A method for preparing an L-glutamic acid as an N-terminal polypeptide

Abstract

This invention discloses a method for preparing a polypeptide with L-glutamic acid as the N-terminus, belonging to the field of polypeptide synthesis. The method includes the following steps: ... N -Benzyloxycarbonyl- L The cyclization reaction of glutamic acid protects its carboxyl group. The carboxyl group of the cyclized product is activated using NHS and DCC, and then reacted with different amino acids to obtain a dipeptide. The carboxyl group of the dipeptide is activated again using NHS and DCC, and then reacted with different amino acids to obtain a tripeptide. The ester group of the obtained dipeptide or tripeptide is hydrolyzed to open the ring, yielding a Cbz-protected dipeptide or tripeptide. The Cbz protecting group in the short peptide is removed using trifluoroacetic acid and a reducing agent to obtain the target dipeptide and tripeptide. Compared with existing technologies, the synthesis method provided by this invention has simple operation steps, high raw material conversion rate, high safety in the preparation process, effectively reduces production costs, and is suitable for industrial-scale production, providing a feasible path for the efficient preparation of short peptides.

Description

Technical Field

[0001] This invention relates to the field of polypeptide synthesis, and more particularly to a method for preparing polypeptides with L-glutamic acid as the N-terminus. Background Technology

[0002] Glutamic acid (Glu), an essential functional amino acid, is widely involved in various physiological processes such as nerve regulation, energy metabolism, intestinal mucosal repair, and skin barrier construction, and has irreplaceable potential application value in the fields of medicine, food, and cosmetics. Using glutamic acid as the N-terminal amino acid, functional short peptides can be formed by linking 1-2 amino acids through peptide bonds: Glu-X type dipeptides formed by linking one amino acid (X), or Glu-XY type tripeptides formed by linking two amino acids (X, Y). These peptides combine the inherent physiological activity of glutamic acid with the structural advantages of small-molecule short peptides. They have small molecular weight, excellent water solubility, high bioavailability, outstanding transdermal performance, strong stability, and low immunogenicity. Functional modular design can be achieved through flexible selection of C-terminal amino acid units (X, Y), showing broad application prospects in multiple fields of medicine, food, and cosmetics.

[0003] In the pharmaceutical field, Glu-X dipeptides and Glu-XY tripeptides play important regulatory roles in neuroprotection, gastrointestinal mucosal repair, liver protection, and immune regulation. They are used as adjunctive interventions or treatments for central nervous system diseases, gastrointestinal diseases, liver metabolic abnormalities, and immunodeficiency. Simultaneously, these short peptides can act as calcium-sensing receptor (CaSR) activators, such as γ-... L -Glutamine- L -Alanine (γ-) L -Glutamyl- L Short peptides containing γ-glutamyl-L-valine and gamma-glutamyl-L-valine are essential for maintaining stable blood calcium levels, which is crucial for human life. Abnormal function of these peptides can lead to various diseases. Therefore, these short peptides have significant potential applications in regulating blood calcium homeostasis and intervening in related diseases. In addition, these short peptides can serve as drug delivery carriers, effectively improving the solubility and membrane permeability of poorly soluble drugs, enhancing targeted drug delivery efficiency, and reducing drug toxicity and side effects. They have significant value in the research and development of pharmaceutical excipients.

[0004] In the food industry, Glu-X dipeptides and Glu-XY tripeptides can be used as natural functional nutritional fortifiers in sports nutrition foods, special medical foods, and functional foods for the middle-aged and elderly to achieve functions such as anti-fatigue, regulating intestinal health, and enhancing the body's immunity. At the same time, these short peptides are heat-resistant and acid- and alkali-resistant, and can be used as natural umami peptides to replace traditional chemical flavorings in various food processing to improve food flavor and product quality. They can also be used as natural antioxidant additives to delay food oxidation and deterioration and extend the shelf life of food.

[0005] In the cosmetics industry, Glu-X dipeptide and Glu-XY tripeptide can be used as high-end effective active ingredients, exerting effects such as moisturizing and water-locking, skin barrier repair, anti-oxidation, anti-photoaging, whitening and brightening, and anti-inflammatory and soothing. They are widely used in repair cosmetics, anti-aging cosmetics, whitening cosmetics, and post-medical aesthetic repair products. At the same time, their low allergenicity and easy transdermal absorption characteristics are significantly superior to traditional cosmetic active ingredients, and they have extremely high commercial conversion value in the cosmetics industry.

[0006] Currently, although there are some studies on the synthesis of glutamate-derived peptides in existing technologies, the synthesis processes for Glu-X dipeptides and Glu-XY tripeptides still have many shortcomings, making it difficult to meet the needs of large-scale preparation. The specific defects are as follows: First, the synthesis steps are cumbersome, and the introduction and removal of protecting groups are complicated, resulting in low synthesis efficiency and low overall yield of the target product, which is difficult to meet the needs of industrial-scale production; Second, there are many side reactions during the synthesis process, especially the easy formation of peptide bonds with non-target configurations, resulting in insufficient purity of the target peptide, affecting its physiological activity and application effects. Third, existing synthesis processes are highly specific and difficult to adapt to the general synthesis of Glu-X dipeptides and Glu-XY tripeptides with different amino acid combinations (X, Y). Furthermore, they cannot simultaneously meet the multi-scenario application requirements of pharmaceutical-grade purity, food-grade safety, and cosmetic-grade stability. Fourth, some synthesis processes use highly toxic reagents, posing environmental hazards. Moreover, most processes for removing the Cbz protecting group use palladium on carbon / hydrogen conditions, which have a high potential explosion risk. In addition, palladium on carbon is expensive, further increasing production costs and thus limiting the industrialization and application of this type of short peptide.

[0007] In summary, current synthesis technologies for Glu-X dipeptides and Glu-XY tripeptides still have significant shortcomings, failing to meet the demands for large-scale, cost-effective, and multi-scenario applications in the pharmaceutical, food, and cosmetic fields. Therefore, there is an urgent need to develop an efficient, simple, highly selective, cost-controllable, and widely applicable method for synthesizing Glu-X dipeptides or Glu-XY tripeptides. This would fill the technological gaps, address many shortcomings of existing synthesis processes, and have significant practical and industrial value in promoting the industrial development and application of these multifunctional bioactive peptides. Summary of the Invention

[0008] To address the shortcomings of existing technologies, this invention provides a glutamine polypeptide (especially γ-glutamyl peptide) with significant medicinal value. L -Glutamine dipeptide or γ- LAn industrial-scale preparation method for glutamyl tripeptides is presented. These peptides have been shown to possess key pharmacological effects such as regulating calcium-sensitive receptors (CaSRs), and are potential drug candidates for neuroprotection, gastrointestinal mucosal repair, liver protection, and immune modulation. The preparation method provided by this invention solves the technical challenges of high cost and difficulty in large-scale production of active peptides in existing synthetic techniques.

[0009] To solve the above-mentioned technical problems, the technical solution of the present invention is as follows: a method for preparing a polypeptide with L-glutamic acid as the N-terminus, the method comprising the step of synthesizing compound C, the synthetic route and method of compound C being as follows: ; Compound B undergoes a carboxyl activation reaction with NHS and DCC. After the reaction, the mixture is filtered, and the solvent is removed from the filtrate. The filtrate is then dissolved in an aprotic solvent to obtain a solution of compound B-OSu. The compound B-OSu solution is added dropwise to a suspension of amino acids, organic bases, and water to carry out an amide condensation reaction. After the reaction, the solvent is removed, the aqueous phase is extracted, the pH is adjusted, and the mixture is extracted again to remove the solvent and obtain compound C.

[0010] As a further description of the above scheme: the polypeptide is a dipeptide γ-Glu-X, a tripeptide γ-Glu-XY, or a polypeptide composed of three or more amino acid residues; When the polypeptide is a dipeptide γ-Glu-X, its preparation process is as follows: Compound C is subjected to a ring-opening reaction and a Cbz protecting group removal reaction to obtain dipeptide γ-Glu-X; When the polypeptide is a tripeptide γ-Glu-XY, its preparation process and synthetic route are as follows: ; In the synthesis route, R2 and R1 may be the same or different; Compound C undergoes a carboxyl activation reaction with NHS and DCC. After the reaction, the mixture is filtered, and the solvent is removed from the filtrate. The filtrate is then dissolved in an aprotic solvent to obtain a solution of compound C-OSu. The C-OSu solution is added dropwise to a suspension of amino acids, organic base, and water to carry out an amide condensation reaction. After the reaction, the solvent is removed, the aqueous phase is extracted, the pH is adjusted, and the mixture is extracted again to remove the solvent and obtain compound E. Compound E is then subjected to a ring-opening reaction and a Cbz-protecting group removal reaction to prepare the tripeptide γ-Glu-XY. When the polypeptide is a polypeptide composed of three or more amino acid residues, its preparation process is as follows: After the compound E undergoes a carboxyl activation reaction, it continues to undergo an amide condensation reaction with one or more amino acids in sequence until the target peptide chain length is obtained. Finally, the compound M obtained undergoes a ring-opening reaction and a Cbz-removal protecting group removal reaction to obtain a polypeptide composed of three or more amino acid residues.

[0011] As a further description of the above scheme: the operation methods of the ring-opening reaction and the deprotection reaction of Cbz are as follows: Ring-opening reaction: The compound to be ring-opened reacts with a strong base. After the reaction, the solvent is removed, the aqueous phase is extracted, the pH is adjusted, and the solvent is removed to obtain the ring-opened compound. Deprotection reaction: The ring-opening compound undergoes a deprotection reaction with trifluoroacetic acid and a reducing agent. After the reaction, the solvent is removed, the aqueous phase is extracted and retained, and the solvent is removed again to obtain the corresponding polypeptide.

