A method for synthesizing 1,2-bisfatty acyl-sn-glycero-3-phosphatidic acid

Abstract

The application belongs to the technical field of medicine and chemical industry, and particularly relates to a synthesis method of 1,2-bifatty acyl-sn-glycerol-3-phosphatidic acid. The synthesis method of the liposome precursor compound 1,2-bifatty acyl-sn-glycerol-3-phosphatidic acid in the application is to perform a phosphorylation reaction on 1,2-bifatty acyl-sn-glycerol and phosphorus oxychloride as raw materials, and then to perform a hydrolysis reaction to obtain the required 1,2-bifatty acyl-sn-glycerol-3-phosphatidic acid. The two-step phosphorylation-hydrolysis reactions in the whole process can be performed under the bifunctional catalyst system, and the high-efficiency catalytic reaction can be realized without replacing the catalyst, and the product conversion rate is high.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical and chemical technology, specifically relating to a method for synthesizing 1,2-difatty acyl-sn-glycerol-3-phosphatidic acid. Background Technology

[0002] Phosphatidic acid (PA) is a common phospholipid compound and a fundamental component of cell membranes, nuclear membranes, and plasmid membranes in plants and animals, serving as a basic substance for life. In 1927, Chibnall and Channon first isolated phosphatidic acid from cabbage leaves. In 1958, Lowell E. Hokin demonstrated the presence of phosphatidic acid in plant and animal tissues. Although present in small amounts in organisms, phosphatidic acid is an intermediate in the synthesis of glycerides and most phospholipids, and also an important second messenger within cells, finding wide applications in food, pharmaceuticals, and cosmetics.

[0003] 1,2-Difatty acyl-sn-glycerol-3-phosphatidic acid, also known as difatty acyl phosphatidic acid, is a common and important class of phospholipid molecules. Its glycerol backbone has two fatty acyl groups attached to positions 1 and 2, respectively, and a phosphate group attached to position 3. Typical 1,2-difatty acyl-sn-glycerol-3-phosphatidic acids include: 1,2-dispalmitoyl-sn-glycerol-3-phosphatidic acid (DPPA), which is linked to two palmitoyl groups (C16 saturated fatty acid), and 1,2-distearyl-sn-glycerol-3-phosphatidic acid (DSPA), which is linked to two stearoyl groups (C18 saturated fatty acid). 1,2-Difatty acyl-sn-glycerol-3-phosphatidic acids possess amphiphilic and negatively charged properties, playing a crucial role in cell membrane composition, signal transduction, drug delivery, and biological research.

[0004] There are several common methods for preparing difatty acylphosphatidic acid in the prior art. For example, the disclosed method for preparing dipalmitoylphosphatidic acid uses 1,2-dipalmitoyl-sn-glycerol as a raw material, which undergoes esterification and hydrolysis to obtain dipalmitoylphospholipid, followed by purification to obtain dipalmitoylphosphatidic acid. Although this method uses inexpensive raw materials and has mild reaction conditions, the overall conversion rate is not ideal. Therefore, there is a need in the art to develop a synthetic method for difatty acylphosphatidic acid with higher conversion efficiency. Summary of the Invention

[0005] The purpose of this invention is to provide a method for synthesizing liposome precursor compounds, particularly 1,2-difatty acyl-sn-glycerol-3-phosphatidic acid.

[0006] To achieve the above objectives, the present invention provides the following technical solution.

[0007] This invention provides a method for synthesizing 1,2-difatty acyl-sn-glycerol-3-phosphatidic acid, comprising the following steps:

[0008] (1) Using 1,2-difatty acyl-sn-glycerol and phosphorus oxychloride as raw materials, a phosphorylation reaction was carried out in the presence of a bifunctional catalyst to obtain an intermediate reactant containing 1,2-difatty acyl-sn-glycerol-3-phosphate dichloro ester;

[0009] (2) Continue to add hydrolyzing agent to the intermediate reactant to carry out hydrolysis reaction and obtain the desired liposome precursor compound 1,2-difatty acyl-sn-glycerol-phosphatidic acid.

