Aminomethyl resin, preparation method thereof and polypeptide solid-phase synthesis method

Abstract

The invention provides aminomethyl resin, a preparation method thereof and a polypeptide solid-phase synthesis method, and belongs to the technical field of aminomethyl resin. The preparation method of the aminomethyl resin comprises the following steps: mixing resin as shown in a formula 1, a compound as shown in a formula 2 and an organic acid catalyst, and carrying out nucleophilic substitution reaction to prepare a compound as shown in a formula 3; hydrolyzing the compound as shown in the formula 3 in an organic amine solution to prepare aminomethyl resin as shown in a formula 4; the organic amine solution comprises organic amine and a solvent, and the solvent comprises at least one of water and an alcohol solvent. According to the preparation method of the aminomethyl resin, the particle size of the aminomethyl resin can be controlled, the activity of an aminomethyl functional group is improved, and the subsequent cleaning and purifying difficulty of a product is reduced.

Description

Technical Field

[0001] This application relates to the field of aminomethyl resin technology, and in particular to an aminomethyl resin and its preparation method, and a method for solid-phase synthesis of polypeptides. Background Technology

[0002] Peptides are ubiquitous in living organisms, composed of amino acids, and possess a wide range of biological activities and good safety profiles, finding extensive applications in medicine, food, cosmetics, agriculture, and animal husbandry. Amide resin is a commonly used starting resin for solid-phase peptide synthesis. It is typically introduced onto polystyrene (PS) resin via phthalimide methylation. However, subsequent hydrolysis using hydrazine in water or alcohol solutions is often employed to prepare aminoide resin. However, the byproduct 2,3-dihydrophthalo-1,4-dione generated during hydrolysis has low solubility and is difficult to remove. Furthermore, the prepared aminoide resin suffers from uncontrollable particle size and low activity of the aminomethyl functional groups. Summary of the Invention

[0003] Based on this, the main objective of this application is to provide an aminomethyl resin and its preparation method, as well as a solid-phase synthesis method for polypeptides, so as to achieve controllable particle size of aminomethyl resin, improve the activity of aminomethyl functional groups, and reduce the difficulty of subsequent cleaning and purification of products.

[0004] The first aspect of this application provides a method for preparing an aminomethyl resin, comprising the following steps:

[0005] The resin shown in Formula 1, the compound shown in Formula 2, and the organic acid catalyst were mixed and a nucleophilic substitution reaction was carried out to prepare the compound shown in Formula 3.

[0006] The compound represented by Formula 3 was hydrolyzed in an organic amine solution to prepare the aminomethyl resin represented by Formula 4.

[0007] The organic amine solution comprises an organic amine and a solvent, wherein the solvent comprises at least one of water and alcohol solvents;

[0008] Formula 1; Formula 2; Formula 3; Equation 4;

[0009] R1 is selected from hydroxyl, chlorine atom, bromine atom, methoxy or benzyloxy.

[0010] In some embodiments, the organic amine solution contains at least one of methylamine, benzylamine, diethanolamine, and ethanolamine.

[0011] In some embodiments, the alcohol solvent in the organic amine solution includes at least one of ethanol, propanol, and isopropanol.

[0012] In some embodiments, the volume ratio of organic amine to solvent in the organic amine solution is (0.2-1):1.

[0013] In some embodiments, the hydrolysis conditions include a temperature of 90-150°C and a reaction time of 8-20 h.

[0014] In some embodiments, the conditions for the nucleophilic substitution reaction include: a reaction temperature of 20-60°C and a reaction time of 8-20 h.

[0015] In some embodiments, the mass molar ratio of the resin shown in Formula 1 to the compound shown in Formula 2 is 1 g: (1-2) mmol.

[0016] In some embodiments, the organic acid catalyst includes at least one of methanesulfonic acid, trifluoromethanesulfonic acid, and p-toluenesulfonic acid.

[0017] In some embodiments, the mass ratio of the organic acid catalyst to the resin shown in Formula 1 is (0.5-1):1.

[0018] In some embodiments, the resin represented by Formula 1 is prepared by the following method:

[0019] The polymer monomer, crosslinking agent, initiator, dispersant and water are mixed and subjected to suspension polymerization to prepare the resin shown in Formula 1.

[0020] In some embodiments, the polymer monomer includes at least one selected from styrene, α-methylstyrene, chloromethylstyrene, allylbenzene, and allylbenzene.

[0021] In some embodiments, the crosslinking agent includes at least one of divinylbenzene, divinyltoluene, and trivinyltoluene.

[0022] In some embodiments, the initiator includes at least one of azobisisobutyronitrile, benzoyl peroxide, and di-tert-butyl peroxide.

[0023] In some embodiments, the dispersant includes at least one of polyethylene glycol, polyvinylpyrrolidone, and gelatin.

[0024] In some embodiments, the mass ratio of the polymer monomer, the dispersant, and the water is (10-30):(0.5-5):100.

[0025] In some embodiments, the mass ratio of the crosslinking agent to the initiator is (0.5-2):(0.5-2).

