Water-based synthesis method of mu-conotoxin KIIIA with instant wrinkle removal effect

Abstract

The invention discloses a water-based synthesis method of mu-conotoxin KIIIA with an instant wrinkle removal effect, and belongs to the technical field of biological medicines. A solid-phase synthesis method is adopted, H-RINK amide CHEMMATRIX resin is used as a solid-phase synthesis carrier, amino acid residues protected by substituent groups are sequentially condensed in an acetonitrile aqueous solution according to the sequence of amino acids from the C end to the N end of a main chain of mu-conotoxin KIIIA, and reduced crude polypeptide is obtained; and carrying out oxidative cyclization on the reduced crude polypeptide in an aqueous solution containing trihydroxymethyl aminomethane hydrochloride, guanidine hydrochloride, reduced glutathione and oxidized glutathione to obtain the mu-conotoxin KIIIA. The method is simple in process, environment-friendly, low in cost, low in chemical waste yield and high in yield (22-27%).

Description

Technical Field

[0001] The invention belongs to the technical field of biomedicine, and in particular relates to a water-based synthesis method of μ-conotoxin KIIIA with instant wrinkle-removing effect. Background Art

[0002] The information disclosed in this background technology section is only intended to enhance the understanding of the overall background of the invention, and should not necessarily be regarded as an admission or any form of suggestion that the information constitutes the prior art already known to a person skilled in the art.

[0003] Conotoxins are derived from marine cone snails and are also called cono polypeptides. Globally, there are more than 500 species of cone snails, each of which can produce thousands of active polypeptides, and there are differences in the active polypeptides produced by different cone snails. It is speculated that the number of cono polypeptides may be as high as millions, which is undoubtedly a biological polypeptide library with great research value. According to the receptors and physiological activities of cono polypeptides, they can be divided into several subfamilies, which are usually represented by Greek letters plus dashes before the names of conotoxins, mainly including α-, μ-, ψ-, ω-, σ-, γ-, δ-, etc. Most conotoxins are rich in disulfide bonds and have specific molecular structures. They can selectively act on specific ion channels or receptors on the cell membrane, thereby showing a variety of biological effects. In the 1980s, Olivera et al. were the first to successfully isolate conotoxins from cone snails. Due to its unique functions, few side effects, and clear targeting, it has become a focus of attention in pharmacology, biomedicine and other fields. In addition, the magical cone snail also inspired the idea of ​​removing wrinkles and reducing lines. In 2012, Swiss scientists developed conus peptides, opening the "magic door to wrinkle removal" in the field of skin care and medical beauty.

[0004] μ-conotoxin is a popular instant wrinkle-removing peptide in recent years. Compared with toxins from spiders, scorpions, snakes and other animals, its peptide chain is significantly shorter, disulfide bonds are abundant, the molecular structure is compact, and the biological activity is extremely high. It can specifically block Na in muscle fibers. + Ion influx channel Nav1.4 allows Na + The influx of ions is blocked and the muscle action potential cannot be formed, thus relaxing the facial muscles and reducing the formation of wrinkles. As a highly folded peptide, μ-conotoxin has a small molecular weight and strong penetration, and can specifically block the Na + The influx channel Nav1.4 causes Na +The inflow is blocked, which in turn inhibits the formation of muscle action potentials. After acting on the nerve synapses, it will not affect the normal functioning of the nervous system. The target is clear and specific, targeting a variety of voltage-sensitive muscle sodium ion channels, mainly blocking the channel Nav1.4, but can retain 5% of the neuromuscular current conduction in the muscle, thereby naturally relaxing the facial muscles and achieving the effect of removing wrinkles.

[0005] μ-conotoxin KIIIA originates from the Indian Ocean-Pacific region of Conus kinoshitai. It is composed of 16 amino acids and is the shortest known μ-conotoxin. The sequence is:

[0006] C1-C2-N3-C4-S5-S6-K7-W8-C9-R10-D11-H12-S13-R14-C15-C16-NH2, containing three pairs of disulfide bonds, linked in the following ways: Cys1-Cys9, Cys2-Cys15 and Cys4-Cys16. Like other μ-conotoxins in this family, KIIIA can inhibit Nav1.2 and Nav1.4, and can also selectively act on the peripheral analgesic target Nav1.7 with nanomolar affinity. As an advanced version of the classic conus peptide, it is shortened from 22 amino acids to 16 amino acids, with enhanced penetration and stability. It can erase wrinkles and achieve instant wrinkle removal. At 1.5ppm, it has the effect of inhibiting the generation of action potentials. It can achieve the effect of injecting botulinum toxin by applying it. It is expected to replace invasive wrinkle removal methods such as injections, thereby removing wrinkles more safely and effectively, and avoiding potential hazards such as infection and facial paralysis caused by invasive products to the human body. It has great application value in beauty.

[0007] The CAS# of μ-conotoxin KIIIA is 884469-67-4, and its molecular formula is:

[0008] C 70 H 106 N 28 O 22 S6, Exact Mass: 1882.636, MW: 1884.152, chemical structure see Figure 1. There are currently three main ways to obtain μ-conotoxin KIIIA. The first is the direct extraction method: it is directly separated and obtained from the poison tube of wild cone snails. However, the amount obtained in this way is very rare. In addition, in recent years, due to the destruction of the marine ecology, the number of wild cone snails has been greatly reduced. The use of this method will further aggravate the deterioration of cone snail resources. In addition, nuclear waste water pollution may cause potential hazards to the extracted products. The second is biological synthesis: the gene of conotoxin is transferred to microorganisms such as Escherichia coli or yeast to promote its expression. However, the amino acid sequence of conotoxin is relatively short, and there are many post-translational modifications, which makes the subsequent separation and purification quite difficult. The last is the peptide solid phase synthesis method: according to the traditional solid phase synthesis and oxidation ideas, straight-chain peptides are synthesized, and after oxidation and cyclization, peptides with specific disulfide bond linkage are selected for separation and purification. Since KIIIA has three pairs of disulfide bonds and can form 15 isomers, separation and purification are difficult. The use of the differences in the three pairs of Cys side chain protecting groups to achieve site-specific cyclization has the disadvantages of cumbersome steps, numerous side reactions, low yield, poor operability, etc., and cannot be produced on a large scale. For example, Structural and functional insights into the inhibition of human voltage-gated sodium channels by μ-conotoxinKIIIA disulfide isomers uses traditional solid-phase synthesis to prepare μ-conotoxin KIIIA, and uses the differences in the three pairs of Cys side chain protecting groups to achieve site-specific cyclization. The steps are cumbersome and the yield is low, with a yield of only 10%. Summary of the invention

[0009] In order to solve the deficiencies of the prior art, the purpose of the present invention is to provide a water-based synthesis method of μ-conotoxin KIIIA with instant wrinkle removal effect. The method provided by the present invention has the advantages of environmental friendliness, low cost and high yield.

[0010] In order to achieve the above object, the technical solution of the present invention is:

[0011] The first aspect of the present invention provides a method for synthesizing μ-conotoxin KIIIA, comprising:

[0012] The solid phase synthesis method was adopted, H-RINK amide CHEMMATRIX resin was used as a solid phase synthesis carrier, and the amino acid residues protected by the substituents were sequentially condensed in the acetonitrile aqueous solution according to the amino acid sequence from the C-terminus to the N-terminus of the main chain of μ-conotoxin KIIIA to obtain a crude reduced polypeptide;

[0013] The crude polypeptide in the reduced state is oxidized and cyclized in an aqueous solution containing tris(hydroxymethylaminomethane) hydrochloride, guanidine hydrochloride, reduced glutathione and oxidized glutathione to obtain μ-conotoxin KIIIA.

