Method for efficiently coupling amino acid and resin in solid-phase synthesis of polypeptide

Abstract

The invention discloses a method for efficiently coupling amino acid and resin in polypeptide solid-phase synthesis, and relates to the technical field of polypeptide solid-phase synthesis. The specific preparation method comprises the following steps: firstly, swelling resin, and then adding amino acid, a solvent, an acyl chloride compound and alkali for reaction; or mixing and dissolving amino acid and anhydride, adding, and adding alkali for reaction; or mixing and dissolving the amino acid and the condensing agent, then adding the condensing agent and the catalyst dissolving solution, reacting, separating, washing and blocking after the reaction is finished, and finally washing and drying to obtain the amino-acid resin conjugate. According to the method disclosed by the invention, the amino acid can be effectively coupled to the resin, the loading degree is high, the access rate of the amino acid can be effectively improved, the racemization degree of the amino acid is reduced, the production cost is greatly reduced, and industrial large-scale production is facilitated.

Description

Technical Field

[0001] The present invention relates to the technical field of polypeptide solid phase synthesis, and in particular to a method for efficient coupling of amino acids and resins in polypeptide solid phase synthesis. Background Art

[0002] The Fmoc strategy for solid-phase peptide synthesis, a method developed in the 1970s, offers advantages such as mild reaction conditions, few side reactions, and high yields. Currently, it has been widely used in the study of peptide structure and properties. In solid-phase peptide synthesis, Wang resin, a hydroxy resin, is used in 40% of applications and is the most widely used peptide synthesis support. The first step in solid-phase peptide synthesis involves attaching the carboxyl group of the first amino acid to the resin via an ester or amide bond. The efficiency of this first step not only directly affects the yield of the target peptide, but also plays a crucial role in determining the dosage of the next amino acid. Therefore, studying the efficiency of the first step in solid-phase peptide synthesis is crucial.

[0003] The main methods for connecting Fmoc-protected amino acids to hydroxyl resins are the acyl chloride method and the activated ester method. These methods are characterized by long reaction times, low connection efficiency, and a high degree of racemization. Since the cost of the solid-phase support in peptide synthesis generally accounts for 20%-40% of the synthesis cost, such inefficient connection methods inevitably increase the cost of large-scale synthesis. Therefore, a method for efficiently connecting amino acids to resins is still needed. Summary of the Invention

[0004] The purpose of the present invention is to provide a method for efficiently coupling amino acids with resins in solid-phase synthesis of polypeptides, so as to solve the problems of low efficiency of amino acid-resin connection and high degree of racemization in the prior art.

[0005] In order to solve the above technical problems, the present invention specifically provides the following technical solutions: The present invention discloses a method for efficiently coupling amino acids with resin in solid-phase synthesis of polypeptides, comprising: The resin is swelled, and then an amino acid and a solvent are added, and then an acyl chloride compound and a base are added to react. After the reaction is completed, the resin is separated and washed, and then a blocking agent is added to block the ends, and finally washed and dried to obtain an amino acid resin conjugate; or, The resin is swelled, and then the amino acid and acid anhydride are mixed and dissolved and added, and a base is added to react. After the reaction is completed, the mixture is separated and washed, and then a blocking reagent is added to block the ends, and finally washed and dried to obtain an amino acid resin conjugate; or, The resin is swelled, the amino acid and the condensing agent are mixed and dissolved and then added, and then the condensing agent and the catalyst solution are added to react. After the reaction is completed, the mixture is separated and washed, and then a blocking agent is added to block the ends, and finally washed and dried to obtain an amino acid resin conjugate; The molar ratio of the resin to the amino acid is 1:1.5-4.

[0006] Preferably, the resin is a wang resin, and the substitution degree of the wang resin is 0.4-1.2 mmol / g; the amino acids are Fmoc-Ala-OH, Fmoc-Cys(Trt)-OH, Fmoc-Asp(OtBu)-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Phe-OH, Fmoc-Gly-OH, Fmoc-His(Trt)-OH, Fmoc-Ile-OH, Fmoc-Lys(Boc)- OH, Fmoc-Leu-OH, Fmoc-Met-OH, Fmoc-Asn(Trt)-OH, Fmoc-Pro-OH, Fmoc-Gln(Trt)-OH, Fmoc-Arg(Pbf)-O H, at least one of Fmoc-Ser(tBu)-OH, Fmoc-Thr(tBu)-OH, Fmoc-Val-OH, Fmoc-Trp(Boc)-OH and Fmoc-Tyr(tBu)-OH.

[0007] Preferably, the solvent is at least one of N,N-dimethylformamide and dichloromethane, and the ratio of the amino acid to the solvent is 1 mol:3-4 L; the acyl chloride compound is at least one of 2,6-dichlorobenzoyl chloride, 2,4,6-trichlorobenzoyl chloride and 3,5-dichlorobenzoyl chloride; the molar ratio of the amino acid to the acyl chloride compound is 1:0.5-3; the base co-reacting with the acyl chloride compound is pyridine, and the molar ratio of the amino acid to the base is 1:1.5-5.

[0008] Preferably, the acid anhydride is at least one of diisopropyl carbonate and Boc anhydride, and the molar ratio of the amino acid to the acid anhydride is 1:0.5-3; the base co-reacting with the acid anhydride is pyridine, and the molar ratio of the amino acid to the base is 1:1.5-5.

[0009] Preferably, the condensing agent in the mixture of amino acid and condensing agent is 1-hydroxybenzotriazole, and the condensing agent added to the condensing agent is diisopropylcarbodiimide. The molar ratio of the amino acid to 1-hydroxybenzotriazole is 1:0.8-1.2, and the molar ratio of the amino acid to diisopropylcarbodiimide is 1:0.8-1.2. The catalyst solution is obtained by dissolving the catalyst in N,N-dimethylformamide.

[0010] Preferably, the catalyst is at least one of 4-dimethylaminopyridine, 4-dimethylaminopyridine N-oxide, 4-pyrrolidinylpyridine, 5,6,9,10-tetrahydro-4H,8H-pyrido[3,2,1-IJ][1,6]naphthyridine, 4,4,10,10-tetramethyl-5,6,9,10-tetrahydro-4H,8H-pyridyl[3,2,1-IJ][1,6]naphthyridine and pyridine derivatives, and the molar ratio of the amino acid to the catalyst is 1:0.02-0.5; the pyridine derivative is prepared from dimethyl 4-hydroxymethylpyridine-2,6-dicarboxylate, methanol, hydrogen bromide, diisobutyl iminodiacetate and 4-formylphenylboronic acid methyliminodiacetate.

[0011] Pyridine derivatives are prepared from dimethyl 4-hydroxymethylpyridine-2,6-dicarboxylate, methanol, hydrogen bromide, diisobutyl iminodiacetate, and 4-formylphenylboronic acid methyliminodiacetate. The use of pyridine derivatives can more effectively promote the coupling of amino acids with resins, making the coupling more complete and improving reaction efficiency. It also improves reaction stability, ensuring the continuity and efficiency of the coupling process, thereby achieving high loading and amino acid access rates.

[0012] Preferably, the end-capping reagent includes acetic anhydride, N,N-diisopropylethylamine and dichloromethane, the usage ratio of the resin to acetic anhydride is 1 mol: 0.8-1.1 L, the volume ratio of acetic anhydride to N,N-diisopropylethylamine is 1:1-2.5, and the volume ratio of acetic anhydride to dichloromethane is 1:7-9.