[0026] In some embodiments, the conditions for the suspension polymerization reaction include: a stirring speed of 200-400 rpm; a reaction temperature of 70-90°C; and a reaction time of 8-20 h.

[0027] In a second aspect of this application, an aminomethyl resin prepared by the method described in the first aspect is provided.

[0028] A third aspect of this application provides a method for solid-phase synthesis of polypeptides, comprising the following steps:

[0029] Polypeptides are prepared using aminomethyl resin prepared by the method described in the first aspect or aminomethyl resin described in the second aspect as starting materials.

[0030] Compared with traditional technologies, this application has at least the following beneficial effects:

[0031] This application utilizes an organic acid as a catalyst to conduct a nucleophilic substitution reaction to prepare the compound shown in Formula 3. Then, the compound is hydrolyzed in an organic amine solution. By adjusting the composition of the organic amine solution, the particle size of the aminomethyl resin can be controlled, increasing the amino functional group density to meet the needs of different applications. Simultaneously, the hydrolysis byproduct N-hydroxyethyl phthalimide is soluble in the organic amine solution and can be removed without a complex purification process, simplifying the process and requiring less organic solvent for purification. The reaction exhibits good stability and reproducibility during the preparation of the aminomethyl resin, making it suitable for industrial production. Attached Figure Description

[0032] To better describe and illustrate the embodiments or examples provided in this application, reference may be made to one or more accompanying drawings. Additional details or examples used to describe the drawings should not be considered as limiting the scope of any of the disclosed applications, the currently described embodiments or examples, or the best mode of conduct of these applications as currently understood. Furthermore, the same reference numerals denote the same parts throughout the drawings. In the drawings:

[0033] Figure 1 This is a schematic diagram of a solid-phase peptide synthesis method; where R represents resin, Linker represents linker molecule, A represents the first amino acid molecule, and B represents the second amino acid molecule.

[0034] Figure 2 This is the standard curve for the deprotection of Fmoc-Gly-OH. Detailed Implementation

[0035] The present application will be further described in detail below with reference to the embodiments and examples. These embodiments and examples are only for illustrating the present application and are not intended to limit the scope of the present application. The purpose of providing these embodiments and examples is to make the disclosure of the present application more thorough and comprehensive. It should also be understood that the present application can be implemented in many different forms and is not limited to the embodiments and examples described herein. Those skilled in the art can make various modifications or alterations without departing from the spirit of the present application, and the equivalent forms obtained also fall within the protection scope of the present application. In addition, numerous specific details are set forth in the following description to provide a fuller understanding of the present application. It should be understood that the present application can be implemented without one or more of these details.

[0036] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the application. The term "and / or" as used herein includes any and all combinations of one or more of the associated listed items.

[0037] Unless otherwise specified, the term "solution" in this application refers to an aqueous solution, and the term "ethanol" in this application refers to anhydrous ethanol.

[0038] To address the problems of difficult byproduct removal, uncontrollable particle size, and low activity of aminomethyl functional groups in the current method of preparing aminomethyl resin by methylation of phthalimide, which uses hydrazine in water or alcohol solution for hydrolysis, this application uses an organic acid as a catalyst to carry out a nucleophilic substitution reaction to prepare the compound shown in Formula 3. Then, the compound shown in Formula 3 is hydrolyzed in an organic amine solution. By adjusting the composition of the organic amine solution, the particle size of the aminomethyl resin can be controlled, and the amino functional group density of the aminomethyl resin can be increased to meet the needs of different applications. At the same time, the hydrolysis byproduct N-hydroxyethyl phthalimide can be dissolved in the organic amine solution and can be removed without a complicated impurity removal process, making the treatment simple.

[0039] The first aspect of this application provides a method for preparing an aminomethyl resin, comprising the following steps:

[0040] The resin shown in Formula 1, the compound shown in Formula 2, and the organic acid catalyst were mixed and a nucleophilic substitution reaction was carried out to prepare the compound shown in Formula 3.

[0041] The compound represented by Formula 3 was hydrolyzed in an organic amine solution to prepare the aminomethyl resin represented by Formula 4.

[0042] The organic amine solution comprises an organic amine and a solvent, wherein the solvent comprises at least one of water and alcohol solvents;

[0043] Formula 1; Formula 2; Formula 3; Equation 4;

[0044] R1 is selected from hydroxyl (-OH), chlorine atom (-Cl), bromine atom (-Br), methoxy (-OCH3) or benzyloxy (-OBn).

[0045] This application prepares the compound shown in Formula 3 by nucleophilic substitution reaction using an organic acid as a catalyst, and then hydrolyzes the compound shown in Formula 3 in an organic amine solution of a specific composition to prepare aminomethyl resin. This achieves high yield and high active site synthesis of aminomethyl resin, increases the amino functional group density of aminomethyl resin, and makes the hydrolysis byproduct N-hydroxyethyl phthalimide easy to wash away.