[0012] As a further description of the above scheme, the synthetic route and method of compound B are as follows: ; Compound A N -Benzyloxycarbonyl- L - Glutamic acid and paraformaldehyde undergo a cyclization reaction catalyzed by p-toluenesulfonic acid monohydrate. After the reaction, the solvent is removed, the mixture is extracted, and the organic phase is removed to obtain compound B.

[0013] As a further description of the above scheme: the amino acid is selected from valine, valine, alanine, leucine, methionine, phenylalanine, glycine, isoleucine, tryptophan, histidine, glutamine, aspartic acid-4-tert-butyl ester, arginine, cysteine, glutamine, N (e)-Boc- L -Lysine, asparagine, serine, tyrosine, threonine, or proline; The general structural formula of the amino acid is H2N-CH(R1)-COOH or , where R1 represents the side chain group of the amino acid excluding the amino and carboxyl groups, and R1 specifically corresponds to the following: n-propyl for valine, isopropyl for valine, methyl for alanine, isobutyl for leucine, methylthioethyl for methionine, benzyl for phenylalanine, hydrogen for glycine, isopentyl for isoleucine, 3-indolemethyl for tryptophan, and 1-... H -Imidazol-4-ylmethyl, corresponding to glutamine as carbamoylethyl, to aspartic acid-4-tert-butyl ester as tert-butoxycarbonylmethyl, to arginine as 3-guanidinopropyl, and to cysteine ​​as mercaptomethyl. N (e)-Boc- L The form is 4-(tert-butoxycarbonylamino)butyl for lysine, carbamoylmethyl for asparagine, hydroxymethyl for serine, 4-hydroxybenzyl for tyrosine, 1-hydroxyethyl for threonine, and tetrahydropyrrole-2-yl for proline.

[0014] Preferably, the solvent for the carboxyl activation reaction is 1,4-dioxane, tetrahydrofuran, acetonitrile, dichloromethane, etc. N,N - Dimethylformamide (DMF) or dimethyl sulfoxide (DMSO); the molar ratio of the compound to be activated to NHS and DCC is 1:1.0~3.0:1.0~4.0, and the compound to be activated is compound B, compound C, compound E or compound M; The aprotic solvent is selected from tetrahydrofuran, acetonitrile, etc. N,N - Dimethylformamide (DMF) or dimethyl sulfoxide (DMSO); The concentration of the compound B-OSu or C-OSu solution is 1.0~4.0 mmol / mL; The organic base refers to triethylamine or N,N -Diisopropylethylamine; the temperature of the amide condensation reaction is 0~30℃; the reaction time is 2h~15h.

[0015] Preferably, the solvent for the ring-opening reaction is methanol / water, ethanol / water, isopropanol / water, tetrahydrofuran / water, or acetonitrile / water; the strong base is sodium hydroxide, potassium hydroxide, or lithium hydroxide; the temperature of the ring-opening reaction is 0~30℃; the reaction time is 1~6h; and the molar ratio between the compound to be ring-opened and the strong base is 1:2.0~4.0.

[0016] Preferably, the solvent for the deprotection reaction of the Cbz protecting group is dichloromethane, 1,2-dichloroethane, tetrahydrofuran, toluene, or 1,4-dioxane; The reducing agent refers to triethylsilane or triisopropylsilane; The temperature for the deprotection reaction of the Cbz group is 40~80℃; the reaction time is 2~16h; and the molar ratio of the ring-opening compound, trifluoroacetic acid and reducing agent is 1:20~40:10~20.

[0017] Preferably, the pH is adjusted to 0.5-2; the solvent for the cyclization reaction is toluene, xylene, cyclohexane, or n-heptane. The molar ratio of compound A, paraformaldehyde, and p-toluenesulfonic acid monohydrate is 1:1-2:0.008-0.02.

[0018] The present invention also provides the use of the polypeptide in the preparation of polypeptide drugs or health care products.

[0019] As a further optimization of the above scheme, the inventors, through numerous experiments, developed a technical route for preparing γ-Glu-X dipeptides and γ-Glu-XY tripeptides with dual medicinal and edible functions. The process is simple, easy to operate, and yields high results. All reagents used are commonly available and can be prepared at the gram level in the laboratory. The specific method and route are as follows.

[0020]

[0021] The preparation methods for dipeptides and tripeptides include the following steps: Step 1: Compound A undergoes a cyclization reaction with paraformaldehyde and p-toluenesulfonic acid monohydrate using toluene, xylene, cyclohexane, or n-heptane as a solvent. Toluene is removed by concentration under reduced pressure. The mixture is then extracted with ethyl acetate and water, and the organic phase is evaporated to dryness to obtain compound B, which can be used directly in the next step without further purification. The cyclization reaction temperature is 100-120℃; the reaction time is 2-12 hours; and the solvent is any one of toluene, xylene, cyclohexane, or n-heptane. Step 2: Compound B is prepared using 1,4-dioxane, tetrahydrofuran, acetonitrile, dichloromethane, N,N Using dimethylformamide (DMF) or dimethyl sulfoxide (DMSO) as solvents, a carboxyl activation reaction occurs with NHS and DCC. After filtration, the filtrate is evaporated to dryness and then treated with tetrahydrofuran, acetonitrile, ... N,N A solution of compound B-OSu was obtained by dissolving it in dimethylformamide (DMF) or dimethyl sulfoxide (DMSO); the solution of compound B-OSu was added dropwise to a suspension of amino acids, organic base, and water to carry out an amide condensation reaction; the solvent was removed by concentration under reduced pressure; the mixture was extracted with ethyl acetate and water, retaining the aqueous phase; the pH was adjusted to 0.5-2 with hydrochloric acid, and then extracted with ethyl acetate; the solvent was evaporated to dryness to obtain compound C, which can be used directly in the next step without purification; the solvent for carboxyl activation is 1,4-dioxane, tetrahydrofuran, acetonitrile, dichloromethane, etc. N,N - Dimethylformamide (DMF) or dimethyl sulfoxide (DMSO); the reaction solvent for the active ester of compound B-OSu with amino acids is tetrahydrofuran, acetonitrile, N,N - Dimethylformamide (DMF) or dimethyl sulfoxide (DMSO); the temperature of the amide reaction is 0~30℃; the time of the amide reaction is 2 h~15 h; the molar ratio of compound B to NHS and DCC is 1:1.0~3.0:1.0~4.0; the concentration of compound B-OSu in the solution is 1.0~4.0 mmol / mL.

[0022] Step 3: Compound C undergoes a ring-opening reaction with a strong base. The solvent is removed by concentration under reduced pressure, and the mixture is extracted with ethyl acetate and water, retaining the aqueous phase. The pH is adjusted to 0.5-2 with hydrochloric acid, and the mixture is extracted again with ethyl acetate. The solvent is then evaporated to obtain compound D, which can be used directly in the next step without further purification. The solvent for the ring-opening reaction is methanol / water, ethanol / water, isopropanol / water, tetrahydrofuran / water, or acetonitrile / water. The strong base refers to sodium hydroxide, potassium hydroxide, and lithium hydroxide. The temperature of the ring-opening reaction is 0-30℃. The reaction time is 1-6 hours. The molar ratio between the compound to be ring-opened and the strong base is 1:2.0-4.0. Step 4: Compound D undergoes a deprotection reaction with trifluoroacetic acid and a reducing agent to remove the Cbz protecting group. The solvent is removed by concentration under reduced pressure, and the mixture is extracted with ethyl acetate and water. The aqueous phase is retained, and the solvent is evaporated to obtain the dipeptide. The reducing agent is triethylsilane or triisopropylsilane. The temperature of the deprotection reaction is 40-80°C. The reaction time is 2-16 h. The molar ratio of the ring-opened compound, trifluoroacetic acid, and reducing agent is 1:20-40:10-20. The solvent for the deprotection reaction is dichloromethane, 1,2-dichloroethane, tetrahydrofuran, toluene, or 1,4-dioxane.

[0023] Step 5: Compound C is prepared using 1,4-dioxane, tetrahydrofuran, acetonitrile, and dichloromethane. N,N Using dimethylformamide (DMF) or dimethyl sulfoxide (DMSO) as solvents, a carboxyl activation reaction occurs with NHS and DCC. After filtration, the filtrate is evaporated to dryness and then treated with tetrahydrofuran, acetonitrile, ... N,N A solution of compound C-OSu was obtained by dissolving it in dimethylformamide (DMF) or dimethyl sulfoxide (DMSO); the solution of compound C-OSu was added dropwise to a suspension of amino acids, organic base, and water to carry out an amide condensation reaction; the solvent was removed by concentration under reduced pressure; the mixture was extracted with ethyl acetate and water, retaining the aqueous phase; the pH was adjusted to 0.5-2 with hydrochloric acid, and then extracted with ethyl acetate; the solvent was evaporated to dryness to obtain compound E, which can be used directly in the next step without purification; the solvent for the carboxyl activation reaction was 1,4-dioxane, tetrahydrofuran, acetonitrile, dichloromethane, etc. N,N - Dimethylformamide (DMF) or dimethyl sulfoxide (DMSO); the reaction solvent for the C-OSu active ester compound with amino acids is tetrahydrofuran, acetonitrile, N,N-dimethylformamide (DMF) or dimethyl sulfoxide (DMSO); the organic base refers to triethylamine or N,N -Diisopropylethylamine; the temperature of the amide reaction is 0~30 ℃; the reaction time is 2 h~15 h; the molar ratio of compound C to NHS and DCC is 1:1.0~3.0:1.0~4.0; the concentration of compound C-OSu in the solution is 1.0~4.0 mmol / mL.

