Synthesis method of Fmoc-Xaa-Ser (Psi (Me, Me) Pro)-OH pseudo dipeptide

Abstract

The invention discloses a synthesis method of Fmoc-Xaa-Ser (Psi (Me, Me) Pro)-OH pseudo dipeptide, and belongs to the technical field of polypeptide synthesis. The synthesis method comprises the following steps: reacting Fmoc-Xaa-OH with pentafluorophenol and H-Ser-OH, purifying the synthesized Fmoc-Xaa-Ser-OH with R-1-phenylethylamine, and reacting the purified Fmoc-Xaa-Ser-OH with methoxypropene under the catalysis of a thiocyanic acid pyridine derivative to synthesize the Fmoc-Xaa-Ser (Psi (Me, Me) Pro)-OH pseudo dipeptide. The invention discloses a synthesis method of Fmoc-Xaa-Ser (Psi (Me, Me) Pro)-OH pseudo dipeptide, which is high in purity, high in raw material conversion rate and high in target product selectivity.

Description

Technical Field

[0001] The present invention relates to the technical field of polypeptide synthesis, and in particular to a method for synthesizing a Fmoc-Xaa-Ser(Psi(Me,Me)Pro)-OH pseudo dipeptide. Background Art

[0002] Peptides are a class of compounds composed of amino acids connected by peptide bonds. With the development of modern biotechnology, peptide compounds have gradually become a research hotspot due to their high selectivity, high efficiency, and high safety. Peptide compounds are becoming more and more important in the field of biomedicine, and more and more peptide drugs have been developed and used in clinical practice. With the increasing application of peptide drugs, the synthesis technology of peptide compounds is also developing rapidly, especially the maturity of solid-phase synthesis technology based on the Fmoc method, which has laid an important foundation for the development and application of peptide drugs.

[0003] H-Ser-OH, as a conventional amino acid, appears in various biologically active polypeptide compounds and plays a very wide role in the synthesis of pharmaceutical peptides. In solid-phase synthesis based on the Fmoc method, Fmoc-Ser(tBu)-OH is usually used to extend the peptide chain. However, in the synthesis of long-chain peptides, the application of Fmoc-Ser(tBu)-OH has certain limitations. When synthesizing peptide chains containing -Xaa-Ser(tBu)-, the folding and bending of the peptide chain in space will cause difficulties in subsequent coupling. In practical applications, Fmoc-Xaa-Ser(Psi(Me,Me)Pro)-OH is often used instead of the conventional coupling of Fmoc-Xaa-OH and Fmoc-Ser(tBu)-OH, so that the peptide chain can be stretched in space, making it more convenient to synthesize longer peptide chains. Common Fmoc-Xaa-Ser(Psi(Me,Me)Pro)-OH pseudo-dipeptides include Fmoc-Ser(tBu)-Ser(Psi(Me,Me)Pro)-OH, Fmoc-Pro-Ser(Psi(Me,Me)Pro)-OH, etc.

[0004] The synthetic route of Fmoc-Xaa-Ser(Psi(Me,Me)Pro)-OH pseudo-dipeptides is usually to couple Fmoc-Xaa-OH with H-Ser-OH to obtain Fmoc-Xaa-Ser-OH, and then undergo a one-step cyclization to obtain the target pseudo-dipeptide. However, in the actual synthesis of this type of pseudo-dipeptide, there are usually problems such as low reaction conversion rate and difficulty in product purification, which leads to the high price of Fmoc-Xaa-Ser(Psi(Me,Me)Pro)-OH currently on the market, and further leads to excessively high costs for polypeptide active pharmaceutical ingredients using this type of pseudo-dipeptide as raw material.

[0005] Therefore, a more general and easy-to-operate method for synthesizing and purifying Fmoc-Xaa-Ser(Psi(Me,Me)Pro)-OH type pseudo-dipeptide is developed to obtain Fmoc-Xaa-Ser(Psi(Me,Me)Pro)-OH pseudo-dipeptide with low impurity content and high safety, which has great application prospects and economic practical value. Summary of the invention

[0006] The purpose of the present invention is to provide a method for synthesizing a Fmoc-Xaa-Ser(Psi(Me,Me)Pro)-OH pseudo-dipeptide, so as to improve the synthesis purity, raw material conversion rate and selectivity of the Fmoc-Xaa-Ser(Psi(Me,Me)Pro)-OH pseudo-dipeptide.

[0007] The technical solution adopted by the present invention to achieve the above-mentioned purpose is: A method for synthesizing a Fmoc-Xaa-Ser(Psi(Me,Me)Pro)-OH pseudo-dipeptide, comprising: S1. Synthesizing pentafluorophenol ester by reacting Fmoc-Xaa-OH with pentafluorophenol; S2, reacting pentafluorophenol ester with H-Ser-OH in the presence of N,O-bis(trimethylsilyl)acetamide to synthesize Fmoc-Xaa-Ser-OH; S3, purification of Fmoc-Xaa-Ser-OH using R-1-phenylethylamine; S4, reacting Fmoc-Xaa-Ser-OH with methoxypropylene in the presence of a catalyst to synthesize Fmoc-Xaa-Ser(Psi(Me,Me)Pro)-OH; The Xaa is an amino acid; the catalyst includes 4-methylbenzenesulfonic acid pyrimidine, or a thiocyanate pyridine derivative; the thiocyanate pyridine derivative has a thiocyanate group and a pyridine group.

