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CN-122011119-A - Preparation method and application of bionic collagen cyclohexapeptide

CN122011119ACN 122011119 ACN122011119 ACN 122011119ACN-122011119-A

Abstract

The invention provides a preparation method and application of bionic collagen cyclohexapeptide, and belongs to the technical field of biological medicines. The method adopts a liquid phase synthesis strategy, and constructs the bionic collagen cyclohexapeptide shown in the formula (I) with high efficiency and high selectivity through an optimized reaction route and a protecting group strategy. The method overcomes the defects of complex solid phase synthesis process, high cost, complex components of collagen hydrolysate, nonuniform quality and the like, and realizes the large-scale preparation of the high-purity bionic collagen cyclohexapeptide with definite structure and single molecular weight. The prepared bionic collagen cyclohexapeptide has the advantages of bioactivity of collagen peptide and stability of cyclopeptide, and has good application prospect in the fields of skin aging resistance, tissue repair and biological materials. Formula (I)

Inventors

  • Jiang Chengda
  • HU JIALE

Assignees

  • 成都欣肽生物科技有限公司

Dates

Publication Date
20260512
Application Date
20260212

Claims (10)

  1. 1. The preparation method of the bionic collagen cyclohexapeptide is characterized in that the bionic collagen cyclohexapeptide has a structure shown in a formula (I), and the synthesis method is a liquid phase synthesis method and comprises the following steps: Formula (I) (1) Cyclization reaction of Linear hexapeptide precursor Deprotecting amino terminal and carboxyl terminal, and performing intramolecular cyclization reaction to obtain bionic collagen cyclohexapeptide with protected side chain hydroxyl group R 1 is a hydroxyl protecting group; (2) Removing protecting groups, namely removing side chain hydroxyl protecting groups of the bionic collagen cyclohexapeptide obtained in the step (1) under alkaline conditions to obtain the bionic collagen cyclohexapeptide shown in the formula (I).
  2. 2. The synthetic method of claim 1 wherein in step (1), the intramolecular cyclization reaction is carried out by any of the following means: (I) The carboxyl end of the linear hexapeptide precursor is activated into activated ester and the amino end protecting group is removed, and then cyclizing is carried out in an organic solvent, or, (II) removing amino-terminal and carboxyl-terminal protecting groups from the linear hexapeptide precursor to obtain free linear hexapeptide acid, and then directly cyclizing the free linear hexapeptide acid with a condensing agent and a base; in the step (2), the hydroxy group removal protection comprises the following steps of reacting the bionic collagen cyclohexapeptide with an organic solvent and an inorganic base to obtain the bionic collagen cyclohexapeptide shown in the formula (I).
  3. 3. The synthesis method according to claim 2, wherein step (II) comprises the sub-steps of: ① Reacting a linear hexapeptide precursor, an organic solvent, a catalyst and a reducing agent to obtain an intermediate product A R 3 is an amino protecting group; ② Reacting the intermediate A, an activated ester reagent, an activating agent and an organic solvent to obtain an intermediate B R 4 is an activated ester group; ③ Reacting the intermediate product B with an organic solvent and an acid to obtain an intermediate product C X is an organic acid or an inorganic acid; ④ Reacting the intermediate product C, an organic solvent and organic alkali to obtain the bionic collagen cyclohexapeptide with side chain hydroxyl groups protected 。
  4. 4. The synthetic method according to any one of claims 1 to 3, wherein the organic solvents are each independently selected from N, N-dimethylformamide, dichloromethane or methanol, the activators and condensing agents are each independently selected from 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride, O-benzotriazol-tetramethylurea hexafluorophosphate, 2- (7-azobenzotriazol) -N, N, N ', N' -tetramethylurea hexafluorophosphate, ethyl 2-oxime nitriloacetate, the organic bases are each independently selected from N, N-diisopropylethylamine, triethylamine or N-methylmorpholine, the inorganic bases are each independently selected from sodium carbonate, sodium hydroxide, potassium hydroxide or potassium carbonate, the acids are each independently selected from trifluoroacetic acid or 1,4 dioxane solution, the catalysts are each independently selected from palladium carbon or palladium aluminum oxide, the reducing agents are each independently selected from hydrogen, and the activating ester reagents are selected from pentafluorophenol, N-hydroxysuccinimide, N- (benzyloxycarbonyl) succinimidyl-fluoroimide or 9-methylfluoroimide.
