Preparation method of unsaturated fatty acid phosphatidylcholine
Through metal salt-modified silica gel combined with column chromatography technology and freeze-drying process, the separation and purification problems of unsaturated fatty acid phosphatidylcholine are solved, and efficient and low-cost production is achieved.
Patent Information
- Application Number
- CN202410091770.5
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-01-23
- Publication Date
- 2025-07-25
AI Technical Summary
The prior art is difficult to efficiently separate and purify unsaturated fatty acid phosphatidylcholine, resulting in high production costs and unstable product quality.
The unsaturated fatty acid phosphatidylcholine was prepared by using metal salt-modified silica gel combined with column chromatography technology through esterification and freeze-drying. The unsaturated fatty acid phosphatidylcholine was used to improve the resolution by combining metal salt with silica gel, and purified by modified silica gel.
The purity and yield of unsaturated fatty acid phosphatidylcholine is improved, production costs are reduced, and efficient industrial production is achieved.
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Figure CN120365307A_ABST
Abstract
Description
Technical Field
[0001] The present invention relates to the field of synthetic phospholipids, and particularly to a method for preparing unsaturated fatty acid phosphatidylcholine. Background Art
[0002] Phosphatidylcholine (PC) is a class of amphiphilic molecules and can be applied to fat emulsion and lipid nanoparticle formulations. The type of fatty acid in phospholipids has a great influence on the properties of PC such as solubility and phase transition temperature. Therefore, synthetic phospholipids with different functions can be designed. Unsaturated fatty acid phosphatidylcholine (UPC) such as dioleoyl phosphatidylcholine (DOPC) and dierucoyl phosphatidylcholine (DEPC) are applied to the DepoFoam drug delivery system to achieve slow release of drugs. Currently, there are 3 products on the market for the DepoFoam drug delivery system: liposomal cytarabine injection (Depocyt), liposomal morphine sulfate injection (DepoDur), and liposomal bupivacaine injection (Exparel). In addition, other polyunsaturated acid phosphatidylcholines have functions such as regulating blood lipids, removing blood clots, and immunomodulation. Therefore, the development of UPC preparation technology has important application value in the fields of medicine, scientific research, health food, etc.
[0003] In production, due to the presence of unsaturated chains in UPC, the product presents a viscous mass or liquid during the preparation process, which is significantly different from the solid powder form of saturated fatty acid phosphatidylcholine, resulting in difficulty in applying conventional phosphatidylcholine preparation processes to the preparation of UPC. At the same time, UPC containing unsaturated bonds is easily oxidized and requires more stringent control than saturated chain phospholipids in terms of reaction, drying, storage and other conditions. Therefore, how to develop a suitable preparation process for UPC to ensure product quality requirements has become an urgent problem to be solved.
[0004] The synthesis of phosphatidylcholine uses glycerophosphocholine and specific fatty acids as raw materials, and undergoes an esterification reaction under the action of a condensing agent and an acid-binding agent, and is purified by methods such as column chromatography or recrystallization. Due to the similar polarity of some condensing agents and acid-binding agents to UPC, problems such as low resolution occur during column separation, or the situation where UPC and impurities appear alternately, resulting in the need to use a large amount of organic solvents and multiple column separations to obtain high-purity UPC. According to the structural differences between UPC and condensing agents and acid-binding agents, new column chromatography packing materials can be designed to improve the separation degree between the product and impurities. The development of an efficient and widely applicable purification method can promote the industrial production of UPC. Summary of the Invention
[0005] The object of the present invention is to solve the problems existing in the purification and separation of synthetic phosphatidylcholine containing unsaturated chains, such as high difficulty and high cost, and a new purification technical route is designed. The present invention proposes the following series of UPC preparation routes: specific fatty acids, glycerophosphocholine, condensing agents, and acid-binding agents are used as starting materials to synthesize specific UPC; modified silica gel combined with metal salts is used to purify the UPC mixture; the product is dried by freeze-drying.