[0008] The invention uses Fmoc-Xaa-OH to react with pentafluorophenol to synthesize pentafluorophenol ester, and then reacts with H-Ser-OH under the action of N,O-bis(trimethylsilyl)acetamide to synthesize Fmoc-Xaa-Ser-OH, uses R-1-phenylethylamine to purify Fmoc-Xaa-Ser-OH, uses thiocyanate pyridine derivatives as catalysts, and uses alkyl aluminum complexes as co-catalysts to react Fmoc-Xaa-Ser-OH with methoxypropylene to synthesize Fmoc-Xaa-Ser(Psi(Me,Me)Pro)-OH pseudo dipeptide. The invention uses R-1-phenylethylamine to change the salt solubility of Fmoc-Xaa-Ser-OH in a solvent, so that the ammonium salt of Fmoc-Xaa-Ser-OH is precipitated from the solvent, and Fmoc-Xaa-Ser-OH is purified to improve the purity of Fmoc-Xaa-Ser-OH, thereby improving the purity of Fmoc-Xaa-Ser(Psi(Me,Me)Pro)-OH pseudo-dipeptide; and then uses a thiocyanate pyridine derivative as a catalyst and an alkyl aluminum complex as a co-catalyst to strengthen the reaction process and accelerate the reaction rate, thereby improving the raw material conversion rate and the target product selectivity in the process of synthesizing the Fmoc-Xaa-Ser(Psi(Me,Me)Pro)-OH pseudo-dipeptide.

[0009] Preferably, in the preparation of the thiocyanate pyridine derivative, 4,6-dichloro-5-pyrimidinecarboxaldehyde is first reacted with potassium thiocyanate and then with 3-aminomethylpyrimidine to obtain the thiocyanate pyridine derivative.

[0010] More preferably, the molar ratio of 4,6-dichloro-5-pyrimidinecarboxaldehyde to potassium thiocyanate is 1:4-8.

[0011] More preferably, the molar ratio of 4,6-dichloro-5-pyrimidinecarboxaldehyde to 3-aminomethylpyrimidine is 1:2-6.

[0012] Preferably, the preparation of thiocyanate pyridine derivatives is specifically, Weigh potassium thiocyanate and 4,6-dichloro-5-pyrimidinecarboxaldehyde, add glacial acetic acid solution, stir vigorously for 20-60 minutes, add ice water at a temperature of 2-10°C, let stand for 20-60 minutes, add 3-aminomethylpyrimidine, stir at room temperature for 12-36 hours, and remove the solvent by vacuum filtration to obtain a thiocyanate pyridine derivative.

[0013] More preferably, the molar ratio of 4,6-dichloro-5-pyrimidinecarboxaldehyde to potassium thiocyanate is 1:4-8.

[0014] More preferably, the usage ratio of 4,6-dichloro-5-pyrimidinecarboxaldehyde to glacial acetic acid is 1 mmol:1-5 mL.

[0015] More preferably, the usage ratio of 4,6-dichloro-5-pyrimidinecarboxaldehyde to ice water is 1 mmol:10-30 mL.

[0016] More preferably, the molar ratio of 4,6-dichloro-5-pyrimidinecarboxaldehyde to 3-aminomethylpyrimidine is 1:2-6.

[0017] Preferably, the mass ratio of Fmoc-Xaa-OH to pentafluorophenol is 1:0.2-1.

[0018] Preferably, the mass ratio of Fmoc-Xaa-OH to N,O-bis(trimethylsilyl)acetamide is 1:0.5-1.

[0019] Preferably, the mass ratio of H-Ser-OH to N,O-bis(trimethylsilyl)acetamide is 1:2-3.

[0020] Preferably, the mass ratio of Fmoc-Xaa-OH to R-1-phenylethylamine is 1:0.2-0.5.

[0021] Preferably, the mass ratio of Fmoc-Xaa-Ser-OH to the catalyst is 1:0.02-0.1.

[0022] Preferably, the mass ratio of Fmoc-Xaa-Ser-OH to methoxypropylene is 1:0.2-1.

[0023] Preferably, a method for synthesizing a Fmoc-Xaa-Ser(Psi(Me,Me)Pro)-OH pseudo-dipeptide comprises: S1. Weigh Fmoc-Xaa-OH, add dichloromethane to dissolve, stir at room temperature until dissolved, add pentafluorophenol, stir in an ice bath at 2-10°C, add 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride, stir to react, and detect by thin layer chromatography (TLC). After the raw materials have completely reacted, wash with saturated brine for 2-5 times, add anhydrous sodium sulfate to dry, and obtain pentafluorophenol ester.

[0024] S2. Weigh H-Ser-OH and N,O-bis(trimethylsilyl)acetamide, add N,N-dimethylformamide, stir overnight, slowly add pentafluorophenol ester obtained in step S1, use high performance liquid chromatography (HPLC) to control, after the raw materials have been completely consumed, add sodium carbonate, water and dichloromethane, add petroleum ether and stir well, take the water layer, add ethyl acetate to the aqueous phase, adjust the pH to neutral with hydrochloric acid, stir well, separate the organic phase, spin dry the solvent, and obtain Fmoc-Xaa-Ser-OH.

[0025] S3. Dissolve the Fmoc-Xaa-Ser-OH obtained in step S2 with dichloromethane, add R-1-phenylethylamine and stir, solid precipitates, and filter to obtain purified Fmoc-Xaa-Ser-OH.

[0026] S4. Add Fmoc-Xaa-Ser-OH, methoxypropylene and pyridine toluenesulfonate obtained in step S3 to tetrahydrofuran, open condensed water, replace nitrogen in the system 3 times, start heating reflux reaction for 12-24 hours, after the temperature drops to room temperature, pour the reaction solution into sodium carbonate solution, stir, wash the reaction solution with petroleum ether 2-5 times, separate the liquid to obtain the aqueous phase, add ethyl acetate to the aqueous phase, adjust the pH to neutral with hydrochloric acid, separate the liquid to obtain the organic phase, wash with saturated brine, add anhydrous sodium sulfate to dry, spin dry the solvent to obtain Fmoc-Xaa-Ser(Psi(Me,Me)Pro)-OH.

[0027] More preferably, in step S1, Fmoc-Xaa-OH is Fmoc-Ser(tBu)-OH, or Fmoc-Pro-OH.

[0028] More preferably, in step S1, the usage ratio of Fmoc-Xaa-OH to dichloromethane is 1 g: 2-10 mL.

[0029] More preferably, in step S1, the mass ratio of Fmoc-Xaa-OH to pentafluorophenol is 1:0.2-1.

[0030] More preferably, in step S1, the mass ratio of Fmoc-Xaa-OH to 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride is 1:0.2-1.

[0031] More preferably, in step S2, the mass ratio of H-Ser-OH to N,O-bis(trimethylsilyl)acetamide is 1:2-3.