  5. 5. The method of synthesis according to claim 1, wherein the method of preparation of the linear hexapeptide precursor comprises the steps of: (A) Synthesizing a tripeptide unit; (B) Synthesizing a linear hexapeptide precursor; The tripeptide unit has the structure of R 2 is a carboxyl protecting group.
  6. 6. The method of claim 5, wherein in step (a), the synthesizing tripeptide unit comprises the sub-steps of: (a) Dissolving hydroxyproline protected by a protecting group and an alkaline catalyst in an organic solvent, adding glycine protected by the protecting group for coupling reaction, adding a protecting agent, an imidazole compound and a catalyst for upper protection reaction, and purifying to obtain a dipeptide product protected by the protecting group ; (B) Dissolving the dipeptide product protected by the protecting group and a deprotection agent in an organic solvent for deprotection reaction, and purifying to obtain the dipeptide product with the protecting group removed ; (C) Dissolving proline protected by a protecting group in an organic solvent, adding a dipeptide product without the protecting group, performing coupling reaction under an alkaline catalyst, and purifying to obtain a tripeptide unit ; In step (B), the synthetic linear hexapeptide precursor comprises the sub-steps of: (i) Tripeptide protected by protecting group is taken as raw material, and is subjected to deprotection reaction with metal catalyst in organic solvent, and intermediate product 1 is obtained after purification ; (Ii) Dissolving tripeptide protected by protecting group and deprotection agent in organic solvent for deprotection reaction, purifying to obtain intermediate 2 ; (Iii) Dissolving the intermediate product 1 in an organic solvent, adding the intermediate product 2, performing coupling reaction under the condition of an alkaline catalyst, and purifying to obtain a linear hexapeptide precursor 。
  7. 7. The synthetic method according to claim 5, wherein in the step (A) and/or the step (B), the coupling reaction is performed in the presence of a carboxyl activating system selected from one or more of N, N ' -diisopropylcarbodiimide, O-benzotriazol-tetramethylurea hexafluorophosphate, 2- (7-azobenzotriazole) -N, N, N ', N ' -tetramethylurea hexafluorophosphate, ethyl 2-oxime nitriloacetate.
  8. 8. The method according to claim 5, wherein in the step (a), the hydroxyproline protected by the protecting group is Boc-L-hydroxyproline, the carboxyl activating system of the coupling reaction is benzotriazole-N, N, N ', N' -tetramethylurea hexafluorophosphate, the basic catalyst is N, N-diisopropylethylamine, the glycine protected by the protecting group is glycine benzyl ester hydrochloride, the organic solvent is N, N-dimethylformamide, the equivalent ratio of hydroxyproline protected by the protecting group to benzotriazole-N, N, N ', N' -tetramethylurea hexafluorophosphate to N, N-diisopropylethylamine to glycine protected by the protecting group is 1:0.5-1:2-5:1-1.2, the time of the condensation reaction is 1-5 hours, the protecting agent is acetic anhydride, the imidazole compound is imidazole, the catalyst is 4-dimethylaminopyridine, and the equivalent ratio of acetic anhydride to imidazole to dimethylaminopyridine is 1:1-3, the time of the condensation reaction is 1:3-3; in the step (b), the deprotection agent is 1,4 epoxy hexacyclic ring or salt thereof, the organic solvent is methylene dichloride, the equivalent ratio of the dipeptide product protected by the protecting group to the deprotection agent is 1:9-11, the mass-volume ratio of the dipeptide product protected by the protecting group to the organic solvent is 1:0.5-2, and the deprotection reaction time is 0.5-2 hours; In the step (c), the proline protected by the protecting group is Boc-L-proline, the organic solvent is dichloromethane, the alkaline catalyst is triethylamine, the equivalent ratio of the proline protected by the protecting group, the dipeptide product of the deprotecting group, the carboxyl activating system and the alkaline catalyst is 1:1-3:2-4:1-2, the mass volume ratio of the proline protected by the protecting group to the organic solvent is 1:9-11, and the reaction time is 2-6 hours; in the step (i), the metal catalyst is palladium supported on carbon, the equivalent ratio of tripeptide protected by the protecting group to the metal catalyst is 1:0.005-0.02, the organic solvent is methanol, and the reaction time is 3-8 hours.
  9. 9. The synthetic method according to claim 5, wherein the hydroxyl protecting group is acetyl, t-butoxycarbonyl, t-butyldiphenylsilyl or methoxymethyl, the carboxyl protecting group is benzyl, acetyl, t-butoxycarbonyl, t-butyldiphenylsilyl or methoxymethyl, and the amino protecting group is t-butoxycarbonyl, acetyl, t-butyldiphenylsilyl or methoxymethyl; Preferably, the hydroxyl protecting group is acetyl, the carboxyl protecting group is benzyl, and the amino protecting group is t-butoxycarbonyl.
  10. 10. Use of a biomimetic collagen cyclic hexapeptide of formula (I) in the manufacture of a medicament for promoting fibroblast proliferation: Formula (I).

