Preparation method for mu-conotoxin

A technology of cone snail and wrinkle, which is applied in the field of polypeptide product preparation, can solve the problems of high extraction cost and low biological content, and achieve the effects of high total yield, high purity of pure products and short synthesis cycle

Active Publication Date: 2020-01-17
应连心
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Due to its special structure, the product is currently only reported in the literature that it is obtained through biological extraction, the content in the organis

Method used

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  • Preparation method for mu-conotoxin
  • Preparation method for mu-conotoxin
  • Preparation method for mu-conotoxin

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0029] Example 1: Coupling of Fmoc-Cys(Trt)-OH (22nd amino acid)

[0030] Weigh Rink Amide AM Resin (100.00g, loading: 0.6mmol / g) and wash it once with 1000mL dry DMF, drain it, swell in 1000mL DMF for 2 h, and drain it.

[0031] Add 20% piperidine / DMF solution (DBLK solution) for deprotection twice, 1000mL / time, 5min+15min. After the deprotection is completed, wash with DMF 6 times, 1000 mL / time / min, drain, and detect ninhydrin, K+.

[0032] Weigh 70.29 g Fmoc-Cys(Trt)-OH, dissolve 17.84 g HOBt in 1000 mL DMF, add 16.67 g DIC under ice cooling to activate the solution for about 5 min, pour it into the reaction column, stir the reaction at room temperature for 2 h, and take a sample , ninhydrin detection, K-; wash the resin 3 times with DMF, 1000 mL / time / min, and drain.

Embodiment 2

[0033] Example 2: Coupling of Fmoc-Cys(Trt)-OH (21st amino acid)

[0034] Add 20% piperidine / DMF solution (DBLK solution) for deprotection twice, 1000mL / time, 5min+15min. After the deprotection is completed, wash with DMF 6 times, 1000 mL / time / min, drain, and detect ninhydrin, K+.

[0035] Weigh 70.29 g Fmoc-Cys(Trt)-OH, dissolve 17.84 g HOBt in 1000 mL DMF, add 16.67 g DIC under ice cooling to activate the solution for about 5 min, pour it into the reaction column, stir the reaction at room temperature for 2 h, and take a sample , ninhydrin detection, K-; wash the resin 3 times with DMF, 1000 mL / time / min, and drain.

Embodiment 3

[0036] Example 3: Coupling of Fmoc-Arg(Pbf)-OH (20th amino acid)

[0037] Add 20% piperidine / DMF solution (DBLK solution) for deprotection twice, 1000mL / time, 5min+15min. After the deprotection is completed, wash with DMF 6 times, 1000 mL / time / min, drain, and detect ninhydrin, K+.

[0038]Weigh 63.15 g Fmoc-Arg(Pbf)-OH, dissolve 17.84 g HOBt in 1000 mL DMF, add 16.67 g DIC under ice cooling to activate the solution for about 5 min, pour it into the reaction column, stir the reaction at room temperature for 2 h, and take a sample , ninhydrin detection, K-; wash the resin 3 times with DMF, 1000 mL / time / min, and drain.

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Abstract

The invention relates to the technical field of polypeptide product preparation, in particular to a preparation method for mu-conotoxin. The method disclosed by the invention adopts a solid phase andliquid phase combination method to synthesize a mu-conotoxin product. A solid phase synthesis method is adopted for coupling peptides to resin in sequence from a C terminal to an N terminal accordingto the main chain of the mu-conotoxin, then, the peptides are cut from the resin to obtain a linear peptide, a natural oxidation method is adopted to oxidize the linear peptide to obtain mu-conotoxincrude peptide cyclization liquid with three pairs of disulfide bonds, and the crude peptide cyclization liquid is purified to obtain a high-purity refined peptide. By use of the technology disclosed by the invention, the high levels of aspects, including a refined peptide quality and a total yield, can be simultaneously realized by simple technical steps. Compared with a biological extraction technology, the preparation method disclosed by the invention is simple in reaction operation, short in days for construction, low in cost and high in yields, can easily achieve industrialized requirements and has a wide market prospect.

Description

technical field [0001] The invention relates to the technical field of preparation of polypeptide products, in particular to a preparation method of cone snail anti-wrinkle factor. Background technique [0002] Cono snail anti-wrinkle factor, the English name is mu-conotoxin, the specific peptide sequence is as follows: [0003] H-Pyr 1 -Gly 2 -Cys 3 -Cys 4 -Asn 5 -Gly 6 -Pro 7 -Lys 8 -Gly 9 -Cys 10 -Ser 11 -Ser 12 -Lys 13 -Trp 14 -Cys 15 -Arg 16 -Asp 17 -His 18 -Ala 19 -Arg 20 -Cys 21 -Cys 22 -CONH2 (Disulfide Bridge: 3-15, Disulfide Bridge: 4-21; Disulfide Bridge: 10-22) [0004] Cono snail anti-wrinkle protein (mu-conotoxin) belongs to the u-conotoxin family. It was first isolated from marine snail venom and targets a variety of voltage-sensitive sodium channels, mainly blocking Nav1.4 channels. Cono snail anti-wrinkle can eliminate expression skin wrinkles on the human face and is used in the field of cosmetics. [0005] There are 22 amino acids i...

Claims

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Application Information

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IPC IPC(8): C07K14/435C07K1/04C07K1/06C07K1/20C07K1/34C07K1/36
CPCC07K14/43504Y02P20/55
Inventor 应连心
Owner 应连心
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