[0032] More preferably, in step S2, the usage ratio of H-Ser-OH to N,N-dimethylformamide is 1 g: 2-10 mL.

[0033] More preferably, in step S2, the mass ratio of H-Ser-OH to sodium carbonate is 1:1-2.

[0034] More preferably, the usage ratio of sodium carbonate to water in step S2 is 1 g:5-15 mL.

[0035] More preferably, the volume ratio of water to petroleum ether in step S2 is 1:1-3.

[0036] More preferably, in step S2, the volume ratio of petroleum ether to ethyl acetate is 1:1-2.

[0037] More preferably, in step S3, the usage ratio of R-1-phenylethylamine and dichloromethane is 1 g:6-7 mL.

[0038] More preferably, in step S4, the mass ratio of Fmoc-Xaa-Ser-OH to methoxypropylene is 1:0.2-1.

[0039] More preferably, in step S4, the mass ratio of Fmoc-Xaa-Ser-OH to pyridine toluenesulfonate is 1:0.02-0.1.

[0040] More preferably, in step S4, the usage ratio of Fmoc-Xaa-Ser-OH to tetrahydrofuran is 1 g:5-20 mL.

[0041] More preferably, in step S4, the usage ratio of Fmoc-Xaa-Ser-OH to the sodium carbonate solution is 1 g:5-20 mL, and the concentration of the sodium carbonate solution is 0.2-1 g / mL.

[0042] More preferably, in step S4, the usage ratio of Fmoc-Xaa-Ser-OH to ethyl acetate is 1 g:5-20 mL.

[0043] Preferably, in a method for synthesizing Fmoc-Xaa-Ser(Psi(Me,Me)Pro)-OH pseudo-dipeptide, the catalyst pyridine p-toluenesulfonate is replaced with a thiocyanate pyridine derivative. The thiocyanate pyridine derivative promotes the synthesis of amide bonds between the substrate amino acid raw materials by reducing the activation energy, thereby promoting the reaction and improving the raw material conversion rate and target product selectivity in the process of synthesizing Fmoc-Xaa-Ser(Psi(Me,Me)Pro)-OH pseudo-dipeptide.

[0044] Preferably, in a method for synthesizing Fmoc-Xaa-Ser(Psi(Me,Me)Pro)-OH pseudo-dipeptide, an alkyl aluminum complex is additionally added as a co-catalyst on the basis of a thiocyanate pyridine derivative as a catalyst, and the mass ratio of the thiocyanate pyridine derivative to the alkyl aluminum complex is 1:0.5-0.1. The alkyl aluminum complex can strengthen the reaction process and further improve the raw material conversion rate and target product selectivity of the reaction.

[0045] More preferably, the preparation of the alkyl aluminum complex is specifically, Weigh 2-diphenylphosphobenzaldehyde and 2-isopropenylphenylaniline, add methanol and dichloromethane, stir and mix evenly, add p-toluenesulfonic acid, introduce nitrogen for 20-40 minutes, react at 20-30°C for 1-3 days, filter under reduced pressure, add tetrahydrofuran and lithium aluminum tetrahydride, react at 20-30°C for 1-3 days, add deionized water and sodium hydroxide solution for filtration, evaporate under reduced pressure, retain solid precipitate after filtration, add toluene and triisobutylaluminum, introduce nitrogen for 20-40 minutes, stir at 60-100°C for 6-18 hours, filter under reduced pressure, wash with n-hexane 2-5 times, filter under reduced pressure to obtain an alkyl aluminum complex.

[0046] More preferably, the molar ratio of 2-diphenylphosphobenzaldehyde to 2-isopropenylphenylaniline is 1:1-2.

[0047] More preferably, the usage ratio of 2-diphenylphosphobenzaldehyde to methanol is 1 g: 20-40 mL.

[0048] More preferably, the volume ratio of dichloromethane to methanol is 1:2-4.

[0049] More preferably, the mass ratio of 2-diphenylphosphobenzaldehyde to p-toluenesulfonic acid is 1:0.005-0.01.

[0050] More preferably, the usage ratio of 2-diphenylphosphobenzaldehyde to tetrahydrofuran is 1 mmol:5-10 mL.

[0051] More preferably, the molar ratio of 2-diphenylphosphobenzaldehyde to lithium aluminum tetrahydride is 1:2-3.

[0052] More preferably, the usage ratio of 2-diphenylphosphobenzaldehyde to deionized water is 1 g: 0.2-1 mL.

[0053] More preferably, the mass concentration of the sodium hydroxide solution is 5-15%, and the volume ratio of the sodium hydroxide solution to deionized water is 1:1-2.

[0054] More preferably, the volume ratio of tetrahydrofuran to toluene is 1:1-3.

[0055] More preferably, the volume ratio of triisobutylaluminum to toluene is 1:20-40.

[0056] The present invention uses R-1-phenylethylamine to purify Fmoc-Xaa-Ser-OH, and then uses thiocyanate pyridine derivatives as catalysts and alkyl aluminum complexes as co-catalysts to react Fmoc-Xaa-Ser-OH with methoxypropylene to synthesize Fmoc-Xaa-Ser(Psi(Me,Me)Pro)-OH, so it has the following beneficial effects: R-1-phenylethylamine changes the salt solubility of Fmoc-Xaa-Ser-OH in the solvent, so that the ammonium salt of Fmoc-Xaa-Ser-OH is precipitated from the solvent, thereby improving the purity of Fmoc-Xaa-Ser-OH; using thiocyanate pyridine derivatives as catalysts and alkyl aluminum complexes as co-catalysts can strengthen the reaction process, improve the reaction efficiency, and thus improve the raw material conversion rate and selectivity. Therefore, the present invention provides a method for synthesizing Fmoc-Xaa-Ser(Psi(Me,Me)Pro)-OH pseudo-dipeptide with high purity, high raw material conversion rate and high target product selectivity. BRIEF DESCRIPTION OF THE DRAWINGS

[0057] Figure 1 This is the chromatogram of Fmoc-Ser(tBu)-Ser(Psi(Me,Me)Pro)-OH obtained in Example 1.