[0006] To solve the above technical problems, the present invention adopts the following technical solutions:
[0007] A method for preparing unsaturated fatty acid phosphatidylcholine, comprising the following steps:
[0008] A. Mix glycerophosphocholine, unsaturated fatty acid, condensing agent, acid-binding agent, and organic solvent, carry out an esterification reaction, and obtain a mixture;
[0009] B. Mix a metal salt and silica gel, wash and dry it to obtain modified silica gel;
[0010] C. Use the modified silica gel to perform column chromatography separation on the mixture to obtain a crude phosphatidylcholine product;
[0011] D. Dissolve the crude phosphatidylcholine product and carry out freeze-drying to obtain a solid phosphatidylcholine product.
[0012] Preferably, the specific operation of step B includes: mixing the metal salt and the silica gel in an aqueous solution, washing with water and drying to obtain the modified silica gel.
[0013] Preferably, in step B, the molar ratio of the silica gel to the metal salt is 1:0.05 - 0.5.
[0014] Preferably, in step B, the mixing method of the metal salt and the silica gel includes stirring and mixing; the stirring temperature includes 20 - 100 °C; the stirring time includes 0.5 - 24 hours; the metal salt includes transition metal element salts of IB - VIIIB.
[0015] Preferably, in step B, the metal salt includes one or more of iron salt, copper salt, zinc salt, cobalt salt, nickel salt, manganese salt, chromium salt, ruthenium salt.
[0016] Preferably, in step C, the eluent for the column chromatography includes one or more of halogenated alkanes and lower alcohols.
[0017] Preferably, in step D, the solvent for dissolving the crude phosphatidylcholine product includes one or more of chloroform, lower alcohols, and water.
[0018] Preferably, in step A, the weight ratio of glycerophosphocholine to the unsaturated fatty acid, the condensing agent, the acid-binding agent, and the organic solvent is 1:2-3:2-3:2-3:1-90; the conditions for the esterification reaction include: reaction time of 4-24 hours and reaction temperature of 15-80 °C.
[0019] Preferably, in step A, the unsaturated fatty acid includes one or more of fatty acids with 1 or more unsaturated bonds and a carbon number of C12-C24; the condensing agent includes one or more of dicyclohexylcarbodiimide (DCC), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDCI), N,N'-diisopropylcarbodiimide (DIC), 1-hydroxybenzotriazole (HOBT), O-benzotriazol-1-yl-N,N,N',N'-tetramethyluronium tetrafluoroborate (TBTU), 2-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate (HATU); the acid-binding agent includes one or more of pyridine, 4-dimethylaminopyridine (DMAP), triethylamine, N,N-diisopropylethylamine (DIEA), N-hydroxysuccinimide (NHS); the organic solvent includes one or more of n-hexane, dichloromethane, chloroform, dimethyl sulfoxide, ethyl acetate.
[0020] An unsaturated fatty acid phosphatidylcholine product obtained by the preparation method of the above unsaturated fatty acid phosphatidylcholine.
[0021] Compared with the prior art, implementing the present invention has the following beneficial effects:
[0022] The present invention utilizes the structural differences between UPC, the condensing agent, and the acid-binding agent to design a new type of column chromatography packing to improve the separation degree between the product and impurities, and develops an efficient and widely applicable purification method to promote the industrial production of UPC. BRIEF DESCRIPTION OF THE DRAWINGS
[0023] Figure 1 is the technical roadmap of the present invention.
[0024] Figure 2 are the liquid chromatography diagrams of Invention Examples 1-3.
[0025] Figure 3 are the liquid chromatography diagrams of Comparative Examples 1-3. DETAILED DESCRIPTION OF THE EMBODIMENTS
[0026] To make the objectives, technical solutions, and advantages of the present invention clearer, the present invention will be further described in detail below with reference to the accompanying drawings.
