Preparation method of phosphatidylcholine of saturated fatty acid

Abstract

The invention relates to the field of phospholipid synthesis, in particular to a preparation method of phosphatidylcholine of saturated fatty acid, which is characterized by comprising the following steps: A, mixing glycerophosphorylcholine, unsaturated fatty acid, a condensing agent, an acid-binding agent and a reaction solvent, and carrying out esterification reaction to obtain an intermediate mixture; b, mixing the intermediate mixture with a metal salt solution, and then performing liquid separation to obtain an organic phase to form a refined mixture 1; c, mixing the refined mixture 1 with water, extracting, separating liquid to obtain an organic phase, and removing a solvent to obtain a refined mixture 2; and D, mixing and separating out the refined mixture 2 and an organic solvent, and carrying out solid-liquid separation and drying to obtain a phosphatidylcholine product. The invention solves the problems of high purification and separation difficulty, high cost and the like in the existing synthesis of phosphatidylcholine.

Description

Technical Field

[0001] The present invention relates to the field of synthetic phospholipids, and particularly to a method for preparing phosphatidylcholine of saturated fatty acids. Background Art

[0002] Phosphatidylcholine (PC) is a class of amphiphilic molecules that can be used as emulsifiers and structural lipids in pharmaceutical preparations. Synthetic phosphatidylcholine has a single fatty acid chain, such as distearoyl phosphatidylcholine (DSPC), dimyristoyl phosphatidylcholine (DMPC), and dipalmitoyl phosphatidylcholine (DPPC), etc., which have better stability and uniformity, making it easier to control the particle size in the preparation of lipid nanoparticles (LNP), and the prepared liposomes are more stable. PC series phospholipids can be applied to the preparation of chemical drug liposomes and mRNA delivery systems, and have broad application prospects. Therefore, it is of great significance to explore a high-quality PC preparation method suitable for industrialization. Traditional single purification methods using mixed solvents or hot dissolution and cold precipitation are difficult to obtain high-purity products and cannot meet the requirements of injection pharmaceutical excipients. At the same time, due to the low solubility of phosphatidylcholine of saturated fatty acids, column chromatography purification consumes a large amount of solvents in actual production, resulting in too high production costs and difficult to achieve large-scale production.

[0003] The synthesis of phosphatidylcholine generally 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. Commonly used condensing agents include carbodiimides and onium salts, and commonly used acid-binding agents include heterocyclic derivatives such as pyridines and aliphatic amines. During the separation and purification process, the solubility and polarity of some condensing agents and organic bases are similar to those of PC, resulting in difficult separation of the product and reduced yield. Compared with some condensing agents with better water solubility, such as 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDCI), N-hydroxysuccinimide (NHS), etc., the conventional purification method of water extraction will cause emulsification of the organic phase containing phospholipids, resulting in a lower product yield. Summary of the Invention

[0004] The purpose of the present invention is to solve the problems of difficult purification and separation and high cost in the existing synthesis of phosphatidylcholine, and design a technical route for step-by-step purification. The present invention proposes the following series of PC preparation routes: specific PC is synthesized using specific fatty acids, glycerophosphocholine, a condensing agent, and an acid-binding agent as starting materials; a metal salt is added in the purification process to complex with the condensing agent and the acid-binding agent, and is removed together with glycerophosphocholine by water washing; fatty acids are removed using an organic solvent to obtain a PC product. According to the structural and solubility differences between PC and impurities, the present invention can use a novel process to reduce the purification steps and costs and achieve large-scale production.

[0005] To solve the above technical problems, the present invention adopts the following technical solutions:

[0006] A method for preparing phosphatidylcholine of saturated fatty acid, comprising the following steps:

[0007] A. Mix glycerophosphocholine, unsaturated fatty acid, condensing agent, acid-binding agent and reaction solvent, carry out esterification reaction to obtain an intermediate mixture;

[0008] B. Mix the intermediate mixture with a metal salt solution, then separate the liquid by liquid separation to obtain an organic phase, forming refined mixture 1;

[0009] C. Mix and extract the refined mixture 1 with water, then separate the liquid by liquid separation to obtain an organic phase. After removing the solvent, obtain refined mixture 2;

[0010] D. Mix and precipitate the refined mixture 2 with an organic solvent. After solid-liquid separation and drying, obtain a phosphatidylcholine product.

