A method for preparing a high purity polypeptide starting material

Abstract

The application discloses a preparation method of high-purity polypeptide starting material, and belongs to the technical field of polypeptide solid-phase synthesis, and particularly relates to a preparation method of high-purity polypeptide starting material; the preparation method comprises the following steps: taking (2,4-dimethoxybenzyl) amino acid methyl ester as a starting raw material, treating the starting raw material through a selective de-esterification system, and modifying the starting raw material through an amino protecting group to obtain a polypeptide starting material containing racemization impurities; and then, the polypeptide starting material is purified through chiral resolution to obtain a high-purity polypeptide starting material; the (2,4-dimethoxybenzyl) amino acid methyl ester is obtained by reacting L-amino acid methyl ester hydrochloride with 2,4-dimethoxybenzaldehyde. The raw material and the reaction system used in the synthesis process of the polypeptide starting material are optimized, the generation of racemization impurities and other impurities is effectively inhibited, a chiral resolution strategy is introduced, and therefore, the high-purity polypeptide starting material is obtained, and a reliable synthesis path is provided for the large-scale preparation of high-quality polypeptide synthesis.

Description

Technical Field

[0001] This invention relates to the field of peptide solid-phase synthesis technology, and specifically to a method for preparing high-purity peptide starting materials. Background Technology

[0002] Peptide synthesis typically employs solid-phase synthesis strategies. However, during the assembly of certain challenging peptide sequences, peptide resins are prone to condensation polymerization. This phenomenon is primarily caused by intermolecular or intramolecular β-sheet interactions within the peptide chain. These interactions usually occur on the main chain structure, particularly the hydrogen bond network formed between amide hydrogen and carbonyl groups. Once peptide resin condensation polymerization occurs, it directly affects the coupling efficiency of subsequent amino acids, manifesting as incomplete deprotection and hindered coupling reactions. This leads to the formation of severe deletion peptide byproducts, increasing the difficulty of synthesis and significantly reducing the purity of the final crude peptide.

[0003] To inhibit the condensation polymerization of peptide resins, a common strategy is to chemically modify the peptide backbone by introducing a protecting group to block intermolecular hydrogen bonding interactions. 2,4-Dimethoxybenzyl (Dmb) is a widely used protecting group. Especially in difficult peptide sequences containing consecutive alanine (Ala) residues, condensation polymerization often concentrates at adjacent Ala sites. Therefore, amino acid derivatives with pre-introduced Dmb protection, such as Fmoc-(Dmb)Ala-OH or Fmoc-Ala-(Dmb)Ala-OH, can be used as starting materials to protect the backbone at key sites, thereby effectively mitigating the condensation polymerization problem.

[0004] Currently, the introduction of Dmb protecting groups onto Ala residues is mainly achieved through reductive amination, where H-Ala-OH reacts with 2,4-dimethoxybenzaldehyde in the presence of sodium triacetoxyborohydride to generate H-(Dmb)Ala-OH. However, this method is affected by the carboxyl terminus of alanine, resulting in low reaction efficiency and a tendency for isomerization during imine intermediate formation. This leads to racemization of the product, introducing enantiomeric impurities that are difficult to separate, directly affecting the purity of the final product and further negatively impacting the quality of peptide synthesis. Therefore, developing a novel method to effectively reduce the formation of isomerization impurities and prepare Fmoc-(Dmb)Ala-OH and Fmoc-Ala-(Dmb)Ala-OH with high purity and high yield has significant synthetic value and application demand. Summary of the Invention

[0005] The purpose of this invention is to provide a method for preparing high-purity peptide starting materials. By optimizing the raw materials and reaction system used in the synthesis of peptide starting materials, the generation of racemic impurities and other impurities is effectively suppressed. At the same time, a chiral resolution strategy is introduced to prepare high-purity peptide starting materials free of isomerization impurities, thereby obtaining high-purity peptide starting materials.

[0006] The technical solution adopted by the present invention to achieve the above objectives is as follows: A method for preparing a high-purity peptide starting material includes: using (2,4-dimethoxybenzyl) amino acid methyl ester as the starting material, selectively deesterifying and modifying with amino protecting groups to obtain a peptide starting material containing racemic impurities, followed by chiral resolution and purification to obtain a high-purity peptide starting material; (2,4-dimethoxybenzyl) amino acid methyl ester is obtained by reacting L-amino acid methyl ester hydrochloride with 2,4-dimethoxybenzaldehyde; the molar ratio of L-amino acid methyl ester hydrochloride to 2,4-dimethoxybenzaldehyde is 1:0.5-1.5.

[0007] This invention uses L-amino acid methyl ester hydrochloride (H-Xaa-OMe) as the starting material. Under the action of sodium triacetoxyborohydride, it undergoes a reductive amination reaction with 2,4-dimethoxybenzaldehyde to efficiently obtain (2,4-dimethoxybenzyl)amino acid methyl ester (H-(Dmb)Xaa-OMe). Subsequently, selective deesterification and amino protecting group modification are performed to suppress side reactions and reduce the formation of racemic impurities, thereby significantly improving the purity and overall yield of the prepared peptide starting material containing racemic impurities. Further chiral resolution and purification yields a high-purity peptide starting material. The high-purity peptide starting material prepared by this invention can serve as a special modification strategy in solid-phase peptide synthesis. By introducing Dmb protecting groups into the main chain, it effectively inhibits peptide resin condensation and improves the yield and purity of crude peptides with difficult peptide sequences.

[0008] Preferably, the selective deesterification system comprises a calcium chloride solution and an aqueous sodium hydroxide solution; the volume ratio of the calcium chloride solution to the aqueous sodium hydroxide solution is 1:0.1-0.5. This invention combines calcium chloride and sodium hydroxide solutions in the selective deesterification system. By adjusting the alkalinity of the reaction environment, it effectively mitigates side reactions that may be triggered by high concentrations of sodium hydroxide and suppresses the formation of impurities caused by the removal of the Fmoc protecting group under strongly alkaline conditions, thereby effectively improving the purity and overall yield of the target product.

[0009] More preferably, the calcium chloride solution is a solution formed by dissolving calcium chloride in an aqueous isopropanol solution, wherein the concentration of calcium chloride in the calcium chloride solution is 0.5-1 mol / L; and the volume fraction of isopropanol in the aqueous isopropanol solution is 65-75%.

[0010] Preferably, the purity of the polypeptide starting material containing racemic impurities is ≥90%, and the total yield is ≥43%; the content of racemic impurities in the polypeptide starting material is ≤8%.

[0011] Preferably, the chiral resolution and purification reagent includes (1R,2S)-2-amino-1,2-diphenylethanol or S-1-α-phenylethylamine.

