Preparation method of mu-conopeptide refined peptide

Abstract

The invention belongs to the technical field of polypeptide chemical synthesis, and provides a preparation method of mu-conopeptide refined peptide, which comprises the following steps: S1, sequentially coupling amino acids containing protecting groups by adopting a solid-phase synthesis technology to prepare mu-conopeptide resin, then cutting the resin and removing the protecting groups of amino acid side chains to obtain mu-conopeptide resin; the preparation method comprises the following steps of: obtaining a [mu]-conopeptide linear peptide, namely H-Pyr-Gly-Cys-Cys-Asn-Gly-Pro-Lys-Gly-Cys-Ser-Lys-Trp-Cys-Arg-Asp-His-Ala-Arg-Cys-NH2, and carrying out ultrasonic treatment to obtain the [mu]-conopeptide linear peptide. S2, dissolving the [mu]-conopeptide linear peptide in a 1-butyl-3-methylimidazolium dihydrogen phosphate ion solution, adding sodium sulfate, and carrying out air oxidation pairing ring formation to obtain [mu]-conopeptide crude peptide; and S3, purifying the [mu]-conopeptide by using an ultrafiltration method in combination with a high performance liquid chromatography to obtain the [mu]-conopeptide refined peptide. According to the [mu]-conopeptide cyclization method, expensive reagents are not used, the cost is relatively low, the folding rate of the conopeptide is high in correctness, and the improved purification method is suitable for large-scale production.

Description

Technical Field

[0001] The invention belongs to the technical field of polypeptide chemical synthesis, and particularly relates to a method for preparing a refined μ-conopeptide. Background Art

[0002] Cono peptides, also known as conotoxins, are a class of small molecule polypeptide toxins secreted by the marine mollusk cone snail. Cones live in tropical and subtropical waters and secrete these toxins through venom glands when hunting, quickly paralyzing their prey. Cono peptides are usually composed of 10 to 30 amino acids, with a complex structure, mostly formed into a ring by disulfide bonds. There are many types of cono peptides, and each toxin produced by cone snails has a unique molecular structure and biological activity. Its mechanism of action is diverse, and it can specifically bind to and regulate various ion channels and receptors. It is an important tool for studying neuroscience, pharmacology and biotechnology.

[0003] Cono peptides are a class of small molecule polypeptides secreted by conus, usually composed of 10 to 30 amino acids, with multiple disulfide bonds, forming a stable ring structure, which makes it highly stable under extreme pH values ​​and temperatures. Its molecular weight is generally between 1000 and 3000 Daltons, with good water solubility and specific three-dimensional structure.

[0004] μ-conopeptide mainly acts on voltage-gated sodium channels (VGSCs), blocking the influx of sodium ions into nerve cells, thereby interfering with nerve signal transmission. This mechanism gives it a strong paralytic and analgesic effect. It is also used in the cosmetic field to relax facial muscles and reduce wrinkles and fine lines, with an effect similar to botulinum toxin but more gentle. Its unique physicochemical properties and biological activity give it a wide range of application potential in pharmacology and cosmetic science.

[0005] Conopeptides are obtained mainly through three methods: extraction from cone snail venom glands, chemical synthesis and biosynthesis. The biosynthesis method uses genetic engineering to modify microorganisms to express conopeptide precursor proteins, and then enzymatic treatment is performed to generate active conopeptides. The extraction of natural conopeptides faces the problems of scarce resources and low extraction efficiency. The number of cone snails is limited and the venom gland extraction process is complicated, resulting in low yields. Although the biosynthesis method can ensure the accurate folding and correct molecular conformation of the product, the technology is not yet mature and cannot meet the needs of industrialization.

