Polypeptide solid-phase synthesis carrier and preparation method thereof

Abstract

The invention belongs to the technical field of functional polymer material preparation, and particularly relates to a polypeptide solid-phase synthesis carrier and a preparation method thereof.The preparation method comprises the following steps that a water phase is prepared, and the water phase comprises purified water and a dispersing agent; preparing an oil phase, wherein the oil phase comprises a monovinyl compound, a crosslinking monomer, a functional monomer and an initiator; adding the oil phase into the water phase, carrying out polymerization reaction to obtain a base ball, and introducing amino or hydroxyl onto the base ball to obtain a polypeptide solid-phase synthesis carrier; the used crosslinking monomer comprises at least one of allyl ether, ethylene glycol diallyl ether and diethylene glycol divinyl ether. The polypeptide solid-phase synthesis carrier prepared by the method has the properties of high swelling and good mechanical strength, so that a reaction reagent is easy to diffuse, the purity and the yield of polypeptide synthesis are improved, and the production cost of polypeptide is reduced.

Description

Technical Field

[0001] The invention belongs to the technical field of functional polymer material preparation, and specifically relates to a polypeptide solid phase synthesis carrier and a preparation method thereof. Background Art

[0002] With the rapid development of combinatorial chemistry, polymer carriers are increasingly widely used in organic synthesis. Especially in the fields of peptide synthesis and nucleic acid synthesis, the emergence of solid-phase synthesis methods has accelerated the synthesis of peptides, and has been applied to the production of multiple drugs. The basic process of peptide solid-phase synthesis is as follows: functional groups such as amino groups or hydroxyl groups are introduced into an insoluble carrier, and these groups react with the carboxyl groups of α-amino-protected amino acids to connect the amino acids to the carrier in the form of covalent bonds. Then the α-amino protecting group is removed, and the next amino-protected amino acid monomer is added after washing. Repeat the above steps until the desired polypeptide sequence is synthesized. Because of its outstanding advantages: excess reaction reagents can be used to complete the reaction; the product of each step is fixed on a solid-phase carrier that is insoluble in any solvent, and the desired product and other excess reagents, catalysts and other small molecule reagents can be separated by simple filtration; in all operation steps before separating the product from the solid-phase carrier, the solid-phase carrier is always in the same container without any mechanical loss; it is easy to automate.

[0003] The performance of the solid phase synthesis carrier is one of the key factors for the successful solid phase synthesis. In order to have a large space to accommodate the growing longer peptide chain during the synthesis process and to make the reagents diffuse easily to the reaction site, the carrier used needs to have good swelling properties. In the prior art, the carrier generally uses a polystyrene gel carrier with a low cross-linking degree of about 1% to 2%, which has a high swelling degree in solvents such as dichloromethane and dimethylformamide. The cross-linking agent used in the preparation of low-cross-linked polystyrene gel carriers is divinylbenzene, but the divinylbenzene molecule is small, and the cross-linking bridge formed will be shorter, which makes the cross-linking network poor in stretchability; and divinylbenzene is a rigid structure, which will make the overall flexibility of the synthesized carrier worse. After multiple swellings, the mechanical strength of the carrier will deteriorate, and it will break, causing the screen plate of the peptide synthesis equipment to be blocked. The prior art also discloses a method for preparing a new solid phase synthesis carrier with high swelling performance using diethylene glycol dimethacrylate or triethylene glycol dimethacrylate as a crosslinking agent. The prepared carrier has good reaction kinetics, but the crosslinking agent used contains an ester bond, which is unstable in strong acid and strong base conditions, limiting its scope of use. The prior art also discloses a low crosslinking microsphere carrier in the solid phase synthesis of polypeptides, which has excellent swelling properties, good reaction kinetics, and is not easy to produce fragments; but it uses 1,4-bis(p-vinylbenzyloxybutane) as a crosslinking agent. The structure of 1,4-bis(p-vinylbenzyloxybutane contains a benzene ring. During the chloromethylation process, the benzene ring is prone to side reactions that change the carrier structure. Summary of the invention

[0004] Therefore, the technical problem to be solved by the present invention is to overcome the defects of the solid phase synthesis carrier with excellent swelling property or poor mechanical strength or instability in the prior art, thereby providing a polypeptide solid phase synthesis carrier and a preparation method thereof.

[0005] To this end, the present invention provides the following technical solutions.

[0006] The invention provides a preparation method of a polypeptide solid phase synthesis carrier, comprising the following steps: S1: preparing an aqueous phase, wherein the aqueous phase comprises purified water and a dispersant; S2: preparing an oil phase, wherein the oil phase comprises a monovinyl compound, a crosslinking monomer, a functional monomer and an initiator; wherein the crosslinking monomer comprises at least one of allyl ether, ethylene glycol diallyl ether and diethylene glycol divinyl ether; S3: adding the oil phase to the aqueous phase, performing a polymerization reaction, and obtaining a base ball; and S4: introducing an amino group or a hydroxyl group into the base ball, and obtaining the polypeptide solid phase synthesis carrier.

[0007] Optionally, the dispersant is a water-soluble polymer.

[0008] Optionally, the monovinyl compound is an aromatic monovinyl compound.

[0009] Optionally, the functional monomer refers to a monomer having both a double bond capable of free radical polymerization and a halogenated group or other group that can be converted into a hydroxyl group or an amino group through a reaction.

[0010] Optionally, the initiator is an organic peroxide or an azo compound.

[0011] Optionally, the dispersant includes at least one of polyvinyl alcohol, hydroxyethyl cellulose, hydroxyethyl methyl cellulose, carboxymethyl cellulose, methyl cellulose, ethyl cellulose, sodium polyacrylate, and polyvinyl pyrrolidone.

[0012] Optionally, the monovinyl compound includes styrene and benzene ring substituted derivatives thereof, wherein the substituent is an alkyl or alkoxy group containing 1 to 5 carbon atoms.

[0013] Optionally, the functional monomer includes at least one of acyloxystyrene and its derivatives, haloalkylstyrene monomers, and 4-vinylphenyl ester monomers.

[0014] Optionally, the functional monomers include 4-acetoxystyrene, benzoyloxystyrene, 4-(4-bromobutyl)styrene, p-chloromethylstyrene, 4-vinylbenzoic acid methyl ester, and 4-vinylphenylacetic acid ethyl ester.

[0015] Optionally, the initiator includes at least one of benzoyl peroxide, lauroyl peroxide, tert-butyl peroxy-2-ethylhexanoate, azobisisobutyronitrile, azobisisovaleronitrile, and azobisisoheptanoonitrile.

[0016] Preferably, the dispersant is polyvinyl alcohol.

[0017] Preferably, the monovinyl compound is styrene.

[0018] Preferably, the functional monomer is p-chloromethylstyrene.

[0019] Preferably, the initiator is benzoyl peroxide.

[0020] Preferably, the mass ratio of the oil phase to the water phase is 1:3 to 1:20.

[0021] Optionally, in the oil phase, based on the total mass of the monovinyl compound, the cross-linking monomer and the functional monomer, the monovinyl compound accounts for 65% to 99.5%.