[0024] Step 6: Compound E undergoes a ring-opening reaction with a strong base. The solvent is removed by concentration under reduced pressure, and the mixture is extracted with ethyl acetate and water, retaining the aqueous phase. The pH is adjusted to 0.5-2 with hydrochloric acid, and the mixture is extracted again with ethyl acetate. The solvent is then evaporated to obtain compound F, which can be used directly in the next step without further purification. The solvent for the ring-opening reaction is methanol / water, ethanol / water, isopropanol / water, tetrahydrofuran / water, or acetonitrile / water. The strong base is sodium hydroxide, potassium hydroxide, or lithium hydroxide. The temperature of the ring-opening reaction is 0-30℃. The reaction time is 1-6 hours. The molar ratio between the compound to be ring-opened and the strong base is 1:2.0-4.0. Step 7: Compound F undergoes a deprotection reaction with trifluoroacetic acid and a reducing agent to remove the Cbz protecting group. The solvent is removed by concentration under reduced pressure, and the mixture is extracted with ethyl acetate and water. The aqueous phase is retained, and the solvent is evaporated to obtain the tripeptide. The reducing agent is triethylsilane or triisopropylsilane. The temperature of the deprotection reaction is 40-80°C; the reaction time is 2-16 h; the molar ratio of the ring-opened compound, trifluoroacetic acid, and reducing agent is 1:20-40:10-20; the solvent for the deprotection reaction is dichloromethane, 1,2-dichloroethane, tetrahydrofuran, toluene, or 1,4-dioxane.

[0025] Compared with the prior art, the present invention has the following beneficial effects: The present invention uses commercially available N-benzyloxycarbonyl-L-glutamic acid as the starting material. After a one-step cyclization reaction with paraformaldehyde to protect the carboxyl and amino groups, the carboxyl group is activated with NHS and DCC and reacted with different amino acids. After hydrolysis and ring opening, the Cbz protecting group is removed with trifluoroacetic acid and triisopropylsilane to obtain a dipeptide. Then, using the unhydrolyzed and ring-opened dipeptide as an intermediate, the carboxyl group is activated with NHS and DCC, reacted with different amino acids, and after hydrolysis and ring opening, the Cbz protecting group is removed with trifluoroacetic acid and a reducing agent to obtain a tripeptide.

[0026] The preparation method provided by this invention is simple in technical route and convenient in operation, with a simple post-processing procedure. It not only has a high raw material conversion rate, but also effectively maintains the optical purity of dipeptides and tripeptides, avoiding optical isomer recombinant rearrangement. The preparation process is highly safe, and the reagents used are all commonly used industrial reagents that are easy to obtain and inexpensive, which significantly reduces the preparation cost. At the same time, it can easily achieve gram-level preparation of dipeptides and tripeptides under laboratory conditions, which is easy to scale up for industrial use. It provides a reliable technical path for the efficient and low-cost preparation of dipeptides and tripeptides. Attached Figure Description

[0027] Figure 1 For dipeptide-1 (γ-Glu-Ala) 1 H NMR spectrum; Figure 2 For tripeptide-1 (γ-Glu-Ala-Gly) 1 H NMR spectrum. Detailed Implementation

[0028] The technical solutions of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. The described embodiments are only some embodiments of the present invention, not all embodiments, and the protection scope of the present invention is not limited to the following embodiments. All raw materials and reagents used in the present invention are commonly used reagents and are commercially available. The present invention has no special limitations. The dipeptide prepared by the present invention is γ- L -Glutamine dipeptide, the prepared tripeptide is γ-L -Glutamine tripeptide.

[0029] Example 1 N 5 -(( S )-1-Carboxyethyl)- L - Glutamine Synthesis

[0030] Step 1: Take N -Benzyloxycarbonyl- L -Glutamic acid (compound A) (140.60 g, 0.50 mol, 1.00 eq), paraformaldehyde 22.50 g (0.75 mol, 1.50 eq), and p-toluenesulfonic acid monohydrate (TsOH·H2O, 1.00 g) were added to a 1 L round-bottom flask. Then, 500 mL of toluene was added to the system. The flask was connected to an oil-water separator, and the system was heated to reflux for 3 h. TLC analysis showed that the starting material was almost completely consumed. Reflux was continued for another 2 h before further processing. The system was cooled to room temperature, diluted with ethyl acetate, washed twice with saturated sodium chloride solution, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to obtain compound B (140.00 g, yield 95%, yellow oil). No purification was required; it was directly added to the next reaction step.

[0031] Step 2: Take compound B (10.00 g, 25.48 mmol) and N 3.52 g (30.58 mmol) of hydroxysuccinimide (NHS) was added to a 1 L round-bottom flask, along with 20 mL of 1,4-dioxane. Then, under nitrogen protection, the following was added... N,N '-Dicyclohexylcarbodiimide (DCC) (7.86 g, 38.22 mmol) showed the formation of a white precipitate (DCU). The reaction system was allowed to react overnight at room temperature (reaction time ≥ 12 h). The white solid was filtered off using a sintered glass funnel (or Buchner funnel), washed with dioxane, and the filtrate was retained. The solvent was removed by concentration under reduced pressure, and the residue was then dissolved in 10 mL of tetrahydrofuran (THF). This solution was added dropwise through a dropping funnel to an aqueous solution (5 mL) containing alanine (L-Ala-OH) (2.72 g, 30.58 mmol) and Et3N (3.87 g, 38.22 mmol). After the addition was complete, the reaction was allowed to proceed for another 3 h at room temperature. THF was removed under reduced pressure, and the mixture was extracted twice with ethyl acetate. The aqueous phase was retained, and the organic phase was washed once with water. The pH of the system was then adjusted to less than 2 with 6N HCl, extracted twice with ethyl acetate, washed twice with saturated sodium chloride solution, dried over anhydrous sodium sulfate, and the solvent was removed under reduced pressure to obtain compound C (7.43 g, yield 80%, yellow oil).

[0032] Step 3: Take compound C (2.00 g, 5.49 mmol, 1.00 eq.) obtained in the previous step and add it to 10 mL of methanol and 3 mL of water. Add lithium hydroxide monohydrate (460.72 mg, 10.98 mmol, 2.00 eq.) under ice bath conditions. Then, raise the temperature to room temperature and react for 3 h. Concentrate under reduced pressure to remove methanol. Extract with ethyl acetate and water, retain the aqueous phase. Adjust the pH to 1 with 3N HCl and extract with ethyl acetate again, retain the organic phase. Wash with water and concentrate to obtain compound D (1.43 g, yield 74%, colorless oil).

[0033] Step 4: Compound D (1.43 g, 4.06 mmol, 1 eq.) obtained in the previous step was added to 10 mL of trifluoroacetic acid and 5 mL of DCM, along with triisopropylsilane (9.64 g, 12.48 mL, 60.90 mmol, 15 eq.). The mixture was reacted at 60 °C for 12 h. The solvent was evaporated, and the residue was dissolved in water and extracted three times with ethyl acetate. The aqueous phase was retained and concentrated to give the product dipeptide-1 (γ-Glu-Ala) (708 mg, yield 80%, colorless solid). 1 H NMR (400 MHz, DMSO- d 6 ) δ12.47 (s, 2H), 8.53 (d, J = 69.8 Hz, 2H), 8.32 (d, J = 7.2 Hz, 1H), 4.19 (p, J =7.2 Hz, 1H), 3.89 (t, J = 6.5 Hz, 1H), 2.41 – 2.25 (m, 2H), 2.05 – 1.94 (m,2H), 1.26 (d, J = 7.3 Hz, 3H), the proton spectrum is as follows Figure 1 As shown.

