Method for synthesizing blue copper peptide in full liquid phase

Abstract

The invention discloses a blue copper peptide full liquid phase synthesis method in the technical field of polypeptide synthesis. The blue copper peptide full liquid phase synthesis method comprises the following steps: S1, synthesizing Fmoc-His (Trt)-Lys (Boc)-OtBu. HCl; s2, H-His (Trt)-Lys (Boc)-OtBu is synthesized, and H-His (Trt)-Lys (Boc)-OtBu is S3, Fmoc-Gly-His (Trt)-Lys (Boc)-OtBu is synthesized, and Fmoc-Gly-His (Trt)-Lys (Boc)-OtBu is synthesized; s4, Gly-His-Lys is synthesized, and Gly-His-Lys is synthesized; and S5, synthesizing Gly-His-Lys-Cu. The full-liquid-phase synthesis method of the copper-blue peptide is reasonable in structural design, combines a Boc strategy and an Fmoc strategy, synthesizes the copper-blue peptide by using a full-liquid-phase method, and has the advantages that the synthesis process is convenient to treat, the yield is increased, the cost is reduced, and the method is green and environment-friendly.

Description

Technical Field

[0001] The invention relates to the technical field of polypeptide synthesis, and in particular to a method for fully liquid-phase synthesis of a blue copper peptide. Background Art

[0002] Blue copper peptides are often used in beauty products such as wrinkle removal, anti-aging, and repair. With its extremely small molecular weight, easy absorption, no irritation, no burden, high activity, and high efficacy, it has become the most high-end ingredient with the most effective effect in reversing skin age. Blue copper peptide series products are currently widely used in China. They are not only used for wrinkle removal and anti-aging functions in high-end cosmetics, but also for skin repair after laser beauty. In addition, they also include oral repair such as mouthwash and wound healing treatment after skin damage. They are safe, efficient, and effective, and have good market prospects.

[0003] CN113150066A discloses a method for synthesizing a blue copper peptide, wherein chloroacetyl chloride is reacted with histidine to generate chloroacetyl-L-histidine, which is then reacted with N-hydroxysuccinimide to obtain Mca-His-OSu, which is then reacted with protected lysine to obtain Mca-His-Lys(TFA)-OH, which is then reacted with ammonia water to obtain GHK, and copper peptides are obtained by reacting with copper acetate, copper sulfate or copper chloride. CN111808165A also discloses a method for synthesizing a GHK copper peptide, which uses copper acetate, copper hydroxide and a mixture thereof as a complexing agent, which is different from the present application in that the post-treatment method is different. The comparative document specification

[0008] has disclosed that the use of the above-mentioned complexing agent can avoid the use of resin, and the molar ratio of CHK tripeptide to copper salt is 1:1. For this reason, we propose a method for the full liquid phase synthesis of a blue copper peptide. Summary of the invention

[0004] The object of the present invention is to provide a method for the full liquid phase synthesis of blue copper peptides to solve the problems raised in the above background technology.

[0005] To achieve the above object, the present invention provides the following technical solution: a method for the full liquid phase synthesis of blue copper peptides, comprising the following steps: S1. Synthesis of Fmoc-His(Trt)-Lys(Boc)-OtBu.HCl; S2, synthesis of H-His(Trt)-Lys(Boc)-OtBu; S3, synthesis of Fmoc-Gly-His(Trt)-Lys(Boc)-OtBu; S4. Synthesis of Gly-His-Lys; S5. Synthesis of Gly-His-Lys-Cu.