[0027] Example 1
[0028] As Figure 1As shown, weigh 56.4 g (0.2 mol) of oleic acid, 24.4 g of DMAP (0.2 mol), 25.7 g (0.1 mol) of glycerophosphocholine, 41.2 g (0.2 mol) of DCC and 400 ml of chloroform into a round-bottom flask, and react at 50 °C for 16 hours. After the reaction is completed, filter the reaction solution to obtain the filtrate as mixture 1 for standby.
[0029] Weigh another 35 g of ferric chloride and dissolve it in 100 ml of pure water. Add 700 g of silica gel and stir at 25 °C for 2 h, then filter by suction, and wash the filter residue with a small amount of water. After drying, modified silica gel 1 is obtained. Weigh 700 g of modified silica gel 1 and pack it in a chromatography column, and add mixture 1 onto the column. Use an eluent of 8 L of chloroform:methanol with a volume ratio of 2:1, and concentrate the eluent to obtain a paste. Dissolve the paste in 60 ml of absolute ethanol and obtain DOPC solid by freeze-drying. The mass of the solid is 69.31 g, and the purity detected by liquid chromatography is 99.6%, and the yield is 87.8%.
[0030] Comparative Example 1
[0031] Weigh 56.4 g (0.2 mol) of oleic acid, 24.4 g of DMAP (0.2 mol), 25.7 g (0.1 mol) of glycerophosphocholine, 41.2 g (0.2 mol) of DCC and 400 ml of chloroform into a round-bottom flask, and react at 50 °C for 16 hours. After the reaction is completed, filter the reaction solution to obtain the filtrate as mixture 1-1 for standby.
[0032] Weigh 700 g of silica gel and pack it in a chromatography column, and add mixture 1-1 onto the column. Use an eluent of 18 L of chloroform:methanol with a volume ratio of 3:1, and concentrate the eluent to obtain a paste. Dissolve the paste in 60 ml of absolute ethanol and obtain DOPC solid by freeze-drying. The mass of the solid is 62.18 g, and the purity detected by liquid chromatography is 98.4%, and the yield is 77.8%.
[0033] By comparing Example 1 and Comparative Example 1, the modified silica gel prepared with ferric chloride in Example 1 can selectively improve the binding ability to the condensing agent and the acid-binding agent, and use a higher-polarity eluent to improve the elution and purification speed of DOPC, reducing the consumption of organic solvents.
[0034] Example 2
[0035] As Figure 1 shown, weigh 68.1 g (0.2 mol) of erucic acid, 24.4 g of CDI (0.2 mol), 25.7 g (0.1 mol) of glycerophosphocholine, 41.2 g (0.2 mol) of DBU and 400 ml of chloroform into a round-bottom flask, and react at 30 °C for 14 hours. After the reaction is completed, mixture 2 is obtained.
[0036] Another 50 g of copper sulfate was dissolved in 100 ml of pure water, 700 g of silica gel was added, stirred at 35 °C for 3 h, then filtered by suction, and the filter residue was washed with a small amount of water and dried to obtain modified silica gel 2. 700 g of modified silica gel 2 was weighed and filled in a chromatography column, and mixture 2 was loaded onto the column. A 9 L eluent with a volume ratio of chloroform to methanol of 2:1 was used, and the eluent was concentrated to obtain a paste. The paste was dissolved in 60 ml of 95% ethanol and freeze-dried to obtain DEPC solid. The mass of the powder was 68.3 g, and the purity detected by liquid chromatography was: 98.7%, and the yield was 85.9%.
[0037] Comparative Example 2
[0038] 68.1 g (0.2 mol) of erucic acid, 24.4 g of CDI (0.2 mol), 25.7 g (0.1 mol) of glycerophosphocholine, 41.2 g (0.2 mol) of DBU and 400 ml of chloroform were weighed and placed in a round-bottomed flask, and reacted at 30 °C for 14 hours. After the reaction, mixture 2-1 was obtained.
