Full liquid phase synthesis method of oligopeptide-1
Through the full liquid phase synthesis method, combined with the Boc and Fmoc strategies, innovative synthesis sequence and customized amino acid units are adopted to solve the problems of high cost and many by-products of the existing GHK tripeptide synthesis method, and high-purity and low-cost GHK tripeptide synthesis is achieved, suitable for large-scale production and has the advantages of green and environmental protection.
Patent Information
- Application Number
- CN202411554313.1
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-11-04
- Publication Date
- 2025-03-18
- Estimated Expiration
- Not applicable · inactive patent
AI Technical Summary
The existing GHK tripeptide synthesis method is costly and has many by-products, and is not suitable for large-scale production in factories.
The whole liquid phase synthesis method is adopted, through the combination of Boc strategy and Fmoc strategy, innovative synthesis sequence and customized amino acid units are adopted, which avoids the racemic defects of His during the synthesis process, simplifies the treatment after the reaction, and reduces the amount of solvents and raw materials.
It improves the purity of intermediate products, reduces production costs, and reduces the generation of by-products. It is suitable for large-scale production in factories, and has mild reaction conditions and is green and environmentally friendly.
Abstract
Description
Technical Field
[0001] The invention relates to the technical field of polypeptide synthesis, and in particular to a full liquid phase synthesis method of oligopeptide-1. Background Art
[0002] Oligopeptide-1 (tripeptide-1) (GHK) is an endogenous collagen tripeptide that exists in tissue fluids such as plasma. It can regulate skin regeneration, enhance the differentiation and proliferation of epidermal basal cells, promote wound healing and tissue regeneration, increase collagen and mucopolysaccharides, stimulate the synthesis of core proteoglycan, promote blood vessel growth and nerve growth, and has antioxidant and anti-inflammatory effects.
[0003] At present, the synthesis method of GHK is mainly classical solid phase synthesis. Solid phase synthesis is to extend the protected amino acids from the C-terminus to the N-terminus one by one, and obtain a GHK product with a purity higher than 90% through condensation, deprotection, re-condensation, cutting resin, centrifugation, freeze drying and high-performance liquid phase preparation. The market price of the resin required in this method is too expensive, the exchange activity is small, and the condensing agent and base used in its synthesis process are obviously excessive, resulting in an increase in the cost of GHK raw materials, and it is not suitable for large-scale production in factories. Therefore, it is of great significance to reduce the production cost of GHK tripeptide and ensure the stability and safety of the GHK synthesis process.
[0004] Xiong Ying et al. disclosed a method for synthesizing a GHK tripeptide (Master's thesis, Xiamen University, 2007). The method uses glycine, lysine and histidine as raw materials to prepare N-Boc-Gly-OH, His(trt) and Lys(Boc), respectively, and then synthesizes N-Boc-Gly-His(trt)–OH in sequence, and then synthesizes N-Boc-Gly-His(trt)-Lys(Boc)–OH, and then removes the protection to obtain the GHK tripeptide. The whole process of this method is simple to operate, but the disadvantage is that the yield of synthesizing N-Boc-Gly-His(trt)–OH is low, only 3 6.5%, the purity is also low, and resin is needed in the synthesis process, which is not suitable for large-scale factory production. In addition, what is finally obtained is the trifluoroacetate of GHK tripeptide, not the GHK tripeptide product, which does not meet the actual needs. Patent CN111732628A discloses a method for synthesizing GHK tripeptide, which is synthesized from the N-terminus to the C-terminus in sequence. The cost of His is high, and when Boc-Gly-His(Trt)-OSu is synthesized from Boc-Gly-OSu and Trt-His-OH, it is easy to racemize and difficult to react completely, and the side reactions increase. For this reason, we proposed a full liquid phase synthesis method for oligopeptide-1. Summary of the invention
[0005] The purpose of the present invention is to provide a full liquid phase synthesis method for oligopeptide-1 to solve the problems of high cost and more by-products raised in the above-mentioned background technology.
[0006] To achieve the above object, the present invention provides the following technical solution: a full liquid phase synthesis method of oligopeptide-1, 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.
[0007] 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.
[0008] 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.
[0009] 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.
[0010] 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.
[0011] Compared with the prior art, the invention has the following beneficial effects: the full liquid phase synthesis method of the oligopeptide-1 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 environmental protection, 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 oligopeptide-1, which has the advantages of convenient treatment during the synthesis process, improved yield, reduced cost, and green and environmental protection. DETAILED DESCRIPTION
[0012] Embodiment 1: The present invention provides a technical solution: a full liquid phase synthesis method of oligopeptide-1, (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%.
[0013] (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%.
[0014] (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%.
[0015] (4) Synthesis of H-Gly-His-Lys-OH 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%.
[0016] Embodiment 2: The present invention provides a technical solution: a full liquid phase synthesis method of oligopeptide-1, (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%.
[0017] (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%.
[0018] (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 vacuum dry to obtain the product with a purity of 89.1 and a yield of 90%.
[0019] 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 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 the solid, and vacuum dried to obtain a product with a purity of 95.1 and a yield of 98%.
[0020] Embodiment three: The present invention provides a technical solution: a full liquid phase synthesis method of oligopeptide-1, (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%.
[0021] (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%.
[0022] (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 90% and a yield of 89.7%.
[0023] (4) Synthesis of H-Gly-His-Lys-OH 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 the solid, and vacuum dried to obtain the product with a purity of 96% and a yield of 98.3%.
[0024] Comparative Example 1 (3) Synthesis of Boc-Gly-His(Trt)-Lys(Boc)-OtBu.HCl Weigh H-His(Trt)-Lys(Boc)-OtBu (80 mmol) and Boc-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 (110 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 81% and a yield of 80%.
Claims
1. A full liquid phase synthesis method of oligopeptide-1, 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.
2. The all-liquid phase synthesis method of oligopeptide-1 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 all-liquid phase synthesis method of oligopeptide-1 according to claim 1, characterized in that: 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.
4. The all-liquid phase synthesis method of oligopeptide-1 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 all-liquid phase synthesis method of oligopeptide-1 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.