Synthesis method of 1-palmitoyl-2-oleoyl lecithin

Abstract

The present invention discloses a method for synthesizing 1-palmitoyl-2-oleoyl lecithin. Using cis-1,3-O-benzylidene glycerol as the starting material, the exposed hydroxyl group is protected with TBSCl, and DMDO is added for ring-opening reaction. It is coupled with DHP HM Resin through the catalysis of PPTS for solid-phase synthesis. The TBS protecting group is removed by an organic solvent containing TFA, condensed with palmitic acid, and then the benzyl protecting group is removed by trimethylsilyl iodide and condensed with elaidic acid. After drying, it is reacted and cleaved in an alcohol mixed solvent of PPTS and DCE, and finally, 2-chloro-2-oxo-1,3,2-dioxaphospholane, triethylamine, and choline are added to obtain POPC. The method of the present invention can improve the product purity and yield, enhance the reaction efficiency, and is suitable for industrial production.

Description

Technical Field

[0001] The invention relates to the field of pharmaceutical chemicals, and in particular to a method for synthesizing 1-palmitoyl-2-oleoyl lecithin. Background Art

[0002] The chemical name of 1-palmitoyl-2-oleoylphosphatidylcholine is 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine, abbreviated as POPC, and CAS number is 26853-31-6. 1-palmitoyl-2-oleoylphosphatidylcholine is a complex lipid molecule consisting of a glyceride backbone, two fatty acyl chains (one oleoyl and one palmitoyl) and a phosphate group. Its structural formula is:

[0003]

[0004] POPC plays an indispensable role in the structure and function of biomembranes. Biomembranes are an important component of cells. They not only provide the necessary structural support for cells, but also regulate the transport and exchange of substances inside and outside cells. As one of the main components of biomembranes, POPC plays an important role in maintaining the stability of biomembranes and regulating membrane permeability.

[0005] In addition, POPC is also involved in the preparation of liposomes. Liposomes are artificially prepared membrane structures that are widely used in drug carriers, gene therapy, and vaccine preparation. POPC has become an ideal material for preparing liposomes due to its excellent membrane-forming ability and biocompatibility. By adjusting the ratio of POPC to other phospholipids, liposomes with different physical and chemical properties can be prepared to meet the research needs of different fields.

[0006] POPC also plays a key role in the study of lipid bilayer properties. The lipid bilayer is the basic structure of biological membranes, and its stability, fluidity, and interaction with other substances all affect the normal function of cells. Through the study of POPC lipid bilayers, scientists can gain a deeper understanding of the structure and function of biological membranes and provide a theoretical basis for the treatment and prevention of related diseases.

[0007] POPC can also carry long-chain omega-3 fatty acids EPA and DHA into cell membranes, which is essential for maintaining cell elasticity and function. These fatty acids have multiple physiological activities in the human body, such as regulating blood lipids, anti-inflammatory, and anti-oxidation, and play an important role in preventing cardiovascular diseases and improving immunity.

[0008] In the prior art, the document Synthesis of Lysophosphatidylcholine and Mixed Phosphatidylcholine J. Org. Chem. 2022, 87, 8194-8197 provides a method for synthesizing 1-palmitoyl-2-oleoyl phosphatidylcholine, firstly protecting all hydroxyl groups with different protecting groups, then removing the protecting groups in turn, reacting with phosphoinositol, palmitic acid, elaidic acid, etc., to generate the target product POPC. There is also the possibility that the unprotected hydroxyl group on the 2-carbon may react with BzCl, which leads to side reactions in the subsequent reaction process, so that the purity of the product is reduced and the yield is reduced; the entire synthesis route has eleven steps, and the post-processing steps are cumbersome. NaH is used when benzyl bromide protects the hydroxyl group, the Grignard reagent EtMgBr is used when the benzoyl protecting group is removed, and the hydrogenation reaction is performed when the benzyl group is removed. These reactions have special requirements for reaction equipment and reaction conditions, and have high requirements for industrial production. Summary of the invention

[0009] The invention aims to provide a new synthesis method of 1-palmitoyl-2-oleoyl phosphatidylcholine, improve the purity and yield of the product and improve the reaction efficiency.