[0039] 700 g of silica gel was weighed and filled in a chromatography column, and mixture 2-1 was loaded onto the column. A 9 L eluent with a volume ratio of chloroform to methanol of 2:1 was used, and the eluent was concentrated to obtain a paste. The paste was dissolved in 60 ml of 95% ethanol and freeze-dried to obtain a solid. The mass of the powder was 37.8 g, and the purity detected by liquid chromatography was: 99.4%, and the yield was 41.6%.
[0040] By comparing Example 2 with Comparative Example 2, when the eluent with the same concentration and volume was added, Example 2 used modified silica gel to improve the resolution and avoid the situation of alternating elution of the condensing agent and the acid-binding agent.
[0041] Example 3
[0042] As Figure 1 shown, 65.7 g (0.2 mol) of docosahexaenoic acid, 76.0 g of HATU (0.2 mol), 25.7 g (0.1 mol) of glycerophosphocholine, 25.8 g (0.2 mol) of DIEA and 1 L of dichloromethane were weighed and placed in a round-bottomed flask, and reacted at 40 °C for 14 hours. After the reaction, mixture 3 was obtained.
[0043] Another 40 g of cobalt nitrate was dissolved in 100 ml of pure water, 700 g of silica gel was added, stirred at 35 °C for 3 h, then filtered by suction, and the filter residue was washed with a small amount of water and dried to obtain modified silica gel 3. 700 g of modified silica gel 3 was weighed and filled in a chromatography column, and mixture 2 was loaded onto the column. A 10 L eluent with a volume ratio of dichloromethane to methanol of 2:1 was used, and the eluent was concentrated to obtain a paste. The paste was dissolved in 60 ml of anhydrous methanol and freeze-dried to obtain DHPC solid. The mass of the powder was 68.9 g, and the purity detected by liquid chromatography was: 98.2%, and the yield was 78.1%.
[0044] Comparative Example 3
[0045] Weigh 65.7 g of docosahexaenoic acid (0.2 mol), 76.0 g of HATU (0.2 mol), 25.7 g (0.1 mol) of glycerophosphocholine, 25.8 g (0.2 mol) of DIEA and 1 L of dichloromethane into a round-bottom flask, and react at 40 °C for 14 hours. After the reaction, mixture 3-1 is obtained.
[0046] Weigh another 40 g of calcium chloride and dissolve it in 150 ml of pure water. Add 700 g of silica gel and stir at 35 °C for 3 h, then filter by suction. Wash the filter residue with a small amount of water and dry it to obtain modified silica gel 3-1. Weigh 700 g of modified silica gel 3 and pack it in a chromatography column, and add mixture 3-1 onto the column. Use an eluent of 10 L of dichloromethane:methanol with a volume ratio of 2:1, and concentrate the eluent to obtain a paste. Dissolve the paste in 60 ml of anhydrous methanol and obtain DHPC solid by freeze-drying. The mass of the powder is 48.7 g, and the purity detected by liquid chromatography is 98.8%, and the yield is 54.8%.
[0047] In Comparative Example 3, calcium chloride, a metal salt other than transition metal elements of IB-VIIIB, was added. Under the same operation, Comparative Example 3 could not effectively use the condensing agent and the acid-binding agent. Experiments show the necessity of the selection of metal salt types for the successful implementation of this technical solution. Metal salts of main group elements such as calcium salts and magnesium salts will cause a significant decrease in the effect.
[0048] Effect Example 1
[0049] The liquid chromatography detection of the examples of the present invention is as Figure 2 shown, and the liquid chromatography detection of the comparative examples is as Figure 3 shown. The data comparison between the examples and the comparative examples is shown in Table 1:
[0050] Table 1
[0051] Variety Condensing agent + acid-binding agent Example Purity (%) Yield (%) Comparative example Purity (%) Yield (%) DOPC DCC + DMAP Example 1 99.6 87.8 Comparative example 1 99.8 78.4 DEPC CDI + DBU Example 2 98.7 85.9 Comparative example 2 99.4 41.6 DHPC HATU + DIEA Example 3 98.2 78.1 Comparative example 3 98.8 54.8
[0052] As can be seen from Table 1, for Examples 1-3 using the process of the present invention, there is a significant improvement in the yield compared to Comparative Examples 1-3.
