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Preparation method of conus anti-wrinkle serum

A technology of cone snail and wrinkle, which is applied to the preparation method of peptides, chemical instruments and methods, animal/human peptides, etc., can solve the problems of low purity of linear crude peptides, high pressure on environmental protection, and complicated operations, and is suitable for large-scale Effects of large-scale production, increased yield, and simple operation process

Active Publication Date: 2021-12-03
汉肽生物医药集团有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] Patent CN112279905A proposes to use Fmoc solid-phase method to synthesize conus anti-wrinkle factor, selectively oxidize first to form a pair of disulfide bonds, and then form the remaining two pairs of disulfide bonds through natural oxidation, and select dipeptide fragments for coupling to increase the yield; This method is more complicated to operate, and the use of high-concentration mercaptoethanol has a greater pressure on environmental protection
[0007] In a word, in the process of preparing conus anti-wrinkle protein, there is a technical problem that the purity of the linear crude peptide is low, which leads to a low overall yield

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0042] Example 1: Synthesis of Fmoc-Gly-Cys(tButhio)-OH

[0043] Accurately weigh 2.09Kg (10mol) of H-Cys(tButio)-OH into a 50L reaction kettle, add 12L of 10% sodium carbonate aqueous solution, and dissolve under stirring; after the solution is clear, add Fmoc-Gly-OSu 3.94kg dropwise at low temperature (10mol) / 12L tetrahydrofuran solution, stirred and reacted, and the end point was monitored by TLC; after concentration under reduced pressure, acid adjustment, ethyl acetate extraction, drying, and crystallization, the dipeptide monomer Fmoc-Gly-Ser(tBu) with a purity of 99.1% was obtained -OH 3.86kg, yield 76.2%.

Embodiment 2

[0044] Embodiment 2: Synthesis of Fmoc-Asn(Trt)-Gly-OH

[0045] Accurately weigh 0.90kg (12mol) of glycine into a 50L reaction kettle, add 12L of 10% sodium carbonate aqueous solution, and dissolve under stirring; 12L tetrahydrofuran solution, stirred and reacted, and the end point was monitored by TLC; after concentration under reduced pressure, acid adjustment, ethyl acetate extraction, drying, and crystallization, the dipeptide monomer Fmoc-Asn(Trt)-Gly-OH with a purity of 99.2% was obtained 4.78kg , Yield 73.2%

Embodiment 3

[0046] Embodiment 3: the preparation of peptide resin

[0047] Accurately weigh 600g of Fmoc-Cys(tButio)-Rink amide AM-resins (synthesis scale: 270mmol) with a degree of substitution of 0.45mmol / g and place it in a 50L synthesis column, add 5L of DCM to swell for 30min; Wash twice, add 5L of 20% piperidine / DMF solution for deprotection twice, react for 10min and 10min respectively; then wash twice with 5.6L DMF, DCM, DMF respectively; Fmoc-Arg(Pbf)-OH, Fmoc-Ala-OH, Fmoc-His(Trt)-OH, Fmoc-Asp(OtBu)-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Cys(tButhio)-OH , Fmoc-Trp(Boc)-OH, Fmoc-Lys(Boc)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Gly-Cys(tButhio)-OH, Fmoc- Lys(Boc)-OH, Fmoc-Pro-OH, Fmoc-Asn(Trt)-Gly-OH, Fmoc-Cys(tButio)-OH, Fmoc-Gly-Cys(tButio)-OH, Boc-Pyr-OH Linked to obtain 1738.7g of linear peptide resin;

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PUM

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Abstract

The invention discloses a method for preparing conus anti-wrinkle serum through solid-liquid combination, and belongs to the field of polypeptide synthesis. The method comprises the following steps of: (a) performing solid-phase synthesis on linear peptide resin; (b) performing splitting decomposition to obtain linear peptide with protected sulfydryl; and (c) removing a sulfydryl side chain protecting group, performing one-step oxidation to obtain three pairs of disulfide bonds, obtaining the conus anti-wrinkle serum, and performing purification and freeze drying to obtain fine peptide. According to the method, dipeptide monomers Fmoc-Gly-Cys (tButhio)-OH and Fmoc-Asn (Trt)-Gly-OH are adopted for the first time to perform solid-phase synthesis on the conus anti-wrinkle serum, and meanwhile, a strategy that stable tButhio protects cysteine sulfydryl under a TFA splitting decomposition condition is introduced, so that the purity of the linear peptide can be effectively improved, the yield of a final product is increased, the amplification of the synthesis scale is facilitated, and the production cost is reduced.

Description

technical field [0001] The invention relates to the field of polypeptide synthesis, in particular to a preparation method of cone snail anti-wrinkle factor. [0002] technical background [0003] 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. Cosmetically, it has an instant anti-wrinkle effect; acts as an instant relaxant to create a smoother skin appearance. Cone snail anti-wrinkle provides effective muscle relaxation but not complete stiffness and paralysis, and retains 5% of muscle neuromuscular electrical conduction, so it provides a natural rejuvenation effect. Cono snail anti-wrinkle protein is a highly folded peptide with strong penetrating power and high stability. It targets muscles to achieve relaxation. [0004] Cono snail anti-wrinkle protein is composed of 22 amino acids, the specific seque...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/435C07K1/36C07K1/34C07K1/16C07K1/06C07K1/04
CPCC07K14/43504Y02P20/55
Inventor 殷世清刘焕赠翟涛杜传强
Owner 汉肽生物医药集团有限公司