[0010] The present invention is achieved through the following technical solutions:

[0011] A method for synthesizing 1-palmitoyl-2-oleoyl phosphatidylcholine, comprising the following steps:

[0012] Step 1: TBSCl reacts with imidazole and cis-1,3-O-benzylpropylene glycol in a solvent to protect the exposed hydroxyl group on cis-1,3-O-benzylpropylene glycol to generate intermediate 1;

[0013] Step 2: Intermediate 1 reacts with DMDO in a solvent to form intermediate 2;

[0014] Step 3: Intermediate 2 is catalyzed by PPTS and reacted with DHP HM Resin in a solvent to generate intermediate 3 resin;

[0015] Step 4: Add the intermediate 3 resin to a solid phase synthesis column, remove the TBS protecting group on the intermediate 3 resin by reacting with an organic solvent containing TFA, and wash the resin with DMF;

[0016] Step 5: Dissolve palmitic acid and a condensing agent in DMF and add the mixture to a solid phase synthesis column to react with the resin obtained in step 4, and wash the obtained resin with DMF;

[0017] Step 6: adding trimethylsilyl iodide and DCM to a solid phase synthesis column to react, removing the benzyl protecting group on the resin obtained in step 5, and washing the resin with DMF;

[0018] Step 7: dissolving the elaidic acid and the condensing agent in DMF and adding them to the solid phase synthesis column to react with the resin obtained in step 6, washing the resin with DMF, and shrinking and drying with methanol;

[0019] Step 8: The dried resin in step 7 is reacted and cracked in an alcohol solvent mixed with PPTS and DCE to obtain intermediate 4;

[0020] Step nine: The intermediate 4 is reacted with 2-chloro-2-oxo-1,3,2-dioxaphospholane and triethylamine in a solvent, and then choline is added to react to generate the target product POPC.

[0021] To optimize the above technical solutions, the specific measures taken also include:

[0022] In step 1, the reaction molar ratio of cis-1,3-O-benzylpropylene glycol to imidazole and TBSCl is 1:2-3:1.5-2, and the reaction temperature is 20-25° C.; the solvent is DMF or DCM.

[0023] In step 2, the reaction molar ratio of intermediate 1 to DMDO is 1:3-4, the reaction temperature is 0-5° C.; the solvent is at least one of acetone, tetrahydrofuran, and DCM.

[0024] In step 3, the reaction molar ratio of DHP HM Resin to intermediate 2 and PPTS is 1:2-3:0.3-0.5, the reaction temperature is 60-80° C., and the solvent is DCE or chloroform.

[0025] In step 4, the reaction conditions for removing the TBS protecting group on the resin with an organic solvent containing TFA are bubbling reaction at room temperature; the organic solvent is selected from DCM or DCE; the content of TFA in the organic solvent is 0.1%-0.5% v / v; the volume / mass ratio of the organic solvent containing TFA to the intermediate 3 is 4~7:1 ml / g.

[0026] The reaction molar ratio of the intermediate 3 resin added in step 4 to the palmitic acid added in step 5 is 1:2~3; in step 5, the reaction temperature is 20~35°C, and the condensing agent is selected from at least one of DIC and DMAP, DCC and DMAP, PyBOP and HOBt.

[0027] The reaction molar ratio of the intermediate 3 resin added in step 4 to the trimethylsilane iodide added in step 6 is 1:1.5~2; in step 6, the reaction temperature is 20~35°C.

[0028] The reaction molar ratio of the intermediate 3 resin added in step 4 to the elaidic acid added in step 7 is 1:2-3; in step 7, the reaction temperature is 20-35° C., and the condensing agent is selected from at least one of DIC / DMAP, DCC / DMAP, and PyBOP / HOBt.

[0029] The reaction molar ratio of the intermediate 3 resin added in step 4 to the PPTS added in step 8 is 1:2-3; in step 8, the reaction temperature is 60-80° C.; the alcohol solvent is selected from at least one of MeOH, PrOH, and BuOH.

[0030] In step nine, the intermediate 4 reacts with 2-chloro-2-oxo-1,3,2-dioxaphospholane, triethylamine and choline in a molar ratio of 1:2~3:2~3:6~8; the solvent is selected from at least one of tetrahydrofuran, toluene and DCM.

[0031] The reaction process is as follows:

[0032]

[0033] Compared with the prior art, the present invention has the following beneficial effects:

[0034] The invention proposes a new synthetic route for synthesizing 1-palmitoyl-2-oleoyl phosphatidylcholine, which comprises the following steps: using cis-1,3-O-benzyl propylene glycol as a starting material, protecting the exposed hydroxyl group with TBSCl, adding DMDO for ring opening reaction, coupling with DHP HM Resin through PPTS catalysis, performing solid phase synthesis, removing the TBS protective group through an organic solvent containing TFA, condensing with palmitic acid, removing the benzyl protective group through trimethyl iodosilane, condensing with elaidic acid, drying the resin, reacting and cracking in a mixed solvent of PPTS and DCE alcohols, and finally adding 2-chloro-2-oxy-1,3,2-dioxaphospholane, triethylamine and choline for reaction to obtain POPC.