[0046] In some embodiments, the resin represented by Formula 1 is prepared by the following method:

[0047] The polymer monomer, crosslinking agent, initiator, dispersant and water are mixed and subjected to suspension polymerization to prepare the resin shown in Formula 1.

[0048] In some embodiments, the polymer monomer includes at least one selected from styrene, α-methylstyrene, chloromethylstyrene, allylbenzene, and allylbenzene.

[0049] In some embodiments, the crosslinking agent includes at least one of divinylbenzene, divinyltoluene, and trivinyltoluene.

[0050] In some embodiments, the initiator includes at least one of azobisisobutyronitrile, benzoyl peroxide, and di-tert-butyl peroxide.

[0051] In some embodiments, the dispersant includes at least one of polyethylene glycol, polyvinylpyrrolidone, and gelatin.

[0052] In some embodiments, the polymer monomer is styrene, the crosslinking agent is divinylbenzene, and the resin represented by Formula 1 is prepared by the following method:

[0053] Styrene, divinylbenzene, initiator, dispersant and water are mixed and subjected to suspension polymerization to prepare the resin shown in Formula 1. The reaction formula is as follows:

[0054] .

[0055] In some embodiments, the mass ratio of the polymer monomer, the dispersant, and the water is (10-30):(0.5-5):100, which can be 10:0.5:100, 10:5:100, 16.67:1:100, 30:0.5:100, or 30:5:100.

[0056] In some embodiments, the mass ratio of the crosslinking agent to the initiator is (0.5-2):(0.5-2), which can be 0.5:2, 0.5:1, 0.5:0.5, 1.3:1, 1:0.5 or 2:0.5.

[0057] In some embodiments, the resin represented by Formula 1 is prepared by the following method:

[0058] A polymer monomer solution is prepared by mixing polymer monomers, crosslinking agents, and initiators.

[0059] An aqueous phase is prepared by mixing a dispersant with water;

[0060] The polymer monomer solution and the aqueous phase are mixed and subjected to suspension polymerization to prepare the resin shown in Formula 1.

[0061] In some embodiments, the resin represented by Formula 1 is a microsphere resin.

[0062] In some embodiments, the D90 particle size of the microsphere resin is 100-200 μm, and can be 100 μm, 110 μm, 120 μm, 130 μm, 140 μm, 150 μm, 160 μm, 170 μm, 180 μm, 190 μm or 200 μm.

[0063] In some embodiments, the conditions for preparing an aqueous phase by mixing the dispersant and water include: a mixing speed of 100-500 rpm, which can be 100 rpm, 200 rpm, 300 rpm, 400 rpm or 500 rpm; and a temperature of 30-50°C, which can be 30°C, 35°C, 40°C, 45°C or 50°C.

[0064] In some embodiments, methylene blue solution is used as an indicator in the preparation steps of the resin shown in Formula 1.

[0065] In some embodiments, in the preparation step of the microsphere resin, methylene blue solution is used as an indicator, and the dispersant, methylene blue solution and water are mixed to prepare an aqueous phase.

[0066] In some embodiments, the mass ratio of the polymer monomer to the aqueous phase (10-30):100 can be 10:100, 15:100, 16.5:100, 20:100, 25:100 or 30:100.

[0067] In some embodiments, the mass fraction of the crosslinking agent in the polymer monomer solution is 0.5%-2%, which can be 0.5%, 1%, 1.3%, 1.5% or 2%.

[0068] In some embodiments, the initiator in the polymer monomer solution has a mass fraction of 0.5-2%, which can be 0.5%, 0.8%, 0.98%, 1%, 1.5% or 2%.

[0069] In some embodiments, the mass fraction of the dispersant in the aqueous phase is 0.5-5%, which can be 0.5%, 0.99%, 1%, 2%, 3%, 4% or 5%.

[0070] In some embodiments, the conditions for the suspension polymerization reaction include: a stirring speed of 200-400 rpm, which can be 200 rpm, 250 rpm, 300 rpm, 350 rpm or 400 rpm; a reaction temperature of 70-90°C, which can be 70°C, 75°C, 80°C, 85°C or 90°C; and a reaction time of 8-20 h, which can be 8 h, 10 h, 12 h, 16 h, 20 h or 24 h.

[0071] In some embodiments, the conditions for the nucleophilic substitution reaction include: a reaction temperature of 20-60°C, which can be 20°C, 22°C, 24°C, 26°C, 27°C, 28°C, 30°C, 32°C, 35°C, 40°C, 45°C, 50°C, 55°C, or 60°C; and a reaction time of 8-20 hours, which can be 8 hours, 10 hours, 12 hours, 16 hours, 20 hours, or 24 hours.

[0072] In some embodiments, the mass molar ratio of the resin shown in Formula 1 to the compound shown in Formula 2 is 1g:(1-2)mmol, which can be 1g:1mmol, 1g:1.1mmol, 1g:1.5mmol, 1g:1.8mmol or 1g:2mmol.

