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CN-122011156-A - Weight-losing peptide with high activity and preparation method and application thereof

CN122011156ACN 122011156 ACN122011156 ACN 122011156ACN-122011156-A

Abstract

The application relates to the technical field of biological peptides, and discloses a high-activity weight-losing peptide, a preparation method and application thereof, wherein the weight-losing peptide is prepared from Arg34-GLP-1 (7-37) main chain, an acylating agent, an N-acetyl-L-arginine chaotropic regulator, a sodium benzoate interface regulator, a zinc salt masking agent and a reaction solvent. The preparation method comprises the steps of carrying out in-situ coordination masking on the N-terminal of the polypeptide by utilizing zinc ions in a water phase, then carrying out solvent polarity switching by feeding a reaction solvent to construct a homogeneous reaction system, carrying out high-selectivity acylation reaction, and finally carrying out metal decomplexing and deprotection treatment. The application utilizes the synergistic effect of metal coordination locking and a chaotropic regulator to effectively solve the technical problems of serious N-terminal competitive side reaction, easy racemization of histidine and easy aggregation and precipitation of polypeptide in a mixed solvent in the modification process, and the obtained product has high regioselectivity and complete bioactivity.

Inventors

  • GUO CHANGBAO
  • XIN JING
  • Mou Chunli

Assignees

  • 济南健丰化工有限公司

Dates

Publication Date
20260512
Application Date
20260210

Claims (10)

  1. 1. A high-activity slimming peptide is characterized by being prepared from Arg34-GLP-1 (7-37) polypeptide main chain, an acylating agent, a non-nucleophilic chaotropic regulator, an interface regulator, a metal masking agent and a reaction solvent; The metal masking agent is used in an amount of 1.8-2.2 mol parts and the acylating agent is used in an amount of 1.15-1.3 mol parts based on 1mol part of Arg34-GLP-1 (7-37) polypeptide main chain; The concentration of the non-nucleophilic chaotropic regulator in the reaction composition is 0.3-0.5 mol/L, and the concentration of the interface regulator in the reaction composition is 50-100 mmol/L.
  2. 2. The weight loss peptide of claim 1 wherein the non-nucleophilic chaotropic modulator is N-acetyl-L-arginine; the interface regulator is sodium benzoate; The metal masking agent is zinc acetate or zinc acetate dihydrate; The acylating agent is stearyl diacid mono-tert-butyl ester-N-hydroxysuccinimide ester.
  3. 3. The weight loss peptide of claim 1 wherein the reaction solvent is a mixed solvent of water and N-methylpyrrolidone, wherein the volume ratio of N-methylpyrrolidone to water is from 50:50 to 60:40.
  4. 4. A process for the preparation of a highly active slimming peptide according to any one of claims 1 to 3, comprising the steps of: S1, dissolving Arg34-GLP-1 (7-37) polypeptide main chain in water, adding a non-nucleophilic chaotropic regulator and an interface regulator, and regulating pH to be alkaline to obtain a precursor solution; s2, in-situ metal masking, namely adding a metal masking agent into the precursor solution, and reacting to form a metal-peptide complex; s3, solvent polarity switching, namely, adding an organic solvent into the metal-peptide complex in a flowing way to construct a homogeneous reaction system; s4, high-selectivity acylation reaction, namely adding an acylating agent into the S3 system for reaction; s5, quenching and decomplexing, namely adding a quenching agent to terminate the reaction after the reaction is finished, and adding a metal decomplexing agent to remove metal ions; S6, post-treatment and deprotection, namely filtering and concentrating the product obtained in the step S5, removing side chain protecting groups, and purifying to obtain the target slimming peptide.
  5. 5. The method according to claim 4, wherein in step S1, the concentration of the Arg34-GLP-1 (7-37) polypeptide main chain is 10.0-15.0 g/L, and the pH is adjusted to 7.8-8.2.
  6. 6. The method for preparing a slimming peptide according to claim 4, wherein in the step S2, the molar ratio of zinc ions to polypeptides in the metal masking agent is 1.8:1-2.2:1, and the method of adding is dropwise adding, and stirring is performed for 30-45 min at 20-25 ℃ after dropwise adding.
  7. 7. The method according to claim 4, wherein in step S3, the organic solvent is fed to a volume ratio of N-methylpyrrolidone to the accumulated water phase in the system of 50:50 to 60:40, and the temperature of the system is controlled to be 20-25 ℃ during the feeding.
  8. 8. The method according to claim 4, wherein in step S4, the acylating agent is added dropwise, the pH value after the dropwise is maintained at 7.8-8.2, and the reaction time is 45-90 min.
  9. 9. The method according to claim 4, wherein in step S5, the quencher is glycine, the metal decomplexing agent is disodium edentate, and the molar ratio of disodium edentate to the added metal masking agent is 1.5:1-2.0:1; In the step S6, the method for removing the side chain protecting group is to add trifluoroacetic acid into the concentrated intermediate solution to make the volume percentage of TFA reach 90%, and react for 30min at room temperature to remove the side chain tert-butyl ester.
  10. 10. Use of a highly active weight loss peptide according to any one of claims 1-3 in the manufacture of a medicament for the prevention or treatment of obesity.