[0035] During the reaction process of this synthesis method, only one hydroxyl group is always exposed to react, which reduces the generation of by-products and improves the purity and yield of the product.

[0036] This synthesis method introduces the resin DHP HM Resin for solid phase synthesis reaction. The post-treatment conditions during the reaction are simple, and only DMF is needed to rinse the resin, which greatly improves the reaction efficiency.

[0037] The benzyl removal reaction in this synthesis method uses trimethylsilyl iodide to replace the hydrogen and Pd / C system, and the hydrogen and Pd / C system have high requirements for equipment; while the reaction conditions of the method of the present application are mild, the equipment requirements are low, and the operation is safe; in the solid phase synthesis, the benzyl removal by trimethylsilyl iodide only requires the resin to be rinsed multiple times with DMF to remove the by-products, and the yield is high, which is suitable for industrial production. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] Figure 1 : HPLC chromatogram of 1-palmitoyl-2-oleoyl phosphatidylcholine prepared in Example 1.

[0039] Figure 2 : The HPLC chromatogram of 1-palmitoyl-2-oleoyl phosphatidylcholine prepared in Example 2.

[0040] Figure 3 : HPLC chromatogram of 1-palmitoyl-2-oleoyl phosphatidylcholine prepared in Example 3. DETAILED DESCRIPTION

[0041] The above contents of the present invention are further described in detail below in the form of embodiments, but this should not be understood as the scope of the above subject matter of the present invention being limited to the following embodiments, and all technologies realized based on the above contents of the present invention belong to the scope of the present invention.

[0042] The experimental methods used in the following examples are all conventional methods unless otherwise specified, and the reagents, methods and equipment used are all conventional reagents, methods and equipment in the technical field unless otherwise specified.

[0043] The abbreviations, English terms and notes in the present invention are shown in Table 1:

[0044] Table 1 Abbreviations, English terms and notes

[0045]

[0046] Comparative Example 1: Synthesis of POPC in the literature (Synthesis of Lysophosphatidylcholine and Mixed Phosphatidylcholine J. Org. Chem. 2022, 87, 8194-8197)

[0047] Step 1: R-acetone glycerol acetal is reacted with benzyl bromide to obtain intermediate 1, and the hydroxyl group of R-acetone glycerol acetal is protected;

[0048] Step 2: Intermediate 1 is reacted in 1M HCl in THF solution to remove the propylidene to obtain intermediate 2;

[0049] Step 3: Intermediate 2 reacts with benzoyl chloride to selectively protect the hydroxyl group on the 3-carbon to obtain intermediate 3;

[0050] Step 4: Intermediate 3 reacts with tert-butyldimethylsilyl chloride to protect the hydroxyl group on the 2-carbon to obtain intermediate 4;

[0051] Step 5: The intermediate 4 is treated with the Grignard reagent ethylmagnesium bromide to remove the benzoyl protecting group on the 3-carbon to obtain the intermediate 5;

[0052] Step 6: The unprotected hydroxyl group of intermediate 5 reacts with inositol phosphate to obtain intermediate 6;

[0053] Step 7: Intermediate 6 reacts with trimethylamine to obtain intermediate 7;

[0054] Step 8: intermediate 7 is debenzylated under the action of palladium carbon and hydrogen to obtain intermediate 8;

[0055] Step 9: Intermediate 8 undergoes condensation reaction with palmitic acid to obtain intermediate 9;

[0056] Step 10: The hydroxyl protecting group on the carbon 2 of intermediate 9 was removed in the presence of 0.1% TFA aqueous solution to obtain intermediate 10;

[0057] Step 11: The intermediate 10 undergoes a condensation reaction with elaidic acid to obtain the product POPC.

[0058] The reaction process is as follows:

[0059]

[0060] This synthetic route first protects all hydroxyl groups with different protecting groups, then removes the protecting groups in turn, and reacts with phosphate inositol, palmitic acid, elaidic acid, etc. to generate the target product POPC. However, in step 3, during the reaction of intermediate 2 with benzoyl chloride, the unprotected hydroxyl group on the 2-carbon may also react with BzCl, resulting in side reactions in the subsequent reaction process, which reduces the purity of the product and the yield. The entire synthetic route has eleven steps, and the post-processing steps are cumbersome. NaH is used when benzyl bromide protects the hydroxyl group, the Grignard reagent EtMgBr is used when removing the benzoyl protecting group, and a hydrogenation reaction is performed when removing the benzyl group. These reactions have special requirements for reaction equipment and reaction conditions, and have high requirements for industrial production, which increases the production cost.