[0034] Table 1. Preparation methods of Examples 1-9 of the present invention serial number name Preparation process Example 1 Dipeptide-1 <![CDATA[ N 5 -(( S )-1-Carboxyethyl)- L -Glutamine; N 5 -(( S )-1-carboxyethyl)- L -glutamine]]> As shown in Example 1 above. Example 2 Dipeptide-2 <![CDATA[ N 5 -(( S )-1-Carboxy-2-methylpropyl)- L -Glutamine; N 5 -(( S )-1-carboxy-2-methylpropyl)- L -glutamine]]> Similar to Example 1, toluene in step 1 was replaced with xylene, alanine in step 2 was replaced with valine, the carboxyl-activating solvent 1,4-dioxane was replaced with tetrahydrofuran, the solvent for the reaction of compound B-OSu active ester with amino acids was replaced with acetonitrile, methanol / water in step 3 was replaced with ethanol / water, and dichloromethane in step 4 was replaced with 1,2-dichloroethane. The reaction temperature was 65°C, and the reaction time was 10 h. Example 3 Dipeptide-3 <![CDATA[ N 5 -(( S )-1-Carboxybutyl)- L -Glutamine; N 5 -(( S )-1-carboxybutyl)- L -glutamine]]> Similar to Example 1, toluene in step 1 was replaced with cyclohexane, alanine in step 2 was replaced with valine, the carboxyl-activating solvent 1,4-dioxane was replaced with acetonitrile, the solvent for the reaction of compound B-OSu active ester with amino acids was replaced with dimethylformamide (DMF), methanol / water in step 3 was replaced with isopropanol / water, and dichloromethane in step 4 was replaced with tetrahydrofuran. The reaction temperature was 70 °C, and the reaction time was 12 h. Example 4 Dipeptide-4 ()-2-(()-4-amino-4-carboxybutanamido)-5-methylhexanoic acid; ()-2-(()-4-amino-4-carboxybutanamido)-5-methylhexanoic acid Similar to Example 1, toluene in step 1 was replaced with n-heptane, alanine in step 2 was replaced with leucine, the carboxyl-activating solvent 1,4-dioxane was replaced with dichloromethane, the solvent for the reaction of compound B-OSu active ester with amino acids was replaced with dimethyl sulfoxide (DMSO), methanol / water in step 3 was replaced with tetrahydrofuran / water, and dichloromethane in step 4 was replaced with toluene. The reaction temperature was 80 °C, and the reaction time was 6 h. Example 5 Dipeptide-5 <![CDATA[ N 5 -(( S )-1-Carboxy-3-(methylthio)propyl)- L -Glutamine; N 5 -(( S )-1-carboxy-3-(methylthio)propyl)- L -glutamine]]> Similar to Example 1, alanine in step 2 was replaced with methionine, the carboxyl-activating solvent 1,4-dioxane was replaced with dimethylformamide (DMF), methanol / water in step 3 was replaced with acetonitrile / water, and dichloromethane in step 4 was replaced with 1,4-dioxane. The reaction temperature was 75 °C, and the reaction time was 12 h. Example 6 Dipeptide-6 <![CDATA[ N 5 -(( S )-1-Carboxy-3-Phenypropyl)- L -Glutamine; N 5 -(( S )-1-carboxy-3-phenylpropyl)- L -glutamine]]> Similar to Example 1, alanine in step 2 was replaced with phenylalanine, and the carboxyl-activated solvent 1,4-dioxane was replaced with dimethyl sulfoxide (DMSO). Example 7 Dipeptide-7 <![CDATA[ N 5 -(carboxymethyl)- L -Glutamine; N 5 -(carboxymethyl)- L -glutamine]]> Similar to Example 1, alanine in step 2 was replaced with glycine. Example 8 Dipeptide-8 <![CDATA[ N 5 -((1 S ,2 R )-1-Carboxy-2-methylbutyl)- L -Glutamine; N 5 -((1 S ,2 R )-1-carboxy-2-methylbutyl)- L -glutamine]]> Similar to Example 1, in step 2, alanine was replaced with isoleucine, 1,4-dioxane was replaced with acetonitrile, tetrahydrofuran was replaced with dimethylformamide (DMF), and the reaction temperature in step 4 was 65 °C for 10 h. Example 9 Dipeptide-9 <![CDATA[ N 5 -(( S )-1-Carboxy-2-(1H-indol-3-yl)ethyl)- L -Glutamine; N 5 -(( S )-1-carboxy-2-(1H-indol-3-yl)ethyl)- L -glutamine]]> Similar to Example 1, alanine in step 2 was replaced with tryptophan, the solvent 1,4-dioxane was replaced with tetrahydrofuran, the pH was adjusted to 2, the reaction temperature in step 4 was 70 °C, and the reaction time was 8 h. Example 2 N 5 -(( S )-1-Carboxy-2-methylpropyl)- L - Glutamine Synthesis Using a method similar to that used in Example 1 of this invention to prepare dipeptide-1, alanine in step 2 was replaced with valine, the carboxyl-activating solvent 1,4-dioxane was replaced with tetrahydrofuran, the solvent for the reaction of compound B-OSu active ester with amino acids was replaced with acetonitrile, methanol / water in step 3 was replaced with ethanol / water, and dichloromethane in step 4 was replaced with 1,2-dichloroethane. The reaction temperature was 65 °C, and the reaction time was 10 h. After hydrolysis and removal of the Cbz protecting group, dipeptide-2 (γ-Glu-Val) was obtained with a yield of 75%. 1 H NMR (400 MHz, DMSO- d 6) δ 12.36 (s, 2H), 8.41 (s, 2H), 8.18 (d, J = 8.4Hz, 1H), 4.14 (dd, J = 8.5, 5.7 Hz, 1H), 3.85 (t, J = 6.4 Hz, 1H), 2.37 (dd, J =14.8, 6.5 Hz, 2H), 2.08 – 1.99 (m, 2H), 0.97 (dd, J = 7.0, 2.8 Hz, 1H), 0.89(s, 3H), 0.87(s, 3H).

[0035] Example 3 N 5 -(( S )-1-Carboxybutyl)- L - Glutamine Synthesis The preparation of dipeptide-1 was carried out using a method similar to that in Example 1 of this invention, except that alanine in step 2 was replaced with valine, the carboxyl-activating solvent 1,4-dioxane was replaced with acetonitrile, and the solvent for the reaction of compound B-OSu active ester with amino acids was replaced with [other solvent not specified]. N,N Dimethylformamide (DMF), methanol / water in step 3 was replaced with isopropanol / water, dichloromethane in step 4 was replaced with tetrahydrofuran, the reaction temperature was 70 °C, the reaction time was 12 h, and after hydrolysis and removal of the Cbz protecting group, dipeptide-3 (γ-Glu-Nva) was obtained with a yield of 60%. 1 H NMR (400 MHz, DMSO- d 6) δ 8.93 (s, 2H), 8.54 – 8.44 (m, 1H), 4.12 (d, J = 5.3 Hz, 1H), 2.37 (d, J = 4.8 Hz, 1H), 2.28 (q, J= 7.9 Hz, 2H), 1.85 (q, J = 7.2 Hz, 2H), 1.70 – 1.50 (m, 2H), 1.31 (q, J = 7.6 Hz, 2H), 0.86 (t, J = 7.3 Hz, 3H).

[0036] Example 4 ( S )-2-(( S Synthesis of 4-amino-4-carboxybutamido-5-methylhexanoic acid Using a method similar to that used in Example 1 of this invention to prepare dipeptide-1, alanine in step 2 was replaced with leucine, the carboxyl-activating solvent 1,4-dioxane was replaced with dichloromethane, the reaction solvent of compound B-OSu active ester with amino acids was replaced with dimethyl sulfoxide (DMSO), methanol / water in step 3 was replaced with tetrahydrofuran / water, and dichloromethane in step 4 was replaced with toluene. The reaction temperature was 80 °C, and the reaction time was 6 h. After hydrolysis and removal of the Cbz protecting group, dipeptide-4 (γ-Glu-Leu) was obtained with a yield of 70%. 1 H NMR (400 MHz, DMSO- d 6) δ 12.76 (s, 2H), 8.34 (s, 2H), 8.27(d, J = 7.9 Hz, 1H), 4.20 (dd, J = 14.2, 8.7 Hz, 1H), 3.92 (s, 1H), 2.41 – 2.24(m, 2H), 1.99 (t, J = 6.6 Hz, 2H), 1.72 – 1.55 (m, 2H), 1.55 – 1.38 (m, 2H), 0.91 (d, J = 3.7 Hz, 1H), 0.89 (d, J = 6.7 Hz, 3H), 0.84 (d, J = 6.4 Hz, 3H).

[0037] Example 5 N 5 -(( S )-1-Carboxy-3-(methylthio)propyl)- L - Glutamine Synthesis The preparation of dipeptide-1 was carried out using a method similar to that in Example 1 of this invention, except that alanine in step 2 was replaced with methionine, and the carboxyl-activating solvent 1,4-dioxane was replaced with [other solvent]. N,NDimethylformamide (DMF), methanol / water in step 3 was replaced with acetonitrile / water, dichloromethane in step 4 was replaced with 1,4-dioxane, the reaction temperature was 75℃, the reaction time was 12 h, and after hydrolysis and removal of Cbz protecting group, dipeptide-5 (γ-Glu-Met) was obtained with a yield of 65%. 1 H NMR (400 MHz, DMSO- d 6) δ 8.46 (s, 2H), 8.33 (d, J = 7.7 Hz, 1H), 4.35 – 4.30 (m, 1H), 3.77 (s, 1H), 3.39 – 3.23 (m, 2H), 2.81 (s, 3H), 2.47 (d, J = 7.3 Hz, 2H), 2.35 (d, J = 9.6 Hz, 2H), 1.99 (s, 2H).

[0038] Example 6 N 5 -(( S )-1-Carboxy-3-Phenypropyl)- L - Glutamine Synthesis Using a method similar to that used in Example 1 of this invention to prepare dipeptide-1, alanine in step 2 was replaced with phenylalanine, and the carboxyl-activated solvent 1,4-dioxane was replaced with dimethyl sulfoxide (DMSO). After hydrolysis, ring opening, and removal of the Cbz protecting group, dipeptide-6 (γ-Glu-Phe) was obtained with a yield of 85%. 1 H NMR (400 MHz, DMSO- d 6) δ 8.86 (s, 2H), 8.39 (d, J = 8.1 Hz, 1H), 7.28 – 7.22 (m, 5H), 4.43 – 4.37 (m, 1H), 3.64 (d, J =6.7 Hz, 1H), 3.06 (dd, J = 13.8, 4.8 Hz, 2H), 2.84 (dd, J = 13.9, 9.8 Hz, 2H), 2.24 (t, J = 7.8 Hz, 2H), 1.87 (dt, J = 19.4, 6.8 Hz, 2H).

[0039] Example 7 N 5 -(carboxymethyl)-L - Glutamine Synthesis Using a method similar to that used in Example 1 of this invention to prepare dipeptide-1, alanine in step 2 was replaced with glycine, and methanol / water in step 3 was replaced with tetrahydrofuran / water. After hydrolysis and removal of the Cbz protecting group, dipeptide-7 (γ-Glu-Gly) was obtained with a yield of 57%. 1 H NMR (400 MHz, DMSO- d 6) δ 12.64 (s, 2H), 8.48 (s, 2H), 8.33(t, J = 5.9 Hz, 1H), 3.88 (d, J = 5.2 Hz, 1H), 3.74 (d, J = 5.9 Hz, 2H), 2.43 –2.31 (m, 2H), 2.04 – 1.96 (m, 2H).

