Synthesis method of dipalmitoyl phosphatidic acid
Patent Information
- Application Number
- CN202511801049.1
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-12-02
- Publication Date
- 2026-03-13
AI Technical Summary
The existing methods for preparing dipalmitoyl phosphatidic acid have harsh conditions, cumbersome post-processing procedures, low yield, high environmental pressure, and complex processes.
Phospholipase D is used to catalyze the enzyme reaction of dipalmitoylphosphatidylcholine, combined with the purification process, including dissolution, filtration, and recrystallization, to simplify the reaction route and improve the yield.
This method achieves the synthesis of dipalmitoyl phosphatidyl acid, which features simple process conditions, easy operation, short route, high repeatability, and high overall yield, making it suitable for industrial production.
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Abstract
Description
Technical Field
[0001] This invention relates to the field of compound preparation, and more specifically, to a method for synthesizing dipalmitoylphosphatidic acid. Background Technology
[0002] Dipalmitoyl phosphatidyl acid (DPPA), also known as 1,2-hexadecanoyl-sn-glycerol-3-phosphate (sodium salt), is an important member of the synthetic phospholipid family, with its technological applications primarily focused on the fields of medicine and materials science. Dipalmitoyl phosphatidyl acid (DPPA) is composed of a dipalmitoyl hydrophobic chain and a choline hydrophilic head group, enabling it to self-assemble into a stable lipid bilayer. This property makes it an ideal material for constructing drug delivery systems such as liposomes and nanocarriers. In the field of drug delivery, DPPA can be used to prepare liposomes with high encapsulation efficiency through thin-film dispersion or pH gradient methods, significantly improving the solubility and targeting of hydrophobic drugs. For example, it is used as a core excipient in the antitumor drug verteporfen liposome (Visudyne). Furthermore, its biocompatibility advantages have made it a focus of attention in the development of gene therapy vectors and vaccine adjuvants. At the basic research level, DPPA's phase transition temperature (approximately 23°C) is close to physiological conditions, making it a classic model molecule for simulating the dynamic behavior of cell membranes, used to study membrane protein interactions and drug transmembrane mechanisms. With the development of precision medicine, DPPA can be further optimized for carrier performance through chemical modification, showing great potential in cutting-edge fields such as tumor targeting and transdermal drug delivery.
[0003] The existing preparation methods for dipalmitoyl phosphatidyl acid are subject to harsh conditions, have drawbacks such as cumbersome post-processing procedures, low yield, high environmental pressure, and complex processes. Summary of the Invention
[0004] In view of this, the present invention aims to provide a method for synthesizing dipalmitoylphosphatidic acid to solve the problems of low yield, high environmental pressure and complex process in the prior art.
[0005] To solve the above-mentioned technical problems, this application is implemented as follows:
[0006] This invention provides a method for synthesizing dipalmitoylphosphatidic acid, comprising the following steps:
[0007] (1) Dipalmitoylphosphatidylcholine is reacted with enzymes under the action of phospholipase to obtain crude dipalmitoylphosphatidyl acid;
[0008] (2) Dipalmitoyl phosphatidyl acid crude product was purified to obtain dipalmitoyl phosphatidyl acid.
[0009] In this invention, the phospholipase includes phospholipase D.
[0010] In this invention, phospholipase D is an enzyme that catalyzes the hydrolysis of the ester bond between phosphate and organic bases (such as choline, ethanolamine, etc.) in phospholipid molecules, and its hydrolysis products are phosphatidic acid and organic bases. It can achieve hydrolysis under very mild conditions. This invention uses this phospholipase to hydrolyze dipalmitoylphosphatidylcholine into crude dipalmitoylphosphatidyl acid, which simplifies the reaction route and is suitable for industrial production.
[0011] The synthesis method according to the present invention has the characteristics of simple process conditions, easy operation, short route, high repeatability, high overall yield, and is conducive to industrial production.
[0012] Preferably, in the above-mentioned method for synthesizing dipalmitoylphosphatidic acid, the enzyme-catalyzed reaction process includes:
[0013] Dipalmitoylphosphatidylcholine, phospholipase, acetic acid (providing a weakly acidic environment for the enzymatic reaction), and the first solvent are mixed to carry out the enzymatic catalytic reaction.