[0061] The present application proposes a new synthesis method of 1-palmitoyl-2-oleoyl phosphatidylcholine. During the reaction process, only one hydroxyl group is exposed to react, which reduces the generation of by-products and improves the purity and yield of the product. By introducing resin DHP HM Resin for solid phase synthesis reaction, the post-treatment conditions during the reaction are simple, and only DMF is needed to rinse the resin, which greatly improves the reaction efficiency. Trimethyl iodosilane is used to replace hydrogen and Pd / C system in the benzyl removal reaction. See the following Examples 1-3 for details.

[0062] Embodiment 1:

[0063] The synthesis of POPC is carried out according to the following steps:

[0064] (1) Synthesis of intermediate 1:

[0065] Add 180g of cis-1,3-O-benzylpropylene glycol, 136g of imidazole and 500ml of DMF into a reaction bottle, stir and cool to 0°C; add 226.1g of TBSCl and stir at 20-25°C for 12 hours; imidazole is an easy leaving group, which increases the activity of the acylation reaction and also acts as an acid binding agent.

[0066] Post-treatment: The reaction solution was added to 2000 ml of a mixture of ice water and n-hexane = 1:1 (v / v), extracted 3 times with 1000 ml of EA × 3, the organic phases were combined, 100 g of anhydrous sodium sulfate was added and dried for 1 hour, filtered, and the filtrate was concentrated to dryness under reduced pressure at 50-60 ° C to obtain 279.44 g of intermediate 1 with a yield of 95%.

[0067] (2) Synthesis of intermediate 2

[0068] Add 279 g of intermediate 1, 210.56 g of DMDO and 1000 ml of acetone into a reaction bottle, and stir at 0-5°C for 5 hours.

[0069] Post-treatment: concentrate to dryness under reduced pressure at 50-60°C to obtain a crude product; the crude product is purified by column chromatography (EA:n-hexane=0:1-1:5) to obtain 224.72 g of intermediate 2 with a yield of 80%.

[0070] (3) Synthesis of intermediate 3

[0071] 50 g of DHP HM Resin (substitution degree 1.0 mmol / g), 29.65 g of intermediate 2, 3.77 g of PPTS and 250 ml of DCE were added to a reaction bottle, and the mixture was stirred at 70° C. for 2 hours.

[0072] Post-treatment: The reaction solution was cooled to room temperature, filtered, and the resin was dried in vacuo at 20-25°C to dryness to obtain 360.1 g of the intermediate with a substitution degree of 0.57 mmol / g.

[0073] (4) Solid phase synthesis 1: Removal of TBS protecting group

[0074] Weigh 50 g (0.57 mmol / g) of intermediate 3, add it to a peptide solid phase synthesis tube, add 250 ml of DMF × 2 and wash twice (nitrogen bubbling, 1 min each time, and drain after washing). Add 250 ml of DMF to the peptide solid phase synthesis tube, bubble nitrogen for 30 min, and drain the liquid after full swelling.

[0075] Add 250 ml of 0.1% TFA solution in DCM (volume ratio), bubble nitrogen for 1 hour, and drain the liquid; wash 6 times with 250 ml of DMF (bubble nitrogen for 1 min each time, and drain after washing).

[0076] (5) Solid phase synthesis II: Palmitic acid condensation

[0077] Weigh 24.15g of palmitic acid, 10.79g of DIC, and 250ml of DMF, add them to a beaker, stir to dissolve, add them to a polypeptide solid phase synthesis tube, add 0.7g of DMAP, bubble with nitrogen for 2 hours, reaction temperature 22-25°C, drain the liquid; wash 6 times with 250ml of DMF × 6 (bubble with nitrogen, 1min each time, and drain after washing).

[0078] (6) Solid Phase Synthesis III: Removal of Benzyl

[0079] Weigh 11.4 g of trimethylsilyl iodide, add it to a beaker, add 250 ml of DCM to dilute the trimethylsilyl iodide, add it to the polypeptide solid phase synthesis tube, bubble with nitrogen for 1 hour, reaction temperature 22~25°C, and drain the liquid; wash 6 times with DMF 250 ml×6 (nitrogen bubbling, 1 min each time, and drain after washing); remove the benzyl group from trimethylsilyl iodide in solid phase synthesis, only need to use DMF to rinse the resin multiple times to remove the by-product.