[0010] Specifically, the method for synthesizing 1,2-difatty acyl-sn-glycerol-3-phosphatidic acid includes liposome precursor compounds comprising 1,2-bisstearoyl-sn-glycerol-3-phosphatidic acid or 1,2-dispalmitoyl-sn-glycerol-3-phosphatidic acid;

[0011] Therefore, the 1,2-difatty acyl-sn-glycerol used as a raw material includes 1,2-bisstearoyl-sn-glycerol or 1,2-dispalmitoyl-sn-glycerol.

[0012] Specifically, in the method for synthesizing 1,2-difatty acyl-sn-glycerol-3-phosphatidic acid, the bifunctional catalyst includes a porous zeolite active support and an ionic liquid active component supported on the surface of the porous zeolite active support.

[0013] The cation of the ionic liquid is 1,1,3,3-tetramethylguanidine;

[0014] The anion of the ionic liquid is 1-(2-hydroxyethyl)imidazolium.

[0015] Specifically, in the method for synthesizing 1,2-difatty acyl-sn-glycerol-3-phosphatidic acid, the amount of the bifunctional catalyst used is 20-30 wt% of the total mass of the 1,2-difatty acyl-sn-glycerol and phosphorus oxychloride.

[0016] Specifically, in the method for synthesizing 1,2-difatty acyl-sn-glycerol-3-phosphatidic acid, the molar ratio of 1,2-difatty acyl-sn-glycerol to phosphorus oxychloride is 1:1-4.

[0017] Specifically, in the method for synthesizing 1,2-difatty acyl-sn-glycerol-3-phosphatidic acid, the solvent for the phosphorylation reaction includes one or more of benzene, cyclohexane, n-hexane, and dichloromethane.

[0018] Specifically, in the method for synthesizing 1,2-difatty acyl-sn-glycerol-3-phosphatidic acid, the phosphorylation reaction is carried out at a temperature of 40-60°C for a reaction time of 3-6 hours.

[0019] Specifically, in the method for synthesizing 1,2-difatty acyl-sn-glycerol-3-phosphatidic acid, the hydrolysing agent includes water, sodium hydroxide solution, or sodium carbonate solution.

[0020] Specifically, in the method for synthesizing 1,2-difatty acyl-sn-glycerol-3-phosphatidic acid, the amount of hydrolysing agent used is 20-50 wt% of the total mass of the 1,2-difatty acyl-sn-glycerol and phosphorus oxychloride.

[0021] Specifically, in the method for synthesizing 1,2-difatty acyl-sn-glycerol-3-phosphatidic acid, the hydrolysis reaction is carried out at a temperature of 25-50°C for 5-8 hours.

[0022] Compared with the prior art, the beneficial effects of the present invention are:

[0023] The method for synthesizing the liposome precursor compound 1,2-bisfatty acyl-sn-glycerol-3-phosphatidic acid of the present invention involves a phosphorylation reaction using 1,2-bisfatty acyl-sn-glycerol and phosphorus oxychloride as raw materials, followed by a hydrolysis reaction to obtain the desired 1,2-bisfatty acyl-sn-glycerol-3-phosphatidic acid. Both phosphorylation-hydrolysis reactions in the entire process can be carried out in the bifunctional catalyst system, achieving high-efficiency catalytic reactions without the need to change the catalyst, and resulting in a high product conversion rate. Detailed Implementation

[0024] The technical solutions of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0025] The present invention provides a method for synthesizing a liposome precursor compound, namely 1,2-difatty acyl-sn-glycerol-3-phosphatidic acid, in the following embodiments, specifically including the following steps:

[0026] (1) Using 1,2-difatty acyl-sn-glycerol and phosphorus oxychloride as raw materials, a phosphorylation reaction was carried out in the presence of a bifunctional catalyst to obtain an intermediate reactant containing 1,2-difatty acyl-sn-glycerol-3-phosphate dichloro ester;

[0027] (2) Continue to add hydrolyzing agent to the intermediate reactant to carry out hydrolysis reaction and obtain the desired liposome precursor compound 1,2-difatty acyl-sn-glycerol-phosphatidic acid.

