Synthesis method of phosphatidyl ethanolamine

Abstract

The invention relates to the field of synthesis of phospholipids, and discloses a synthesis method of phosphatidyl ethanolamine, which comprises the following steps: reacting O-phosphoethanolamine with an alkaline substance to obtain O-phosphoethanolamine metal salt, refining, and reacting with an amino protective agent to obtain amino-protected O-phosphoethanolamine salt; the preparation method comprises the following steps: reacting di-fatty glyceride with trichloroacetonitrile to form an imine intermediate; o-phosphoethanolamine salt with amino protection is added into the imine intermediate, and phosphatidyl ethanolamine with amino protection is obtained; and after deprotection, refining to obtain 98% phosphatidyl ethanolamine. The method is mild in reaction condition, good in selectivity, low in production cost and beneficial to industrial mass production.

Description

Technical Field

[0001] The invention relates to the field of synthetic phospholipids, and in particular to a method for synthesizing phosphatidylethanolamine. Background Art

[0002] Phosphatidylethanolamine PE has special physiological functions and surface properties, and is widely used in the fields of food and medicine. In the food industry, PE can be used to make health foods, which have the effects of improving memory, repairing nerve cells, promoting brain development and improving immunity. In the pharmaceutical industry, PE can be used as a drug carrier. In recent years, the application of PE series phospholipids in long-circulating liposomes, polymer micelles, long-circulating nanoparticles and other drug carriers has achieved rapid development. It can be seen that PE series phospholipids have a very broad development prospect, and it is of great significance to explore the preparation method of high-quality PE suitable for industrialization.

[0003] At present, there are two main methods for synthesizing PE series phospholipids: (1) using phosphorus oxychloride to react with diglycerol and ethanolamine respectively [200710089940.2]. (2) using 1,3-cycloglycerophosphate to react with ethanolamine to prepare glycerophosphoethanolamine [202011421108.X]. However, some of the raw materials used in these two preparation methods are expensive, and the reagents such as acyl chloride and phosphorus oxychloride are highly corrosive and toxic, which can easily damage production equipment and pollute the environment, making it difficult to achieve industrial production. Summary of the invention

[0004] The purpose of the present invention is to solve the problems of difficulty and high cost in the synthesis of existing PE series phospholipids. The present invention proposes the following PE synthesis route: first, O-phosphoethanolamine is reacted with an alkaline substance to obtain O-phosphoethanolamine salt (PE-K), and then after the amine group is protected, it is reacted with the imine of 1,2-diglyceride, and finally the amine group is removed to obtain phosphatidylethanolamine.

[0005] The advantages of the above-mentioned synthetic route are: (1) the synthetic raw materials are relatively cheap and easy to obtain; (2) the reaction conditions are mild and the selectivity is good; (3) the equipment requirements are relatively low and it is easy to realize industrial production.

[0006] In order to solve the above technical problems, the present invention adopts the following technical solutions:

[0007] A method for synthesizing phosphatidylethanolamine comprises the following steps:

[0008] A. Adding alkaline substances to O-phosphoethanolamine to react, thereby obtaining O-phosphoethanolamine salt;

[0009] B. adding an amine protecting agent to the O-phosphoethanolamine salt to obtain an O-phosphoethanolamine salt with a protecting group;

[0010] C. Adding a catalyst to diglyceride and reacting with trichloroacetonitrile, and obtaining an imine intermediate after purification;

[0011] D. adding the O-phosphoethanolamine salt with a protecting group to the imine intermediate to obtain phosphatidylethanolamine with a protecting group;

[0012] E. Deprotecting and refining the phosphatidylethanolamine with a protected group to obtain phosphatidylethanolamine.

[0013] Preferably, in step A, the molar ratio of the O-phosphoethanolamine to the alkaline substance is 1:1.1-2.5; in step B, the molar ratio of the O-phosphoethanolamine salt to the amine protecting agent is 1:1-2; in step C, the molar ratio of the diglyceride, the catalyst, and the trichloroacetonitrile is 1:1-2:2-6; in step D, the molar ratio of the O-phosphoethanolamine salt with a protecting group to the imine intermediate is 1:0.7-2; in step E, the deprotection reaction includes a reaction with a deprotecting agent.