[0080] (7) Solid phase synthesis IV: condensation of oleic acid

[0081] Weigh 21.92g of elaidic acid, 10.79g of DIC, and 250ml of DMF, add them to a beaker, stir and dissolve, add them to a polypeptide solid phase synthesis tube, add 0.7g of DMAP, bubble nitrogen for 2 hours, reaction temperature 22-25°C, drain the liquid; wash 6 times with 250ml of DMF (bubble nitrogen, 1min each time, drain after washing).

[0082] Add MeOH 250 ml × 3 and wash three times (nitrogen bubbling, the three times are 5 min, 5 min, 10 min respectively, and then pump out after washing); dry with nitrogen to obtain 65.3 g of dry resin.

[0083] (8) Synthesis of intermediate 4

[0084] 65.3 g of dry resin, 15 g of PPTS, 100 ml of DCE and 500 ml of MeOH were added to a reaction bottle, and the mixture was stirred at 60° C. for 2 hours.

[0085] Post-treatment: The reaction solution was cooled and filtered, and the filtrate was concentrated to dryness under reduced pressure at 40-50 °C to obtain 14.2 g of intermediate 4 with a yield of 83.73%.

[0086] (9) Synthesis of POPC

[0087] Add 14 g of intermediate 4 and 100 ml of tetrahydrofuran to a reaction bottle, add 0.3 g of DMAP, add 7 g of triethylamine, stir and cool to 0°C, add 7 g of 2-chloro-2-oxo-1,3,2-dioxaphospholane dropwise; after the addition is complete, stir and react at room temperature for 5 hours; add 15 g of choline, stir and react at 45-50°C for 24 hours.

[0088] Post-treatment: The reaction solution was cooled to room temperature, filtered, and the filtrate was concentrated to dryness under reduced pressure at 40-50°C to obtain a crude product; the crude product was purified by column chromatography (DCM:MeOH=10:1-1:1) to obtain POPC 14.5 g, as shown in the chromatogram. Figure 1 , HPLC purity: 97.12%, yield: 81.07%.

[0089] Embodiment 2:

[0090] The synthesis of POPC is carried out according to the following steps:

[0091] (1) Synthesis of intermediate 1:

[0092] Add 180 g of cis-1,3-O-benzylpropylene glycol, 204 g of imidazole and 500 ml of DCM into a reaction bottle, stir and cool to 0°C; add 301 g of TBSCl, stir and react at 20-25°C for 12 hours.

[0093] Post-treatment: The reaction solution was added to 2000 ml of a mixture of ice water and n-hexane = 1:1 (v / v), extracted 3 times with 1000 ml of EA × 3, the organic phases were combined, 100 g of anhydrous sodium sulfate was added and dried for 1 hour, filtered, and the filtrate was concentrated to dryness under reduced pressure at 50-60 ° C to obtain 281.2 g of intermediate 1 with a yield of 95.6%.

[0094] (2) Synthesis of intermediate 2

[0095] Add 280 g of intermediate 1, 281.5 g of DMDO and 1000 ml of tetrahydrofuran into a reaction bottle, and stir at 0-5°C for 5 hours.

[0096] Post-treatment: concentrate to dryness under reduced pressure at 50-60°C to obtain a crude product; the crude product is purified by column chromatography (EA:n-hexane=0:1-1:5) to obtain 221.3 g of intermediate 2 with a yield of 78.5%.

[0097] (3) Synthesis of intermediate 3

[0098] 50 g of DHP HM Resin (substitution degree 1.0 mmol / g), 44 g of intermediate 2, 6 g of PPTS and 250 ml of DCE were added into a reaction bottle and stirred at 80° C. for 2 hours.

[0099] Post-treatment: The reaction solution was cooled to room temperature, filtered, and the resin was dried in vacuo at 20-25°C to dryness to obtain 361.2 g of the intermediate with a substitution degree of 0.61 mmol / g.

[0100] (4) Solid phase synthesis 1: Removal of TBS protecting group

[0101] Weigh 50 g (0.61 mmol / g) of intermediate 3, add it to a peptide solid phase synthesis tube, add 250 ml of DMF × 2 and wash twice (nitrogen bubbling, 1 min each time, and remove after washing); add 250 ml of DMF to the peptide solid phase synthesis tube, bubble nitrogen for 30 min, and drain the liquid after sufficient swelling.

[0102] Add 250 ml of 0.2% TFA solution in DCM (volume ratio), bubble nitrogen for 1 hour, and drain the liquid; wash 6 times with 250 ml of DMF (bubble nitrogen for 1 min each time, and drain after washing).

