified GPC is a highly viscous substance,
In the present invention, GPC can be impregnated into a polymer-supported and immobilized azabicyclo-based catalyst for easy handling and efficient conversion to lysophosphatidylcholine, although it can be used as it is dispersed in the reaction system. The impregnating method is as follows. A predetermined amount of a polymer-supported immobilized catalyst is added to a methanol solution of purified GPC and dispersed well, and then the methanol is removed under reduced pressure and dried to obtain a powdered impregnated G.
It can be used as a PC. It can also be used as a known GPC cadmium chloride complex, but in the present invention, it is not always necessary to use it in the form of a cadmium chloride complex by impregnation with a polymer-supported immobilized catalyst. When cadmium chloride is not used, handling is safe, and a cadmium recovery step is not required, so that it can be manufactured by a more streamlined method.

The acyl group of the acylimidazole used as another raw material in the present invention is a natural or synthetic saturated or unsaturated fatty acid residue. Fatty acids are preferably those having 8 to 24 carbon atoms, such as lauric acid, myristic acid, palmitic acid, stearic acid, oleic acid, linoleic acid, linolenic acid, arachidonic acid, eicosapentaenoic acid, docosahexaenoic acid and the like, or molecules. Synthetic fatty acids having a polymerizable group therein, such as 2,4-octadecadienoic acid and a phenyl group, can be used.

Acylimidazole is a fatty acid imidazo-
Chem., 182, 2183-2192 (1981) described by P. Ferruti et al., A method of condensing a fatty acid and imidazole with dicyclohexylcarbodiimide in chloroform, and S. Murata. By Che
m. Lett., 1819-1820 (1983) .A method of reacting a fatty acid with 1,1′-oxazaryldiimidazole in chloroform or dimethylformamide is used, and the structure of the target lysophosphatidylcholine is used. The raw material of any acyl imidazole can be provided according to the above.

The polymer-supported and immobilized azabicyclo catalyst used in the present invention is obtained by bonding and supporting and immobilizing an azabicyclo compound represented by the formula [I] as a side chain to a crosslinked resin. Examples of the crosslinking resin include vinyl monomers such as styrene, α-methylstyrene, vinyltoluene, acrylic acid or its ester, methacrylic acid or its ester, and divinylbenzene, ethylene glycol diacrylate, ethylene glycol dimethacrylate, and the like. And a crosslinkable comonomer. X represents an alkylene group, a phenylene group itself, or these groups are -COO-, -CONH-, -CO-, -O-, -NH-, -S-
And the like, to form a divalent group.

The number of the azabicyclo compound-containing side chains represented by the formula [I] bonded to the crosslinked resin is not particularly limited, but a base having a base content of 0.5 to 5 mmol / g per polymer is preferred.

These polymer-supported fixed catalysts are preferably used in the form of particles such as polymer beads.

The methylene chain of the azabicyclo compound of the polymer-supported immobilized azabicyclo catalyst is represented by the formula [I]:
a, b, and c are each independently selected from integers from 1 to 15. Anything other than those combinations is difficult to manufacture. Specifically, 1,5-diazabicyclo
[4,3,0] non-5-ene, 1,5-diazabicyclo [4,4,0] -5-
Decene, 1,8-diazabicyclo [5,4,0] -7-undecene,
1,10-diazabicyclo [7,4,0] -9-tridecene, 1,6-diazabicyclo [5,3,0] -6-decene, 1,6-diazabicyclo [5,
5,0] -6-dodecene, 1,5,7-triazabicyclo [4,4,0] -5-
Decene can be used.

These polymer-supported immobilized azabicyclo catalysts can be synthesized by reacting a lithiated or sodium azabicyclo compound with a chloromethylated polymer {Makromol. Chem., 185, 2117-21).
24 (1984), J. Macromol. Sci. Chem., A 29, 248-261 (199
2}.