[0073] In some embodiments, the organic acid catalyst includes at least one of methanesulfonic acid, trifluoromethanesulfonic acid, and p-toluenesulfonic acid.

[0074] In some embodiments, the mass ratio of the organic acid catalyst to the resin shown in Formula 1 is (0.5-1):1, which can be 0.5:1, 0.6:1, 0.7:1, 0.8:1, 0.9:1 or 1:1.

[0075] In some embodiments, the step of carrying out the nucleophilic substitution reaction includes:

[0076] The resin shown in Formula 1, the compound shown in Formula 2, the organic acid catalyst, and the first organic solvent were mixed and a nucleophilic substitution reaction was carried out to prepare the compound shown in Formula 3.

[0077] The first organic solvent includes at least one of dichloromethane, dichloroethane, benzene, and toluene;

[0078] The volume-to-mass ratio of the first organic solvent to the resin shown in Formula 1 is (5-10) mL:1g, which can be 5mL:1g, 6mL:1g, 7mL:1g, 8mL:1g, 9mL:1g or 10mL:1g.

[0079] In some embodiments, the organic amine solution contains at least one of methylamine, benzylamine, diethanolamine, and ethanolamine.

[0080] In some embodiments, the alcohol solvent in the organic amine solution includes at least one of ethanol, propanol, and isopropanol.

[0081] In some embodiments, the volume ratio of the organic amine to the solvent in the organic amine solution is (0.2-1):1, which can be 0.2:1, 0.3:1, 0.4:1, 0.5:1, 0.6:1, 0.7:1, 0.8:1, 0.9:1 or 1:1.

[0082] In some embodiments, the hydrolysis conditions include: a reaction temperature of 90-150°C, which can be 90°C, 100°C, 110°C, 120°C, 130°C, 140°C or 150°C; and a reaction time of 8-20h, which can be 8h, 10h, 12h, 16h, 20h or 24h.

[0083] In some embodiments, after hydrolyzing the N-methylphthalimide-modified resin intermediate in an organic amine solution, a cleaning step is further included, wherein the cleaning conditions include cleaning with ethanol and / or water.

[0084] In a second aspect of this application, an aminomethyl resin prepared by the method described in the first aspect is provided.

[0085] A third aspect of this application provides a method for solid-phase synthesis of polypeptides, comprising the following steps:

[0086] Polypeptides are prepared using aminomethyl resin prepared by the method described in the first aspect or aminomethyl resin described in the second aspect as starting materials.

[0087] Solid-phase peptide synthesis (SPPS) involves the repeated addition of amino acids, such as... Figure 1 As shown, Figure 1 In this method, R represents resin; Linker represents the linker molecule; A represents the first amino acid molecule; and B represents the second amino acid molecule. The carboxyl group of the first amino acid molecule A of the target peptide is coupled to a solid-phase support (resin-linker complex) containing the linker molecule. After deprotection of the amino group, it is coupled to the second amino acid molecule B. This process is repeated (coupling → washing → deprotection → neutralization and washing → next round of coupling) to achieve the desired peptide chain. Solid-phase peptide synthesis greatly simplifies post-processing steps, yields higher yields and product purity, and has seen significant development and application in the synthesis of bioactive peptides in recent decades.

[0088] In some embodiments, the polypeptide solid-phase synthesis method includes the following steps:

[0089] The Fmoc solid-phase polypeptide synthesis method is used to prepare polypeptides using aminomethyl resin prepared by the preparation method described in the first aspect or aminomethyl resin described in the second aspect as starting materials.

[0090] The embodiments of this application will be described in detail below with reference to examples. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of this application. For experimental methods in the following embodiments where specific conditions are not specified, please refer to the guidelines given in this application, or follow experimental manuals or conventional conditions in the art, or follow the conditions recommended by the manufacturer, or refer to experimental methods known in the art.

[0091] Example 1

[0092] The preparation method of aminomethyl resin is as follows:

[0093] ;

[0094] In Formula 2, R1 is a hydroxyl group;

[0095] In a 250 mL three-necked flask, add 120 mL of deionized water, 0.1 g of 0.1 wt% methylene blue solution, and 1.2 g of dispersant (0.9 g of polyvinylpyrrolidone and 0.3 g of polyethylene glycol). Stir at 40 °C and 200 rpm until the solution is clear and transparent to prepare the aqueous phase. In a beaker, add 20 g of polymer monomer (styrene), 0.52 g of crosslinking agent (divinylbenzene, 80% purity), and 0.2 g of initiator (benzoyl peroxide). Mix well to prepare the polymer monomer solution. Add the polymer monomer solution to the aqueous phase, stir at 250 rpm, and heat to 80 °C for suspension polymerization. React for 12 h. After the reaction, cool to 60 °C, filter, wash twice with hot water and twice with ethanol, and dry to prepare microsphere resin (as shown in Formula 1).