[0012] Preferably, the L-amino acid methyl ester hydrochloride is any one of L-alanine methyl ester hydrochloride, L-glycine methyl ester hydrochloride, L-valine methyl ester hydrochloride, L-leucine methyl ester hydrochloride, L-isoleucine methyl ester hydrochloride, L-phenylalanine methyl ester hydrochloride, L-tryptophan methyl ester hydrochloride, L-tyrosine methyl ester hydrochloride, L-aspartic acid methyl ester hydrochloride, L-asparagine methyl ester hydrochloride, L-glutamic acid methyl ester hydrochloride, L-lysine methyl ester hydrochloride, L-glutamine methyl ester hydrochloride, L-methionine methyl ester hydrochloride, L-serine methyl ester hydrochloride, L-threonine methyl ester hydrochloride, L-cysteine ​​methyl ester hydrochloride, L-proline methyl ester hydrochloride, L-histidine methyl ester hydrochloride, and L-arginine methyl ester hydrochloride.

[0013] Preferably, the amino protecting group includes an Fmoc protecting group, a Boc protecting group, or a Cbz protecting group.

[0014] Preferably, the high-purity peptide starting material is Fmoc-(Dmb)Ala-OH and / or Fmoc-Ala-(Dmb)Ala-OH.

[0015] Preferably, the preparation method of (2,4-dimethoxybenzyl)amino acid methyl ester (H-(Dmb)Xaa-OMe) is as follows: L-amino acid methyl ester hydrochloride (H-Xaa-OMe·HCl), N,N-diisopropylethylamine (DIEA), 2,4-dimethoxybenzaldehyde, and dichloromethane (DCM) were mixed and stirred at room temperature. Then, sodium triacetoxyborohydride was slowly added, and the reaction was carried out at room temperature. The reaction was confirmed by thin-layer chromatography (TLC) and mass spectrometry (MS). Water was added to quench the reaction, and the organic and aqueous phases were separated. The organic phase was extracted and washed 1-5 times with saturated sodium bicarbonate solution. The organic phase was separated and dried with anhydrous sodium sulfate. The solvent was removed from the organic phase by rotary evaporation to obtain a pale yellow oil. Finally, the product was purified by column chromatography to obtain (2,4-dimethoxybenzyl)amino acid methyl ester (H-(Dmb)Xaa-OMe).

[0016] More preferably, the L-amino acid methyl ester hydrochloride is any one of L-alanine methyl ester hydrochloride, L-glycine methyl ester hydrochloride, L-valine methyl ester hydrochloride, L-leucine methyl ester hydrochloride, L-isoleucine methyl ester hydrochloride, L-phenylalanine methyl ester hydrochloride, L-tryptophan methyl ester hydrochloride, L-tyrosine methyl ester hydrochloride, L-aspartic acid methyl ester hydrochloride, L-asparagine methyl ester hydrochloride, L-glutamic acid methyl ester hydrochloride, L-lysine methyl ester hydrochloride, L-glutamine methyl ester hydrochloride, L-methionine methyl ester hydrochloride, L-serine methyl ester hydrochloride, L-threonine methyl ester hydrochloride, L-cysteine ​​methyl ester hydrochloride, L-proline methyl ester hydrochloride, L-histidine methyl ester hydrochloride, and L-arginine methyl ester hydrochloride.

[0017] More preferably, the molar ratio of H-Xaa-OMe·HCl to DIEA is 1:1-2.

[0018] More preferably, the molar ratio of H-Xaa-OMe·HCl to 2,4-dimethoxybenzaldehyde is 1:0.5-1.5.

[0019] More preferably, the ratio of H-Xaa-OMe·HCl to DCM is 1 mmol: 2-10 mL.

[0020] More preferably, the molar ratio of H-Xaa-OMe·HCl to sodium triacetoxyborohydride is 1:2-6.

[0021] More preferably, the volume ratio of DCM to water is 1:0.5-1.5.

[0022] Preferably, the method for preparing peptide starting materials containing racemic impurities is as follows: The selective deesterification system was stirred for 10-30 min, and H-(Dmb)Xaa-OMe was added to continue the reaction. After the reaction was confirmed by TLC, fluorenemethyloxycarbonyl chloride (Fmoc-Cl) was slowly added at 0°C. After the reaction was confirmed by TLC, the mixture was extracted 1-5 times with ethyl acetate, and the organic phase was retained and dried over anhydrous sodium sulfate. The dried phase was then purified by column chromatography to obtain Fmoc-(Dmb)Xaa-OH containing racemic impurities. The selective deesterification system consisted of calcium chloride solution and sodium hydroxide aqueous solution.

[0023] More preferably, the calcium chloride solution is a solution formed by dissolving calcium chloride in an aqueous isopropanol solution, wherein the concentration of calcium chloride in the calcium chloride solution is 0.5-1 mol / L; and the volume fraction of isopropanol in the aqueous isopropanol solution is 65-75%.

[0024] More preferably, the concentration of the sodium hydroxide aqueous solution is 0.2-1 mol / L, and the volume ratio of the calcium chloride solution to the sodium hydroxide aqueous solution is 1:0.1-0.5.

[0025] More preferably, the ratio of sodium hydroxide aqueous solution to H-(Dmb)Xaa-OMe is 1 mL: 0.5-1.5 mmol.

[0026] More preferably, the molar ratio of H-(Dmb)Xaa-OMe to Fmoc-Cl is 1:0.5-1.5.

[0027] Preferably, the method for preparing a polypeptide starting material containing racemic impurities includes: The selective deesterification system was stirred for 20 min, and then Fmoc-Xaa-(Dmb)Xaa-OMe was added to react. The reaction was confirmed by TLC, and the mixture was extracted 1-5 times with ethyl acetate. The organic phase was retained and dried over anhydrous sodium sulfate. The solution was then evaporated to dryness and purified by column chromatography to obtain Fmoc-Xaa-(Dmb)Xaa-OH containing racemic impurities. The selective deesterification system consisted of calcium chloride solution and sodium hydroxide aqueous solution.

[0028] More preferably, the calcium chloride solution is a solution formed by dissolving calcium chloride in an aqueous isopropanol solution, wherein the concentration of calcium chloride in the calcium chloride solution is 0.5-1 mol / L; and the volume fraction of isopropanol in the aqueous isopropanol solution is 65-75%.

[0029] More preferably, the concentration of the sodium hydroxide aqueous solution is 0.2-1 mol / L, and the volume ratio of the calcium chloride solution to the sodium hydroxide aqueous solution is 1:0.1-0.5.

[0030] More preferably, the ratio of sodium hydroxide aqueous solution to Fmoc-Xaa-(Dmb)Xaa-OMe is 1 mL: 0.2-0.8 mmol.

