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Solid-phase synthesis method of antibacterial peptide Iseganan

A solid-phase synthesis, Esgenan technology, applied in the preparation methods of peptides, chemical instruments and methods, peptides, etc., can solve the problem of unable to meet the requirements of large-scale industrial production, unable to ensure the correct pairing of disulfide bonds, increase after Deal with problems such as difficulty, and achieve the effects of high yield, low pollution and few by-products

Inactive Publication Date: 2013-04-03
NANJING UNIV OF TECH +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In this method, the high concentration of TFA used in the removal of fully protected linear peptides on the solid phase will also remove part of the Acm protecting group at the same time, which cannot ensure the correct pairing of disulfide bonds
Ring formation in the liquid phase requires a high degree of dilution to prevent the formation of disulfide bonds between molecules, thus increasing the difficulty of post-processing and reducing production efficiency, which cannot meet the requirements of large-scale industrial production

Method used

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  • Solid-phase synthesis method of antibacterial peptide Iseganan
  • Solid-phase synthesis method of antibacterial peptide Iseganan
  • Solid-phase synthesis method of antibacterial peptide Iseganan

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0052] Fmoc-Arg(Pbf)-Gly-Gly-Leu-Cys(Acm)-Tyr(tBu)-Cys(Mmt)-Arg(Pbf)-Gly-Arg-Phe-Cys(Mmt)-Val-Cys(Acm) Preparation and Analysis of -Val-Gly-Arg(Pbf)-Amino Resin

[0053] (1) Preparation of H-Arg(Pbf)-amino resin

[0054] Weigh 2 g of Rink Amide resin with a substitution degree of 0.6 mmol / g in a solid phase reactor, add 10 mL of DCM to swell for 20 min, then add 10 mL of 20% piperidine in DMF, react for 5 min, wash with DMF once, add 10 mL of 20% piperidine DMF solution of pyridine, reacted for 15min. Drain. Add Fmoc-Arg(Pbf)-OH (3.6mmol), HOBt (4.3mmol), DIC (4.3mmol), DMF (15mL) into the solid phase reactor, and react at 20°C for 2h. Wash and dry the resin to obtain Fmoc-Arg(Pbf)-resin. Add 10 mL of blocking reagent (acetic anhydride (mmol): DIPEA (mmol): DMF = 1:1:8) to the resin, react for 1 h, block the remaining amino groups, wash with DCM, MeOH and DMF several times, and drain. Add 20% PIP / DMF solution and react for 20 minutes to remove the Fmoc protecting group, w...

Embodiment 2

[0065]

[0066] Weigh 3 g of the linear peptide resin obtained in step (2) of Example 1, add 30 mL of 1% TFA in DCM:DMSO=2:1 (volume ratio) solution to a solid phase reactor, and shake at 25°C for 4 hours. Drain. Wash with DMF and anhydrous methanol three times each, and drain. got Cys 5-14 A ring peptide resin:

[0067]

[0068] (2) Cys Glam 5-14 Analysis of a cyclic crude peptide

[0069] Weigh 0.5 g of the peptide resin obtained in step (1) of Example 2, add 5 mL of DCM to the solid phase reactor, swell for 10 min, and drain. Add 10 mL of 20% piperidine in DMF, react for 5 min, wash once with DMF, add 10 mL of 20% piperidine in DMF, and react for 15 min. Drain. They were washed twice with DMF and three times with anhydrous methanol, and drained. Prepare 5 mL of lysate, and the volume ratio of each component is TFA: sulfide anisole: TIS: anisole: water = 90: 2.5: 2.5: 2.5: 2.5. After shaking and reacting at 25° C. for 3 h, the reaction solution was poured into ...

Embodiment 3

[0070] The preparation and analysis of embodiment 3 Estherglam

[0071]

[0072]

[0073] Weigh 2 g of the peptide resin obtained in step (1) of Example 2, add 10 mL of DCM to the solid phase reactor, swell for 10 min, and drain. Add 10 mL of DMF. Dissolve 655.2 mg of iodine in 10 mL of MeOH and add it to the peptide resin. The mixture is shaken and reacted for 4 hours at room temperature, washed with DMF and anhydrous methanol three times each, and drained. got Cys 9-12 , Cys 5-14 Bicyclic peptide resin 1.78g.

[0074] (2) Preparation of crude peptide of Esserglynan

[0075] Add 10 mL of 20% piperidine in DMF to the peptide resin obtained in step (1) of Example 3, react for 5 min, wash once with DMF, add 10 mL of 20% piperidine in DMF, and react for 15 min. Drain. They were washed twice with DMF and three times with anhydrous methanol, and drained. Prepare 20 mL of lysate, and the volume ratio of each component is TFA: sulfide anisole: TIS: anisole: water = 90: 2...

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Abstract

The invention relates to a solid-phase synthesis method of an antibacterial peptide Iseganan containing two pairs of disulfide bonds, which comprises the following steps: (1) according to a solid-phase synthesis method, sequentially coupling an amino resin used as a raw material with amino acids to synthesize a protected 17 peptide resin; (2) removing weak acid sensitive protecting groups, and then oxidizing to generate a first pair of disulfide bonds; (3) using iodine to remove Acm protecting groups, and cyclizing to generate a second pair of disulfide bonds; and (4) finally cracking from the resin, removing the protecting groups, and purifying to obtain the Iseganan antibacterial peptide. The route provided by the invention has the advantages of short operation steps, simple post treatment, fewer byproducts, high yield and low pollution, and is suitable for large-scale industrial production.

Description

technical field [0001] The invention relates to the technical field of polypeptide drug synthesis, in particular to a solid-phase synthesis method of the antibacterial peptide eucygnan containing multiple pairs of disulfide bonds. Background technique [0002] Iseganan (Iseganan, whose amino acid sequence is shown below) is an analogue of porcine neutrophil peptide-1 (Protegrin-1, PG-1), which can bind to bacterial extracellular components such as lipopolysaccharide or lipid membrane acid And so on, cause the bacteria to swell, burst, and kill the bacteria by destroying the cell membrane. It has inhibitory effects on Gram-positive and negative bacteria, fungi, and yeasts. The MIC for Gram-positive bacteria is 0.13-0.16 μg / mL, and the MIC for Gram-negative bacteria related to oral mucositis is 0.063-8 μg / mL. IntraBiotics is developing the formulation of Essergnan. A Phase III clinical trial involving 323 patients showed that Essergnan can prevent oral mucositis. Symptoms s...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K7/08C07K1/06C07K1/04
CPCY02P20/55
Inventor 董海军毛怡春叶青缪李鹏梁泽民苏贤斌
Owner NANJING UNIV OF TECH
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