[0011] Preferably, in step B, the molar ratio of the metal salt in the metal salt solution to the glycerophosphocholine is 1:0.15 - 3.

[0012] By adding a clarified metal salt solution, a water-soluble good chelate formed with excessive raw materials and impurities is formed, so as to realize the separation from PC.

[0013] Preferably, in step B, the mixing method of the intermediate mixture and the metal salt solution includes stirring and mixing; the stirring temperature is 20 - 100 °C; the stirring time is 0.5 - 24 hours.

[0014] 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 ruthenium salt.

[0015] Preferably, in step C, the volume ratio of the organic solvent to water is 1:0.1 - 2; the mixing extraction is repeated 1 - 4 times; the method of removing the solvent includes evaporation to dryness under reduced pressure.

[0016] Preferably, in step D, the mixing method of the mixing and precipitation includes stirring and mixing; the stirring temperature is 15 - 80 °C; the stirring time is 4 - 24 hours; the method of solid-liquid separation includes filtration.

[0017] Preferably, in step D, the mass ratio of the refined mixture 2 to the organic solvent is 1:1 - 20; the mixing and precipitation is repeated 1 - 4 times; the organic solvent includes one or more of n-hexane, octane, dichloromethane, chloroform, ethyl acetate, acetone, tetrahydrofuran, methyl tert-butyl ether, methanol, ethanol, water.

[0018] Preferably, in step A, the weight ratio of glycerophosphocholine to the saturated fatty acid, the condensing agent, the acid-binding agent, and the reaction solvent is 1:2-3:2-3:2-3:1-90; the conditions for the esterification reaction include: a reaction time of 4-24 hours and a reaction temperature of 15-80°C.

[0019] Preferably, in step A, the unsaturated fatty acid includes one or more 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-N,N,N',N'-tetramethyluronium tetrafluoroborate (TBTU), 2-(7-azabenzotriazol)-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] A phosphatidylcholine product of the saturated fatty acid obtained by the preparation method of the phosphatidylcholine of the above-mentioned saturated fatty acid.

[0021] Compared with the prior art, implementing the present invention has the following beneficial effects:

[0022] In the present invention, since most condensing agents and acid-binding agents are Lewis base reagents containing nitrogen atoms, they can be combined with a suitable Lewis acid reagent. Metal ions are typical Lewis acid molecules and can provide 4-6 coordination sites. Theoretically, one metal ion can combine with 1-6 Lewis base molecules. After the condensing agent and the acid-binding agent are coordinated with the metal ion, a water-soluble good chelate is formed, which can be separated from the lipid-soluble PC by washing with water. An efficient and widely applicable purification process is established to improve the production efficiency of PC industrialization. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 It is the technical roadmap of the present invention.

[0024] Figure 2 It is the liquid chromatogram of Invention Examples 1-4.

[0025] Figure 3 It is the liquid chromatogram of Comparative Examples 1-4. DETAILED DESCRIPTION OF THE INVENTION

[0026] To make the objectives, technical solutions, and advantages of the present invention more clear, the present invention will be further described in detail below with reference to the accompanying drawings.

[0027] Example 1

[0028] As Figure 1 shown, weigh 568 g (2 mol) of stearic acid, 244 g of DMAP (2 mol), 257 g (1 mol) of glycerophosphocholine, 412 g (2 mol) of DCC, and 4 L of chloroform into a round-bottom flask, and react at 50 °C for 16 hours. Separately, weigh 152 g of ferrous sulfate and dissolve it in 800 ml of pure water for later use. After the reaction is completed, filter the reaction mixture, add the prepared 400 mL of ferrous sulfate solution to the flask, stir at 25 °C for 0.5 hours, and then let it stand for liquid separation. Add the remaining 400 mL of ferrous sulfate to the organic phase, stir at 25 °C for 0.5 hours, and then let it stand for liquid separation. Extract the organic phase with 200 mL of purified water and then perform liquid separation. After concentration, a light yellow solid is obtained. Add 1 L of ethyl acetate to the solid and perform pulping at 50 °C for 1 hour, filter to obtain a white filter residue, and dry to obtain DSPC solid. The mass of the powder is 654.2 g, the purity detected by liquid chromatography is 99.8%, and the yield is 82.9%.