[0014] After obtaining the imine intermediate in step C, the O-phosphoethanolamine salt with a protecting group can be directly added thereto without separation.

[0015] Preferably, in step A, the alkaline substance includes one or more of potassium carbonate, potassium bicarbonate, potassium hydroxide, sodium carbonate, sodium bicarbonate, and sodium hydroxide; in step B, the amino protecting group includes one or more of Boc protecting group, Fmoc protecting group, and Cbz protecting group; in step B, the fatty acid chain of the diglyceride includes one or more of C10 to C24, and the catalyst includes 1,8-diazabicycloundec-7-ene; in step E, the specific operation of reacting with a deprotecting agent for deprotection includes: dissolving the phosphatidylethanolamine with a protecting group in a deprotection reaction solvent and a mixed solution of the deprotecting agent, and stirring the reaction.

[0016] The alkaline substance is generally potassium salt, sodium salt or its hydroxide. Other metal alkalines have poor solubility in ester solvents, poor reaction effect or even no reaction, making it difficult to convert O-phosphoethanolamine into O-phosphoethanolamine salt.

[0017] Preferably, in step A, the O-phosphoethanolamine is first dissolved in water before the reaction; in step B, the amino protecting agent is first dissolved in a protecting agent solvent before the reaction, including one or more of dioxane, tetrahydrofuran, and dimethylformamide; in step D, the O-phosphoethanolamine salt with a protecting group is first dissolved in a reaction solvent before the reaction, and the reaction solvent includes one or more of chloroform and dichloromethane; in step E, the deprotection reaction solvent includes one or more of chloroform and dichloromethane, and the deprotecting agent includes one or more of trifluoroacetic acid, acetic acid, and piperidine.

[0018] Preferably, in step A, the amount of water includes 2 to 10 times the mass of the O-phosphoethanolamine; in step B, the volume ratio of the amount of the protective agent solvent to the water includes 1:1 to 4; in step D, the volume mass ratio of the O-phosphoethanolamine salt with a protecting group to the reaction solvent includes 1g:5 to 10ml; in step E, the volume ratio of the deprotection reaction solvent to the deprotecting agent includes 3 to 9:1.

[0019] The amino protective agent can be directly added to water, or dissolved in a protective agent solvent first. The protective agent is first dissolved in a protective agent solvent and then added to water containing ethanolamine phosphate, which is better than adding the protective agent directly to water for reaction, and is also better than adding the protective agent directly to a mixture of water and protective agent solvent. The protective agent solvent has a solubilizing effect on the amino protective agent, but adding too much will cause the precipitation of alkaline substances, resulting in a decrease in pH, which is not conducive to the reaction of the protective group. Therefore, the amount of protective agent solvent should be kept in a suitable ratio with water.

[0020] Preferably, in step A, the reaction conditions include continuous stirring at an ice bath or room temperature for 0.5 to 1 hour; in step B, the reaction conditions include continuous stirring at room temperature for 4 to 12 hours; in step C, the reaction conditions include continuous stirring at room temperature for 6 to 18 hours; in step D, the reaction conditions include continuous stirring at room temperature for 2 to 8 hours. In step E, the reaction conditions include continuous stirring at room temperature for 20-30 minutes.

[0021] Step A releases reaction heat during the reaction process. When preparing in large quantities, an ice bath can lower the temperature of the entire system and make the reaction proceed more safely.

[0022] Preferably, in step E, the specific operation of the purification includes: using a solvent to pulp and filter the deprotected sample to obtain the phosphatidylethanolamine with a purity greater than 98%.

[0023] Preferably, in step E, the beating time is 0.5 to 1 h.

[0024] Preferably, in step E, the solvent comprises one or more of acetone and ethyl acetate.

[0025] A phosphatidylethanolamine product obtained by the above-mentioned synthesis method of phosphatidylethanolamine.

[0026] Compared with the prior art, the present invention has the following beneficial effects:

[0027] The present invention has the following characteristics: (1) the raw materials for synthesis are relatively cheap and readily available; (2) the reaction conditions are mild and the selectivity is good; and (3) the requirements for equipment are relatively low and industrial production is easy to realize. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 Schematic diagram of the reaction process of Example 1 of the present invention.

[0029] Figure 2 Schematic diagram of the reaction process of Example 2 of the present invention.

