Synthesis method of high-purity copper peptide

Abstract

The application provides a synthesis method of high-purity copper peptide and belongs to the technical field of polypeptide synthesis. The method comprises the following steps: taking o-phthaloylglycyl chloride and L-histidine protected by hexamethyldisilazane as raw materials to react to obtain Pht-Gly-His-OH; protecting Pht-Gly-His-OH by Boc anhydride to obtain Pht-Gly-His(Boc)-OH; activating the carboxyl group of Pht-Gly-His(Boc)-OH to obtain Pht-Gly-His(Boc)-OSu; allowing Pht-Gly-His(Boc)-OSu to react with H-Lys(Boc)-OH to obtain Pht-Gly-His(Boc)-Lys(Boc)-OH; removing the Boc protecting group to obtain Pht-Gly-His-Lys-OH; and finally removing the o-phthaloyl group to obtain GHK.

Description

Technical Field

[0001] This invention belongs to the field of polypeptide synthesis technology, specifically a method for synthesizing high-purity copper peptides. Background Technology

[0002] Glycyl-L-histyl-L-lysine (GHK) is a naturally occurring tripeptide found in human blood plasma, and its copper complex, copper peptide, is an important metallopeptide complex in vivo. Copper peptides play multiple biological functions in tissue repair, extracellular matrix remodeling, anti-inflammatory regulation, and redox homeostasis.

[0003] The discovery of copper peptides dates back to 1973 when Loren Pickart isolated the tripeptide GHK from human plasma, which exhibited hepatocyte proliferation-promoting activity, and found that it existed in the form of a copper complex. This discovery sparked a surge of research into endogenous bioactive copper peptides. GHK has a simple amino acid sequence—glycine (Gly), histidine (His), and lysine (Lys) linked in sequence—but it is precisely this simple structure that endows it with complex biological functions.

[0004] The multifunctionality of copper peptides stems from their unique coordination chemistry. The imidazole ring of the histidine residue and the terminal amino group of glycine together form a tripentate ligand, which interacts with Cu... 2+ A stable planar quadrilateral coordination configuration is formed, while the amino group of the lysine side chain participates in the recognition of the target cell receptor. This structure allows the copper peptide to act as both a "signal peptide" to regulate gene expression and a "carrier peptide" to precisely deliver copper ions to target cells.

[0005] In recent years, the application boundaries of copper peptides have been continuously expanding. In the cosmetics field, it is hailed as the "golden peptide for anti-wrinkle"; in the field of regenerative medicine, it has shown the potential to promote wound healing, hair follicle regeneration, and nerve repair; and the latest research has extended its application to bone tissue engineering and the prevention and treatment of neurodegenerative diseases.

[0006] Traditional methods for synthesizing GHK primarily rely on classical solid-phase synthesis, which is hampered by high costs and limitations for industrial production. Existing reports on liquid-phase synthesis of GHK can be broadly categorized into two types: those employing condensing agents throughout the synthesis process and those completely eliminating condensing agents. The first type, using condensing agents, is relatively expensive, and the removal of byproducts generated by the condensing agents is a significant challenge. The second type, eliminating condensing agents, suffers from insufficient transesterification activity, potentially leading to side reactions and impurities that negatively impact the final product's quality and yield.

[0007] For example, patent application number CN202010593858.9 discloses a method for synthesizing GHK tripeptide, including the following steps:

[0008] (1) Under the action of sodium hydroxide, glycine reacts with Boc anhydride in 1,4-dioxane. After the reaction is completed, Boc-Gly-OH is obtained by post-treatment.

[0009] (2) Under the action of triethylamine, histidine reacts with dimethyldichlorosilane in dichloromethane. After the reaction is completed, a dichloromethane solution containing triphenylchloromethane and triethylamine are added to continue the reaction. After the reaction is completed, the reaction is purified.

[0010] (3) Lysine or its salt reacts with Boc-anhydride in a mixed solvent of acetone and water under the action of sodium bicarbonate and copper sulfate pentahydrate. After the reaction is completed, Boc-Lys-OH is obtained by purification.

[0011] (4) Under the action of DCC, the Boc-Gly-OH obtained in step (1) reacts with Hosu in 1,4-dioxane. After the reaction is completed, Boc-Gly-Osu is obtained by purification.

[0012] (5) Under the action of DIPEA, Boc-Gly-OSu from step (4) and Trt-His-OH from step (2) react in DMF. After the reaction is completed, Boc-Gly-His(Trt)-OH is obtained by purification.

[0013] (6) Under the action of DCC, Boc-Gly-His(Trt)-OH from step (5) reacts with Hosu in dioxane. After the reaction is completed, Boc-Gly-His(Trt)-Osu is obtained after post-treatment.

[0014] (7) Under the action of sodium bicarbonate, Boc-Lys-OH from step (3) and Boc-Gly-His(Trt)-OSu from step (6) react in a mixed solvent of tetrahydrofuran and water. After the reaction is completed, Boc-Gly-His(Trt)-Lys(Boc)-OH is obtained by purification.

[0015] (8) Under the action of triethylsilane, Boc-Gly-His(Trt)-Lys(Boc)-OH and trifluoroacetic acid in step (7) react in dichloromethane. After the reaction is completed, GHK tripeptide trifluoroacetate is obtained by purification.

[0016] (9) The GHK tripeptide trifluoroacetate from step (8) was dissolved in a mixed solution of ethyl acetate and methanol, and then diethylamine was added until a white precipitate was formed. The mixture was filtered, the filter cake was collected, and the GHK tripeptide was purified to obtain the GHK tripeptide.

[0017] In the aforementioned patent, condensing agents were used in steps (4)-(6). The use of condensing agents and the removal of the generated condensing agent byproducts led to a decrease in yield and a relatively high cost. From the Boc-Gly-OSu step, the total yield was only about 60%, which is low. The purity of the obtained GHK tripeptide was only 96%. Summary of the Invention

[0018] This invention provides a simple, low-cost, and high-purity synthetic method for copper peptides using inexpensive and readily available glycine, L-histidine, and H-Lys(Boc)-OH as raw materials. The method is as follows: This invention provides a method for synthesizing high-purity copper peptides, and the reaction formula for the synthesis is as follows: .

[0019] Includes the following steps: S1: Synthesis of Pht-Gly-His-OH L-histidine protected by phthaloylglycyl chloride and hexamethyldisilazane was reacted at room temperature. After the reaction was completed, the solution was concentrated, and then an alcoholic solution of base A was added to the concentrate. Pht-Gly-His-OH was obtained by solid-liquid separation.

[0020] S2: Synthesis of Pht-Gly-His-Lys-OH Pht-Gly-His-OH was obtained by protecting Pht-Gly-His(Boc)-OH with Boc anhydride; Pht-Gly-His(Boc)-OH was then reacted with N-hydroxysuccinimide in the presence of a condensing agent to activate the carboxyl group, yielding Pht-Gly-His(Boc)-OSu; Pht-Gly-His(Boc)-OSu was then reacted with H-Lys(Boc)-OH at room temperature to obtain Pht-Gly-His(Boc)-Lys(Boc)-OH; finally, the Boc protecting group was removed at room temperature in the presence of a deprotecting reagent. After the reaction was completed, the aqueous layer was extracted, concentrated under reduced pressure, and then alkali solution B was added to crystallize Pht-Gly-His-Lys-OH.