[0029] Comparative Example 1

[0030] Weigh 568 g (2 mol) of stearic acid, 244 g of DMAP (2 mol), 257 g (1 mol) of glycerophosphocholine, 412 g (2 mol) of DCC, and 4 L of chloroform into a round-bottom flask, and react at 50 °C for 16 hours. After the reaction is completed, filter the reaction solution, concentrate the filtrate, and perform silica gel column chromatography. Use an eluent of 80 L of chloroform:methanol with a volume ratio of 3:1, concentrate the eluent, and dry the solid to obtain a powder. The mass of the powder is 619.1 g, the purity detected by liquid chromatography is 99.8%, and the yield is 78.4%

[0031] By comparing Example 1 and Comparative Example 1, although the product yields and purities of the two purification methods are the same, only 1 L of ethyl acetate and 1 L of water are used in the purification process of Example 1, which is much lower than the 80 L of organic solvents used in the prior art in column chromatography in Comparative Example 1. Example 1 effectively reduces the usage amount of organic solvents and the purification time.

[0032] Example 2

[0033] As Figure 1As shown, 456 g (2 mol) of myristic acid, 270 g of HOBT (2 mol), 257 g (1 mol) of glycerophosphocholine, 284 g (2 mol) of EDCI and 4 L of chloroform were weighed and placed in a round-bottom flask, and the reaction was carried out at 50 °C for 16 hours. Separately, 159 g of copper sulfate was dissolved in 1000 ml of pure water for later use. After the reaction was completed, 500 mL of the prepared copper sulfate solution was added to the flask, stirred at 25 °C for 0.5 hour, and then allowed to stand for liquid separation. The organic phase was then added with the remaining 500 mL of copper sulfate, stirred at 25 °C for 0.5 hour, and then allowed to stand for liquid separation. The organic phase was extracted with 200 mL of purified water and then separated, and analyzed by thin-layer chromatography. After concentration, a light yellow solid was obtained. The solid was slurried in 1 L of acetone for 1 hour, filtered to obtain a white filter residue, and dried to obtain DMPC solid. The mass of the powder was 529.4 g, the purity detected by liquid chromatography was 98.8%, and the yield was 67.4%

[0034] Comparative Example 2

[0035] 456 g (2 mol) of myristic acid, 270 g of HOBT (2 mol), 257 g (1 mol) of glycerophosphocholine, 284 g (2 mol) of EDCI and 4 L of chloroform were weighed and placed in a round-bottom flask, and the reaction was carried out at 50 °C for 16 hours. The organic phase was washed twice with 1000 mL of 5% dilute hydrochloric acid solution, then extracted once with 1000 mL of saturated brine, and monitored by thin-layer chromatography. The organic phase was concentrated to obtain a light yellow solid, which was slurried in 1 L of acetone for 1 hour, filtered to obtain a white filter residue, and dried to obtain DMPC solid. The mass of the powder was 436.4 g, the purity detected by liquid chromatography was 90.4%, and the yield was 43.7%

[0036] By comparing Example 2 and Comparative Example 2, for the water-soluble condensing agent and acid-binding agent, Comparative Example 2 purified by washing with water according to the existing technology. According to thin-layer chromatography detection, more water was needed to effectively extract the condensing agent, but the final liquid phase results showed that the purification operation of washing with water failed to effectively remove all the condensing agent. At the same time, the use of more water led to a larger emulsion layer, resulting in a lower yield of phospholipids.