[0014] Phospholipases are a class of enzymes that catalyze the hydrolysis of the ester bonds between phosphate groups and organic bases (such as choline and ethanolamine) in phospholipid molecules, thus promoting the hydrolysis reaction. The reaction equation for the hydrolysis reaction is shown below:
[0015]
[0016] Preferably, in the above-mentioned method for synthesizing dipalmitoylphosphatidic acid, the weight ratio of dipalmitoylphosphatidylcholine, the phospholipase, and the acetic acid is 1:(2-3):(0.09-0.15). Within this range, the enzyme solution exhibits the highest enzymatic reaction efficiency; below this range, the conversion rate is low; above this range, the conversion rate shows virtually no improvement. Within this range, the acetic acid exhibits the highest enzymatic reaction efficiency; below this range, the conversion rate is low; above this range, high acidity leads to decreased enzyme activity and a low conversion rate.
[0017] The weight ratio of dipalmitoylphosphatidylcholine to the first solvent is 1:(5-8);
[0018] The first solvent includes chloroform and / or dichloroform.
[0019] In this invention, by dissolving the raw materials in the first solvent, the hydrolysis reaction can be better promoted.
[0020] Preferably, in the above-mentioned method for synthesizing dipalmitoylphosphatidic acid, the temperature of the enzyme-catalyzed reaction is 20-30°C, and the time of the enzyme-catalyzed reaction is 4-8 hours.
[0021] Preferably, in the above-mentioned method for synthesizing dipalmitoylphosphatidic acid, after the enzyme-catalyzed reaction is completed, the method further includes:
[0022] Add sodium hydroxide solution to the obtained product and stir for 2-4 hours; then allow to stand and separate into layers.
[0023] After separation, ethanol is added to the organic phase, and the mixture is stirred for 0.5–1 hour.
[0024] After cooling to below 15°C, stir for 2-4 hours, filter, and dry under reduced pressure to obtain the crude product.
[0025] Preferably, in the above-mentioned method for synthesizing dipalmitoylphosphatidic acid, the purification process includes:
[0026] The crude dipalmitoyl phosphatidyl acid was dissolved in a mixed solvent of dichloromethane, acetone, and ethanol, cooled, and then crystallized. Solid-liquid separation and vacuum drying were then performed.
[0027] In this invention, the purification process can effectively remove unreacted raw materials and some of the byproducts generated by the reaction. Compared with traditional column chromatography, this method is simpler and easier to operate.
[0028] Preferably, the mass ratio of the crude dipalmitoyl phosphatidyl acid, the dichloromethane, the acetone, and the ethanol is 1:(5-10):(5-10):(2:4).
[0029] Preferably, in the above-mentioned method for synthesizing dipalmitoylphosphatidic acid, the dissolution temperature is 30-40°C, the first dispersion time is 0.5-1 hour, and the dissolution rotation speed is 50-100 rpm.
[0030] Preferably, in the above-mentioned purification method of dipalmitoyl phosphatidic acid, the temperature of the first cooling crystallization is 10-20°C, the time of the cooling crystallization is 1-2 hours, and the rotation speed of the second dispersion is 80-120 rpm.
[0031] Preferably, in the above-mentioned purification method of dipalmitoyl phosphatidyl acid, the conditions for vacuum drying are: pressure of -0.07 to -0.1 MPa, temperature of 30 to 40°C, and time of 8 to 10 hours.
[0032] The beneficial technical effects of the present invention through the above technical solution are as follows:
[0033] This invention overcomes the shortcomings of existing methods for preparing dipalmitoyl phosphatidyl acid, such as harsh conditions, cumbersome post-processing, low yield, and high production cost. It provides a purification and preparation method for dipalmitoyl phosphatidyl acid. This invention uses dipalmitoyl phosphatidylcholine as raw material, which undergoes an enzymatic reaction followed by a salt formation reaction. The resulting product is dissolved, filtered, and recrystallized to obtain high-purity dipalmitoyl phosphatidyl acid. The reaction conditions are mild, the operation is safe and simple, the route is short, the post-processing is simple, and the yield is high, making it suitable for industrial production. Attached Figure Description
[0034] The accompanying drawings are provided to further illustrate the invention and form part of the specification. They are used together with the following detailed description to explain the invention, but do not constitute a limitation thereof.