[0014] In some embodiments of the present invention, the amino acid sequence from the C-terminus to the N-terminus of the μ-conotoxin KIIIA main chain is:

[0015] C1-C2-N3-C4-S5-S6-K7-W8-C9-R10-D11-H12-S13-R14-C15-C16-NH2.

[0016] In some embodiments of the present invention, the method for preparing a reduced polypeptide crude peptide comprises: using H-RINK amide CHEMMATRIX resin as a solid phase synthesis carrier, sequentially condensing Smoc-L-Cys(Trt)-OH, Smoc-L-Cys(Trt)-OH, Smoc-L-Arg(Pbf)-OH, Smoc-L-Ser(tBu)-OH, Smoc-L-His(Trt)-OH, Smoc-L-Asp(otBu)-OH, Smoc-L-Arg(Pbf)-OH from the C-terminus to the N-terminus. bf)-OH, Smoc-L-Cys(Trt)-OH, Smoc-L-Trp(Boc)-OH, Smoc-L-Lys(Boc)-OH, Smoc-L-Ser(tBu)-OH, Smoc-L-Ser(tBu)-OH, Smoc-L-Cys(Trt)-OH, Smoc-L-Asn(Trt)-OH, Smoc-L-Cys(Trt)-OH, Smoc-L-Cys(Trt)-OH, and the reduced crude polypeptide was obtained after cleavage and drying.

[0017] In some embodiments of the present invention, the method for preparing a crude reduced polypeptide comprises:

[0018] Take H-RINK amide CHEMMATRIX resin and swell it in purified water, and then wash it with purified water;

[0019] The first amino acid Smoc-L-Cys(Trt)-OH and a condensing agent are added to an acetonitrile aqueous solution for activation, and then added to the swollen H-RINK amide CHEMATRIX resin for reaction to prepare Smoc-L-Cys(Trt)-OH-resin, and the Smoc protecting group is removed to obtain L-Cys(Trt)-OH-resin;

[0020] Activate Smoc-L-Cys(Trt)-OH, Smoc-L-Arg(Pbf)-OH, Smoc-L-Ser(tBu)-OH, Smoc-L-His(Trt)-OH, Smoc-L-Asp(otBu)-OH, Smoc-L-Arg(Pbf)-OH, Smoc-L-Cys(Trt)-OH, Smoc-L-Trp(Boc)-OH, Smoc-L-Lys(Boc)-OH, Smoc-L-Ser(tBu)-OH, Smoc-L-Ser(tBu)-OH, Smoc-L-Cys(Trt)-OH, Smoc-L-Asn(Trt)-OH, Smoc-L-Cys(Trt)-OH, Smoc-L-Cys(Trt)-OH;

[0021] The activated Smoc-L-Cys(Trt)-OH was added to the L-Cys(Trt)-OH-resin for condensation. After the condensation was completed, the Smoc protecting group was removed, and then Smoc-L-Arg(Pbf)-OH, Smoc-L-Ser(tBu)-OH, Smoc-L-His(Trt)-OH, Smoc-L-Asp(otBu)-OH, Smoc-L-Arg(Pbf)-OH, Smoc-L-Cys(Trt ...Ser(tBu)-OH, Smoc-L-His(Trt)-OH, Smoc-L-Ser(tBu)-OH, Smoc-L-His(Trt)-OH, Smoc-L-Ser(tBu)-OH, Smoc-L-Ser(tBu)-OH, Smoc-L-His(Trt)-OH, Smoc-L-Ser(tBu)-OH, Smoc-L-Ser(tBu)-OH, Smoc-L-His(Trt)-OH, Smoc-L-Ser(tBu)-OH, Smoc-L-Ser(t Smoc-L-Trp(Boc)-OH, Smoc-L-Lys(Boc)-OH, Smoc-L-Ser(tBu)-OH, Smoc-L-Ser(tBu)-OH, Smoc-L-Cys(Trt)-OH, Smoc-L-Asn(Trt)-OH, Smoc-L-Cys(Trt)-OH, Smoc-L-Cys(Trt)-OH, after each condensation of an amino acid, the Smoc protecting group is removed to finally obtain a reduced polypeptide resin;

[0022] The reduced polypeptide resin is cleaved and dried to obtain the reduced polypeptide crude peptide.

[0023] It should be noted that when different amino acids are activated separately, the activation method is the same as the first amino acid, that is, the amino acid and the condensing agent are added to the acetonitrile aqueous solution for activation.

[0024] In some embodiments of the present invention, the H-RINK amide CHEMMATRIX resin has a degree of substitution of 0.4-0-6 mmol / g.

[0025] It should be noted that the term substitution degree refers to how many millimoles of active reaction sites are loaded in each resin. For example, the substitution degree is 0.4-0-6mmol / g, that is, each gram of H-RINK amide CHEMMATRIX resin contains 0.4-0-6mmol of active reaction sites. The amount of other ingredients (such as amino acids, condensing agents, etc.) used in the preparation process can be determined based on the content of active reaction sites in the resin used.

[0026] In some embodiments of the present invention, the swelling method of H-RINK amide CHEMMATRIX resin includes: adding H-RINK amide CHEMMATRIX resin to purified water to swell for 1.5 to 2.5 hours, and then washing with purified water twice.

[0027] In some embodiments of the present invention, the volume fraction of acetonitrile in the acetonitrile aqueous solution is 5-10%.

[0028] In some embodiments of the present invention, during the activation process, the molar ratio of the 1st to 7th amino acids Smoc-L-Cys(Trt)-OH, Smoc-L-Cys(Trt)-OH, Smoc-L-Asn(Trt)-OH, Smoc-L-Cys(Trt)-OH, Smoc-L-Ser(tBu)-OH, Smoc-L-Ser(tBu)-OH, and Smoc-L-Lys(Boc)-OH to the resin is 2.5-3.0; the 8th to 16th amino acids Smoc-L-Cys(Trt)-OH, Smoc-L-Asn(Trt)-OH, Smoc-L-Cys(Trt)-OH, Smoc-L-Ser(tBu)-OH, and Smoc-L-Lys(Boc)-OH are 2.5-3.0; The molar ratios of Smoc-L-Trp(Boc)-OH, Smoc-L-Cys(Trt)-OH, Smoc-L-Arg(Pbf)-OH, Smoc-L-Asp(otBu)-OH, Smoc-L-His(Trt)-OH, Smoc-L-Ser(tBu)-OH, Smoc-L-Arg(Pbf)-OH, Smoc-L-Cys(Trt)-OH, and Smoc-L-Cys(Trt)-OH relative to the resin are all 2.0-2.5.