[0058] Figure 2 The chromatogram of Fmoc-Pro-Ser(Psi(Me,Me)Pro)-OH obtained in Example 6 DETAILED DESCRIPTION The present invention is further described in detail below in conjunction with specific embodiments, and the examples provided are only for illustrating the present invention, rather than for limiting the scope of the present invention. The examples provided below can be used as a guide for further improvements by those of ordinary skill in the art, and do not constitute a limitation of the present invention in any way.

[0059] The experimental methods in the following examples are conventional methods unless otherwise specified. The materials, reagents, etc. used in the following examples are commercially available unless otherwise specified.

[0060] Embodiment 1: S1. Synthesis of Fmoc-Ser(tBu)-Ser(Psi(Me,Me)Pro)-OH, including: S11. Weigh Fmoc-Ser(tBu)-OH, add dichloromethane to dissolve, stir at room temperature until dissolved, add pentafluorophenol, stir in an ice bath at 5°C, add 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride, stir to react, detect by thin layer chromatography (TLC), and after the raw materials have reacted completely, wash twice with saturated brine, add anhydrous sodium sulfate to dry, and obtain pentafluorophenol ester. The dosage ratio of Fmoc-Ser(tBu)-OH and dichloromethane is 1g:5mL; the mass ratio of Fmoc-Ser(tBu)-OH and pentafluorophenol is 1:0.53; the mass ratio of Fmoc-Ser(tBu)-OH and 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride is 1:0.6.

[0061] S12, weigh H-Ser-OH and N,O-bis(trimethylsilyl)acetamide, add N,N-dimethylformamide, stir overnight, slowly add pentafluorophenol ester obtained in step S11, use high performance liquid chromatography (HPLC) to control, after the raw materials have been completely consumed, add sodium carbonate, water and dichloromethane, add petroleum ether and stir well, take the water layer, add ethyl acetate to the aqueous phase, adjust the pH to neutral with hydrochloric acid, stir well, separate the organic phase, spin dry the solvent, and obtain Fmoc-Ser(tBu)-Ser-OH. The mass ratio of H-Ser-OH and N,O-bis(trimethylsilyl)acetamide is 1:2.5; the dosage ratio of H-Ser-OH and N,N-dimethylformamide is 1g:5mL; the mass ratio of H-Ser-OH and sodium carbonate is 1:1.6; the dosage ratio of sodium carbonate and water is 1g:9mL; the volume ratio of water and petroleum ether is 1:2; the volume ratio of petroleum ether and ethyl acetate is 1:1.

[0062] S13, dissolving the Fmoc-Ser(tBu)-Ser-OH obtained in step S12 with dichloromethane, adding R-1-phenylethylamine and stirring, solid precipitated, filtered, and obtained the purified product Fmoc-Ser(tBu)-Ser-OH. The amount ratio of R-1-phenylethylamine to dichloromethane is 1 g:6.25 mL.

[0063] S14. Add Fmoc-Ser(tBu)-Ser-OH, methoxypropylene and pyridine toluenesulfonate obtained in step S13 to tetrahydrofuran, open condensed water, replace nitrogen in the system 3 times, start heating reflux reaction for 12 hours, after the temperature drops to room temperature, pour the reaction solution into sodium carbonate solution and stir, wash the reaction solution twice with petroleum ether, separate the liquid and take the aqueous phase, add ethyl acetate to the aqueous phase, adjust the pH to neutral with hydrochloric acid, separate the liquid and take the organic phase, wash with saturated brine, add anhydrous sodium sulfate to dry, spin dry the solvent to obtain Fmoc-Ser(tBu)-Ser(Psi(Me,Me)Pro)-OH. The mass ratio of Fmoc-Ser(tBu)-Ser-OH and methoxypropylene is 1:0.3; the mass ratio of Fmoc-Ser(tBu)-Ser-OH and pyridine toluenesulfonate is 1:0.053; the dosage ratio of Fmoc-Ser(tBu)-Ser-OH and tetrahydrofuran is 1g:10mL; the concentration of sodium carbonate solution is 0.35g / mL, the dosage ratio of Fmoc-Ser(tBu)-Ser-OH and sodium carbonate solution is 1g:10mL; the dosage ratio of Fmoc-Ser(tBu)-Ser-OH and ethyl acetate is 1g:10mL.

[0064] Embodiment 2: S1. Preparation of thiocyanatopyridine derivatives, comprising: Weigh potassium thiocyanate and 4,6-dichloro-5-pyrimidinecarboxaldehyde, add glacial acetic acid solution, stir vigorously for 30 minutes, add ice water at 4°C, let stand for 30 minutes, add 3-aminomethylpyrimidine, stir at room temperature for 24 hours, remove the solvent by vacuum filtration, and obtain thiocyanate pyridine derivatives. The molar ratio of 4,6-dichloro-5-pyrimidinecarboxaldehyde to potassium thiocyanate is 1:6; the dosage ratio of 4,6-dichloro-5-pyrimidinecarboxaldehyde to glacial acetic acid is 1mmol:2mL; the dosage ratio of 4,6-dichloro-5-pyrimidinecarboxaldehyde to ice water is 1mmol:20mL; the molar ratio of 4,6-dichloro-5-pyrimidinecarboxaldehyde to 3-aminomethylpyrimidine is 1:4.

[0065] S2, the synthesis of Fmoc-Ser(tBu)-Ser(Psi(Me,Me)Pro)-OH, except that pyridine p-toluenesulfonate is replaced by the thiocyanate pyridine derivative synthesized in step S1 of this embodiment, and other conditions are the same as those in Example 1.

[0066] Embodiment 3: S1. Preparation of thiocyanate pyridine derivatives, same as in Example 2.

[0067] S2. The synthesis of Fmoc-Ser(tBu)-Ser(Psi(Me,Me)Pro)-OH was carried out under the same conditions as in Example 1 except that the mass ratio of Fmoc-Ser(tBu)-Ser-OH and the thiocyanate pyridine derivative was changed to 1:0.025.

[0068] Embodiment 4: S1. Preparation of thiocyanate pyridine derivatives, same as in Example 2.