[0022] Optionally, in the oil phase, based on the total mass of the monovinyl compound, the cross-linking monomer and the functional monomer, the cross-linking monomer accounts for 0.5% to 5%.

[0023] Optionally, in the oil phase, based on the total mass of the monovinyl compound, the cross-linking monomer and the functional monomer, the functional monomer accounts for 0 to 30%.

[0024] Preferably, in the oil phase, the weight of the initiator is 0.5% to 5% of the total weight of the monovinyl compound, the crosslinking monomer, and the functional monomer.

[0025] Preferably, in the aqueous phase, the weight of the dispersant is 0.2% to 5% of the weight of the aqueous phase.

[0026] Preferably, in the oil phase, based on the total weight of the monovinyl compound, the cross-linking monomer and the functional monomer, the monovinyl compound accounts for 77% to 99%.

[0027] Preferably, in the oil phase, based on the total mass of the monovinyl compound, the crosslinking monomer and the functional monomer, the crosslinking monomer accounts for 1% to 3%.

[0028] Preferably, in the oil phase, based on the total weight of the monovinyl compound, the cross-linking monomer and the functional monomer, the functional monomer accounts for 0% to 20%.

[0029] Optionally, when the functional monomer accounts for 0% of the monomer weight percentage in the oil phase, after step S3 and before step S4, the step of introducing a halogenated group or other groups that can be converted into hydroxyl or amino groups by reaction to the base ball is also included. The operation of introducing each group is a conventional operation in the art. Taking the introduction of a halogenated group as an example, typically and non-limitingly, the specific operation is: add 30-80g of the base ball obtained in step S3 to a 1L reactor equipped with a stirrer and a thermometer, add 250-800ml of dichloromethane, and start stirring; reduce the temperature in the reactor to -10-0°C; add 15-50g of chloromethyl ether to the reactor and continue stirring; add 1.5-10g of tin tetrachloride to the reactor; keep the temperature in the reactor at -5-5°C, and terminate the reaction after 2h of reaction. Drain the solution in the reactor, wash it 3 times with dichloromethane (each time adding 250-800ml), and then put the carrier into a 45°C oven to dry to obtain a chloromethylated base ball.

[0030] Optionally, in step S3, the polymerization reaction temperature is 50-90° C. and the time is 6-24 hours.

[0031] Preferably, in step S3, the polymerization reaction temperature is 68-85° C. and the time is 8-16 hours.

[0032] The preparation method of the polypeptide solid phase synthesis carrier provided by the present invention may specifically include: preparing an aqueous phase, adding a certain amount of purified water into a reactor, and adding a dispersant whose weight percentage is 0.2% to 5% of the weight of the aqueous phase, dissolving and setting aside; controlling the mass ratio of the oil phase to the aqueous phase to be 1:3 to 1:20, preparing the oil phase, weighing a monovinyl compound, a crosslinking monomer, a functional monomer and an initiator, wherein, based on the total mass of the monovinyl compound, the crosslinking monomer and the functional monomer, the monovinyl compound accounts for 65% to 99.5%, the crosslinking monomer accounts for 0.5% to 5%, the functional monomer accounts for 0% to 30%, and the amount of the initiator is 0.5% to 5% of the total mass of the monovinyl compound, the crosslinking monomer and the functional monomer, and mixing evenly; adding the oil phase into the reactor, stirring and heating to 50 to 90° C., reacting for 6 to 24 hours, washing after the reaction is completed, sieving out a product with a particle size of 35 to 300 μm, and drying to obtain a base ball.

[0033] In the preparation method of the polypeptide solid phase synthesis carrier provided by the present invention, the method of introducing amino groups or hydroxyl groups into the base ball through halogenated groups or other groups that can be converted into hydroxyl groups or amino groups through reactions is a conventional method.

[0034] Taking the halide group contained on the base ball as an example, typically but not limitingly, the method for introducing a hydroxyl group into the base ball containing a chloro group is as follows: 30 to 50 g of the base ball and 300 to 500 ml of N, N-dimethylformamide are added to a 1L reactor equipped with a condenser, a stirrer and a thermometer, and stirring is started; then 15 to 100 g of p-hydroxybenzyl alcohol is added, the temperature is raised to 50 to 60° C. and the reaction is carried out for 12 to 16 hours; after the reaction is completed, it is cooled to room temperature, and then washed with N, N-dimethylformamide twice, washed with purified water until neutral, and then washed with anhydrous ethanol three times and filtered, and vacuum dried to obtain a solid phase synthesis carrier containing a functional group of hydroxyl.

[0035] Taking the halide contained on the base ball as an example, typically but not limitedly, the method for introducing an amino group into the base ball containing a chloro group is as follows: 30 to 50 g of the base ball and 300 to 500 ml of N, N-dimethylformamide are added to a 1L reactor equipped with a condenser, a stirrer and a thermometer, and stirring is started; then 10 to 100 g of potassium phthalimide is added, the temperature is raised to 90 to 95° C. and the reaction is carried out for 12 to 16 hours; after the reaction is completed, it is cooled to room temperature, washed with N, N-dimethylformamide twice, washed with purified water until neutral, and then washed with anhydrous ethanol three times and filtered, and returned to the reactor; Add 100-200g of anhydrous ethanol and 20-50g of hydrazine hydrate to the reactor, heat to 65-75°C and react for 12-16h; after the reaction, wash with ethanol / purified water solution with a volume ratio of 50:50 for 3 times, wash with purified water until neutral, wash with anhydrous ethanol for 3 times and filter dry, and put back into the reactor; add 100-200g of anhydrous ethanol and 30-50g of concentrated hydrochloric acid to the reactor, heat to 50-60°C and react for 4-6h; after the reaction, cool to room temperature, wash with water until neutral, and then vacuum dry to obtain a solid phase synthesis carrier containing an amino functional group.

[0036] The present invention also provides a polypeptide solid phase synthesis carrier prepared by the above preparation method.

[0037] Optionally, the particle size of the polypeptide solid phase synthesis carrier is 35-300 μm. Preferably, the particle size of the polypeptide solid phase synthesis carrier is 75-150 μm. In this way, in the device, the carrier will not cause clogging of the sieve plate due to too small particle size, and the diffusion speed of the reagent during the reaction is also guaranteed.

