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Solid-phase preparation method of liraglutide

A technology for solid-phase preparation and liraglutide is applied in the field of solid-phase preparation of liraglutide, which can solve the problems of serious resin shrinkage, high technical difficulty, and many similar impurities.

Active Publication Date: 2013-09-18
HYBIO PHARMA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] At present, Novo Nordisk mainly uses yeast to produce liraglutide through genetic recombination technology; however, due to the need for genetic recombination technology, the technology is difficult and the cost is relatively high. 34 - The side chain of GLP-1(7-37)-OH is unprotected, and N α -hexadecanoyl-Glu(ONSu)-OtBu will produce more impurities during the reaction, and the loss will be greater
It is also reported that the solid-phase step-by-step coupling method is used for preparation. Since there are many hydrophobic amino acids in the sequence of liraglutide, the resin shrinks severely during the step-by-step coupling, and the reaction is incomplete, resulting in more impurities in the crude peptide and products with similar properties. , HPLC preparation is difficult and the yield is low

Method used

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Examples

Experimental program
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Effect test

Embodiment 1

[0047] The preparation of embodiment 1Fmoc-Gly-Wang Resin

[0048] Weighing 100g of Wang Resin with a degree of substitution of 0.803mmol / 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 (35.81g, 120.5mmol) and HOBt (17.9g, 132.5mmol) was dissolved in DMF, after ice bath for 10min, DIC (16.72g, 132.5mmol) was added to activate for 3-5min, and then added to the reaction column, and DMAP (1.47g, 12.05mmol) was added at the same time, and stirred for 2h under nitrogen, Drain the reaction liquid, wash 4 times with DMF, wash 3 times with DCM, add acetic anhydride (164g, 160.6mmol) and pyridine (127g, 160.6mmol) in DCM solution to block for 8h, drain off the blocking solution, wash with DMF 6 times, and wash with DCM Twice, MeOH shrinks and dries to obtain Fmoc-Gly-Wang Resin with a substitution degree of 0.36mmol / g.

Embodiment 2

[0049] Embodiment 2 The preparation of the first polypeptide fragment-the first resin

[0050]Weigh 13.89g of Fmoc-Gly-Wang Resin with a degree of substitution of 0.36mmol / g and add it to the solid-phase reaction column, wash twice with DMF, swell with DMF for 30min, wash twice with DMF, and add Fmoc-Arg(pbf)-OH( 9.73g, 15mmol) and HOBt (2.13g, 15.8mmol) were dissolved in DMF, ice-bathed for 10min, added DIC (1.98g, 15.8mmol) to activate for 3-5min, then added to the solid-phase reaction column, stirred for 2h under nitrogen, The ninhydrin detection reaction is complete, remove the reaction solution, wash 3 times with DMF, deprotect 20% DBLK (6+6min), and wash 6 times with DMF;

[0051] Dissolve Fmoc-Gly-OH (4.46g, 15mmol) and HOBt (2.13g, 15.8mmol) in DMF, ice-bath for 10min, add DIC (1.98g, 15.8mmol) to activate for 3-5min, then add to the solid-phase reaction column During the reaction, the reaction was stirred under nitrogen for 2 hours, and the reaction was detected by n...

Embodiment 3

[0053] Example 3 The second polypeptide fragment whose sequence is shown in SEQ ID No.2 is Fmoc-Thr(tBu)-Phe-Thr(tBu)-Ser(tBu)-Asp(OtBu)-Val-Ser(tBu)-Ser Preparation of (tBu)-Tyr(tBu)-Leu-Glu(OtBu)-Gly-Gln(trt)-Ala-Ala-Lys(Alloc)-Glu(OtBu)-Phe-Ile-OH

[0054] Weighing 46.5g of 2-CTC Resin with a degree of substitution of 0.86mmol / g was added to the solid-phase reaction column, washed twice with DMF, swelled with DMF for 30min, washed twice with DMF, and Fmoc-Ile-OH (42.4g, 120mmol) and DIPEA (15.5g, 120mmol) were dissolved in DMF, ice-bathed for 10min, and then added to the solid-phase reaction column. After stirring for 5 minutes under nitrogen, 15.5g of DIPEA was added again, and the reaction was continued for 1h, and 25.6g of MeOH was added to seal for 20 minutes. Dry the reaction solution, wash with DMF 6 times to obtain Fmoc-Ile-2-CTC Resin;

[0055] Add 20% DBLK to Fmoc-Ile-2-CTC Resin for deprotection (6+6min), wash 6 times with DMF, Fmoc-Phe-OH (46.5g, 120mmol), HOBt ...

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Abstract

The invention relates to the field of polypeptide synthesis and in particular relates to a solid-phase preparation method of liraglutide. The preparation method has the beneficial effects that difficult sequences or single fragments containing the difficult sequences are connected to resins; amino acid connection is carried out by adopting a stepwise coupling method in other regions; the purity and yields of coarse peptides are obviously improved; and the cost is relatively low.

Description

technical field [0001] The invention relates to the field of polypeptide synthesis, in particular to a solid-phase preparation method of liraglutide. Background technique [0002] With the improvement of living standards and changes in lifestyle, the incidence of diabetes in my country has been increasing year by year in recent years. Diabetes is caused by genetic factors, immune dysfunction, microbial infection and its toxins, free radical toxins, mental factors and other pathogenic factors acting on the body, resulting in hypofunction of pancreatic islets, insulin resistance, etc., and then causing sugar, protein, fat, A series of metabolic disorder syndromes such as water and electrolytes, clinically characterized by hyperglycemia, typical cases may appear polyuria, polydipsia, polyphagia, weight loss and other symptoms, that is, "three more and one less" symptoms, once the control is not good It will cause serious complications, leading to failure and lesions in the kid...

Claims

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Application Information

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IPC IPC(8): C07K14/605C07K1/06C07K1/04
Inventor 潘俊锋刘建马亚平袁建成
Owner HYBIO PHARMA
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