[0006] The current chemical synthesis method of cono peptides usually uses solid-phase peptide synthesis to obtain linear peptides, which are then oxidized in a liquid aqueous solution to form corresponding disulfide bonds. Although this method can accurately control the amino acid sequence, during the oxidation process, the currently used aqueous solution oxidation method usually needs to be oxidized at a very low concentration to solve the side reactions of disulfide bond mismatch and disulfide bond formation between molecules. The oxidation concentration is usually 1 mg / mL, and the oxidation time is more than 72 hours. This oxidation method will undoubtedly produce a large amount of waste liquid, which brings huge challenges to environmental protection. In addition, the synthesis process also requires a lot of resources and time, which limits large-scale production and wide application. Summary of the invention

[0007] The present invention aims to solve the defects of the prior art conopeptide synthesis method and provide a method for preparing μ-conopeptide by chemical synthesis. The method significantly improves the purity and yield of μ-conopeptide, and the side reaction products are easier to remove, especially in the disulfide bond oxidation ring step, the mismatch is reduced, thereby avoiding the problem of reduced synthesis yield and reduced purity of crude peptide caused by mismatch.

[0008] The invention provides a method for preparing refined μ-conopeptide peptide.

[0009] The method for preparing the refined μ-conopeptide peptide comprises the following steps:

[0010] S1: The μ-conopeptide resin is prepared by sequentially coupling amino acids containing protective groups using solid phase synthesis technology, and then the resin is cut and the amino acid side chain protective groups are removed to obtain the μ-conopeptide linear peptide:

[0011] H-Pyr-Gly-Cys-Cys-Asn-Gly-Pro-Lys-Gly-Cys-Ser-Ser-Lys-Trp-Cys-Arg-Asp-His-Ala-Arg-Cys-Cys-NH2;

[0012] S2: dissolving the μ-conopeptide linear peptide in a 1-butyl-3-methylimidazole dihydrogen phosphate ion solution and adding sodium sulfate to perform air oxidation to pair the peptides into rings, thereby obtaining a μ-conopeptide crude peptide;

[0013] S3: Purify the crude μ-cono peptide using ultrafiltration combined with high performance liquid chromatography to obtain refined μ-cono peptide.

[0014] Further, step S1 includes:

[0015] S11: Preparation of μ-conopeptide linear peptide resin using solid phase synthesis technology:

[0016] H-Pyr-Gly-Cys(X)-Cys(X)-Asn(trt)-Gly-Pro-Lys(Boc)-Gly-Cys(X)-Ser(tBu)-Ser(tBu)-Lys(Boc)-Tr p(Boc)-Cys(X)-Arg(pbf)-Asp(tBu)-His(trt)-Ala-Arg(pbf)-Cys(X)-Cys(X)-Y, wherein X is selected from one of trt and Mmt, and Y is a resin selected from Rink amide AM-Resin;

[0017] S12: using a cleavage reagent prepared with TFA, TIS, EDT, phenol and water in a volume ratio of 95:2.5:2.5:0.5:0.5 to perform cleavage to prepare a μ-conopeptide main chain linear peptide.

[0018] Furthermore, the solvent of the 1-butyl-3-methylimidazolium dihydrogen phosphate ion solution in step S2 is 0.1M phosphate buffer, and the concentration of 1-butyl-3-methylimidazolium dihydrogen phosphate ion is 0.1-0.5M.

[0019] Furthermore, the 0.1M phosphate buffer described in step S2 is a phosphate buffer with sodium dihydrogen phosphate: disodium hydrogen phosphate = 1:9, and the concentration of the 1-butyl-3-methylimidazole dihydrogen phosphate ion is 0.2M.

[0020] Furthermore, in step S2, after the μ-cono peptide linear peptide is dissolved in 1-butyl-3-methylimidazole dihydrogen phosphate ion solution, the concentration of the μ-cono peptide linear peptide is 10 mg / mL to 200 mg / mL, and after the sodium sulfate is added, the concentration of the sodium sulfate is 0.01 to 0.1 M, the reaction pH is 6.5 to 8.5, the reaction temperature is 16 to 36° C., and the reaction time is 12 to 24 hours.

[0021] Furthermore, in step S2, after the μ-cono peptide linear peptide is dissolved in 1-butyl-3-methylimidazole dihydrogen phosphate ion solution, the concentration of the cono peptide linear peptide is 10-200 mg / mL, and after the sodium sulfate is added, the final concentration of the sodium sulfate is 0.05 M, the reaction pH is 7.4±0.5, and the reaction temperature is 25-36°C.