[0006] Preferably, the step S1 specifically includes: Take Fmoc-His(Trt)-OH,H-Lys(Boc)-OtBu.HCl as the amino acid monomer, add a condensing agent, an activating agent, an organic base and a solvent to react in an ice-water bath. After the reaction is completed, filter the filtrate, concentrate it, add 0.5M hydrochloric acid solution to precipitate, filter the solid, and vacuum dry it to obtain the product; The condensing agent includes any one of DCC, BOP, PyBOP, HBTU, HATU, and EDC.HCl; The activator includes any one of HOBt, HOSu, HOAt, and HOOBt; The organic base is any one of DIEA and TEA; The solvent includes any one of DMF, DCM, NMP, THF, and dioxane; The molar ratio of the Fmoc-His(Trt)-OH to the H-Lys(Boc)-OtBu.HCl is 1:1.05-1:1.5; preferably 1:1.1; The molar ratio of the H-Lys(Boc)-OtBu.HCl to the condensing agent, the activating agent and the organic base is 1:1:1:1.

[0007] Preferably, the step S2 specifically includes: Dissolve the Fmoc-His(Trt)-Lys(Boc)-OtBu.HCl obtained in step S1 in diethylamine, concentrate, add an ether solvent to precipitate, filter to obtain a solid, and dry in vacuo; The ether solvent includes petroleum ether.

[0008] Preferably, the step S3 specifically includes: Using Fmoc-Gly-OH and H-His(Trt)-Lys(Boc)-OtBu prepared in step S2 as amino acid monomers, adding a condensing agent, an activating agent, an organic base and a solvent to react under ice-water bath conditions, filtering the filtrate after the reaction is completed, concentrating, adding 0.5M hydrochloric acid solution to precipitate, filtering the solid, and vacuum drying to obtain the product; The condensing agent includes any one of DCC, BOP, PyBOP, HBTU, HATU, and EDC.HCl; The activator includes any one of HOBt, HOSu, HOAt, and HOOBt; The organic base is any one of DIEA and TEA; The solvent includes any one of DMF, DCM, NMP, THF, and dioxane; The molar ratio of the H-His(Trt)-Lys(Boc)-OtBu to the Fmoc-Gly-OH is 1:1.05-1:1.5; The molar ratio of the Fmoc-Gly-OH to the condensing agent, the activating agent and the organic base is 1:1:1:1.

[0009] Preferably, the step S4 specifically includes: The Fmoc-Gly-His(Trt)-Lys(Boc)-OtBu obtained in step S3 is dissolved in the lysate, concentrated, and then slurried with an ether solvent, filtered to obtain a solid, and vacuum dried to obtain the solid; The lysate is TFA:DCM:TIS=60:30:10; The ether solvent includes any one of petroleum ether or isopropyl ether.

[0010] Preferably, the step S5 specifically includes: The Gly-His-Lys obtained in step S4 is dissolved in water, and a divalent copper salt (CuCl2, CuAc2, Cu(OH)2, etc.) is added, the molar ratio of Gly-His-Lys to the divalent copper salt is 1:1.1-1:5, 5% ammonia water is used to adjust the pH to 8±0.1 in an ice bath, and the mixture is diluted with purified water, treated with a weakly acidic cation exchange resin, concentrated after elution, and freeze-dried to obtain a blue copper peptide product; The divalent copper salt includes any one of CuCl2, CuAc2, and Cu(OH)2.

[0011] Preferably, the molar ratio of Gly-His-Lys to the divalent copper salt is 1:2.

[0012] Compared with the prior art, the invention has the following beneficial effects: the method for the full liquid phase synthesis of the blue copper peptide adopts a combination of the Boc strategy and the Fmoc strategy, adopts an innovative synthesis sequence, and a customized amino acid unit, thereby avoiding the defect of His being easily racemized during the synthesis process, facilitating the treatment after the reaction, and removing the excess reactant by a simple water washing or extraction operation, without the need for complex and costly processes such as column passing, thereby improving the purity of the intermediate product, without the need for expensive solid phase carriers, greatly reducing the amount of solvents and raw materials required, and greatly reducing the cost, without using any easily toxic reagents, without requiring hydrogenation reaction, without involving any dangerous chemical reactions, with mild reaction conditions, green and environmentally friendly, high product yield and purity, and the crude product purity can reach more than 95%, thereby adopting a combination of the Boc strategy and the Fmoc strategy, and using the full liquid phase method to synthesize the blue copper peptide, which has the advantages of convenient treatment of the synthesis process, improved yield, reduced cost, and green and environmentally friendly. DETAILED DESCRIPTION