[0038] The polypeptide solid phase synthesis carrier provided by the present invention is used to prepare polypeptides in a conventional method in the field. Typically and non-limitingly, the specific steps are as follows (taking the solid phase carrier containing a hydroxyl functional group, wang solid phase synthesis carrier, as an example):

[0039] (1) Coupling of the first amino acid

[0040] Add an appropriate amount of solid phase synthesis carrier to the reactor, add dichloromethane or N,N-dimethylformamide in an amount of 8-15 ml per gram of carrier to swell for 30 minutes, and then filter the solution; weigh Fmoc protected amino acid (1.5-2.5 times, based on the amount of solid phase synthesis carrier), 1-hydroxybenzotriazole (HOBt, 1.5-2.5 times, based on the amount of solid phase synthesis carrier) in a beaker, add an appropriate amount of N,N-dimethylformamide solution to stir and dissolve, and then add N,N'-diisopropylcarbodiimide (DIC, 1.5 ~2.5 times, based on the amount of the substance of the solid phase synthesis carrier) and 4-dimethylaminopyridine (DMAP, 0.1-0.3 times, based on the amount of the substance of the solid phase synthesis carrier) are mixed evenly; the mixed reaction solution is added into the reactor and reacted at room temperature for 2-3 hours; after the reaction is completed, the solution is filtered and dried, and washed three times with N,N-dimethylformamide (8-15 ml per gram of carrier), dichloromethane (8-15 ml per gram of carrier), and N,N-dimethylformamide (8-15 ml per gram of carrier) in sequence, each time for 30 minutes, and filtered after washing.

[0041] (2) Removal of Fmoc protecting group

[0042] Add 20% piperidine / N,N-dimethylformamide solution (v / v, 8-15 ml per gram of carrier) to the reactor and react at room temperature for 30-60 minutes; after the reaction, filter the solution and wash the solid phase synthesis carrier three times with N,N-dimethylformamide (8-15 ml per gram of carrier), dichloromethane (8-15 ml per gram of carrier), and N,N-dimethylformamide (8-15 ml per gram of carrier) in sequence, each time for 30 minutes, and filter after washing.

[0043] (3) Amino acid coupling

[0044] Weigh Fmoc protected amino acid (1.5-2.5 times, based on the amount of substance of solid phase synthesis carrier) and HOBt (1.5-2.5 times, based on the amount of substance of solid phase synthesis carrier) in a beaker, add appropriate amount of N,N-dimethylformamide solution and stir to dissolve, then add DIC (1.5-2.5 times, based on the amount of substance of solid phase synthesis carrier) to the beaker and mix well; add the well mixed reaction solution to the reactor and react at room temperature for 2-3h; after the reaction is completed, filter the solution and wash the solid phase synthesis carrier with N,N-dimethylformamide (8-15ml per gram of carrier), dichloromethane (8-15ml per gram of carrier) three times in sequence, each time for 30min, and filter after washing.

[0045] (4) Removal of Fmoc protecting group

[0046] Add 20% piperidine / N,N-dimethylformamide solution (v / v, 8-15 ml per gram of carrier) to the reactor and react at room temperature for 30-60 minutes; after the reaction, filter the solution and wash the solid phase synthesis carrier three times with N,N-dimethylformamide (8-15 ml per gram of carrier), dichloromethane (8-15 ml per gram of carrier), and N,N-dimethylformamide (8-15 ml per gram of carrier) in sequence, each time for 30 minutes, and filter after washing.

[0047] (5) Peptide chain extension

[0048] Repeat steps (3) and (4) until the target polypeptide is obtained; wash the solid phase synthesis carrier loaded with the polypeptide with methanol (8-15 ml per gram of carrier) three times, each time for 30 minutes, filter dry after washing, and place the carrier in a vacuum oven to dry for 16-24 hours.

[0049] (6) Cracking

[0050] Weigh the dried solid phase synthesis carrier loaded with polypeptide into the reactor, add the cutting reagent (10-20 ml per gram of carrier), the cutting reagent is trifluoroacetic acid: thioanisole: water = 95:2.5:2.5 (v / v / v), react at room temperature for 2-4 hours, cut the polypeptide from the solid phase synthesis carrier, filter, collect the filtrate, wash the carrier twice with trifluoroacetic acid (10-20 ml per gram of carrier), collect and combine the filtrate to obtain a cutting solution containing polypeptide.

[0051] (7) Precipitation

[0052] Add icy ether to the cutting solution containing the polypeptide (8-10 ml of icy ether per ml of cutting solution), place at 0-4°C for 12-18 hours to precipitate the polypeptide, and obtain a crude polypeptide.

[0053] The beneficial effects of the present invention are:

[0054] The present invention provides a preparation method of a polypeptide solid phase synthesis carrier, comprising the following steps: S1: preparing an aqueous phase, wherein the aqueous phase comprises purified water and a dispersant; S2: preparing an oil phase, wherein the oil phase comprises a monovinyl compound, a crosslinking monomer, a functional monomer and an initiator; wherein the crosslinking monomer comprises at least one of allyl ether, ethylene glycol diallyl ether and diethylene glycol divinyl ether; S3: adding the oil phase to the aqueous phase, performing a polymerization reaction, and obtaining a base ball; S4: introducing an amino group or a hydroxyl group to the base ball, and obtaining a polypeptide solid phase synthesis carrier. The obtained polypeptide solid phase synthesis carrier has the properties of high swelling and good mechanical strength, and while making the reaction reagent easy to diffuse, its service life is increased and the cost is reduced. At least one of allyl ether, ethylene glycol diallyl ether and diethylene glycol divinyl ether is used as a cross-linking agent. Their molecular chains are relatively long and have certain hydrophilicity, which can improve the swelling performance of the polypeptide solid phase synthesis carrier, facilitate the diffusion of the reaction reagents, and improve the reaction efficiency; the structure is stable, and no side reactions will occur during the functionalization process of the carrier or the synthesis of polypeptides, resulting in changes in the microstructure of the carrier or the introduction of impurities; the polymerization speed is relatively slow, which is conducive to improving the uniformity of the chemical structure inside the carrier and forming uniformly distributed active sites; the uniform distribution of active sites avoids the mutual influence of polypeptide chains during the polypeptide synthesis process, which leads to the occurrence of carrier breakage, and improves the mechanical strength of the carrier.

[0055] In the preparation method of the polypeptide solid phase synthesis carrier provided by the present invention, the mass ratio of the oil phase to the water phase is specified to be 1:3 to 1:20, which can reduce the adhesion between the carriers during the preparation process, improve the conduction of polymerization heat, and improve the equipment utilization rate, thereby improving production efficiency. DETAILED DESCRIPTION

[0056] The following examples are provided for a better understanding of the present invention, but are not intended to limit the best mode of implementation, nor to limit the content and protection scope of the present invention. Any product identical or similar to the present invention obtained by anyone under the inspiration of the present invention or by combining the features of the present invention with other prior arts shall fall within the protection scope of the present invention.

[0057] If no specific experimental steps or conditions are specified in the examples, the conventional experimental steps or conditions described in the literature in the field can be used. If no manufacturer is specified for the reagents or instruments used, they are all conventional reagent products that can be obtained commercially.

