CN116143867A

CN116143867A - Method for preparing GHK tripeptide and blue copper peptide without condensing agent

Info

Publication number: CN116143867A
Application number: CN202310144991.XA
Authority: CN
Inventors: 王圣克; 刘书华; 潘四毫; 李正华
Original assignee: Shanghai Ruikang Biotechnology R & D Co ltd
Current assignee: Shanghai Ruikang Biotechnology R & D Co ltd
Priority date: 2023-02-21
Filing date: 2023-02-21
Publication date: 2023-05-23

Abstract

The invention belongs to the technical field of biological medicines and cosmetics, and particularly relates to a method for preparing GHK tripeptide and blue copper peptide without condensing agents. The method of the invention comprises the following steps: step 1, reacting methyl histidine and chloroacetyl chloride to obtain a product Mca-His-OMe; step 2, reacting Mca-His-OMe with trifluoroacetyl lysine to obtain a product Mca-His-Lys (TFA) -OH; and 3, under the action of ammonia water, carrying out ammonolysis reaction on Mca-His-Lys (TFA) -OH to obtain the GHK tripeptide. The method of the invention does not use condensing agent, can effectively reduce the production cost, has simple and efficient steps, higher yield and product purity, and is easy to realize commercial mass production. Therefore, the invention has good application prospect.

Description

Method for preparing GHK tripeptide and blue copper peptide without condensing agent

Technical Field

The invention belongs to the technical field of biological medicines and cosmetics, and particularly relates to a method for preparing GHK tripeptide and blue copper peptide without condensing agents.

Background

Blue copper peptide is a compound composed of copper ions and tripeptide-1 (GHK), and appears blue in appearance. They are conventionally called blue copper peptides (GHK-Cu), also known as blue copper peptides, tripeptide-1 copper. Blue copper peptide belongs to small molecule protein, and is the earliest discovered peptide. After entering the skin, the blue copper peptide releases signals, induces fibroblasts to produce collagen, and promotes collagen generation. In addition, blue copper peptides can carry copper ions into the interior of cells, which release copper ions upon encountering enzymes, which are a very important component of many enzymatic reaction processes. Blue copper peptide has the function of promoting the generation of proteoglycan (GAG), which is an important component constituting extracellular matrix, and can make skin full, moist and elastic. Proteoglycans are deficient and the skin can relax, age, dry, and rough.

Tripeptide-1 is used as a key ligand of the blue copper peptide and determines the quality of the blue copper peptide. Currently, the synthesis method of GHK is mainly solid phase synthesis. The classical procedure for solid phase synthesis is mainly to fix the C-terminus of the first amino acid with a resin, thus extending one by one from the N-terminus. The high-purity GHK product is prepared through condensation, deprotection, recondensing, resin cutting, centrifugation, freeze-drying and high-efficiency liquid phase. The cost of solid phase synthesis is high, and the condensing agent and solvent used in the synthesis process are consumed in large amounts, so that the method is not suitable for large-scale commercial production. Therefore, a synthetic process for producing GHK tripeptide at low cost, and being safe, stable and green is needed.

By designing an organic synthesis route, the reaction requiring the use of a condensing agent is avoided, and the method is one of the feasible means for reducing the production cost of the GHK tripeptide. However, current methods for tripeptide-1 and copper blue peptides report that there is little to no condensing agent strategy. However, there are still some problems in the synthesis methods reported so far without using condensing agents, for example, patent CN 107778349a discloses a method for synthesizing GHK acetate at low cost, which deprotects Tit-Gly-His (Tit) -Lys (Trt) -OH in acetic acid and triisopropylsilane to generate GHK acetate. Although the method can reduce the cost of producing GHK acetate, too many acetate ions are generated in the synthesis of GHK-Cu, which is unfavorable for the synthesis and application of blue copper peptide and causes barriers to the commercial mass production of further blue copper peptide.