The production of lysophosphatidylcholine according to the present invention is carried out in a solvent in the presence of an acyl imidazole in the presence of GP.
The reaction is carried out by adding C and a polymer-supported fixed catalyst or a polymer-supported fixed catalyst impregnated with GPC and reacting with stirring.

In the present invention, lysophosphatidylcholine is produced by the catalysis of an azabicyclo compound supported and immobilized on a polymer. However, even if an azabicyclo compound not supported and immobilized on a polymer is used as it is, Is mostly phosphatidylcholine, and the yield of lysophosphatidylcholine is extremely low. Further, in the present invention, since the polymer-supported immobilized catalyst is used, separation of the product, recovery and reuse of the catalyst are easy.

As the solvent, for example, chloroform, carbon tetrachloride, 1,1,1-trichloroethane, 1,1,1,2-tetrachloroethane, benzene, toluene, xylene, hexane, heptane, tetrahydrofuran, 1,4 -Dioxane, dimethylsulfoxide, dimethylformamide, dimethylacetamide, N-methylpyrrolidone, hexamethylphosphorylamide and the like can be used alone or in combination.

The amount of acylimidazole used relative to GPC is 0.5 equivalent or more (based on the hydroxyl group in GPC), preferably 2 to 4 equivalents. The reaction yield is low below the above range, and the consumption of acylimidazole above the above range is low. Many are uneconomical.

The amount of the polymer-supported fixed catalyst is preferably 0.5 to 5 equivalents, more preferably 2 to 4 equivalents (based on the hydroxyl group in GPC) with respect to GPC. When the amount of the polymer-supported immobilized catalyst with respect to GPC is less than the above range, the reaction yield of lysophosphatidylcholine is low, and the by-product of phosphatidylcholine tends to increase accordingly, and when it exceeds the above range, a large excess is charged. However, the reaction yield of lysophosphatidylcholine does not increase so much and the polymer-supported immobilized catalyst is wasted.

The amount of the solvent is preferably such that uniform stirring is possible, and is selected according to the charging ratio of the acyl imidazole, GPC, and the polymer-supported and immobilized catalyst. The reaction temperature is preferably from room temperature to 80 ° C., and the reaction time may be from 1 to 72 hours.

After the completion of the reaction, the polymer-supported immobilized catalyst and the precipitate are separated by filtration, and the lysophosphatidylcholine of high purity can be obtained by purifying the precipitate more easily than in the conventional method. The polymer-supported immobilized catalyst can be easily separated from the reaction system by filtration or the like, and when used in the form of particles, can be easily separated from the precipitate by washing with water, sieving or the like. The separated polymer-supported immobilized catalyst can be used repeatedly for lysophosphatidylcholine production.

[0031]

According to the present invention, by using a polymer-supported immobilized azabicyclo catalyst, lysophosphatidylcholine can be produced selectively and in a high yield by selectively suppressing a large amount of lecithin which is usually produced in a conventional method. Can be. Further, both the reaction and the purification are easy, and the amount of the fatty acid used can be reduced as compared with the conventional method using a fatty acid anhydride. This is advantageous particularly in the production of lysophosphatidylcholine containing a fatty acid which is difficult to produce. Furthermore, since the catalyst can be easily recovered from the reaction system by polymer-supported immobilization, the purification efficiency of the obtained lysophosphatidylcholine is improved, and the regeneration and reuse of the recovered polymer-supported immobilized catalyst can be improved. It is possible and economical. Furthermore, by reacting GPC after impregnation with the polymer-supported immobilized catalyst, heavy metals such as cadmium which have been conventionally used do not always need to be used, and in this case, purification of lysophosphatidylcholine is facilitated and cadmium recovery is facilitated. This eliminates the need for a pollution treatment process, and can be a simple and safe process. Thus, the present invention can produce safe lysophosphatidylcholine at high yield and at low cost.