[0030] Figure 3 Schematic diagram of the reaction process of Example 3 of the present invention. DETAILED DESCRIPTION

[0031] In order 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.

[0032] Example 1

[0033] like Figure 1 As shown, 70.5 g (0.5 mol) of O-phosphoethanolamine was taken, dissolved in 250 ml of water, 75.9 g (0.55 mol) of potassium carbonate was added, and stirred for 0.8 hours under ice bath to obtain potassium salt of O-phosphoethanolamine (PE-K). 218 g (1 mol) of Boc anhydride was dissolved in 150 ml of dioxane, and added to the PE-K aqueous solution, stirred for 12 hours to obtain crude Boc-phosphoethanolamine potassium salt (Boc-PE-K crude product). Boc-PE-K was concentrated to dryness, dissolved in 200 ml of ethanol, filtered, and concentrated to obtain 158.14 g (0.499 mol) of Boc-PE-K.

[0034] 312 g (0.5 mol) of 1,2-stearate glyceryl was dissolved in 1 L of dichloromethane, 304 g of 1,8-diazabicycloundec-7-ene (DBU, 1 mol) and 416 g of trichloroacetonitrile (2.89 mol) were added, and the mixture was stirred at room temperature for 9 h to obtain a crude imine intermediate.

[0035] 158.14 g of Boc-PE-K (0.499 mol) was dissolved in 800 ml of dichloromethane, added to the prepared imine, and stirred at room temperature for 3 h to obtain crude Boc-DSPE. 200 ml of trifluoroacetic acid was added, stirred at room temperature for 30 min, concentrated, and 1 L of acetone was added for pulping. After suction filtration, the pulping was repeated twice and dried to obtain 266.8 g of DSPE (purity 98.6%) with a yield of 71.3%.

[0036] Example 2

[0037] like Figure 2 As shown, 70.5 g (0.5 mol) of O-phosphoethanolamine was taken, dissolved in 700 ml of water, 67.2 g (1.2 mol) of potassium hydroxide was added, and stirred for 0.5 hours under ice bath to obtain potassium salt of O-phosphoethanolamine (PE-K). 154 g (0.6 mol) of Fmoc-Cl was dissolved in 350 ml of tetrahydrofuran, and added to the PE-K aqueous solution, stirred for 4 hours to obtain crude potassium salt of Fmoc-phosphoethanolamine (crude Fmoc-PE-K). Fmoc-PE-K was concentrated to dryness, dissolved in 200 ml of ethanol, filtered, concentrated again, suspended in 200 ml of acetone, and filtered to obtain 218.6 g (0.49 mol) of Fmoc-PE-K.

[0038] Take 284 g (0.5 mol) of 1,2-palmitoylglycerol and dissolve it in 0.5 L of chloroform, add 152 g of 1,8-diazabicycloundec-7-ene (DBU, 0.5 mol) and 288 g of trichloroacetonitrile (2 mol), and stir at room temperature for 9 hours to obtain a crude imine intermediate.

[0039] 218.6 g (0.49 mol) of Fmoc-PE-K was dissolved in 1.1 L of chloroform, added to the prepared imine, and stirred at room temperature for 6 h to obtain crude Fmoc-DPPE. 500 ml of piperidine was added, stirred at room temperature for 30 min, concentrated, and 1 L of ethyl acetate was added for slurrying. The mixture was repeated twice. After filtration, 229.9 g of DPPE (purity 98.4%) was obtained, with a yield of 66.5%.

[0040] Example 3

[0041] like Figure 3As shown, 141g (1 mol) of O-phosphoethanolamine was taken, dissolved in 700ml of water, 63.6g (0.6mol) of sodium carbonate was added, and stirred for reaction for 1 hour under ice bath to obtain sodium salt of O-phosphoethanolamine (PE-Na). 187g (1.1mol) of Cbz-Cl was dissolved in 200ml of dimethylformamide, and added to the PE-Na aqueous solution, stirred for reaction for 4h, and Cbz-phosphoethanolamine potassium salt crude product (Cbz-PE-Na crude product) was obtained. Cbz-PE-Na was concentrated to dryness, dissolved in 400ml of ethanol, filtered, concentrated again, suspended in 400ml of acetone, and filtered to obtain 258.3g (0.9mol) of Cbz-PE-Na.