[0013] Preferably, the reaction temperature for adding the amino acid, solvent, acyl chloride compound and base to react is 20-30° C., and the reaction time is 3-5 h.

[0014] Preferably, the amino acid and the acid anhydride are mixed and dissolved, and then added, and the base is added to react at a reaction temperature of 20-30° C. and a reaction time of 3-5 h.

[0015] Preferably, the reaction temperature for the addition of the condensing agent and the catalyst solution is 20-30° C., and the reaction time is 3-5 h.

[0016] The present invention discloses a method for efficiently coupling amino acids with resins in solid-phase synthesis of polypeptides, specifically comprising: The resin is swollen for 20-40 minutes, and then the liquid is removed. Then, amino acid, solvent, acyl chloride compound and base are added to react at 20-30°C for 2-6 hours. After the reaction is completed, separation and washing are performed, and then a blocking reagent is added to block for 20-40 minutes, 1-3 times, and finally washed and dried to obtain an amino acid resin conjugate.

[0017] Preferably, the resin is a wang resin, and the degree of substitution of the wang resin is 0.4-1.2 mmol / g.

[0018] Preferably, the reagent used for resin swelling is at least one of N,N-dimethylformamide and dichloromethane.

[0019] Preferably, the amino acid is at least one of Fmoc-Ala-OH, Fmoc-Cys(Trt)-OH, Fmoc-Asp(OtBu)-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Phe-OH, Fmoc-Gly-OH, Fmoc-His(Trt)-OH, Fmoc-Ile-OH, Fmoc-Lys(Boc)-OH, Fmoc-Leu-OH, Fmoc-Met-OH, Fmoc-Asn(Trt)-OH, Fmoc-Pro-OH, Fmoc-Gln(Trt)-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Thr(tBu)-OH, Fmoc-Val-OH, Fmoc-Trp(Boc)-OH and Fmoc-Tyr(tBu)-OH.

[0020] Preferably, the molar ratio of the resin to the amino acid is 1:1.5-4.

[0021] Preferably, the solvent is at least one of N,N-dimethylformamide and dichloromethane, and the ratio of the amino acid to the solvent is 1 mol: 3-4 L.

[0022] Preferably, the acyl chloride compound is at least one of 2,6-dichlorobenzoyl chloride, 2,4,6-trichlorobenzoyl chloride and 3,5-dichlorobenzoyl chloride; and the molar ratio of the amino acid to the acyl chloride compound is 1:0.5-3.

[0023] Preferably, the base is pyridine; and the molar ratio of the amino acid to the base is 1:1.5-5.

[0024] Preferably, the end-capping reagent includes acetic anhydride, N,N-diisopropylethylamine and dichloromethane, the usage ratio of the resin to acetic anhydride is 1 mol: 0.8-1.1 L, the volume ratio of acetic anhydride to N,N-diisopropylethylamine is 1:1-2.5, and the volume ratio of acetic anhydride to dichloromethane is 1:7-9.

[0025] Preferably, the reagent used for washing after the reaction is dichloromethane.

[0026] Preferably, the reagent used for washing after end-capping is methanol.

[0027] The present invention discloses a method for efficiently coupling amino acids with resins in solid-phase synthesis of polypeptides, specifically comprising: After the resin is swollen for 20-40 minutes, the liquid is removed, and then the amino acid and acid anhydride are mixed and dissolved and added to the swollen resin. A base is added and reacted at 20-30°C for 2-6 hours. After the reaction is completed, the mixture is separated and washed, and then a blocking reagent is added to block for 20-40 minutes, 1-3 times, and finally washed and dried to obtain the amino acid resin conjugate.

[0028] Preferably, the resin is a wang resin, and the degree of substitution of the wang resin is 0.4-1.2 mmol / g.

[0029] Preferably, the reagent used for resin swelling is at least one of N,N-dimethylformamide and dichloromethane.

[0030] Preferably, the amino acid is at least one of Fmoc-Ala-OH, Fmoc-Cys(Trt)-OH, Fmoc-Asp(OtBu)-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Phe-OH, Fmoc-Gly-OH, Fmoc-His(Trt)-OH, Fmoc-Ile-OH, Fmoc-Lys(Boc)-OH, Fmoc-Leu-OH, Fmoc-Met-OH, Fmoc-Asn(Trt)-OH, Fmoc-Pro-OH, Fmoc-Gln(Trt)-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Thr(tBu)-OH, Fmoc-Val-OH, Fmoc-Trp(Boc)-OH and Fmoc-Tyr(tBu)-OH.

[0031] Preferably, the molar ratio of the resin to the amino acid is 1:1.5-4.

[0032] Preferably, the reagent used to dissolve the amino acid and the acid anhydride is at least one of N,N-dimethylformamide and dichloromethane, and the ratio of the amino acid to the solvent is 1 mol: 3-4 L.

[0033] Preferably, the acid anhydride is at least one of diisopropyl carbonate and Boc anhydride, and the molar ratio of the amino acid to the acid anhydride is 1:0.5-3.

[0034] Preferably, the base is pyridine, and the molar ratio of the amino acid to the base is 1:1.5-5.

[0035] Preferably, the end-capping reagent includes acetic anhydride, N,N-diisopropylethylamine and dichloromethane, the usage ratio of the resin to acetic anhydride is 1 mol: 0.8-1.1 L, the volume ratio of acetic anhydride to N,N-diisopropylethylamine is 1:1-2.5, and the volume ratio of acetic anhydride to dichloromethane is 1:7-9.

[0036] Preferably, the reagent used for washing after the reaction is dichloromethane.

[0037] Preferably, the reagent used for washing after end-capping is methanol.

[0038] The present invention discloses a method for efficiently coupling amino acids with resins in solid-phase synthesis of polypeptides, specifically comprising: The resin is swollen for 20-40 minutes, and then the liquid is removed. Then, the amino acid and the condensing agent are mixed and dissolved and added to the swollen resin. Then, the condensing agent and the catalyst solution are added to react at 20-30°C for 2-6 hours. After the reaction is completed, the mixture is separated and washed. Then, a blocking reagent is added to block for 20-40 minutes, and the blocking is performed 1-3 times. Finally, the amino acid resin conjugate is obtained after washing and drying.

[0039] Preferably, the resin is a wang resin, and the degree of substitution of the wang resin is 0.4-1.2 mmol / g.

[0040] Preferably, the reagent used for resin swelling is at least one of N,N-dimethylformamide and dichloromethane.

[0041] Preferably, the amino acid is at least one of Fmoc-Ala-OH, Fmoc-Cys(Trt)-OH, Fmoc-Asp(OtBu)-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Phe-OH, Fmoc-Gly-OH, Fmoc-His(Trt)-OH, Fmoc-Ile-OH, Fmoc-Lys(Boc)-OH, Fmoc-Leu-OH, Fmoc-Met-OH, Fmoc-Asn(Trt)-OH, Fmoc-Pro-OH, Fmoc-Gln(Trt)-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Thr(tBu)-OH, Fmoc-Val-OH, Fmoc-Trp(Boc)-OH and Fmoc-Tyr(tBu)-OH.

[0042] Preferably, the molar ratio of the resin to the amino acid is 1:1.5-4.

[0043] Preferably, the condensing agent in the mixture of amino acid and condensing agent is 1-hydroxybenzotriazole, and the molar ratio of the amino acid to 1-hydroxybenzotriazole is 1:0.8-1.2.