[0031] More preferably, the preparation method of Fmoc-Xaa-(Dmb)Xaa-OMe is as follows: H-(Dmb)Xaa-OMe, DCM, and triethylamine were mixed, and the mixture was evacuated 1-5 times and purged with nitrogen 1-5 times. Fmoc-Xaa-Cl was slowly added dropwise at 0-4℃. After the addition was complete, the reaction was carried out at room temperature. The reaction was confirmed by TLC by adding water to quench the reaction. The organic phase was separated by DCM extraction, dried with anhydrous sodium sulfate, and the solvent was removed by rotary evaporation to obtain Fmoc-Xaa-(Dmb)Xaa-OMe.

[0032] More preferably, the ratio of H-(Dmb)Xaa-OMe to DCM is 1 mmol: 2-3 mL.

[0033] More preferably, the molar ratio of H-(Dmb)Xaa-OMe to triethylamine is 1:2-4.

[0034] More preferably, the molar ratio of H-(Dmb)Xaa-OMe and Fmoc-Xaa-Cl is 1:0.5-1.5.

[0035] More preferably, the preparation method of Fmoc-Xaa-Cl is as follows: Fmoc-Xaa-OH, DCM, and N,N-dimethylformamide (DMF) were mixed, and the mixture was evacuated 1-5 times and purged with nitrogen 1-5 times. Oxaloyl chloride was slowly added dropwise at 0°C. After the addition was complete, the reaction was allowed to proceed at room temperature. The reaction was confirmed by TLC, and the solvent was removed by rotary evaporation to obtain Fmoc-Xaa-Cl. The molar ratio of Fmoc-Xaa-OH to DCM was 1 mmol: 5-10 mL; the molar ratio of Fmoc-Xaa-OH to DMC was 1:0.05-0.15; and the molar ratio of Fmoc-Xaa-OH to oxalyl chloride was 1:2-4. Fmoc-Xaa-OH is Fmoc-Ala-OH, Fmoc-Gly-OH, Fmoc-Val-OH, Fmoc-Leu-OH, Fmoc-Ile-OH, Fmoc-Phe- OH, Fmoc-Trp(Boc)-OH, Fmoc-Tyr(tBu)-OH, Fmoc-Asp(OtBu)-OH, Fmoc-Asn(Trt)-OH, Fmoc-Glu( OtBu)-OH, Fmoc-Lys(Boc)-OH, Fmoc-Gln(Trt)-OH, Fmoc-Met-OH, Fmoc-Ser(tBu)-OH, Fmoc-Thr( tBu)-OH, Fmoc-Cys(Trt)-OH, Fmoc-Pro(Boc)-OH, Fmoc-His(Trt)-OH, or Fmoc-Arg(Pbf)-OH.

[0036] Preferably, the purification method for high-purity peptide starting materials is as follows: The polypeptide starting material containing racemic impurities, chiral resolution and purification reagent, and ethyl acetate are mixed and stirred until dissolved. The mixture is stirred for 1-5 hours at room temperature, filtered to retain the solid, washed with ethyl acetate, and then mixed with phosphoric acid aqueous solution and ethyl acetate for 10-60 minutes. The organic phase is separated and dried with anhydrous sodium sulfate. The solvent is removed by rotary evaporation. This step is repeated 1-3 times to obtain the purified high-purity polypeptide starting material.

[0037] More preferably, the chiral resolution and purification reagent includes (1R,2S)-2-amino-1,2-diphenylethanol or S-1-α-phenylethylamine.

[0038] More preferably, the mass ratio of the polypeptide starting material containing racemic impurities to (1R,2S)-2-amino-1,2-diphenylethanol is 1:0.1-1.

[0039] More preferably, the ratio of the polypeptide starting material containing racemic impurities to ethyl acetate is 1 g: 10-50 mL.

[0040] More preferably, the mass concentration of the phosphoric acid aqueous solution is 2-10%.

[0041] More preferably, the volume ratio of the aqueous phosphoric acid solution to ethyl acetate is 1:0.5-1.5.

[0042] The present invention also discloses the use of L-amino acid methyl ester hydrochloride in the preparation of polypeptide starting materials containing racemic impurities with 2,4-dimethoxybenzyl protection, wherein the purity of the polypeptide starting materials containing racemic impurities is ≥90% and the total yield is ≥43%; and the content of racemic impurities in the polypeptide starting materials containing racemic impurities is ≤8%.

[0043] The present invention also discloses the use of a selective deesterification system composed of calcium chloride solution and sodium hydroxide aqueous solution in the preparation of high-purity peptide synthesis starting materials, wherein the volume ratio of calcium chloride solution to sodium hydroxide aqueous solution is 1:0.1-0.5; the calcium chloride solution is a solution formed by dissolving calcium chloride in isopropanol aqueous solution, wherein the concentration of calcium chloride in the calcium chloride solution is 0.5-1 mol / L; and the volume fraction of isopropanol in the isopropanol aqueous solution is 65-75%.

[0044] This invention addresses the problem of low reductive amination efficiency by using L-amino acid methyl ester hydrochloride H-Xaa-OMe as the starting material. It reacts with 2,4-dimethoxybenzaldehyde in the presence of sodium triacetoxyborohydride to obtain (2,4-dimethoxybenzyl)amino acid methyl ester H-(Dmb)Xaa-OMe. Selective deesterification and amino protecting group modification yield a peptide starting material containing racemic impurities. This is then purified by chiral resolution to obtain a high-purity peptide starting material. The selective deesterification system includes calcium chloride solution and sodium hydroxide solution. Therefore, this invention has the following beneficial effects: The raw materials and reaction system used in the synthesis process are optimized, effectively suppressing the generation of racemic impurities and other impurities, so that the total yield of the prepared peptide starting material containing racemic impurities is ≥43%, the purity is ≥90%, and the content of racemic impurities is ≤8%, thereby significantly improving the efficiency of chiral resolution and purification. High-purity peptide starting materials can be obtained through simple chiral resolution and purification steps, which not only shortens the process flow, but also reduces reagent consumption and operation time, and has good process economy and scalability. Attached Figure Description

[0045] Figure 1The preparation route for H-(Dmb)Ala-OMe is shown.

[0046] Figure 2 This describes the preparation route for Fmoc-(Dmb)Ala-OH containing racemic impurities.

[0047] Figure 3 The purification route for Fmoc-(Dmb)Ala-OH is shown.

[0048] Figure 4 The preparation route for Fmoc-Ala-(Dmb)Ala-OMe.

[0049] Figure 5 This describes the preparation route for Fmoc-Ala-(Dmb)Ala-OH containing racemic impurities.

[0050] Figure 6 The purification route for Fmoc-Ala-(Dmb)Ala-OH.

[0051] Figure 7 The spectra are those of Fmoc-(Dmb)Ala-OH before and after chiral resolution and purification.

[0052] Figure 8 The spectrum of purified Fmoc-(Dmb)Ala-OH is shown.

[0053] Figure 9 The graphs are those of Fmoc-Ala-(Dmb)Ala-OH before and after chiral separation and purification.