[0058] In the present invention, the method for determining the chlorine content in the polypeptide solid phase synthesis carrier is modified based on "GB 5009.44-2016 Determination of Chloride in Food", and the specific method is as follows:

[0059] (1) Preparation of carrier samples

[0060] Wash the dried solid-phase synthesis carrier with methanol (30-50 ml per gram of carrier) three times, wash with acetone (30-50 ml per gram of carrier) until there is no white precipitate detected by silver nitrate, wash with ethanol (30-50 ml per gram of carrier) three times and filter dry, and bake the carrier under an infrared lamp for 20 minutes. Use a balance with an accuracy of 0.0001g to weigh two portions of 0.2g of solid-phase synthesis carrier and place them in a silver crucible, add 2g of sodium hydroxide and 5g of potassium nitrate to the silver crucible; change the crucible cover, place the silver crucible on an electric furnace and burn it, remove the silver crucible after about 5 minutes of blue smoke, and cool it; rinse the cooled silver crucible with hot ultrapure water (about 80°C), transfer all the solids to a handle evaporator, add 1 drop of phenolphthalein indicator, stir, add a small amount of concentrated nitric acid, and add 1-2ml of excess after the red color fades;

[0061] (2) Titration

[0062] The titration was performed according to the Volhard method for chlorine content titration in GB 5009.44-2016 Determination of Chloride in Food, and the chlorine content of the solid phase synthetic carrier was calculated.

[0063] In the present invention, the method for determining the degree of substitution, i.e., the content of amino or hydroxyl groups in the polypeptide solid phase synthesis carrier is: reacting with fluorenylmethoxycarbonyl-L-leucine (Fmoc-Leu-OH), then removing the Fmoc protecting group, determining the amount of Fmoc removed by colorimetry, and then calculating the content of amino or hydroxyl groups in the solid phase synthesis carrier. Typically, but not limited to, specifically: accurately weigh 1.0 g of the solid phase synthesis carrier, suspend it in 10 ml of N,N-dimethylformamide solvent, and then add Fmoc-Leu-OH 1g, 1g of benzotriazole-N,N,N',N'-tetramethyluronium hexafluorophosphate (HBTU) and 1ml of N,N-diisopropylethylamine (DIEA), stirred at room temperature for 2h; after the reaction, washed with N,N-dimethylformamide (10-20ml each time, washed 3 times) and methanol (10-20ml each time, washed 3 times), and then dried to obtain a solid phase synthesis carrier preloaded with Fmoc-Leu; 0.1g of preloaded Fmoc- The solid phase synthesis carrier of Leu is suspended in a 20% piperidine / N,N-dimethylformamide (v / v) solution, shaken at room temperature for 30 minutes, filtered, the filtrate is collected, washed with N,N-dimethylformamide and the filtrate is collected; the filtrates are combined and fixed to volume, and the absorbance at 300nm is measured after dilution by an appropriate multiple; the above Fmoc removal reaction is repeated through a series of known concentrations of Fmoc-Leu-OH and the absorbance is measured to make a standard curve; the content of amino or hydroxyl groups in the peptide solid phase synthesis carrier is calculated through the standard curve.

[0064] Example 1

[0065] This embodiment provides a method for preparing a polypeptide solid phase synthesis carrier, comprising the following steps:

[0066] (1) Prepare the aqueous phase: add 2 L of purified water to a 3 L reactor equipped with a condenser, a stirrer and a thermometer, then add 20 g of polyvinyl alcohol and dissolve for later use;

[0067] (2) preparing the oil phase: weigh 240 g of styrene, 4.5 g of allyl ether, 55.5 g of p-chloromethylstyrene and 3 g of benzoyl peroxide, and mix them evenly;

[0068] (3) Add the oil phase to the reactor, start stirring, raise the temperature to 75°C and polymerize for 12 hours. After the reaction is completed, wash with hot water, sieve and collect the product with a particle size of 75 to 150 μm, and vacuum dry to obtain a base ball with a chlorine content of 1.2 mmol / g;

[0069] (4) Add 50 g of the base ball obtained in (3) and 500 ml of N,N-dimethylformamide to a 1 L reactor equipped with a condenser, a stirrer and a thermometer, and start stirring; add 30 g of potassium phthalimide, raise the temperature to 95° C. and react for 16 h; after the reaction is completed, cool to room temperature, wash twice with N,N-dimethylformamide, then wash with purified water until neutral, and finally wash three times with anhydrous ethanol and filter dry, and return to the reactor; add 200 g of potassium phthalimide to the reactor. 50g of anhydrous ethanol and 50g of hydrazine hydrate, heated to 75℃ and reacted for 16 hours; after the reaction, washed 3 times with ethanol / purified water solution with a volume ratio of 50:50, washed with purified water until neutral, washed 3 times with anhydrous ethanol and filtered, and put back into the reactor; added 200g of anhydrous ethanol and 50g of concentrated hydrochloric acid to the reactor, heated to 60℃ and reacted for 6h; after the reaction, cooled to room temperature, washed with water until neutral, and then vacuum dried to obtain a solid phase synthesis carrier containing amino groups. After testing, the amino content of the obtained amino solid phase synthesis carrier is 1.15mmol / g.

[0070] Example 2

[0071] This embodiment provides a method for preparing a polypeptide solid phase synthesis carrier, comprising the following steps:

[0072] (1) Prepare the aqueous phase: add 2 L of purified water to a 3 L reactor equipped with a condenser, a stirrer and a thermometer, then add 10 g of polyvinyl alcohol and dissolve for later use;

[0073] (2) preparing the oil phase: weigh 250 g of styrene, 9 g of ethylene glycol diallyl ether, 41 g of p-chloromethylstyrene and 3 g of benzoyl peroxide, and mix them evenly;

[0074] (3) Add the oil phase to the reactor, start stirring, raise the temperature to 80°C and polymerize for 10 hours. After the reaction is completed, wash with hot water, sieve and collect the product with a particle size of 75 to 150 μm, and vacuum dry to obtain a base ball with a chlorine content of 0.9 mmol / g;

[0075] (4) Add 50 g of the base ball obtained in (3) and 500 ml of N,N-dimethylformamide to a 1 L reactor equipped with a condenser, a stirrer and a thermometer, and start stirring; then add 30 g of p-hydroxybenzyl alcohol, raise the temperature to 50°C and react for 16 hours; after the reaction is completed, cool to room temperature, then wash with N,N-dimethylformamide twice, wash with purified water until neutral, and then wash with anhydrous ethanol three times and filter the solid phase synthesis carrier, vacuum dry, and obtain a solid phase synthesis carrier containing a hydroxyl functional group. After testing, the hydroxyl content of the obtained hydroxyl solid phase synthesis carrier is 0.85 mmol / g.

[0076] Example 3

[0077] This embodiment provides a method for preparing a polypeptide solid phase synthesis carrier, comprising the following steps:

[0078] (1) Prepare the aqueous phase: add 2 L of purified water to a 3 L reactor equipped with a condenser, a stirrer and a thermometer, then add 10 g of polyvinyl alcohol and dissolve for later use;

[0079] (2) preparing the oil phase: weigh 210 g of styrene, 14 g of diethylene glycol divinyl ether, 76 g of p-chloromethylstyrene and 3 g of benzoyl peroxide, and mix them evenly;

[0080] (3) Add the oil phase to the reactor, start stirring, and heat to 85°C for polymerization for 8 hours. After the reaction is completed, wash with hot water, sieve and collect the product with a particle size of 75 to 150 μm, and vacuum dry to obtain a base ball with a chlorine content of 1.6 mmol / g;

[0081] (4) Add 50 g of the base ball obtained in (3) and 500 ml of N,N-dimethylformamide to a 1L reactor equipped with a condenser, a stirrer and a thermometer, and start stirring; then add 30 g of p-hydroxybenzyl alcohol, raise the temperature to 50°C and react for 16 hours; after the reaction is completed, cool to room temperature, then wash with N,N-dimethylformamide twice, wash with purified water until neutral, and then wash with anhydrous ethanol three times and filter the solid phase synthesis carrier, vacuum dry, and obtain a solid phase synthesis carrier containing a hydroxyl functional group. After testing, the hydroxyl content of the obtained hydroxyl solid phase synthesis carrier is 1.4 mmol / g.