[0069] S2. Preparation of alkyl aluminum complex, comprising: Weigh 2-diphenylphosphobenzaldehyde and 2-isopropylenephenylaniline, add methanol and dichloromethane, stir and mix evenly, add p-toluenesulfonic acid, introduce nitrogen for 30 minutes, react at 25°C for 2 days, filter under reduced pressure, add tetrahydrofuran and lithium aluminum tetrahydride, react at 25°C for 2 days, add deionized water and sodium hydroxide solution for filtration, evaporate under reduced pressure, retain the solid precipitate after filtration, add toluene and triisobutylaluminum, introduce nitrogen for 30 minutes, stir at 80°C for 12 hours, filter under reduced pressure, wash with n-hexane 3 times, filter under reduced pressure to obtain an alkyl aluminum complex. The molar ratio of 2-diphenylphosphobenzaldehyde to 2-isopropenylphenylaniline is 1:1; the dosage ratio of 2-diphenylphosphobenzaldehyde to methanol is 1 g:25 mL; the volume ratio of dichloromethane to methanol is 1:3; the mass ratio of 2-diphenylphosphobenzaldehyde to p-toluenesulfonic acid is 1:0.007; the dosage ratio of 2-diphenylphosphobenzaldehyde to tetrahydrofuran is 1 mmol:6 mL; the molar ratio of 2-diphenylphosphobenzaldehyde to lithium aluminum tetrahydride is 1:2.4; the dosage ratio of 2-diphenylphosphobenzaldehyde to deionized water is 1 g:0.3 mL; the mass concentration of sodium hydroxide solution is 10%, and the volume ratio of sodium hydroxide solution to deionized water is 1:1; the volume ratio of tetrahydrofuran to toluene is 1:2; and the volume ratio of triisobutylaluminum to toluene is 1:30.

[0070] S3. Synthesis of Fmoc-Ser(tBu)-Ser(Psi(Me,Me)Pro)-OH, including: S31. Weigh Fmoc-Ser(tBu)-OH, add dichloromethane to dissolve, stir at room temperature until dissolved, add pentafluorophenol, stir in an ice bath at 5°C, add 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride, stir to react, detect by thin layer chromatography (TLC), and after the raw materials have reacted completely, wash twice with saturated brine, add anhydrous sodium sulfate to dry, and obtain pentafluorophenol ester. The dosage ratio of Fmoc-Ser(tBu)-OH and dichloromethane is 1g:5mL; the mass ratio of Fmoc-Ser(tBu)-OH and pentafluorophenol is 1:0.53; the mass ratio of Fmoc-Ser(tBu)-OH and 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride is 1:0.6.

[0071] S32. Weigh H-Ser-OH and N,O-bis(trimethylsilyl)acetamide, add N,N-dimethylformamide, stir overnight, slowly add pentafluorophenol ester obtained in step S31, and use high performance liquid chromatography (HPLC) to control. After the raw materials have been completely consumed, add sodium carbonate, water and dichloromethane, add petroleum ether and stir well, take the water layer, add ethyl acetate to the aqueous phase, adjust the pH to neutral with hydrochloric acid, stir well, separate the organic phase, dry the solvent, and obtain Fmoc-Ser(tBu)-Ser-OH. The mass ratio of H-Ser-OH and N,O-bis(trimethylsilyl)acetamide is 1:2.5; the dosage ratio of H-Ser-OH and N,N-dimethylformamide is 1g:5mL; the mass ratio of H-Ser-OH and sodium carbonate is 1:1.6; the dosage ratio of sodium carbonate and water is 1g:9mL; the volume ratio of water and petroleum ether is 1:2; the volume ratio of petroleum ether and ethyl acetate is 1:1.

[0072] S33, dissolving the Fmoc-Ser(tBu)-Ser-OH obtained in step S32 with dichloromethane, adding R-1-phenylethylamine and stirring, solid precipitates, and filtering to obtain the purified product Fmoc-Ser(tBu)-Ser-OH. The amount ratio of R-1-phenylethylamine to dichloromethane is 1 g:6.25 mL.

[0073] S34. Add Fmoc-Ser(tBu)-Ser-OH, methoxypropylene, thiocyanate pyridine derivative and alkyl aluminum complex obtained in step S33 to tetrahydrofuran, open condensed water, replace nitrogen in the system 3 times, start heating reflux reaction for 12 hours, after the temperature drops to room temperature, pour the reaction solution into sodium carbonate solution and stir, wash the reaction solution twice with petroleum ether, separate the aqueous phase, add ethyl acetate to the aqueous phase, adjust the pH to neutral with hydrochloric acid, separate the organic phase, wash with saturated brine, add anhydrous sodium sulfate to dry, spin-dry the solvent to obtain Fmoc-Ser(tBu)-Ser(Psi(Me,Me)Pro)-OH. The mass ratio of Fmoc-Ser(tBu)-Ser-OH and methoxypropylene is 1:0.3; the mass ratio of Fmoc-Ser(tBu)-Ser-OH and thiocyanate pyridine derivative is 1:0.053; the mass ratio of thiocyanate pyridine derivative and alkyl aluminum complex is 1:0.1; the amount ratio of Fmoc-Ser(tBu)-Ser-OH and tetrahydrofuran is 1g:10mL; the concentration of sodium carbonate solution is 0.35g / mL, the amount ratio of Fmoc-Ser(tBu)-Ser-OH and sodium carbonate solution is 1g:10mL; the amount ratio of Fmoc-Ser(tBu)-Ser-OH and ethyl acetate is 1g:10mL.

[0074] Embodiment 5: S1. Preparation of thiocyanate pyridine derivatives, same as in Example 2.

[0075] S2. Preparation of alkyl aluminum complex, same as Example 4.

[0076] S3. The synthesis of Fmoc-Ser(tBu)-Ser(Psi(Me,Me)Pro)-OH was carried out under the same conditions as in Example 4 except that the mass ratio of the thiocyanate pyridine derivative and the alkyl aluminum complex was changed to 1:0.05.

[0077] Embodiment 6: S1. Synthesis of Fmoc-Pro-Ser(Psi(Me,Me)Pro)-OH. Except for replacing Fmoc-Ser(tBu)-OH with Fmoc-Pro-OH•H2O in step S11, other conditions are the same as those in Example 1.