[0029] It should be noted that the term "molar ratio relative to the resin" refers to the molar ratio of the amino acid to the active reaction sites contained in the resin. For example, the molar ratio of the first amino acid Smoc-L-Cys(Trt)-OH relative to the resin is 2.5-3, that is, the ratio of the amount of the first amino acid Smoc-L-Cys(Trt)-OH to the amount of the active reaction site in the resin is 2.5-3. It can also be understood as an equivalent ratio, wherein the amount of the active reaction site in the resin is 1 equivalent, and the amount of the first amino acid Smoc-L-Cys(Trt)-OH is 2.5-3.0 equivalents. For example, if the amount of the active reaction site in the resin is 10 mmol, the amount of the first amino acid Smoc-L-Cys(Trt)-OH is (2.5-3)*10 mmol.

[0030] In some embodiments of the present invention, the condensing agent consists of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC-HCl), ethyl 2-oximecyanoacetate (Oxyma) and sodium bicarbonate;

[0031] The molar ratio of EDC-HCl, Oxyma and sodium bicarbonate to the resin is (4-6):(2-4):(2-4).

[0032] It should be noted that the term "molar ratio of EDC-HCl, Oxyma and sodium bicarbonate relative to the resin" refers to the ratio of the amount of EDC-HCl, Oxyma and sodium bicarbonate divided by the amount of active reaction site in the resin. For example, when the amount of active reaction site in the resin is 10 mmol, the molar ratio of EDC-HCl, Oxyma and sodium bicarbonate relative to the resin is (4-6):(2-4):(2-4), the amount of EDC-HCl is (4-6)*10 mmol, the amount of Oxyma is (2-4)*10 mmol, and the amount of sodium bicarbonate is (2-4)*10 mmol.

[0033] In some embodiments of the present invention, the condensation reaction temperature is 20-40° C. and the time is 1-3 h.

[0034] In some embodiments of the present invention, the reagent for removing the Smoc protecting group is a mixed solution of sodium hydroxide and ethanolamine;

[0035] The mixed solution of sodium hydroxide and ethanolamine is obtained by mixing 0.5-1.0M sodium hydroxide and 25-30% by mass ethanolamine solution in a volume ratio of (0.9-1.1):(0.9-1.1), preferably 1:1.

[0036] In some embodiments of the present invention, the method for removing the Smoc protecting group comprises: adding 0.5-1.0M sodium hydroxide / 25-30% ethanolamine solution to remove Smoc twice, the two removals are carried out at 10-30°C for 20 minutes and 30 minutes respectively; and then washing the resin with purified water until the pH reaches 6.5-7.5.

[0037] In some embodiments of the present invention, a lysis solution is added when cleaving the reduced polypeptide resin, wherein the lysis solution is a 15-20% by volume hexafluoroisopropanol dichloromethane solution, and the amount of the lysis solution is 9-11 times the volume of the reduced polypeptide resin.

[0038] In some embodiments of the present invention, the preparation method of μ-conotoxin KIIIA comprises:

[0039] Add 80-100 times the weight of purified water to the reduced polypeptide crude peptide, stir until dissolved; add tris(hydroxymethylaminomethane) hydrochloride, stir until dissolved, the final concentration is 100-120mM; add guanidine hydrochloride, stir until dissolved, the final concentration is 0.2-0.25M; add reduced glutathione, stir until dissolved, the final concentration is 1.0-1.2mM; add oxidized glutathione, stir until dissolved, the final concentration is 0.2-0.25mM; react at 20-30°C with stirring overnight; adjust the pH to 4-6; filter, purify and freeze-dry to obtain μ-conotoxin KIIIA.

[0040] It should be noted that the term final concentration refers to the final concentration of the compound in the oxidative cyclization system. For example, the final concentration in the above “add tris(hydroxymethyl)aminomethane hydrochloride, stir until dissolved, and the final concentration is 100-120 mM” means that the concentration of tris(hydroxymethyl)aminomethane hydrochloride in the oxidative cyclization system is 100-120 mM.

[0041] In some embodiments of the present invention, the filtration is performed using a 0.45 μm filter membrane.

[0042] In some embodiments of the present invention, the purification operation includes: purification by reverse phase high performance liquid chromatography, mobile phase A is formic acid aqueous solution, mobile phase B is acetonitrile, replacement liquid C is ammonium acetate solution, and the wavelength is 220nm.

[0043] In some embodiments of the present invention, the purification operation includes: vacuum filtering the filtered oxidative cyclization reaction liquid, purifying the filtrate using a C18 preparative column by reverse phase high performance liquid chromatography, the mobile phase A is 0.1% formic acid aqueous solution, the mobile phase B is acetonitrile, and the replacement liquid C is 20 g / L ammonium acetate solution.

[0044] The beneficial effects of the present invention are:

[0045] The present invention provides a water-based synthesis method of μ-conotoxin KIIIA with instant wrinkle removal effect, and the method is environmentally friendly: by using water-based solvents, green condensing agents, new green resins, and acetonitrile aqueous solution as solvents in the entire coupling process, the dependence on harmful chemical solvents in traditional solid-phase peptide synthesis is significantly reduced, thereby reducing the impact on the environment. The method is cost-effective: due to the reduction of the use of expensive and harmful solvents and the improvement of the atomic economy of the synthesis process, the cost of peptide production is greatly reduced, and the yield is between 22-27%, which is greater than the yield of traditional solid-phase synthesis (~10%, J.Biol.Chem. (2022) 298 (3) 101728). The method reduces chemical waste: The synthesis method of the present invention reduces the generation of chemical waste, making it in line with the current global trend of sustainable development and environmental protection. The method is simplified in process: compared with the traditional solid-phase peptide method, the present invention avoids the use of three pairs of Cys different side chain protecting groups to achieve localized cyclization, and adopts the one-pot method of oxidative cyclization system for directional cyclization, and the synthesis process is more simplified and efficient, which is conducive to industrial production. In summary, the present invention not only provides an environmentally friendly, economical and efficient method for synthesizing μ-conotoxin KIIIA polypeptide, but also provides a feasible technical solution for the synthesis of similar complex polypeptides. BRIEF DESCRIPTION OF THE DRAWINGS

[0046] The accompanying drawings in the specification, which constitute a part of the present invention, are used to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute improper limitations on the present invention.

[0047] Figure 1 is the chemical structural formula of μ-conotoxin KIIIA polypeptide;

[0048] Figure 2 This is the HPLC purity chart of the crude peptide after oxidation cyclization in Example 2 of the present invention;

[0049] Figure 3 This is the HPLC purity chart of μ-conotoxin KIIIA after purification in Example 2 of the present invention;

[0050] Figure 4 This is the HPLC purity chart of the crude peptide after oxidation cyclization in Example 4 of the present invention;

[0051] Figure 5 This is the HPLC purity chart of μ-conotoxin KIIIA after purification in Example 4 of the present invention;

[0052] Figure 6 This is the HPLC purity chart of the crude peptide after oxidation cyclization in Example 6 of the present invention;

[0053] Figure 7This is the HPLC purity chart of μ-conotoxin KIIIA after purification in Example 6 of the present invention;

[0054] Figure 8 This is the mass spectrum of μ-conotoxin KIIIA obtained in Example 6 of the present invention;

[0055] Fig. 9 This is the mass spectrum of μ-conotoxin KIIIA after disulfide bond reduction obtained in Example 6 of the present invention;

[0056] Fig.10 This is a cell viability curve of μ-conotoxin KIIIA prepared in Example 6 of the present invention;

[0057] Fig.11 This is a graph showing the first depolarization pulse result of μ-conotoxin KIIIA prepared in Example 6 of the present invention;

[0058] Fig.12 This is a diagram showing the second depolarization pulse result of μ-conotoxin KIIIA prepared in Example 6 of the present invention. DETAILED DESCRIPTION

[0059] The present application is further described below in conjunction with specific examples. It should be understood that these examples are only used to illustrate the present application and are not intended to limit the scope of the present application. The experimental methods in the following examples without specifying specific conditions are usually carried out under conventional conditions or according to the conditions recommended by the manufacturer.