[0022] Furthermore, in step S3, during the ultrafiltration process, the pressure is set to 0.2-0.4 MPa, the flow rate is set to 20 mL / min, the retained liquid is collected, and refined peptides are obtained after liquid phase purification.

[0023] The main advantages of the present invention are:

[0024] 1. The present invention uses a 1-butyl-3-methylimidazole dihydrogen phosphate ion system in the oxidative cyclization process of μ-conopeptide. The system can provide a mild oxidation system environment, improve the dispersibility of conopeptide in the reaction medium, and contribute to a uniform oxidation folding process; the dihydrogen phosphate ion can also provide stable oxidation conditions, which are suitable for the formation and stability of disulfide bonds of conopeptide, and contribute to the correct spatial structure folding; the unique polarity and ionic interaction of ionic liquids can improve the selectivity of the reaction, reduce the occurrence of side reactions and misfolding, and thus improve the correct folding rate of conopeptide. In addition, the 1-butyl-3-methylimidazole dihydrogen phosphate ion has low volatility and low toxicity, is environmentally friendly and safe, meets the requirements of green chemistry, and the by-products generated during the cyclization process are easier to remove.

[0025] 2. The oxidation is carried out in the oxidation system of the ionic liquid of the present invention, and the correct pairing of three pairs of disulfide bonds of μ-conopeptide can be completed under the condition of higher reaction concentration, which greatly reduces the generation of oxidation waste liquid in the traditional oxidation method and is more environmentally friendly.

[0026] 3. The reaction of the present invention in ionic liquid can accelerate the formation of disulfide bonds. The reaction time is faster than that of traditional oxidation methods, which greatly improves production efficiency.

[0027] 3. The μ-conopeptide cyclization method of the present invention does not use expensive reagents, has low cost, and the improved purification method is suitable for large-scale production. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 This is the liquid chromatogram of μ-cono crude peptide.

[0029] Figure 2 The liquid chromatogram of the purified product.

[0030] Figure 3 This is the changing trend of the inhibition rate of μ-cono peptide stock solution on elastase in the concentration range of 0.5 to 25 ppm.

[0031] Figure 4 This is the changing trend of the inhibition rate of hyaluronidase by μ-cono peptide stock solution in the concentration range of 0.5 to 25 ppm. DETAILED DESCRIPTION

[0032] In order to make the above-mentioned objects, features and advantages of the present invention more obvious and understandable, the specific implementation modes of the present invention are described in detail below with reference to the accompanying drawings, but they should not be construed as limiting the applicable scope of the present invention.

[0033] Example 1 Preparation of Fmoc-Cys(trt)-AM-Resin

[0034] Take 98.04g of Rink amide AM-Resin with a substitution degree of 0.51mmol / g and add it into a solid phase reactor. Add N,N-dimethylformamide (DMF) to swell for 30min. After drying, add 20% piperidine-DMF solution, stir with nitrogen for reaction for 5min and then dry. Add 20% piperidine-DMF solution again, stir for reaction for 10min and then dry. Add DMF to wash and dry. Wash 6 times in total and dry for use.

[0035] Weigh 87.86g (3eq) of Fmoc-Cys(trt)-OH and 24.32g (1.2eq) of HOBT and add them to the swollen resin. Add 28mL of DIC at the same time, stir under nitrogen for two hours, wash twice with DMF, and then use DBLK to remove the Fmoc protecting group for 15 minutes. After the end, wash five times with DMF to obtain Fmoc-Cys(trt)-AM-Resin.

[0036] Example 2 Preparation of μ-cono linear peptide

[0037] Take Fmoc-Cys(trt)-AM-Resin in Implementation Case 1, refer to the steps of Implementation Case 1, and couple the subsequent amino acids in the order shown in the table below to obtain μ-cono peptide linear peptide resin.

