Large-scale preparation and purification method and application of mu-conopeptide

A purification method and cono peptide technology, which is applied to the preparation method of peptides, chemical instruments and methods, cosmetic preparations, etc., can solve problems such as high cost, difficult extraction, and failure to meet market demand, so as to improve efficiency and operate Simple, Pain-Free Effects

Active Publication Date: 2020-03-20
珠海市维琪科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Among them, the extraction yield of natural products is limited, the process of directly separating and extracting conotoxin from cono snails is complicated, difficult to extract, and high in cost, and the biological synthesis process is not mature, so it is difficult to realize the industrialization of conotoxin and cannot Meet the needs of the market

Method used

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  • Large-scale preparation and purification method and application of mu-conopeptide
  • Large-scale preparation and purification method and application of mu-conopeptide
  • Large-scale preparation and purification method and application of mu-conopeptide

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1B

[0052] Example 1 Boc-pGlu-Gly-Cys(Trt)-Cys(Trt)-Asn(Trt)-Gly-Pro-Lys(Boc)-Gly-Cys(Trt)-Ser(tBu)-Lys(Boc)-Ser

[0053] Preparation of (tBu)-Trp(Boc)-Cys(Trt)-Arg(Pbf)-Asp(OtBu)-His(Trt)-Ala-Arg(Pbf)-Cys(Trt)-Cys(Trt)-Amino Resin

[0054] 1.1 Resin swelling

[0055] Add 75g of Rink AM resin with a degree of substitution of 0.7 into the solid-phase column, add 500ml of DMF to completely immerse the resin, then blow in nitrogen, start stirring at 100r / min, stir for 5min, and then drain the solvent for 5min. Then add 500ml of DMF, completely immerse the resin, pass nitrogen gas, start stirring, 100r / min, stir for 30min, and drain the solvent. Continue to add 500ml of DMF, completely immerse the resin, pass nitrogen gas, start stirring, 100r / min, and drain the solvent for 5min to complete the swelling.

[0056] 1.2 De-Fmoc

[0057] Add 500ml of 20% piperidine / DMF, blow in nitrogen gas, stir the reaction for 5min, and drain it for 3min. Then add 500ml of 20% piperidine / DMF, blow ...

Embodiment 2

[0070] Example 2H-pGlu-Gly-Cys-Cys-Asn-Gly-Pro-Lys-Gly-Cys-Ser-Lys-Ser-Trp-Cys-Arg-Asp-His-Ala-Arg-Cys-

[0071] Cys-NH 2 preparation of

[0072] 2.1 Reagent pretreatment

[0073] Put TFA, isopropyl ether, and ether into the refrigerator for more than 4 hours. Phenol in 60°C water bath for 2h.

[0074] 2.2 Cracking operation

[0075] Weigh 90g of the resin to be cracked and put it into a 1L round bottom flask.

[0076] Measure 15.7ml of ethanedithiol, 31.5ml of sulfide anisole, 15.7ml of water, 535.5ml of TFA, and 31.5ml of phenol, mix them evenly, and pour them into a 1L round bottom flask filled with resin, magnetic stirring, 500r / min, the reaction time is 2.5h.

[0077] After 2.5 hours of cleavage reaction, the lysate was concentrated below 38°C for 1 hour, and the lysate resin was vacuum filtered with a sand core filter funnel, and dried for 1 minute. Add 15ml TFA to wash the resin, and drain it for 1min. Add 15ml TFA again to wash the resin, and drain it for 1min...

Embodiment 3

[0084]

[0085] 3.1 Preparation of 0.1mol / L buffer solution

[0086] Weigh 102g of Tris into a 10L serum bottle, add 8.5L of purified water, stir, and adjust the pH to 8.2 to obtain a 0.1mol / L Tris buffer.

[0087] 3.2 Oxidative folding operation

[0088] After the crude peptide was taken out from the vacuum desiccator and weighed (about 30 g), it was immediately put into a buffer solution in a 10 L serum bottle, and stirred for 16 hours. The color of the solution is clear and bright yellow. The buffer solution of the crude product of μ-conopeptide formed by oxidative folding to form three pairs of disulfide bonds was obtained.

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Abstract

The invention discloses a large-scale preparation and purification method and application of mu- conopeptide. The large-scale preparation and purification method of the mu-conopeptide comprises the steps of preparation of linear peptide amino resin, cracking, linear peptide folding and crude peptide purification. Amino resin with the substitution degree of 0.6-0.8 is adopted, and the synthesis efficiency is improved; the amino acid is sequentially coupled through solid-phase synthesis to synthesize protected 22-peptide amino resin, and cracking and concentration are carried out; three pairs ofdisulfide bonds are formed by one-step folding through air oxidation, no extra oxidizing agent needs to be added in the folding process, and the method is simple to operate, low in cost and suitablefor industrial mass production; crude peptide is prepared and purified through HPLC, and the mu- conopeptide with the purity larger than 97% is obtained. The invention further provides the applicationof the mu- conopeptide in preparing wrinkle-removing cosmetics. An effect of injecting botulinum toxin can be achieved through simple smearing, and side effects such as pain and facial paralysis caused by botulinum toxin injection can be avoided.

Description

technical field [0001] The invention belongs to the field of polypeptide drugs, and relates to a large-scale preparation and purification method and application of μ-cono peptide. Background technique [0002] Conotoxin is a mixed toxin secreted by the venom tube of the marine gastropod mollusk (Conus) and the venom gland on the inner wall of the poison sac. An active polypeptide compound with high specificity for neuroreceptors. Compared with other natural peptide toxins, conotoxins have outstanding advantages such as small molecular weight, stable structure, high activity, high selectivity and easy synthesis. According to its conserved signal peptide sequence and disulfide bond skeleton, conotoxins can be divided into more than 20 superfamilies such as A, M, O, P, S, T, I, V, Y, J, D, C and L , and according to their pharmacological targets, they can be further divided into pharmacological families such as α, μ, ω, κ, δ, ψ, σ, ρ, γ, vasopressin, convulsants and sleep pep...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/435C07K1/04C07K1/06C07K1/20A61K8/64A61Q19/08
CPCA61K8/64A61Q19/08C07K14/43504
Inventor 丁文锋孙新林
Owner 珠海市维琪科技有限公司
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