[0013] Embodiment 1: The present invention provides a technical solution: a method for synthesizing blue copper peptide in full liquid phase. (1) Synthesis of Fmoc-His(Trt)-Lys(Boc)-OtBu.HCl Fmoc-His(Trt)-OH (100mmol), H-Lys(Boc)-OtBu.HCl (110mmol), and HOBt (110mmol) were weighed in a 1000ml round-bottom flask, and DMF 50ml and THF 400ml were added to dissolve the mixture. The mixture was placed in an ice-water bath for 30min, and TEA (110mmol) and DCC (110mmol) were added. The mixture was stirred for 30min and then the ice bath was removed. After 3h, TLC was used to monitor that the reaction of Fmoc-His(Trt)-OH was complete. After the reaction was completed, the solid was removed by filtration, and the filtrate was concentrated to a small amount. 0.5M hydrochloric acid solution (1000ml) was added to precipitate the solid, which was filtered out and dried in vacuo to obtain the product with a purity of 96.6% and a yield of 103.5%.

[0014] (2) Synthesis of H-His(Trt)-Lys(Boc)-OtBu The compound obtained in step (1) was dissolved in 500 ml of diethylamine, concentrated to a small amount, and precipitated by adding petroleum ether. The solid was filtered out and vacuum dried to obtain a product with a purity of 97.2% and a yield of 99.2%.

[0015] (3) Synthesis of Fmoc-Gly-His(Trt)-Lys(Boc)-OtBu.HCl Weigh H-His(Trt)-Lys(Boc)-OtBu (80 mmol) and Fmoc-Gly-OH (88 mmol), HOBt (88 mmol) obtained in step (2) into a 1000 ml round-bottom flask, add 400 ml of THF, dissolve, place in an ice-water bath for 30 min, add DCC (88 mmol), stir for 30 min and remove the ice bath. After 3 h, TLC monitors that the reaction of H-His(Trt)-Lys(Boc)-OtBu is complete. After the reaction is complete, filter out the solid, take the filtrate, concentrate to a small amount, add 1000 ml of 0.5 M hydrochloric acid solution, precipitate the solid, filter out the solid, and dry in vacuo to obtain the product with a purity of 89.9% and a yield of 90.2%.

[0016] (4) Synthesis of H-Gly-His-Lys-OH.2TFA The compound obtained in step (3) was dissolved in 500 ml of cleavage solution (TFA: DCM: TIS = 60: 30: 10), concentrated to a small amount, added with isopropyl ether to precipitate, filtered to obtain a solid, and vacuum dried to obtain a product with a purity of 98.1% and a yield of 86.8%.

[0017] (5) Synthesis of H-Gly-His-Lys-Cu The compound 12 g (20 mmol) obtained in step (4) was dissolved in 500 ml of purified water, and 5.4 g (40 mmol) of CuCl2 was added and stirred to dissolve. The pH was adjusted to 8±0.1 with 5% aqueous ammonia. D151 ion exchange resin to be loaded was prepared separately, the sample was loaded, and then eluted with 1% aqueous ammonia. The eluate was collected and concentrated, and 10 times of anhydrous ethanol was added. The solution was placed in a refrigerator and frozen overnight to precipitate crystals. The solid was filtered and dried in vacuo at 25°C for 10 h to obtain the obtained product. The purity was 98.6% and the yield was 65.6%.