[0096] 15g of the microsphere resin shown in Formula 1 and 150mL of dichloromethane were added to a 250mL three-necked flask and stirred for 15min to ensure uniform dispersion and full swelling of the microsphere resin. Then, 2.92g of the compound shown in Formula 2 (N-hydroxymethyl phthalimide, where R1 is a hydroxyl group in Formula 2) and 15g of organic acid catalyst (methanesulfonic acid) were added sequentially. The mixture was reacted at room temperature (25℃) for 12h, filtered, washed once with dichloromethane and once with ethanol, and dried to prepare the compound shown in Formula 3.

[0097] Add 150 mL of organic amine solution (ethanolic solution of ethanolamine, wherein the mass ratio of ethanolamine to ethanol is 20:80) to a 250 mL three-necked flask, then add 15 g of the compound shown in Formula 3, and stir at 110 °C to carry out hydrolysis for 12 h; after the reaction is completed, cool and filter, wash twice with ethanol, dry, and prepare aminomethyl resin (as shown in Formula 4).

[0098] Example 2

[0099] The preparation method of aminomethyl resin in Example 2 is basically the same as that in Example 1, except that the organic amine solution "ethanolic solution of ethanolamine, wherein the mass ratio of ethanolamine to ethanol is 20:80" is replaced with an equal amount of "organic amine solution (aqueous solution of ethanolamine, wherein the mass ratio of ethanolamine to water is 20:80)".

[0100] Microsphere resin and aminomethyl resin were prepared according to the method in Example 1.

[0101] Example 3

[0102] The preparation method of aminomethyl resin in Example 3 is basically the same as that in Example 1, except that: the organic acid catalyst "methanesulfonic acid" is replaced with an equal amount of "trifluoromethanesulfonic acid"; and the organic amine solution "ethanolamine in ethanol solution, wherein the mass ratio of ethanolamine to ethanol is 20:80" is replaced with an equal amount of "methylamine in ethanol solution, wherein the mass ratio of methylamine to ethanol is 20:80".

[0103] Microsphere resin and aminomethyl resin were prepared according to the method in Example 1.

[0104] Example 4

[0105] The preparation method of aminomethyl resin in Example 4 is basically the same as that in Example 3, except that the organic amine solution "ethanol solution of methylamine, wherein the mass ratio of methylamine to ethanol is 20:80" is replaced with an equal amount of "aqueous solution of methylamine, wherein the mass ratio of methylamine to water is 20:80".

[0106] Microsphere resin and aminomethyl resin were prepared according to the method in Example 3.

[0107] Example 5

[0108] The preparation method of aminomethyl resin in Example 5 is basically the same as that in Example 1, except that: the organic acid catalyst "methanesulfonic acid" is replaced with an equal amount of "p-toluenesulfonic acid"; and the organic amine solution "ethanolamine in ethanol solution, wherein the mass ratio of ethanolamine to ethanol is 20:80" is replaced with an equal amount of "diethanolamine in ethanol solution, wherein the mass ratio of diethanolamine to ethanol is 20:80".

[0109] Microsphere resin and aminomethyl resin were prepared according to the method in Example 1.

[0110] Example 6

[0111] The preparation method of the aminomethyl resin in Example 6 is basically the same as that in Example 5, except that the organic amine solution "diethanolamine in ethanol, wherein the mass ratio of diethanolamine to ethanol is 20:80" is replaced with an equal amount of "diethanolamine in aqueous solution, wherein the mass ratio of diethanolamine to water is 20:80".

[0112] Microsphere resin and aminomethyl resin were prepared according to the method in Example 5.

[0113] Comparative Example 1

[0114] The preparation method of the aminomethyl resin in Comparative Example 1 is basically the same as that in Example 1, except that the organic amine solution "ethanol amine in ethanol solution, wherein the mass ratio of ethanolamine to ethanol is 20:80" is replaced with an equal amount of "ethanol amine in toluene solution, wherein the mass ratio of ethanolamine to toluene is 20:80".

[0115] Microsphere resin and aminomethyl resin were prepared according to the method in Example 1.

[0116] Comparative Example 2

[0117] The preparation method of the aminomethyl resin in Comparative Example 2 is basically the same as that in Example 1, except that the organic amine solution "ethanol amine in ethanol solution, wherein the mass ratio of ethanol amine to ethanol is 20:80" is replaced with an equal amount of "ethanol amine in dichloromethane solution, wherein the mass ratio of ethanol amine to dichloromethane is 20:80".

[0118] Microsphere resin and aminomethyl resin were prepared according to the method in Example 1.

[0119] Comparative Example 3

[0120] The preparation method of the aminomethyl resin in Comparative Example 3 is basically the same as that in Example 3, except that the organic amine solution "ethanol solution of methylamine, wherein the mass ratio of methylamine to ethanol is 20:80" is replaced with an equal amount of "toluene solution of methylamine, wherein the mass ratio of methylamine to toluene is 20:80".

[0121] Microsphere resin and aminomethyl resin were prepared according to the method in Example 3.