[0021] S3: GHK Synthesis In solvent D, under the action of a phthaloyl-removing reagent, Pht-Gly-His-Lys-OH is dephthaloylated to obtain GHK. The phthaloyl-removing reagent is selected from hydrazine hydrate, n-butylamine, or n-propylamine, etc., and solvent D is selected from water, methanol, or ethanol, etc. When the phthaloyl-removing reagent is selected from hydrazine hydrate, solvent D is selected from water. After the reaction is complete, the pH is adjusted to 3.8-4.4 with acid, the phthaloyl hydrazine is removed by filtration, the filtrate is concentrated to dryness under reduced pressure, the pH is adjusted to 7.0-8.0 with alkali, and crude GHK is obtained by solvent crystallization. When the phthaloyl-removing reagent is selected from n-butylamine or n-propylamine, solvent D is selected from methanol or ethanol. After the reaction is complete, solvent D is removed by reduced pressure concentration, water is added, and the pH is adjusted to 3.8-4.4 with acid. The aqueous layer is extracted, concentrated to dryness under reduced pressure, the pH is adjusted to 7.0-8.0 with alkali, and crude GHK is obtained by solvent crystallization.

[0022] S4: GHK Synthesis GHK is complexed with copper salts to obtain copper peptides.

[0023] Specifically, step S1 includes: S101: In a solvent, glycine and phthalic anhydride react under the catalysis of a base to give phthaloylglycine. Then, thionyl chloride is added dropwise at 60-70℃ to give phthaloylglycyl chloride. The molar ratio of glycine, phthalic anhydride, and thionyl chloride is 1.0:1.0-1.05:1.0-1.5; the mass ratio of glycine to base is 1.0:0.0001-0.001.

[0024] S102: In a solvent, L-histidine and hexamethyldisilazane react under acid catalysis via reflux to yield a protected histidine compound. The molar ratio of L-histidine to hexamethyldisilazane is 1.0:1.5-2.0; the mass ratio of L-histidine to acid is 1.0:0.0001-0.001.

[0025] S103: At room temperature, phthaloylglycyl chloride reacts with histidine protecting compound. After the reaction is complete, the mixture is concentrated, and an alcoholic solution of base A is added to the concentrate. Solid-liquid separation yields Pht-Gly-His-OH. This process simultaneously achieves deprotection and purification. The molar ratio of histidine protecting compound to phthaloylglycyl chloride is calculated based on the molar ratio of L-histidine to glycine: L-histidine to glycine is 1.0:1.0-1.2. The molar ratio of histidine protecting compound to base A is calculated based on the molar ratio of L-histidine to base A: L-histidine to base A is 1.0:0.9-1.2.

[0026] Among them, base A is selected from sodium bicarbonate, sodium carbonate, potassium carbonate, sodium hydroxide, potassium hydroxide, triethylamine or imidazole, etc., preferably imidazole.

[0027] The alcohol is selected from methanol, ethanol or isopropanol, etc., and is preferably methanol.

[0028] Steps S101 and S102 are standard procedures, and detailed descriptions are omitted in this embodiment. The solvents (such as toluene, benzene, xylene, DMF, etc.) used in steps S101 and S102 may be the same or different, but toluene can be used in both cases. Specifically, the base can be triethylamine, and the acid can be concentrated sulfuric acid.

[0029] Specifically, step S2 includes: S201: Synthesis of Pht-Gly-His(Boc)-OH In water and aprotic solvent A, at room temperature, Boc anhydride reacts with Pht-Gly-His-OH in the presence of an acid-binding agent to yield Pht-Gly-His(Boc)-OH. The molar ratio of Pht-Gly-His-OH, Boc anhydride, and the acid-binding agent is 1.0:1.0-1.2:2.0-2.5.

[0030] S202: Synthesis of Pht-Gly-His(Boc)-OSu In aprotic solvent B, at room temperature, Pht-Gly-His(Boc)-OH reacts with N-hydroxysuccinimide in the presence of a condensing agent, activating the carboxyl group to yield Pht-Gly-His(Boc)-OSu. The molar ratio of Pht-Gly-His(Boc)-OH, N-hydroxysuccinimide, and the condensing agent is 1.0:1.0-1.2:1.0-1.2.

[0031] S203: Synthesis of Pht-Gly-His(Boc)-Lys(Boc)-OH In an aprotic solvent C, at room temperature and under the action of base C, Pht-Gly-His(Boc)-OSu and H-Lys(Boc)-OH condense to give Pht-Gly-His(Boc)-Lys(Boc)-OH. The molar ratio of Pht-Gly-His(Boc)-OSu, H-Lys(Boc)-OH, and base C is 1.0:1.0-1.1:2.0-2.5.

[0032] S204: Synthesis of Pht-Gly-His-Lys-OH Under the action of a Boc-removing protecting agent, Pht-Gly-His(Boc)-Lys(Boc)-OH undergoes Boc-removing protection at room temperature. After the reaction is complete, the aqueous layer is extracted, concentrated under reduced pressure, and then crystallized with alkali B solution to obtain Pht-Gly-His-Lys-OH. The mass ratio of Pht-Gly-His(Boc)-Lys(Boc)-OH to the Boc-removing protecting agent is 1.0:1.0-10.0, and the molar ratio of Pht-Gly-His(Boc)-Lys(Boc)-OH to alkali B is 1.0:1.8-2.2.

[0033] In step S2, aprotic solvent A, aprotic solvent B, and aprotic solvent C may be the same or different.

[0034] The acid-binding agent is selected from sodium bicarbonate, sodium carbonate, potassium carbonate, sodium hydroxide, potassium hydroxide, or triethylamine, etc., with sodium bicarbonate being preferred.

[0035] Among them, base B is selected from sodium bicarbonate, sodium carbonate, potassium carbonate, sodium hydroxide, potassium hydroxide, triethylamine or imidazole, etc., preferably sodium carbonate.

[0036] Among them, base C is selected from sodium bicarbonate, sodium carbonate, potassium carbonate, sodium hydroxide, potassium hydroxide or triethylamine, etc., preferably triethylamine.

[0037] The aprotic solvent A is selected from acetone, tetrahydrofuran, acetonitrile, dichloromethane, DMF or DMSO, etc., preferably tetrahydrofuran.

[0038] The aprotic solvent B is selected from acetone, tetrahydrofuran, acetonitrile, dichloromethane, DMF or DMSO, etc., preferably dichloromethane.

[0039] The aprotic solvent C is selected from acetone, tetrahydrofuran, acetonitrile, dichloromethane, DMF or DMSO, etc., preferably dichloromethane.

[0040] The condensing agent is selected from DCC, DIC, EDCI, HBTU or PyBOP, etc., with DIC being preferred.

[0041] The Boc-deprotecting reagent is selected from trifluoroacetic acid, hydrochloric acid, tetrahydrofuran solution of hydrogen chloride, dichloromethane solution of hydrogen chloride, or ethyl acetate solution of hydrogen chloride, etc., preferably hydrochloric acid.

[0042] Specifically, step S3 includes: In solvent D, under the action of a phthaloyl-removing reagent, Pht-Gly-His-Lys-OH undergoes the removal of the phthaloyl group to obtain GHK. The molar ratio of Pht-Gly-His-Lys-OH to the phthaloyl-removing reagent is 1:1-10; solvent D is selected from water, methanol, or ethanol, etc., and the phthaloyl-removing reagent is selected from hydrazine hydrate, n-butylamine, or n-propylamine, etc.

[0043] In one embodiment, when the phthaloyl removal reagent is selected from hydrazine hydrate, the solvent D is selected from water. After the reaction is completed, the pH is adjusted to 3.8-4.4 with hydrochloric acid, the phthaloyl hydrazine is removed by filtration, the filtrate is concentrated to dryness under reduced pressure, the pH is adjusted to 7.0-8.0 with ammonia, ethanol is added for crystallization to obtain crude GHK, and then purified GHK is obtained by crystallization with water and ethanol.