[0078] Embodiment 7: S1. Preparation of thiocyanate pyridine derivatives, same as in Example 2.

[0079] S2. Synthesis of Fmoc-Pro-Ser(Psi(Me,Me)Pro)-OH. Except for replacing pyridine p-toluenesulfonate with the thiocyanate pyridine derivative synthesized in step S1 of this example, other conditions are the same as those in Example 6.

[0080] Embodiment 8: S1. Preparation of thiocyanate pyridine derivatives, same as in Example 2.

[0081] S2. The synthesis of Fmoc-Pro-Ser(Psi(Me,Me)Pro)-OH was carried out under the same conditions as in Example 6 except that the mass ratio of Fmoc-Pro-Ser-OH to the thiocyanate pyridine derivative was changed to 1:0.025.

[0082] Embodiment 9: S1. Preparation of thiocyanate pyridine derivatives, same as in Example 2.

[0083] S2. Preparation of alkyl aluminum complex, same as Example 4.

[0084] S3. Synthesis of Fmoc-Pro-Ser(Psi(Me,Me)Pro)-OH. Except for replacing Fmoc-Ser(tBu)-OH with Fmoc-Pro-OH•H2O in step S31, other conditions are the same as those in Example 4.

[0085] Embodiment 10: S1. Preparation of thiocyanate pyridine derivatives, same as in Example 3.

[0086] S2. Preparation of alkyl aluminum complex, same as Example 5.

[0087] S3. The synthesis of Fmoc-Pro-Ser(Psi(Me,Me)Pro)-OH was carried out under the same conditions as in Example 9 except that the mass ratio of the thiocyanate pyridine derivative and the alkyl aluminum complex was changed to 1:0.05.

[0088] Comparative Example 1: S1. Preparation of alkyl aluminum complex, same as Example 4.

[0089] S2. The synthesis of Fmoc-Ser(tBu)-Ser(Psi(Me,Me)Pro)-OH was carried out under the same conditions as in Example 4 except that no thiocyanate pyridine derivative was added.

[0090] Comparative Example 2: S1. Preparation of alkyl aluminum complex, same as Example 4.

[0091] S2. The synthesis of Fmoc-Pro-Ser(Psi(Me,Me)Pro)-OH was carried out under the same conditions as in Example 9 except that no thiocyanate pyridine derivative was added.

[0092] Experimental example: 1. Material Characterization Fmoc-Ser(tBu)-Ser(Psi(Me,Me)Pro)-OH synthesized in Example 1 and Fmoc-Pro-Ser(Psi(Me,Me)Pro)-OH synthesized in Example 6 were collected and identified by RP-HPLC. The chromatographic conditions were as follows: 1525 HPLC system; Jupiter C18 chromatographic column (250 mm×4.6 mm); Waters 1489 UV detector, wavelength 220 nm; mobile phase A: 0.1% trifluoroacetic acid solution; mobile phase B: 0.1% trifluoroacetic acid-acetonitrile solution; flow rate 1 mL / min; sample mass concentration 10 mg / mL; injection volume 20 μL; linear elution gradient: 0-2 min, 5%A; 2-10 min, 5%-15%A; 10-12 min, 15%-5%A.

[0093] According to the RP-HPLC results, the purity of the product Fmoc-Ser(tBu)-Ser-OH and the final product Fmoc-Ser(tBu)-Ser(Psi(Me,Me)Pro)-OH synthesized in Example 1 after purification by R-1-phenylethylamine, and the purity of the product Fmoc-Pro-Ser-OH and the final product Fmoc-Pro-Ser(Psi(Me,Me)Pro)-OH synthesized in Example 6 after purification by R-1-phenylethylamine were calculated. Purity (%) = (target protein peak area / target protein peak height) / (total peak area / total peak height) × 100%.

[0094] Figure 1 The chromatogram of Fmoc-Ser(tBu)-Ser(Psi(Me,Me)Pro)-OH obtained in Example 1, the retention time of Fmoc-Ser(tBu)-Ser(Psi(Me,Me)Pro)-OH is 24.843 min; Figure 2 The chromatogram of Fmoc-Pro-Ser(Psi(Me,Me)Pro)-OH obtained in Example 6 shows that the retention time of Fmoc-Pro-Ser(Psi(Me,Me)Pro)-OH is 18.732 min. This indicates that the method of the present invention can synthesize Fmoc-Xaa-Ser(Psi(Me,Me)Pro)-OH.

[0095] The purity of the product Fmoc-Ser(tBu)-Ser-OH obtained by Example 1 after purification by R-1-phenylethylamine was 99.1%, and the purity of the final product Fmoc-Ser(tBu)-Ser(Psi(Me,Me)Pro)-OH was 98.7%; the purity of the product Fmoc-Pro-Ser-OH obtained by Example 6 after purification by R-1-phenylethylamine was 98.6%, and the purity of the final product Fmoc-Pro-Ser(Psi(Me,Me)Pro)-OH was 99.1%. This shows that the method of the present invention can obtain high-purity intermediate product Fmoc-Xaa-Ser-OH and final product Fmoc-Xaa-Ser(Psi(Me,Me)Pro)-OH.

[0096] 3. Determination of raw material conversion rate The intermediate product Fmoc-Xaa-Ser-OH purified by R-1-phenylethylamine was prepared according to the methods of Examples 1-9 and Comparative Examples 1-2, and the content of Fmoc-Xaa-Ser-OH was recorded. Fmoc-Xaa-Ser-OH was used as a raw material, and Fmoc-Xaa-Ser(Psi(Me,Me)Pro)-OH was prepared according to the methods of Examples 1-9 and Comparative Examples 1-2, and the raw material conversion rate of Fmoc-Xaa-Ser-OH was calculated. Raw material conversion rate (%) = (initial raw material content - product content) / product content × 100%. The measurement results are shown in Tables 1 and 2.