[0044] Preferably, the condensing agent added again is diisopropylcarbodiimide, and the molar ratio of the amino acid to the diisopropylcarbodiimide is 1:0.8-1.2.

[0045] Preferably, the catalyst solution is obtained by dissolving the catalyst in N,N-dimethylformamide, and the catalyst is at least one of 4-dimethylaminopyridine, 4-dimethylaminopyridine N-oxide, 4-pyrrolidinylpyridine, 5,6,9,10-tetrahydro-4H,8H-pyrido[3,2,1-IJ][1,6]naphthyridine, 4,4,10,10-tetramethyl-5,6,9,10-tetrahydro-4H,8H-pyridyl[3,2,1-IJ][1,6]naphthyridine and pyridine derivatives.

[0046] More preferably, the ratio of the catalyst to N,N-dimethylformamide is 1 mol:0.4-1 L.

[0047] Preferably, the molar ratio of the amino acid to the catalyst is 1:0.02-0.5.

[0048] Preferably, the end-capping reagent includes acetic anhydride, N,N-diisopropylethylamine and dichloromethane, the usage ratio of the resin to acetic anhydride is 1 mol: 0.8-1.1 L, the volume ratio of acetic anhydride to N,N-diisopropylethylamine is 1:1-2.5, and the volume ratio of acetic anhydride to dichloromethane is 1:7-9.

[0049] Preferably, the reagent used for washing after the reaction is dichloromethane.

[0050] Preferably, the reagent used for washing after end-capping is methanol.

[0051] The present invention discloses a method for preparing a pyridine derivative, which specifically comprises: Dimethyl 4-hydroxymethylpyridine-2,6-dicarboxylate is mixed with methanol, followed by the addition of potassium borohydride. The mixture is reacted at 30-40°C for 2-6 hours. After completion of the reaction, the pH is adjusted to 5-6.5, then to 7.5-8.5, followed by separation and purification to obtain the pyridinolate. Hydrogen bromide is added to the pyridinolate, followed by the dropwise addition of acetic anhydride. The mixture is reacted at 100-120°C for 2-5 hours. After completion of the reaction, the mixture is subjected to rotary evaporation, the pH is adjusted to 7.5-8.5, the solid is isolated, and the solid is dried to obtain the pyridine intermediate. Diisobutyl iminodiacetate, 4-formylphenylboronic acid methyliminodiacetate, sodium carbonate, and acetonitrile are added to the pyridine intermediate. The mixture is reacted at 25-35°C for 2-4 hours. After completion of the reaction, the mixture is rotary evaporated, ethyl acetate and water are added, the organic layer is separated, washed, and dried to obtain the pyridine derivative.

[0052] Preferably, the usage ratio of dimethyl 4-hydroxymethylpyridine-2,6-dicarboxylate to methanol is 1 g: 12-20 ml.

[0053] Preferably, the mass ratio of dimethyl 4-hydroxymethylpyridine-2,6-dicarboxylate to potassium borohydride is 1:0.5-1.

[0054] Preferably, the ratio of pyridinolate to hydrogen bromide is 1 g: 5-10 ml.

[0055] Preferably, the ratio of pyridinolate to acetic anhydride is 1 g: 20-30 ml.

[0056] Preferably, the mass ratio of the pyridine intermediate to diisobutyl iminodiacetate is 1:0.6-1.

[0057] Preferably, the mass ratio of the pyridine intermediate to 4-formylphenylboronic acid methyliminodiacetate is 1:0.4-0.9.

[0058] Preferably, the mass ratio of the pyridine intermediate to the sodium carbonate is 1:3-5-4.5.

[0059] Preferably, the usage ratio of the pyridine intermediate to acetonitrile is 1 g:30-50 ml.

[0060] Preferably, the volume ratio of ethyl acetate to water is 1:0.3-1.

[0061] Preferably, the washing agent is a saturated sodium chloride solution, which consists of sodium chloride and water.

[0062] More preferably, in the process of efficient coupling of amino acids with resins of the present invention, in addition to using 4-pyrrolidinopyridine and pyridine derivatives, N,9-diphenyl-9H-carbazole-2-amine can also be used. The synergistic use of N,9-diphenyl-9H-carbazole-2-amine can significantly optimize the coupling reaction, promote the binding of amino acids to resins, improve the efficiency and selectivity of the reaction, and thus further improve the loading degree and amino acid access rate.

[0063] Preferably, the molar ratio of the amino acid to the N,9-diphenyl-9H-carbazole-2-amine is 1:0.02-0.07.

[0064] Compared with the prior art, the present invention has the following beneficial effects: The present invention provides a method for efficiently coupling amino acids to resins in solid-phase peptide synthesis. First, the resin is swollen, and then an amino acid, a solvent, an acyl chloride compound, and a base are added for reaction; alternatively, the amino acid and an acid anhydride are mixed and dissolved before being added, and a base is added for reaction; alternatively, the amino acid and a condensing agent are mixed and dissolved before being added, and then the condensing agent and a catalyst solution are added for reaction. After the reaction is completed, the mixture is separated, washed, and capped, and finally washed and dried to obtain an amino acid resin conjugate. The method of the present invention can effectively couple amino acids to the resin, has a high loading density, and can effectively increase the access rate of the amino acids, reduce the degree of racemization of the amino acids, greatly reduce production costs, and facilitate industrial scale-up production. BRIEF DESCRIPTION OF THE DRAWINGS

[0065] To more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for the embodiments or the description of the prior art. Obviously, the drawings described below are merely exemplary, and those skilled in the art can derive other implementation drawings based on the provided drawings without inventive effort.

[0066] Figure 1 This is the result diagram of the determination of the isomer ratio; Figure 2 This is a graph showing the results of determining the proportion of Fmoc-Gly-Gly-Wang resin in Fmoc-Gly-Wang resin. DETAILED DESCRIPTION

[0067] The following will clearly and completely describe the technical solutions in the embodiments of the present invention in conjunction with the accompanying drawings. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0068] The following first describes the concepts involved in this application with reference to the accompanying drawings. It should be noted that the following description of each concept is intended only to make the content of this application easier to understand and does not limit the scope of protection of this application. At the same time, the embodiments and features in the embodiments of this application can be combined with each other unless there is a conflict. The following detailed description of this application will be made with reference to the accompanying drawings and in conjunction with the embodiments.

[0069] The abbreviations used in the specification and claims have the following meanings:

[0070] Example 1: Coupling of amino acids to resin: Wang resin was first immersed in dichloromethane to swell for 30 minutes. After swelling, the liquid was filtered. Fmoc-Arg(Pbf)-OH and dichloromethane were added to the swollen Wang resin, followed by 2,6-dichlorobenzoyl chloride and pyridine. The mixture was reacted at 25°C for 4 hours, then separated and washed three times with dichloromethane. After washing, acetic anhydride, N,N-diisopropylethylamine, and dichloromethane were added for end-capping for 30 minutes, and this was repeated twice. After end-capping, the resin was washed four times with methanol and dried to obtain Fmoc-Arg(Pbf)-Wang resin. The degree of substitution of wang resin is 0.8 mmol / g, the molar ratio of wang resin to Fmoc-Arg(Pbf)-OH is 1:2, the ratio of Fmoc-Arg(Pbf)-OH to dichloromethane added during the reaction is 1 mol:3.75 L, the molar ratio of Fmoc-Arg(Pbf)-OH to 2,6-dichlorobenzoyl chloride is 1:1, and the molar ratio of Fmoc-Arg(Pbf)-OH to pyridine is 1:2. For each end-capping step, the ratio of wang resin to acetic anhydride is 1 mol:0.95 L, the volume ratio of acetic anhydride to N,N-diisopropylethylamine is 1:1.84, and the volume ratio of acetic anhydride to dichloromethane is 1:7.89.