[0044] In another embodiment, when the phthaloyl removal reagent is selected from n-butylamine or n-propylamine, the solvent D is selected from methanol or ethanol. After the reaction is completed, solvent D is removed by vacuum concentration, water is added, and the pH is adjusted to 3.8-4.4 with hydrochloric acid. The mixture is extracted with dichloromethane to obtain an aqueous layer. The aqueous layer is concentrated to dryness under vacuum, and the pH is adjusted to 7.0-8.0 with ammonia. Ethanol is added to crystallize and obtain crude GHK. Then, purified GHK is obtained by crystallization with water and ethanol.

[0045] Step S4 specifically includes: complexing GHK and copper salt in water at room temperature to obtain copper peptides. The molar ratio of GHK to copper salt is 1.0:0.5-1.5.

[0046] The copper salt is selected from copper acetate, copper acetate monohydrate, copper sulfate, copper sulfate pentahydrate, copper chloride dihydrate, or copper chloride, etc., with copper chloride dihydrate being preferred.

[0047] Preferably, in step S1, a one-pot method can be used to complete the three-step reaction to obtain the intermediate Pht-Gly-His-OH, in which case the corresponding solvent is the same, and the raw materials are added in the order of steps S101-S103 (added after the corresponding reaction is completed). In step S2, a one-pot method can be used to complete the four-step reaction to obtain the intermediate Pht-Gly-His-Lys-OH, in which case the corresponding solvent is the same.

[0048] Specifically, the present invention provides a method for synthesizing high-purity copper peptides, the method comprising the following steps: (1) Synthesis of Pht-Gly-His-OH a) Synthesis of phthaloylglycyl chloride In a solvent, glycine and phthalic anhydride react under alkaline catalysis via reflux to yield phthaloylglycine. After the reaction is complete, the temperature is lowered to 60-70℃, thionyl chloride is added dropwise, and the mixture is kept at this temperature until the reaction is complete, yielding phthaloylglycyl chloride. The molar ratio of glycine, phthalic anhydride, and thionyl chloride is 1.0:1.00-1.05:1.0-1.5; the mass ratio of glycine to alkaline is 1.0:0.0001-0.001.

[0049] b) Synthesis of histidine protected compounds In a solvent, L-histidine and hexamethyldisilazane react under acid catalysis via reflux to yield a protected histidine compound. The molar ratio of L-histidine to hexamethyldisilazane is 1.0:1.5-2.0; the mass ratio of L-histidine to acid is 1.0:0.0001-0.001.

[0050] c) Synthesis of Pht-Gly-His-OH Phthaloylglycyl chloride and histidine protecting compound react at room temperature. After the reaction is complete, the mixture is concentrated, and then an alcoholic solution of base A is added to the concentrate. Solid-liquid separation yields Pht-Gly-His-OH. Base A is selected from sodium bicarbonate, sodium carbonate, potassium carbonate, sodium hydroxide, potassium hydroxide, triethylamine, or imidazole, etc.; the alcohol is selected from methanol, ethanol, or isopropanol, etc.; the molar ratio of histidine protecting compound to phthaloylglycyl chloride is calculated based on the molar ratio of L-histidine to glycine: the molar ratio of L-histidine to glycine is 1.0:1.0-1.2; the molar ratio of histidine protecting compound to base A is calculated based on the molar ratio of L-histidine to base A: the molar ratio of L-histidine to base A is 1.0:0.9-1.2.

[0051] (2) Synthesis of Pht-Gly-His-Lys-OH a) Synthesis of Pht-Gly-His(Boc)-OH In water, Pht-Gly-His-OH is reacted dropwise with a solution of aprotic solvent A (Boc anhydride) in the presence of an acid-binding agent to yield Pht-Gly-His(Boc)-OH at room temperature. The molar ratio of Pht-Gly-His-OH, Boc anhydride, and the acid-binding agent is 1.0:1.0-1.2:2.0-2.5. The acid-binding agent is selected from sodium bicarbonate, sodium carbonate, potassium carbonate, sodium hydroxide, potassium hydroxide, or triethylamine, etc.; the aprotic solvent A is selected from acetone, tetrahydrofuran, acetonitrile, dichloromethane, DMF, or DMSO, etc.

[0052] b) Synthesis of Pht-Gly-His(Boc)-OSu Pht-Gly-His(Boc)-OH and N-hydroxysuccinimide react at room temperature in an aprotic solvent B to yield Pht-Gly-His(Boc)-OSu under the action of a condensing agent. The molar ratio of Pht-Gly-His(Boc)-OH, N-hydroxysuccinimide, and the condensing agent is 1.0:1.0-1.2:1.0-1.2. The aprotic solvent B is selected from acetone, tetrahydrofuran, acetonitrile, dichloromethane, DMF, or DMSO, etc.; the condensing agent is selected from DCC, DIC, EDCI, HBTU, or PyBOP, etc.

[0053] c) Synthesis of Pht-Gly-His(Boc)-Lys(Boc)-OH In an aprotic solvent C, H-Lys(Boc)-OH reacts with Pht-Gly-His(Boc)-OSu at room temperature under the action of base C to yield Pht-Gly-His(Boc)-Lys(Boc)-OH. The molar ratio of Pht-Gly-His(Boc)-OSu, H-Lys(Boc)-OH, and base C is 1.0:1.0-1.2:2.0-2.5. The aprotic solvent C is selected from acetone, tetrahydrofuran, acetonitrile, dichloromethane, DMF, or DMSO, etc.; the base C is selected from sodium bicarbonate, sodium carbonate, potassium carbonate, sodium hydroxide, potassium hydroxide, or triethylamine, etc.

[0054] d) Synthesis of Pht-Gly-His-Lys-OH At room temperature, Pht-Gly-His(Boc)-Lys(Boc)-OH reacts with a Boc-removing reagent. After the reaction is complete, the mixture is extracted to obtain an aqueous layer, which is concentrated under reduced pressure. A base B solution is added, precipitating a large amount of solid. The solid is then filtered to obtain Pht-Gly-His-Lys-OH. The Boc-removing reagent is selected from hydrochloric acid, tetrahydrofuran solution of hydrogen chloride, dichloromethane solution of hydrogen chloride, ethyl acetate solution of hydrogen chloride, or trifluoroacetic acid, etc.; the mass ratio of Pht-Gly-His(Boc)-Lys(Boc)-OH to the Boc-removing reagent is 1.0:1.0-10.0; the molar ratio of Pht-Gly-His(Boc)-Lys(Boc)-OH to base B is 1.0:1.8-2.2; and base B is selected from sodium bicarbonate, sodium carbonate, potassium carbonate, sodium hydroxide, potassium hydroxide, triethylamine, or imidazole, etc.

[0055] (3) Synthesis of GHK In solvent D, Pht-Gly-His-Lys-OH is dephthalated by a phthaloyl-removing reagent to obtain GHK. The molar ratio of Pht-Gly-His-Lys-OH to the phthaloyl-removing reagent is 1:1.0-1.5; solvent D is selected from water, methanol, or ethanol; and the phthaloyl-removing reagent is selected from hydrazine hydrate, n-butylamine, or n-propylamine.

[0056] When the phthaloyl removal reagent is selected from hydrazine hydrate, solvent D is selected from water. After the reaction is completed, the pH is adjusted to 3.8-4.4 with acid, the phthaloyl hydrazine is removed by filtration, the filtrate is concentrated to dryness under reduced pressure, the pH is adjusted to 7.0-8.0 with alkali, and crude GHK is obtained by solvent crystallization. Then, purified GHK is obtained by crystallization with water and ethanol.