[0060] Unless otherwise defined, all professional and scientific terms used herein have the same meanings as those familiar to those skilled in the art. The reagents or raw materials used in this application can be purchased through conventional channels. Unless otherwise specified, the reagents or raw materials used in this application are used in a conventional manner in the art or in accordance with the product instructions. In addition, any method and material similar to or equivalent to the content described herein can be applied to the method of this application. The preferred implementation methods and materials described in the text are for demonstration purposes only.

[0061] The corresponding Chinese meanings of the English abbreviations of substances appearing in the claims and specification of the present invention are shown in the following table:

[0062]

[0063]

[0064] Example 1: Preparation of reduced μ-conotoxin KIIIA polypeptide

[0065] 1. Resin swelling: Take 20 g of H-RINK amide CHEMMATRIX resin (substitution degree is 0.5 mmol / g, total active reaction sites are 10 mmol, 1 equivalent) and add 200 mL of purified water to swell the resin for 2 hours, and then drain the solvent; then wash the resin twice with 100 mL of purified water and drain the solvent.

[0066] 2. Preparation of Smoc-L-Cys(Trt)-OH-resin: Add Smoc-L-Cys(Trt)-OH (21 mmol, 2.1 equivalents) and EDC-HCl (50 mmol, 5 equivalents) / Oxyma (30 mmol, 3 equivalents) / sodium bicarbonate (30 mmol, 3 equivalents) to an appropriate amount of 10% acetonitrile aqueous solution, mix and activate for 20 minutes, then add to the resin and react at 30°C for 3 hours; repeat the above operation to obtain Smoc-L-Cys(Trt)-OH-resin.

[0067] 3. Removal of Smoc protecting group: Add 100 mL of 0.8 M sodium hydroxide / 28% ethanolamine solution at 30°C to remove the Smoc protecting group twice, with reaction times of 20 minutes and 30 minutes respectively; then wash the resin with purified water until the pH reaches about 7, and drain the solvent to obtain L-Cys(Trt)-OH-resin.

[0068] 4. Activation of amino acids and peptide fragments: Smoc-L-Cys(Trt)-OH (21 mmol, 2.1 equivalents), Smoc-L-Arg(Pbf)-OH (21 mmol, 2.1 equivalents), Smoc-L-Ser(tBu)-OH (21 mmol, 2.1 equivalents), Smoc-L-His(Trt)-OH (21 mmol, 2.1 equivalents), Smoc-L-Asp(otBu)-OH (21 mmol, 2.1 equivalents), Smoc-L-Arg(Pbf)-OH (21 mmol, 2.1 equivalents), Smoc-L-Cys(Trt)-OH (21 mmol, 2.1 equivalents), Smoc-L-Trp(Boc)-OH (21 mmol, 2.1 equivalents), Smoc-L-Lys(Boc)- OH (26 mmol, 2.6 equiv.), Smoc-L-Ser(tBu)-OH (26 mmol, 2.6 equiv.), Smoc-L-Ser(tBu)-OH (26 mmol, 2.6 equiv.), Smoc-L-Cys(Trt)-OH (26 mmol, 2.6 equiv.), Smoc-L-Asn(Trt)-OH (26 mmol, 2.6 equiv.), Smoc-L-Cys(Trt)-OH (26 mmol, 2.6 equiv.) and Smoc-L-Cys(Trt)-OH (26 mmol, 2.6 equiv.) were added to an appropriate amount of 10% acetonitrile aqueous solution with EDC-HCl (50 mmol, 5 equiv.) / Oxyma (30 mmol, 3 equiv.) / sodium bicarbonate (30 mmol, 3 equiv.) and reacted at 25 °C for 20 min.

[0069] 5. Coupling of amino acids and peptide fragments with L-Cys(Trt)-OH-resin: Activated Smoc-L-Cys(Trt)-OH, Smoc-L-Arg(Pbf)-OH, Smoc-L-Ser(tBu)-OH, Smoc-L-His(Trt)-OH, Smoc-L-Asp(otBu)-OH, Smoc-L-Arg(Pbf)-OH, Smoc-L-Cys(Trt)-OH, Smoc-L-Trp(Boc)-OH, Smoc-L-Lys(Boc)-OH, Smoc-L-Ser(t Bu)-OH, Smoc-L-Ser(tBu)-OH, Smoc-L-Cys(Trt)-OH, Smoc-L-Asn(Trt)-OH, Smoc-L-Cys(Trt)-OH and Smoc-L-Cys(Trt)-OH are sequentially added to the resin from which the Smoc protecting group has been removed (the amino acids are condensed sequentially, one activated amino acid is condensed each time, and the Smoc protecting group is removed after each condensation of an amino acid), the amino acid condensation reaction is carried out at 30°C for 2 hours, the reaction progress is monitored by ninhydrin colorimetric reaction, and finally μ-conotoxin KIIIA reduced polypeptide resin is obtained.

[0070] 6. Cleavage of μ-conotoxin KIIIA reduced state polypeptide: Add 10 times the volume of cleavage reagent (18% hexafluoroisopropanol dichloromethane solution) to the peptide resin, react at 25°C for 3 hours, filter, and concentrate the filtrate to dryness to obtain 25.0 g of μ-conotoxin KIIIA reduced state crude peptide.

[0071] Example 2: Cyclization and purification of μ-conotoxin KIIIA

[0072] 1. Add 25.0 g of the crude peptide from Example 1 to 2.5 L of purified water and stir until dissolved.

[0073] 2. Add 47.3 g of tris(hydroxymethyl)aminomethane hydrochloride) and stir until dissolved. The final concentration is 120 mM.

[0074] 3. Add 59.7 g of guanidine hydrochloride and stir until dissolved. The final concentration is 250 mM.

[0075] 4. Add 0.92 g of reduced glutathione and stir until dissolved. The final concentration is 1.2 mM.

[0076] 5. Add 0.38 g of oxidized glutathione and stir until dissolved. The final concentration is 0.25 mM.

[0077] 6. Stir the reaction at 20-30°C for 16 hours. HPLC detection shows that the cyclization is complete and the purity after cyclization is 60.7%. Figure 2, add acetic acid to adjust the pH to 5; filter through a 0.45 μm filter membrane.

[0078] 7. The crude peptide was purified by reverse phase HPLC using a C18 preparative column. The mobile phase A was 0.1% formic acid aqueous solution, the mobile phase B was acetonitrile, and the replacement liquid C was 20 g / L ammonium acetate solution. The UV detection wavelength was 220 nm. After concentration and freeze drying, 4.2 g of pure product was obtained with a purity of 97.7%. Figure 3 , yield 22.1%.

[0079] Example 3: Preparation of reduced μ-conotoxin KIIIA polypeptide

[0080] 1. Resin swelling: Take 40 g of H-RINK amide CHEMMATRIX resin (substitution degree is 0.5 mmol / g, total active reaction sites are 20 mmol, 1 equivalent) and add 400 mL of purified water to swell the resin for 2 hours, and then drain the solvent; then wash the resin twice with 100 mL of purified water and drain the solvent.