[0032]

DESCRIPTION OF THE PREFERRED EMBODIMENTS The present invention will be specifically described below based on embodiments. Synthesis Example 1 (Synthesis of catalyst) 500 ml equipped with stirring blade, reflux condenser and nitrogen gas inlet
Anhydrous tetrahydrofuran (THF) 300m in a four-necked flask
l and 1,8-diazabicyclo [5,4,0] -7-undecene (DBU)
30.4 g (200 mmol, manufactured by San Apro Co., Ltd.) was added, and the mixture was cooled to -78 ° C under a dry nitrogen gas atmosphere. After stirring, 120 ml (192 mmol) of n-butyllithium (hexane solution) was added dropwise over 1.5 hours with stirring, followed by stirring for 2 hours to complete the lithiation reaction of DBU. Next, 20.0 g of chloromethylated polystyrene polymer (Cl content: 1.91 mmol / g, 2 mol% crosslinked with divinylbenzene, manufactured by Seimi Chemical Co., Ltd.) was added, and the mixture was slowly returned to room temperature and stirred for 36 hours. below freezing,
After adding 30 ml of methanol and separating the phases, polymer beads were added.
Is filtered through a glass filter, and methanol, methanol
Water / water (volume ratio: 1/1), acetone and THF in this order.
Vacuum dried. Purified polystyrene polymer-supported immobilization DB
17.5 g of U catalyst (base content: 1.91 mmol / g) were obtained.

Synthesis Example 2 (Synthesis of catalyst) 500 ml equipped with a stirring blade, a reflux condenser, and a nitrogen gas inlet.
In a four-necked flask, 300 ml of anhydrous THF and 27.8 g of 1,5,7-triazabicyclo [4,4,0] -5-decene (TBD) (200 mmol, Fluka C
hemie AG) and cooled to −78 ° C. in a dry nitrogen gas atmosphere. 5.3 g (221 mmol) of sodium hydride with stirring
Was added and stirring was continued for 3 hours to complete the sodium reaction of TBD. Chloromethylated polystyrene polymer-20.0 g
(Cl content: 1.91mmol / g, 2mol% divinylbenzene cross-linked,
(Manufactured by Seimi Chemical Co., Ltd.), and the mixture was slowly returned to room temperature, followed by stirring for 48 hours. Under ice cooling, methanol
After adding 50 ml and separating the phases, the polymer beads were filtered through a glass filter, washed with methanol, methanol / water (volume ratio: 1/1), acetone and THF in this order, and vacuumed. Dried. Purified polystyrene polymer-supported TBD catalyst 1
7.1 g (base content: 1.91 mmol / g) were obtained.

Synthesis Example 3 (Synthesis of GPC) Commercially available egg yolk lecithin (trade name: PL-100, Cupy Corporation)
200g silica gel (70-200mesh, Merck) 3
Using liter, chloroform / methanol / water = 65/25 /
Column separation was performed using 4 (volume ratio) as an eluent. Rf = 0.39
The phosphatidylcholine (PC) fraction (developing agent: chloroform / methanol / water = 65/25/4) was concentrated, and 65.6 g of one spot of pure PC was obtained by thin-layer chromatography. This is Ether 50
After dissolving in 0 ml, add 100 ml of tetrabutylammonium hydroxide (10% methanol solution) while stirring under ice cooling.
Dropped in minutes. The resulting highly viscous precipitate was obtained by decantation and washed with ether. Dissolve in 50 ml of methanol,
It was added to 500 ml of acetone cooled to 5 ° C. The resulting precipitate was obtained by decantation, and this operation was repeated once more, and GPC1 from which methanol had been completely removed.
7.6 g were obtained.