[0057] In this process, when the phthaloyl removal reagent is selected from n-butylamine or n-propylamine, the solvent D is selected from methanol or ethanol. After the reaction is completed, the solvent D is removed by vacuum concentration, water is added, and the pH is adjusted to 3.8-4.4 with acid. The aqueous layer is extracted and concentrated to dryness under vacuum. The pH is adjusted to 7.0-8.0 with alkali. Crude GHK is obtained by solvent crystallization, and then purified GHK is obtained by crystallization with water and ethanol.

[0058] (4) Synthesis of copper peptides In water, GHK and copper salt complex at room temperature to obtain copper peptides. The molar ratio of GHK to copper salt is 1.0:0.5-1.5; wherein the copper salt is selected from copper acetate, copper acetate monohydrate, copper sulfate, copper sulfate pentahydrate, copper chloride dihydrate, or copper chloride, etc.

[0059] Preferably, the present invention provides a method for synthesizing high-purity copper peptides, the method comprising the following steps: (1) Synthesis of Pht-Gly-His-OH a) Synthesis of phthaloylglycyl chloride In toluene, glycine and phthalic anhydride react under triethylamine catalysis via reflux to yield phthaloylglycine. After the reaction is complete, the temperature is lowered to 60-70℃, thionyl chloride is added dropwise, and the mixture is kept at this temperature. After the reaction is complete, the mixture is concentrated to dryness under reduced pressure, and toluene is added to obtain a phthaloylglycyl chloride solution. The molar ratio of glycine, phthalic anhydride, and thionyl chloride is 1.0:1.00-1.05:1.0-1.5; the mass ratio of glycine to triethylamine is 1.0:0.0001-0.001.

[0060] b) Synthesis of histidine protected compounds In toluene, L-histidine and hexamethyldisilazane react under concentrated sulfuric acid catalysis via reflux to yield a protected histidine compound. The molar ratio of L-histidine to hexamethyldisilazane is 1.0:1.5-2.0; the mass ratio of L-histidine to sulfuric acid is 1.0:0.0001-0.001.

[0061] c) Synthesis of Pht-Gly-His-OH Phthalylglycyl chloride solution and histidine protectant were reacted at room temperature. After the reaction was complete, the mixture was concentrated, and then an imidazole methanol solution was added to the concentrate. Solid-liquid separation was performed to obtain Pht-Gly-His-OH. The molar ratio of histidine protectant to phthaloylglycyl chloride was calculated based on the molar ratio of L-histidine to glycine: L-histidine to glycine was 1.0:1.0-1.2; the molar ratio of histidine protectant to imidazole was calculated based on the molar ratio of L-histidine to imidazole: L-histidine to imidazole was 1.0:0.9-1.2.

[0062] (2) Synthesis of Pht-Gly-His-Lys-OH a) Synthesis of Pht-Gly-His(Boc)-OH Pht-Gly-His-OH was reacted in water with a solution of Boc anhydride in tetrahydrofuran by dropwise addition of sodium bicarbonate at room temperature. After the reaction was complete, tetrahydrofuran was removed by vacuum distillation, followed by extraction with dichloromethane. The organic layer was washed twice with water and dried to obtain Pht-Gly-His(Boc)-OH. The molar ratio of Pht-Gly-His-OH, Boc anhydride, and sodium bicarbonate was 1.0:1.0-1.2:2.0-2.5.

[0063] b) Synthesis of Pht-Gly-His(Boc)-OSu Pht-Gly-His(Boc)-OH and N-hydroxysuccinimide react in dichloromethane at room temperature under the action of DIC to yield Pht-Gly-His(Boc)-OSu. The molar ratio of Pht-Gly-His(Boc)-OH, N-hydroxysuccinimide, and DIC is 1.0:1.0-1.2:1.0-1.2.

[0064] c) Synthesis of Pht-Gly-His(Boc)-Lys(Boc)-OH In dichloromethane, H-Lys(Boc)-OH reacts with Pht-Gly-His(Boc)-OSu in the presence of triethylamine at room temperature. After the reaction is complete, the mixture is washed with water, and the organic layer is concentrated under reduced pressure to obtain Pht-Gly-His(Boc)-Lys(Boc)-OH. The molar ratio of Pht-Gly-His(Boc)-OSu, H-Lys(Boc)-OH, and triethylamine is 1.0:1.0-1.2:2.0-2.5.

[0065] d) Synthesis of Pht-Gly-His-Lys-OH At room temperature, Pht-Gly-His(Boc)-Lys(Boc)-OH reacts with hydrochloric acid. After the reaction is complete, the aqueous phase is extracted with dichloromethane to obtain an aqueous layer. The layer is concentrated under reduced pressure, and sodium carbonate solution is added, precipitating a large amount of solid. The mixture is then cooled and filtered to obtain Pht-Gly-His-Lys-OH. The mass ratio of Pht-Gly-His(Boc)-Lys(Boc)-OH to hydrochloric acid is 1.0:1.0-10.0; the molar ratio of Pht-Gly-His(Boc)-Lys(Boc)-OH to sodium carbonate is 1.0:1.8-2.2.

[0066] (3) Synthesis of GHK In water, under the action of hydrazine hydrate, Pht-Gly-His-Lys-OH loses the phthaloyl group. After the reaction is complete, the pH is adjusted to 3.8-4.4 with hydrochloric acid, the phthaloyl hydrazine is removed by filtration, the filtrate is concentrated to dryness under reduced pressure, the pH is adjusted to 7.0-8.0 with ammonia, ethanol is added for crystallization, solid-liquid separation is performed to obtain crude GHK, and then purified GHK is obtained by crystallization with water and ethanol.

[0067] (4) Synthesis of copper peptides In water, GHK and copper chloride dihydrate complexed at room temperature. After the reaction was complete, the mixture was concentrated under reduced pressure to a paste, ethanol was added, and the solid-liquid mixture was separated to obtain copper peptides. The molar ratio of GHK to copper chloride dihydrate was 1.0:0.5-1.5.

[0068] In peptide synthesis, the amino acid protection strategies (there are many protecting groups for amino and carboxyl groups) and the acid-amine condensation methods (there are also many acid-amine condensation methods) have a significant impact on reaction conditions, purification processes, yield, purity, optical rotation, impurity composition, and suitability for industrial production. Although current technologies generally employ amino and carboxyl protection for amino acids and acid-amine condensation methods that activate carboxyl groups for peptide synthesis, the sheer number of amino acid protecting groups and the different carboxyl activation methods within acid-amine condensation can lead to a wide range of results, potentially resulting in substantial differences.

[0069] This patent has the following advantages: (1) This invention uses glycine, L-histidine and H-Lys(Boc)-OH as raw materials. Among them, glycine and L-histidine are unprotected amino acids. Compared with most synthesis methods, which use protected glycine and protected histidine as raw materials, the raw material cost will be much lower.

[0070] (2) In the synthesis method of GHK of the present invention, the acid-amine condensation method of the intermediate Pht-Gly-His-OH in the first half adopts the acyl chloride method, which directly uses phthaloylglycyl chloride and histidine protecting agent to obtain it, thus avoiding the use of high-cost condensing agents in this step. Although the synthesis of the intermediate Pht-Gly-His-Lys-OH in the second half of the synthesis method of GHK adopts the active ester method, which uses condensing agents (subsequent purification is simple, yield is 97.6%, purity is 99.2%), the reaction conditions are mild, the selectivity is high, there are few impurities, and it is easy to purify in the later stage to obtain high-purity GHK. Compared with the method that does not use condensing agents at all, when the amine ester exchange is used in the second half, the methyl ester has low activity, the reaction temperature of the amine ester exchange is high, there are many side reactions and many impurities, which is a major challenge to the final purification and yield of GHK.