[0071] Example 2: Coupling of amino acids with resin: The coupling of amino acids with resin was performed in accordance with Example 1, except that the molar ratio of Fmoc-Arg(Pbf)-OH to 2,6-dichlorobenzoyl chloride in this embodiment was 1:2, and the molar ratio of Fmoc-Arg(Pbf)-OH to pyridine was 1:4. Other conditions and parameters were the same as in Example 1.

[0072] Example 3: Coupling of amino acid to resin: The coupling of amino acid to resin was performed in the same manner as in Example 2, except that Fmoc-Arg(Pbf)-OH was replaced with Fmoc-Phe-OH. Other conditions and parameters were the same as in Example 2.

[0073] Example 4: Coupling of amino acids to resin: First, immerse Wang resin in dichloromethane and swell it for 30 minutes. After swelling, filter out the liquid. Then, dissolve Fmoc-Arg(Pbf)-OH and Boc anhydride in dichloromethane and add them to the swollen Wang resin. Pyridine is then added and the mixture is reacted at 25°C for 4 hours. The mixture is then separated and washed three times with dichloromethane. After washing, end-capping is performed with acetic anhydride, N,N-diisopropylethylamine, and dichloromethane for 30 minutes. This is repeated twice. After end-capping, the resin is washed four times with methanol and dried to obtain Fmoc-Arg(Pbf)-Wang resin. The degree of substitution of wang resin is 0.8 mmol / g, the molar ratio of wang resin to Fmoc-Arg(Pbf)-OH is 1:2, the molar ratio of Fmoc-Arg(Pbf)-OH to Boc anhydride is 1:1, the ratio of Fmoc-Arg(Pbf)-OH to dichloromethane added during the reaction is 1 mol:3.75 L, the molar ratio of Fmoc-Arg(Pbf)-OH to pyridine is 1:1, and for each end-capping step, the ratio of wang resin to acetic anhydride is 1 mol:0.95 L, the volume ratio of acetic anhydride to N,N-diisopropylethylamine is 1:1.84, and the volume ratio of acetic anhydride to dichloromethane is 1:7.89.

[0074] Example 5: Coupling of amino acid with resin: The coupling of amino acid with resin was different from that in Example 4, except that Boc anhydride was replaced with diisopropyl carbonate. Other conditions and parameters were the same as those in Example 2.

[0075] Example 6: Coupling of amino acids to resin: The coupling of amino acids to resin was performed in the same manner as in Example 4, except that the molar ratio of Fmoc-Arg(Pbf)-OH to Boc anhydride was 1:2, and the molar ratio of Fmoc-Arg(Pbf)-OH to pyridine was 1:2. Other conditions and parameters were the same as in Example 4.

[0076] Example 7: Coupling of amino acids with resin: The coupling of amino acids with resin is different from that in Example 4, except that the molar ratio of Wang resin to Fmoc-Arg(Pbf)-OH in this example is 1:3, and other conditions and parameters are the same as in Example 4.

[0077] Example 8: Coupling of amino acid to resin: The coupling of amino acid to resin was performed in the same manner as in Example 7, except that Fmoc-Arg(Pbf)-OH was replaced with Fmoc-Phe-OH. Other conditions and parameters were the same as in Example 7.

[0078] Example 9: Coupling of amino acid to resin: The coupling of amino acid to resin was performed in the same manner as in Example 7, except that Fmoc-Arg(Pbf)-OH was replaced with Fmoc-Gly-OH. Other conditions and parameters were the same as in Example 7.

[0079] Example 10: Coupling of amino acids with resin: First, immerse the Wang resin in dichloromethane and swell it for 30 minutes. After swelling, filter out the liquid. Then dissolve Fmoc-Arg(Pbf)-OH and 1-hydroxybenzotriazole in dichloromethane and add them to the swollen Wang resin. Then add diisopropylcarbodiimide. Then dissolve 4-pyrrolidinopyridine in N,N-dimethylformamide and add it dropwise. React at 25°C for 4 hours. After the reaction, separate and wash with dichloromethane three times. After washing, add acetic anhydride, N,N-diisopropylethylamine and dichloromethane for end-capping for 30 minutes, and repeat the end-capping twice. After end-capping, wash it with methanol four times and finally dry to obtain Fmoc-Arg(Pbf)-Wang resin. The degree of substitution of the Wang resin is 0.8mmol / g The molar ratio of Wang resin to Fmoc-Arg(Pbf)-OH was 1:2, the molar ratio of Fmoc-Arg(Pbf)-OH to 1-hydroxybenzotriazole was 1:1, the molar ratio of Fmoc-Arg(Pbf)-OH to dichloromethane added during the reaction was 1 mol:3.75 L, the molar ratio of Fmoc-Arg(Pbf)-OH to diisopropylcarbodiimide was 1:1, and Fmo The molar ratio of c-Arg(Pbf)-OH to 4-pyrrolidinylpyridine was 1:0.1, and the ratio of 4-pyrrolidinylpyridine to N,N-dimethylformamide was 1 mol:0.75 L. For each end-capping step, the ratio of Wang resin to acetic anhydride was 1 mol:0.95 L, the volume ratio of acetic anhydride to N,N-diisopropylethylamine was 1:1.84, and the volume ratio of acetic anhydride to dichloromethane was 1:7.89.

[0080] Example 11: Coupling of amino acid to resin: The coupling of amino acid to resin was performed in the same manner as in Example 10, except that 4-pyrrolidinylpyridine was replaced with 4-dimethylaminopyridine N-oxide. Other conditions and parameters were the same as in Example 10.

[0081] Example 12: Coupling of amino acid to resin: The coupling of amino acid to resin was similar to that in Example 10, except that 4-pyrrolidinylpyridine was replaced with 4-dimethylaminopyridine. Other conditions and parameters were the same as those in Example 10.

[0082] Example 13: Coupling of amino acid to resin: The coupling of amino acid to resin was performed in the same manner as in Example 10, except that 4-dimethylaminopyridine was replaced with 5,6,9,10-tetrahydro-4H,8H-pyrido[3,2,1-IJ][1,6]naphthyridine. Other conditions and parameters were the same as in Example 10.

[0083] Example 14: Coupling of amino acids to resin: The coupling of amino acids to resin was performed as in Example 10, except that 4-dimethylaminopyridine was replaced with 4,4,10,10-tetramethyl-5,6,9,10-tetrahydro-4H,8H-pyridyl[3,2,1-IJ][1,6]naphthyridine. Other conditions and parameters were the same as in Example 10.

[0084] Example 15: Coupling of amino acid with resin: The coupling of amino acid with resin was different from that in Example 10, except that the molar ratio of Wang resin to Fmoc-Arg(Pbf)-OH was 1:3, and other conditions and parameters were the same as in Example 10.

[0085] Example 16: Coupling of amino acid to resin: The coupling of amino acid to resin was performed in the same manner as in Example 15, except that Fmoc-Arg(Pbf)-OH was replaced with Fmoc-Phe-OH. Other conditions and parameters were the same as in Example 15.

[0086] Example 17: Coupling of amino acid and resin: The coupling of amino acid and resin was different from that in Example 16, except that the dichloromethane used to swell the resin was replaced by N,N-dimethylformamide. Other conditions and parameters were the same as in Example 16.