Example 1 0.26 g (1 mmol) of GPC obtained in Synthesis Example 3 and 2.0 g (base content: 3.82 mmol) of a polystyrene polymer-supported and immobilized DBU catalyst obtained in Synthesis Example 1 were suspended in 20 ml of dehydrated methanol. After turbidity, the methanol was slowly removed with a rotary evaporator, and then dried in a desiccator containing phosphorus pentoxide for one day and night, and a GPC-impregnated polystyrene polymer-supported immobilized DBU catalyst was used.
2.20 g were obtained. Dehydrated chloroform 30m in 100ml flask
l, myristoyl imidazole 3.34 g (1.2 mmol), the above-mentioned GPC-impregnated polystyrene polymer supported and immobilized DBU catalyst 1.
81 g (GPC content: 0.8 mmol, base content: 3.1 mmol) were sequentially added, and stirring was continued at room temperature for 5 hours. After filtering off the solid content, chloroform / methanol / water = 65/25/4 using the filtrate as eluent.
(Volume ratio) using silica gel column to obtain 0.25 g of lysomiristoyl phosphatidylcholine and 0.07 g of dimyristoyl phosphatidylcholine. The yield of this lyso-form was equivalent to 67.5% in terms of GPC charged, and the lyso-form / diacyl-form ratio was 54/10 (molar ratio). The results of these products, such as IR, MNR, mass spectrometry, and TLC, were consistent with the standard products. Further, the obtained lysomiristoyl phosphatidylcholine was confirmed to be 1-myristoyl-2-lyso-sn-3-phosphatidylcholine having the stereospecific number position -1 by the method for confirming the stereospecific number position described in the analysis example. Was.

Comparative Example 1 In a 100 ml flask, 30 ml of dehydrated chloroform, 3.34 g (1.2 mmol) of myristoyl imidazole, GPC obtained in Synthesis Example 3
0.21 g (0.8 mmol) and 0.47 g (3.1 mmol) of DBU were sequentially added, and stirring was continued at room temperature for 5 hours. After the solid content was filtered off, methanol was added to the filtrate, and chloroform / methanol = 1/1.
(Volume ratio). Use this solution as an ion exchange resin
Amberlite 200C "(registered trademark, ROHM & HAR-
And the DBU was removed. Concentrate the solution and use chloroform / methanol / water = 65/2 as eluent.
Purification on a silica gel column using 5/4 (volume ratio) yielded 0.12 g of dimyristoyl phosphatidylcholine (yield to GPC: 22.0%), but the target lysomiristoyl phosphatidylcholine was only traces.

Example 2 0.26 g (1 mmol) of GPC obtained in Synthesis Example 3 and 2.0 g (base content: 3.82 mmol) of a polystyrene polymer-supported and fixed TBD catalyst obtained in Synthesis Example 2 were suspended in 20 ml of dehydrated methanol. After turbidity, methanol was slowly removed with a rotary evaporator, and then vacuum-dried overnight in a desiccator containing phosphorus pentoxide, and GPC-impregnated polystyrene polymer-supported immobilized TBD catalyst.
2.22 g was obtained. Dehydrated chloroform 25m in 100ml flask
l, Oleylimidazole 0.67 g (2.0 mmol), GPC
1.13 g of impregnated polystyrene polymer supported TBD catalyst
(GPC content: 0.5 mmol, base content: 1.91 mmol) were sequentially added, and stirring was continued at room temperature for 8 hours. After filtering off the solid content, chloroform / methanol / water = 65/25/4 using the filtrate as eluent.
(Volume ratio) to purify with a silica gel column to obtain 0.21 g of lysooleyl phosphatidylcholine and 0.04 g of dioleyl phosphatidylcholine. The yield of this lyso form corresponds to 80.5% in terms of charged GPC, and the lyso form / diacyl form ratio
= 8/1 (molar ratio). The obtained lysooleyl phosphatidylcholine was 1-oleyl-2-lyso-sn-3-phosphatidylcholine of stereospecific number position -1 as a result of measurement by the method of the analysis example.