[0054] Figure 10 The spectrum of purified Fmoc-Ala-(Dmb)Ala-OH is shown. Detailed Implementation

[0055] The present invention will now be described in further detail with reference to specific embodiments. The given embodiments are merely illustrative of the invention and not intended to limit its scope. The embodiments provided below can serve as a guide for further improvements by those skilled in the art and do not constitute a limitation on the invention in any way.

[0056] Unless otherwise specified, the experimental methods used in the following examples are conventional methods. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0057] Example 1: The preparation method and route of H-(Dmb)Ala-OMe are as follows: Figure 1 As shown, including, L-alanine methyl ester hydrochloride (H-Ala-OMe·HCl), N,N-diisopropylethylamine (DIEA), 2,4-dimethoxybenzaldehyde, and dichloromethane (DCM) were mixed and stirred at room temperature. Then, sodium triacetoxyborohydride was slowly added, and the reaction was carried out at room temperature. The reaction was confirmed by thin-layer chromatography (TLC) and mass spectrometry (MS). Water was added to quench the reaction, and the organic and aqueous phases were separated. The organic phase was extracted and washed three times with saturated sodium bicarbonate solution, and dried with anhydrous sodium sulfate. The solvent in the organic phase was removed by rotary evaporation to obtain a pale yellow oily substance. Finally, H-(Dmb)Ala-OMe was obtained by column chromatography. The molar ratio of H-Ala-OMe·HCl to DIEA is 1:1.5; the molar ratio of H-Ala-OMe·HCl to 2,4-dimethoxybenzaldehyde is 1:1.1; the volume ratio of H-Ala-OMe·HCl to DCM is 1 mmol:4 mL; the molar ratio of H-Ala-OMe·HCl to sodium triacetoxyborohydride is 1:4; and the volume ratio of DCM to water is 1:1.

[0058] The preparation method of Fmoc-(Dmb)Ala-OH containing racemic impurities, the preparation route is as follows: Figure 2 As shown, including, Calcium chloride solution and sodium hydroxide aqueous solution were mixed and stirred for 20 min. H-(Dmb)Ala-OMe was added to continue the reaction. After the reaction was confirmed by TLC, fluorene methyloxycarbonyl chloride (Fmoc-Cl) was slowly added at 0 °C. After the reaction was confirmed by TLC, the mixture was extracted three times with ethyl acetate. The organic phase was retained and dried with anhydrous sodium sulfate. The solution was then evaporated to dryness and purified by column chromatography to obtain Fmoc-(Dmb)Ala-OH containing racemic impurities. The calcium chloride solution is a solution formed by dissolving calcium chloride in an aqueous isopropanol solution. The concentration of calcium chloride in the calcium chloride solution is 0.8 mol / L; the volume fraction of isopropanol in the aqueous isopropanol solution is 70%; the concentration of sodium hydroxide aqueous solution is 0.5 mol / L; the volume ratio of calcium chloride solution to sodium hydroxide aqueous solution is 1:0.2; the volume ratio of sodium hydroxide aqueous solution to H-(Dmb)Ala-OMe is 1 mL:1 mmol; and the molar ratio of H-(Dmb)Ala-OMe to Fmoc-Cl is 1:1.

[0059] The purification method and preparation route of Fmoc-(Dmb)Ala-OH are as follows: Figure 3 As shown, including, Fmoc-(Dmb)Ala-OH containing racemic impurities, (1R,2S)-2-amino-1,2-diphenylethanol, and ethyl acetate were mixed and stirred until dissolved. The mixture was stirred for 3 hours at room temperature, filtered, and the solid was retained. The solid was washed with ethyl acetate, and then an aqueous solution of phosphoric acid and ethyl acetate were added and stirred for 30 minutes. The organic phase was separated and dried over anhydrous sodium sulfate. The solvent was removed by rotary evaporation. This step was repeated once to obtain purified Fmoc-(Dmb)Ala-OH. The mass ratio of Fmoc-(Dmb)Ala-OH to (1R,2S)-2-amino-1,2-diphenylethanol was 1:0.37; the volume ratio of Fmoc-(Dmb)Ala-OH to ethyl acetate was 1 g:20 mL; the mass concentration of the phosphoric acid aqueous solution was 5%, and the volume ratio of the phosphoric acid aqueous solution to ethyl acetate was 1:1. ¹H NMR (400 MHz, CD₃OD): δ 8.27 (s, 1H), 7.76–7.59 (m, 3H), 7.45–7.15 (m, 6H), 6.47–6.32 (m, 2H), 4.49–4.11 (m, 6H), 3.75 (s, 6H), 1.26 (d, J=7.2 Hz, 3H). HRMS (ESI-TOF) calcd for C 27 H 27 NNaO6 + ([M+Na)) + ): 484.1731; found: 484.1706. Example 2: The preparation method of H-(Dmb)Ala-OMe is the same as in Example 1.

[0060] Methods for preparing Fmoc-Ala-Cl include, Fmoc-Ala-OH, DCM, and N,N-dimethylformamide (DMF) were mixed, evacuated three times, and purged with nitrogen three times. Oxaloyl chloride was slowly added dropwise at 0°C. After the addition was complete, the reaction was carried out at room temperature. The reaction was confirmed by TLC, and the solvent was removed by rotary evaporation to obtain Fmoc-Ala-Cl. The molar ratio of Fmoc-Ala-OH to DCM was 1 mmol:7.6 mL; the molar ratio of Fmoc-Ala-OH to DMC was 1:0.1; and the molar ratio of Fmoc-Ala-OH to oxalyl chloride was 1:3.

[0061] The preparation method and route of Fmoc-Ala-(Dmb)Ala-OMe are as follows: Figure 4 As shown, including, H-(Dmb)Ala-OMe, DCM, and triethylamine were mixed, and the mixture was evacuated three times and purged with nitrogen three times. Fmoc-Ala-Cl was slowly added dropwise at 0°C. After the addition was complete, the reaction was allowed to proceed at room temperature. The reaction was confirmed by TLC, and water was added to quench the reaction. The organic phase was separated by extraction with DCM, dried over anhydrous sodium sulfate, and the solvent was removed by rotary evaporation to obtain Fmoc-Ala-(Dmb)Ala-OMe. The molar ratio of H-(Dmb)Ala-OMe to DCM was 1 mmol: 2.5 mL; the molar ratio of H-(Dmb)Ala-OMe to triethylamine was 1:3; and the molar ratio of H-(Dmb)Ala-OMe to Fmoc-Ala-Cl was 1:1.