[0037] Example 3

[0038] As Figure 1As shown, weigh 624 g (2 mol) of arachidic acid, 270 g of HOBT (2 mol), 257 g (1 mol) of glycerophosphocholine, 642 g (2 mol) of TBTU, 258 g (2 mol) of DIEA and 4 L of chloroform into a round-bottom flask, and react at 50 °C for 16 hours. Separately, weigh 309 g of nickel sulfate and dissolve it in 1000 ml of pure water for later use. After the reaction is completed, add the prepared 500 mL of nickel sulfate solution to the flask, stir at 25 °C for 0.5 hours, and then let it stand for liquid separation. Add the remaining 500 mL of nickel sulfate to the organic phase, stir at 25 °C for 0.5 hours, and then let it stand for liquid separation. The organic phase is extracted with 200 mL of purified water and then separated, and analyzed by thin-layer chromatography. After concentration, a white solid is obtained. The solid is slurried in 1 L of ethyl acetate for 1 hour, filtered to obtain a white solid, and dried to obtain DAPC solid. The mass of the powder is 516.0 g, the purity detected by liquid chromatography is: 98.1%, and the yield is 65.7%

[0039] Comparative Example 3

[0040] Weigh 624 g (2 mol) of arachidic acid, 270 g of HOBT (2 mol), 257 g (1 mol) of glycerophosphocholine, 642 g (2 mol) of TBTU, 258 g (2 mol) of DIEA and 4 L of chloroform into a round-bottom flask, and react at 50 °C for 16 hours. After the reaction is completed, concentrate the filtrate to obtain a yellow paste, add 5 L of acetone and slurry for 1 hour, filter to obtain a white filter residue, and monitor using thin-layer chromatography. The filter residue is slurried again in 5 L of methyl tert-butyl ether for 1 hour, monitored by thin-layer chromatography, and filtered to obtain a white filter residue. After drying, the mass of the DAPC powder is 583.6 g, the purity detected by liquid chromatography is: 88.4%, and the yield is 65.6%

[0041] By comparing Example 3 and Comparative Example 3, in Comparative Example 3, the unreacted arachidic acid can be effectively removed by slurrying the reaction product with an organic solvent. However, thin-layer chromatography monitoring shows that the prior art has poor removal effects on glycerophosphocholine and the condensing agent, and a large amount of organic solvents are used. The purity of the final product in Comparative Example 3 is lower than 98%, failing to meet the product quality requirements.

[0042] Example 4

[0043] As Figure 1As shown, 681 g (2 mol) of palmitic acid, 258 g of DIEA (2 mol), 257 g (1 mol) of glycerophosphocholine, 760 g (2 mol) of HATU and 4 L of chloroform were weighed into a round-bottom flask and reacted at 50 °C for 16 hours. Another 136 g of zinc chloride was dissolved in 1200 ml of pure water for later use. After the reaction, 600 mL of the prepared zinc chloride solution was added to the flask, stirred at 25 °C for 1 hour, and then left to stand for liquid separation. The organic phase was then added with the remaining 600 mL of zinc chloride, stirred at 35 °C for 1 hour, and left to stand for liquid separation. The organic phase was extracted with 200 mL of purified water, separated, and analyzed by thin-layer chromatography. After concentration, a white solid was obtained. The solid was added to 1 L of absolute ethanol and slurried for 1 hour, and the white filter residue was obtained by filtration. After drying, the DPPC solid was obtained. The mass of the powder was 625.7 g, the purity detected by liquid chromatography was 99.1%, and the yield was 85.3%

[0044] Comparative Example 4

[0045] 681 g (2 mol) of palmitic acid, 258 g of DIEA (2 mol), 257 g (1 mol) of glycerophosphocholine, 760 g (2 mol) of HATU and 4 L of dichloromethane were weighed into a round-bottom flask and reacted at 50 °C for 16 hours. Another 117 g of sodium chloride was dissolved in 1000 ml of pure water for later use. After the reaction, 600 mL of the prepared sodium chloride solution was added to the flask, stirred at 25 °C for 1 hour, and then left to stand for liquid separation. The organic phase was then added with the remaining 600 mL of sodium chloride, stirred at 35 °C for 1 hour, and left to stand for liquid separation. The organic phase was extracted with 200 mL of purified water, separated, and analyzed by thin-layer chromatography. After concentration, a white solid was obtained. The solid was added to 1 L of absolute ethanol and slurried for 1 hour, and the white filter residue was obtained by filtration. After drying, the DPPC solid was obtained. The mass of the powder was 649.7 g, the purity detected by liquid chromatography was 78.5%, and the yield was 69.5%

[0046] By comparing Example 4 with Comparative Example 4, in Comparative Example 4, sodium chloride was used instead of zinc chloride, and the former could not coordinate with the Lewis base, resulting in a significant decrease in the scavenging effect of the condensing agent. Therefore, under other identical conditions, Comparative Example 4 could not effectively purify the DPPC crude product, proving that the addition of metal ions is an indispensable step in the technical solution of the present invention.

