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Solid-phase fragment method for the synthesis of exenatide

A technology of exenatide and solid-phase synthesis, which is applied in the direction of peptides, specific peptides, hormone peptides, etc., can solve the problems of low purity and yield, excessive waste liquid, and many impurities

Active Publication Date: 2020-04-07
NANJING HUAWE MEDICINE TECH DEV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0012] The technical problem to be solved by the present invention is to provide a new moxa compound for the problems of many impurities, low purity and yield, high cost, cumbersome operation steps, excessive waste liquid, and unfavorable industrial production in the existing synthesis process. Synthetic method of senatide

Method used

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  • Solid-phase fragment method for the synthesis of exenatide
  • Solid-phase fragment method for the synthesis of exenatide
  • Solid-phase fragment method for the synthesis of exenatide

Examples

Experimental program
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Effect test

Embodiment 1

[0068] Example 1: Synthetic Fragment [1-19] Resin

[0069] Fmoc-Rink Amide MBHA resin (degree of substitution 0.312mmol / g, 10g) was put into a solid-phase reaction bottle, swollen with DCM for 30 minutes, washed twice with DMF, and the solution was drained. Add 20% piperidine in DMF solution at room temperature, deprotect the reaction twice, the time is 5min and 15min respectively, drain the solution, wash the resin twice with DMF and IPA alternately, wash twice with DMF, wash twice with DCM, and drain , to obtain a deprotected resin. Add Fmoc-Ser(tBu)-OH (3.59g) and 50mL DMF to the dry reaction flask successively to obtain a solution. Add HOBt (1.52g) and DIC (1.42g) in an ice bath, and stir for 10 minutes to obtain an activated solution. Add the above solution into the solid-phase reaction vial, and stir for 3 hours until the ninhydrin detection coupling reaction ends. Drained, the resin was washed three times with appropriate amount of DMF, and washed three times with ...

Embodiment 2

[0071] Embodiment two: synthetic fragment-resin peptide Weigh 50 g of 2-CTC resin with a substitution degree of 1.1 mmol / g, add it to a solid-phase reaction bottle, and wash it twice with DCM. Add 107.05g of Fmoc-Arg(pbf)-OH and 500mL of DCM to the solid-phase reaction flask in sequence, and after stirring for 5 minutes, slowly add 42.65g of DIEA dropwise, and stir for 2 hours after dropping. Add 100mL methanol and stir for 30min. Wash 3 times with DMF, twice with DCM, twice with methanol, and drain. After vacuum drying, 76.51 g of Fmoc-Arg(pbf)-CTC resin was obtained, and the degree of substitution was measured to be 0.865 mmol / g. Add the resin into the solid phase reactor, add 500mL DMF, stir for 20 minutes, and drain. Add 400 mL of 20% piperidine in DMF, stir at room temperature for 5 min, and drain; add 400 mL of 20% piperidine in DMF, stir for another 15 min at room temperature, and drain. The resin was alternately washed twice with DMF and IPA, twice with DMF, and t...

Embodiment 3

[0072] Embodiment three: synthetic polypeptide fragment one 78.56 g of the resin obtained in Example 2 was added to 1000 mL of 20% TFE in DCM, and stirred for 2 hours. The resin was removed by filtration, and the filtrate was collected and concentrated under reduced pressure to remove the solvent. Add 200mL of DCM to the residue to dissolve, wash with salt water three times, dry the organic phase with anhydrous sodium sulfate for 30min, concentrate under reduced pressure to about 100mL of DCM, add 1000mL of glacial ether for precipitation and crystallization, filter, suck dry, and vacuum dry to obtain 27.36g Fragment 1, yield 86%, purity 97%, mass spectrum 1187.7 (M-H) - .

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Abstract

The invention provides a solid-phase synthesis method of exenatide. In the method described in the invention, according to peptide sequences of the main chain of exenatide, amino acid sequences are divided into a basic fragment [1-19] resin and two fragments, and the three short chain polypeptides can be separately synthesized first. The basic fragment [1-19] resin is subjected to coupling with afragment 1, amino acids are coupled continually in a stepwise manner to obtain a fragment [1-35] resin, and finally, the fragment [1-35] resin is subjected to coupling with a fragment 2 to finish allamino acid linkage, followed by pyrolysis, purification and salification, thereby obtaining the target product, with structures of each fragment and each fragment resin as described in the specification. The method is simple in operation, and the obtained product is high in purity, low in cost and conducive to industrial production.

Description

technical field [0001] The invention relates to the field of polypeptide solid-phase synthesis, in particular to the synthesis of exenatide by a solid-phase fragment method. Background technique [0002] Exenatide is a polypeptide composed of 39 amino acids. According to the amino acid sequence from the C-terminal to the N-terminal of the main chain of exenatide, its sequence is: [0003] His 39 -Gly 38 -Glu 37 -Gly 36 -Thr 35 -Phe 34 -Thr 33 -Ser 32 -Asp 31 -Leu 30 -Ser 29 -Lys 28 -Gln 27 -Met 26 -Glu 25 -Glu 24 -Glu 23 -Ala 22 -Val 21 -Arg 20 -Leu 19 -Phe 18 -Ile 17 -Glu 16 -Trp 15 -Leu 14 -Lys 13 -Asn 12 -Gly 11 -Gly 10 -Pro 9 -Ser 8 -Ser 7 -Gly 6 -Ala 5 -Pro 4 -Pro 3 -Pro 2 -Ser 1 . The amino acid sequence of exenatide has 53% homology with human glucagon-like peptide-1 (GLP-1), has a high affinity with the GLP-1 receptor, and also has a significant hypoglycemic effect. -1 has the same physiological function. Stimulates the body t...

Claims

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Application Information

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IPC IPC(8): C07K14/575C07K1/06C07K1/04
CPCC07K14/57563Y02P20/55
Inventor 张孝清许雷鸣邹正才宋志春包金远
Owner NANJING HUAWE MEDICINE TECH DEV
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