[0122] Comparative Example 4

[0123] The preparation method of the aminomethyl resin in Comparative Example 4 is basically the same as that in Example 3, except that the organic amine solution "ethanol solution of methylamine, wherein the mass ratio of methylamine to ethanol is 20:80" is replaced with an equal amount of "dichloromethane solution of methylamine, wherein the mass ratio of methylamine to dichloromethane is 20:80".

[0124] Microsphere resin and aminomethyl resin were prepared according to the method in Example 3.

[0125] Comparative Example 5

[0126] The preparation method of the aminomethyl resin in Comparative Example 5 is basically the same as that in Example 1, except that the organic acid catalyst "methanesulfonic acid" is replaced with an equal amount of "sulfuric acid".

[0127] Microsphere resin and aminomethyl resin were prepared according to the method in Example 1.

[0128] Comparative Example 6

[0129] The preparation method of the aminomethyl resin in Comparative Example 6 is basically the same as that in Example 2, except that the organic acid catalyst "methanesulfonic acid" is replaced with an equal amount of "sulfuric acid".

[0130] Microsphere resin and aminomethyl resin were prepared according to the method in Example 2.

[0131] Comparative Example 7

[0132] The preparation method of the aminomethyl resin in Comparative Example 7 is basically the same as that in Example 5, except that the organic amine solution "diethanolamine in ethanol, wherein the mass ratio of diethanolamine to ethanol is 20:80" is replaced with an equal amount of "diethanolamine in toluene, wherein the mass ratio of diethanolamine to toluene is 20:80".

[0133] Microsphere resin and aminomethyl resin were prepared according to the method in Example 5.

[0134] Comparative Example 8

[0135] The preparation methods of the aminomethyl resin in Comparative Example 8 and Example 5 are basically the same, except that the organic amine solution "diethanolamine in ethanol, wherein the mass ratio of diethanolamine to ethanol is 20:80" is replaced with an equal amount of "diethanolamine in dichloromethane, wherein the mass ratio of diethanolamine to dichloromethane is 20:80".

[0136] Microsphere resin and aminomethyl resin were prepared according to the method in Example 5.

[0137] Experimental Example 1

[0138] The particle size of the microsphere resin and aminomethyl resin prepared in Examples 1-6 and Comparative Examples 1-8 and the surface functional group density of the aminomethyl resin were determined.

[0139] (1) The particle size (D10, D50 and D90) of the microsphere resin and aminomethyl resin prepared in Examples 1-6 and Comparative Examples 1-8 was measured using the LS-909Ⅱ laser particle size analyzer of China Eurotech Corporation, and the particle size change rate after the microsphere resin was prepared into aminomethyl resin was calculated.

[0140] Particle size testing conditions: dispersion medium: deionized water (refractive index 1.33); sample material: polystyrene latex (refractive index 1.596); shading rate: 5%~15%; background sampling time: 10s; single sampling time: 10s; analysis range: 0.02μm~3000μm.

[0141] (2) The surface functional group density of the aminomethyl resins prepared in Examples 1-6 and Comparative Examples 1-8 was tested using the Fmoc synthesis method. The specific operation is as follows:

[0142] Resin swelling: Weigh 1.0g of aminomethyl resin, add 10mL of DMF (N,N-dimethylformamide) and soak for 20min to ensure that the resin is fully swollen. Remove the DMF to prepare the swollen aminomethyl resin.

[0143] Synthesis of Fmoc-Gly-OH resin: Based on the molar amount of N-hydroxymethyl phthalimide added during the preparation of aminomethyl resin (the molar ratio of N-hydroxymethyl phthalimide to Fmoc-Gly-OH is 1:1), Fmoc-Gly-OH (N-fluorenemethoxycarbonylglycine), HOBT (1-hydroxybenzotriazole), and DIC (diisopropylcarbodiimide) in a molar ratio of 1:2.5:2.5 were weighed and added to 10 mL of DMF. The mixture was stirred and pre-reacted at room temperature (25℃) for 20 min. Then, it was added to the swollen aminomethyl resin and stirred and reacted at room temperature (25℃) for 2 h. After the reaction was completed, the resin was dried under vacuum and washed three times with 10 mL of DCM (dichloromethane). The resin was then dried under vacuum at 30±5℃ to obtain Fmoc-Gly-OH resin.

[0144] Ninhydrin color development and Fmoc protecting group removal: Take a small amount of Fmoc-Gly-OH resin and add it to 2 mL of ninhydrin color development solution (20% ninhydrin ethanol solution: 80% phenol ethanol solution = 1:1 (v:v)). Develop the color at 120℃ for 3 min and observe whether the resin surface changes color or blue spots appear. Take 0.5 g of Fmoc-Gly-OH resin and add it to 5 mL of 20% piperidine DMF solution. React for 30 min to remove the protecting group and collect the protecting group solution.