[0047] Effect Example 1

[0048] 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:

[0049] Table 1

[0050]

[0051] As can be seen from Table 1, the process of the present invention is applicable to the preparation of various different saturated fatty acid phosphatidylcholines. According to the differences in specific fatty acids, Examples 1 to 4 have significantly improved in terms of purity or yield compared to Comparative Examples 1 to 4.

[0052] 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 method for preparing phosphatidylcholine of saturated fatty acid, characterized in that, It includes the following steps: A. Mix glycerophosphocholine, unsaturated fatty acid, condensing agent, acid-binding agent and reaction solvent, carry out an esterification reaction to obtain an intermediate mixture; B. Mix the intermediate mixture with a metal salt solution, then separate the liquid to obtain an organic phase, forming refined mixture 1; C. Mix and extract the refined mixture 1 with water, then separate the liquid to obtain an organic phase, and after removing the solvent, obtain refined mixture 2; D. Mix and precipitate the refined mixture 2 with an organic solvent, and after solid-liquid separation and drying, obtain a phosphatidylcholine product.

2. The preparation method of phosphatidylcholine of saturated fatty acid according to claim 1, characterized in that, In step B, the molar ratio of the metal salt in the metal salt solution to the glycerophosphocholine is 1:0.15 - 3.

3. The preparation method of phosphatidylcholine of saturated fatty acid according to claim 1, characterized in that, In step B, the mixing method of the intermediate mixture and the metal salt solution includes stirring and mixing; the stirring temperature is 20 - 100 °C; the stirring time is 0.5 - 24 hours.

4. The preparation method of phosphatidylcholine of saturated fatty acid according to claim 1, characterized in that, 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.

5. The method for preparing phosphatidylcholine of saturated fatty acid according to claim 1, wherein In step C, the volume ratio of the refined mixture 1 to water is 1:0.1 - 2; the mixing and extraction are repeated 1 - 4 times; the method of removing the solvent includes drying under reduced pressure.

6. The preparation method of phosphatidylcholine of saturated fatty acid according to claim 1, characterized in that, In step D, the mixing method of the mixing and precipitation includes stirring and mixing; the stirring temperature is 15 - 80 °C; the stirring time is 4 - 24 hours; the method of solid-liquid separation includes filtration.

7. The preparation method of phosphatidylcholine of saturated fatty acid according to claim 1, characterized in that, In step D, the mass ratio of the refined mixture 2 to the organic solvent is 1:1 - 20; the mixing and precipitation are repeated 1 - 4 times; the organic solvent includes one or more of n-hexane, octane, dichloromethane, chloroform, ethyl acetate, acetone, tetrahydrofuran, methyl tert-butyl ether, methanol, ethanol, water.

8. The preparation method of phosphatidylcholine of saturated fatty acid according to claim 1, characterized in that, In step A, the weight ratio of the glycerophosphocholine to the saturated fatty acid, the condensing agent, the acid-binding agent, the reaction solvent is 1:2 - 3:2 - 3:2 - 3:1 - 90; the conditions of the esterification reaction include: reaction time 4 - 24 hours, reaction temperature 15 - 80 °C.

9. The preparation method of phosphatidylcholine of saturated fatty acid according to claim 1, characterized in that, In step A, the saturated fatty acid includes one or more of fatty acids with carbon numbers C12 - C24; 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 reaction solvent includes one or more of n-hexane, dichloromethane, chloroform, dimethyl sulfoxide, ethyl acetate.

10. A saturated fatty acid phosphatidylcholine product obtained by the preparation method of the saturated fatty acid phosphatidylcholine according to claim 1.

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