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Method for preparing protein-polyamino acid cyclic conjugate

A polyamino acid and protein technology, applied in the field of biomedicine, can solve problems such as accelerated clearance, low immunogenicity, and accumulated toxicity

Active Publication Date: 2017-07-07
PEKING UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Polyethylene glycol has the advantages of low immunogenicity and good water solubility. However, with the wide application of polyethylene glycol in cosmetics, food, pharmaceuticals and other fields, most people have produced polyethylene glycol antibodies in their bodies, which will instead Accelerated blood clearance of PEG-modified protein drugs
What's more serious is that polyethylene glycol is not degradable in the human body, and long-term use will lead to accumulated toxicity

Method used

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  • Method for preparing protein-polyamino acid cyclic conjugate
  • Method for preparing protein-polyamino acid cyclic conjugate
  • Method for preparing protein-polyamino acid cyclic conjugate

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0177] Example 1: Synthesis of polyamino acid P1 for protein coupling

[0178]

[0179] L-glutamic acid (ethylene glycol) in the glove box 3 Ester-N-carboxylide anhydride (N1, 40.0 mg, 0.131 mmol, 20 equiv) was dissolved in anhydrous N,N-dimethylformamide (1.0 mL) and added trimethylsilylthiophenol in DMF Solution (13.6μL×0.5M, 1.0 equivalent), stirred at room temperature for 25 hours, then poured the reaction solution into 40mL ether solution, and the white precipitate was the product. Centrifuge to obtain a solid product, pour out the supernatant and rinse the product with 40 mL of ether again, pour the supernatant after centrifugation to obtain a solid and dry it in a vacuum oven to obtain 30 mg of a colorless transparent colloidal solid (yield 72%), which is stored in - 20 ℃ refrigerator.

[0180] Poly-L-glutamic acid (ethylene glycol) with an average degree of polymerization of 7 was synthesized in a manner similar to the above method 3 Esters, simply add L-glutam...

Embodiment 2

[0181] Example 2: Synthesis of block polyamino acid P2 for protein cyclization

[0182]

[0183] L-glutamic acid (ethylene glycol) in the glove box 3Ester-N-carboxylide anhydride (N1, 13.0 mg, 0.044 mmol, 7 equiv) was dissolved in anhydrous N,N-dimethylformamide (1.0 mL) and trimethylsilylthiophenol in DMF was added Solution (13.1μL×0.5M, 1.0 equivalent), stirred at room temperature for 25 hours, then added glycine-N-carboxy internal anhydride (N2, 2.0mg, 0.0197mmol, 3 equivalents), continued to stir at room temperature for 2 hours, post-treatment method Same as Example 1.

Embodiment 3

[0184] Example 3: Synthesis of polyamino acid P4 for protein coupling

[0185]

[0186] Ethyl phosphate-L-tyrosine N-carboxylidene anhydride (N3, 100 mg, 0.291 mmol, 20 equiv) was dissolved in anhydrous N,N-dimethylformamide (2.0 mL) in a glove box, and A DMF solution of trimethylsilylthiophenol (29.1 μL×0.5M, 1.0 equivalent) was added, stirred at room temperature for 25 hours, and the post-treatment method was the same as in Example 1.

[0187] The above polyamino acid (P3, 80mg, 0.0133mmol, 1.0eq) was dissolved in dichloromethane (1.5mL), and triethylamine (300μL, 2.1mmol) and trimethylbromosilane (360μL, 2.7mmol) were added sequentially . The reaction solution was heated to 60°C and stirred for 8 hours. After the solvent was spin-dried on a rotary evaporator, the product was dissolved in deionized water (4mL), and EDTA (10mg) was added, and the pH was adjusted with aqueous sodium hydroxide solution (1M). to neutral, and dialyzed against aqueous sodium chloride (1000D...

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Abstract

The invention relates to field of biological medicines, in particular to a method for preparing a site specific protein-polyamino acid cyclic conjugate. The method comprises the following steps: (1) using an initiator to initiate N-carboxyl anhydride and glycine N-carboxyl anhydride to be polymerized to obtain block polyamino acid of which the C-terminal has a phenyl thioester structure and the N-terminal has a polyglycine block structure; (2) mixing the obtained block polyamino acid with proteins of which the N-terminal has cysteine and the C-terminal has an LPXaTG sequence, and performing native chemical ligation and transpeptidase reaction continuously to realize cyclization of proteins, wherein Xa is any one of amino acids. The protein-polyamino acid cyclic conjugate prepared by the invention has the characteristics of polypeptide main chain structure, good biocompatibility, low immunogenicity and the like, and is very suitable for modification of protein drugs, thereby being capable of being used for manufacturing the protein drugs.

Description

technical field [0001] The present disclosure relates to the field of biomedicine, in particular to a method for preparing a site-specific protein-polyamino acid cyclic conjugate. Background technique [0002] Protein drugs (such as antibodies, interferons) have been widely used in the field of biomedicine, such as targeted therapy, clinical diagnosis, etc. Compared with other drugs, protein drugs have better specificity, quick effect and low toxicity. However, its disadvantages such as poor stability and short blood circulation time limit the efficacy and application of such drugs to a certain extent. [0003] At present, the most researched system for improving protein stability and prolonging its blood circulation time is the protein-polymer hybrid system. The blood circulation time of protein drugs is relatively short due to the degradation of proteases in the body. Therefore, grafting polymer materials with better water solubility on the surface of proteins has a cert...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K47/64C08H1/00A61K38/00
CPCC08G69/42C08H1/00A61K38/21A61K39/395A61K38/00A61K47/645C07K14/56C07K14/71C07K14/43595C07K2319/00C07K2319/50A61P31/12A61P35/00A61P37/00A61P37/04A61P5/00C07K1/10C07K14/001
Inventor 吕华袁劲松侯颖钦周雨
Owner PEKING UNIV
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