[0062] The preparation method of Fmoc-Ala-(Dmb)Ala-OH containing racemic impurities, the preparation route is as follows: Figure 5 As shown, including, Calcium chloride solution and sodium hydroxide aqueous solution were mixed and stirred for 20 min. Fmoc-Ala-(Dmb)Ala-OMe was added to react. The reaction was confirmed by TLC spotting. The mixture was extracted three times with ethyl acetate, and the organic phase was retained and dried over anhydrous sodium sulfate. The solution was then evaporated to dryness and purified by column chromatography to obtain Fmoc-Ala-(Dmb)Ala-OH containing racemic impurities. The calcium chloride solution was formed by dissolving calcium chloride in an aqueous isopropanol solution at a concentration of 0.8 mol / L. The isopropanol solution contained 70% isopropanol by volume. The sodium hydroxide aqueous solution had a concentration of 0.5 mol / L, and the volume ratio of calcium chloride solution to sodium hydroxide solution was 1:0.2. The volume ratio of sodium hydroxide solution to Fmoc-Ala-(Dmb)Ala-OH was 1 mL:0.45 mmol.

[0063] Purification method and preparation route of Fmoc-Ala-(Dmb)Ala-OH are as follows: Figure 6 As shown, including, Fmoc-Ala-(Dmb)Ala-OH containing racemic impurities, S-1-α-phenylethylamine, and ethyl acetate were mixed and stirred until dissolved. The mixture was stirred for 3 hours at room temperature, filtered to retain the solid, washed with ethyl acetate, and then an aqueous solution of phosphoric acid and ethyl acetate were added and stirred for 30 minutes. The organic phase was separated and dried over anhydrous sodium sulfate. The solvent was removed by rotary evaporation. This step was repeated once to obtain purified Fmoc-Ala-(Dmb)Ala-OH. The mass ratio of Fmoc-Ala-(Dmb)Ala-OH to S-1-α-phenylethylamine was 1:0.2; the volume ratio of Fmoc-Ala-(Dmb)Ala-OH to ethyl acetate was 1 g:20 mL; the mass concentration of the phosphoric acid aqueous solution was 5%, and the volume ratio of the phosphoric acid aqueous solution to ethyl acetate was 1:1. ¹H NMR (400 MHz, CDCl₃): δ 8.51 (s, 1H), 7.75 (d, J=7.6 Hz, 2H), 7.62 (dd, J=7.2, 1.6 Hz, 2H), 7.40–7.37 (m, 2H), 7.32–7.26 (m, 2H), 7.13 (d, J=8.8 Hz, 1H), 6.44–6.40 (m, 2H), 6.10 (d, J=8.0 Hz, 1H),4.86-4.78 (m, 1H), 4.59-4.38 (m, 3H), 4.34-4.32 (m, 2H), 4.23-4.19 (m, 1H),3.77 (s, 3H), 3.75 (s, 3H), 1.41 (d, J=6.8 Hz, 3H), 1.33 (d, J=7.2 Hz, 3H).HRMS (ESI-TOF) calcd for C 30 H 33 N2O7 + ([M+H)) + ): 533.2282; found: 533.2284. Comparative Example 1: Methods for preparing H-(Dmb)Ala-OH include, L-alanine (H-Ala-OH), N,N-diisopropylethylamine (DIEA), 2,4-dimethoxybenzaldehyde, and dichloromethane (DCM) were mixed and stirred at room temperature. Then, sodium triacetoxyborohydride was slowly added, and the reaction was carried out at room temperature. The reaction was monitored by HPLC to confirm that it had stopped and that there was still a raw material remaining. Water was added to quench the reaction, and the organic and aqueous phases were separated. The organic phase was extracted and washed three times with saturated sodium bicarbonate solution. The organic phase was separated and dried with anhydrous sodium sulfate. The solvent in the organic phase was removed by rotary evaporation to obtain a pale yellow oily substance. Finally, the product was purified by column chromatography to obtain H-(Dmb)Ala-OH. The molar ratio of H-Ala-OH to DIEA is 1:1.5; the molar ratio of H-Ala-OH to 2,4-dimethoxybenzaldehyde is 1:1.1; the volume ratio of H-Ala-OH to DCM is 1 mmol:4 mL; the molar ratio of H-Ala-OH to sodium triacetoxyborohydride is 1:4; and the volume ratio of DCM to water is 1:1.

[0064] The preparation method of Fmoc-(Dmb)Ala-OH containing racemic impurities is the same as that in Example 1, except that H-(Dmb)Ala-OMe is replaced with H-(Dmb)Ala-OH.

[0065] The purification method for Fmoc-(Dmb)Ala-OH is the same as that in Example 1, except that Fmoc-(Dmb)Ala-OH is replaced with Fmoc-(Dmb)Ala-OH containing racemic impurities prepared in this example, and all other conditions are the same as in Example 1.

[0066] Comparative Example 2: The preparation method of H-(Dmb)Ala-OMe is the same as in Example 1.

[0067] The preparation method of Fmoc-(Dmb)Ala-OH containing racemic impurities is the same as that of Example 1, except that calcium chloride solution was not used.

[0068] The purification method for Fmoc-(Dmb)Ala-OH is the same as that in Example 1, except that Fmoc-(Dmb)Ala-OH is replaced with Fmoc-(Dmb)Ala-OH containing racemic impurities prepared in this example, and all other conditions are the same as in Example 1.

[0069] Comparative Example 3: The preparation method of H-(Dmb)Ala-OH is the same as that of Comparative Example 1.

[0070] The preparation method of Fmoc-Ala-Cl is the same as in Example 2.

[0071] The preparation methods of Fmoc-Ala-(Dmb)Ala-OH include, H-(Dmb)Ala-OH, DCM, and triethylamine were mixed, and the mixture was evacuated three times and purged with nitrogen three times. Fmoc-Ala-Cl was slowly added dropwise at 0°C. After the addition was complete, the reaction was allowed to proceed at room temperature. The reaction was confirmed by TLC, and water was added to quench the reaction. The organic phase was separated by extraction with DCM, dried with anhydrous sodium sulfate, and the solvent was removed by rotary evaporation. The residue was purified by column chromatography to obtain Fmoc-Ala-(Dmb)Ala-OH containing racemic impurities. The molar ratio of H-(Dmb)Ala-OH to DCM was 1 mmol: 2.5 mL; the molar ratio of H-(Dmb)Ala-OH to triethylamine was 1:3; and the molar ratio of H-(Dmb)Ala-OH to Fmoc-Ala-Cl was 1:1.

[0072] The purification method for Fmoc-Ala-(Dmb)Ala-OH is the same as that in Example 2, except that Fmoc-Ala-(Dmb)Ala-OH is replaced with Fmoc-Ala-(Dmb)Ala-OH prepared in this comparative example, and all other conditions are the same as in Example 2.

[0073] Comparative Example 4: The preparation method of H-(Dmb)Ala-OMe is the same as in Example 1.

[0074] The preparation method of Fmoc-Ala-Cl is the same as in Example 2.

[0075] The preparation method of Fmoc-Ala-(Dmb)Ala-OMe is the same as in Example 2.

[0076] The preparation method of Fmoc-Ala-(Dmb)Ala-OH containing racemic impurities is the same as that of Example 2, except that calcium chloride solution was not used.