[0071] (3) The use of TMS protecting groups to protect the amino, carboxyl, and imidazole groups of L-histidine effectively prevents histidine racemization. Simultaneously, Boc protection of Pht-Gly-His-OH yields Pht-Gly-His(Boc)-OH, which, upon carboxyl activation and condensation with H-Lys(Boc)-OH, also effectively prevents histidine racemization. Final amino acid analysis showed HisR / S < 0.001.

[0072] (4) This patent optimizes the screening process by using phthaloyl as the first-step protecting group. The resulting intermediates Pht-Gly-His-OH and Pht-Gly-His-Lys-OH are good solids, avoiding the formation of oily or semi-solid products that are not easy to solidify by intermediates such as Boc protecting groups. This allows for effective purification and control of impurities, creating conditions for obtaining high-purity GHK and copper peptides. It also avoids the use of high-cost Fmoc protecting groups and Cbz protecting groups that require special catalytic hydrogenolysis equipment.

[0073] (5) The key first and second steps both adopt the "one-pot method" to obtain the key intermediates Pht-Gly-His-OH and Pht-Gly-His-Lys-OH, which is simple to operate. (6) Compared to most synthetic methods that ultimately remove the Boc protecting group from trifluoroacetic acid, hydrochloric acid, or tetrahydrofuran solution of hydrogen chloride to obtain GHK, this method directly yields GHK in a 3-molecule anion form, such as the trihydrochloride of GHK, i.e., GHK.3HCl. Since its solubility in organic solvents is very low, while its solubility in water is too high, which is not conducive to crystallization and purification, the applicant found that the free form of GHK has excellent solubility in water but poor solubility in organic solvents. Therefore, this invention utilizes the solubility characteristics of different salt forms of GHK. By controlling the pH value, GHK is controlled in a 1-anion salt form, i.e., the GHK.HCl form. A pH of 7.0-8.0 effectively avoids the problems of excessive solubility in the 3-molecule anion salt form, the 2-molecule anion salt form, and the free form, thus obtaining the monohydrochloride form of GHK, GHK.HCl. GHK.HCl is easy to crystallize, thereby solving the purification problem of GHK.

[0074] In summary, this patent directly yields an easily separable and processable solid in step 1 (with a yield close to 99%), with a purity greater than 99%. In step 2, it also directly yields an easily separable and processable solid (with a yield close to 98%), with a purity greater than 99%. This high yield and high purity product is extremely rare in organic synthesis. In step 3, purification with only ethanol achieves a purity of 99.5% and a yield of over 91%, while maintaining an extremely low histidine racemization. The final product, copper peptide, can be easily purified (using ethanol; this patent uses ethanol for purification in multiple steps, resulting in low cost and suitability for industrial production) to achieve a purity of 99.6%. Detailed Implementation

[0075] To further illustrate the present invention, the following detailed description of the embodiments is provided in conjunction with the present invention, but these descriptions should not be construed as limiting the scope of protection of the present invention.

[0076] Example 1: Synthesis of Pht-Gly-His-OH a) Synthesis of phthaloylglycyl chloride Add 80.0g glycine, 159.4g phthalic anhydride, and 450.0g toluene to a four-necked flask, then add 0.04g triethylamine. Heat to reflux and react. After the reaction is complete, cool to 60-70℃ and add 152.1g thionyl chloride dropwise. After the addition is complete, keep warm until the reaction is complete, concentrate under reduced pressure to dryness, and then add 750g toluene to obtain a phthaloylglycyl chloride solution.

[0077] b) Synthesis of histidine protected compounds 150g L-histidine, 280g hexamethyldisilazane, 300g toluene and 0.075g concentrated sulfuric acid were added to a four-necked flask. The mixture was heated to reflux and the reaction was completed. The mixture was then cooled to room temperature to obtain the histidine protected compound.

[0078] c) Synthesis of Pht-Gly-His-OH At room temperature, phthaloylglycyl chloride solution was added dropwise to histidine protecting agent. After the reaction was complete, a peptide synthesis solution was obtained. The peptide synthesis solution was then concentrated under reduced pressure. After concentration, 65.8 g of imidazole in 600 g of methanol solution was added, and a large amount of solid precipitated by stirring. Pht-Gly-His-OH was obtained by filtration and dried to obtain 326.9 g, with a yield of 98.8% and a purity of 99.2%.

[0079] Example 2: Synthesis of Pht-Gly-His-Lys-OH.HCl a) Synthesis of Pht-Gly-His(Boc)-OH Add 320.0 g Pht-Gly-His-OH, 1000.0 g water, and 172.8 g sodium bicarbonate to a four-necked flask. At room temperature, add 244.8 g of a 250 g tetrahydrofuran solution of Boc anhydride dropwise. After the reaction is complete, concentrate under reduced pressure to remove the tetrahydrofuran. Extract with 1000 g dichloromethane to obtain the organic layer. Use 500 g of [unspecified substance] to [unspecified product]. Wash twice with water and dry with anhydrous magnesium sulfate to obtain a dichloromethane solution of Pht-Gly-His(Boc)-OH.

[0080] b) Synthesis of Pht-Gly-His(Boc)-OSu To the above Pht-Gly-His(Boc)-OH dichloromethane solution, add 118.4 g of N-hydroxysuccinimide, and at room temperature, add 130.0 g of DIC in 150 g of dichloromethane solution dropwise. After the reaction is complete, Pht-Gly-His(Boc)-OSu dichloromethane solution is obtained.

[0081] c) Synthesis of Pht-Gly-His(Boc)-Lys(Boc)-OH Add 1000g of dichloromethane, 253.3g of H-Lys(Boc)-OH, and 208.1g of triethylamine to a four-necked flask. Add Pht-Gly-His(Boc)-OSu dichloromethane solution dropwise at room temperature. After the reaction is complete, use 1000g of... Wash three times with water to obtain an organic layer, concentrate under reduced pressure to dryness to obtain Pht-Gly-His(Boc)-Lys(Boc)-OH.

[0082] d) Synthesis of Pht-Gly-His-Lys-OH Pht-Gly-His(Boc)-Lys(Boc)-OH and 800g of 30% hydrochloric acid were added to a four-necked flask. After the reaction was complete, 500g of [a solution was used]. The aqueous phase was extracted three times with dichloromethane to remove the byproduct DIU (a byproduct of DIC), and the aqueous layer was concentrated to dryness under reduced pressure to remove excess hydrochloric acid. Then, 550g of an aqueous solution of 99.1g sodium carbonate was added dropwise, and a large amount of solid precipitated from the system. The mixture was cooled to 0-10℃ to crystallize, filtered, and 462.5g of Pht-Gly-His-Lys-OH.HCl was obtained, with a yield of 97.6% and a purity of 99.2%.

[0083] Example 3: Synthesis of GHK.HCl 400 g of water, 35.3 g of hydrazine hydrate, and 220 g of Pht-Gly-His-Lys-OH.HCl were added to a four-necked flask. The mixture was heated to 75-85 °C. After the reaction was complete, the pH was adjusted to 3.8-4.4 with hydrochloric acid. The byproduct phthalyl hydrazide was removed by filtration. The filtrate was concentrated to dryness under reduced pressure. The pH was adjusted to 7.0-8.0 with ammonia. 600 g of anhydrous ethanol was added dropwise, and crystallization was performed. The mixture was then filtered to obtain crude GHK. The crude product was crystallized from water and anhydrous ethanol to obtain 149.0 g of purified GHK.HCl, with a yield of 91.1% and a purity of 99.5%. The amino acid analysis showed His R / S = 0.0006 and Lys R / S = 0.