[0097] Table 1 Raw material conversion rate of Fmoc-Ser(tBu)-Ser-OH (%)

[0098] As can be seen from Table 1, the raw material conversion rate of Example 2-3 is higher than that of Example 1 because, in the synthesis of Fmoc-Ser(tBu)-Ser(Psi(Me,Me)Pro)-OH, Example 1 uses pyridine p-toluenesulfonate as a catalyst, while Example 2-3 uses a thiocyanate pyridine derivative to replace pyridine p-toluenesulfonate; the raw material conversion rate of Example 2 is higher than that of Example 3 because, in the synthesis of Fmoc-Ser(tBu)-Ser(Psi(Me,Me)Pro)-OH, Example 3 reduces the amount of thiocyanate pyridine derivatives used. This shows that the use of thiocyanate pyridine derivatives as catalysts can effectively improve the raw material conversion rate of Fmoc-Ser(tBu)-Ser(Psi(Me,Me)Pro)-OH. The raw material conversion rate of Example 4-5 is higher than that of Example 2 and Comparative Example 1 because in the synthesis of Fmoc-Ser(tBu)-Ser(Psi(Me,Me)Pro)-OH, Examples 4-5 not only use thiocyanate pyridine derivatives to replace pyridine p-toluenesulfonate as a catalyst, but also use an alkyl aluminum complex as a co-catalyst; Example 2 only uses thiocyanate pyridine derivatives, and Comparative Example 1 only uses an alkyl aluminum complex. The raw material conversion rate of Example 4 is higher than that of Example 5 because in the synthesis of Fmoc-Ser(tBu)-Ser(Psi(Me,Me)Pro)-OH, Example 5 reduces the amount of alkyl aluminum complex used. This shows that in the synthesis of Fmoc-Ser(tBu)-Ser(Psi(Me,Me)Pro)-OH, further using an alkyl aluminum complex as a co-catalyst can further improve the raw material conversion rate of Fmoc-Ser(tBu)-Ser(Psi(Me,Me)Pro)-OH.

[0099] Table 2 Raw material conversion rate of Fmoc-Pro-Ser-OH (%)

[0100] As can be seen from Table 2, the raw material conversion rate of Example 7-8 is higher than that of Example 6 because, in the synthesis of Fmoc-Pro-Ser(Psi(Me,Me)Pro)-OH, Example 6 uses pyridine p-toluenesulfonate as a catalyst, while Examples 7-8 use thiocyanate pyridine derivatives to replace pyridine p-toluenesulfonate; the raw material conversion rate of Example 7 is higher than that of Example 8 because, in the synthesis of Fmoc-Pro-Ser(Psi(Me,Me)Pro)-OH, Example 8 reduces the amount of thiocyanate pyridine derivatives used. This shows that the use of thiocyanate pyridine derivatives as catalysts can effectively improve the raw material conversion rate of Fmoc-Pro-Ser(Psi(Me,Me)Pro)-OH. The raw material conversion rate of Examples 9-10 is higher than that of Examples 7 and Comparative Example 1 because in the synthesis of Fmoc-Pro-Ser(Psi(Me,Me)Pro)-OH, Examples 9-10 not only use thiocyanate pyridine derivatives to replace pyridine p-toluenesulfonate as a catalyst, but also use an alkyl aluminum complex as a co-catalyst; Example 7 only uses thiocyanate pyridine derivatives, and Comparative Example 1 only uses alkyl aluminum complexes. The raw material conversion rate of Example 9 is higher than that of Example 10 because in the synthesis of Fmoc-Pro-Ser(Psi(Me,Me)Pro)-OH, Example 10 reduces the amount of alkyl aluminum complex used. This shows that in the synthesis of Fmoc-Pro-Ser(Psi(Me,Me)Pro)-OH, further using an alkyl aluminum complex as a co-catalyst can further improve the raw material conversion rate of Fmoc-Pro-Ser(Psi(Me,Me)Pro)-OH.

[0101] 3. Determination of the selectivity of Fmoc-Xaa-Ser(Psi(Me,Me)Pro)-OH Fmoc-Xaa-Ser(Psi(Me,Me)Pro)-OH was prepared according to the methods of Examples 1-9 and Comparative Examples 1-2, respectively, and the selectivity of Fmoc-Xaa-Ser(Psi(Me,Me)Pro)-OH was calculated. Selectivity (%) = Fmoc-Xaa-Ser(Psi(Me,Me)Pro)-OH content in the product / total content of the product × 100%. The measurement results are shown in Tables 3 and 4.

[0102] Table 3 Selectivity of Fmoc-Ser(tBu)-Ser(Psi(Me,Me)Pro)-OH (%)

[0103] As can be seen from Table 3, the selectivity of Example 2-3 is higher than that of Example 1 because, in the synthesis of Fmoc-Ser(tBu)-Ser(Psi(Me,Me)Pro)-OH, Example 1 uses pyridine p-toluenesulfonate as a catalyst, while Example 2-3 uses a thiocyanate pyridine derivative to replace pyridine p-toluenesulfonate; the selectivity of Example 2 is higher than that of Example 3 because, in the synthesis of Fmoc-Ser(tBu)-Ser(Psi(Me,Me)Pro)-OH, Example 3 reduces the amount of thiocyanate pyridine derivative used. This shows that the use of thiocyanate pyridine derivatives as catalysts can effectively improve the selectivity of Fmoc-Ser(tBu)-Ser(Psi(Me,Me)Pro)-OH. The selectivity of Example 4-5 is higher than that of Example 2 and Comparative Example 1 because in the synthesis of Fmoc-Ser(tBu)-Ser(Psi(Me,Me)Pro)-OH, Examples 4-5 not only use thiocyanate pyridine derivatives to replace pyridine p-toluenesulfonate as a catalyst, but also use an alkyl aluminum complex as a co-catalyst; Example 2 only uses thiocyanate pyridine derivatives, and Comparative Example 1 only uses an alkyl aluminum complex. The selectivity of Example 4 is higher than that of Example 5 because in the synthesis of Fmoc-Ser(tBu)-Ser(Psi(Me,Me)Pro)-OH, Example 5 reduces the amount of alkyl aluminum complex used. This shows that in the synthesis of Fmoc-Ser(tBu)-Ser(Psi(Me,Me)Pro)-OH, further using an alkyl aluminum complex as a co-catalyst can further improve the selectivity of Fmoc-Ser(tBu)-Ser(Psi(Me,Me)Pro)-OH.