[0082] Example 4

[0083] This embodiment provides a method for preparing a polypeptide solid phase synthesis carrier, comprising the following steps:

[0084] (1) Prepare the aqueous phase: add 2 L of purified water to a 3 L reactor equipped with a condenser, a stirrer and a thermometer, then add 10 g of polyvinyl alcohol and dissolve for later use;

[0085] (2) preparing the oil phase: weigh 270 g of styrene, 6 g of ethylene glycol diallyl ether, 24 g of p-chloromethylstyrene and 3 g of benzoyl peroxide, and mix them evenly;

[0086] (3) Add the oil phase into the reactor, start stirring, raise the temperature to 80°C and polymerize for 10 hours. After the reaction is completed, wash with hot water, sieve and collect the product with a particle size of 75 to 150 μm, and vacuum dry to obtain a base ball with a chlorine content of 0.5 mmol / g;

[0087] (4) Add 50 g of the base ball obtained in (3) and 500 ml of N,N-dimethylformamide to a 1 L reactor equipped with a condenser, a stirrer and a thermometer, and start stirring; add 30 g of potassium phthalimide, raise the temperature to 95° C. and react for 16 h; after the reaction is completed, cool to room temperature, wash twice with N,N-dimethylformamide, then wash with purified water until neutral, and finally wash three times with anhydrous ethanol and filter dry, and return to the reactor; add 200 g of potassium phthalimide to the reactor. 50g of anhydrous ethanol and 50g of hydrazine hydrate, heated to 75℃ and reacted for 16 hours; after the reaction, washed 3 times with ethanol / purified water solution with a volume ratio of 50:50, washed with purified water until neutral, washed 3 times with anhydrous ethanol and filtered, and put back into the reactor; added 200g of anhydrous ethanol and 50g of concentrated hydrochloric acid to the reactor, heated to 60℃ and reacted for 6h; after the reaction, cooled to room temperature, washed with water until neutral, and then vacuum dried to obtain a solid phase synthesis carrier containing amino groups. After testing, the amino content of the obtained amino solid phase synthesis carrier is 0.45mmol / g.

[0088] Example 5

[0089] This embodiment provides a method for preparing a polypeptide solid phase synthesis carrier, comprising the following steps:

[0090] (1) Prepare the aqueous phase: add 2 L of purified water to a 3 L reactor equipped with a condenser, a stirrer and a thermometer, then add 10 g of polyvinyl alcohol and dissolve for later use;

[0091] (2) preparing the oil phase: weigh 240 g of styrene, 10 g of diethylene glycol divinyl ether, 50 g of p-chloromethylstyrene and 3 g of benzoyl peroxide, and mix them evenly;

[0092] (3) Add the oil phase into the reactor, start stirring, raise the temperature to 80°C and polymerize for 6 hours. After the reaction is completed, wash with hot water, sieve and collect the product with a particle size of 75 to 150 μm, and vacuum dry to obtain a base ball with a chlorine content of 1.0 mmol / g;

[0093] (4) Add 50 g of the base ball obtained in (3) and 500 ml of N,N-dimethylformamide to a 1 L reactor equipped with a condenser, a stirrer and a thermometer, and start stirring; then add 30 g of p-hydroxybenzyl alcohol, raise the temperature to 50°C and react for 16 hours; after the reaction is completed, cool to room temperature, then wash with N,N-dimethylformamide twice, wash with purified water until neutral, and then wash with anhydrous ethanol three times and filter the solid phase synthesis carrier, vacuum dry, and obtain a solid phase synthesis carrier containing a hydroxyl functional group. After testing, the hydroxyl content of the obtained hydroxyl solid phase synthesis carrier is 0.93 mmol / g.

[0094] Example 6

[0095] This embodiment provides a method for preparing a polypeptide solid phase synthesis carrier, comprising the following steps:

[0096] (1) Prepare the aqueous phase: add 2 L of purified water to a 3 L reactor equipped with a condenser, a stirrer and a thermometer, then add 10 g of polyvinyl alcohol and dissolve for later use;

[0097] (2) Prepare the oil phase: weigh 290 g of styrene, 10 g of ethylene glycol diallyl ether and 3 g of benzoyl peroxide and mix them evenly;

[0098] (3) Add the oil phase into the reactor, start stirring, raise the temperature to 80°C and polymerize for 12 hours. After the reaction is completed, wash with hot water, sieve and collect the product with a particle size of 75 to 150 μm, and vacuum dry to obtain base balls;

[0099] (4) Add 50g of the base ball obtained in (3) to a 1L reactor equipped with a stirrer and a thermometer, add 450ml of dichloromethane, and start stirring; lower the temperature in the reactor to -5°C; add 30g of chloromethyl ether to the reactor and continue stirring; add 3g of tin tetrachloride to the reactor; keep the temperature in the reactor at 0-5°C, and react for 2h before the reaction is terminated. Drain the solution in the reactor, wash it 3 times with dichloromethane and filter it, then place it in a 45°C oven to dry, and obtain a chloromethylated base ball with a chlorine content of 0.79mmol / g.

[0100] (5) Add 50 g of the chloromethylated modified base ball obtained in (4) and 500 ml of N,N-dimethylformamide to a 1L reactor equipped with a condenser, a stirrer and a thermometer, and start stirring; then add 30 g of p-hydroxybenzyl alcohol, raise the temperature to 50°C and react for 16 hours; after the reaction is completed, cool to room temperature, then wash with N,N-dimethylformamide twice, wash with purified water until neutral, and then wash with anhydrous ethanol three times and filter the solid phase synthesis carrier, vacuum dry, and obtain a solid phase synthesis carrier containing a hydroxyl functional group. After testing, the hydroxyl content of the obtained hydroxyl solid phase synthesis carrier is 0.72 mmol / g.