[0028] The method for synthesizing the liposome precursor compound 1,2-bisfatty acyl-sn-glycerol-3-phosphatidic acid of the present invention involves a phosphorylation reaction using 1,2-bisfatty acyl-sn-glycerol and phosphorus oxychloride as raw materials, followed by a hydrolysis reaction to obtain the desired 1,2-bisfatty acyl-sn-glycerol-3-phosphatidic acid. Both phosphorylation-hydrolysis reactions in the entire process can be carried out in the bifunctional catalyst system, achieving high-efficiency catalytic reactions without the need to change the catalyst, and resulting in a high product conversion rate.

[0029] In some specific embodiments, the liposome precursor compound includes 1,2-distearyl-sn-glycero-3-phosphatidic acid or 1,2-dispalmitoyl-sn-glycero-3-phosphatidic acid;

[0030] In some specific embodiments, the 1,2-difatty acyl-sn-glycerol used as a raw material includes 1,2-bisstearoyl-sn-glycerol or 1,2-dispalmitoyl-sn-glycerol.

[0031] In some specific embodiments, the bifunctional catalyst includes a porous zeolite active support and an ionic liquid active component supported on the surface of the porous zeolite active support.

[0032] The cation of the ionic liquid is 1,1,3,3-tetramethylguanidine;

[0033] The anion of the ionic liquid is 1-(2-hydroxyethyl)imidazolium.

[0034] The ionic liquid is a system composed of the cation 1,1,3,3-tetramethylguanidine and the anion 1-(2-hydroxyethyl)imidazole. Its preparation method can refer to the general ionic liquid synthesis methods in the prior art. For example, it can be obtained by neutralization reaction in a solvent system such as acetonitrile using equimolar amounts of 1,1,3,3-tetramethylguanidine and 1-(2-hydroxyethyl)imidazole as raw materials, at room temperature and pressure, for a reaction time of about 18-24 hours.

[0035] The bifunctional catalyst can be obtained by impregnating porous zeolite as an active carrier in the ionic liquid using a conventional impregnation method.

[0036] As a feasible implementation method, the preparation method of the bifunctional catalyst includes: mixing and dispersing the synthesized ionic liquid with solvents such as acetone, then fully impregnating the porous zeolite support in the mixture, and performing impregnation loading under ultrasonic-assisted treatment. After the porous zeolite support is placed in the ionic liquid solution for static treatment for 24 hours, it is dried.

[0037] In some specific embodiments, the amount of the bifunctional catalyst is 20-30 wt% of the total mass of the 1,2-difatty acyl-sn-glycerol and phosphorus oxychloride.

[0038] In some specific embodiments, the molar ratio of 1,2-difatty acyl-sn-glycerol to phosphorus oxychloride is 1:1-4.

[0039] In some specific embodiments, the solvent for the phosphorylation reaction includes one or more of benzene, cyclohexane, n-hexane, and dichloromethane.

[0040] In some specific embodiments, the phosphorylation reaction is carried out at a temperature of 40-60°C for a time of 3-6 hours.

[0041] In some specific embodiments, the hydrolysant includes water, sodium hydroxide solution, or sodium carbonate solution.

[0042] In some specific embodiments, the amount of the hydrolysant is 20-50 wt% of the total mass of the 1,2-difatty acyl-sn-glycerol and phosphorus oxychloride.

[0043] In some specific embodiments, the hydrolysis reaction is carried out at a temperature of 25-50°C for 5-8 hours.

[0044] Preparation Example

[0045] This preparation example is used to prepare the bifunctional catalyst.

[0046] At room temperature and pressure, 1,1,3,3-tetramethylguanidine and 1-(2-hydroxyethyl)imidazole were reacted in a 1:1 molar ratio in a sufficient amount of acetonitrile solvent for 24 hours. The reaction products were collected, and the acetonitrile and water were removed by rotary evaporation to obtain the desired ionic liquid.