Therefore, there is a need in the art to develop a new synthesis route, which is simple and efficient, and suitable for commercial large-scale production of tripeptide-1 and blue copper peptide without condensing agent, so as to meet the market demand for blue copper peptide and downstream products thereof.

Disclosure of Invention

Aiming at the problems of the prior art, the invention provides a method for preparing GHK tripeptide and blue copper peptide without condensing agent, and aims to provide a novel synthetic route, which realizes a simple and efficient organic synthesis method of GHK tripeptide and blue copper peptide suitable for commercial mass production on the premise of not using condensing agent.

A preparation method of GHK tripeptide comprises the following synthesis steps:

the method specifically comprises the following steps:

step 1, reacting methyl histidine and chloroacetyl chloride to obtain a product Mca-His-OMe;

step 2, reacting Mca-His-OMe with trifluoroacetyl lysine to obtain a product Mca-His-Lys (TFA) -OH;

and 3, under the action of ammonia water, carrying out ammonolysis reaction on Mca-His-Lys (TFA) -OH to obtain the GHK tripeptide.

Preferably, in the step 1, the dosage ratio of the histidine methyl ester to the chloroacetyl chloride is 1 (1-3); the reaction is carried out under the action of a base selected from at least one of sodium hydroxide or potassium carbonate; the reaction is carried out in a solvent selected from at least one of tetrahydrofuran, dioxane or dimethyltetrahydrofuran.

Preferably, in step 1, the reaction conditions are: the reaction temperature is 0-10 ℃, the pH of the reaction is 10-12, and the reaction time is 5-8 hours.

Preferably, in the step 2, the dosage ratio of Mca-His-OMe to trifluoroacetyl lysine is 1 (1-2); the reaction is carried out under the action of a base selected from triethylamine, pyridine or 4-dimethylaminopyridine; the reaction is carried out in a solvent selected from 1, 4-dioxane, tetrahydrofuran or dichloromethane.

Preferably, in step 2, alkali is added until the solution is clear; the reaction conditions are as follows: the reaction temperature is 60-80 ℃ and the reaction time is 12-24 hours.

Preferably, in step 3, mca-His-Lys (TFA) -OH is used in a molar ratio of 1 (10-100) to ammonia.

Preferably, in step 3, the reaction conditions are: mca-His-Lys (TFA) -OH is added into ammonia water at 0-10 ℃, the temperature is raised to 50-70 ℃ for reaction, and the reaction time is 12-24 hours.

The invention also provides a preparation method of the blue copper peptide, which comprises the following steps:

step a, preparing GHK tripeptide according to the preparation method;

step b, the GHK tripeptide is combined with Cu ²⁺ And (3) salt mixing reaction to obtain the blue copper peptide.

Preferably, in step b, the reaction conditions are: GHK tripeptide and Cu-containing ²⁺ The salt of GHK tripeptide and Cu are used in an amount sufficient to ²⁺ The molar ratio of (2) is 1 (0.5-3), the reaction pH is 6-7, and the reaction time is 3-7 hours.

Preferably, in the step b, the method for separating and obtaining the blue copper peptide comprises the following steps: slowly dripping ethanol, cooling the reaction system to 0-10 ℃, filtering and drying.

In the present invention, abbreviations of the respective compounds have the following chemical structural formulas:

the invention designs a new synthesis route aiming at the aim of synthesizing the GHK tripeptide and the blue copper peptide without condensing agent, and optimizes the reaction conditions of each step in a preferable scheme. The invention solves the problem of high cost for producing GHK tripeptide and blue copper peptide in the prior art, and has good application prospect.

It should be apparent that, in light of the foregoing, various modifications, substitutions and alterations can be made herein without departing from the spirit and scope of the invention as defined by the appended claims.

The above-described aspects of the present invention will be described in further detail below with reference to specific embodiments in the form of examples. It should not be understood that the scope of the above subject matter of the present invention is limited to the following examples only. All techniques implemented based on the above description of the invention are within the scope of the invention.

