CN114014889A

CN114014889A - Method for synthesizing phosphatidylcholine by using solid phase carrier

Info

Publication number: CN114014889A
Application number: CN202111411799.XA
Authority: CN
Inventors: 于文渊; 吉民; 王冬冬; 刘来富; 胡海燕
Original assignee: Jiangsu Dongnan Nano Material Co ltd
Current assignee: Jiangsu Dongnan Nano Material Co ltd
Priority date: 2021-11-25
Filing date: 2021-11-25
Publication date: 2022-02-08

Abstract

The invention provides a method for artificially synthesizing phosphatidylcholine by using a solid phase carrier, which comprises the steps of firstly melting the glycerophosphatidylcholine, then uniformly adsorbing the glycerophosphatidylcholine by using active carbon, then carrying out acylation reaction with fatty acid chloride, and filtering and recrystallizing the obtained product to obtain the high-purity phosphatidylcholine. The phosphatidylcholine prepared by the method can be used as a pharmaceutic adjuvant for various medicines such as injection, tablets, capsules and the like.

Description

Method for synthesizing phosphatidylcholine by using solid phase carrier

Technical Field

The invention belongs to the field of pharmaceutical chemicals, and particularly relates to a method for synthesizing phosphatidylcholine by using a solid phase carrier and application thereof.

Background

Phospholipid is an important component of a biological membrane, a polar end containing phosphate radicals has hydrophilicity, and two longer non-polar ends of hydrocarbon chains have lipophilicity, and the unique physicochemical property enables the phospholipid to form a closed bilayer in a water medium to become a biological membrane skeleton, so that the phospholipid plays a vital role in the liposome technology. However, natural phospholipid as liposome material has the disadvantages of short service life, easy oxidation, poor stability, etc., and the application is limited.

Phosphatidylcholine is an important artificially synthesized phospholipid, is mainly applied to the preparation of liposome, and is an indispensable additive auxiliary material in liposome pharmaceutical preparations.

The common methods for preparing phosphatidylcholine in the prior art are various, wherein natural substances are subjected to countercurrent extraction, the purity after extraction is relatively high, but the yield is low, the extracting agents are usually acetonitrile, ethanol and the like, the selectivity is low, and the material waste is serious. The purity of the phosphatidylcholine obtained by supercritical extraction is about 70%, and the problems of low purity, low yield, high equipment requirement, large investment and the like exist. The method for separating phosphatidylcholine by column chromatography and membrane has the defects of non-regeneration, high cost, small treatment capacity and the like.

Disclosure of Invention

The invention overcomes the defects of harsh conditions, complex post-treatment procedures, serious environmental pollution, lower reaction conversion rate, lower yield, high production cost and unsuitability for industrial production of the existing preparation method of the phosphatidylcholine, and provides a method for synthesizing the phosphatidylcholine by solid phase. The activated carbon is cheap and easy to obtain, the reaction condition is mild, the operation is safe, the post-treatment is simple, the yield is high, and the obtained phosphatidylcholine has high purity and is suitable for industrial production.

In order to achieve the purpose, the main technical scheme provided by the invention is as follows:

a method for solid phase synthesis of phosphatidylcholine, comprising the steps of:

(1) melting the glycerophosphatidylcholine, and uniformly adsorbing the glycerophosphatidylcholine by using active carbon;

(2) the choline alfoscerate adsorbed by the active carbon and the fatty acid chloride are subjected to acylation reaction in the presence of alkali, and then the phosphatidylcholine is purified.

The hydrophobic end of the phosphatidylcholine is a saturated or unsaturated fatty hydrocarbon chain. Including but not limited to common phosphatidylcholines such as Distearoylphosphatidylcholine (DSPC), Dipalmitoylphosphatidylcholine (DPPC), erucylphosphatidylcholine (DEPC), Dimyristoylphosphatidylcholine (DMPC), Hydrogenated Soybean Phosphatidylcholine (HSPC), etc., other fatty acid structured phosphatidylcholines are also contemplated by the present invention.