[0081] 2. Preparation of Smoc-L-Cys(Trt)-OH-resin: Add Smoc-L-Cys(Trt)-OH (44 mmol, 2.2 equivalents) and EDC-HCl (5 equivalents) / Oxyma (3 equivalents) / sodium bicarbonate (3 equivalents) to an appropriate amount of 10% acetonitrile, mix and activate for 20 minutes, then add to the resin and react at 30°C for 3 hours; repeat the above operation to obtain Smoc-L-Cys(Trt)-OH-resin.

[0082] 3. Removal of Smoc protecting group: Add 200 mL of 0.8 M sodium hydroxide / 28% ethanolamine solution at 30°C to remove the Smoc protecting group twice, with reaction times of 20 minutes and 30 minutes respectively; then wash the resin with purified water until the pH reaches about 7, and drain the solvent to obtain L-Cys(Trt)-OH-resin.

[0083] 4. Activation of amino acids and peptide fragments: Smoc-L-Cys(Trt)-OH (44 mmol, 2.2 equivalents), Smoc-L-Arg(Pbf)-OH (44 mmol, 2.2 equivalents), Smoc-L-Ser(tBu)-OH (44 mmol, 2.2 equivalents), Smoc-L-His(Trt)-OH (44 mmol, 2.2 equivalents), Smoc-L-Asp(otBu)-OH (44 mmol, 2.2 equivalents), Smoc-L-Arg(Pbf)-OH (44 mmol, 2.2 equivalents), Smoc-L-Cys(Trt)-OH (44 mmol, 2.2 equivalents), Smoc-L-Trp(Boc)-OH (44 mmol, 2.2 equivalents), Smoc-L-Lys(Boc)-OH -OH (56 mmol, 2.8 equiv.), Smoc-L-Ser(tBu)-OH (56 mmol, 2.8 equiv.), Smoc-L-Ser(tBu)-OH (56 mmol, 2.8 equiv.), Smoc-L-Cys(Trt)-OH (56 mmol, 2.8 equiv.), Smoc-L-Asn(Trt)-OH (56 mmol, 2.8 equiv.), Smoc-L-Cys(Trt)-OH (56 mmol, 2.8 equiv.) and Smoc-L-Cys(Trt)-OH (56 mmol, 2.8 equiv.) were added to an appropriate amount of 10% acetonitrile and reacted at 25°C for 20 minutes.

[0084] 5. Coupling of amino acids and peptide fragments with L-Cys(Trt)-OH-resin: Activated Smoc-L-Cys(Trt)-OH, Smoc-L-Arg(Pbf)-OH, Smoc-L-Ser(tBu)-OH, Smoc-L-His(Trt)-OH, Smoc-L-Asp(otBu)-OH, Smoc-L-Arg(Pbf)-OH, Smoc-L-Cys(Trt)-OH, Smoc-L-Trp(Boc)-OH, Smoc-L-Lys(Boc)-OH, Smoc-L-Ser(t Bu)-OH, Smoc-L-Ser(tBu)-OH, Smoc-L-Cys(Trt)-OH, Smoc-L-Asn(Trt)-OH, Smoc-L-Cys(Trt)-OH and Smoc-L-Cys(Trt)-OH are sequentially added to the resin from which the Smoc protecting group has been removed (the amino acids are condensed sequentially, one activated amino acid is condensed each time, and the Smoc protecting group is removed after each condensation of an amino acid), the amino acid condensation reaction is carried out at 30°C for 2 hours, the reaction progress is monitored by ninhydrin colorimetric reaction, and finally μ-conotoxin KIIIA reduced polypeptide resin is obtained.

[0085] 6. Cleavage of μ-conotoxin KIIIA reduced peptide: Add 10 times the volume of cleavage reagent (18% hexafluoroisopropanol dichloromethane solution) to the peptide resin, react at 25°C for 3 hours, filter, and concentrate the filtrate to dryness to obtain 53.1g of μ-conotoxin KIIIA reduced crude peptide.

[0086] Example 4: Cyclization and purification of μ-conotoxin KIIIA

[0087] 1. Add 53.1 g of crude peptide from Example 3 to 5 L of purified water and stir until dissolved.

[0088] 2. Add 86.7 g of tris(hydroxymethyl)aminomethane hydrochloride) and stir until dissolved. The final concentration is 110 mM.

[0089] 3. Add 105.0 g of guanidine hydrochloride and stir until dissolved. The final concentration is 220 mM.

[0090] 4. Add 1.69 g of reduced glutathione and stir until dissolved. The final concentration is 1.1 mM.

[0091] 5. Add 0.67 g of oxidized glutathione and stir until dissolved. The final concentration is 0.22 mM.

[0092] 6. Stir the reaction at 20-30°C for 24 hours. HPLC detection shows that the cyclization is complete and the purity after cyclization is 67.1%. Figure 4, add acetic acid to adjust the pH to 5; filter through a 0.45 μm filter membrane.

[0093] 7. The crude peptide was purified by reverse phase HPLC using a C18 preparative column. The mobile phase A was 0.1% formic acid aqueous solution, the mobile phase B was acetonitrile, and the replacement liquid C was 20 g / L ammonium acetate solution. The UV detection wavelength was 220 nm. After concentration and freeze drying, 9.3 g of pure product was obtained with a purity of 96.6%. Figure 5 , yield 24.7%.

[0094] Example 5: Preparation of reduced μ-conotoxin KIIIA polypeptide

[0095] 1. Resin swelling: Take 100 g of H-RINK amide CHEMMATRIX resin (substitution degree is 0.5 mmol / g, total active reaction sites are 50 mmol, 1 equivalent) and add 1000 mL of purified water to swell the resin for 2 hours, and then drain the solvent; then wash the resin twice with 100 mL of purified water and drain the solvent.

[0096] 2. Preparation of Smoc-L-Cys(Trt)-OH-resin: Add Smoc-L-Cys(Trt)-OH (110 mmol, 2.2 equivalents) and EDC-HCl (5 equivalents) / Oxyma (3 equivalents) / sodium bicarbonate (3 equivalents) to an appropriate amount of 10% acetonitrile, mix and activate for 20 minutes, then add to the resin and react at 30°C for 3 hours; repeat the above operation to obtain Smoc-L-Cys(Trt)-OH-resin.

[0097] 3. Removal of Smoc protecting group: Add 500 mL of 0.8 M sodium hydroxide / 28% ethanolamine solution at 30°C to remove the Smoc protecting group twice, with reaction times of 20 minutes and 30 minutes respectively; then wash the resin with purified water until the pH reaches about 7, and drain the solvent to obtain L-Cys(Trt)-OH-resin.