[0040] Example 8 N 5 -((1 S ,2 R )-1-Carboxy-2-methylbutyl)- L - Glutamine Synthesis The preparation of dipeptide-1 was carried out using a method similar to that in Example 1 of this invention, except that alanine in step 2 was replaced with isoleucine, 1,4-dioxane was replaced with acetonitrile, and tetrahydrofuran was replaced with [other solvent]. N,N Dimethylformamide (DMF) was reacted at 65 °C for 10 h in step 4. After hydrolysis and removal of the Cbz protecting group, dipeptide-8 (γ-Glu-Ile) was obtained with a yield of 74%. 1 H NMR (400 MHz, DMSO- d 6) δ 8.80 (s, 2H), 8.28 (d, J = 8.3 Hz, 1H), 4.16 (dd, J = 8.3, 5.9 Hz, 1H), 3.67 (t, J = 6.5 Hz, 1H), 2.35 (t, J = 7.7 Hz, 2H), 1.94 (dd, J = 15.6, 8.6 Hz, 2H), 1.82 – 1.72 (m, 1H), 1.45 – 1.34 (m, 1H), 1.24 – 1.14 (m, 1H), 0.86 (s, 3H), 0.84 (d, J= 2.1 Hz, 3H).

[0041] Example 9 N 5 -(( S )-1-Carboxyl-2-(1 H -indol-3-yl)ethyl)- L - Glutamine Synthesis Using a method similar to that used in Example 1 of this invention to prepare dipeptide-1, alanine in step 2 was replaced with tryptophan, the solvent 1,4-dioxane was replaced with tetrahydrofuran, the pH was adjusted to 2, the reaction temperature in step 4 was 70°C, the reaction time was 8 h, and after hydrolysis and removal of the Cbz protecting group, dipeptide-9 (γ-Glu-Trp) was obtained with a yield of 86%. 1 H NMR (400 MHz, DMSO- d 6) δ 12.72 (s, 2H), 8.46 (d, J = 8.0 Hz, 1H), 8.26 (s, 4H), 7.20 (s, 1H), 7.07 (dd, J = 18.6, 5.8 Hz, 2H), 6.74 (s, 1H), 4.36 – 4.29 (m, 1H), 3.93 (s,1H), 3.30 – 3.19 (m, 2H), 2.40 – 2.29 (m, 2H), 2.06 – 1.98 (m, 2H).

[0042] Example 10 N 5 -(( S )-1-((carboxymethyl)amino)-1-oxopropane-2-yl)- L - Glutamine Synthesis

[0043] Step 1: Take the (3-(( S )-3-((benzyloxy)carbonyl)-5-oxooxazolidine-4-yl)propionyl)- L -Alanine ((3-(( S )-3-((benzyloxy)carbonyl)-5-oxooxazolidin-4-yl)propanoyl)-L-alanine, compound C (5.00 g, 13.72 mmol) and N1.89 g (16.46 mmol) of hydroxysuccinimide (NHS) was added to a 1 L round-bottom flask, along with 15 mL of 1,4-dioxane. Then, under nitrogen protection, the following was added: N,N '-Dicyclohexylcarbodiimide (DCC) (4.25 g, 20.58 mmol) showed the formation of a white precipitate (DCU). The system was reacted overnight at room temperature (reaction time ≥ 12 h). The white solid was filtered off using a sintered glass funnel (or Buchner funnel), washed with dioxane, and the filtrate was retained. The solvent was removed by concentration under reduced pressure, and the residue was then dissolved in 50 mL of tetrahydrofuran (THF). This solution was added dropwise through a dropping funnel to an aqueous solution (3 mL) containing glycine (1.24 g, 16.46 mmol) and Et3N (2.08 g, 20.58 mmol). After the addition was complete, the reaction was carried out at room temperature for another 3 h. THF was removed under reduced pressure, and the mixture was extracted twice with ethyl acetate. The aqueous phase was retained, and the organic phase was washed once with water. The pH of the system was then adjusted to less than 2 with 6N HCl, extracted twice with ethyl acetate, washed twice with saturated sodium chloride solution, dried over anhydrous sodium sulfate, and the solvent was removed under reduced pressure to obtain compound E (4.39 g, yield 76%, yellow oil).

[0044] Step 2: Take compound E (4.39 g, 10.43 mmol, 1.00 eq.) obtained in the previous step and add it to 10 mL of methanol and 3 mL of water. Add lithium hydroxide monohydrate (874 mg, 20.83 mmol, 2.00 eq.) under ice bath conditions. Then, raise the temperature to room temperature and react for 3 h. Remove methanol by concentration under reduced pressure. Extract with ethyl acetate and water, retain the aqueous phase. Adjust the pH to 1 with 3N HCl and extract with ethyl acetate again, retain the organic phase. Wash with water and concentrate to obtain compound F (3.33 g, yield 78%, colorless oil).

[0045] Step 3: Compound F (3.33 g, 8.13 mmol, 1 eq.) obtained in the previous step was added to 10 mL of trifluoroacetic acid and 5 mL of DCM, along with triisopropylsilane (19.31 g, 24.98 mL, 121.95 mmol, 15 eq.). The reaction was carried out at 60 °C for 12 h. The solvent was evaporated, and the residue was dissolved in water and extracted three times with ethyl acetate. The aqueous phase was retained and concentrated to obtain the product tripeptide-1 (γ-Glu-Ala-Gly) (1.57 g, yield 70%, colorless solid). 1 H NMR (400 MHz, DMSO- d 6) δ 8.56 (s, 2H), 8.32 (d, J= 7.5 Hz, 1H), 8.22 (d, J = 7.4 Hz, 1H), 4.19 (t, J = 7.3 Hz, 1H), 3.85 (s, 1H), 3.74 (s, 2H), 2.33 (q, J = 7.0, 5.9 Hz, 2H), 2.02– 1.95 (m, 2H), 1.26 (d, J = 7.3 Hz, 3H), the proton spectrum is as follows Figure 2 As shown.