Preferably, the mass ratio of the glycerophosphatidylcholine to the activated carbon in the step (1) is 1: 1-1: 5, preferably 1: 3.

preferably, the method of the invention specifically comprises the following steps:

(1) heating the choline glycerophosphate to be molten (preferably 80 ℃) under the protection of nitrogen, adding powdered activated carbon into the molten choline glycerophosphate, stirring (1-2 hours) to enable the choline glycerophosphate to be fully and uniformly adsorbed by the activated carbon, and cooling (preferably 20 ℃);

(2) adding a reaction solvent and alkali into the choline alfoscerate adsorbed by the activated carbon, adding fatty acid chloride, and reacting to obtain phosphatidylcholine;

and R represents the fatty chain group of fatty acid chloride.

Preferably, the base in step (2) is selected from one or more of sodium carbonate, potassium carbonate, sodium bicarbonate, potassium bicarbonate, sodium hydroxide, potassium hydroxide, calcium hydroxide, triethylamine and sodium hydride, and triethylamine is more preferred.

Preferably, the reaction solvent in step (2) is one or more selected from dichloromethane, trichloromethane, 1, 2-dichloroethane, tetrahydrofuran, dioxane, ethyl acetate and N, N-dimethylformamide, and more preferably trichloromethane.

Preferably, the fatty acid chloride includes, but is not limited to, one or more of fatty acid chlorides such as stearoyl chloride, palmitoyl chloride, erucic acid chloride, oleic acid chloride, myristic acid chloride, lauric acid chloride, linolenic acid chloride and the like.

The method of the invention further comprises the step (3): filtering the reaction solution, cooling the filtrate, stirring, crystallizing, filtering and drying.

The invention also aims to provide the phosphatidylcholine prepared by the method, and the HPLC purity of the phosphatidylcholine is more than or equal to 99.0%.

The phosphatidylcholine obtained by the invention can be used for preparing medical injection or pharmaceutic adjuvant.

Compared with the prior art, the invention has the following advantages:

1. in the prior art, choline alfoscerate is white granular solid, and reacts with fatty acid chloride in a heterogeneous phase for a long time, so that the reaction is incomplete, impurities such as lysophosphatidylcholine are easily generated, and the purity is low. If high-purity phosphatidylcholine is desired, a purification method such as column chromatography is needed, and the yield is low. The invention utilizes the characteristics of large specific surface area and easy adsorption of the activated carbon, so that choline alfoscerate is fully and uniformly adsorbed on the microporous surface of the activated carbon by the activated carbon in a melting state, thereby greatly increasing the reaction surface area of the choline alfoscerate, further increasing the molecular collision probability in the reaction with fatty acid chloride, reducing the generation of lysophospholipid, being beneficial to purification methods such as recrystallization and the like, and obviously improving the reaction yield and the product purity.

2. The unreacted solid choline alfoscerate in the method is absorbed in the activated carbon, can be separated from the reaction product phosphatidylcholine in the reaction solvent by filtration, and is simple and convenient to operate.

3. The technological raw materials of the phosphatidylcholine are cheap, the reaction time is short, the reaction conversion rate is high, the purification steps are few, the solvent is used a little, the controllability is good, the operability is strong, the cost is greatly reduced, the defects of low yield, high cost and complicated operation in the prior art are overcome, the large-scale industrial production is easy, the economic benefit is high, the yield can reach more than 85 percent, and the purity is more than or equal to 99 percent. The product obtained by the invention has stable quality, and meets the standards of medical injection and pharmaceutic adjuvant through degerming and vacuum drying.

Drawings

FIG. 1 shows DSPC obtained in example 1¹H-NMR spectrum.

FIG. 2 shows the spectrum of DSPC MS obtained in example 1.

FIG. 3 shows DPPC obtained in example 2¹H-NMR spectrum.

FIG. 4 is the spectrum of DPPC MS obtained in example 2.

FIG. 5 shows DEPC obtained in example 3¹H-NMR spectrum.

FIG. 6 is the DEPC MS spectrum obtained in example 3.

FIG. 7 shows DMPC obtained in example 4¹H-NMR spectrum.

FIG. 8 is a spectrum of DMPC MS obtained in example 4.

FIG. 9 shows DOPC obtained in example 5¹H-NMR spectrum.

FIG. 10 is a spectrum of DOPC MS obtained in example 5.

Detailed Description

The technical solutions of the present invention are described below with specific examples, but the scope of the present invention is not limited thereto.