[0035] Figure 1 This is a liquid phase content detection chromatogram of the dipalmitoyl phosphatidyl acid product prepared in Example 1. Detailed Implementation
[0036] Unless otherwise specified, all embodiments and optional embodiments of this application can be combined to form new technical solutions.
[0037] Unless otherwise specified, all technical features and optional technical features of this application may be combined to form new technical solutions.
[0038] The present invention will be further described in detail below through examples. All raw materials used in the examples are commercially available.
[0039] In the following examples and comparative examples, the phospholipase was phospholipase D, purchased from Shanghai Yunluo Biotechnology Co., Ltd.
[0040] Example 1
[0041] The synthesis of dipalmitoylphosphatidic acid includes the following steps:
[0042] 50g dipalmitoylphosphatidylcholine, 150g phospholipase, 5g acetic acid, and 300g chloroform were mixed and subjected to an enzymatic reaction at 30℃ for 8 hours. After the reaction was completed, 200g of 5% sodium hydroxide solution was added and stirred for 2-4 hours. The mixture was allowed to stand and separate into layers. 50g of ethanol was added to the organic phase and stirred for 0.5-1 hour. The mixture was then cooled to below 15℃ and stirred for 2 hours. The mixture was filtered, and the resulting product was dried under reduced pressure (25℃, -0.09MPa) to obtain 41.5g of crude dipalmitoylphosphatidyl acid, with a yield of 91%.
[0043] 40g of crude dipalmitoyl phosphatidyl acid was added to a glass reaction flask, followed by 200g of dichloromethane, 200g of acetone, and 105g of ethanol. The mixture was stirred at 34°C and 60rpm to dissolve the solution. The temperature was then lowered to 12°C, and the mixture was stirred at 80rpm for 1 hour to induce crystallization. The solid was then filtered and dried under reduced pressure at 30°C for 8 hours to obtain 36g of finished dipalmitoyl phosphatidyl acid, with a yield of 90%.
[0044] The liquid phase content detection chromatogram of the dipalmitoyl phosphatidyl acid product obtained in Example 1 above is shown below. Figure 1 As shown. By Figure 1 It can be seen that the present invention has successfully synthesized dipalmitoylphosphatidic acid.
[0045] Example 2
[0046] 50g dipalmitoylphosphatidylcholine, 150g phospholipase, 4.7g acetic acid, and 300g chloroform were mixed and subjected to an enzyme-catalyzed reaction at 30℃ for 8 hours. After the reaction was completed, 200g of 5% sodium hydroxide solution was added and stirred for 3 hours. The mixture was allowed to stand and separate into layers. 50g of ethanol was added to the organic phase and stirred for 1 hour. The mixture was then cooled to below 15℃ and stirred for 3 hours. The mixture was filtered, and the resulting product was dried under reduced pressure at 30℃ to obtain 41.2g of crude dipalmitoylphosphatidyl acid, with a yield of 90%.
[0047] Add 40g of crude dipalmitoyl phosphatidyl acid to a glass reaction flask, then add 200g of dichloromethane, 200g of acetone, and 100g of ethanol. Dissolve the solution by stirring at 50-100 rpm at 30°C. Then cool the mixture to 10-20°C and stir at 80-120 rpm for 1-2 hours to induce crystallization. Filter the solid to obtain the final product. Dry the solid under reduced pressure (30-40°C) for 8-10 hours to obtain 36.2g of finished dipalmitoyl phosphatidyl acid, with a yield of 90.5%.
[0048] Example 3
[0049] 50g dipalmitoylphosphatidylcholine, 130g phospholipase, 5.0g acetic acid, and 300g chloroform were mixed and subjected to an enzyme-catalyzed reaction at 30℃ for 8 hours. After the reaction was completed, 200g of 5% sodium hydroxide solution was added and stirred for 2 hours. The mixture was allowed to stand and separate into layers. 50g of ethanol was added to the organic phase and stirred for 1 hour. The mixture was then cooled to below 15℃ and stirred for 3 hours. The mixture was filtered, and the resulting product was dried under reduced pressure at 30℃ to obtain 41.8g of crude dipalmitoylphosphatidyl acid, with a yield of 91.5%.
