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Preparation method of Hainantoxin-III

A Hainan tarantula and toxin technology, applied in the field of solid-phase peptide synthesis, can solve the problems of limited research and application, difficulty in obtaining natural toxins, etc.

Inactive Publication Date: 2015-09-09
HUNAN INSTITUTE OF SCIENCE AND TECHNOLOGY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] Hainan tarantula toxin III, as a highly specific voltage-gated sodium channel Nav1.7 inhibitor, is expected to form a new type of analgesic drug. However, the natural toxin of Hainan tarantula toxin III is very difficult to obtain, which limits its Research and application, the present invention provides a large-scale preparation method for the preparation of Hainan tarantula toxin III

Method used

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  • Preparation method of Hainantoxin-III
  • Preparation method of Hainantoxin-III

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0026] Example 1 Synthesis of Hainan Tarantula Toxin III Peptide Chain

[0027] Weigh 20 grams of Rink Amide resin with a substitution value of 0.5 mmol / g, expand it in DMF for 30 minutes, drain it, add 30 mL of 20% piperidine for deprotection for 15 minutes, drain it, add 30 mL of DMF to wash once, add 20% piperidine 30 ml was deprotected for 10 minutes, washed 5 times with DMF, and completed Fmoc-Leu-OH, Fmoc-Tyr(tBu)-OH, Fmoc-Val-OH, Fmoc-Lys(Boc)- OH, Fmoc-Cys(Trt)-OH, Fmoc-Trp(Boc)-OH, Fmoc-Lys(Boc)-OH, Fmoc-His(Boc)-OH, Fmoc-Lys(Boc)-OH, Fmoc-Ser (tBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Cys(Trt)-OH, Fmoc-Ala-OH, Fmoc-Tyr(tBu)-OH, Fmoc-Asn(Trt)-OH, Fmoc- Pro-OH, Fmoc-Cys(Mmt)-OH, Fmoc-Cys(Trt)-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Asn(Trt)-OH, Fmoc-Lys(Boc)-OH, Fmoc -Gly-OH, Fmoc-Pro-OH, Fmoc-Thr(tBu)-OH, Fmoc-Cys(Trt)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Asp(OtBu)-OH, Fmoc-Gly Coupling of -OH, Fmoc-Phe-OH, Fmoc-Gly-OH, Fmoc-Lys(Boc)-OH, Fmoc-Cys(Mmt)-OH, Fmoc-Gly-OH. The NMM / HATU / HOBt system...

Embodiment 2

[0028] Example 2 Hainan Tarantula Toxin III Peptide Resin Removes Mmt Protection

[0029] Example 1 The synthetic Hainan tarantula toxin III peptide resin was deprotected with 20% piperidine / DMF, washed 5 times with DMF, washed 3 times with DCM, dried with nitrogen, added 600 ml of 3% TFA / DCM solution, and reacted After 10 minutes, remove the solution, wash 5 times with DMF, wash 5 times with DCM, and blow dry with nitrogen.

Embodiment 3

[0030] Example 3 Hainan Tarantula toxin III peptide resin is oxidized by air oxidation to form a pair of disulfide bonds

[0031] Add 1000 milliliters of 0.1 M phosphate buffer solution to the Hainan arachnid toxin III peptide resin obtained in Example 2, and add 1,000 ml of 0.1 M phosphate buffer solution to the pH value of 7.5. During the reaction, the air is ventilated, the reaction temperature is room temperature, and the reaction time is determined according to the oxidation process. After 24 hours of reaction, the peptide resin was taken for detection. The peptide resin was washed with water and then added to the DTNB solution. The end point of the reaction was that the solution did not turn yellow.

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Abstract

The invention provides a preparation method of Hainantoxin-III and belongs to the field of polypeptide and protein preparation. The Hainantoxin-III is synthesized with a Fmoc solid-phase peptide synthesis method, synthetic resin adopts Rink amide resin, cysteine in the second position and cysteine in the seventeenth position adopt Fmoc-Cys(Mmt)-OH, cysteine in other positions adopts Fmoc-Cys(Trt)-OH, Mnt protection removal is performed by adopting 3% TFA (trifluoroacetic acid) after polypeptide chain synthesis ends, disulfide bonds of cysteine in the second position and cysteine in the seventeenth position are formed with an air oxidation method, polypeptide is removed from the resin through pyrolysis after a first pair of disulfide bonds is formed, a second pair of disulfide bonds and a third pair of disulfide bonds are formed with a solid-phase DTNB (5,5'-Dithiobis-(2-nitrobenzoic acid)) oxidation method, and the Hainantoxin-III with biological activity is finally formed. The biological activity and the natural toxic activity of Hainantoxin-III prepared with the method are consistent, and the synthetic efficiency is high.

Description

technical field [0001] The invention relates to solid-phase polypeptide synthesis, in particular to a solid-phase preparation method of Hainan tarantula toxin III. Background technique [0002] Hainan tarantula toxin III is a polypeptide toxoid molecule isolated from Hainan tarantula, 33 amino acid residues, molecular weight 3609 Da, three pairs of disulfide bonds, Hainan tarantula toxin III is a TTX-S type sodium Channel inhibitors have highly selective inhibitory activity on the voltage-gated sodium channel Nav1.7, and Hainan tarantula toxin III is expected to be developed into a specific Nav1.7 voltage-gated sodium channel inhibitor. [0003] The voltage-gated sodium channel Nav1.7 plays a very important role in the conduction of neuropathic pain and inflammatory pain. Inhibiting the activity of the voltage-gated sodium channel Nav1.7 can inhibit the generation of pain. Therefore, the development of voltage-gated sodium channels Nav1.7 highly selective inhibitors ar...

Claims

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Application Information

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IPC IPC(8): C07K14/435C07K1/06C07K1/04
CPCC07K14/43518
Inventor 刘宇
Owner HUNAN INSTITUTE OF SCIENCE AND TECHNOLOGY
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