[0103] (5) Solid phase synthesis II: Palmitic acid condensation

[0104] Weigh 18 g palmitic acid, 18 g DCC, and 250 ml DMF, add to a beaker, stir to dissolve, add to a polypeptide solid phase synthesis tube, add 0.8 g DMAP, bubble with nitrogen for 2 hours, reaction temperature 22-25°C, drain the liquid; wash 6 times with 250 ml DMF × 6 (bubble with nitrogen, 1 min each time, and drain after washing).

[0105] (6) Solid Phase Synthesis III: Removal of Benzyl

[0106] Weigh 9.2 g of trimethylsilyl iodide, add it to a beaker, add 250 ml of DCM to dilute, add it to the peptide solid phase synthesis tube, bubble nitrogen for 1 hour, reaction temperature 25-28 ° C, drain the liquid; wash 6 times with 250 ml of DMF × 6 (nitrogen bubbling, 1 min each time, drain after washing).

[0107] (7) Solid phase synthesis IV: condensation of oleic acid

[0108] Weigh 16 g of elaidic acid, 18 g of DCC, and 250 ml of DMF, add them to a beaker, stir to dissolve, add them to a polypeptide solid phase synthesis tube, add 0.8 g of DMAP, bubble with nitrogen for 2 hours, reaction temperature 22-25°C, and drain the liquid; wash 6 times with 250 ml of DMF (bubble with nitrogen, 1 min each time, and drain after washing).

[0109] Add MeOH 250 ml × 3 and wash three times (nitrogen bubbling, the three times are 5 min, 5 min, 10 min respectively, and then pump out after washing); dry with nitrogen to obtain 66.1 g of dry resin.

[0110] (8) Synthesis of intermediate 4

[0111] 66.1 g of dry resin, 22 g of PPTS, 100 ml of DCE and 500 ml of PrOH were added to a reaction bottle, and the mixture was stirred at 80° C. for 2 hours.

[0112] Post-treatment: The reaction solution was cooled and filtered, and the filtrate was concentrated to dryness under reduced pressure at 40-50 °C to obtain 15.3 g of intermediate 4 with a yield of 84.3%.

[0113] (9) Synthesis of POPC

[0114] Add 15 g of intermediate 4 and 100 ml of tetrahydrofuran to a reaction bottle, add 0.4 g of DMAP, add 5.1 g of triethylamine, stir and cool to 0°C, add 10.5 g of 2-chloro-2-oxo-1,3,2-dioxaphospholane dropwise; after the addition is complete, stir and react at room temperature for 5 hours; add 21 g of choline, stir and react at 45-50°C for 24 hours.

[0115] Post-treatment: The reaction solution was cooled to room temperature, filtered, and the filtrate was concentrated to dryness under reduced pressure at 40-50°C to obtain a crude product; the crude product was purified by column chromatography (DCM:MeOH=10:1-1:1) to obtain POPC 16.2 g, as shown in the chromatogram. Figure 2 , HPLC purity: 97.23%, yield: 84.54%.

[0116] Embodiment 3:

[0117] The synthesis of POPC is carried out according to the following steps:

[0118] (1) Synthesis of intermediate 1:

[0119] Add 180 g of cis-1,3-O-benzylpropylene glycol, 170 g of imidazole, and 500 ml of DCM to a reaction bottle, stir and cool to 0° C. Add 256 g of TBSCl, and stir and react at 20-25° C. for 12 hours.

[0120] Post-treatment: The reaction solution was added to 2000 ml of a mixture of ice water and n-hexane = 1:1 (v / v), extracted 3 times with 1000 ml of EA × 3, the organic phases were combined, 100 g of anhydrous sodium sulfate was added and dried for 1 hour, filtered, and the filtrate was concentrated to dryness under reduced pressure at 50-60 ° C to obtain 275.26 g of intermediate 1 with a yield of 93.6%.

[0121] (2) Synthesis of intermediate 2

[0122] Add 275 g of intermediate 1, 242 g of DMDO and 1000 ml of acetone into a reaction bottle, and stir at 0-5°C for 5 hours.

[0123] Post-treatment: concentrate to dryness under reduced pressure at 50-60°C to obtain a crude product; the crude product is purified by column chromatography (EA:n-hexane=0:1-1:5) to obtain 224.1 g of intermediate 2 with a yield of 80.9%.

[0124] (3) Synthesis of intermediate 3

[0125] 50 g of DHP HM Resin (substitution degree 1.0 mmol / g), 37 g of intermediate 2, 5 g of PPTS and 250 ml of DCE were added into a reaction bottle and stirred at 60° C. for 2 hours.