[0046] Table 2 Preparation methods of Examples 10-20 of the present invention serial number name Preparation process Example 10 Tripeptide-1 <![CDATA[ N 5 -(( S )-1-((carboxymethyl)amino)-1-oxopropane-2-yl)- L -Glutamine; N 5 -(( S )-1-((carboxymethyl)amino)-1-oxopropan-2-yl)- L -glutamine]]> As shown in Example 10 above. Example 11 Tripeptide-2 <![CDATA[ N 5 -(( S )-1-((carboxymethyl)amino)-3-methyl-1-oxobutane-2-yl)- L -Glutamine; N 5 -(( S )-1-((carboxymethyl)amino)-3-methyl-1-oxobutan-2-yl)- L -glutamine]]> Similar to Example 10, (3-(()-3-((benzyloxy)carbonyl)-5-oxooxazolidin-4-yl)propanoyl)--alanine was replaced with (3-(()-3-((benzyloxy)carbonyl)-5-oxooxazolidin-4-yl)propanoyl)--valine, the solvent 1,4-dioxane was replaced with tetrahydrofuran, the solvent for the reaction of the C-OSu active ester with the amino acid was replaced with acetonitrile, methanol / water in step 3 was replaced with ethanol / water, dichloromethane in step 4 was replaced with 1,2-dichloroethane, the reaction temperature was 65°C, and the reaction time was 10 h. Example 12 Tripeptide-3 <![CDATA[ N 5 -(( S )-1-((carboxymethyl)amino)-1-oxopentane-2-yl)- L -Glutamine; N 5 -(( S )-1-((carboxymethyl)amino)-1-oxopentan-2-yl)- L -glutamine]]> Similar to Example 10, (3-(()-3-((benzyloxy)carbonyl)-5-oxooxazolidin-4-yl)propanoyl)-alanine was replaced with (3-(()-3-((benzyloxy)carbonyl)-5-oxooxazolidin-4-yl)propanamido)pentanoic acid, tetrahydrofuran was replaced with acetonitrile, the solvent for the reaction of compound C-OSu active ester with amino acids was replaced with dimethylformamide (DMF), methanol / water in step 3 was replaced with isopropanol / water, dichloromethane in step 4 was replaced with tetrahydrofuran, the reaction temperature was 70 °C, and the reaction time was 12 h. Example 13 Tripeptide-4 <![CDATA[ N 5 -(( S )-1-((carboxymethyl)amino)-5-methyl-1-oxohexane-2-yl)- L -Glutamine; N 5 -(( S )-1-((carboxymethyl)amino)-5-methyl-1-oxohexan-2-yl)- L -glutamine]]> Similar to Example 10, (3-(()-3-((benzyloxy)carbonyl)-5-oxooxazolidin-4-yl)propanamido)--alanine was replaced with (()-2-(3-(()-3-((benzyloxy)carbonyl)-5-oxooxazolidin-4-yl)propanamido)-5-methylhexanoic acid, the solvent 1,4-dioxane was replaced with acetonitrile, the solvent for the reaction of compound C-OSu active ester with amino acids was replaced with dimethyl sulfoxide (DMSO), methanol / water in step 3 was replaced with tetrahydrofuran / water, dichloromethane in step 4 was replaced with toluene, the reaction temperature was 80 °C, and the reaction time was 6 h. Example 14 Tripeptide-5 <![CDATA[ N 5 -(( S )-1-((carboxymethyl)amino)-4-(methylthio)-1-oxobutane-2-yl)- L -Glutamine; N 5 -(( S )-1-((carboxymethyl)amino)-4-(methylthio)-1-oxobutan-2-yl)- L -glutamine]]> Similar to Example 10, (3-(()-3-((benzyloxy)carbonyl)-5-oxooxazolidin-4-yl)propanoyl)--alanine was replaced with (3-(()-3-((benzyloxy)carbonyl)-5-oxooxazolidin-4-yl)propanoyl)--methionine (3-(()-3-((benzyloxy)carbonyl)-5-oxooxazolidin-4-yl)propanoyl)-L-methionine, the solvent 1,4-dioxane was replaced with dimethylformamide (DMF), methanol / water in step 3 was replaced with acetonitrile / water, and dichloromethane in step 4 was replaced with 1,4-dioxane. The reaction temperature was 75 °C, and the reaction time was 12 h. Example 15 Tripeptide-6 <![CDATA[ N 5 -(( S )-1-((carboxymethyl)amino)-1-oxo-4-phenylbut-2-yl)- L -Glutamine; N 5 -(( S )-1-((carboxymethyl)amino)-1-oxo-4-phenylbutan-2-yl)- L -glutamine]]> Similar to Example 10, (3-(()-3-((benzyloxy)carbonyl)-5-oxooxazolidin-4-yl)propanamido)-alanine was replaced with (()-2-(3-(()-3-((benzyloxy)carbonyl)-5-oxooxazolidin-4-yl)propanamido)-4-phenylbutanoic acid (()-2-(3-(()-3-((benzyloxy)carbonyl)-5-oxooxazolidin-4-yl)propanamido)-4-phenylbutanoic acid, and the solvent 1,4-dioxane was replaced with dimethyl sulfoxide (DMSO). Example 16 Tripeptide-7 <![CDATA[ N 5 -(2-((carboxymethyl)amino)-2-oxoethyl)- L -Glutamine; N 5 -(2-((carboxymethyl)amino)-2-oxoethyl)- L -glutamine]]> Similar to Example 10, (3-(()-3-((benzyloxy)carbonyl)-5-oxooxazolidin-4-yl)propanoyl)-alanine was replaced with (()-(3-(3-((benzyloxy)carbonyl)-5-oxooxazolidin-4-yl)propanoyl)glycine (()-(3-(3-((benzyloxy)carbonyl)-5-oxooxazolidin-4-yl)propanoyl)glycine) Example 17 Tripeptide-8 <![CDATA[ N 5 -((2 S ,3 R )-1-((carboxymethyl)amino)-3-methyl-1-oxopent-2-yl)- L -Glutamine; N 5 -((2 S ,3 R )-1-((carboxymethyl)amino)-3-methyl-1-oxopentan-2-yl)- L -glutamine]]> Similar to Example 10, (3-(()-3-((benzyloxy)carbonyl)-5-oxooxazolidin-4-yl)propanoyl)-alanine was replaced with (3-(()-3-((benzyloxy)carbonyl)-5-oxooxazolidin-4-yl)propanoyl)-alloisoleucine Example 18 Tripeptide-9 <![CDATA[ N 5 -(( S )-1-((carboxymethyl)amino)-3-(1H-indol-3-yl)-1-oxopropane-2-yl)- L -Glutamine; N 5 -(( S )-1-((carboxymethyl)amino)-3-(1H-indol-3-yl)-1-oxopropan-2-yl)- L -glutamine]]> Similar to Example 10, (3-(()-3-((benzyloxy)carbonyl)-5-oxooxazolidin-4-yl)propanoyl)-L-alanine was replaced with (3-(()-3-((benzyloxy)carbonyl)-5-oxooxazolidin-4-yl)propanoyl)-tryptophan Example 19 Tripeptide-10 <![CDATA[ N 5 -(( S )-1-((( R )-1-Carboxy-2-(1H-indol-3-yl)ethyl)amino)-1-oxopropane-2-yl)- L -Glutamine; N 5 -(( S )-1-((( R )-1-carboxy-2-(1H-indol-3-yl)ethyl)amino)-1-oxopropan-2-yl)- L -glutamine]]> Similar to Example 10, glycine was replaced with tryptophan, the solvent 1,4-dioxane was replaced with tetrahydrofuran, the pH was adjusted to 2, the reaction temperature in step 4 was 70 °C, and the reaction time was 8 h. Example 20 Tripeptide-11 <![CDATA[ N 5 -(( S )-1-((( R )-1-Carboxy-2-methylpropyl)amino)-5-methyl-1-oxohexane-2-yl)- L -Glutamine; N 5 -(( S )-1-((( R )-1-carboxy-2-methylpropyl)amino)-5-methyl-1-oxohexan-2-yl)- L -glutamine]]> Similar to Example 10, (3-(()-3-((benzyloxy)carbonyl)-5-oxooxazolidin-4-yl)propanamido)--alanine was replaced with (()-2-(3-(()-3-((benzyloxy)carbonyl)-5-oxooxazolidin-4-yl)propanamido)-5-methylhexanoic acid, and glycine was replaced with valine. Example 11 N 5 -(( S )-1-((carboxymethyl)amino)-3-methyl-1-oxobutane-2-yl)- L - Glutamine Synthesis Using a method similar to that used in Example 10 of this invention to prepare tripeptide-1, the (3-(( S )-3-((benzyloxy)carbonyl)-5-oxooxazolidine-4-yl)propionyl)- L -Alanine is replaced with (3-(( S )-3-((benzyloxy)carbonyl)-5-oxooxazolidine-4-yl)propionyl)- L -Valine, the solvent 1,4-dioxane was replaced with tetrahydrofuran, the solvent for the reaction of compound C-OSu active ester with amino acids was replaced with acetonitrile, methanol / water in step 3 was replaced with ethanol / water, dichloromethane in step 4 was replaced with 1,2-dichloroethane, the reaction temperature was 65 °C, the reaction time was 10 h, after hydrolysis and removal of Cbz protecting group, tripeptide-2 (γ-Glu-Val-Gly) was obtained with a yield of 73%. 1 H NMR (400 MHz, DMSO- d 6) δ 12.82 (s, 2H), 8.33 (d, J = 7.5 Hz, 2H), 8.29 (s, 1H), 8.16 (d, J = 8.4 Hz, 1H), 4.17 (ddd, J = 21.0,8.7, 6.2 Hz, 1H), 3.95 – 3.86 (m, 1H), 3.75 (dd, J= 14.7, 6.0 Hz, 1H), 2.48 –2.29 (m, 2H), 2.05 (dd, J = 12.7, 6.9 Hz, 1H), 2.02 – 1.92 (m, 2H), 0.98 (dd, J =6.9, 4.7 Hz, 1H), 0.89 (s, 3H), 0.87 – 0.64 (m, 3H).

[0047] Example 12 N 5 -(( S )-1-((carboxymethyl)amino)-1-oxopentane-2-yl)- L - Glutamine Synthesis Using a method similar to that used in Example 10 of this invention to prepare tripeptide-1, the (3-(( S )-3-((benzyloxy)carbonyl)-5-oxooxazolidine-4-yl)propionyl)- L -Alanine is replaced with (3-(( S )-3-((benzyloxy)carbonyl)-5-oxooxazolidine-4-yl)propionyl)- L -Alanine, the solvent tetrahydrofuran is replaced with acetonitrile, and the solvent for the reaction of compound C-OSu active ester with amino acids is replaced with... N,N Dimethylformamide (DMF), in step 3 methanol / water was replaced with isopropanol / water, in step 4 dichloromethane was replaced with tetrahydrofuran, the reaction temperature was 70 °C, the reaction time was 12 h, after hydrolysis and removal of Cbz protecting group, tripeptide-3 (γ-Glu-Nva-Gly) was obtained with a yield of 76%. 1 H NMR (400 MHz, DMSO- d 6) δ 8.05 (s, 1H), 7.35 (s, 2H), 7.33 (s, 1H), 5.02 (s, 2H), 4.23 (s, 1H), 3.64 (s, 1H), 2.21 (s, 2H), 1.88 (d, J = 24.7 Hz, 2H), 1.56 (d, J = 55.1 Hz, 2H), 1.39 – 1.24 (m,2H), 0.84 (d, J = 7.1 Hz, 3H).

[0048] Example 13 N 5 -(( S)-1-((carboxymethyl)amino)-5-methyl-1-oxohexane-2-yl)- L - Glutamine Synthesis The method used is similar to that used in Example 10 of this invention to prepare tripeptide-1, except that in step 1, (3-((S)-3-((benzyloxy)carbonyl)-5-oxooxazolidine-4-yl)propionyl)-L-alanine is replaced with ( S )-2-(3-(( S )-3-((benzyloxy)carbonyl)-5-oxooxazolidine-4-yl)propamido)-5-methylhexanoic acid was prepared by replacing 1,4-dioxane with acetonitrile, replacing dimethyl sulfoxide (DMSO) with dimethyl sulfoxide (DSO) with dimethyl sulfoxide (DSO) with dimethyl sulfoxide (DSO) with dimethyl sulfoxide (DSO) with dimethyl sulfoxide (DSO) in step 3, replacing methanol / water with tetrahydrofuran / water with dichloromethane in step 4, and replacing dichloromethane with toluene with DSO. The reaction temperature was 80 °C and the reaction time was 6 h. After hydrolysis and removal of the Cbz protecting group, tripeptide-4 (γ-Glu-Leu-Gly) was obtained with a yield of 65%. 1 H NMR (400 MHz, DMSO- d 6) δ12.33 (s, 2H), 8.34 (s, 2H), 8.25 (t, J = 5.9 Hz, 1H), 8.17 (d, J = 8.3 Hz, 1H), 4.37 – 4.28 (m, 1H), 3.92 (s, 1H), 3.70 (s, 2H), 2.41 – 2.24 (m, 2H), 2.00(dt, J = 15.8, 7.4 Hz, 2H), 1.60 (p, J = 6.5 Hz, 1H), 1.52 – 1.42 (m, 2H), 0.91(d, J = 6.6 Hz, 1H), 0.88 (d, J = 6.6 Hz, 3H), 0.84 (d, J = 6.5 Hz, 3H).