[0042] Take 400 g (1 mol) of 1,2-decyl glyceride and dissolve it in 1.5 L of dichloromethane, add 608 g of 1,8-diazabicycloundec-7-ene (DBU, 2 mol) and 432 g of trichloroacetonitrile (3 mol) and stir at room temperature for 18 h to obtain a crude imine intermediate.

[0043] 258.3 g (0.9 mol) of Cbz-PE-Na was dissolved in 1 L of dichloromethane, added to the prepared imine, and stirred at room temperature for 6 h to obtain crude Cbz-DDPE. 500 ml of trifluoroacetic acid was added, stirred at room temperature for 30 min, concentrated, and 1.5 L of ethyl acetate was added for slurrying. The mixture was repeated twice. After filtration, 315.4 g of DDPE (purity 98.2%) was obtained, with a yield of 60.3%.

[0044] Comparative Example 1

[0045] Mix 0.20ml (3.3mmol) ethanolamine, 0.75ml (5.4mmol) triethylamine and 10ml chloroform, add 1.07g (3.8mmol) triphenylmethane and stir for 4h to obtain ethanolamine with amine protective agent. Place 0.60ml (4.3mmol) triethylamine, 0.37ml (4.0mmol) phosphorus oxychloride and 20ml chloroform in an ice bath and pass nitrogen for protection. Dissolve 2.0g (3.2mmol) distearic acid glycerol in 20ml chloroform and add dropwise to the reaction bottle. Stir for 3h, then heat to room temperature and continue stirring for 4h. Then add ethanolamine with amine protective agent and continue to react for 4h. Then, chloroform-trifluoroacetic acid (3:1) was added, the pH was adjusted to 1, and the reaction mixture was stirred at room temperature for 10 min to remove the protection. A sodium carbonate aqueous solution was added and stirred until the pH was 6-7. The organic layer was concentrated and separated by column chromatography. The mixture was eluted with chloroform-ethanol-water (400:100:5) to obtain 0.50 g of DSPE (purity 98.5%) with a yield of 20.0%.

[0046] Comparative Example 2

[0047] Take 5g of the phospholipid mixture, dissolve it with 20ml of chloroform, add 0.015g of triethylamine and 4g of triphenylmethane, stir and react at room temperature for 2 hours to obtain the intermediate mixture 1 containing Tr-PE, and recover the chloroform. Use 20ml of methanol to dissolve the intermediate mixture 1, add 0.5g of sodium methoxide, place it at 50℃ and stir for alcoholysis for 4 hours to obtain the intermediate mixture 2 containing GPC and Tr-GPE. After silica gel column chromatography and elution with chloroform-ethanol-water (400:100:5), 0.71 g of Tr-GPE was obtained, which was dissolved in 10 ml of chloroform, and 0.96 g of stearic acid, 0.45 g of DMAP and 0.76 g of DCC were added. The mixture was stirred at 50°C for reaction. After the reaction was completed, 1.41 g of Tr-DSPE was obtained after silica gel column chromatography and elution with chloroform-ethanol-water (400:100:5). The mixture was dissolved in 14 ml of chloroform-trifluoroacetic acid (3:1), and deprotected by stirring at room temperature. A sodium carbonate aqueous solution was added and stirred to pH 6-7. The organic layer was taken and 20 ml of acetone was added for crystallization to obtain 0.95 g of DSPE (purity 99.2%) with a yield of 19%.

[0048] By comparing Example 1 with Comparative Example 1 and Comparative Example 2, the yield of the synthesis method of the present invention is much higher than that of Comparative Example 1 using the traditional process, and the raw materials have the characteristics of selectivity, no acidic and corrosive gas generation, etc. Compared with phosphorus oxychloride, Example 1 is simpler, the steps are shorter, the production process is simpler, and it is conducive to industrial production.

[0049] The above disclosure is only the preferred embodiment of the present invention, which cannot be used to limit the scope of the present invention. Therefore, equivalent changes made according to the claims of the present invention are still within the scope of the present invention.