[0126] Post-treatment: The reaction solution was cooled to room temperature, filtered, and the resin was dried in vacuo at 20-25°C to dryness to obtain 359.4 g of the intermediate with a substitution degree of 0.54 mmol / g.

[0127] (4) Solid phase synthesis 1: Removal of TBS protecting group

[0128] Weigh 50 g (0.54 mmol / g) of intermediate 3, add it to a peptide solid phase synthesis tube, add 250 ml of DMF × 2 and wash twice (nitrogen bubbling, 1 min each time, and remove after washing); add 250 ml of DMF to the peptide solid phase synthesis tube, bubble nitrogen for 30 min, and drain the liquid after sufficient swelling.

[0129] Add 250 ml of 0.5% TFA solution in DCM (volume ratio), bubble nitrogen for 1 hour, and drain the liquid; wash 6 times with 250 ml of DMF (bubble nitrogen for 1 min each time, and drain after washing).

[0130] (5) Solid phase synthesis II: Palmitic acid condensation

[0131] Weigh 19 g palmitic acid, 42 g PyBOP, 10 g HOBt, and 250 ml DMF, add to a beaker and stir to dissolve, add 10 g DIPEA, stir, add to a polypeptide solid phase synthesis tube, bubble with nitrogen for 2 hours, reaction temperature 22-25°C, drain the liquid; wash 6 times with 250 ml DMF × 6 (nitrogen bubbling, 1 min each time, drain after washing).

[0132] (6) Solid Phase Synthesis III: Removal of Benzyl

[0133] Weigh 9.2 g of trimethylsilyl iodide, add it to a beaker, add 250 ml of DCM to dilute, add it to the peptide solid phase synthesis tube, bubble with nitrogen for 1 hour, reaction temperature 22-25 °C, drain the liquid; wash 6 times with 250 ml of DMF (bubble with nitrogen, 1 min each time, drain after washing).

[0134] (7) Solid phase synthesis IV: condensation of oleic acid

[0135] Weigh 17.3g of oleic acid, 42g of PyBOP, 10g of HOBt, and 250ml of DMF, add them to a beaker and stir to dissolve, add 10g of DIPEA and stir. Add them to the peptide solid phase synthesis tube, bubble nitrogen for 2 hours, reaction temperature 22-25℃, drain the liquid; wash 6 times with 250ml of DMF (bubble nitrogen, 1min each time, and drain after washing).

[0136] Add MeOH 250 ml × 3 and wash three times (nitrogen bubbling, the three times are 5 min, 5 min, 10 min respectively, and then pump out after washing); dry with nitrogen to obtain 65.5 g of dry resin.

[0137] (8) Synthesis of intermediate 4

[0138] 65.5 g of dry resin, 17 g of PPTS, 100 ml of DCE and 500 ml of BuOH were added to a reaction bottle, and the mixture was stirred at 80° C. for 2 hours.

[0139] Post-treatment: The reaction solution was cooled and filtered, and the filtrate was concentrated to dryness under reduced pressure at 40-50 °C to obtain 13.5 g of intermediate 4 with a yield of 84.04%.

[0140] (9) Synthesis of POPC

[0141] Add 13 g of intermediate 4 and 100 ml of DCM to a reaction bottle, add 0.3 g of DMAP, add 5.5 g of triethylamine, stir and cool to 0°C, add 7.8 g of 2-chloro-2-oxo-1,3,2-dioxaphospholane dropwise; after the addition is complete, stir and react at room temperature for 5 hours; add 16 g of choline, stir and react at 45-50°C for 24 hours.

[0142] Post-treatment: The reaction solution was cooled to room temperature, filtered, and the filtrate was concentrated to dryness under reduced pressure at 40-50°C to obtain a crude product; the crude product was purified by column chromatography (DCM:MeOH=10:1-1:1) to obtain POPC 13.3 g, as shown in the chromatogram. Figure 3 , HPLC purity: 97.23%, yield: 80.08%.

[0143] The above description is only a preferred embodiment of the present invention and does not limit the present invention in any form. Any technician familiar with the profession, without departing from the scope of the technical solution of the present invention, according to the technical essence of the present invention, any simple modification, equivalent replacement and improvement made to the above embodiment still falls within the protection scope of the technical solution of the present invention.