[0145] Volume adjustment with protecting group solution and UV-Vis spectrophotometer testing:

[0146] Preparation of standard absorbance curves for Fmoc-Gly-OH deprotection: 2.0 mmol of Fmoc-Gly-OH was dissolved in 5 mL of 20% piperidine DMF solution. The reaction was carried out at room temperature for 30 min to remove the protecting group. The protecting group solution was collected by filtration and diluted to 50 mL with acetonitrile to prepare a 40 mmol / L protecting group stock solution (based on the concentration of Fmoc-Gly-OH). The protecting group stock solution was then diluted with acetonitrile to prepare protecting group stock solutions of 36 mmol / L, 32 mmol / L, 28 mmol / L, 24 mmol / L, 20 mmol / L, 16 mmol / L, 12 mmol / L, 8 mmol / L, and 4 mmol / L, respectively, to prepare standard solutions. The absorbance value was detected at the 301 nm UV absorption peak. The concentration range of Fmoc-Gly-OH was plotted on the x-axis and the absorbance value on the y-axis. The prepared concentration range was 4-40 mmol / L. Standard absorbance curve of Fmoc-Gly-OH deprotected group at mmol / L.

[0147] The collected protecting group solution was diluted to 50 mL (0.05 L) with acetonitrile to prepare the test protecting group solution. The absorbance value of the test protecting group solution was measured at the 301 nm UV absorption peak. Each sample was tested in triplicate, and the average absorbance was calculated and substituted into the Fmoc-Gly-OH deprotection standard absorbance curve (e.g., Figure 2 As shown, the concentration of Fmoc-Gly-OH was calculated using y = 0.0386x + 0.0029, and the grafting density of Fmoc-Gly-OH and the surface amino functional group density of the aminomethyl resin were further calculated.

[0148] The formula for calculating the Fmoc-Gly-OH grafting density is:

[0149] C Fmoc-Gly-OH = c1 × v / m1;

[0150] Among them, C Fmoc-Gly-OHThe grafting density of Fmoc-Gly-OH is expressed in mmol / g.

[0151] c1 is the concentration of Fmoc-Gly-OH, in mmol / L;

[0152] v represents the volume of the protective group solution to be tested, in L;

[0153] m1 represents the mass of Fmoc-Gly-OH resin, expressed in grams.

[0154] The surface amino functional group density (unit: mmol / g) of aminomethyl resin was calculated based on the Fmoc-Gly-OH grafting density. The calculation formula is as follows:

[0155] The surface amino functional group density of aminomethyl resin = 1000 × C Fmoc-Gly-OH × m2 / (1000 - M ×C Fmoc-Gly-OH );

[0156] Where M is the molecular weight of Fmoc-Gly-OH (279.31 mg / mmol), in mg / mmol;

[0157] C Fmoc-Gly-OH The grafting density of Fmoc-Gly-OH is expressed in mmol / g.

[0158] m2 represents the mass of aminomethyl resin, in grams.

[0159] 1000 is the conversion factor from mg to g.

[0160] The measurement results are shown in Tables 1 and 2.

[0161] Table 1. Particle size of microsphere resin and aminomethyl resin

[0162]

[0163]

[0164] Table 2. Surface functional group density and ninhydrin color development results of aminomethyl resin.

[0165]

[0166] " / " indicates that it was not measured.

[0167] The results in Tables 1 and 2 show that, compared with Comparative Examples 1-8, the microsphere resin and aminomethyl resin prepared in Examples 1-6 of this application did not show significant changes in particle size, but had a high amino density. This indicates that the aminomethyl resin of this application can maintain stable particle size before and after aminomethyl functionalization, improving the structural stability and mechanical strength of the resin in solvent. Meanwhile, in the Fmoc synthesis method test, the Fmoc-Gly-OH resin prepared from the aminomethyl resin of Examples 1-6 showed a negative result in ninhydrin color development, and there was no color change or blue spots on the resin surface, indicating that the amino groups on the surface of the aminomethyl resin were completely reacted. This shows that the preparation method of the aminomethyl resin of this application can improve the activity of the aminomethyl resin.

[0168] The active functional groups on the resin surface in solid-phase peptide synthesis are the sites for attaching the first amino acid. Insufficient activity leads to incomplete coupling between the first amino acid and the resin, resulting in some peptide chains failing to elongate properly and forming numerous short peptide impurities. Uneven distribution of peptide chains on the resin surface also increases the likelihood of interactions between adjacent peptide chains, especially in cyclic peptide synthesis, easily forming dimers and other polymerization impurities. This results in insufficient peptide yield, reduced purity, and even structural defects such as local deletions and sequence disorderes. Therefore, ninhydrin is commonly used in solid-phase peptide synthesis to detect the incompleteness of the reaction and confirm whether further synthesis can proceed. A positive ninhydrin colorimetric result indicates that the peptide chains could not elongate properly during solid-phase synthesis, indicating structural defects and low application value of aminomethyl resin.

[0169] Comparing Examples 1-6, Comparative Examples 1-4, and Comparative Examples 7-8, it can be seen that when Comparative Examples 1-4 and 7-8 were hydrolyzed using toluene or dichloromethane solutions of organic amines, the particle size of the resin before and after aminomethyl functionalization changed significantly, exceeding 10%. However, when Examples 1-6 were hydrolyzed using alcoholic solvents (ethanol) or aqueous solutions of organic amines, the particle size change rate of the resin before and after aminomethyl functionalization did not exceed 5%. This indicates that using alcoholic solvents or aqueous solutions of organic amines for hydrolysis in this application can maintain stable resin particle size and improve the structural stability and mechanical strength of the resin.