[0050] 40g of crude dipalmitoyl phosphatidyl acid was added to a glass reaction flask, followed by 200g of dichloromethane, 200g of acetone, and 100g of ethanol. The mixture was stirred at 30°C and 50-100 rpm to dissolve the solution. The temperature was then lowered to 20°C, and the mixture was stirred at 100 rpm for 2 hours to induce crystallization. The solid was then filtered and dried under reduced pressure at 35°C and -0.08 MPa for 8 hours to obtain 35.8g of finished dipalmitoyl phosphatidyl acid, with a yield of 89.5%.
[0051] Example 4
[0052] 50g dipalmitoylphosphatidylcholine, 115g phospholipase, 5.0g acetic acid, and 290g chloroform were mixed and subjected to an enzyme-catalyzed reaction at 30℃ for 7 hours. After the reaction was completed, 200g of 5% sodium hydroxide solution was added and stirred for 2 hours. The mixture was allowed to stand and separate into layers. 50g of ethanol was added to the organic phase and stirred for 1 hour. The mixture was then cooled to below 15℃ and stirred for 2 hours. The mixture was filtered, and the resulting product was dried under reduced pressure at 30℃ to obtain 40.6g of crude dipalmitoylphosphatidyl acid, with a yield of 88.9%.
[0053] 40g of crude dipalmitoyl phosphatidyl acid was added to a glass reaction flask, followed by 200g of dichloromethane, 200g of acetone, and 110g of ethanol. The mixture was stirred at 40℃ for 50-100 rpm to dissolve the solution. The temperature was then lowered to 15℃, and the mixture was stirred at 100 rpm for 2 hours to induce crystallization. The solid was then filtered and dried under reduced pressure at 30℃ and -0.08 MPa for 10 hours to obtain 36.5g of finished dipalmitoyl phosphatidyl acid, with a yield of 91%.
[0054] Example 5
[0055] 50g dipalmitoylphosphatidylcholine, 100g phospholipase, 4.7g acetic acid, and 300g chloroform were mixed and subjected to an enzyme-catalyzed reaction at 30℃ for 8 hours. After the reaction was completed, 200g of 5% sodium hydroxide solution was added and stirred for 3 hours. The mixture was allowed to stand and separate into layers. 50g of ethanol was added to the organic phase and stirred for 1 hour. The mixture was then cooled to below 15℃ and stirred for 3 hours. The mixture was filtered, and the resulting product was dried under reduced pressure at 30℃ to obtain 41.2g of crude dipalmitoylphosphatidyl acid, with a yield of 90%.
[0056] 40g of crude dipalmitoyl phosphatidyl acid was added to a glass reaction flask, followed by 200g of dichloromethane, 200g of acetone, and 100g of ethanol. The mixture was stirred at 30°C and 50-100 rpm to dissolve the solution. The temperature was then lowered to 10°C, and the mixture was stirred at 80-120 rpm for 1-2 hours to induce crystallization. The solid was then filtered and dried under reduced pressure at 30°C and -0.08 MPa for 10 hours to obtain 36.2g of finished dipalmitoyl phosphatidyl acid, with a yield of 90.5%.
[0057] Comparative Example 1
[0058] 50g dipalmitoylphosphatidylcholine, 80g phospholipase, 5g acetic acid, and 300g chloroform were mixed and subjected to an enzyme-catalyzed reaction at 30℃ for 8 hours. After the reaction was completed, 200g of 5% sodium hydroxide solution was added and stirred for 3 hours. The mixture was allowed to stand and separate into layers. 50g of ethanol was added to the organic phase and stirred for 1 hour. The mixture was then cooled to below 15℃ and stirred for 3 hours. The mixture was filtered, and the resulting product was dried under reduced pressure at 30℃ to obtain 31.2g of crude dipalmitoylphosphatidyl acid, with a yield of 68%.