[0098] 4. Activation of amino acids and peptide fragments: Smoc-L-Cys(Trt)-OH (110 mmol, 2.2 equivalents), Smoc-L-Arg(Pbf)-OH (110 mmol, 2.2 equivalents), Smoc-L-Ser(tBu)-OH (110 mmol, 2.2 equivalents), Smoc-L-His(Trt)-OH (110 mmol, 2.2 equivalents), Smoc-L-Asp(otBu)-OH (110 mmol, 2.2 equivalents), Smoc-L-Arg(Pbf)-OH (110 mmol, 2.2 equivalents), Smoc-L-Cys(Trt)-OH (110 mmol, 2.2 equivalents), Smoc-L-Trp(Boc)-OH (110 mmol, 2.2 equivalents), Smoc-L-Lys(Boc)-OH -OH (140 mmol, 2.8 equiv.), Smoc-L-Ser(tBu)-OH (140 mmol, 2.8 equiv.), Smoc-L-Ser(tBu)-OH (140 mmol, 2.8 equiv.), Smoc-L-Cys(Trt)-OH (140 mmol, 2.8 equiv.), Smoc-L-Asn(Trt)-OH (140 mmol, 2.8 equiv.), Smoc-L-Cys(Trt)-OH (140 mmol, 2.8 equiv.) and Smoc-L-Cys(Trt)-OH (140 mmol, 2.8 equiv.) were added to an appropriate amount of 10% acetonitrile and reacted at 25°C for 20 minutes.

[0099] 5. Coupling of amino acids and peptide fragments with L-Cys(Trt)-OH-resin: Activated Smoc-L-Cys(Trt)-OH, Smoc-L-Arg(Pbf)-OH, Smoc-L-Ser(tBu)-OH, Smoc-L-His(Trt)-OH, Smoc-L-Asp(otBu)-OH, Smoc-L-Arg(Pbf)-OH, Smoc-L-Cys(Trt)-OH, Smoc-L-Trp(Boc)-OH, Smoc-L-Lys(Boc)-OH, Smoc-L-Ser(t Bu)-OH, Smoc-L-Ser(tBu)-OH, Smoc-L-Cys(Trt)-OH, Smoc-L-Asn(Trt)-OH, Smoc-L-Cys(Trt)-OH and Smoc-L-Cys(Trt)-OH are sequentially added to the resin from which the Smoc protecting group has been removed (the amino acids are condensed sequentially, one activated amino acid is condensed each time, and the Smoc protecting group is removed after each condensation of an amino acid), the amino acid condensation reaction is carried out at 30°C for 2 hours, the reaction progress is monitored by ninhydrin colorimetric reaction, and finally μ-conotoxin KIIIA reduced polypeptide resin is obtained.

[0100] 6. Cleavage of μ-conotoxin KIIIA reduced peptide: Add 10 times the volume of cleavage reagent (18% hexafluoroisopropanol dichloromethane solution) to the peptide resin, react at 25°C for 3 hours, filter, and concentrate the filtrate to dryness to obtain 126.0 g of μ-conotoxin KIIIA reduced crude peptide.

[0101] Example 6: Cyclization and purification of μ-conotoxin KIIIA

[0102] 1. Add 126.0 g of the crude peptide from Example 5 to 12.5 L of purified water and stir until dissolved.

[0103] 2. Add 206.8 g of tris(hydroxymethyl)aminomethane hydrochloride) and stir until dissolved. The final concentration is 105 mM.

[0104] 3. Add 250.8 g of guanidine hydrochloride and stir until dissolved. The final concentration is 210 mM.

[0105] 4. Add 3.84 g of reduced glutathione and stir until dissolved. The final concentration is 1.0 mM.

[0106] 5. Add 1.53 g of oxidized glutathione and stir until dissolved. The final concentration is 0.20 mM.

[0107] 6. Stir the reaction at 20-30°C for 40 hours. HPLC detection shows that the cyclization is complete and the purity after cyclization is 73.7%. Figure 6, add acetic acid to adjust the pH to 4-6; filter through a 0.45μm filter membrane.

[0108] 7. The crude peptide was purified by reverse phase HPLC using a C18 preparative column. The mobile phase A was 0.1% formic acid aqueous solution, the mobile phase B was acetonitrile, and the replacement liquid C was 20 g / L ammonium acetate solution. The UV detection wavelength was 220 nm. After concentration and freeze drying, 25.1 g of μ-conotoxin KIIIA pure product was obtained with a purity of 98.3%. Figure 7 , yield 26.6%.

[0109] Example 7: Identification of disulfide bond connection mode of μ-conotoxin KIIIA

[0110] Molecular weight analysis: Weigh the μ-conotoxin KIIIA prepared in Example 6, dissolve it in ultrapure water to a concentration of about 0.5 mg / mL, divide it into two portions, one portion is directly loaded, and the other portion is reduced by adding an appropriate amount of dithiothreitol solution at 37° C. for 30 minutes.

[0111] Disulfide bond analysis: Weigh the μ-conotoxin KIIIA prepared in Example 6, add water to dilute the sample to a concentration of 1.4 mg / mL, adjust the sample pH to 3.0 with 10% formic acid aqueous solution, add a final concentration of 10 mM tris(2-carboxyethyl)phosphine solution and let it stand at 25°C for 10 minutes. Then add 100 μL of 1-cyano-4-dimethylaminopyridine tetrafluoroborate solution and let it stand at 25°C for 15 minutes, then immediately desalt with a C18 desalting column and spin dry, and finally cleave under alkaline conditions, add an appropriate amount of 1M ammonia solution to dissolve it and let it stand at 25°C for 1 hour. After the reaction, the sample was immediately spun dry and re-dissolved in 50 μL 0.1 M tri(2-carboxyethyl)phosphine. The sample was allowed to react at 37°C for 30 minutes before being loaded for detection. A water / acetonitrile mobile phase system was used, and liquid phase separation was performed using a surface-charged hybrid particle C18 chromatographic column. The high-resolution mass spectrometry method was used to detect the primary mass number of the cleavage peptide and match the HCD secondary fragment mass number to identify the cleavage peptide sequence and confirm the disulfide bond connection mode.

[0112] After molecular weight confirmation, the intact molecular weight results before reduction are shown in Figure 8 The molecular weight after reduction is Fig. 9 Before reduction, the primary parent ion of the sample has a divalent ion peak of (M+2H) 2+ The molecular weight (M+H)+ after reduction was changed to 1889.6808, with a molecular weight difference of 6Da, indicating that the sample did not form aggregates and the molecule may contain 3 pairs of disulfide bonds.

[0113] After disulfide bond identification, the monovalent parent ions (M+H) were detected at different retention times. +954.3216, 1003.3570, 249.0473, 338.1649, divalent parent ion (M+2H) 2+ 746.2971 and trivalent parent ion (M+3H) 3+ The ion peak is 534.6809, and the secondary fragment b / y ions of each parent ion match well. From the b / y ions, it can be seen that 954.3216 is the parent ion of the derivatized cleavage peptide segment Cys-Cys-Asn-Cys-Ser-Ser-Lys-Trp-NH2, 1003.3571 is the parent ion of the derivatized cleavage peptide segment Cys-Arg-Asp-His-Ser-Arg-Cys-Cys-NH2, and m / z 954.3216 and 1003.3571 are two chemical cleavage fragment peaks formed by the opening of the disulfide bond Cys1-Cys9 in the partially reduced state.