Comparative Example 2 In a 100 ml flask, 25 ml of dehydrated chloroform, 0.67 g (2.0 mmol) of oleoyl imidazole, and 0.1% of GPC obtained in Synthesis Example 3
3 g (0.5 mmol) and 0.27 g (1.94 mmol) of TBD were sequentially added, and stirring was continued at room temperature for 8 hours. After the solid content was filtered off, methanol was added to the filtrate, and chloroform / methanol = 1/1.
(Volume ratio). This solution was ion-exchanged with Amberlite 200C (trade name, manufactured by Rohm and Haas Company).
To remove the TBD. The solution was concentrated and purified with a silica gel column using chloroform / methanol / water = 65/25/4 (volume ratio) as eluent to obtain 0.37 g of dioleoylphosphatidylcholine (yield to GPC: 94.1%). Although it was obtained, the target lyso-oleoyl phosphatidylcholine was only traces.

Example 3 10.0 g of GPC obtained in Synthesis Example 3 and 15 g of cadmium chloride were added to 30 ml of water.
Was dissolved in 250 ml of cold ethanol, and the resulting precipitate was again dissolved in 20 ml of water and added dropwise to the cold ethanol. The resulting crystals were collected by filtration to give the GPC-CdCl ₂ complex body 16.5g by overnight vacuum drying at 80 ° C.. This GPC-CdCl ₂ Four 30 ml three-necked flasks each containing 0.11 g (0.25 mmol) of the complex and 10 ml of dimethyl sulfoxide were prepared, and the polystyrene polymer-supported and immobilized DBU catalyst / GPC hydroxyl group ratio = 1, 2, 3, 4 (Equivalent ratio), a polystyrene polymer-supported and immobilized DBU catalyst was charged. 0.51 g (1.5 mmol) of palmitol imidazole dissolved in a mixed solvent of 5 ml of dimethyl sulfoxide and 5 ml of chloroform was simultaneously added to each of these flasks.
The reaction was carried out at 40 ° C for 5 hours at the same stirring speed. 1-Lisopalmitoyl phosphatidylcholine and dipalmitoyl phosphatidylcholine having stereospecific number position-1 were obtained, and the ratio between them was determined by a TLC-FID analyzer (Iatroscan MK-5, manufactured by Jatron). The results are shown in Table 1.

[0040]

[Table 1]

Example 4 A polystyrene polymer-supported immobilization was performed in the same manner as in Example 3 except that the GPC-CdCl ₂ complex prepared in Example 3 was used and the catalyst was replaced with a polystyrene polymer-supported and immobilized TBD catalyst.
The ratio of lysopalmitoyl phosphatidylcholine and dipalmitoyl phosphatidylcholine to lysopalmitoyl phosphatidylcholine and dipalmitoyl phosphatidylcholine under each condition of hydroxyl ratio of TBD catalyst / GPC = 1, 2, 3, 4 (equivalent ratio)
FID analyzer (Yatron, Iatroscan MK)
-5). The results are shown in Table 2.

[0042]

[Table 2]

Analysis Example (Confirmation of Stereospecific Number Position) To the lysophosphatidylcholine obtained in Examples 1 to 4, lauric anhydride and 4-dimethylaminopyridine were added to synthesize diacylphosphatidylcholine. Subsequently, the fatty acid at the stereospecific number-2 position was hydrolyzed with phospholipase A2. This fatty acid was methyl-esterified with boron trifluoride-methanol, and the result of GC analysis showed that methyl laurate was detected in an amount of 95% or more, so that the lysophosphatidylcholine of the obtained example was converted into a stereospecific number- It was confirmed to be 1-acyl-2-lyso-sn-3-phosphatidylcholine.

──────────────────────────────────────────────────続 き Continued on the front page (58) Field surveyed (Int. Cl. ⁷ , DB name) C07F 9/10 B01J 31/06 C07B 61/00 300 CA (STN) REGISTRY (STN)

Claims (1)

(57) [Claims] 1. An azabicyclo compound represented by the formula [I] bonded as a side chain to a crosslinked resin, and glycerophosphocholine and acylimidazole are added in the presence of a polymer-supported and immobilized azabicyclo-based catalyst. And lysophosphatidylcholine. Embedded image (Wherein, X represents a phenylene group, an alkylene group or a divalent group containing these groups, Y represents CH or N 2, a, b,
c represents an integer from 1 to 15. )