[0101] Comparative Example 1

[0102] This comparative example provides a method for preparing a polypeptide solid phase synthesis carrier, comprising the following steps:

[0103] (1) Prepare the aqueous phase: add 2 L of purified water to a 3 L reactor equipped with a condenser, a stirrer and a thermometer, then add 20 g of polyvinyl alcohol and 30 g of sodium chloride and dissolve them for later use;

[0104] (2) preparing the oil phase: weighing 250 g of styrene, 5 g of divinylbenzene (the weight percentage of divinylbenzene is 63%), 45 g of p-chloromethylstyrene and 3 g of benzoyl peroxide, and mixing them evenly;

[0105] (3) Add the oil phase into the reactor, start stirring, raise the temperature to 80°C and polymerize for 10 hours. After the reaction is completed, wash with hot water, sieve and collect the product with a particle size of 75 to 150 μm, and vacuum dry to obtain a base ball with a chlorine content of 1.0 mmol / g;

[0106] (4) Add 50 g of the base ball obtained in (3) and 500 ml of N,N-dimethylformamide to a 1 L reactor equipped with a condenser, a stirrer and a thermometer, and start stirring; add 30 g of potassium phthalimide, raise the temperature to 95° C. and react for 16 h; after the reaction is completed, cool to room temperature, wash twice with N,N-dimethylformamide, then wash with purified water until neutral, and finally wash three times with anhydrous ethanol and filter dry, and return to the reactor; add 200 g of potassium phthalimide to the reactor. 50g of anhydrous ethanol and 50g of hydrazine hydrate, heated to 75℃ and reacted for 16 hours; after the reaction, washed 3 times with ethanol / purified water solution with a volume ratio of 50:50, washed with purified water until neutral, washed 3 times with anhydrous ethanol and filtered, and put back into the reactor; added 200g of anhydrous ethanol and 50g of concentrated hydrochloric acid to the reactor, heated to 60℃ and reacted for 6h; after the reaction, cooled to room temperature, washed with water until neutral, and then vacuum dried to obtain a solid phase synthesis carrier containing amino groups. After testing, the amino content of the obtained amino solid phase synthesis carrier is 0.90mmol / g.

[0107] Comparative Example 2

[0108] This comparative example provides a method for preparing a polypeptide solid phase synthesis carrier, comprising the following steps:

[0109] (1) Prepare the aqueous phase: add 2 L of purified water to a 3 L reactor equipped with a condenser, a stirrer and a thermometer, then add 20 g of polyvinyl alcohol and 30 g of sodium chloride and dissolve them for later use;

[0110] (2) preparing the oil phase: weighing 250 g of styrene, 5 g of divinylbenzene (the weight percentage of divinylbenzene is 63%), 45 g of p-chloromethylstyrene and 3 g of benzoyl peroxide, and mixing them evenly;

[0111] (3) Add the oil phase into the reactor, start stirring, raise the temperature to 80°C and polymerize for 10 hours. After the reaction is completed, wash with hot water, sieve and collect the product with a particle size of 75 to 150 μm, and vacuum dry to obtain a base ball with a chlorine content of 1.0 mmol / g;

[0112] (4) Add 50g of the base ball obtained in (4) and 500ml of N,N-dimethylformamide to a 1L reactor equipped with a condenser, a stirrer and a thermometer, and start stirring; then add 30g of p-hydroxybenzyl alcohol, raise the temperature to 50°C and react for 16h; after the reaction is completed, cool to room temperature, then wash with N,N-dimethylformamide twice, wash with purified water until neutral, and then wash with anhydrous ethanol three times and filter the solid phase synthesis carrier, vacuum dry, and obtain a solid phase synthesis carrier containing a hydroxyl functional group. After testing, the hydroxyl content of the obtained hydroxyl solid phase synthesis carrier is 0.87mmol / g.

[0113] Comparative Example 3

[0114] This embodiment provides a method for preparing a polypeptide solid phase synthesis carrier, comprising the following steps:

[0115] (1) Prepare the aqueous phase: add 2 L of purified water to a 3 L reactor equipped with a condenser, a stirrer and a thermometer, then add 20 g of polyvinyl alcohol and dissolve for later use;

[0116] (2) preparing the oil phase: weigh 240 g of styrene, 4.5 g of diethylene glycol dimethacrylate, 55.5 g of p-chloromethylstyrene and 3 g of benzoyl peroxide, and mix them evenly;

[0117] (3) Add the oil phase to the reactor, start stirring, raise the temperature to 75°C and polymerize for 12 hours. After the reaction is completed, wash with hot water, sieve and collect the product with a particle size of 75 to 150 μm, and vacuum dry to obtain a solid phase synthesis carrier with a chlorine content of 1.2 mmol / g;

[0118] (4) Add 50 g of the solid phase synthesis support obtained in (3) and 500 ml of N,N-dimethylformamide to a 1 L reactor equipped with a condenser, a stirrer and a thermometer, and start stirring; add 30 g of potassium phthalimide, raise the temperature to 95° C. and react for 16 h; after the reaction, cool to room temperature, wash twice with N,N-dimethylformamide, then wash with purified water until neutral, and finally wash three times with anhydrous ethanol and filter dry, then return to the reactor; add 20 0g of anhydrous ethanol and 50g of hydrazine hydrate, heated to 75℃ and reacted for 16 hours; after the reaction, washed 3 times with ethanol / purified water solution with a volume ratio of 50:50, washed with purified water until neutral, washed 3 times with anhydrous ethanol and filtered, and put back into the reactor; added 200g of anhydrous ethanol and 50g of concentrated hydrochloric acid to the reactor, heated to 60℃ and reacted for 6h; after the reaction, cooled to room temperature, washed with water until neutral, and then vacuum dried to obtain a solid phase synthesis carrier containing amino groups. After testing, the amino content of the obtained amino solid phase synthesis carrier is 1.13mmol / g.

[0119] Comparative Example 4

[0120] This embodiment provides a method for preparing a polypeptide solid phase synthesis carrier, comprising the following steps:

[0121] (1) Prepare the aqueous phase: add 2 L of purified water to a 3 L reactor equipped with a condenser, a stirrer and a thermometer, then add 20 g of polyvinyl alcohol and dissolve for later use;

[0122] (2) Prepare the oil phase: weigh 295.5 g of p-chloromethylstyrene, 4.5 g of allyl ether and 3 g of benzoyl peroxide, and mix them evenly;

[0123] (3) Add the oil phase to the reactor, start stirring, raise the temperature to 75°C and polymerize for 12 hours. After the reaction is completed, wash with hot water, sieve and collect the product with a particle size of 75 to 150 μm, and vacuum dry to obtain a base ball with a chlorine content of 6.3 mmol / g;

[0124] (4) Add 50 g of the base ball obtained in (3) and 500 ml of N,N-dimethylformamide to a 1 L reactor equipped with a condenser, a stirrer and a thermometer, and start stirring; add 100 g of potassium phthalimide, raise the temperature to 95° C. and react for 16 h; after the reaction is completed, cool to room temperature, wash twice with N,N-dimethylformamide, then wash with purified water until neutral, and finally wash three times with anhydrous ethanol and filter dry, and return to the reactor; add 200 g of potassium phthalimide to the reactor. 50g of anhydrous ethanol and 50g of hydrazine hydrate, heated to 75℃ and reacted for 16 hours; after the reaction, washed 3 times with ethanol / purified water solution with a volume ratio of 50:50, washed with purified water until neutral, washed 3 times with anhydrous ethanol and filtered, and put back into the reactor; added 200g of anhydrous ethanol and 50g of concentrated hydrochloric acid to the reactor, heated to 60℃ and reacted for 6h; after the reaction, cooled to room temperature, washed with water until neutral, and then vacuum dried to obtain a solid phase synthesis carrier containing amino groups. After testing, the amino content of the obtained amino solid phase synthesis carrier is 5.5mmol / g.