[0059] Add 30g of crude dipalmitoyl phosphatidyl acid to a glass reaction flask, then add 150g of dichloromethane, 150g of acetone, and 65g of ethanol. Dissolve the solution by stirring at 50-100 rpm at 30°C. Then cool the mixture to 10-20°C and stir at 80-120 rpm for 1-2 hours to induce crystallization. Filter the solid to obtain the final product. Dry the solid under reduced pressure at 30°C and -0.08 MPa for 10 hours to obtain 25.2g of finished dipalmitoyl phosphatidyl acid, with a yield of 84%.
[0060] Comparative Example 2
[0061] 50g dipalmitoylphosphatidylcholine, 120g phospholipase, 2g acetic acid, and 300g chloroform were mixed and subjected to an enzyme-catalyzed reaction at 30°C for 8 hours. After the reaction was completed, 200g of 5% sodium hydroxide solution was added and stirred for 3 hours. The mixture was allowed to stand and separate into layers. 50g of ethanol was added to the organic phase and stirred for 1 hour. The mixture was then cooled to below 15°C and stirred for 3 hours. The mixture was filtered, and the resulting product was dried under reduced pressure at 30°C to obtain 25g of crude dipalmitoylphosphatidyl acid, with a yield of 54.7%.
[0062] 20g of crude dipalmitoyl phosphatidyl acid was added to a glass reaction flask, followed by 80g of dichloromethane, 80g of acetone, and 80g of ethanol. The mixture was stirred at 30°C and 50-100 rpm to dissolve the solution. The temperature was then lowered to 10°C-20°C, and the mixture was stirred at 80-120 rpm for 1-2 hours to induce crystallization. The solid was then filtered and dried under reduced pressure (30-40°C) for 8-10 hours to obtain 16.4g of finished dipalmitoyl phosphatidyl acid, with a yield of 82%.
[0063] Comparative Example 3
[0064] 50g dipalmitoylphosphatidylcholine, 100g phospholipase, 15g acetic acid, and 300g chloroform were mixed and subjected to an enzyme-catalyzed reaction at 30℃ for 8 hours. After the reaction was completed, 200g of 5% sodium hydroxide solution was added and stirred for 3 hours. The mixture was allowed to stand and separate into layers. 50g of ethanol was added to the organic phase and stirred for 1 hour. The mixture was then cooled to below 15℃ and stirred for 3 hours. The mixture was filtered, and the resulting product was dried under reduced pressure at 30℃ to obtain 23.5g of crude dipalmitoylphosphatidyl acid, with a yield of 51.4%.
[0065] 20g of crude dipalmitoyl phosphatidyl acid was added to a glass reaction flask, followed by 200g of dichloromethane, 200g of acetone, and 100g of ethanol. The mixture was stirred at 30°C and 50-100 rpm to dissolve the solution. The temperature was then lowered to 10°C-20°C, and the mixture was stirred at 80-120 rpm for 1-2 hours to induce crystallization. The solid was then filtered and dried under reduced pressure (30-40°C) for 8-10 hours to obtain 17.1g of finished dipalmitoyl phosphatidyl acid, with a yield of 85.5%.
[0066] Comparative Example 4
[0067] 50g dipalmitoylphosphatidylcholine, 200g phospholipase, 5.0g acetic acid, and 300g chloroform were mixed and subjected to an enzyme-catalyzed reaction at 30℃ for 7 hours. After the reaction was completed, 200g of 5% sodium hydroxide solution was added and stirred for 2 hours. The mixture was allowed to stand and separate into layers. 50g of ethanol was added to the organic phase and stirred for 1 hour. The mixture was then cooled to below 15℃ and stirred for 2 hours. The mixture was filtered, and the resulting product was dried under reduced pressure at 30℃ to obtain 41.5g of crude dipalmitoylphosphatidyl acid, with a yield of 91%.
[0068] 40g of crude dipalmitoyl phosphatidyl acid was added to a glass reaction flask, followed by 200g of dichloromethane, 200g of acetone, and 110g of ethanol. The mixture was stirred at 40°C and 50-100 rpm to dissolve the solution. The temperature was then lowered to 15°C, and the mixture was stirred at 100 rpm for 2 hours to induce crystallization. The solid was then filtered and dried under reduced pressure at 30°C for 8-10 hours to obtain 36.4g of finished dipalmitoyl phosphatidyl acid, with a yield of 91%.