[0114] Similarly, according to the b / y ions, 534.6809 is the trivalent parent ion of the derivatized cleavage peptide Cys-Asn-Cys-Ser-Ser-Lys-Trp-Cys-Arg-Asp-His-Ser-Arg-NH2, and 249.0473 is the parent ion of the cleavage peptide Cys-Cys-NH2, which are mainly two chemical cleavage fragment peaks formed by the opening of the disulfide bond Cys2-Cys15 in the partially reduced state. The b / y ion matching showed that 338.1649 was the 1-valent parent ion of the fragmentation peptide Cys-Cys-Asn-NH2, and the 2-valent ion peak of the fragmentation peptide Cys-Ser-Ser-Lys-Trp-Cys-Arg-Asp-His-Ser-Arg-Cys-NH2 was 746.2971, with parent ions m / z = 338.1649 and 746.2971. (2+) The two ion peaks correspond to the chemical cleavage fragment peaks formed by the partial reduction and opening of the disulfide bond Cys4-Cys16. The theoretical and measured molecular weights of the detected components are summarized in Table 1.

[0115] Table 1 Summary of the theoretical and measured molecular weight test results of each component of the sample conus peptide

[0116]

[0117]

[0118] In summary, the μ-conotoxin KIIIA prepared in Example 6 contains three pairs of intramolecular disulfide bonds, and the identified disulfide bond connection modes are Cys1-Cys9, Cys2-Cys15 and Cys4-Cys16.

[0119] Example 8: μ-conotoxin KIIIA cytotoxicity test experiment

[0120] The μ-conotoxin KIIIA prepared in Example 6 was prepared into a 1500 ppm stock solution with purified water, and the stock solution was diluted to obtain solutions with concentration gradients (%, v / v) of 0.05%, 0.1%, 0.15625%, 0.3125%, 0.625%, 1%, 1.25%, 2.5%, 5% and 10%.

[0121] Cell inoculation: Inoculate cells into 96-well plates, 95 μL / well; add samples to cells, incubate at 37°C, 5% CO2 for 24 hours; add Cell Counting-Lite 2.0 Luminescent Cell Viability Assay to each well, shake for 2 minutes, and incubate at room temperature for 30 minutes; read data on a microplate reader, and the obtained sample cell viability curve is shown in Fig.10 .

[0122] According to the MTT results, it is believed that the μ-conotoxin KIIIA prepared in Example 6 does not show obvious cytotoxicity within a concentration range of 10% (150 ppm) based on Nav1.4-CHO cells.

[0123] Example 9: Experiment on the inhibition of activation of Nav1.4 sodium channel by μ-conotoxin KIIIA

[0124] The μ-conotoxin KIIIA prepared in Example 6 was prepared into a 1500 ppm stock solution with purified water, and the stock solution was diluted to obtain solutions with concentration gradients of 0.1%, 1% and 10% (%, v / v).

[0125] Cell culture

[0126] Maintenance medium: CHO cells are cultured in HAM'S / F-12 medium containing 10% fetal bovine serum at 37°C and a carbon dioxide concentration of 5%. Cell passaging: Discard the liquid and wash once with PBS, add 1mL of 0.25%-Trypsin-EDTA solution, and incubate at 37°C for 1.5min. When the cells detach from the bottom of the dish, add 5mL of complete medium preheated at 37°C. Gently blow the cell suspension with a pipette to separate the aggregated cells, transfer to a sterile centrifuge tube, centrifuge at 1000rpm for 5min, and collect the cells. Expansion or maintenance culture: Inoculate the cells in a 6cm cell culture dish, with 2.5×10 cells inoculated in each cell culture dish. 5 cells (final volume: 5 mL). To maintain the electrophysiological activity of the cells, the cell density did not exceed 80%. Before the patch clamp test, 0.25%-Trypsin-EDTA was used to separate the cells. 8×10 3The cells were plated on coverslips and cultured in 24-well plates (final volume: 500 μL). After 18 hours, the assay was performed.

[0127] Patch clamp assay

[0128] The voltage stimulation scheme for whole-cell patch clamp recording of Nav1.4 current is as follows: After the whole-cell seal is formed, the cell voltage is clamped at -120mV. First, the voltage is stepped from -110mV to -20mV in 10mV steps and maintained for 5s, and then a 0mV depolarizing pulse is given to measure the peak of its inward current to obtain the half-inactivation voltage. The resting state and half-inactivation state of the sodium current are detected using a double pulse mode. First, the first depolarizing pulse is given to 0mV for 20ms to detect the resting sodium current. Then, the conditional voltage is adjusted to the half-inactivation voltage between the two depolarizing pulses and maintained for 5s, and then the voltage is restored to -120mV and maintained for 20ms to allow the channels that are not bound to the compound and in the inactivated state to recover. A second depolarizing pulse is given to 0mV for 20ms to detect the sodium current in the half-inactivation state. Finally, the clamping voltage was restored to -120 mV, and data were repeatedly collected at intervals of 20 s to observe the effect of the sample on the peak sodium current in two different states.

[0129] Data analysis

[0130] First, the peak current value of each sample concentration (Peak current compound) and the peak current value of the blank control (Peak current Control) were normalized, and then the inhibition rate of each sample concentration under different conditions was calculated, that is, Inhibition% = (1-(Peak current compound / Peak current Control) * 100%. The mean (Mean) and standard deviation (SD) of the inhibition rate of each concentration were calculated, and the data were expressed as Mean ± SD.

[0131] like Fig.11 , Fig.12 As shown, based on Nav1.4-CHO cells, compared with the control group, the first depolarizing pulse inhibition rates of μ-conotoxin KIIIA prepared in Example 6 at concentrations of 0.1% (1.5ppm), 1.0% (15ppm), and 10% (150ppm) (v / v) were 80.85%, 90.56%, and 94.69%, respectively, and the second depolarizing pulse inhibition rates were 85.13%, 94.27%, and 96.27%, respectively, indicating that μ-conotoxin KIIIA can inhibit the activation of Nav1.4 sodium ion channels at the corresponding concentrations.

[0132] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A method for synthesizing μ-conotoxin KIIIA, characterized in that: include: The solid phase synthesis method was adopted, H-RINK amide CHEMMATRIX resin was used as a solid phase synthesis carrier, and the amino acid residues protected by the substituents were sequentially condensed in the acetonitrile aqueous solution according to the amino acid sequence from the C-terminus to the N-terminus of the main chain of μ-conotoxin KIIIA to obtain a crude reduced polypeptide; The crude polypeptide in the reduced state is oxidized and cyclized in an aqueous solution containing tris(hydroxymethylaminomethane) hydrochloride, guanidine hydrochloride, reduced glutathione and oxidized glutathione to obtain μ-conotoxin KIIIA.

2. The method according to claim 1, characterized in that: The amino acid sequence from the C-terminus to the N-terminus of the μ-conotoxin KIIIA main chain is: C1-C2-N3-C4-S5-S6-K7-W8-C9-R10-D11-H12-S13-R14-C15-C16-NH2.

3. The method according to claim 1, characterized in that The preparation method of the reduced polypeptide crude peptide comprises: using H-RINK amide CHEMMATRIX resin as a solid phase synthesis carrier, sequentially condensing Smoc-L-Cys(Trt)-OH, Smoc-L-Cys(Trt)-OH, Smoc-L-Arg(Pbf)-OH, Smoc-L-Ser(tBu)-OH, Smoc-L-His(Trt)-OH, Smoc-L-Asp(otBu)-OH, Smoc-L-Arg(Pbf)-OH from the C-terminus to the N-terminus , Smoc-L-Cys(Trt)-OH, Smoc-L-Trp(Boc)-OH, Smoc-L-Lys(Boc)-OH, Smoc-L-Ser(tBu)-OH, Smoc-L-Ser(tBu)-OH, Smoc-L-Cys(Trt)-OH, Smoc-L-Asn(Trt)-OH, Smoc-L-Cys(Trt)-OH, Smoc-L-Cys(Trt)-OH, and the reduced crude polypeptide was obtained after cleavage and drying.