[0084] Example 4: Synthesis of GHK.HCl Add 400g methanol, 158.7g n-butylamine, and 220g Pht-Gly-His-Lys-OH.HCl to a four-necked flask. Heat to reflux and react. After the reaction is complete, concentrate under reduced pressure to remove the solvent and excess n-butylamine. Add 400g water and adjust the pH to 3.8-4.4 with hydrochloric acid. Use 500g... 3. Dichloromethane extraction yielded an aqueous layer, which was concentrated to dryness under reduced pressure. The pH was adjusted to 7.0-8.0 with ammonia, and 600g of anhydrous ethanol was added dropwise. Crystallization was performed, and the product was filtered to obtain crude GHK. The crude product was crystallized from water and anhydrous ethanol to obtain 147.5g of purified GHK.HCl, with a yield of 90.2% and a purity of 99.6%. Amino acid determination showed His R / S = 0.0005 and Lys R / S = 0.

[0085] Example 5: Synthesis of copper peptides 140.0g GHK.HCl and water were added to a four-necked flask, followed by 63.4g of copper chloride dihydrate. The mixture was stirred until dissolved and concentrated under reduced pressure to a paste. Ethanol was added to precipitate a large amount of solid. The mixture was filtered to obtain 160.3g of copper peptide, with a yield of 98.4% and a purity of 99.6%.

[0086] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A method for synthesizing high-purity copper peptides, characterized in that, Includes the following steps: S1: Synthesis of Pht-Gly-His-OH L-histidine protected by phthaloylglycyl chloride and hexamethyldisilazane was reacted at room temperature. After the reaction was completed, the solution was concentrated, and then an alcoholic solution of base A was added to the concentrate. Pht-Gly-His-OH was obtained by solid-liquid separation. S2: Synthesis of Pht-Gly-His-Lys-OH Pht-Gly-His-OH was obtained by protecting Pht-Gly-His(Boc)-OH with Boc anhydride; Pht-Gly-His(Boc)-OH was then reacted with N-hydroxysuccinimide in the presence of a condensing agent to activate the carboxyl group, yielding Pht-Gly-His(Boc)-OSu; Pht-Gly-His(Boc)-OSu was then reacted with H-Lys(Boc)-OH at room temperature to obtain Pht-Gly-His(Boc)-Lys(Boc)-OH; finally, the Boc protecting group was removed at room temperature in the presence of a deprotecting reagent. After the reaction was complete, the aqueous layer was extracted, concentrated under reduced pressure, and then crystallized with alkali solution B to obtain Pht-Gly-His-Lys-OH. S3: GHK Synthesis In solvent D, under the action of a phthaloyl-removing reagent, Pht-Gly-His-Lys-OH is used to remove phthaloyl groups to obtain GHK. The phthaloyl-removing reagent is selected from hydrazine hydrate, n-butylamine, or n-propylamine, and solvent D is selected from water, methanol, or ethanol. When the phthaloyl-removing reagent is selected from hydrazine hydrate, solvent D is selected from water. After the reaction is complete, the pH is adjusted to 3.8-4.4 with acid, the phthaloyl hydrazine is removed by filtration, the filtrate is concentrated to dryness under reduced pressure, the pH is adjusted to 7.0-8.0 with alkali, and crude GHK is obtained by solvent crystallization. When the phthaloyl-removing reagent is selected from n-butylamine or n-propylamine, solvent D is selected from methanol or ethanol. After the reaction is complete, solvent D is removed by reduced pressure concentration, water is added, and the pH is adjusted to 3.8-4.4 with acid. The aqueous layer is extracted, concentrated to dryness under reduced pressure, the pH is adjusted to 7.0-8.0 with alkali, and crude GHK is obtained by solvent crystallization. S4: GHK Synthesis GHK is complexed with copper salts to obtain copper peptides.

2. The method for synthesizing high-purity copper peptide according to claim 1, characterized in that, Step S1 specifically includes: S101: In a solvent, glycine and phthalic anhydride react under the catalysis of an alkali to give phthaloylglycine, followed by the dropwise addition of thionyl chloride at 60-70°C to give phthaloylglycyl chloride; the molar ratio of glycine, phthalic anhydride and thionyl chloride is 1.0:1.0-1.05:1.0-1.5; the mass ratio of glycine to alkali is 1.0:0.0001-0.001; S102: In a solvent, L-histidine and hexamethyldisilazane react under acid catalysis by reflux to obtain a histidine protected compound; the molar ratio of L-histidine to hexamethyldisilazane is 1.0:1.5-2.0; the mass ratio of L-histidine to acid is 1.0:0.0001-0.001; S103: At room temperature, phthaloylglycyl chloride and histidine protectant react. After the reaction is complete, the mixture is concentrated, and an alcoholic solution of base A is added to the concentrate. Solid-liquid separation is performed to obtain Pht-Gly-His-OH. The molar ratio of the histidine protectant to phthaloylglycyl chloride is calculated based on the molar ratio of L-histidine to glycine: the molar ratio of L-histidine to glycine is 1.0:1.0-1.

2. The molar ratio of the histidine protectant to base A is calculated based on the molar ratio of L-histidine to base A: the molar ratio of L-histidine to base A is 1.0:0.9-1.

2.

3. The method for synthesizing high-purity copper peptide according to claim 2, characterized in that, The base A is selected from sodium bicarbonate, sodium carbonate, potassium carbonate, sodium hydroxide, potassium hydroxide, triethylamine, or imidazole; the alcohol is selected from methanol, ethanol, or isopropanol.

4. The method for synthesizing high-purity copper peptide according to claim 2, characterized in that, Step S2 specifically includes: S201: Synthesis of Pht-Gly-His(Boc)-OH In water and aprotic solvent A, at room temperature, Boc anhydride reacts with Pht-Gly-His-OH in the presence of an acid-binding agent to yield Pht-Gly-His(Boc)-OH, wherein the molar ratio of Pht-Gly-His-OH, Boc anhydride, and acid-binding agent is 1.0:1.0-1.2:2.0-2.5; S202: Synthesis of Pht-Gly-His(Boc)-OSu In aprotic solvent B, at room temperature, Pht-Gly-His(Boc)-OH and N-hydroxysuccinimide are reacted with a condensing agent to activate the carboxyl group, yielding Pht-Gly-His(Boc)-OSu. The molar ratio of Pht-Gly-His(Boc)-OH, N-hydroxysuccinimide, and the condensing agent is 1.0:1.0-1.2:1.0-1.

2. S203: Synthesis of Pht-Gly-His(Boc)-Lys(Boc)-OH Pht-Gly-His(Boc)-OSu and H-Lys(Boc)-OH condense in an aprotic solvent C at room temperature and under the action of base C to obtain Pht-Gly-His(Boc)-Lys(Boc)-OH; the molar ratio of Pht-Gly-His(Boc)-OSu, H-Lys(Boc)-OH and base C is 1.0:1.0-1.1:2.0-2.5; S204: Synthesis of Pht-Gly-His-Lys-OH Under the action of a Boc-removing protecting agent, Pht-Gly-His(Boc)-Lys(Boc)-OH loses its Boc protecting group at room temperature. After the reaction is complete, an aqueous layer is extracted, concentrated under reduced pressure, and then a base B solution is added to crystallize Pht-Gly-His-Lys-OH. The mass ratio of Pht-Gly-His(Boc)-Lys(Boc)-OH to the Boc-removing protecting agent is 1.0:1.0-10.0, and the molar ratio of Pht-Gly-His(Boc)-Lys(Boc)-OH to base B is 1.0:1.8-2.

2.