Description

Preparation method and application of bionic collagen cyclohexapeptide Technical Field The invention belongs to the technical field of biological medicine, and particularly relates to a preparation method and application of bionic collagen cyclohexapeptide. Background Collagen is the most abundant structural protein in animals, and its molecular structure is usually represented by (Gly-X-Y) n repeat sequence, wherein X site is mainly proline (Pro), Y site is mainly hydroxyproline (Hyp), so Gly-Pro-Hyp is the most representative repeat unit in collagen. Researches show that the complete macromolecular glue is difficult to be absorbed through skin due to the fact that the molecular weight is too high, and the collagen peptide with low molecular weight has good transdermal performance and has multiple anti-aging effects of repairing skin injury, improving skin moisture content, enhancing skin elasticity, reducing wrinkles and the like. Collagen peptides containing hydroxyproline are considered as key components that exert these biological activities (Seong S H et al, J Cosmet Dermatol, 2024; wang L et al, curr Opin Food Sci, 2023). In addition, collagen peptide has been demonstrated to have the effects of promoting cell proliferation, supporting angiogenesis and tissue repair (Ohara H et al, J Dermatol, 2010; zhang Z et al, J Sci Food Agric, 2011), and has important application value in the fields of cell culture, medical repair and skin care. Currently related studies and applications focus mainly on linear collagen peptides. However, linear polypeptides are susceptible to oxidation and hydrolysis during storage and use, and their membrane permeability and structural stability remain to be improved, limiting their bioavailability and long-term efficacy. Compared with linear peptide, cyclic peptide has the characteristics of limited conformation, high enzymolysis stability, excellent binding efficiency with target point, good biocompatibility and the like, and is attracting attention in the development of active ingredients of medicines and cosmetics. The cyclic peptide can penetrate the skin barrier more effectively, and through simulating the combination of endogenous signal molecules and cell surface receptors, the pathways of collagen synthesis, melanin generation inhibition, free radical neutralization and the like are regulated, so that potential advantages are presented in the aspects of aging resistance, whitening, oxidation resistance and the like. Early cyclic peptides are mainly extracted from plants, but natural sources have low cyclic peptide content, complex extraction process, high cost, resource and environment limitation, and are difficult to meet the large-scale requirements. The current cyclic peptide synthesis mainly depends on a solid phase synthesis strategy, namely, gradually coupling and synthesizing a linear precursor on resin, and then performing solution phase cyclization after cleavage and purification. The whole process has complicated steps, high price of protecting amino acid monomer is needed, raw material investment is excessive to ensure coupling efficiency, and the purification and treatment are carried out for many times, so that the production cost is high, the process is complex, and the large-scale preparation and application of the amino acid monomer are restricted. In addition, the preparation of cyclic peptides by microbial fermentation is difficult to introduce unnatural amino acids such as hydroxyproline, and the consistency of product quality is difficult to control. Another common route to collagen peptide acquisition is the hydrolysis of native collagen. However, the product obtained by the method is a mixture of peptide fragments with different chain lengths and structures, has complex components and wide molecular weight distribution, may have pathogen residue risk, and is difficult to obtain single cyclic collagen peptide with definite structure and high purity. Therefore, developing a synthetic method which is simple and convenient in process, controllable in cost, suitable for large-scale preparation and capable of accurately constructing the hydroxyproline unit-containing cyclopeptide becomes a technical problem to be solved in the field. In particular, how to efficiently and selectively realize cyclization in a liquid phase system and overcome side reactions possibly caused by hydroxy of a hydroxyproline side chain is one of the key challenges in the synthesis process of the bionic collagen cyclohexapeptide. Disclosure of Invention The invention aims to provide a preparation method and application of bionic collagen cyclic hexapeptide, and aims to overcome the defects that the cyclic peptide is high in preparation cost, complex in process and difficult to synthesize the hydroxyproline unit-containing bionic cyclic peptide in large scale in the prior art. The specific technical scheme of the invention is as follows: The invention provides a preparation metho