[0087] Example 18: Coupling of amino acid to resin: The coupling of amino acid to resin was performed in the same manner as in Example 15, except that Fmoc-Arg(Pbf)-OH was replaced with Fmoc-Gly-OH. Other conditions and parameters were the same as in Example 15.

[0088] Example 19: Preparation of pyridine derivatives: Dimethyl 4-hydroxymethylpyridine-2,6-dicarboxylate was mixed with methanol, followed by the addition of potassium borohydride. The mixture was reacted at 35°C for 4 hours. After the reaction, the pH was adjusted to 6 and then to 8. The reaction solution was separated and purified by column chromatography to obtain the pyridinolate. Hydrogen bromide was added to the pyridinolate, followed by the dropwise addition of acetic anhydride. The reaction was continued at 110°C for 3 hours. After the reaction, the mixture was rotary evaporated, the pH was adjusted to 8, and the solid was separated. The solid was dried to obtain the pyridine intermediate. Diisobutyl iminodiacetate, 4-formylphenylboronic acid methyliminodiacetate, sodium carbonate, and acetonitrile were added to the pyridine intermediate. The mixture was reacted at 30°C for 3 hours. After the reaction, the mixture was rotary evaporated, ethyl acetate and water were added, the organic layer was separated, washed with saturated sodium chloride solution, and finally dried to obtain the pyridine derivative. The mass ratio of 4-hydroxymethylpyridine-2,6-dicarboxylic acid dimethyl ester to methanol is 1 g:15 ml, the mass ratio of 4-hydroxymethylpyridine-2,6-dicarboxylic acid dimethyl ester to potassium borohydride is 1:0.7, the mass ratio of pyridinolate to hydrogen bromide is 1 g:8.5 ml, the mass ratio of pyridinolate to acetic anhydride is 1 g:25 ml, the mass ratio of pyridine intermediate to diisobutyl iminodiacetate is 1:0.8, the mass ratio of pyridine intermediate to 4-formylphenylboronic acid methyliminodiacetate is 1:0.6, the mass ratio of pyridine intermediate to sodium carbonate is 1:3.5, the mass ratio of pyridine intermediate to acetonitrile is 1 g:40 ml, the volume ratio of ethyl acetate to water is 1:0.5, and the saturated sodium chloride solution consists of sodium chloride and water.

[0089] Coupling of amino acids to resin: First, immerse the Wang resin in dichloromethane and swell it for 30 minutes. After swelling, filter out the liquid. Then, dissolve Fmoc-Arg(Pbf)-OH and 1-hydroxybenzotriazole in dichloromethane and add them to the swollen Wang resin. Diisopropylcarbodiimide is then added. Then, dissolve 4-pyrrolidinylpyridine and a pyridine derivative in N,N-dimethylformamide and add them dropwise. The reaction is allowed to proceed at 20-30°C for 4 hours. After completion of the reaction, separate the mixture and wash it three times with dichloromethane. After washing, add acetic anhydride, N,N-diisopropylethylamine, and dichloromethane to cap the mixture for 30 minutes. Repeat this process twice. After capping, wash it four times with methanol and dry it to obtain the Fmoc-Arg(Pbf)-Wang resin. The degree of substitution of wang resin is 0.8 mmol / g, the molar ratio of wang resin to Fmoc-Arg(Pbf)-OH is 1:2, the molar ratio of Fmoc-Arg(Pbf)-OH to 1-hydroxybenzotriazole is 1:1, the ratio of Fmoc-Arg(Pbf)-OH to dichloromethane added during the reaction is 1 mol:3.75 L, the molar ratio of Fmoc-Arg(Pbf)-OH to diisopropylcarbodiimide is 1:1, and the molar ratio of Fmoc-Arg(Pbf)-OH to 1-hydroxybenzotriazole is 1:1. f) The molar ratio of Fmoc-Arg(Pbf)-OH to 4-pyrrolidinylpyridine is 1:0.1, the molar ratio of Fmoc-Arg(Pbf)-OH to pyridine derivative is 1:0.05, and the ratio of 4-pyrrolidinylpyridine to N,N-dimethylformamide is 1 mol:0.75 L; for each end-capping, the ratio of Wang resin to acetic anhydride is 1 mol:0.95 L, the volume ratio of acetic anhydride to N,N-diisopropylethylamine is 1:1.84, and the volume ratio of acetic anhydride to dichloromethane is 1:7.89.

[0090] Example 20: The preparation of pyridine derivatives is the same as in Example 19.

[0091] Coupling of amino acid and resin: The coupling of amino acid and resin was different from that in Example 19, except that the molar ratio of Fmoc-Arg(Pbf)-OH to the pyridine derivative was 1:0.07, and other conditions and parameters were the same as in Example 19.

[0092] Example 21: The preparation of pyridine derivatives is the same as in Example 19.

[0093] Coupling of amino acids with resin: First, immerse the Wang resin in dichloromethane and swell it for 30 minutes. After swelling, filter out the liquid. Then, dissolve Fmoc-Arg(Pbf)-OH and 1-hydroxybenzotriazole in dichloromethane and add them to the swollen Wang resin. Then add diisopropylcarbodiimide. Then, dissolve 4-pyrrolidinylpyridine, pyridine derivatives and N,9-diphenyl-9H-carbazole-2-amine in N,N-dimethylformamide and add them dropwise. React at 20-30°C for 4 hours. After the reaction, separate and wash with dichloromethane three times. After washing, add acetic anhydride, N,N-diisopropylethylamine and dichloromethane for end-capping for 30 minutes, and repeat the end-capping twice. After end-capping, wash it with methanol four times and finally dry to obtain Fmoc-Arg(Pbf)-Wang resin. The degree of substitution of Wang resin is 0.8mmol / g. The molar ratio of Wang resin to Fmoc-Arg(Pbf)-OH was 1:2, the molar ratio of Fmoc-Arg(Pbf)-OH to 1-hydroxybenzotriazole was 1:1, the molar ratio of Fmoc-Arg(Pbf)-OH to dichloromethane added during the reaction was 1 mol:3.75 L, the molar ratio of Fmoc-Arg(Pbf)-OH to diisopropylcarbodiimide was 1:1, the molar ratio of Fmoc-Arg(Pbf)-OH to 4-pyrrolidinylpyridine was 1:0.1, and the molar ratio of Fmoc-Arg(Pbf)-OH to 1-hydroxybenzotriazole was 1:1. The molar ratio of g(Pbf)-OH to pyridine derivatives was 1:0.05, the molar ratio of Fmoc-Arg(Pbf)-OH to N,9-diphenyl-9H-carbazole-2-amine was 1:0.03, and the ratio of 4-pyrrolidinylpyridine to N,N-dimethylformamide was 1 mol:0.75 L. For each end-capping step, the ratio of wang resin to acetic anhydride was 1 mol:0.95 L, the volume ratio of acetic anhydride to N,N-diisopropylethylamine was 1:1.84, and the volume ratio of acetic anhydride to dichloromethane was 1:7.89.

[0094] Example 22: The preparation of pyridine derivatives is the same as in Example 19.

[0095] Coupling of amino acids with resin: The coupling of amino acids with resin is different from that in Example 21, except that the molar ratio of Fmoc-Arg(Pbf)-OH to N,9-diphenyl-9H-carbazole-2-amine is 1:0.05, and other conditions and parameters are the same as in Example 21.