[0077] The purification method for Fmoc-Ala-(Dmb)Ala-OH is the same as that in Example 2, except that the Fmoc-Ala-(Dmb)Ala-OH containing racemic impurities is replaced with the Fmoc-Ala-(Dmb)Ala-OH containing racemic impurities prepared in this example, and all other conditions are the same as in Example 2.

[0078] Experimental example: 1. Yield The yields of H-(Dmb)Ala-OMe and Fmoc-(Dmb)Ala-OH containing racemic impurities prepared in Example 1; Fmoc-Ala-(Dmb)Ala-OH containing racemic impurities prepared in Example 2; H-(Dmb)Ala-OH and Fmoc-(Dmb)Ala-OH containing racemic impurities prepared in Comparative Example 1; Fmoc-(Dmb)Ala-OH containing racemic impurities prepared in Comparative Example 2; Fmoc-Ala-(Dmb)Ala-OH containing racemic impurities prepared in Comparative Example 3; and Fmoc-Ala-(Dmb)Ala-OH containing racemic impurities prepared in Comparative Example 4 were determined. The yield was calculated using the formula: Yield (%) = Actual yield / Theoretical yield × 100%.

[0079] Table 1 - Intermediate Yields

[0080] The results are shown in Table 1. The yield of H-(Dmb)Ala-OMe prepared in this invention is significantly higher than that of H-(Dmb)Ala-OH. This difference may be related to the chemical structure of the starting materials in the synthetic route. Specifically, H-(Dmb)Ala-OMe was synthesized using H-Ala-OMe·HCl as the starting material, while H-(Dmb)Ala-OH was synthesized using H-Ala-OH. It is evident that when introducing a Dmb protecting group onto the Ala residue, using H-Ala-OMe·HCl as the starting material can effectively promote the reductive amination reaction and effectively suppress side reactions, thereby significantly improving the final yield of the target product.

[0081] Table 2 - Overall Yield of Fmoc-(Dmb)Ala-OH Containing Racemic Impurities

[0082] The results are shown in Table 2. The overall yield of Fmoc-(Dmb)Ala-OH containing racemic impurities prepared in Example 1 was significantly higher than that in Comparative Example 1. This is due to the difference in the raw materials used. Example 1 used H-(Dmb)Ala-OMe as the raw material, while Comparative Example 1 used H-(Dmb)Ala-OH. This indicates that using H-(Dmb)Ala-OMe as the raw material to prepare Fmoc-(Dmb)Ala-OH containing racemic impurities is beneficial to improving the overall yield of the target product. This may be because the H-(Dmb)Ala-OH raw material contains free carboxyl groups. In the activation and condensation reaction system, the α-position of the amino acid is more acidic, and the carboxyl group is more reactive than the ester group, leading to severe racemization and oligomerization reactions, resulting in the formation of complex impurities, which significantly reduces the overall yield of the target product. In contrast, the methyl ester group in H-(Dmb)Ala-OMe effectively blocks the activity of the carboxyl group, inhibits the occurrence of side reactions, and ensures the progress of the main reaction, thereby obtaining the target product with a high overall yield.

[0083] The overall yield of Fmoc-(Dmb)Ala-OH containing racemic impurities prepared in Example 1 was significantly higher than that in Comparative Example 2. This is due to the difference in the selective deesterification system. Example 1 used a combined system of calcium chloride and sodium hydroxide solutions, while Comparative Example 2 used only sodium hydroxide solution. This may be because the strongly alkaline system using sodium hydroxide solution alone could lead to a side reaction in which some products undergo the removal of the Fmoc protecting group, regenerating H-(Dmb)Ala-OH, thus affecting the overall yield of the target product Fmoc-(Dmb)Ala-OH. Using a weakly alkaline demethylation system of calcium chloride / sodium hydroxide solution in the selective deesterification system is beneficial for suppressing side reactions and improving the yield.

[0084] Therefore, in the preparation of Fmoc-(Dmb)Ala-OH containing racemic impurities, using H-Ala-OMe·HCl as the starting material, introducing a Dmb protecting group onto its Ala residue to obtain the intermediate H-(Dmb)Ala-OMe, and then carrying out subsequent reactions in a selective deesterification system composed of calcium chloride solution and sodium hydroxide solution, is beneficial to improving the overall yield of the target product Fmoc-(Dmb)Ala-OH containing racemic impurities.

[0085] Table 3 - Overall Yield of Fmoc-Ala-(Dmb)Ala-OH Containing Racemic Impurities

[0086] The results are shown in Table 3. The overall yield of Fmoc-Ala-(Dmb)Ala-OH containing racemic impurities prepared in Example 2 was significantly higher than that in Comparative Example 3. This is due to the difference in the raw materials. In Example 2, H-(Dmb)Ala-OMe was used as the raw material. After reacting with Fmoc-Ala-Cl, Fmoc-Ala-OH containing racemic impurities was obtained through selective deesterification. In contrast, Comparative Example 3 used H-(Dmb)Ala-OH as the raw material. This indicates that using H-(Dmb)Ala-OMe as the raw material to prepare Fmoc-Ala-(Dmb)Ala-OH containing racemic impurities is beneficial to improving the overall yield of the target product. This may be because the carboxyl group of H-(Dmb)Ala-OH may be highly reactive during the condensation process, easily undergoing side reactions such as cross-condensation and ester formation. H-(Dmb)Ala-OMe can reduce the occurrence of side reactions, thereby significantly improving the overall yield of the target product.

[0087] The overall yield of Fmoc-Ala-(Dmb)Ala-OH containing racemic impurities prepared in Example 2 was 43.1%, while Comparative Example 4 did not yield the target product Fmoc-Ala-(Dmb)Ala-OH. This was due to the difference in the selective deesterification system. Example 2 used a composite system of calcium chloride solution and sodium hydroxide solution, while Comparative Example 4 used only sodium hydroxide solution. This may be because the strongly alkaline system of using sodium hydroxide solution alone can cause a side reaction of deprotection of the Fmoc protecting group, generating H-Ala-(Dmb)Ala-OH, which in turn affects the formation of the target product Fmoc-Ala-(Dmb)Ala-OH. Introducing calcium chloride solution into the selective deesterification system helps to regulate the reaction environment, mitigate the alkaline effect, thereby inhibiting side reactions and increasing the yield.

[0088] Therefore, in the preparation of Fmoc-Ala-(Dmb)Ala-OH containing racemic impurities, using H-Ala-OMe·HCl as the starting material, introducing a Dmb protecting group onto its Ala residue to obtain the intermediate H-(Dmb)Ala-OMe, and then reacting it with Fmoc-Ala-Cl, followed by subsequent reactions in a selective deesterification system composed of calcium chloride solution and sodium hydroxide solution, is beneficial to improving the overall yield of the target product Fmoc-Ala-(Dmb)Ala-OH containing racemic impurities.