4. The method according to claim 1, characterized in that The method for preparing the reduced polypeptide crude peptide comprises: Take H-RINK amide CHEMMATRIX resin and swell it in purified water, and then wash it with purified water; The first amino acid Smoc-L-Cys(Trt)-OH and a condensing agent are added to an acetonitrile aqueous solution for activation, and then added to the swollen H-RINK amide CHEMATRIX resin for reaction to prepare Smoc-L-Cys(Trt)-OH-resin, and the Smoc protecting group is removed to obtain L-Cys(Trt)-OH-resin; Activate Smoc-L-Cys(Trt)-OH, Smoc-L-Arg(Pbf)-OH, Smoc-L-Ser(tBu)-OH, Smoc-L-His(Trt)-OH, Smoc-L-Asp(otBu)-OH, Smoc-L-Arg(Pbf)-OH, Smoc-L-Cys(Trt)-OH, Smoc-L-Trp(Boc)-OH, Smoc-L-Lys(Boc)-OH, Smoc-L-Ser(tBu)-OH, Smoc-L-Ser(tBu)-OH, Smoc-L-Cys(Trt)-OH, Smoc-L-Asn(Trt)-OH, Smoc-L-Cys(Trt)-OH, Smoc-L-Cys(Trt)-OH; The activated Smoc-L-Cys(Trt)-OH was added to the L-Cys(Trt)-OH-resin for condensation. After the condensation was completed, the Smoc protecting group was removed, and then Smoc-L-Arg(Pbf)-OH, Smoc-L-Ser(tBu)-OH, Smoc-L-His(Trt)-OH, Smoc-L-Asp(otBu)-OH, Smoc-L-Arg(Pbf)-OH, Smoc-L-Cys(Trt ...Ser(tBu)-OH, Smoc-L-His(Trt)-OH, Smoc-L-Ser(tBu)-OH, Smoc-L-His(Trt)-OH, Smoc-L-Ser(tBu)-OH, Smoc-L-Ser(tBu)-OH, Smoc-L-His(Trt)-OH, Smoc-L-Ser(tBu)-OH, Smoc-L-Ser(tBu)-OH, Smoc-L-His(Trt)-OH, Smoc-L-Ser(tBu)-OH, Smoc-L-Ser(t Smoc-L-Trp(Boc)-OH, Smoc-L-Lys(Boc)-OH, Smoc-L-Ser(tBu)-OH, Smoc-L-Ser(tBu)-OH, Smoc-L-Cys(Trt)-OH, Smoc-L-Asn(Trt)-OH, Smoc-L-Cys(Trt)-OH, Smoc-L-Cys(Trt)-OH, after each condensation of an amino acid, the Smoc protecting group is removed to finally obtain a reduced polypeptide resin; The reduced polypeptide resin is cleaved and dried to obtain the reduced polypeptide crude peptide.

5. The method according to claim 4, characterized in that The H-RINK amide CHEMMATRIX resin substitution degree is 0.4-0-6mmol / g; Preferably, the swelling method of the H-RINK amide CHEMMATRIX resin comprises: adding the H-RINK amide CHEMMATRIX resin into purified water to swell for 1.5 to 2.5 hours, and then washing twice with purified water.

6. The method according to claim 4, characterized in that During the activation process, the molar ratio of the 1st to 7th amino acids Smoc-L-Cys(Trt)-OH, Smoc-L-Cys(Trt)-OH, Smoc-L-Asn(Trt)-OH, Smoc-L-Cys(Trt)-OH, Smoc-L-Ser(tBu)-OH, Smoc-L-Ser(tBu)-OH, and Smoc-L-Lys(Boc)-OH to the resin was 2.5-3.0; the 8th to 16th amino acids Smoc-L-T The molar ratio of rp(Boc)-OH, Smoc-L-Cys(Trt)-OH, Smoc-L-Arg(Pbf)-OH, Smoc-L-Asp(otBu)-OH, Smoc-L-His(Trt)-OH, Smoc-L-Ser(tBu)-OH, Smoc-L-Arg(Pbf)-OH, Smoc-L-Cys(Trt)-OH, and Smoc-L-Cys(Trt)-OH to the resin was 2.0-2.5; Preferably, the volume fraction of acetonitrile in the acetonitrile aqueous solution is 5 to 10%.

7. The method according to claim 4, characterized in that The condensing agent is composed of 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride, ethyl 2-oxime cyanoacetate and sodium bicarbonate; wherein the molar ratio of 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride, ethyl 2-oxime cyanoacetate and sodium bicarbonate relative to the resin is (4-6):(2-4):(2-4); Preferably, the condensation reaction temperature is 20-40°C and the time is 1-3h.

8. The method according to claim 4, characterized in that The reagent for removing the Smoc protecting group is a mixed solution of sodium hydroxide and ethanolamine; The mixed solution of sodium hydroxide and ethanolamine is obtained by mixing 0.5-1.0M sodium hydroxide and 25-30% ethanolamine solution in a volume ratio of (0.9-1.1):(0.9-1.1); Preferably, the method for removing the Smoc protecting group comprises: adding 0.5-1.0M sodium hydroxide / 25-30% ethanolamine solution to remove Smoc twice, the two removals are respectively carried out at 10-30°C for 20 minutes and 30 minutes; and then washing the resin with purified water until the pH reaches 6.5-7.

5.

9. The method according to claim 4, characterized in that When the reduced polypeptide resin is cleaved, a cleavage solution is added, wherein the cleavage solution is a hexafluoroisopropanol dichloromethane solution with a volume fraction of 15-20%, and the amount of the cleavage solution is 9-11 times the volume of the reduced polypeptide resin.

10. The method according to claim 1, characterized in that The preparation method of μ-conotoxin KIIIA comprises: Add 80-100 times the weight of purified water to the reduced crude polypeptide, stir until dissolved; add tris(hydroxymethyl)aminomethane hydrochloride, stir until dissolved, the final concentration is 100-120mM; add guanidine hydrochloride, stir until dissolved, the final concentration is 0.2-0.25M; add reduced glutathione, stir until dissolved, the final concentration is 1.0-1.2mM; add oxidized glutathione, stir until dissolved, the final concentration is 0.2-0.25mM; react at 20-30°C, stir overnight; adjust the pH to 4-6; filter, purify, and freeze-dry to obtain μ-conotoxin KIIIA; Preferably, the filtration is carried out using a 0.45 μm filter membrane; Preferably, the purification operation comprises: purification by reverse phase high performance liquid chromatography, mobile phase A is formic acid aqueous solution, mobile phase B is acetonitrile, replacement liquid C is ammonium acetate solution, and the wavelength is 220nm; Further preferably, the purification operation includes: vacuum filtering the filtered oxidative cyclization reaction liquid, purifying the filtrate using a C18 preparative column by reverse phase high performance liquid chromatography, the mobile phase A is 0.1% formic acid aqueous solution, the mobile phase B is acetonitrile, and the replacement liquid C is 20 g / L ammonium acetate solution.

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