Claims

1. A method for synthesizing 1-palmitoyl-2-oleoyl phosphatidylcholine, characterized in that: The following steps are involved: Step 1: TBSCl reacts with imidazole and cis-1,3-O-benzylpropylene glycol in a solvent to protect the exposed hydroxyl group on cis-1,3-O-benzylpropylene glycol to generate intermediate 1; Step 2: Intermediate 1 reacts with DMDO in a solvent to form intermediate 2; Step 3: Intermediate 2 is catalyzed by PPTS and reacted with DHP HM Resin in a solvent to generate intermediate 3 resin; Step 4: Add the intermediate 3 resin to a solid phase synthesis column, remove the TBS protecting group on the intermediate 3 resin by reacting with an organic solvent containing TFA, and wash the resin with DMF; Step 5: Dissolve palmitic acid and a condensing agent in DMF and add the mixture to a solid phase synthesis column to react with the resin obtained in step 4, and wash the obtained resin with DMF; Step 6: adding trimethylsilyl iodide and DCM to a solid phase synthesis column to react, removing the benzyl protecting group on the resin obtained in step 5, and washing the resin with DMF; Step 7: dissolving the elaidic acid and the condensing agent in DMF and adding them to the solid phase synthesis column to react with the resin obtained in step 6, washing the resin with DMF, and shrinking and drying with methanol; Step 8: The dried resin in step 7 is reacted and cracked in an alcohol solvent mixed with PPTS and DCE to obtain intermediate 4; Step 9: The intermediate 4 reacts with 2-chloro-2-oxo-1,3,2-dioxaphospholane and triethylamine in a solvent, and then choline is added to react to generate the target product POPC; the reaction process is as follows: 。 2. The method for synthesizing 1-palmitoyl-2-oleoyl phosphatidylcholine according to claim 1, characterized in that: In step 1, the reaction molar ratio of cis-1,3-O-benzylpropylene glycol to imidazole and TBSCl is 1:2-3:1.5-2, and the reaction temperature is 20-25° C.; the solvent is DMF or DCM.

3. The method for synthesizing 1-palmitoyl-2-oleoyl phosphatidylcholine according to claim 1, characterized in that: In step 2, the reaction molar ratio of intermediate 1 to DMDO is 1:3-4, the reaction temperature is 0-5° C.; the solvent is at least one of acetone, tetrahydrofuran, and DCM.

4. The method for synthesizing 1-palmitoyl-2-oleoyl phosphatidylcholine according to claim 1, characterized in that: In step 3, the reaction molar ratio of DHP HM Resin to intermediate 2 and PPTS is 1:2-3:0.3-0.5, the reaction temperature is 60-80° C., and the solvent is DCE or chloroform.

5. The method for synthesizing 1-palmitoyl-2-oleoyl phosphatidylcholine according to claim 1, characterized in that: In step 4, the reaction conditions for removing the TBS protecting group on the resin with an organic solvent containing TFA are bubbling reaction at room temperature; the organic solvent is selected from DCM or DCE; the content of TFA in the organic solvent is 0.1%-0.5% v / v; the volume / mass ratio of the organic solvent containing TFA to the intermediate 3 is 4~7:1 ml / g.

6. The method for synthesizing 1-palmitoyl-2-oleoyl phosphatidylcholine according to claim 1, characterized in that: The reaction molar ratio of the intermediate 3 resin added in step 4 to the palmitic acid added in step 5 is 1:2-3; in step 5, the reaction temperature is 20-35°C.

7. The method for synthesizing 1-palmitoyl-2-oleoyl phosphatidylcholine according to claim 1, characterized in that: The reaction molar ratio of the intermediate 3 resin added in step 4 to the trimethylsilane iodide added in step 6 is 1:1.5~2; in step 6, the reaction temperature is 20~35°C.

8. The method for synthesizing 1-palmitoyl-2-oleoyl phosphatidylcholine according to claim 1, characterized in that: The reaction molar ratio of the intermediate 3 resin added in step 4 to the elaidic acid added in step 7 is 1:2-3; in step 7, the reaction temperature is 20-35°C.

9. The method for synthesizing 1-palmitoyl-2-oleoyl phosphatidylcholine according to claim 1, characterized in that: The reaction molar ratio of the intermediate 3 resin added in step 4 to the PPTS added in step 8 is 1:2-3; in step 8, the reaction temperature is 60-80° C.; the alcohol solvent is selected from at least one of MeOH, PrOH, and BuOH.

10. The method for synthesizing 1-palmitoyl-2-oleoyl phosphatidylcholine according to claim 1, characterized in that: In step nine, the intermediate 4 reacts with 2-chloro-2-oxo-1,3,2-dioxaphospholane, triethylamine and choline in a molar ratio of 1:2~3:2~3:6~8; the solvent is selected from at least one of tetrahydrofuran, toluene and DCM.

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