Claims

1. A method for synthesizing phosphatidylethanolamine, characterized in that: The steps include: A. Adding alkaline substances to O-phosphoethanolamine to react, thereby obtaining O-phosphoethanolamine salt; B. adding an amine protecting agent to the O-phosphoethanolamine salt to obtain an O-phosphoethanolamine salt with a protecting group; C. Adding a catalyst to diglyceride and reacting with trichloroacetonitrile to obtain an imine intermediate; D. adding the O-phosphoethanolamine salt with a protecting group to the imine intermediate to obtain phosphatidylethanolamine with a protecting group; E. Deprotecting and refining the phosphatidylethanolamine with a protected group to obtain phosphatidylethanolamine.

2. The method for synthesizing phosphatidylethanolamine according to claim 1, characterized in that: In step A, the molar ratio of the O-phosphoethanolamine to the alkaline substance is 1:1.1-2.5; in step B, the molar ratio of the O-phosphoethanolamine salt to the amine protective agent is 1:1-2; in step C, the molar ratio of the diglyceride, the catalyst, and the trichloroacetonitrile is 1:1-2:2-6; in step D, the molar ratio of the O-phosphoethanolamine salt with a protecting group to the imine intermediate is 1:0.7-2; in step E, the deprotection reaction includes one or more of hydrogenation deprotection and deprotection by reaction with a deprotecting agent.

3. The method for synthesizing phosphatidylethanolamine according to claim 2, characterized in that: In step A, the alkaline substance includes one or more of potassium carbonate, potassium bicarbonate, potassium hydroxide, sodium carbonate, sodium bicarbonate, and sodium hydroxide; in step B, the amino protecting group includes one or more of Boc protecting group, Fmoc protecting group, and Cbz protecting group; in step B, the fatty acid chain of the diglyceride includes one or more of C10 to C24, and the catalyst includes 1,8-diazabicycloundec-7-ene; in step E, the specific operation of reacting with a deprotecting agent for deprotection includes: dissolving the phosphatidylethanolamine with a protecting group in a deprotection reaction solvent and a mixed solution of the deprotecting agent, and stirring the reaction.

4. The method for synthesizing phosphatidylethanolamine according to claim 3, characterized in that: In step A, the O-phosphoethanolamine is first dissolved in water before the reaction; in step B, the amino protecting agent is first dissolved in a protecting agent solvent before the reaction, and the protecting agent solvent includes one or more of dioxane, tetrahydrofuran, and dimethylformamide; in step D, the O-phosphoethanolamine salt with a protecting group is first dissolved in a reaction solvent before the reaction, and the reaction solvent includes one or more of chloroform and dichloromethane; in step E, the deprotection reaction solvent includes one or more of chloroform and dichloromethane, and the deprotecting agent includes one or more of trifluoroacetic acid, acetic acid, and piperidine.

5. The method for synthesizing phosphatidylethanolamine according to claim 4, characterized in that: In step A, the amount of water is 2 to 10 times the mass of the O-phosphoethanolamine; in step B, the volume ratio of the amount of the protective agent solvent to the water is 1:1 to 4; in step D, the volume mass ratio of the O-phosphoethanolamine salt with a protecting group to the reaction solvent is 1g:5 to 10ml; in step E, the volume ratio of the deprotection reaction solvent to the deprotecting agent is 3 to 9:

1.

6. The method for synthesizing phosphatidylethanolamine according to claim 1, characterized in that: In step A, the reaction conditions include continuous stirring in an ice bath or at room temperature, and the reaction time is 0.5 to 1 hour; in step B, the reaction conditions include continuous stirring at room temperature, and the reaction time is 4 to 12 hours; in step C, the reaction conditions include continuous stirring at room temperature, and the reaction time is 6 to 18 hours; in step D, the reaction conditions include continuous stirring at room temperature, and the time is 2 to 8 hours; in step E, the reaction conditions include continuous stirring at room temperature, and the time is 20 to 30 minutes.

7. The method for synthesizing phosphatidylethanolamine according to claim 1, characterized in that: In step E, the specific operation of the purification includes: using a solvent to pulp and filter the deprotected sample to obtain the phosphatidylethanolamine with a purity greater than 98%.

8. The method for synthesizing phosphatidylethanolamine according to claim 7, characterized in that: In step E, the beating time is 0.5 to 1 hour.

9. The method for synthesizing phosphatidylethanolamine according to claim 7, characterized in that: In step E, the solvent includes one or more of acetone and ethyl acetate.

10. A phosphatidylethanolamine product obtained by the method for synthesizing phosphatidylethanolamine according to claim 1.

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