Description

Weight-losing peptide with high activity and preparation method and application thereof Technical Field The invention relates to the technical field of biological peptides, in particular to a weight-losing peptide with high activity, and a preparation method and application thereof. Background Glucagon-like peptide-1 (GLP-1) analogs have become important drugs for the treatment of obesity and type 2 diabetes due to their remarkable hypoglycemic and weight-reducing effects. To overcome the short half-life of native GLP-1, it is often necessary to modify the long chain fatty acid side chains at specific lysine residues in the polypeptide backbone to increase its binding capacity to plasma albumin. However, in the process of chemically modifying the polypeptide main chain represented by Arg34-GLP-1 (7-37), a serious synthetic chemical challenge is faced. Arg34-GLP-1 (7-37) polypeptide comprises a plurality of nucleophilic active sites, wherein alpha-amino and imidazolyl of N-terminal histidine (His 7) have similar nucleophilic reactivity with epsilon-amino of target modification site lysine. In conventional acylation reaction systems, it is difficult for the acylating agent to distinguish the above sites, leading to a very competitive acylation reaction at the N-terminus, resulting in N-terminal monoacylation or N, ε -bisacylation impurities. The physical and chemical properties of the position isomer impurities are highly similar to those of the target product, so that the position isomer impurities are extremely difficult to separate by a conventional chromatographic means, and the yield and purity of the final product are seriously affected. Furthermore, in order to increase the nucleophilicity of lysine side chains, the reaction is usually carried out under alkaline conditions, but this introduces a new risk that the chiral center of the N-terminal histidine is thermodynamically unstable in alkaline environments, and proton extraction is very likely to occur, leading to configuration inversion, i.e., conversion from the biologically active L-form to the inactive D-form, and this racemization side reaction directly leads to loss of the biological activity of the drug. To solve the above-mentioned regioselectivity problem, the prior art attempts to protect with complexes of transition metal ions (e.g., zinc ions) with N-terminal histidines. However, this strategy presents significant solvent compatibility barriers in practical applications. Since long chain fatty acid derivatives required for modification are generally strongly hydrophobic, a high proportion of organic solvents (such as N-methylpyrrolidone) must be used for dissolution. However, the metal-polypeptide complex belongs to a hydrophilic salt structure, when the proportion of an organic solvent in the system is increased, the dielectric constant of the solution is reduced, and the metal-polypeptide complex is extremely easy to desolvate to generate hydrophobic aggregation and even precipitation. The phase separation phenomenon not only damages the uniformity of a reaction system and hinders the mass transfer of the reaction, but also can cause the protection failure of metal ions due to precipitation, thereby further causing N-terminal side reaction or physical loss of a polypeptide main chain. Therefore, how to maintain the colloid stability of the metal-polypeptide coordination structure and inhibit the racemization of the N-terminal while ensuring the solubility of the long-chain acylating agent is a key technical problem to be solved in the preparation of the high-activity weight-reducing peptide at present. Disclosure of Invention Aiming at the defects of the prior art, the invention provides a weight-losing peptide with high activity, a preparation method and application thereof, and solves the technical problems of serious N-terminal competitive side reaction, easy racemization of histidine and poor solvent compatibility of a reaction system in the polypeptide side chain modification process, which leads to polypeptide aggregation and precipitation. In order to achieve the aim, the first aspect of the invention provides a high-activity slimming peptide, which is prepared from Arg34-GLP-1 (7-37) polypeptide main chain, an acylating agent, a non-nucleophilic chaotropic regulator, an interface regulator, a metal masking agent and a reaction solvent; the metal masking agent is used in an amount of 1.8-2.2 mol parts and the acylating agent is used in an amount of 1.15-1.3 mol parts based on 1mol part of Arg34-GLP-1 (7-37) polypeptide main chain; The concentration of the non-nucleophilic chaotropic regulator in the reaction composition is 0.3-0.5 mol/L, and the concentration of the interface regulator in the reaction composition is 50-100 mmol/L. By adopting the technical scheme, the invention constructs a reaction system with specific ionic strength and coordination environment, and realizes high-selectivity modification through the chemical sy