[0047] Take 10g of the ionic liquid prepared above, add an appropriate amount of acetone solvent and mix and disperse thoroughly. Take another 20g of porous zeolite particles as a carrier and completely immerse them in the above mixed solution. Impregnate and load the carrier under ultrasonic assistance for about 30 minutes. Then, continue to place the porous zeolite carrier in the above mixed solution and let it stand for 24 hours. Filter the loaded porous zeolite carrier and dry it at low temperature until all the acetone evaporates to obtain the desired catalyst. Example 1

[0048] This embodiment uses 1,2-dispalmitoyl-sn-glycerol as a starting material to synthesize 1,2-dispalmitoyl-sn-glycerol-3-phosphatidic acid, specifically including the following steps:

[0049] (1) 1,2-bispalmitoyl-sn-glycerol and phosphorus oxychloride were taken as raw materials in a molar ratio of 1:2 and added to an appropriate amount of dichloromethane solvent. A bifunctional catalyst (preparation example) accounting for 25 wt% of the total amount of 1,2-bispalmitoyl-sn-glycerol and phosphorus oxychloride was added and mixed thoroughly. The system temperature was controlled at 50°C and the reaction was carried out for 6 h to obtain an intermediate reactant containing 1,2-bispalmitoyl-sn-glycerol-3-phosphate dichloro ester. The product structure was found to be correct according to the detection method described in Chinese Patent CN111253434A.

[0050] (2) Continue to add water dropwise to the reaction product of step (1) as a hydrolysant, and control the amount of water to be 50 wt% of the total mass of 1,2-bispalmitoyl-sn-glycerol and phosphorus oxychloride. After stirring and hydrolyzing at 30°C for 6 hours, collect the reaction product and dry it. According to the detection method described in Chinese Patent CN111253434A, the product structure is correct. Example 2

[0051] This embodiment uses 1,2-distearyl-sn-glycerol as a starting material to synthesize 1,2-distearyl-sn-glycerol-3-phosphatidic acid, specifically including the following steps:

[0052] (1) 1,2-bisstearoyl-sn-glycerol and phosphorus oxychloride were taken as raw materials in a molar ratio of 1:3 and added to an appropriate amount of dichloromethane solvent. A bifunctional catalyst (preparation example) accounting for 25 wt% of the total amount of 1,2-bisstearoyl-sn-glycerol and phosphorus oxychloride was added and mixed thoroughly. The system temperature was controlled at 50°C and the reaction was carried out for 6 h to obtain an intermediate reactant containing 1,2-bisstearoyl-sn-glycerol-3-phosphate dichloro ester.

[0053] (2) Continue to add water dropwise to the reaction product of step (1) as a hydrolysant, and control the amount of water to be 40 wt% of the total mass of the 1,2-bisstearoyl-sn-glycerol and phosphorus oxychloride. After stirring and hydrolyzing at 40°C for 6 hours, collect the reaction product and dry it. Example 3

[0054] This embodiment uses 1,2-distearyl-sn-glycerol as a starting material to synthesize 1,2-distearyl-sn-glycerol-3-phosphatidic acid, specifically including the following steps:

[0055] (1) 1,2-bisstearoyl-sn-glycerol and phosphorus oxychloride were taken as raw materials in a 1:1 molar ratio and added to an appropriate amount of dichloromethane solvent. A bifunctional catalyst (preparation example) accounting for 20 wt% of the total amount of 1,2-bisstearoyl-sn-glycerol and phosphorus oxychloride was added and mixed thoroughly. The system temperature was controlled at 40°C and the reaction was carried out for 3 h to obtain an intermediate reactant containing 1,2-bisstearoyl-sn-glycerol-3-phosphate dichloro ester.