[0096] Comparative Example 1: The preparation of pyridine derivatives is the same as in Example 19.

[0097] Coupling of amino acid and resin: The coupling of amino acid and resin was different from that in Example 19, except that the mass ratio of Fmoc-Arg(Pbf)-OH to the pyridine derivative was 1:0.005, and other conditions and parameters were the same as in Example 19.

[0098] Comparative Example 2: The preparation of pyridine derivatives is the same as in Example 19.

[0099] Coupling of amino acid with resin: The coupling of amino acid with resin was different from that in Example 21 except that no pyridine derivative was used. Other conditions and parameters were the same as in Example 21.

[0100] Experimental Example 1: The loading degree of amino acids attached to the resin was measured and calculated, i.e., the number of millimoles of amino acids attached per gram of resin in the amino acid resin conjugate. A 5ml piperidine solution was shaken for 30 minutes, then methanol was added to dilute to 50ml. 1ml was removed and diluted to 25ml with methanol and mixed thoroughly as a blank sample. 30mg of Fmoc-Leu-OH was mixed with 5ml piperidine solution and shaken for 30 minutes. Methanol was added to dilute to 50ml. 1ml was removed and diluted to 25ml with methanol and mixed thoroughly as a standard sample. 100mg of the prepared amino acid resin conjugate was mixed with 5ml piperidine solution and shaken for 30 minutes to remove the Fmoc protecting group. The liquid was separated and methanol was added to dilute to 50ml. 1ml was removed and diluted to 25ml with methanol and mixed thoroughly as a sample solution. The piperidine solution is composed of piperidine and N,N-dimethylformamide in a volume ratio of 1:4. The prepared amino acid resin conjugates include the Fmoc-Arg(Pbf)-Wang resin, Fmoc-Cys(Trt)-Wang resin, and Fmoc-Gly-Wang resin prepared in Examples 1-22 and Comparative Example 1. The absorbance of the blank, standard, and sample solutions was measured at 300 nm. Loading degree = standard mass × absorbance of the sample to be tested × 1000 / (standard molecular weight × standard absorbance × sample mass).

[0101] Table 1 Loading degree measurement results

[0102] The loading degrees of the amino acid resin conjugates prepared in Examples 1-22 and Comparative Examples 1-2 were measured, and the results are shown in Table 1. Comparison between Example 1 and Example 2 shows that increasing the amount of 2,6-dichlorobenzoyl chloride and pyridine increases the loading degree; comparison between Example 1 and Example 3 shows that the loading degree is higher when Fmoc-Phe-OH is coupled to Wang resin; Compared with Example 4, Example 1 shows that the use of Boc anhydride for the coupling of Fmoc-Arg(Pbf)-OH with wang resin has a higher loading degree; compared with Example 5, Example 4 shows that the loading degree is higher when Boc anhydride is used compared with diisopropyl carbonate; compared with Example 6, Example 4 shows that reducing the amount of Boc anhydride used will reduce the loading degree; compared with Example 7, Example 4 shows that increasing the amount of Fmoc-Arg(Pbf)-OH, Boc anhydride and pyridine used together can improve the loading degree; compared with Example 8, Example 7 shows that the coupling of Fmoc-Phe-OH with wang resin using the anhydride method has a higher loading degree; compared with Example 9, Example 7 shows that the loading degree is also higher when Fmoc-Gly-OH is coupled with wang resin; Comparing Example 10 with Example 11, it is shown that when 4-pyrrolidinylpyridine is used as a catalyst, the effect is better than that of 4-dimethylaminopyridine N-oxide, and the loading degree is higher; comparing Example 10 with Examples 12-14, it is shown that when 4-dimethylaminopyridine is used as a catalyst, the effect is the best, and the loading degree is the highest compared with other catalysts; comparing Example 10 with Example 15, it is shown that Fmoc-Arg(Pbf)-OH , 1-hydroxybenzotriazole, diisopropylcarbodiimide and 4-pyrrolidinopyridine use amount increase, can effectively improve the loading degree; Example 15 is compared with Example 16, it is shown that Fmoc-Phe-OH is better when coupled with wang resin by catalysis, and the loading degree is higher; Example 16 is compared with Example 17, it is shown that swelling of wang resin with dichloromethane is better than that with N,N-dimethylformamide, and the loading degree is higher; Example 15 is compared with Example 18, it is shown that Fmoc-Gly-OH is also better when coupled with wang resin by catalysis, and the loading degree is higher; Example 10 is compared with Example 19, it is shown that using 4-pyrrolidinopyridine together with a pyridine derivative as a catalyst can improve the loading degree; Example 19 is compared with Example 20, it is shown Increasing the amount of pyridine derivatives used can further improve the loading degree; compared with Example 21, Example 19 shows that on the basis of using 4-pyrrolidinopyridine and pyridine derivatives, the use of N,9-diphenyl-9H-carbazole-2-amine can also improve the loading degree; compared with Example 22, Example 21 shows that increasing the amount of N,9-diphenyl-9H-carbazole-2-amine used can also further improve the loading degree; compared with Comparative Example 1, Example 19 shows that the amount of pyridine derivatives used needs to be in an appropriate range. If the amount is too low, there is no obvious effect on improving the loading degree; compared with Comparative Example 2, Example 21 shows that N,9-diphenyl-9H-carbazole-2-amine needs to be used together with a pyridine derivative. Using N,9-diphenyl-9H-carbazole-2-amine alone has no obvious effect on improving the loading degree.

[0103] Experimental Example 2: Amino acid incorporation rate testing. Compared to Experimental Example 1, incorporation rate = amino acid loading on the resin × weight of the amino acid-resin conjugate / resin substitution degree before coupling × resin weight before coupling. The resin substitution degree before coupling was 0.800 mmol / g.