[0069] Test Example 1
[0070] Comparative Example 1 Comparative Example 2 Comparative Example 3 Comparative Example 4 crude product yield 68％ 54.7％ 51.4％ 91％ Finished product yield 84％ 82％ 85.％ 91％
[0071] Comparative Example 1: The enzyme solution weight ratio was 1.6, resulting in low conversion rate of the enzymatic reaction and low yield of crude product.
[0072] Comparative Example 2: The weight proportion of acetic acid was relatively low, resulting in a low conversion rate of the enzymatic reaction and a low yield of crude product.
[0073] Comparative Example 3: The acetic acid weight ratio was relatively high, resulting in a low conversion rate of the enzymatic reaction and a low yield of crude product.
[0074] Comparative Example 1: The enzyme solution weight ratio was 4, the enzymatic reaction conversion rate was low, the crude product yield was 91%, and there was no significant increase.
[0075] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.
Claims
1. A method for synthesizing dipalmitoylphosphatidic acid, characterized in that, Includes the following steps: (1) Dipalmitoylphosphatidylcholine is reacted with enzymes under the action of phospholipase to obtain crude dipalmitoylphosphatidyl acid; (2) Dipalmitoyl phosphatidyl acid crude product was purified to obtain dipalmitoyl phosphatidyl acid.
2. The method for synthesizing dipalmitoylphosphatidyl acid according to claim 1, characterized in that, The enzyme-catalyzed reaction process includes: Dipalmitoylphosphatidylcholine, phospholipase, acetic acid, and a first solvent are mixed to carry out an enzyme-catalyzed reaction.
3. The method for synthesizing dipalmitoylphosphatidyl acid according to claim 2, characterized in that, The weight ratio of dipalmitoylphosphatidylcholine, the phospholipase, and the acetic acid is 1:(2-3):(0.09-0.15). The weight ratio of dipalmitoylphosphatidylcholine to the first solvent is 1:(5-8); The first solvent includes chloroform and / or dichloroform.
4. The method for synthesizing dipalmitoylphosphatidic acid according to claim 1, characterized in that, The enzyme-catalyzed reaction is carried out at a temperature of 20–30°C for 4–8 hours.
5. The method for synthesizing dipalmitoylphosphatidyl acid according to claim 1, characterized in that, After the enzyme-catalyzed reaction is completed, the following steps are also included: Add sodium hydroxide solution to the obtained product, stir for 2-4 hours, and then let it stand to separate into layers; After separation, ethanol is added to the organic phase, and the mixture is stirred for 0.5–1 hour. After cooling to below 15°C, stir for 2-4 hours, filter, and dry under reduced pressure to obtain the crude product.
6. The method for synthesizing dipalmitoylphosphatidyl acid according to claim 1, characterized in that, The refining process includes: The crude dipalmitoyl phosphatidyl acid was dissolved in a mixed solvent of dichloromethane, acetone, and ethanol. After cooling, crystallization was continued, followed by solid-liquid separation and vacuum drying.
7. The method for synthesizing dipalmitoylphosphatidyl acid according to claim 6, characterized in that, The mass ratio of the crude dipalmitoyl phosphatidyl acid, the dichloromethane, the acetone, and the ethanol is 1:(5-10):(5-10):(2:4).
8. The method for synthesizing dipalmitoylphosphatidyl acid according to claim 6, characterized in that, The dissolution temperature is 30-40°C, the first dispersion time is 0.5-1 hour, and the dissolution rotation speed is 50-100 rpm.
9. The method for synthesizing dipalmitoylphosphatidyl acid according to claim 6, characterized in that, The temperature for the first cooling crystallization is 10-20°C, the time for the cooling crystallization is 1-2 hours, and the rotation speed for the second dispersion is 80-120 rpm.
10. The method for synthesizing dipalmitoylphosphatidyl acid according to claim 6, characterized in that, The conditions for vacuum drying are: pressure of -0.07 to -0.1 MPa, temperature of 30 to 40°C, and time of 8 to 10 hours.