[0018] Embodiment 2: The present invention provides a technical solution: a method for synthesizing blue copper peptide in full liquid phase. (1) Synthesis of Fmoc-His(Trt)-Lys(Boc)-OtBu.HCl Fmoc-His(Trt)-OH (100mmol), H-Lys(Boc)-OtBu.HCl (105mmol), HOAt (105mmol) were weighed in a 1000ml round-bottom flask, and DMF 50ml and THF 400ml were added to dissolve the mixture. The mixture was placed in an ice-water bath for 30min, and DIEA (105mmol) and BOP (105mmol) were added. The mixture was stirred for 30min and the ice bath was removed. After 3h, TLC was used to monitor that the reaction of Fmoc-His(Trt)-OH was complete. After the reaction was completed, the solid was removed by filtration, the filtrate was concentrated to a small amount, 0.5M hydrochloric acid solution 1000ml was added to precipitate the solid, the solid was filtered out, and the solid was dried in vacuo to obtain the product with a purity of 95.8% and a yield of 101.5%.

[0019] (2) Synthesis of H-His(Trt)-Lys(Boc)-OtBu The compound obtained in step (1) was dissolved in 500 ml of diethylamine, concentrated to a small amount, and precipitated by adding petroleum ether. The solid was filtered and dried in vacuo to obtain a product with a purity of 96.2% and a yield of 98.7%.

[0020] (3) Synthesis of Fmoc-Gly-His(Trt)-Lys(Boc)-OtBu.HCl Weigh H-His(Trt)-Lys(Boc)-OtBu (80 mmol) and Fmoc-Gly-OH (84 mmol), HOAt (84 mmol) obtained in step (2) into a 1000 ml round-bottom flask, add 400 ml of THF, dissolve, place in an ice-water bath for 30 min, add BOP (84 mmol), stir for 30 min and remove the ice bath. After 3 h, TLC monitors that the reaction of H-His(Trt)-Lys(Boc)-OtBu is complete. After the reaction is complete, filter out the solid, take the filtrate, concentrate to a small amount, add 1000 ml of 0.5 M hydrochloric acid solution, precipitate the solid, filter out the solid, and dry in vacuo to obtain the product with a purity of 88.9% and a yield of 90.1%.

[0021] The condensing agent includes any one of DCC, BOP, PyBOP, HBTU, HATU, and EDC.HCl; The activator includes any one of HOBt, HOSu, HOAt, and HOOBt; The organic base is any one of DIEA and TEA; The solvent includes any one of DMF, DCM, NMP, THF, and dioxane; (4) Synthesis of H-Gly-His-Lys-OH.2TFA The compound obtained in step (3) was dissolved in 500 ml of cleavage solution (TFA: DCM: TIS = 60: 30: 10), concentrated to a small amount, added with isopropyl ether to precipitate, filtered to obtain a solid, and vacuum dried to obtain a product with a purity of 97.1% and a yield of 85.8%.

[0022] (5) Synthesis of H-Gly-His-Lys-Cu The compound 12 g (20 mmol) obtained in step (4) was dissolved in 500 ml of purified water, and 22.1 g (22 mmol) of Cu(OH) was added and stirred to dissolve. The pH was adjusted to 8±0.1 with 5% aqueous ammonia. D151 ion exchange resin to be loaded was prepared separately, the sample was loaded, and then eluted with 1% aqueous ammonia. The eluate was collected and concentrated, and 10 times of anhydrous ethanol was added. The solution was placed in a refrigerator and frozen overnight to precipitate crystals. The solid was filtered and dried in vacuo at 25°C for 10 h to obtain the obtained product. The purity was 97.6% and the yield was 64.6%.

[0023] Embodiment three: The present invention provides a technical solution: a method for synthesizing blue copper peptide in full liquid phase. (1) Synthesis of Fmoc-His(Trt)-Lys(Boc)-OtBu.HCl Fmoc-His(Trt)-OH (100mmol), H-Lys(Boc)-OtBu.HCl (150mmol), HOSu (150mmol) were weighed in a 1000ml round-bottom flask, and DMF 50ml and THF 400ml were added to dissolve the mixture. The mixture was placed in an ice-water bath for 30min, and DIEA (150mmol) and HATU (150mmol) were added. The mixture was stirred for 30min and the ice bath was removed. After 3h, TLC was used to monitor the complete reaction of Fmoc-His(Trt)-OH. After the reaction was completed, the solid was removed by filtration, the filtrate was concentrated to a small amount, 0.5M hydrochloric acid solution (1000ml) was added to precipitate the solid, the solid was filtered out, and the solid was dried in vacuo to obtain the product with a purity of 96.3% and a yield of 101.9%.