Drawings

FIG. 1 is an HPLC detection chart of tripeptide-1 synthesized in example 1;

FIG. 2 is a HPLC detection chart of the blue copper peptide synthesized in example 2;

FIG. 3 is a mass spectrum of the blue copper peptide synthesized in example 3.

Detailed Description

The raw materials and equipment used in the invention are all known products and are obtained by purchasing commercial products.

Example 1 preparation of GHK tripeptide

1. Synthesis of Mca-His-OMe (chloroacetyl-L-histidine methyl ester)

In a 500ml three-necked flask equipped with a thermometer and a stirrer, 16.9g of methyl histidine (100 mmol), 100ml of water and 20ml of tetrahydrofuran are sequentially added, the pH is regulated to 10 by using 50% sodium hydroxide solution, 100ml of tetrahydrofuran solution of 16.8g of chloroacetyl chloride (150 mmol) is dropwise added into the feed liquid, the pH is controlled to be 10 in the whole reaction process, the reaction is continued for 3 hours after the completion of dropwise adding of the chloroacetyl chloride solution, the pH is regulated to be 7 by using hydrochloric acid, ethyl acetate (150 ml of 3) is added for extraction, drying, filtration and concentration are carried out, and 22.8g of Mca-His-OMe crude solid is obtained, and the crude product yield is 93.1%;

synthesis of Mca-His-Lys (TFA) -OH (chloroacetyl-L-histidine-trifluoroacetyl lysine)

In a 500ml three-necked flask equipped with a thermometer, stirrer, 22g of Mca-His-OMe (90 mmol), 100ml of 1, 4-dioxane, 13.6g of triethylamine (135 mmol) were added dropwise under stirring, the temperature was controlled at 30-35℃and then a solution of trifluoroacetyl lysine (24.2 g,100 mmol) in 1, 4-dioxane (100 ml) was added. After the dripping is finished, slowly heating to 60-70 ℃ for reaction for 6 hours. After the reaction, the reaction solution was concentrated, the pH was adjusted to 2-3 with 6M aqueous HCl, extracted with ethyl acetate (150 ml. Times.3), the aqueous phase was extracted with n-butanol (150 ml. Times.3), the n-butanol phases were combined, dried, filtered, and concentrated to a solid to give 35.6g of crude Mca-His-Lys (TFA) -OH in a yield of 87.0%;

synthesis of H-Gly-His-Lys-OH (GHK) (L-Glycine-L-histidine-L-lysine)

35.6g of Mca-His-Lys (TFA) -OH crude product obtained in the last step is added into a 500ml three-necked flask with a thermometer and a stirrer, 200ml of concentrated ammonia water is slowly added dropwise at the internal temperature of 0-10 ℃, a closed container is sealed, the temperature is raised to 50 ℃, and the reaction is carried out for 12-24 hours until the substrate is completely ammonolyzed, and H-Gly-His-Lys-OH is generated by conversion. After the reaction, the mixture was concentrated under reduced pressure, ethanol was added to remove water to a foamy solid, then 30ml of methanol and 50ml of ethyl acetate were added to recrystallize, and the mixture was filtered, and the off-white solid was rinsed with 20ml of a mixed solvent of methanol and ethyl acetate (methanol: ethyl acetate=1:3) and dried to give 23.7g of GHK tripeptide, and the purity of HPLC (HPLC profile shown in fig. 1) was 99%.

The nuclear magnetic data of the GHK tripeptide prepared in this example are as follows: ¹ H NMR(400MHz,D ₂ o) δ (ppm) 7.66 (d, j=1.2 hz, 1H), 6.88 (d, j=1.2 hz, 1H), 4.55 (dd, j=7.5, 6.1hz, 1H), 4.03 (dd, j=7.8, 5.3hz, 1H), 3.59-3.53 (m, 2H), 3.05-2.94 (m, 2H), 2.88 (t, j=7.6 hz, 2H), 1.73-1.52 (m, 4H), 1.28-1.20 (m, 2H), whereby it can be determined that the product prepared in this example is indeed GHK tripeptide.