5. The method for synthesizing high-purity copper peptide according to claim 4, characterized in that, The acid-binding agent is selected from sodium bicarbonate, sodium carbonate, potassium carbonate, sodium hydroxide, potassium hydroxide, or triethylamine; the base B is selected from sodium bicarbonate, sodium carbonate, potassium carbonate, sodium hydroxide, potassium hydroxide, triethylamine, or imidazole; the base C is selected from sodium bicarbonate, sodium carbonate, potassium carbonate, sodium hydroxide, potassium hydroxide, or triethylamine; the aprotic solvent A is selected from acetone, tetrahydrofuran, acetonitrile, dichloromethane, DMF, or DMSO; the aprotic solvent B is selected from acetone, tetrahydrofuran, acetonitrile, dichloromethane, DMF, or DMSO; the aprotic solvent C is selected from acetone, tetrahydrofuran, acetonitrile, dichloromethane, DMF, or DMSO; the condensing agent is selected from DCC, DIC, EDCI, HBTU, or PyBOP; the deBoc protecting group removal reagent is selected from trifluoroacetic acid, hydrochloric acid, a tetrahydrofuran solution of hydrogen chloride, a dichloromethane solution of hydrogen chloride, or an ethyl acetate solution of hydrogen chloride.

6. The method for synthesizing high-purity copper peptide according to claim 1, characterized in that, Step S3 specifically includes: In solvent D, under the action of a phthaloyl-removing reagent, Pht-Gly-His-Lys-OH undergoes the removal of the phthaloyl group to obtain GHK; the molar ratio of Pht-Gly-His-Lys-OH to the phthaloyl-removing reagent is 1:1-10; solvent D is selected from water, methanol, or ethanol, and the phthaloyl-removing reagent is selected from hydrazine hydrate, n-butylamine, or n-propylamine; When the phthaloyl removal reagent is selected from hydrazine hydrate, solvent D is selected from water. After the reaction is completed, the pH is adjusted to 3.8-4.4 with hydrochloric acid, the phthaloyl hydrazine is removed by filtration, the filtrate is concentrated to dryness under reduced pressure, the pH is adjusted to 7.0-8.0 with ammonia, ethanol is added to crystallize and crude GHK is obtained. Then, the purified GHK is obtained by crystallization with water and ethanol. When the phthaloyl removal reagent is selected from n-butylamine or n-propylamine, the solvent D is selected from methanol or ethanol. After the reaction is completed, the solvent D is removed by vacuum concentration, water is added, and the pH is adjusted to 3.8-4.4 with hydrochloric acid. The mixture is extracted with dichloromethane to obtain an aqueous layer. The aqueous layer is concentrated to dryness under vacuum, and the pH is adjusted to 7.0-8.0 with ammonia. Ethanol is added to crystallize and obtain crude GHK. Then, the purified GHK is obtained by crystallization with water and ethanol.

7. The method for synthesizing high-purity copper peptide according to claim 1, characterized in that, Step S4 specifically includes: complexing GHK and copper salt in water at room temperature to obtain copper peptide; the molar ratio of GHK and copper salt is 1.0:0.5-1.5, and the copper salt is selected from copper acetate, copper acetate monohydrate, copper sulfate, copper sulfate pentahydrate, copper chloride dihydrate, or copper chloride.

8. The method for synthesizing high-purity copper peptide according to claim 4, characterized in that, In step S1, a one-pot method can be used to complete three steps of reaction to obtain the intermediate Pht-Gly-His-OH; in step S2, a one-pot method can be used to complete four steps of reaction to obtain the intermediate Pht-Gly-His-Lys-OH.

9. The method for synthesizing high-purity copper peptide according to claim 1, characterized in that, The method includes the following steps: (1) Synthesis of Pht-Gly-His-OH a) Synthesis of phthaloylglycyl chloride In a solvent, glycine and phthalic anhydride react under alkaline catalysis by reflux to yield phthaloylglycine. After the reaction is complete, the temperature is lowered to 60-70℃, thionyl chloride is added dropwise, and the mixture is kept at this temperature. After the reaction is complete, phthaloylglycyl chloride is obtained. The molar ratio of glycine, phthalic anhydride, and thionyl chloride is 1.0:1.00-1.05:1.0-1.5; the mass ratio of glycine to alkaline is 1.0:0.0001-0.

001. b) Synthesis of histidine protected compounds In a solvent, L-histidine and hexamethyldisilazane react under acid catalysis via reflux to obtain a histidine protected compound, wherein the molar ratio of L-histidine to hexamethyldisilazane is 1.0:1.5-2.0; and the mass ratio of L-histidine to acid is 1.0:0.0001-0.

001. c) Synthesis of Pht-Gly-His-OH Phthaloylglycyl chloride and histidine protectant react at room temperature. After the reaction is complete, the mixture is concentrated, and then an alcoholic solution of base A is added to the concentrate. Solid-liquid separation yields Pht-Gly-His-OH. Base A is selected from sodium bicarbonate, sodium carbonate, potassium carbonate, sodium hydroxide, potassium hydroxide, triethylamine, or imidazole; the alcohol is selected from methanol, ethanol, or isopropanol; the molar ratio of histidine protectant to phthaloylglycyl chloride is calculated based on the molar ratio of L-histidine to glycine: the molar ratio of L-histidine to glycine is 1.0:1.0-1.2; the molar ratio of histidine protectant to base A is calculated based on the molar ratio of L-histidine to base A: the molar ratio of L-histidine to base A is 1.0:0.9-1.

2. (2) Synthesis of Pht-Gly-His-Lys-OH a) Synthesis of Pht-Gly-His(Boc)-OH In water, Pht-Gly-His-OH is reacted dropwise with a solution of aprotic solvent A of Boc anhydride under the action of an acid-binding agent, at room temperature, to yield Pht-Gly-His(Boc)-OH; the molar ratio of Pht-Gly-His-OH, Boc anhydride, and acid-binding agent is 1.0:1.0-1.2:2.0-2.5; the acid-binding agent is selected from sodium bicarbonate, sodium carbonate, potassium carbonate, sodium hydroxide, potassium hydroxide, or triethylamine; the aprotic solvent A is selected from acetone, tetrahydrofuran, acetonitrile, dichloromethane, DMF, or DMSO; b) Synthesis of Pht-Gly-His(Boc)-OSu Pht-Gly-His(Boc)-OH and N-hydroxysuccinimide react at room temperature in an aprotic solvent B to yield Pht-Gly-His(Boc)-OSu; the molar ratio of Pht-Gly-His(Boc)-OH, N-hydroxysuccinimide, and the condensing agent is 1.0:1.0-1.2:1.0-1.2; the aprotic solvent B is selected from acetone, tetrahydrofuran, acetonitrile, dichloromethane, DMF, or DMSO; the condensing agent is selected from DCC, DIC, EDCI, HBTU, or PyBOP. c) Synthesis of Pht-Gly-His(Boc)-Lys(Boc)-OH In an aprotic solvent C, H-Lys(Boc)-OH reacts with Pht-Gly-His(Boc)-OSu at room temperature under the action of base C to obtain Pht-Gly-His(Boc)-Lys(Boc)-OH; the molar ratio of Pht-Gly-His(Boc)-OSu, H-Lys(Boc)-OH and base C is 1.0:1.0-1.2:2.0-2.5; the aprotic solvent C is selected from acetone, tetrahydrofuran, acetonitrile, dichloromethane, DMF or DMSO; the base C is selected from sodium bicarbonate, sodium carbonate, potassium carbonate, sodium hydroxide, potassium hydroxide or triethylamine; d) Synthesis of Pht-Gly-His-Lys-OH At room temperature, Pht-Gly-His(Boc)-Lys(Boc)-OH reacts with a Boc-removing reagent. After the reaction is complete, the mixture is extracted to obtain an aqueous layer, which is concentrated under reduced pressure. A base B solution is added, precipitating a large amount of solid. The solid is then filtered to obtain Pht-Gly-His-Lys-OH. The Boc-removing reagent is selected from hydrochloric acid, tetrahydrofuran solution of hydrogen chloride, dichloromethane solution of hydrogen chloride, ethyl acetate solution of hydrogen chloride, or trifluoroacetic acid. The mass ratio of Pht-Gly-His(Boc)-Lys(Boc)-OH to the Boc-removing reagent is 1.0:1.0-10.