[0170] Comparing Examples 1-2 and Comparative Examples 5-6, it can be seen that in the nucleophilic substitution reaction, compared with using sulfuric acid as a catalyst, the organic acid catalyst used in the examples of this application increased the amino group density from 0.76-0.78 mmol / g to 0.86-0.89 mmol / g, an increase of 10%-17%; the Fmoc-Gly-OH grafting density increased from 0.45-0.46 mmol / g to 0.60-0.61 mmol / g, an increase of 33%. This indicates that the use of organic acid catalysts in this application significantly improves the grafting density of the prepared aminomethyl resin compared to traditional Friedel-Crafts inorganic acid catalysts.

[0171] The technical features of the above embodiments can be combined in any way. For the sake of brevity, not all possible combinations of the technical features in the above embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0172] The embodiments described above are merely illustrative of several implementation methods of this application, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the patent application. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of this application, and these all fall within the protection scope of this application. Therefore, the protection scope of this patent application should be determined by the appended claims.

Claims

1. A method for preparing an aminomethyl resin, characterized in that, Includes the following steps: The resin shown in Formula 1, the compound shown in Formula 2, and the organic acid catalyst were mixed and a nucleophilic substitution reaction was carried out to prepare the compound shown in Formula 3. The compound represented by Formula 3 was hydrolyzed in an organic amine solution to prepare the aminomethyl resin represented by Formula 4. The organic amine solution comprises an organic amine and a solvent, wherein the solvent comprises at least one of water and alcohol solvents; Formula 1; Formula 2; Formula 3; Equation 4; R1 is selected from hydroxyl, chlorine atom, bromine atom, methoxy or benzyloxy.

2. The method for preparing aminomethyl resin according to claim 1, characterized in that, The organic amine solution satisfies at least one of the following characteristics: (1) The organic amine includes at least one of methylamine, benzylamine, diethanolamine and ethanolamine; (2) The alcohol solvent includes at least one of ethanol, propanol and isopropanol; (3) In the organic amine solution, the volume ratio of organic amine to solvent is (0.2-1):

1.

3. The method for preparing aminomethyl resin according to claim 1 or 2, characterized in that, It meets at least one of the following characteristics: (1) The conditions for hydrolysis include: reaction temperature of 90-150℃; reaction time of 8-20h; (2) The conditions for nucleophilic substitution reaction include: reaction temperature of 20-60℃; reaction time of 8-20h.

4. The method for preparing aminomethyl resin according to claim 1 or 2, characterized in that, The mass molar ratio of the resin shown in Formula 1 to the compound shown in Formula 2 is 1 g: (1-2) mmol.

5. The method for preparing aminomethyl resin according to claim 1 or 2, characterized in that, It meets at least one of the following characteristics: (1) The organic acid catalyst includes at least one of methanesulfonic acid, trifluoromethanesulfonic acid and p-toluenesulfonic acid; (2) The mass ratio of the organic acid catalyst to the resin shown in Formula 1 is (0.5-1):

1.

6. The method for preparing aminomethyl resin according to claim 1, characterized in that, The resin shown in Formula 1 is prepared by the following method: The polymer monomer, crosslinking agent, initiator, dispersant and water are mixed and subjected to suspension polymerization to prepare the resin shown in Formula 1.

7. The method for preparing aminomethyl resin according to claim 6, characterized in that, It meets at least one of the following characteristics: (1) The polymer monomer includes at least one of styrene, α-methylstyrene, chloromethylstyrene, allylbenzene and allylbenzene; (2) The crosslinking agent includes at least one of divinylbenzene, divinyltoluene, and trivinyltoluene; (3) The initiator includes at least one of azobisisobutyronitrile, benzoyl peroxide and di-tert-butyl peroxide; (4) The dispersant includes at least one of polyethylene glycol, polyvinylpyrrolidone and gelatin.

8. The method for preparing aminomethyl resin according to claim 6 or 7, characterized in that, It meets at least one of the following characteristics: (1) The mass ratio of the polymer monomer, the dispersant, and the water is (10-30):(0.5-5):100; (2) The mass ratio of the crosslinking agent to the initiator is (0.5-2):(0.5-2); (3) The conditions for suspension polymerization reaction include: stirring speed of 200-400 rpm; reaction temperature of 70-90℃; and reaction time of 8-20h.

9. An aminomethyl resin prepared by the method for preparing aminomethyl resin according to any one of claims 1-8.

10. A method for solid-phase synthesis of polypeptides, characterized in that, Includes the following steps: A polypeptide is prepared using the aminomethyl resin prepared by the method of any one of claims 1-8 or the aminomethyl resin of claim 9 as the starting material.

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