Example 2 preparation of blue copper peptide

This example uses the method of example 1 to prepare GHK tripeptides, followed by the following steps:

in a flask with pure water, 5g GHK tripeptide (14.7 mmol) was added in turn, pH was adjusted to 6-7 with hydrochloric acid, equivalent copper chloride (1.97 g,14.7 mmol) was added, after 1 hour of reaction, 100ml ethanol was slowly added dropwise, the reaction system was cooled to 5℃and kept warm for 2 hours, filtered, rinsed with 10ml ethanol, and vacuum dried to give 5.11g of blue copper peptide, yield 86.2%, and HPLC (HPLC profile is shown in FIG. 2) purity 99%.

The mass spectrum data of the blue copper peptide product prepared in this example is shown in fig. 3, and the mass spectrum peak with m/z value of 341.2 is the mass spectrum signal of the blue copper peptide. From this, it was confirmed that the product prepared in this example was indeed a blue copper peptide.

According to the embodiment, the invention provides a novel synthesis method of the GHK tripeptide and the blue copper peptide, which does not use a condensing agent, can effectively reduce the production cost, has the advantages of simple and efficient steps, higher yield and product purity, and is easy to realize commercial mass production. Therefore, the invention has good application prospect.

Claims

1. A preparation method of GHK tripeptide is characterized by comprising the following synthesis steps:

the method specifically comprises the following steps:

2. The method of manufacturing according to claim 1, wherein: in the step 1, the dosage ratio of the histidine methyl ester to the chloroacetyl chloride is 1 (1-3); the reaction is carried out under the action of a base selected from at least one of sodium hydroxide or potassium carbonate; the reaction is carried out in a solvent selected from at least one of tetrahydrofuran, dioxane or dimethyltetrahydrofuran.

3. The method of manufacturing according to claim 1, wherein: in step 1, the reaction conditions are as follows: the reaction temperature is 0-10 ℃, the pH of the reaction is 10-12, and the reaction time is 5-8 hours.

4. The method of manufacturing according to claim 1, wherein: in the step 2, the dosage ratio of Mca-His-OMe to trifluoroacetyl lysine is 1 (1-2); the reaction is carried out under the action of a base selected from triethylamine, pyridine or 4-dimethylaminopyridine; the reaction is carried out in a solvent selected from 1, 4-dioxane, tetrahydrofuran or dichloromethane.

5. The method of manufacturing according to claim 1, wherein: in the step 2, adding alkali until the solution is clear; the reaction conditions are as follows: the reaction temperature is 60-80 ℃ and the reaction time is 12-24 hours.

6. The method of manufacturing according to claim 1, wherein: in step 3, mca-His-Lys (TFA) -OH and ammonia are used in a molar ratio of 1 (10-100).

7. The method of manufacturing according to claim 1, wherein: in step 3, the reaction conditions are as follows: mca-His-Lys (TFA) -OH is added into ammonia water at 0-10 ℃, the temperature is raised to 50-70 ℃ for reaction, and the reaction time is 12-24 hours.

8. The preparation method of the blue copper peptide is characterized by comprising the following steps:

step a, preparing GHK tripeptide according to the preparation method of any one of claims 1-7;

9. The method of preparing as claimed in claim 8, wherein: in step b, the reaction conditions are as follows: GHK tripeptide and Cu-containing ²⁺ The salt of GHK tripeptide and Cu are used in an amount sufficient to ²⁺ The molar ratio of (2) is 1 (0.5-3), the reaction pH is 6-7, and the reaction time is 3-7 hours.

10. The method of preparing as claimed in claim 8, wherein: in the step b, the method for separating and obtaining the blue copper peptide comprises the following steps: slowly dripping ethanol, cooling the reaction system to 0-10 ℃, filtering and drying.