[0104] Table 2 Results of amino acid accession rate determination

[0105] The amino acid accession rates on the amino acid resin conjugates prepared in Examples 1-22 and Comparative Examples 1-2 were measured, and the results are shown in Table 2. Comparison between Example 1 and Example 2 shows that increasing the amount of 2,6-dichlorobenzoyl chloride and pyridine improves the accession rate; comparison between Example 1 and Example 3 shows that the coupling efficiency of Fmoc-Phe-OH with Wang resin is better and the accession rate is higher. Compared with Example 4, Example 1 shows that the use of Boc anhydride for the coupling of Fmoc-Arg (Pbf) -OH with wang resin has a higher access rate; Compared with Example 5, Example 4 shows that when Boc anhydride is used, the coupling effect of Fmoc-Arg (Pbf) -OH with wang resin is better and the access rate is higher when compared with diisopropyl carbonate; Compared with Example 6, Example 4 shows that a reduction in the amount of Boc anhydride used will reduce the access rate; Compared with Example 7, Example 4 shows that a joint increase in the amount of Fmoc-Arg (Pbf) -OH, Boc anhydride and pyridine used can improve the access rate; Compared with Example 8, Example 7 shows that the coupling efficiency of Fmoc-Phe-OH with wang resin using the anhydride method is better and the access rate is higher; Compared with Example 9, Example 7 shows that the coupling efficiency of Fmoc-Gly-OH with wang resin is also better and the access rate is higher. Comparing Example 10 with Example 11, it is shown that when 4-pyrrolidinylpyridine is used as a catalyst, the effect is better than that of 4-dimethylaminopyridine N-oxide, and the accession rate is higher; comparing Example 10 with Examples 12-14, it is shown that when 4-dimethylaminopyridine is used as a catalyst, the effect is the best, and the accession rate is the highest compared with other catalysts; comparing Example 10 with Example 15, it is shown that Fmoc-Arg(Pbf)-OH , 1-hydroxybenzotriazole, diisopropylcarbodiimide and 4-pyrrolidinopyridine usage, can effectively improve the access rate; Example 15 is compared with Example 16, indicating that the efficiency of Fmoc-Phe-OH and wang resin coupling by catalysis is better; Example 16 is compared with Example 17, indicating that swelling of wang resin with dichloromethane is better than that with N,N-dimethylformamide, and the access rate is higher; Example 15 is compared with Example 18, indicating that the efficiency of Fmoc-Gly-OH and wang resin coupling by catalysis is also better, and the access rate is higher; Example 10 is compared with Example 19, indicating that the use of 4-pyrrolidinopyridine and a pyridine derivative as a catalyst can improve the access rate; Example 19 is compared with Example 20, indicating that the pyridine derivative The increase in the amount of biological use can further improve the access rate; compared with Example 21, Example 19 shows that on the basis of using 4-pyrrolidinopyridine and pyridine derivatives, the use of N,9-diphenyl-9H-carbazole-2-amine can also improve the access rate; compared with Example 22, Example 21 shows that the increase in the amount of N,9-diphenyl-9H-carbazole-2-amine used can also further improve the access rate; compared with Comparative Example 1, Example 19 shows that the amount of pyridine derivatives used needs to be in an appropriate range. If the amount is too low, there will be no obvious effect on improving the access rate; compared with Comparative Example 2, Example 21 shows that N,9-diphenyl-9H-carbazole-2-amine needs to be used together with pyridine derivatives. The use of N,9-diphenyl-9H-carbazole-2-amine alone has no obvious effect on improving the access rate.

[0106] Experimental Example 3: The isomer ratio analysis method is as follows: Reference Substance 1 Solution: Accurately weigh 20 mg of Fmoc-Phe-OH and place it in a 10 mL volumetric flask. Dissolve it in a diluent and quantitatively dilute it to a solution containing 2 mg per mL. This is the Reference Substance 1 solution. The diluent is an acetonitrile-water solution, which is a mixture of acetonitrile and water in a 1:1 volume ratio.

[0107] Reference 2 solution: Accurately weigh 20 mg of Fmoc-D-Phe-OH and place it in a 10 mL volumetric flask. Dissolve it in a diluent and quantitatively dilute it to a solution containing 2 mg per mL. This is the reference 2 solution. The diluent is an acetonitrile-water solution, which is a mixture of acetonitrile and water in a 1:1 volume ratio.

[0108] Mobile phase: TFA aqueous solution is used as mobile phase A, and acetonitrile is used as mobile phase B. To prepare the TFA aqueous solution, accurately measure 1 ml of TFA into a 1000 ml volumetric flask, add water to the volume and shake well, and precisely filter through a 0.45 μm microporous filter membrane.

[0109] Detection method: NanoChrom ChromCroe 120 C18 3μm 4.6mm×250mm was used as the chromatographic column, the detection wavelength was 215nm, TFA aqueous solution was used as the mobile phase A, and acetonitrile was used as the mobile phase B for gradient elution. The gradient elution program was as follows: at 0 min, mobile phase A was 90 vol%, mobile phase B was 10 vol%; at 5 min, mobile phase A was 90 vol%, mobile phase B was 10 vol%; at 7 min, mobile phase A was 50 vol%, mobile phase B was 50 vol%; at 40 min, mobile phase A was 35 vol%, mobile phase B was 65 vol%; at 42 min, mobile phase A was 10 vol%, mobile phase B was 90 vol%; at 55 min, mobile phase A was 10 vol%, mobile phase B was 90 vol%; at 56 min, mobile phase A was 90 vol%, mobile phase B was 10 vol%; at 57 min, mobile phase A was 90 vol%, mobile phase B was 10 vol% vol%; column temperature 50°C, flow rate 1.0 ml / min, and injection volume of the test solution 20 μL. The resolution between the main peak and each impurity should be no less than 1.5. The test sample in the test solution was derived from the Fmoc-Phe-Wang resin in the test example, and the coupled product was cleaved from the Fmoc-Phe-Wang resin.

[0110] Determination of isomer ratio: Fmoc-Phe-OH is prone to racemization during coupling. Example 3 couples Fmoc-Phe-OH to Wang resin using the acyl chloride method, Example 8 couples Fmoc-Phe-OH to Wang resin using the acid anhydride method, and Examples 16 and 17 couple Fmoc-Phe-OH to Wang resin using the catalytic method. The isomer ratio of the Fmoc-Phe-Wang resin obtained after coupling is determined.

[0111] The results are as follows Figure 1As shown, compared with Example 3, Example 8 and Example 16, it is shown that the degree of racemization is relatively low when Fmoc-Phe-OH is coupled to the wang resin by a catalytic method; compared with Example 17, Example 16 shows that the degree of racemization of the wang resin when swollen with dichloromethane is lower than that when swollen with N,N-dimethylformamide.

[0112] Experimental Example 4: The analysis method for the proportion of Fmoc-Gly-Gly-Wang resin is as follows: Reference Substance 1 Solution: Accurately weigh 20 mg of Fmoc-Gly-OH and place it in a 10 mL volumetric flask. Dissolve it in a diluent and quantitatively dilute it to a solution containing 2 mg per mL. This is the Reference Substance 1 solution. The diluent is an acetonitrile-water solution, which is a mixture of acetonitrile and water in a 1:1 volume ratio.

[0113] Reference 2 Solution: Accurately weigh 20 mg of Fmoc-Gly-Gly-OH into a 10 mL volumetric flask. Dissolve and quantitatively dilute with diluent to a solution containing 2 mg per mL. This is reference 2 solution. The diluent is acetonitrile-water solution, which is a mixture of acetonitrile and water in a 1:1 volume ratio.

[0114] Mobile phase: TFA aqueous solution is used as mobile phase A, and acetonitrile is used as mobile phase B. To prepare the TFA aqueous solution, accurately measure 1 ml of TFA into a 1000 ml volumetric flask, add water to the volume and shake well, and precisely filter through a 0.45 μm microporous filter membrane.

[0115] Detection method: A NanoChrom ChromCroe 120 C18 3μm, 4.6mm×250mm column was used with a detection wavelength of 215nm. Mobile phase A was TFA aqueous solution, and mobile phase B was acetonitrile. Gradient elution was performed as follows: at 0 minutes, mobile phase A was 65 vol%, mobile phase B was 35 vol%; at 40 minutes, mobile phase A was 55 vol%, mobile phase B was 45 vol%; at 42 minutes, mobile phase A was 10 vol%, mobile phase B was 90 vol%; at 50 minutes, mobile phase A was 10 vol%, mobile phase B was 90 vol%; at 52 minutes, mobile phase A was 90 vol%, mobile phase B was 10 vol%; at 60 minutes, mobile phase A was 90 vol%, mobile phase B was 10 vol%. The column temperature was 50°C, the flow rate was 1.0 mL / min, and the sample injection volume was 20 μL. The resolution between the main peak and each impurity should be no less than 1.5. The sample in the sample solution was derived from the Fmoc-Gly-Wang resin or Fmoc-Gly-Gly-Wang resin described in the test examples. The coupled product was cleaved from the Fmoc-Gly-Wang resin or Fmoc-Gly-Gly-Wang resin.