[0024] (2) Synthesis of H-His(Trt)-Lys(Boc)-OtBu The compound obtained in step (1) was dissolved in 500 ml of diethylamine, concentrated to a small amount, and precipitated by adding petroleum ether. The solid was filtered and dried in vacuo to obtain a product with a purity of 96.2% and a yield of 98.7%.

[0025] (3) Synthesis of Fmoc-Gly-His(Trt)-Lys(Boc)-OtBu.HCl Weigh H-His(Trt)-Lys(Boc)-OtBu (80 mmol) and Fmoc-Gly-OH (120 mmol), HOSu (120 mmol) obtained in step (2) into a 1000 ml round-bottom flask, add 400 ml of THF, dissolve, place in an ice-water bath for 30 min, add HATU (120 mmol), stir for 30 min and remove the ice bath. After 3 h, monitor the completion of the reaction of H-His(Trt)-Lys(Boc)-OtBu by TLC. After the reaction is complete, filter out the solid, take the filtrate, concentrate to a small amount, add 1000 ml of 0.5 M hydrochloric acid solution, precipitate the solid, filter out the solid, and dry in vacuo to obtain the product with a purity of 87.9% and a yield of 88.2%.

[0026] (4) Synthesis of H-Gly-His-Lys-OH.2TFA The compound obtained in step (3) was dissolved in 500 ml of cleavage solution (TFA: DCM: TIS = 60: 30: 10), concentrated to a small amount, added with isopropyl ether to precipitate, filtered to obtain a solid, and vacuum dried to obtain a product with a purity of 97.1% and a yield of 84.8%.

[0027] (5) Synthesis of H-Gly-His-Lys-Cu The compound 12 g (20 mmol) obtained in step (4) was dissolved in 500 ml of purified water, and CuAc 29.1 g (100 mmol) was added and stirred to dissolve. The pH was adjusted to 8±0.1 with 5% aqueous ammonia. D151 ion exchange resin to be loaded was prepared separately, the sample was loaded, and then eluted with 1% aqueous ammonia. The eluate was collected and concentrated, and 10 times of anhydrous ethanol was added. The solution was placed in a refrigerator and frozen overnight to precipitate crystals. The solid was filtered and dried in vacuo at 25°C for 10 h to obtain the obtained product. The purity was 96.6% and the yield was 63.6%.

[0028] Comparative Example 1 (3) Synthesis of Boc-Gly-His(Trt)-Lys(Boc)-OtBu.HCl Weigh H-His(Trt)-Lys(Boc)-OtBu (80mmol) and Fmoc-Gly-OH (88mmol), HOBt (88mmol) obtained in step (2) into a 1000ml round-bottom flask, add 400ml of THF, dissolve, place in an ice-water bath for 30min, add DCC (110mmol), stir for 30min and remove the ice bath. After 3h, monitor the complete reaction of H-His(Trt)-Lys(Boc)-OtBu by TLC. After the reaction is complete, filter out the solid, take the filtrate, concentrate to a small amount, add 1000ml of 0.5M hydrochloric acid solution, precipitate the solid, filter out the solid, and dry in vacuo to obtain the product. 85.9%, yield 95.2%.

Claims

1. A method for the full liquid phase synthesis of blue copper peptides, characterized in that: The following steps are involved: S1. Synthesis of Fmoc-His(Trt)-Lys(Boc)-OtBu.HCl; S2, synthesis of H-His(Trt)-Lys(Boc)-OtBu; S3, synthesis of Fmoc-Gly-His(Trt)-Lys(Boc)-OtBu; S4. Synthesis of Gly-His-Lys; S5. Synthesis of Gly-His-Lys-Cu.