[0049] Example 14 N 5 -(( S )-1-((carboxymethyl)amino)-4-(methylthio)-1-oxobutane-2-yl)- L - Glutamine Synthesis Using a method similar to that used in Example 10 of this invention to prepare tripeptide-1, the (3-(( S)-3-((benzyloxy)carbonyl)-5-oxooxazolidine-4-yl)propionyl)- L -Alanine is replaced with (3-(( S )-3-((benzyloxy)carbonyl)-5-oxooxazolidine-4-yl)propionyl)- L -Methionine, solvent 1,4-dioxane replaced with N,N Dimethylformamide (DMF), in step 3 methanol / water was replaced with acetonitrile / water, in step 4 dichloromethane was replaced with 1,4-dioxane, the reaction temperature was 75℃, the reaction time was 12 h, after hydrolysis and removal of Cbz protecting group, tripeptide-5 (γ-Glu-Met-Gly) was obtained with a yield of 68%. 1 H NMR (400 MHz, DMSO- d 6) δ 12.81 (s, 2H), 8.42 (s, 2H), 8.36 (d, J = 5.5 Hz, 1H), 8.28 (d, J = 5.9 Hz, 1H), 7.49 (s, 2H), 4.35 (dd, J = 8.4, 5.0 Hz, 1H), 3.92(s, 1H), 3.83 – 3.67 (m, 2H), 2.47 (d, J = 1.5 Hz, 2H), 2.41 – 2.24 (m, 2H), 2.03 (s, 3H), 1.98 (d, J = 9.2 Hz, 2H).

[0050] Example 15 N 5 -(( S )-1-((carboxymethyl)amino)-1-oxo-4-phenylbut-2-yl)- L - Glutamine Synthesis Using a method similar to that used in Example 10 of this invention to prepare tripeptide-1, the (3-(( S )-3-((benzyloxy)carbonyl)-5-oxooxazolidine-4-yl)propionyl)- L -Alanine is replaced with ( S )-2-(3-(( S )-3-((benzyloxy)carbonyl)-5-oxooxazolidin-4-yl)propamido)-4-phenylbutyric acid, with the solvent 1,4-dioxane replaced by dimethyl sulfoxide (DMSO), was hydrolyzed and deprotected by Cbz to give tripeptide-6 (γ-Glu-Phe-Gly) in 70% yield. 1H NMR (400MHz, DMSO- d 6) δ 12.60 (s, 2H), 8.47 (s, 2H), 8.28 (d, J = 8.8 Hz, 1H), 8.12 (d, J = 5.9 Hz, 1H), 7.35 – 7.27 (m, 2H), 7.24 – 7.15 (m, 3H), 4.58 – 4.49 (m,1H), 3.78 (d, J = 5.9 Hz, 2H), 3.74 (d, J = 6.3 Hz, 1H), 3.05 (dd, J = 13.7, 3.9Hz, 2H), 2.73 (dd, J = 13.8, 10.5 Hz, 2H), 2.22 (t, J = 7.1 Hz, 2H), 1.90 – 1.80(m, 2H).

[0051] Example 16 N 5 -(2-((carboxymethyl)amino)-2-oxoethyl)- L - Glutamine Synthesis Using a method similar to that used in Example 10 of this invention to prepare tripeptide-1, the (3-(( S )-3-((benzyloxy)carbonyl)-5-oxooxazolidine-4-yl)propionyl)- L -Alanine is replaced with ( S )-(3-(3-((benzyloxy)carbonyl)-5-oxooxazolidin-4-yl)propionyl)glycine, after hydrolysis and removal of the Cbz protecting group, yielded tripeptide-7 (γ-Glu-Gly-Gly) in 64% yield. 1 H NMR (400 MHz, DMSO- d 6) δ 8.56 (s, 2H), 8.33 (t, J = 5.9 Hz, 1H),7.92 (s, 1H), 3.83 (s, 1H), 3.74 (d, J = 5.7 Hz, 2H), 3.62 (d, J = 11.8 Hz, 2H), 2.40 – 2.30 (m, 2H), 2.06 – 1.98 (m, 2H).

[0052] Example 17N 5 -((2 S ,3 R )-1-((carboxymethyl)amino)-3-methyl-1-oxopent-2-yl)- L - Glutamine Synthesis Using a method similar to that used in Example 10 of this invention to prepare tripeptide-1, the (3-(( S )-3-((benzyloxy)carbonyl)-5-oxooxazolidine-4-yl)propionyl)- L -Alanine is replaced with (3-(( S )-3-((benzyloxy)carbonyl)-5-oxooxazolidine-4-yl)propionyl)- L -Isoleucine, after hydrolysis and removal of the Cbz protecting group, yielded tripeptide-8 (γ-Glu-Ile-Gly) in 78% yield. 1 H NMR (400 MHz, DMSO- d 6) δ 8.38 (t, J = 5.8 Hz, 1H), 8.09 (d, J = 8.9 Hz, 1H), 4.36 (dd, J = 9.1, 5.1 Hz, 1H), 4.23 – 4.17 (m, 1H), 3.74 (s,2H), 2.40 – 2.34 (m, 2H), 1.98 – 1.92 (m, 2H), 1.48 – 1.38 (m, 1H), 1.10 (dt, J = 15.3, 7.1 Hz, 2H), 0.86 (s, 3H), 0.82 (d, J = 7.3 Hz, 3H).

[0053] Example 18 N 5 -(( S )-1-((carboxymethyl)amino)-3-(1 H -indol-3-yl)-1-oxopropane-2-yl)- L - Glutamine Synthesis Using a method similar to that used in Example 10 of this invention to prepare tripeptide-1, the (3-(( S )-3-((benzyloxy)carbonyl)-5-oxooxazolidine-4-yl)propionyl)- L -Alanine is replaced with (3-(( S )-3-((benzyloxy)carbonyl)-5-oxooxazolidine-4-yl)propionyl)- L-Tryptophan, solvent 1,4-dioxane was replaced with tetrahydrofuran, pH was adjusted to 2, the reaction temperature in step 3 was 70 °C, the reaction time was 8 h, and after hydrolysis and removal of Cbz protecting group, tripeptide-9 (γ-Glu-Trp-Gly) was obtained with a yield of 75%. 1 H NMR (400 MHz, DMSO- d 6) δ 8.39 (d, J = 6.2 Hz, 1H), 8.30(s, 2H), 8.27 (s, 1H), 7.16 (dd, J = 33.2, 16.2 Hz, 4H), 6.94 – 6.83 (m, 2H), 4.44 (d, J = 7.3 Hz, 1H), 3.94 (s, 1H), 3.70 (s, 2H), 3.32 – 3.21 (m, 2H), 2.32(d, J = 18.3 Hz, 2H), 2.01 (d, J = 3.8 Hz, 2H).

[0054] Example 19 N 5 -(( S )-1-((( R )-1-Carboxyl-2-(1 H -indol-3-yl)ethyl)amino)-1-oxopropane-2-yl)- L - Glutamine Synthesis Using a method similar to that used in Example 10 of this invention to prepare tripeptide-1, glycine was replaced with tryptophan in step 1, the solvent 1,4-dioxane was replaced with tetrahydrofuran, the pH was adjusted to 2, the reaction temperature in step 4 was 70 °C, the reaction time was 8 h, and after hydrolysis and removal of the Cbz protecting group, tripeptide-10 (γ-Glu-Ala-Trp) was obtained with a yield of 72%. 1 H NMR (400 MHz, DMSO-) d 6) δ 12.38 (s, 2H), 8.44 (s, 2H), 8.30 (s, 1H), 8.21 (d, J =7.0 Hz, 1H), 8.11 (s, 1H), 7.24 (t, J = 6.4 Hz, 1H), 7.16 (t, J = 7.8 Hz, 2H),7.02 – 6.89 (m, 2H), 4.33 (d, J= 5.4 Hz, 1H), 3.91 (s, 1H), 3.70 (q, J = 10.2,9.1 Hz, 1H), 3.30 (s, 2H), 2.40 – 2.23 (m, 2H), 2.07 – 1.93 (m, 2H), 1.24 (d, J = 3.1 Hz, 3H).

[0055] Example 20 N 5 -(( S )-1-((( R )-1-Carboxy-2-methylpropyl)amino)-5-methyl-1-oxohexane-2-yl)- L - Glutamine Synthesis Using a method similar to that used in Example 10 of this invention to prepare tripeptide-1, the (3-(( S )-3-((benzyloxy)carbonyl)-5-oxooxazolidine-4-yl)propionyl)- L -Alanine is replaced with ( S )-2-(3-(( S )-3-((benzyloxy)carbonyl)-5-oxooxazolidine-4-yl)propamido)-5-methylhexanoic acid, with glycine replaced by valine, was hydrolyzed and deprotected by Cbz to obtain tripeptide-11 (γ-Glu-Leu-Val) in 68% yield. 1 H NMR (400 MHz, DMSO- d 6) δ12.98 (s, 2H), 8.29 (s, 2H), 8.26 (d, J = 7.9 Hz, 1H), 8.15 (d, J = 8.2 Hz,1H), 4.18 – 4.08 (m, 1H), 3.92 (s, 1H), 2.42 – 2.24 (m, 2H), 2.04 (q, J = 6.7Hz, 1H), 1.98 (dd, J = 8.7, 6.5 Hz, 2H), 1.61 (dt, J = 13.5, 6.6 Hz, 1H), 1.44(t, J = 7.3 Hz, 1H), 1.01 – 0.96 (m, 1H), 0.86 (ddt, J = 14.7, 8.3, 4.4 Hz, 12H).