[0056] (2) Continue to add water dropwise to the reaction product of step (1) as a hydrolysant, and control the amount of water to be 20wt% of the total mass of the 1,2-bisstearoyl-sn-glycerol and phosphorus oxychloride. After stirring and hydrolyzing at 25°C for 8 hours, collect the reaction product and dry it. Example 4

[0057] This embodiment uses 1,2-dispalmitoyl-sn-glycerol as a starting material to synthesize 1,2-dispalmitoyl-sn-glycerol-3-phosphatidic acid, specifically including the following steps:

[0058] (1) 1,2-bispalmitoyl-sn-glycerol and phosphorus oxychloride were taken as raw materials in a molar ratio of 1:4 and added to an appropriate amount of dichloromethane solvent. A bifunctional catalyst (preparation example) accounting for 30 wt% of the total amount of 1,2-bispalmitoyl-sn-glycerol and phosphorus oxychloride was added and mixed thoroughly. The system temperature was controlled at 60°C and the reaction was carried out for 5 h to obtain an intermediate reactant containing 1,2-bispalmitoyl-sn-glycerol-3-phosphate dichloro ester.

[0059] (2) Continue to add water dropwise to the reaction product of step (1) as a hydrolysant, and control the amount of water to be 30 wt% of the total mass of the 1,2-bispalmitoyl-sn-glycerol and phosphorus oxychloride. After stirring and hydrolyzing at 50°C for 5 hours, collect the reaction product and dry it.

[0060] Comparative Example 1

[0061] The preparation method of 1,2-bispalmitoyl-sn-glycerol-3-phosphatidic acid in this comparative example is the same as that in Example 1, except that an equal amount of triethylamine is used instead of the bifunctional catalyst for the reaction.

[0062] Comparative Example 2

[0063] The preparation method of 1,2-bispalmitoyl-sn-glycerol-3-phosphatidic acid in this comparative example is the same as that in Example 1, except that the anionic part of the ionic liquid in the bifunctional catalyst is selected as 2-ethylimidazole, and the synthesis method of the ionic liquid is the same as that in the preparation example.

[0064] Comparative Example 3

[0065] The preparation method of 1,2-bispalmitoyl-sn-glycerol-3-phosphatidic acid in this comparative example is the same as that in Example 1, except that the anionic part of the ionic liquid in the bifunctional catalyst is selected as 2-aminoimidazole, and the synthesis method of the ionic liquid is the same as that in the preparation example.

[0066] Comparative Example 4

[0067] The preparation method of 1,2-bispalmitoyl-sn-glycerol-3-phosphatidic acid in this comparative example is the same as that in Example 1, except that the anionic part of the ionic liquid in the bifunctional catalyst is selected as 2-nitroimidazole, and the synthesis method of the ionic liquid is the same as that in the preparation example.

[0068] Comparative Example 5

[0069] The preparation method of 1,2-bispalmitoyl-sn-glycerol-3-phosphatidic acid in this comparative example is the same as that in Example 1, except that the cationic part of the ionic liquid in the bifunctional catalyst is selected as choline hydroxide, and the synthesis method of the ionic liquid is the same as that in the preparation example.

[0070] Experimental Example

[0071] In this experimental example, the content of the final product 1,2-dispalmitoyl-sn-glycero-3-phosphatidic acid was determined under Example 1 and Comparative Examples 1-5, respectively, and the product yield was calculated according to the following formula:

[0072] Product yield = Product mass / Theoretical product mass × 100%; whereby the theoretical product mass is calculated based on the stoichiometric coefficients of the following chemical reaction equation;

[0073] 1,2-bispalmitoyl-sn-glycerol + POCl3 → 1,2-bispalmitoyl-sn-glycerol-3-phosphate dichloroester + HCl;

[0074] 1,2-Dipalpalmoyl-sn-glycerol-3-phosphate dichloroester + 2H2O → 1,2-Dipalpalmoyl-sn-glycerol-3-phosphatidic acid + 2HCl.

[0075] In this experimental example, the product yield calculation results under the above-mentioned Example 1 and Comparative Examples 1-5 are shown in Table 1 below.

[0076] Table 1 Product Yield Results

[0077] serial number Product yield / % Example 1 97.17 Comparative Example 1 70.45 Comparative Example 2 66.43 Comparative Example 3 75.46 Comparative Example 4 61.24 Comparative Example 5 32.92

[0078] As can be seen, the synthesis method of 1,2-difatty acyl-sn-glycerol-3-phosphatidic acid described in this invention, by screening a suitable bifunctional catalyst, does not require replacing the catalyst during the entire phosphorylation and hydrolysis reaction process. This not only ensures the continuous progress of the reaction but also effectively improves the product yield, which is beneficial to downstream processing.