Example 1

(1) Adding 51.4g of glycerophosphorylcholine into a clean and dry 3000ml three-necked bottle with a mechanical stirrer, replacing nitrogen, starting stirring, heating until the glycerophosphorylcholine is melted, respectively adding 154.2g of different adsorbents (the adsorbents are selected from activated carbon, alumina, silica gel and diatomite) while stirring, and continuously stirring for 1 hour to ensure that the glycerophosphorylcholine is fully and uniformly adsorbed by the adsorbents.

(2) Cooling to room temperature, adding 1500mL of trichloromethane and 60.7g of triethylamine, dropwise adding 181.75g of stearoyl chloride at the temperature of 20 ℃, keeping the temperature and stirring for 1 hour after the dropwise adding is finished, heating to 30-35 ℃, filtering, transferring filtrate into a clean and dry 3000mL three-necked bottle with mechanical stirring, starting stirring, cooling to-5 ℃ for crystallization for 3-4 hours, filtering, leaching a filter cake with a small amount of trichloromethane ice, pumping, and vacuum drying for 24 hours to obtain 137.5g of DSPCs, wherein the yield is 87.0%.

The high performance liquid chromatography purity detection method comprises the following steps:

chromatographic conditions are as follows: using octyl silane bonded silica gel as a filling agent; taking methanol-water (90:3) as a mobile phase, and detecting the wavelength at 214 nm; the flow rate is 0.1 ml/min; the column temperature is 30 ℃; the injection volume was 15. mu.l.

Test solution: a sample of about 30mg is taken, precisely weighed, placed in a 10ml volumetric flask, added with methanol solution and diluted to the scale, shaken up to be used as a test solution.

The determination method comprises the following steps: precisely measuring blank solution and sample solution, injecting into liquid chromatograph, performing area normalization (deducting solvent peak), and recording chromatogram.

In the embodiment, a scheme that alumina, silica gel and diatomite are used as porous adsorption materials is synchronously tested, other conditions except the adsorption materials are consistent, and the obtained result is as follows:

serial number	Adsorbent and process for producing the same	Product weight	Yield of	Purity of
					1	Activated carbon	137.5g	87.0％	99.4％
2	Alumina oxide	98.3g	62.2％	95.3％
					3	Silica gel	100.0g	63.3％	96.6％
4	Diatomite	95.3g	60.3％	97.8％
					5	Is free of	90.5g	57.3％	98.8％

The result shows that when the activated carbon is used as an adsorbing material, the yield and the purity are obviously superior to those of the scheme of alumina, silica gel, diatomite and no adsorbing material, and the yield and the purity of the scheme of the adsorbing materials such as alumina and the like have no obvious advantages compared with the scheme of no adsorbing material. The product DSPC obtained¹The H-NMR spectrum is shown in FIG. 1, and the MS spectrum is shown in FIG. 2.

Example 2

(1) Adding 51.4g of glycerophosphorylcholine into a clean and dry 3000ml three-mouth bottle with mechanical stirring, replacing nitrogen, starting stirring, heating until the glycerophosphorylcholine is melted, adding 154.2g of active carbon while stirring, and continuing stirring for 1 hour to ensure that the glycerophosphorylcholine is fully and uniformly adsorbed by the active carbon.

(2) Cooling to room temperature, adding 1500mL of trichloromethane and 60.7g of triethylamine, dropwise adding 164.92g of palmitoyl chloride at the temperature of 20 ℃, keeping the temperature and stirring for 1 hour after the dropwise adding is finished, heating to 30-35 ℃, filtering, transferring filtrate into a clean and dry 3000mL three-necked bottle with mechanical stirring, starting stirring, cooling to-5 ℃, crystallizing for 3-4 hours, filtering, leaching a small amount of trichloromethane to obtain a filter cake, pumping, and vacuum drying for 24 hours to obtain 130.1g of DPPC with the yield of 88.6%.

The obtained product,¹The H-NMR spectrum is shown in FIG. 3, and the MS spectrum is shown in FIG. 4.

Example 3

(2) Cooling to room temperature, adding 1500mL of trichloromethane and 60.7g of triethylamine, dropwise adding 214.21g of erucyl chloride at the temperature of 20 ℃, keeping the temperature and stirring for 1 hour after the dropwise adding is finished, heating to 30-35 ℃, filtering, transferring filtrate into a clean and dry 3000mL three-necked bottle with mechanical stirring, starting stirring, cooling to-10-0 ℃ for crystallization for 3-4 hours, filtering, leaching a small amount of trichloromethane to obtain a filter cake, pumping, and vacuum drying for 24 hours to obtain 154.7g of DEPC with the yield of 86.1%.