[0056] Example 21 Activity of the polypeptide The dipeptides or tripeptides synthesized in this invention generally possess pharmacological activities and improve food flavor, as shown in Table 3. This invention provides an excellent preparation method for the large-scale production of polypeptide drugs. The preparation method provided by this invention has a simple technical route, is convenient to operate, and has a simple post-processing procedure. It not only has a high raw material conversion rate, but also effectively maintains the optical purity of dipeptides and tripeptides, avoiding optical isomer recombinant rearrangement. The preparation process has high safety, and the reagents used are all commonly used industrial reagents that are readily available and inexpensive, significantly reducing the preparation cost.

[0057] Table 3. Activity of peptides

[0058] peptide Main efficacy / activity γ-Glu-Ala CaSR agonists, inhibitors of PTH secretion γ-Glu-Val CaSR activation, anti-inflammatory, Kokumi γ-Glu-Nva Kokumi flavor (initial flavor profile) γ-Glu-Leu Metabolic intermediates, flavor modifiers γ-Glu-Met High affinity transport substrate γ-Glu-Phe Anti-lipogenesis, promotes CCK / GLP-1 secretion γ-Glu-Gly Neural regulation (NMDA / AMPA), promoting calcium absorption γ-Glu-Ile Kokumi Flavor Activity γ-Glu-Trp Anti-anxiety / depression, anti-inflammatory γ-Glu-Ala-Gly CaSR agonists, metabolic intermediates γ-Glu-Val-Gly Kokumi flavor peptides, CaSR agonists γ-Glu-Nva-Gly Flavor enhancer (mid-to-after flavor profile) γ-Glu-Leu-Gly Kokumi flavor enhanced γ-Glu-Met-Gly Metabolic research tools γ-Glu-Phe-Gly Kokumi / Enhances umami / saltiness, and combats fat production. γ-Glu-Gly-Gly Amino acid metabolism, metal chelation, flavor formation γ-Glu-Ile-Gly Kokumi Flavor and Metabolism Studies γ-Glu-Trp-Gly Anti-anxiety / depressant, anti-inflammatory, antioxidant γ-Glu-Ala-Trp Neuroregulation, Antioxidant γ-Glu-Leu-Val Kokumi / Umami / Salty Enhancement Although embodiments of the invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and their equivalents.

Claims

1. A method for preparing a polypeptide with L-glutamic acid as the N-terminus, characterized in that, The method includes the step of synthesizing compound C, and the synthetic route and method of compound C are as follows: ； Compound B undergoes a carboxyl activation reaction with NHS and DCC. After the reaction, the mixture is filtered, and the solvent is removed from the filtrate. The filtrate is then dissolved in an aprotic solvent to obtain a solution of compound B-OSu. The compound B-OSu solution is added dropwise to a suspension of amino acids, organic bases, and water to carry out an amide condensation reaction. After the reaction, the solvent is removed, the aqueous phase is extracted, the pH is adjusted, and the mixture is extracted again to remove the solvent and obtain compound C.

2. The method for preparing the polypeptide according to claim 1, characterized in that, The polypeptide is a dipeptide γ-Glu-X, a tripeptide γ-Glu-XY, or a polypeptide composed of three or more amino acid residues; When the polypeptide is a dipeptide γ-Glu-X, its preparation process is as follows: Compound C is subjected to a ring-opening reaction and a Cbz protecting group removal reaction to obtain dipeptide γ-Glu-X; When the polypeptide is a tripeptide γ-Glu-XY, its preparation process and synthetic route are as follows: ； In the synthesis route, R2 and R1 may be the same or different; Compound C undergoes a carboxyl activation reaction with NHS and DCC. After the reaction, the mixture is filtered, and the solvent is removed from the filtrate. The filtrate is then dissolved in an aprotic solvent to obtain a solution of compound C-OSu. The C-OSu solution is added dropwise to a suspension of amino acids, organic base, and water to carry out an amide condensation reaction. After the reaction, the solvent is removed, the aqueous phase is extracted, the pH is adjusted, and the mixture is extracted again to remove the solvent and obtain compound E. Compound E is then subjected to a ring-opening reaction and a Cbz-protecting group removal reaction to prepare the tripeptide γ-Glu-XY. When the polypeptide is a polypeptide composed of three or more amino acid residues, its preparation process is as follows: After the compound E undergoes a carboxyl activation reaction, it continues to undergo an amide condensation reaction with one or more amino acids in sequence until the target peptide chain length is obtained. Finally, the compound M obtained undergoes a ring-opening reaction and a Cbz-removal protecting group removal reaction to obtain a polypeptide composed of three or more amino acid residues.

3. The method for preparing the polypeptide according to claim 2, characterized in that, The procedures for the ring-opening reaction and the removal of the Cbz protecting group are as follows: Ring-opening reaction: The compound to be ring-opened reacts with a strong base. After the reaction, the solvent is removed, the aqueous phase is extracted, the pH is adjusted, and the solvent is removed to obtain the ring-opened compound. Deprotection reaction: The ring-opening compound undergoes a deprotection reaction with trifluoroacetic acid and a reducing agent. After the reaction, the solvent is removed, the aqueous phase is extracted and retained, and the solvent is removed again to obtain the corresponding polypeptide.

4. The method for preparing the polypeptide according to any one of claims 1-3, characterized in that, The synthetic route and method for compound B are as follows: ； Compound A N -Benzyloxycarbonyl- L - Glutamic acid and paraformaldehyde undergo a cyclization reaction catalyzed by p-toluenesulfonic acid monohydrate. After the reaction, the solvent is removed, the mixture is extracted, and the organic phase is removed to obtain compound B.

5. The method for preparing the polypeptide according to any one of claims 1-3, characterized in that, The amino acids are selected from valine, valine, alanine, leucine, methionine, phenylalanine, glycine, isoleucine, tryptophan, histidine, glutamine, aspartic acid-4-tert-butyl ester, arginine, cysteine, and glutamine. N (e)-Boc- L -Lysine, asparagine, serine, tyrosine, threonine, or proline; The general structural formula of the amino acid is H2N-CH(R1)-COOH or , where R1 represents the side chain group of the amino acid excluding the amino and carboxyl groups, and R1 specifically corresponds to the following: n-propyl for valine, isopropyl for valine, methyl for alanine, isobutyl for leucine, methylthioethyl for methionine, benzyl for phenylalanine, hydrogen for glycine, isopentyl for isoleucine, 3-indolemethyl for tryptophan, and 1H- for histidine. Imidazol-4-ylmethyl, corresponding to glutamine as carbamoylethyl, to aspartic acid-4-tert-butyl ester as tert-butoxycarbonylmethyl, to arginine as 3-guanidinopropyl, to cysteine ​​as mercaptomethyl, to N(e)-Boc-L-lysine as 4-(tert-butoxycarbonylamino)butyl, to asparagine as carbamoylmethyl, to serine as hydroxymethyl, to tyrosine as 4-hydroxybenzyl, to threonine as 1-hydroxyethyl, and to proline as tetrahydropyrrole-2-ylene.

6. The method for preparing the polypeptide according to claim 2, characterized in that, The solvent for the carboxyl activation reaction is 1,4-dioxane, tetrahydrofuran, acetonitrile, dichloromethane, N,N - Dimethylformamide or dimethyl sulfoxide; the molar ratio of the compound to be activated to NHS and DCC is 1:1.0~3.0:1.0~4.0, and the compound to be activated is compound B, compound C, compound E or compound M; The aprotic solvent is selected from tetrahydrofuran, acetonitrile, etc. N,N -Dimethylformamide or dimethyl sulfoxide; The concentration of the compound B-OSu or C-OSu solution is 1.0~4.0 mmol / mL; The organic base refers to triethylamine or N,N -Diisopropylethylamine; the temperature of the amide condensation reaction is 0~30℃; the reaction time is 2h~15h.

7. The method for preparing the polypeptide according to claim 3, characterized in that, The solvent for the ring-opening reaction is methanol / water, ethanol / water, isopropanol / water, tetrahydrofuran / water, or acetonitrile / water; the strong base is sodium hydroxide, potassium hydroxide, or lithium hydroxide; the temperature of the ring-opening reaction is 0~30℃; the reaction time is 1~6h; and the molar ratio between the compound to be ring-opened and the strong base is 1:2.0~4.

0.

8. The method for preparing the polypeptide according to claim 3, characterized in that, The solvent for the deprotection reaction of the Cbz protecting group is dichloromethane, 1,2-dichloroethane, tetrahydrofuran, toluene, or 1,4-dioxane; The reducing agent refers to triethylsilane or triisopropylsilane; The temperature for the deprotection reaction of the Cbz group is 40~80℃; the reaction time is 2~16h; and the molar ratio of the ring-opening compound, trifluoroacetic acid and reducing agent is 1:20~40:10~20.

9. The method for preparing the polypeptide according to claim 4, characterized in that, Adjust the pH to 0.5-2; the solvent for the cyclization reaction is toluene, xylene, cyclohexane or n-heptane; the molar ratio of compound A, paraformaldehyde and p-toluenesulfonic acid monohydrate is 1:1-2:0.008-0.

02.

10. The use of the polypeptide prepared by the method according to any one of claims 6-9 in the preparation of polypeptide drugs or health care products.

Images