[0104] Table 4 Selectivity of Fmoc-Pro-Ser(Psi(Me,Me)Pro)-OH

[0105] As can be seen from Table 4, the selectivity of Example 7-8 is higher than that of Example 6 because, in the synthesis of Fmoc-Pro-Ser(Psi(Me,Me)Pro)-OH, Example 6 uses pyridine p-toluenesulfonate as a catalyst, while Examples 7-8 use thiocyanate pyridine derivatives to replace pyridine p-toluenesulfonate; the selectivity of Example 7 is higher than that of Example 8 because, in the synthesis of Fmoc-Pro-Ser(Psi(Me,Me)Pro)-OH, Example 8 reduces the amount of thiocyanate pyridine derivatives used. This shows that the use of thiocyanate pyridine derivatives as catalysts can effectively improve the selectivity of Fmoc-Pro-Ser(Psi(Me,Me)Pro)-OH. The selectivity of Examples 9-10 is higher than that of Examples 7 and Comparative Example 1 because, in the synthesis of Fmoc-Pro-Ser(Psi(Me,Me)Pro)-OH, Examples 9-10 not only use thiocyanate pyridine derivatives to replace pyridine p-toluenesulfonate as a catalyst, but also use an alkyl aluminum complex as a co-catalyst; Example 7 only uses thiocyanate pyridine derivatives, and Comparative Example 1 only uses an alkyl aluminum complex. The selectivity of Example 9 is higher than that of Example 10 because, in the synthesis of Fmoc-Pro-Ser(Psi(Me,Me)Pro)-OH, Example 10 reduces the amount of alkyl aluminum complex used. This shows that in the synthesis of Fmoc-Pro-Ser(Psi(Me,Me)Pro)-OH, further using an alkyl aluminum complex as a co-catalyst can further improve the selectivity of Fmoc-Pro-Ser(Psi(Me,Me)Pro)-OH.

[0106] The conventional operations in the operating steps of the present invention are well known to those skilled in the art and will not be described in detail here.

[0107] The embodiments described above provide a detailed description of the technical solutions of the present invention. It should be understood that the above are only specific embodiments of the present invention and are not intended to limit the present invention. Any modifications, supplements or similar substitutions made within the scope of the principles of the present invention should be included in the protection scope of the present invention.

Claims

1. A method for synthesizing a pseudo-dipeptide of Fmoc-Xaa-Ser(Psi(Me,Me)Pro)-OH, comprising: S1. Synthesizing pentafluorophenol ester by reacting Fmoc-Xaa-OH with pentafluorophenol; S2, reacting pentafluorophenol ester with H-Ser-OH in the presence of N,O-bis(trimethylsilyl)acetamide to synthesize Fmoc-Xaa-Ser-OH; S3, purification of Fmoc-Xaa-Ser-OH using R-1-phenylethylamine; S4, reacting Fmoc-Xaa-Ser-OH with methoxypropylene in the presence of a catalyst to synthesize Fmoc-Xaa-Ser(Psi(Me,Me)Pro)-OH; The Xaa is an amino acid; the catalyst includes 4-methylbenzenesulfonic acid pyrimidine, or a thiocyanate pyridine derivative; the thiocyanate pyridine derivative has a thiocyanate group and a pyridine group.

2. A method for synthesizing a Fmoc-Xaa-Ser(Psi(Me,Me)Pro)-OH pseudo-dipeptide according to claim 1, characterized in that: In the preparation of the thiocyanate pyridine derivative, 4,6-dichloro-5-pyrimidinecarboxaldehyde is first reacted with potassium thiocyanate and then with 3-aminomethylpyrimidine to obtain the thiocyanate pyridine derivative.

3. A method for synthesizing a Fmoc-Xaa-Ser(Psi(Me,Me)Pro)-OH pseudo-dipeptide according to claim 2, characterized in that: The molar ratio of the 4,6-dichloro-5-pyrimidinecarboxaldehyde to potassium thiocyanate is 1:4-8.

4. A method for synthesizing a Fmoc-Xaa-Ser(Psi(Me,Me)Pro)-OH pseudo-dipeptide according to claim 2, characterized in that: The molar ratio of the 4,6-dichloro-5-pyrimidinecarboxaldehyde to 3-aminomethylpyrimidine is 1:2-6.

5. The method for synthesizing a Fmoc-Xaa-Ser(Psi(Me,Me)Pro)-OH pseudo-dipeptide according to claim 1, characterized in that: The mass ratio of the Fmoc-Xaa-OH to pentafluorophenol is 1:0.2-1.

6. A method for synthesizing a Fmoc-Xaa-Ser(Psi(Me,Me)Pro)-OH pseudo-dipeptide according to claim 1, characterized in that: The mass ratio of the Fmoc-Xaa-OH to N,O-bis(trimethylsilyl)acetamide is 1:0.5-1.

7. A method for synthesizing a Fmoc-Xaa-Ser(Psi(Me,Me)Pro)-OH pseudo-dipeptide according to claim 1, characterized in that: The mass ratio of the H-Ser-OH to N,O-bis(trimethylsilyl)acetamide is 1:2-3.

8. A method for synthesizing a Fmoc-Xaa-Ser(Psi(Me,Me)Pro)-OH pseudo-dipeptide according to claim 1, characterized in that: The mass ratio of the Fmoc-Xaa-OH to R-1-phenylethylamine is 1:0.2-0.

5.

9. The method for synthesizing a Fmoc-Xaa-Ser(Psi(Me,Me)Pro)-OH pseudo-dipeptide according to claim 1, characterized in that: The mass ratio of the Fmoc-Xaa-Ser-OH to the catalyst is 1:0.02-0.

1.

10. The method for synthesizing a Fmoc-Xaa-Ser(Psi(Me,Me)Pro)-OH pseudo-dipeptide according to claim 1, characterized in that: The mass ratio of the Fmoc-Xaa-Ser-OH to methoxypropylene is 1:0.2-1.

Images