[0038]

[0039] The μ-cono peptide linear peptide resin was dried and added to a reactor, and pre-cooled TFA cleavage solution was added to react for 3 hours. After the reaction was completed, the resin was filtered out, the resin was washed with TFA, the filtrate was combined, and added to anhydrous ether for precipitation. After the solid was precipitated, it was filtered and vacuum dried to obtain the μ-cono peptide linear peptide.

[0040] Example 3 Cyclization of μ-cono linear peptide

[0041] Weigh 13.9g NaH2PO4·2H2O and 14.2g Na2HPO4·7H2O, dissolve in 5L distilled water to prepare phosphate buffer; weigh 47.24g 1-butyl-3-methylimidazole dihydrogen phosphate ion and add it to the prepared phosphate buffer; add the μ-cono peptide linear peptide obtained in Example 2 at a concentration of 60mg / mL; add 0.05M sodium sulfate and stir to disperse, adjust the pH to 7.4 and continue stirring and reacting for 12h to obtain the correctly coordinated μ-cono peptide crude peptide. The purity of the μ-cono peptide crude peptide reaches 88.80%. Figure 1 It is a liquid chromatogram of crude μ-conopeptide, in which the peak area of ​​crude μ-conopeptide (retention time 16.032 min) accounts for 88.80%.

[0042] Example 4 Cyclization of μ-cono linear peptide

[0043] Weigh 13.9g NaH2PO4·2H2O and 14.2g Na2HPO4·7H2O, dissolve in 5L distilled water to prepare phosphate buffer; weigh 23.62g 1-butyl-3-methylimidazole dihydrogen phosphate ion and add it to the prepared phosphate buffer; add the μ-cono linear peptide obtained in Example 2 at a concentration of 10mg / mL; add 0.01M sodium sulfate and stir to disperse, adjust the pH to 7.4 and continue stirring and reacting for 12h to obtain the correctly coordinated μ-cono peptide crude peptide. The purity of the μ-cono peptide crude peptide reaches 85.32%.

[0044] Example 5 Cyclization of μ-cono linear peptide

[0045] Weigh 13.9g NaH2PO4·2H2O and 14.2g Na2HPO4·7H2O, dissolve in 5L distilled water to prepare phosphate buffer; weigh 70.86g 1-butyl-3-methylimidazole dihydrogen phosphate ion and add it to the prepared phosphate buffer; add the μ-cono linear peptide obtained in Example 2 at a concentration of 100mg / mL; add 0.06M sodium sulfate and stir to disperse, adjust the pH to 7.4 and continue stirring and reacting for 16h to obtain the correctly coordinated μ-cono peptide crude peptide. The purity of the μ-cono peptide crude peptide reaches 84.63%.

[0046] Example 6 Cyclization of μ-cono linear peptide

[0047] Weigh 13.9g NaH2PO4·2H2O and 14.2g Na2HPO4·7H2O, dissolve in 5L distilled water to prepare phosphate buffer; weigh 118.11g 1-butyl-3-methylimidazole dihydrogen phosphate ion and add it to the prepared phosphate buffer; add the μ-cono linear peptide obtained in Example 2 at a concentration of 200mg / mL; add 0.1M sodium sulfate and stir to disperse, adjust the pH to 7.4 and continue stirring and reacting for 24h to obtain the correctly coordinated μ-cono peptide crude peptide. The purity of the μ-cono peptide crude peptide reaches 86.32%.

[0048] Example 7 Purification of crude μ-conopeptide

[0049] The reaction solution in Example 3 was subjected to a 3000Da ultrafiltration membrane, loaded into an ultrafiltration device, the pressure was adjusted to 0.2MPa, the flow rate was set to 100mL / min, the retained solution was collected, the filtrate was filtered twice, and the retained solution was finally combined. The retained solution was purified using conventional high performance liquid chromatography to obtain μ-cono peptide.

[0050] Figure 2The liquid chromatogram of the purified product shows that the impurities of the refined peptide are significantly reduced. Only three chromatographic peaks appear at retention times of 16.483 min, 16.9421 min, and 16.917 min, with peak areas of 757 and 1.45 × 10 7 , 4012, among which the chromatographic peak with a retention time of 16.942min is the target chromatographic peak, and the peak area accounts for 99.97%. After purification, the total yield of the product reaches 65%, the purity of the pure product is >99%, and it contains only two single impurities.