[0125] Test Example 1

[0126] The amino-containing solid phase synthesis carriers in Example 1, Example 4 and Comparative Examples 1, 3, and 4 were connected with 4-[(2,4-dimethoxyphenyl)(fluorenylmethoxycarbonyl-amino)methyl]phenoxyacetic acid (Fmoc linker) and used for the synthesis of polypeptide ACP (65-74), and the specific steps were as follows:

[0127] (1) Add 2g of solid phase synthesis carrier, 15ml of N,N-dimethylformamide, and then add 2g of Fmoc linker, 2g of HBTU and 2ml of DIEA to a 25ml round-bottom flask, and stir at room temperature for 2h. After the reaction is completed, wash the carrier with N,N-dimethylformamide (10ml each time, wash 3 times) and methanol (10ml each time, wash 3 times), and then dry the carrier. A solid phase synthesis carrier containing Fmoc linker (i.e., Rink AM carrier) is obtained.

[0128] (2) Place 1 g of the Rink AM carrier obtained in (1) into a 25 ml round-bottom flask for later use;

[0129] (3) Add 10 ml of N,N-dimethylformamide solution into the reactor, stir and swell for 30 min, and then filter the solution;

[0130] (4) Add 10 mL of 20% piperidine / N,N-dimethylformamide solution (v / v) and stir the reaction for 0.5 h to remove the Fmoc protecting group. After the reaction is completed, wash three times with N,N-dimethylformamide (10 ml), dichloromethane (10 ml), and N,N-dimethylformamide (10 ml) in sequence to enter the peptide grafting reaction cycle.

[0131] (5) In a beaker, weigh 1 g of Fmoc-Gly-OH and 1 g of HOBt, add 10 ml of DMF and stir to dissolve, then add 1 ml of DIC to the beaker and mix well;

[0132] (6) Add the solution in step (5) to the reactor and stir the reaction at room temperature for 2 hours; then take 0.001 g and place it in a glass test tube, wash it with ethanol three times, add three drops of 5% ninhydrin solution, and boil it in a boiling water bath for 3 minutes. If the carrier does not change color, it means that the reaction is complete, otherwise continue the reaction; after the reaction is complete, wash it with N,N-dimethylformamide (10 ml), dichloromethane (10 ml), and N,N-dimethylformamide (10 ml) three times in sequence to obtain the Rink AM solid phase synthesis carrier preloaded with Fmoc-Gly amino acid.

[0133] (7) Repeat steps (4), (5), (6) with different Fmoc-amino acids, and finally obtain a solid phase synthesis carrier connected with a polypeptide: Fmoc-Val-Gln-(Trt)-Ala-Ala-Ile-Asp(otBu)-Tyr(tBu)-Ile-Asn(Trt)-Gly-RinkAM carrier. The solid phase synthesis carrier loaded with the polypeptide is washed three times with methanol (20 ml each time), each time for 30 minutes, and then filtered and dried, and the carrier is placed in a vacuum oven to dry for 18 hours.

[0134] The dried carrier was observed under a microscope, and 200 carrier particles were selected to observe their breakage and calculate their breakage rate. The results are shown in Table 1.

[0135] Test Example 2

[0136] The solid phase synthesis carrier containing hydroxyl groups (i.e., wang carrier) in Examples 2, 3, 5, and Comparative Example 2 was applied to the synthesis of polypeptide ACP (65-74), and the specific steps were as follows:

[0137] (1) Place 1 g of a solid phase synthesis support containing hydroxyl groups in a 25 ml round-bottom flask for later use;

[0138] (2) Add 10 ml of dichloromethane to the reactor, stir and swell for 30 min, and then filter the solution;

[0139] (3) In a beaker, weigh 1 g Fmoc-Gly-OH and 1 g HOBt, add 10 ml DMF and stir to dissolve, then weigh 1 ml DIC and add 0.05 g DMAP to the beaker and mix well;

[0140] (4) Add the solution in step (3) to the reactor and stir the reaction at room temperature for 2 hours; after the reaction is complete, wash three times with N, N-dimethylformamide (10 ml), dichloromethane (10 ml), and N, N-dimethylformamide (10 ml) in sequence to obtain a wang solid phase synthesis support preloaded with Fmoc-Gly amino acid;

[0141] (5) Add 10 mL of 20% piperidine / N,N-dimethylformamide solution (v / v) and stir the reaction for 0.5 h to remove the Fmoc protecting group. After the reaction is completed, wash three times with N,N-dimethylformamide (10 ml), dichloromethane (10 ml), and N,N-dimethylformamide (10 ml) in sequence to enter the peptide grafting reaction cycle.

[0142] (6) In a beaker, weigh 1 g of Fmoc-protected amino acid and 1 g of HOBt, add 10 ml of DMF and stir to dissolve, then add 1 ml of DIC to the beaker and mix well;

[0143] (7) Add the solution in step (6) to the reactor and stir the reaction at room temperature for 2 hours; then take 0.001 g and place it in a glass test tube, wash it with ethanol three times, add three drops of 5% ninhydrin solution, and boil it in a boiling water bath for 3 minutes. If the carrier does not change color, it means that the reaction is complete, otherwise continue the reaction; after the reaction is complete, wash it with N,N-dimethylformamide (10 ml), dichloromethane (10 ml), and N,N-dimethylformamide (10 ml) three times in sequence;

[0144] (8) Repeat steps (5), (6), (7) with different Fmoc-amino acids, and finally obtain a solid phase synthesis carrier connected with a polypeptide: Fmoc-Val-Gln-(Trt)-Ala-Ala-Ile-Asp(otBu)-Tyr(tBu)-Ile-Asn(Trt)-Gly-wang carrier. Wash the solid phase synthesis carrier loaded with the polypeptide with methanol (20 ml each time), 30 minutes each time, filter and dry after washing, and place the carrier in a vacuum oven to dry for 18 hours.

[0145] The dried carrier was observed under a microscope, and 200 carrier particles were selected to observe their breakage and calculate their breakage rate. The results are shown in Table 1.

[0146] Test Example 3

[0147] The purity of the polypeptides prepared in Test Example 1 and Test Example 2 was detected, and the specific steps were as follows:

[0148] (1) Take a solid phase synthesis carrier connected with a polypeptide, add a cleavage reagent, the cleavage reagent is a reagent with a volume ratio of trifluoroacetic acid: thioanisole: water = 95:2.5:2.5, add 10 ml of the cleavage reagent per gram of the solid phase synthesis carrier connected with the polypeptide, react at room temperature for 4 hours, cut the polypeptide from the carrier, filter, collect the filtrate, wash twice with 10 ml of trifluoroacetic acid and filter, collect the filtrate and combine.