[0079] It will be apparent to those skilled in the art that the present invention is not limited to the details of the exemplary embodiments described above, and that the invention can be implemented in other specific forms without departing from the spirit or essential characteristics of the invention. Therefore, the embodiments should be considered in all respects as exemplary and non-limiting, and the scope of the invention is defined by the appended claims rather than the foregoing description. Thus, it is intended that all variations falling within the meaning and scope of equivalents of the claims be included within the present invention.

[0080] Furthermore, it should be understood that although this specification describes embodiments, not every embodiment contains only one independent technical solution. This narrative style is merely for clarity. Those skilled in the art should consider the specification as a whole, and the technical solutions in each embodiment can also be appropriately combined to form other embodiments that can be understood by those skilled in the art.

Claims

1. A method for synthesizing 1,2-difatty acyl-sn-glycerol-3-phosphatidic acid, characterized in that, Includes the following steps: (1) Using 1,2-difatty acyl-sn-glycerol and phosphorus oxychloride as raw materials, a phosphorylation reaction was carried out in the presence of a bifunctional catalyst to obtain an intermediate reactant containing 1,2-difatty acyl-sn-glycerol-3-phosphate dichloro ester; The 1,2-difatty acyl-sn-glycerol is 1,2-distearate acyl-sn-glycerol or 1,2-dispalmitoyl-sn-glycerol; The bifunctional catalyst includes a porous zeolite active support and an ionic liquid active component loaded on the surface of the porous zeolite active support. The cation of the ionic liquid is 1,1,3,3-tetramethylguanidine; The anion of the ionic liquid is 1-(2-hydroxyethyl)imidazolium; (2) Continue to add hydrolyzing agent to the intermediate reactant to carry out hydrolysis reaction to obtain the desired liposome precursor compound 1,2-difatty acyl-sn-glycerol-3-phosphatidic acid; The liposome precursor compound 1,2-bisacyl-sn-glycerol-3-phosphatidic acid is 1,2-bisstearoyl-sn-glycerol-3-phosphatidic acid or 1,2-bispalmitoyl-sn-glycerol-3-phosphatidic acid.

2. The method for synthesizing 1,2-difatty acyl-sn-glycerol-3-phosphatidic acid according to claim 1, characterized in that, The amount of the bifunctional catalyst is 20-30 wt% of the total mass of the 1,2-difatty acyl-sn-glycerol and phosphorus oxychloride.

3. The method for synthesizing 1,2-difatty acyl-sn-glycerol-3-phosphatidic acid according to claim 1, characterized in that, The molar ratio of 1,2-difatty acyl-sn-glycerol to phosphorus oxychloride is 1:1-4.

4. The method for synthesizing 1,2-difatty acyl-sn-glycerol-3-phosphatidic acid according to claim 1, characterized in that, The solvent for the phosphorylation reaction is one or more of benzene, cyclohexane, n-hexane, and dichloromethane.

5. The method for synthesizing 1,2-difatty acyl-sn-glycerol-3-phosphatidic acid according to claim 1, characterized in that, The phosphorylation reaction is carried out at a temperature of 40-60℃ for a time of 3-6 hours.

6. The method for synthesizing 1,2-difatty acyl-sn-glycerol-3-phosphatidic acid according to claim 1, characterized in that, The hydrolysing agent is water, sodium hydroxide solution, or sodium carbonate solution.

7. The method for synthesizing 1,2-difatty acyl-sn-glycerol-3-phosphatidic acid according to claim 6, characterized in that, The amount of the hydrolysant is 20-50 wt% of the total mass of the 1,2-difatty acyl-sn-glycerol and phosphorus oxychloride.

8. The method for synthesizing 1,2-difatty acyl-sn-glycerol-3-phosphatidic acid according to claim 1, characterized in that, The hydrolysis reaction is carried out at a temperature of 25-50℃ for 5-8 hours.