[0089] 3. Purity High-performance liquid chromatography (HPLC) was used to determine the following: Fmoc-(Dmb)Ala-OH containing racemic impurities prepared in Example 1, purified Fmoc-(Dmb)Ala-OH prepared in Example 2, and Fmoc-(Dmb)Ala-OH containing racemic impurities prepared in Comparative Example 1. The purity of Fmoc-(Dmb)Ala-OH containing racemic impurities prepared in Comparative Example 2, and the purified Fmoc-(Dmb)Ala-OH; the purity of Fmoc-Ala-(Dmb)Ala-OH containing racemic impurities prepared in Comparative Example 3, and the purified Fmoc-Ala-(Dmb)Ala-OH; and the purity of Fmoc-Ala-(Dmb)Ala-OH containing racemic impurities prepared in Comparative Example 4, and the purified Fmoc-Ala-(Dmb)Ala-OH.

[0090] Table 4 - Purity of Fmoc-(Dmb)Ala-OH

[0091] Figure 7 The spectra before and after chiral resolution and purification of Fmoc-(Dmb)Ala-OH are shown in Table 4. Figure 7 As shown, the purity of Fmoc-(Dmb)Ala-OH containing racemic impurities prepared in Example 1 of this invention is higher than that in Comparative Example 1. This is due to the difference in the raw materials used. Example 1 used H-(Dmb)Ala-OMe as the raw material, while Comparative Example 1 used H-(Dmb)Ala-OH. This may be because the electron-withdrawing ability of the ester group in H-(Dmb)Ala-OMe is weaker than that of the carboxyl group during the reaction, and the acidity at the α-position of the amino acid is weakened, effectively reducing the degree of racemization and thus inhibiting the formation of the racemic impurity Fmoc-D-(Dmb)Ala-OH, resulting in a significant increase in the purity of the target product.

[0092] The purity of Fmoc-(Dmb)Ala-OH containing racemic impurities prepared in Example 1 of this invention is higher than that in Comparative Example 2. This is due to the difference in the selective deesterification system. Example 1 uses a composite system of calcium chloride solution and sodium hydroxide solution, while Comparative Example 2 uses only sodium hydroxide solution. This may be because the strongly alkaline system of using sodium hydroxide solution alone may cause some products to undergo side reactions to remove the Fmoc protecting group, regenerating impurities such as H-(Dmb)Ala-OH, thereby affecting the purity of the target product.

[0093] Figure 8The image shows the chromatogram of purified Fmoc-(Dmb)Ala-OH. During the purification process of Fmoc-(Dmb)Ala-OH, after two chiral resolution purifications, the Fmoc-(Dmb)Ala-OH obtained in Example 1 had a higher purity than Comparative Examples 1 and 2. This may be because the raw materials and reaction system used in the synthesis of Fmoc-(Dmb)Ala-OH in Example 1 effectively suppressed the formation of racemic impurities and other impurities, resulting in higher initial purity and lower impurity content of the product before purification, thereby significantly improving the efficiency of chiral resolution.

[0094] Therefore, in the preparation of Fmoc-(Dmb)Ala-OH, H-Ala-OMe·HCl is used as the starting material. By introducing a Dmb protecting group onto its Ala residue, the intermediate H-(Dmb)Ala-OMe is obtained. Then, it is carried out in a selective deesterification system composed of calcium chloride solution and sodium hydroxide solution. High-purity Fmoc-(Dmb)Ala-OH can be obtained through a simple chiral resolution purification step. This not only shortens the process flow but also reduces reagent consumption and operation time, demonstrating good process economy and scalability.

[0095] Table 5 - Purity of Fmoc-Ala-(Dmb)Ala-OH

[0096] Figure 9 The spectra are those of Fmoc-Ala-(Dmb)Ala-OH before and after chiral resolution and purification. The results are shown in Table 5. Figure 9 As shown, the purity of Fmoc-Ala-(Dmb)Ala-OH containing racemic impurities prepared in Example 2 of this invention is higher than that in Comparative Example 3. This is due to the difference in the raw materials used. In Example 2, H-(Dmb)Ala-OMe was used as the raw material, which reacted with Fmoc-Ala-Cl to obtain Fmoc-Ala-(Dmb)Ala-OMe, which was then selectively deesterified to obtain Fmoc-Ala-(Dmb)Ala-OH containing racemic impurities. In contrast, Comparative Example 3 used H-(Dmb)Ala-OH as the raw material. This may be because the ester group of H-(Dmb)Ala-OMe acts as a temporary steric hindrance protecting group for the α-chiral center during the reaction, effectively reducing the degree of racemization and thus inhibiting the formation of racemic impurities, resulting in a significant increase in the purity of the target product.

[0097] The Fmoc-Ala-(Dmb)Ala-OH containing racemic impurities prepared in Example 2 of this invention has a higher purity than that in Comparative Example 4. This is due to the difference in the selective deesterification system. Example 2 uses a composite system of calcium chloride solution and sodium hydroxide solution, while Comparative Example 4 uses only sodium hydroxide solution. This may be because the strongly alkaline system of using sodium hydroxide solution alone may cause some products to undergo a side reaction of deprotection of the Fmoc protecting group, regenerating impurities such as H-Ala-(Dmb)Ala-OH, thereby affecting the purity of the target product.

[0098] Figure 10 The image shows the chromatogram of purified Fmoc-Ala-(Dmb)Ala-OH. During the purification process of Fmoc-Ala-(Dmb)Ala-OH, after two chiral resolution purifications, the Fmoc-Ala-(Dmb)Ala-OH obtained in Example 2 had a higher purity than Comparative Examples 3 and 4. This may be because the raw materials and reaction system used in the synthesis of Fmoc-Ala-(Dmb)Ala-OH in Example 2 effectively suppressed the formation of racemic impurities and other impurities, resulting in higher initial purity and lower impurity content of the product before purification, thereby significantly improving the efficiency of chiral resolution.

[0099] Therefore, in the preparation of Fmoc-Ala-(Dmb)Ala-OH, H-Ala-OMe·HCl is used as the starting material. By introducing a Dmb protecting group onto its Ala residue, the intermediate H-(Dmb)Ala-OMe is obtained. After reacting with Fmoc-Ala-Cl, subsequent reactions are carried out in a selective deesterification system composed of calcium chloride solution and sodium hydroxide solution. High-purity Fmoc-Ala-(Dmb)Ala-OH can be obtained through a simple chiral resolution purification step. This not only shortens the entire process flow but also reduces reagent consumption and operation time, demonstrating good process economy and scalability.

[0100] The conventional operations in the operation steps of this invention are well known to those skilled in the art and will not be described in detail here.