[0149] (2) Liquid phase analysis was performed using a chromatographic column: ZORBAX SB-C18 (5 μm, 4.6×150 mm, Agilent), a mobile phase of 18% acetonitrile / water (v / v) solution containing 0.05% trifluoroacetic acid (v / v), a flow rate of 0.8 ml / min, and a detection wavelength of 220 nm to detect peptide purity. The results are shown in Table 1.

[0150] Test Example 4

[0151] The swelling degree of the polypeptide solid phase synthesis carrier prepared in the embodiment and the comparative example is detected, and the specific steps are as follows: use a balance with an accuracy of 0.0001g to weigh 2 portions of 2.0g of polypeptide solid phase synthesis carrier samples, respectively put them into 25ml stoppered measuring cylinders, and add dichloromethane (DCM) and N,N-dimethylformamide (DMF) to the scale; stir the carrier and the solvent with a glass rod to fully disperse the carrier; cover the stopper and fully expand for 4h; then slowly stir the carrier with a glass stirring rod to remove bubbles and make the carrier dispersed evenly without agglomeration; take out the stirring rod, knock the measuring cylinder on a table with a rubber pad to make the carrier compact, until the volume of the carrier no longer changes position, and read its volume. The swelling degree is calculated according to the following formula. The results are shown in Table 1.

[0152]

[0153] Table 1

[0154]

[0155]

[0156] By comparing the data of the embodiment and the comparative example, it can be seen that the polypeptide solid phase synthesis carrier prepared by the preparation method of the present invention in the embodiment has good swelling degree and very low breakage rate when preparing polypeptides, which greatly improves the purity of the prepared polypeptides, improves product quality and further reduces costs.

[0157] Obviously, the above embodiments are merely examples for clear explanation, and are not intended to limit the implementation methods. For those skilled in the art, other different forms of changes or modifications can be made based on the above description. It is not necessary and impossible to list all the implementation methods here. The obvious changes or modifications derived therefrom are still within the protection scope of the invention.

Claims

1. A method for preparing a polypeptide solid phase synthesis carrier, characterized in that: The steps include: S1: preparing an aqueous phase, the aqueous phase comprising purified water and a dispersant; S2: preparing an oil phase, wherein the oil phase comprises a monovinyl compound, a crosslinking monomer, a functional monomer and an initiator; The cross-linking monomer includes at least one of allyl ether, ethylene glycol diallyl ether, and diethylene glycol divinyl ether; S3: adding the oil phase into the water phase to undergo polymerization reaction to obtain base balls; S4: Introducing amino groups or hydroxyl groups into the base ball to obtain a polypeptide solid phase synthesis carrier.

2. The method for preparing a polypeptide solid phase synthesis carrier according to claim 1, characterized in that: The dispersant is a water-soluble polymer; and / or, the monovinyl compound is an aromatic monovinyl compound; And / or, the functional monomer refers to a monomer having both a double bond capable of free radical polymerization and a halogenated group or other group that can be converted into a hydroxyl group or an amino group through a reaction; And / or, the initiator is an organic peroxide or an azo compound.

3. The method for preparing a polypeptide solid phase synthesis carrier according to claim 2, characterized in that: The dispersant comprises at least one of polyvinyl alcohol, hydroxyethyl cellulose, hydroxyethyl methyl cellulose, carboxymethyl cellulose, methyl cellulose, ethyl cellulose, sodium polyacrylate and polyvinyl pyrrolidone; and / or, the monovinyl compound includes styrene and its benzene ring substituted derivatives, the substituent being an alkyl or alkoxy group containing 1 to 5 carbon atoms; And / or, the functional monomer includes at least one of acyloxystyrene and its derivatives, haloalkylstyrene monomers, and 4-vinylphenyl ester monomers; Optionally, the functional monomers include 4-acetoxystyrene, benzoyloxystyrene, 4-(4-bromobutyl)styrene, p-chloromethylstyrene, methyl 4-vinylbenzoate, ethyl 4-vinylphenylacetate; And / or, the initiator includes at least one of benzoyl peroxide, lauroyl peroxide, tert-butyl peroxy-2-ethylhexanoate, azobisisobutyronitrile, azobisisovaleronitrile, and azobisisoheptylnitrile.

4. The method for preparing a polypeptide solid phase synthesis carrier according to claim 2 or 3, characterized in that: The dispersant is polyvinyl alcohol; and / or, the monovinyl compound is styrene; And / or, the functional monomer is p-chloromethylstyrene; And / or, the initiator is benzoyl peroxide.

5. The method for preparing a polypeptide solid phase synthesis carrier according to any one of claims 1 to 4, characterized in that: The mass ratio of the oil phase to the water phase is 1:3 to 1:20; and / or, in the oil phase, based on the total weight of the monovinyl compound, the cross-linking monomer, and the functional monomer, the monovinyl compound accounts for 65% to 99.5%; and / or, in the oil phase, the cross-linking monomer accounts for 0.5% to 5% based on the total mass of the monovinyl compound, the cross-linking monomer and the functional monomer; and / or, in the oil phase, the functional monomer accounts for 0 to 30% based on the total mass of the monovinyl compound, the crosslinking monomer and the functional monomer; and / or, in the oil phase, the weight of the initiator is 0.5% to 5% of the total weight of the monovinyl compound, the crosslinking monomer, and the functional monomer; And / or, in the water phase, the weight of the dispersant is 0.2% to 5% of the weight of the water phase.

6. The method for preparing a polypeptide solid phase synthesis carrier according to claim 5, characterized in that: In the oil phase, based on the total weight of the monovinyl compound, the cross-linking monomer, and the functional monomer, the monovinyl compound accounts for 77% to 99%; and / or, in the oil phase, the cross-linking monomer accounts for 1% to 3% based on the total mass of the monovinyl compound, the cross-linking monomer and the functional monomer; And / or, in the oil phase, based on the total weight of the monovinyl compound, the crosslinking monomer and the functional monomer, the functional monomer accounts for 0% to 20%.

7. The method for preparing a polypeptide solid phase synthesis carrier according to claim 5 or 6, characterized in that: When the functional monomer accounts for 0% of the monomer weight percentage in the oil phase, after step S3 and before step S4, the step of introducing halogenated groups or other groups that can be converted into hydroxyl groups or amino groups through reactions into the base ball is also included.

8. The method for preparing a polypeptide solid phase synthesis carrier according to any one of claims 1 to 7, characterized in that: In step S3, the polymerization reaction temperature is 50 to 90° C. and the reaction time is 6 to 24 hours; Preferably, in step S3, the polymerization reaction temperature is 68-85° C. and the time is 8-16 hours.

9. A polypeptide solid phase synthesis carrier obtained by the preparation method according to any one of claims 1 to 8.

10. The polypeptide solid phase synthesis carrier according to claim 9, characterized in that: The particle size of the polypeptide solid phase synthesis carrier is 35 to 300 μm; Preferably, the particle size of the polypeptide solid phase synthesis carrier is 75 to 150 μm.

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