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Method for reducing and/or removing default peptide in solid phase synthesis of polypeptide

A technology of solid-phase synthesis and solid-phase synthesis of peptides, applied in peptide preparation methods, chemical instruments and methods, peptides, etc., to achieve the effects of reducing default peptide impurities, easy access to raw materials, and extensive industrial production value

Active Publication Date: 2017-10-24
HYBIO PHARMA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

This method uses a fully protected peptide fragment containing the default amino acid and its previous amino acid (C-N) for solid-phase synthesis of the polypeptide, and at the same time solves the amino acid default caused by incomplete removal of Fmoc and incomplete condensation reaction, and can be used in the synthesis stage. Default peptide impurity reduced below quality control limit

Method used

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  • Method for reducing and/or removing default peptide in solid phase synthesis of polypeptide
  • Method for reducing and/or removing default peptide in solid phase synthesis of polypeptide
  • Method for reducing and/or removing default peptide in solid phase synthesis of polypeptide

Examples

Experimental program
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Effect test

Embodiment 1

[0058] The preparation of embodiment 1Fmoc-Gly-Wang Resin

[0059] Weighing 40g of Wang Resin with a degree of substitution of 0.75mmol / g was added to the solid-phase reaction column, washed twice with DMF, swelled with DMF for 30min, washed twice with DMF, and Fmoc-Gly-OH (13.38g, 45mmol) and HOBt ( 6.38g, 47.3mmol) was dissolved in DMF, after ice bath for 10min, DIC (5.95g, 47.3mmol) was added to activate for 3-5min, then added to the reaction column, and DMAP (0.55g, 4.5mmol) was added at the same time, stirred for 2h under nitrogen gas, pumped Dry the reaction solution, wash 4 times with DMF, wash 3 times with DCM, add a DCM solution of acetic anhydride (61.2g, 600mmol) and pyridine (47.5g, 600mmol) to block for 8h, remove the blocking solution, wash 6 times with DMF, and wash 2 times with DCM Once, MeOH was shrunk and dried to obtain 43.6g Fmoc-Gly-Wang Resin with a substitution degree of 0.32mmol / g.

Embodiment 2

[0060] The synthesis of embodiment 2 liraglutide peptide resin

[0061] Weighing 31.3g of Fmoc-Gly-Wang Resin with a degree of substitution of 0.32mmol / g was added to the solid-phase reaction column, washed twice with DMF, swelled with DMF for 30min, washed twice with DMF, and Fmoc-Arg(pbf)-OH( 19.46g, 30mmol) and HOBt (4.26g, 31.5mmol) were dissolved in DMF, ice-bathed for 10min, added DIC (3.97g, 31.5mmol) to activate for 3-5min, then added to the solid-phase reaction column, stirred and reacted for 2h under nitrogen, The ninhydrin detection reaction is complete, the reaction solution is taken out, DMF washes 3 times, 20% DBLK deprotection (5+7min), DMF washes 6 times;

[0062] Dissolve Fmoc-Gly-OH (8.92g, 30mmol) and HOBt (4.26g, 31.5mmol) in DMF, ice-bath for 10min, add DIC (3.97g, 31.5mmol) to activate for 3-5min, then add to the solid-phase reaction column During the reaction, the reaction was stirred with nitrogen for 2 hours, and the reaction was detected by ninhydrin...

Embodiment 3

[0064] Example 3 Liraglutide Peptide Resin Cleavage

[0065] Transfer 86.2g of liraglutide peptide resin to a 1000ml single-necked round bottom flask, add 860ml of frozen 2h lysate (TFA:Phenol:H 2 O: EDT = 87.5: 5: 5: 2.5), stirred at room temperature for 2 hours, filtered, the filtrate was added to 8.6L of frozen anhydrous ether, centrifuged, washed and dried to obtain 37.3g of crude peptide, yield: 99.5%, purity : 53.79%, see figure 1 .

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Abstract

The invention relates to the field of polypeptide synthesis and particularly relates to a method for reducing and / or removing a default peptide in solid phase synthesis of a polypeptide. The method adopts an all-protected peptide fragment comprising a default amino acid and an amino acid (C-N) in front of default amino acid in the solid phase synthesis of the polypeptide, the problems of default of the amino acid caused by incomplete removal of Fmoc and incomplete condensation reaction are solved at the same time, and the impurity of the default peptide is reduced to lower than the qualification threshold in the synthesis stage. For the specific performance, the Ala2 default impurity and the Thr5 default impurity in liraglutide are respectively reduced from more than 1% to the nesiritide amount and lower than 0.15%, and the Ser19 default peptide impurity in nesiritide is reduced to the undetectable amount.

Description

technical field [0001] The invention relates to the field of polypeptide synthesis, in particular to a method for reducing and / or removing default peptides in polypeptide solid-phase synthesis. Background technique [0002] In the solid-phase peptide synthesis, some peptides are not completely reacted due to the presence of difficult sequences, and it is easy to form default peptide (non-target peptide) impurities. The properties of some default peptide impurities are very close to the product itself, and the existing separation techniques have little separation effect on them. [0003] In the Fmoc solid-phase synthesis method, the generation of the default peptide is mainly due to the aggregation of the peptide chain, the incomplete removal of the temporary protecting group Fmoc or the incomplete condensation reaction. For incomplete removal of Fmoc, generally 2% DBU can be used to achieve complete removal, but it is accompanied by side reactions. For the incomplete conde...

Claims

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Application Information

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IPC IPC(8): C07K14/605C07K14/58C07K1/16C07K1/06C07K1/04
CPCC07K14/58C07K14/605
Inventor 陈友金宓鹏程陶安进袁建成
Owner HYBIO PHARMA
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