[0116] Determination of the Fmoc-Gly-Gly-Wang resin ratio. When Fmoc-Gly-OH is coupled to Wang resin, Fmoc may fall off, resulting in an additional Gly structure. The Fmoc-Gly-Gly-Wang resin ratio in the Fmoc-Gly-Wang resins prepared in Examples 9 and 18 was determined.

[0117] The results are as follows Figure 2 As shown in the figure, the proportion of Fmoc-Gly-Gly-Wang resin in the Fmoc-Gly-Wang resin prepared in Example 9 is lower than that in Example 18, indicating that when Fmoc-Gly-OH is coupled to Wang resin by a catalytic method, the high alkalinity will increase the shedding of Fmoc and increase the proportion of Fmoc-Gly-Gly-Wang resin.

[0118] The embodiments and / or implementation methods described above are only used to illustrate the preferred embodiments and / or implementation methods for realizing the technology of the present invention, and do not impose any form of limitation on the implementation methods of the technology of the present invention. Any person skilled in the art may make slight changes or modifications to other equivalent embodiments without departing from the scope of the technical means disclosed in the content of the present invention, but they should still be regarded as technologies or embodiments that are essentially the same as the present invention.

[0119] This article uses specific examples to illustrate the principles and implementation methods of this application. The description of the above embodiments is only used to help understand the method and core ideas of this application. The above is only the preferred implementation method of this application. It should be pointed out that due to the limitations of textual expression, there are objectively infinite specific structures. For ordinary technicians in this technical field, without departing from the principles of this application, they can also make several improvements, modifications or changes, and can also combine the above technical features in an appropriate manner; these improvements, modifications, changes or combinations, or the direct application of the inventive concept and technical solution to other occasions without improvement, should be regarded as the scope of protection of this application.

Claims

1. A method for efficiently coupling amino acids to resins in solid-phase peptide synthesis, comprising: The resin is swelled, and then an amino acid and a solvent are added, and then an acyl chloride compound and a base are added to react. After the reaction is completed, the resin is separated and washed, and then a blocking agent is added to block the ends, and finally washed and dried to obtain an amino acid resin conjugate; or, The resin is swelled, and then the amino acid and acid anhydride are mixed and dissolved and added, and a base is added to react. After the reaction is completed, the mixture is separated and washed, and then a blocking reagent is added to block the ends, and finally washed and dried to obtain an amino acid resin conjugate; or, The resin is swelled, the amino acid and the condensing agent are mixed and dissolved and then added, and then the condensing agent and the catalyst solution are added to react. After the reaction is completed, the mixture is separated and washed, and then a blocking agent is added to block the ends, and finally washed and dried to obtain an amino acid resin conjugate; The molar ratio of the resin to the amino acid is 1:1.5-4.

2. The method for efficient coupling of amino acids and resins in solid phase synthesis of polypeptides according to claim 1, characterized in that: The resin is a wang resin, and the substitution degree of the wang resin is 0.4-1.2 mmol / g; the amino acids are Fmoc-Ala-OH, Fmoc-Cys(Trt)-OH, Fmoc-Asp(OtBu)-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Phe-OH, Fmoc-Gly-OH, Fmoc-His(Trt)-OH, Fmoc-Ile-OH, Fmoc-Lys(Boc)- OH, Fmoc-Leu-OH, Fmoc-Met-OH, Fmoc-Asn(Trt)-OH, Fmoc-Pro-OH, Fmoc-Gln(Trt)-OH, Fmoc-Arg(Pbf)-O H, at least one of Fmoc-Ser(tBu)-OH, Fmoc-Thr(tBu)-OH, Fmoc-Val-OH, Fmoc-Trp(Boc)-OH and Fmoc-Tyr(tBu)-OH.

3. The method for efficient coupling of amino acids and resins in solid phase synthesis of polypeptides according to claim 1, characterized in that: The solvent is at least one of N,N-dimethylformamide and dichloromethane, and the ratio of the amino acid to the solvent is 1 mol: 3-4 L; the acyl chloride compound is at least one of 2,6-dichlorobenzoyl chloride, 2,4,6-trichlorobenzoyl chloride and 3,5-dichlorobenzoyl chloride, and the molar ratio of the amino acid to the acyl chloride compound is 1:0.5-3; the base that reacts with the acyl chloride compound is pyridine, and the molar ratio of the amino acid to the base is 1:1.5-5.

4. The method for efficient coupling of amino acids and resins in solid phase synthesis of polypeptides according to claim 1, characterized in that: The acid anhydride is at least one of diisopropyl carbonate and Boc anhydride, and the molar ratio of the amino acid to the acid anhydride is 1:0.5-3; the base reacting with the acid anhydride is pyridine, and the molar ratio of the amino acid to the base is 1:1.5-5.

5. The method for efficient coupling of amino acids and resins in solid phase synthesis of polypeptides according to claim 1, characterized in that: The condensing agent in the mixture of amino acid and condensing agent is 1-hydroxybenzotriazole, the condensing agent further added to the condensing agent is diisopropylcarbodiimide, the molar ratio of the amino acid to 1-hydroxybenzotriazole is 1:0.8-1.2, and the molar ratio of the amino acid to diisopropylcarbodiimide is 1:0.8-1.2; the catalyst dissolving solution is obtained by dissolving the catalyst in N,N-dimethylformamide.

6. The method for efficient coupling of amino acids and resins in solid phase synthesis of polypeptides according to claim 5, characterized in that: The catalyst is at least one of 4-dimethylaminopyridine, 4-dimethylaminopyridine N-oxide, 4-pyrrolidinylpyridine, 5,6,9,10-tetrahydro-4H,8H-pyrido[3,2,1-IJ][1,6]naphthyridine, 4,4,10,10-tetramethyl-5,6,9,10-tetrahydro-4H,8H-pyridyl[3,2,1-IJ][1,6]naphthyridine and pyridine derivatives, and the molar ratio of the amino acid to the catalyst is 1:0.02-0.5; the pyridine derivative is prepared from dimethyl 4-hydroxymethylpyridine-2,6-dicarboxylate, methanol, hydrogen bromide, diisobutyl iminodiacetate and 4-formylphenylboronic acid methyliminodiacetate.

7. The method for efficient coupling of amino acids and resins in solid phase synthesis of polypeptides according to claim 1, characterized in that: The end-capping reagent includes acetic anhydride, N,N-diisopropylethylamine and dichloromethane, and the ratio of resin to acetic anhydride is 1 mol: 0.8-1.1L, the volume ratio of acetic anhydride to N,N-diisopropylethylamine is 1:1-2.5, and the volume ratio of acetic anhydride to dichloromethane is 1:7-9.

8. The method for efficient coupling of amino acids and resins in solid phase synthesis of polypeptides according to claim 1, characterized in that: The reaction temperature for the addition of amino acid, solvent, acyl chloride compound and base is 20-30° C., and the reaction time is 3-5 hours.

9. The method for efficient coupling of amino acids and resins in solid phase synthesis of polypeptides according to claim 1, characterized in that: The amino acid and the acid anhydride are mixed and dissolved, and then added, and the base is added to react at a reaction temperature of 20-30° C. and a reaction time of 3-5 hours.

10. The method for efficient coupling of amino acids and resins in solid phase synthesis of polypeptides according to claim 1, characterized in that: The reaction temperature for the reaction of adding the condensing agent and the catalyst solution is 20-30° C., and the reaction time is 3-5 hours.

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