[0101] The embodiments described above provide a detailed explanation of the technical solutions of the present invention. It should be understood that the above descriptions are merely specific embodiments of the present invention and are not intended to limit the present invention. Any changes and modifications made within the scope of the principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A method for preparing high-purity peptide starting material Fmoc-(Dmb)Ala-OH, characterized in that, include: Preparation of H-(Dmb)Ala-OMe: L-alanine methyl ester hydrochloride H-Ala-OMe in a molar ratio of 1:0.5-1.5 was reacted with 2,4-dimethoxybenzaldehyde in the presence of sodium triacetoxyborohydride to obtain (2,4-dimethoxybenzyl)alanine methyl ester H-(Dmb)Ala-OMe. Preparation of Fmoc-(Dmb)Ala-OH: The H-(Dmb)Ala-OMe was deesterified in a selective deesterification system, and then an amino protecting agent was added for amino protecting group modification to obtain the peptide starting material Fmoc-(Dmb)Ala-OH containing racemic impurities; Preparation of high-purity peptide starting material Fmoc-(Dmb)Ala-OH: The peptide starting material containing racemic impurities, chiral resolution and purification reagent and ethyl acetate are mixed and stirred until dissolved. After stirring at room temperature for 1-5 h, the solid is filtered and washed with ethyl acetate. Phosphoric acid aqueous solution and ethyl acetate are added and stirred for 10-60 min. The organic phase is separated and dried with anhydrous sodium sulfate. The solvent is removed by rotary evaporation. This step is repeated 1-3 times to obtain high-purity peptide starting material Fmoc-(Dmb)Ala-OH. The chiral resolution and purification reagent includes (1R,2S)-2-amino-1,2-diphenylethanol or S-1-α-phenylethylamine; the mass ratio of the polypeptide starting material containing racemic impurities to the chiral resolution and purification reagent is 1:0.1-1; The selective deesterification system comprises a calcium chloride solution and a sodium hydroxide aqueous solution; the volume ratio of the calcium chloride solution to the sodium hydroxide aqueous solution is 1:0.1-0.

5. The calcium chloride solution is a solution formed by dissolving calcium chloride in an aqueous isopropanol solution, with a calcium chloride concentration of 0.5-1 mol / L; the volume fraction of isopropanol in the aqueous isopropanol solution is 65-75%.

2. The preparation method according to claim 1, characterized in that, The purity of the polypeptide starting material containing racemic impurities is ≥90%, and the total yield is ≥43%; the content of racemic impurities in the polypeptide starting material containing racemic impurities is ≤8%.

3. A method for preparing high-purity peptide starting material Fmoc-Ala-(Dmb)Ala-OH, characterized in that, include: Preparation of H-(Dmb)Ala-OMe: L-alanine methyl ester hydrochloride H-Ala-OMe in a molar ratio of 1:0.5-1.5 was reacted with 2,4-dimethoxybenzaldehyde in the presence of sodium triacetoxyborohydride to obtain (2,4-dimethoxybenzyl) amino acid methyl ester H-(Dmb)Ala-OMe. Preparation of Fmoc-Ala-(Dmb)Ala-OMe: H-(Dmb)Ala-OMe, DCM and triethylamine were mixed, vacuumed 1-5 times and purged with nitrogen 1-5 times, and Fmoc-Ala-Cl was slowly added dropwise at 0-4℃. After the addition was complete, the reaction was carried out at room temperature. The reaction was confirmed by TLC spotting. Water was added to quench the reaction, the organic phase was separated by DCM extraction, dried with anhydrous sodium sulfate, and the solvent was removed by rotary evaporation to obtain Fmoc-Ala-(Dmb)Ala-OMe. Preparation of Fmoc-Ala-(Dmb)Ala-OH: The selective deesterification system was mixed and stirred for 20 min, and Fmoc-Ala-(Dmb)Ala-OMe was added to react. The reaction was confirmed by TLC spotting. The organic phase was extracted with ethyl acetate 1-5 times, and the organic phase was retained and dried with anhydrous sodium sulfate. The organic phase was evaporated to dryness and purified by column chromatography to obtain Fmoc-Ala-(Dmb)Ala-OH containing racemic impurities. Preparation of high-purity peptide starting material Fmoc-Ala-(Dmb)Ala-OH: The peptide starting material Fmoc-Ala-(Dmb)Ala-OH containing racemic impurities, chiral resolution and purification reagent and ethyl acetate were mixed and stirred until dissolved. The mixture was stirred at room temperature for 1-5 h, filtered to retain the solid, washed with ethyl acetate, and then phosphoric acid aqueous solution and ethyl acetate were added and stirred for 10-60 min. The organic phase was separated and dried with anhydrous sodium sulfate. The solvent was removed by rotary evaporation. This step was repeated 1-3 times to obtain the purified high-purity peptide starting material Fmoc-Ala-(Dmb)Ala-OH. The preparation method of Fmoc-Ala-Cl is as follows: Fmoc-Ala-OH, DCM and N,N-dimethylformamide are mixed, vacuumed 1-5 times, nitrogen is replaced 1-5 times, oxalyl chloride is slowly added dropwise at 0℃, and the reaction is carried out at room temperature after the addition is completed. The reaction is confirmed by TLC spotting, and the solvent is removed by rotary evaporation to obtain Fmoc-Ala-Cl. The chiral resolution and purification reagent includes (1R,2S)-2-amino-1,2-diphenylethanol or S-1-α-phenylethylamine; the mass ratio of the polypeptide starting material containing racemic impurities to the chiral resolution and purification reagent is 1:0.1-1; The selective deesterification system comprises a calcium chloride solution and a sodium hydroxide aqueous solution; the volume ratio of the calcium chloride solution to the sodium hydroxide aqueous solution is 1:0.1-0.

5. The calcium chloride solution is a solution formed by dissolving calcium chloride in an aqueous isopropanol solution, with a calcium chloride concentration of 0.5-1 mol / L; the volume fraction of isopropanol in the aqueous isopropanol solution is 65-75%.

4. The preparation method according to claim 3, characterized in that, The purity of the polypeptide starting material containing racemic impurities is ≥90%, and the total yield is ≥43%; the content of racemic impurities in the polypeptide starting material containing racemic impurities is ≤8%.

5. The preparation method according to claim 3, characterized in that, When preparing Fmoc-Ala-(Dmb)Ala-OMe, the ratio of H-(Dmb)Xaa-OMe to DCM is 1 mmol: 2-3 mL, the molar ratio of H-(Dmb)Xaa-OMe to triethylamine is 1: 2-4, and the molar ratio of H-(Dmb)Xaa-OMe to Fmoc-Xaa-Cl is 1: 0.5-1.

5.

6. The preparation method according to claim 3, characterized in that, In the preparation method of Fmoc-Ala-Cl, the ratio of Fmoc-Ala-OH to DCM is 1 mmol: 5-10 mL; the molar ratio of Fmoc-Ala-OH to DMC is 1: 0.05-0.15; and the molar ratio of Fmoc-Ala-OH to oxalyl chloride is 1: 2-4.

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