2. The method for all-liquid phase synthesis of a blue copper peptide according to claim 1, characterized in that: The step S1 specifically includes: Take Fmoc-His(Trt)-OH,H-Lys(Boc)-OtBu.HCl as the amino acid monomer, add a condensing agent, an activating agent, an organic base and a solvent to react in an ice-water bath. After the reaction is completed, filter the filtrate, concentrate it, add 0.5M hydrochloric acid solution to precipitate, filter the solid, and vacuum dry it to obtain the product; The condensing agent includes any one of DCC, BOP, PyBOP, HBTU, HATU, and EDC.HCl; The activator includes any one of HOBt, HOSu, HOAt, and HOOBt; The organic base is any one of DIEA and TEA; The solvent includes any one of DMF, DCM, NMP, THF, and dioxane; The molar ratio of the Fmoc-His(Trt)-OH to the H-Lys(Boc)-OtBu.HCl is 1:1.05-1:1.5; The molar ratio of the H-Lys(Boc)-OtBu.HCl to the condensing agent, the activating agent and the organic base is 1:1:1:

1.

3. The method for all-liquid phase synthesis of a blue copper peptide according to claim 1, characterized in that: Step S2 specifically includes: Dissolve the Fmoc-His(Trt)-Lys(Boc)-OtBu.HCl obtained in step S1 in diethylamine, concentrate, add an ether solvent to precipitate, filter to obtain a solid, and dry in vacuo; The ether solvent includes petroleum ether.

4. The method for all-liquid phase synthesis of a blue copper peptide according to claim 1, characterized in that: The step S3 specifically includes: Using Fmoc-Gly-OH and H-His(Trt)-Lys(Boc)-OtBu prepared in step S2 as amino acid monomers, adding a condensing agent, an activating agent, an organic base and a solvent to react under ice-water bath conditions, filtering the filtrate after the reaction is completed, concentrating, adding 0.5M hydrochloric acid solution to precipitate, filtering the solid, and vacuum drying to obtain the product; The condensing agent includes any one of DCC, BOP, PyBOP, HBTU, HATU, and EDC.HCl; The activator includes any one of HOBt, HOSu, HOAt, and HOOBt; The organic base is any one of DIEA and TEA; The solvent includes any one of DMF, DCM, NMP, THF, and dioxane; The molar ratio of the H-His(Trt)-Lys(Boc)-OtBu to the Fmoc-Gly-OH is 1:1.05-1:1.5; The molar ratio of the Fmoc-Gly-OH to the condensing agent, the activating agent and the organic base is 1:1:1:

1.

5. The method for all-liquid phase synthesis of a blue copper peptide according to claim 1, characterized in that: The step S4 specifically includes: The Fmoc-Gly-His(Trt)-Lys(Boc)-OtBu obtained in step S3 is dissolved in the lysate, concentrated, and then slurried with an ether solvent, filtered to obtain a solid, and vacuum dried to obtain the solid; The lysate is TFA:DCM:TIS=60:30:10; The ether solvent includes any one of petroleum ether or isopropyl ether.

6. The method for all-liquid phase synthesis of a blue copper peptide according to claim 1, characterized in that: The step S5 specifically includes: The Gly-His-Lys obtained in step S4 is dissolved in water, and a divalent copper salt (CuCl2, CuAc2, Cu(OH)2, etc.) is added, the molar ratio of Gly-His-Lys to the divalent copper salt is 1:1.1-1:5, 5% ammonia water is used to adjust the pH to 8±0.1 in an ice bath, and the mixture is diluted with purified water, treated with a weakly acidic cation exchange resin, concentrated after elution, and freeze-dried to obtain a blue copper peptide product; The divalent copper salt includes any one of CuCl2, CuAc2, and Cu(OH)2.

7. The method for all-liquid phase synthesis of a blue copper peptide according to claim 1, characterized in that: The molar ratio of Gly-His-Lys to the divalent copper salt is 1:2.