[0053] The above-disclosed are only the preferred embodiments of the present invention. Of course, the scope of the rights of the present invention cannot be limited thereby. Therefore, equivalent changes made according to the claims of the present invention still fall within the scope covered by the present invention.
Claims
1. A preparation method of unsaturated fatty acid phosphatidylcholine, characterized in that, It includes the following steps: A. Mix glycerophosphocholine, unsaturated fatty acid, condensing agent, acid-binding agent and organic solvent, carry out an esterification reaction to obtain a mixture; B. Mix a metal salt and silica gel, wash and dry it to obtain modified silica gel; C. Use the modified silica gel to carry out column chromatography separation on the mixture to obtain a crude phosphatidylcholine product; D. Dissolve the crude phosphatidylcholine product and carry out freeze-drying to obtain a solid phosphatidylcholine product.
2. The preparation method of unsaturated fatty acid phosphatidylcholine according to claim 1, wherein, The specific operation of step B includes: mixing the metal salt and the silica gel in an aqueous solution, washing with water and drying to obtain the modified silica gel.
3. The preparation method of unsaturated fatty acid phosphatidylcholine according to claim 1, wherein In step B, the molar ratio of the silica gel to the metal salt is 1:0.05 - 0.
5.
4. The preparation method of unsaturated fatty acid phosphatidylcholine according to claim 1, characterized in that, In step B, the mixing method of the metal salt and the silica gel includes stirring and mixing; the stirring temperature includes 20 - 100 °C; the stirring time includes 0.5 - 24 hours; the metal salt includes transition metal element salts of IB - VIIIB.
5. The preparation method of unsaturated fatty acid phosphatidylcholine according to claim 1, wherein In step B, the metal salt includes one or more of iron salt, copper salt, zinc salt, cobalt salt, nickel salt, manganese salt, chromium salt, ruthenium salt.
6. The preparation method of unsaturated fatty acid phosphatidylcholine according to claim 1, wherein, In step C, the eluent for the column chromatography includes one or more of halogenated alkanes and lower alcohols.
7. The preparation method of unsaturated fatty acid phosphatidylcholine according to claim 1, wherein In step D, the solvent for dissolving the crude phosphatidylcholine product includes one or more of chloroform, lower alcohols, and water.
8. The preparation method of unsaturated fatty acid phosphatidylcholine according to claim 1, wherein, In step A, the weight ratio of glycerophosphocholine to the unsaturated fatty acid, the condensing agent, the acid-binding agent, and the organic solvent is 1:2 - 3:2 - 3:2 - 3:1 - 90; the conditions for the esterification reaction include: reaction time 4 - 24 hours, reaction temperature 15 - 80 °C.
9. The preparation method of unsaturated fatty acid phosphatidylcholine according to claim 1, characterized in that, In step A, the unsaturated fatty acid includes one or more of fatty acids with a carbon number of C12 - C24 containing more than 1 unsaturated bond; the condensing agent includes one or more of dicyclohexylcarbodiimide, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, N,N'-diisopropylcarbodiimide, 1-hydroxybenzotriazole, O-benzotriazol-N,N,N',N'-tetramethyluronium tetrafluoroborate, 2-(7-azabenzotriazol)-N,N,N',N'-tetramethyluronium hexafluorophosphate; the acid-binding agent includes one or more of pyridine, 4-dimethylaminopyridine, triethylamine, N,N-diisopropylethylamine, N-hydroxysuccinimide; the organic solvent includes one or more of n-hexane, dichloromethane, chloroform, dimethyl sulfoxide, ethyl acetate.
10. An unsaturated fatty acid phosphatidylcholine product obtained by the preparation method of the unsaturated fatty acid phosphatidylcholine according to claim 1.