The obtained product,¹The H-NMR spectrum is shown in FIG. 5, and the MS spectrum is shown in FIG. 6.

Example 4

(2) Cooling to room temperature, adding 1500mL of trichloromethane and 60.7g of triethylamine, dropwise adding 148.09g of tetradecanoic acid chloride at the temperature of 20 ℃, keeping the temperature and stirring for 1 hour after the dropwise adding is finished, heating to 30-35 ℃, filtering, transferring filtrate into a clean and dry 3000mL three-necked bottle with mechanical stirring, starting stirring, cooling to-5 ℃ for crystallization for 3-4 hours, filtering, leaching a small amount of trichloromethane to obtain a filter cake, pumping, and vacuum drying for 24 hours to obtain 122.5g of DMPC with the yield of 90.3%.

The obtained product,¹The H-NMR spectrum is shown in FIG. 7, and the MS spectrum is shown in FIG. 8.

Example 5

(2) Cooling to room temperature, adding 1500mL of trichloromethane and 60.7g of triethylamine, dropwise adding 180.55g of acyl chloride acid at the temperature of 20 ℃, keeping the temperature and stirring for 1 hour after the dropwise adding is finished, heating to 30-35 ℃, filtering, transferring filtrate into a clean and dry 3000mL three-necked bottle with mechanical stirring, starting stirring, cooling to-10-0 ℃ for crystallization for 3-4 hours, filtering, leaching a small amount of trichloromethane to obtain a filter cake, pumping, and vacuum drying for 24 hours to obtain 134.8g of DOPC, wherein the yield is 85.7%.

The obtained product,¹The H-NMR spectrum is shown in FIG. 9, and the MS spectrum is shown in FIG. 10.

Claims

1. A method for synthesizing phosphatidylcholine by using a solid phase carrier is characterized by comprising the following steps:

(1) after the glycerophosphatidylcholine is melted, the glycerophosphatidylcholine is uniformly adsorbed by powdered activated carbon;

(2) and (3) carrying out acylation reaction on the choline alfoscerate adsorbed by the activated carbon and fatty acid chloride in the presence of alkali, and purifying to obtain the phosphatidylcholine.

2. The method according to claim 1, wherein the mass ratio of the glycerophosphatidylcholine to the activated carbon in the step (1) is 1: 1-1: 5.

3. The method according to claim 2, wherein the mass ratio of the glycerophosphatidylcholine to the activated carbon in the step (1) is 1: 3.

4. The method according to claim 1, characterized in that the method is in particular:

(1) heating choline alfoscerate to melt under the protection of nitrogen, adding powdery active carbon under the protection of nitrogen, stirring and adsorbing uniformly, and cooling;

(2) adding a reaction solvent and alkali into the choline alfoscerate adsorbed by the activated carbon, adding fatty acid chloride, and reacting to obtain the phosphatidylcholine.

5. The method according to claim 4, wherein the base in step (2) is selected from one or more of sodium carbonate, potassium carbonate, sodium bicarbonate, potassium bicarbonate, sodium hydroxide, potassium hydroxide, calcium hydroxide, triethylamine and sodium hydride.

6. The method according to claim 4, wherein the reaction solvent in step (2) is one or more selected from dichloromethane, chloroform, 1, 2-dichloroethane, tetrahydrofuran, dioxane, ethyl acetate and N, N-dimethylformamide.

7. The method according to claim 2, wherein the fatty acid chloride is selected from one or more of stearoyl chloride, palmitoyl chloride, erucyl chloride, oleacyl chloride, tetradecanoyl chloride, dodecanoyl chloride, and linoloyl chloride.

8. The process according to claim 4, wherein the reaction solvent in step (2) is selected from chloroform, and the base is selected from triethylamine.

9. The method according to claim 1, characterized by comprising the step (3): filtering the reaction solution, cooling the filtrate, stirring, crystallizing, filtering and drying.

10. A phosphatidylcholine prepared by the method of any one of claims 1 to 9, wherein the purity of the prepared phosphatidylcholine is not less than 99.0%.