0. The molar ratio of Pht-Gly-His(Boc)-Lys(Boc)-OH to base B is 1.0:1.8-2.

2. Base B is selected from sodium bicarbonate, sodium carbonate, potassium carbonate, sodium hydroxide, potassium hydroxide, triethylamine, or imidazole. (3) Synthesis of GHK In solvent D, Pht-Gly-His-Lys-OH is dephthalated by a phthaloyl-removing reagent to obtain GHK; the molar ratio of Pht-Gly-His-Lys-OH to the phthaloyl-removing reagent is 1:1.0-1.5; solvent D is selected from water, methanol or ethanol, and the phthaloyl-removing reagent is selected from hydrazine hydrate, n-butylamine or n-propylamine; When the phthaloyl removal reagent is selected from hydrazine hydrate, solvent D is selected from water. After the reaction is complete, the pH is adjusted to 3.8-4.4 with acid, the phthaloyl hydrazine is removed by filtration, the filtrate is concentrated to dryness under reduced pressure, the pH is adjusted to 7.0-8.0 with alkali, and crude GHK is obtained by solvent crystallization. Then, purified GHK is obtained by crystallization with water and ethanol. When the phthaloyl removal reagent is selected from n-butylamine or n-propylamine, solvent D is selected from methanol or ethanol. After the reaction is complete, solvent D is removed by concentration under reduced pressure, water is added, and the pH is adjusted to 3.8-4.4 with acid. The aqueous layer is extracted, concentrated to dryness under reduced pressure, the pH is adjusted to 7.0-8.0 with alkali, and crude GHK is obtained by solvent crystallization. Then, purified GHK is obtained by crystallization with water and ethanol. (4) Synthesis of copper peptides In water, GHK and copper salt are complexed at room temperature to obtain copper peptides; the molar ratio of GHK to copper salt is 1.0:0.5-1.5, and the copper salt is selected from copper acetate, copper acetate monohydrate, copper sulfate, copper sulfate pentahydrate, copper chloride dihydrate, or copper chloride.

10. The method for synthesizing high-purity copper peptide according to claim 1, characterized in that, The method includes the following steps: (1) Synthesis of Pht-Gly-His-OH a) Synthesis of phthaloylglycyl chloride In toluene, glycine and phthalic anhydride react under triethylamine catalysis via reflux to yield phthaloylglycine. After the reaction is complete, the temperature is lowered to 60-70℃, thionyl chloride is added dropwise, and the mixture is kept at this temperature. After the reaction is complete, the mixture is concentrated to dryness under reduced pressure, and toluene is added to obtain a phthaloylglycyl chloride solution. The molar ratio of glycine, phthalic anhydride, and thionyl chloride is 1.0:1.00-1.05:1.0-1.5; the mass ratio of glycine to triethylamine is 1.0:0.0001-0.

001. b) Synthesis of histidine protected compounds In toluene, L-histidine and hexamethyldisilazane react under concentrated sulfuric acid catalysis by reflux to obtain a histidine protected compound, wherein the molar ratio of L-histidine to hexamethyldisilazane is 1.0:1.5-2.0; and the mass ratio of L-histidine to sulfuric acid is 1.0:0.0001-0.

001. c) Synthesis of Pht-Gly-His-OH Phthaloylglycyl chloride solution and histidine protectant were reacted at room temperature. After the reaction was completed, the mixture was concentrated, and then an imidazole methanol solution was added to the concentrate. Solid-liquid separation was performed to obtain Pht-Gly-His-OH. The molar ratio of histidine protectant to phthaloylglycyl chloride was calculated based on the molar ratio of L-histidine to glycine: L-histidine to glycine was 1.0:1.0-1.2; the molar ratio of histidine protectant to imidazole was calculated based on the molar ratio of L-histidine to imidazole: L-histidine to imidazole was 1.0:0.9-1.

2. (2) Synthesis of Pht-Gly-His-Lys-OH a) Synthesis of Pht-Gly-His(Boc)-OH Pht-Gly-His-OH was reacted in water with sodium bicarbonate by adding a tetrahydrofuran solution of Boc anhydride dropwise at room temperature. After the reaction was complete, tetrahydrofuran was removed by vacuum distillation, followed by extraction with dichloromethane. The organic layer was washed twice with water and dried to obtain Pht-Gly-His(Boc)-OH. The molar ratio of Pht-Gly-His-OH, Boc anhydride, and sodium bicarbonate was 1.0:1.0-1.2:2.0-2.

5. b) Synthesis of Pht-Gly-His(Boc)-OSu Pht-Gly-His(Boc)-OH and N-hydroxysuccinimide react in dichloromethane at room temperature under the action of DIC to obtain Pht-Gly-His(Boc)-OSu; the molar ratio of Pht-Gly-His(Boc)-OH, N-hydroxysuccinimide and DIC is 1.0:1.0-1.2:1.0-1.2; c) Synthesis of Pht-Gly-His(Boc)-Lys(Boc)-OH In dichloromethane, H-Lys(Boc)-OH reacts with Pht-Gly-His(Boc)-OSu in the presence of triethylamine at room temperature. After the reaction is complete, the mixture is washed with water, and the organic layer is concentrated under reduced pressure to obtain Pht-Gly-His(Boc)-Lys(Boc)-OH. The molar ratio of Pht-Gly-His(Boc)-OSu, H-Lys(Boc)-OH, and triethylamine is 1.0:1.0-1.2:2.0-2.

5. d) Synthesis of Pht-Gly-His-Lys-OH At room temperature, Pht-Gly-His(Boc)-Lys(Boc)-OH reacts with hydrochloric acid. After the reaction is complete, the aqueous phase is extracted with dichloromethane to obtain an aqueous layer. The layer is concentrated under reduced pressure, and an aqueous sodium carbonate solution is added, precipitating a large amount of solid. The mixture is then cooled and filtered to obtain Pht-Gly-His-Lys-OH. The mass ratio of Pht-Gly-His(Boc)-Lys(Boc)-OH to hydrochloric acid is 1.0:1.0-10.0; the molar ratio of Pht-Gly-His(Boc)-Lys(Boc)-OH to sodium carbonate is 1.0:1.8-2.

2. (3) Synthesis of GHK In water, under the action of hydrazine hydrate, Pht-Gly-His-Lys-OH loses the phthaloyl group. After the reaction is complete, the pH is adjusted to 3.8-4.4 with hydrochloric acid, the phthaloyl hydrazine is removed by filtration, the filtrate is concentrated to dryness under reduced pressure, the pH is adjusted to 7.0-8.0 with ammonia, ethanol is added for crystallization, solid-liquid separation is performed to obtain crude GHK, and then purified GHK is obtained by crystallization with water and ethanol. (4) Synthesis of copper peptides In water, GHK and copper chloride dihydrate complex at room temperature. After the reaction is complete, the mixture is concentrated under reduced pressure to a paste, ethanol is added, and the solid-liquid separation yields copper peptides. The molar ratio of GHK to copper chloride dihydrate is 1.0:0.5-1.5.