[0051] Example 8 In vitro anti-wrinkle activity of μ-cono peptide

[0052] The μ-cono peptide obtained in Example 4 was prepared into a stock solution with a concentration of 0.5 to 25 ppm, and samples were taken for pretreatment, and elastase solution was added to react at 37°C; substrate was added, and the reaction was kept at 37°C for 60 minutes. After centrifugation, the supernatant was taken and measured at 405 nm with an enzyme marker, and the data was recorded and analyzed. By measuring the inhibition rate of the sample on elastase, it can be reflected whether the sample has anti-wrinkle and firming effects; the higher the elastase inhibition rate, the better the anti-wrinkle and firming effects of the sample.

[0053] Figure 3 The figure shows the variation trend of the inhibition rate of μ-conopeptide essence on elastase in the concentration range of 0.5-25ppm. It can be seen from the figure that the concentration range of 0.5-25ppm can inhibit the activity of elastase, and the inhibition rate is about 8.93%-40.89%. The inhibition rate increases with the increase of sample concentration. Therefore, it can be shown that μ-conopeptide has certain anti-wrinkle and firming effects.

[0054] Example 9 In vitro anti-allergic and soothing activity of μ-cono peptide

[0055] The μ-cono peptide obtained in Example 4 was prepared into a stock solution with a concentration of 0.5 to 25 ppm, and samples were taken for pretreatment, hyaluronidase and substrate were added, and the reaction was kept at 37°C for 60 minutes. The absorbance was measured at 530 nm, and the inhibition rate was calculated. Hyaluronidase is a participant in type I allergic reactions. Hyaluronidase has a strong correlation with inflammation and allergies. Studies have reported that various drugs that release histamine from mast cells can regulate hyaluronidase activity, and some anti-allergic drugs can strongly inhibit hyaluronidase activity. Therefore, inhibition of hyaluronidase activity is used as an indicator for studying anti-allergic effects.

[0056] Figure 4The figure shows the change trend of the inhibition rate of μ-cono peptide stock solution on hyaluronidase in the concentration range of 0.5-25ppm. It can be seen from the figure that within a certain concentration range, μ-cono peptide has a certain inhibitory effect on hyaluronidase, and the inhibition rate is about 4.35% to 32.61%. The inhibition rate increases with the increase of sample concentration. Therefore, it can be shown that μ-cono peptide has a certain soothing effect.

[0057] The above descriptions are merely embodiments of the present invention, and are not intended to limit the patent scope of the present invention. Any slight changes made to the contents of the present invention, or any direct or indirect application in related technical fields, are also included in the patent protection scope of the present invention.

Claims

1. A method for preparing a purified μ-conopeptide peptide, characterized in that: The following steps are involved: S1: μ-conopeptide resin is prepared by sequentially coupling amino acids containing protective groups using solid phase synthesis technology, and then the resin and amino acid side chain protective groups are cleaved to obtain μ-conopeptide linear peptide: H-Pyr-Gly-Cys-Cys-Asn-Gly-Pro-Lys-Gly-Cys-Ser-Ser-Lys-Trp-Cys-Arg-Asp-His-Ala-Arg-Cys-Cys-NH2; S2: dissolving the μ-conopeptide linear peptide in a 1-butyl-3-methylimidazole dihydrogen phosphate ion solution and adding sodium sulfate to perform air oxidation to pair the peptides into rings, thereby obtaining a μ-conopeptide crude peptide; S3: Purify μ-cono peptide using ultrafiltration combined with high performance liquid chromatography to obtain refined μ-cono peptide.

2. The method for preparing the purified μ-conopeptide according to claim 1, characterized in that: Step S1 includes: S11: Preparation of μ-conopeptide resin by solid phase synthesis technique: H-Pyr-Gly-Cys(X)-Cys(X)-Asn(trt)-Gly-Pro-Lys(Boc)-Gly-Cys(X)-Ser(tBu)-Ser(tBu)-Lys(Boc)-Tr p(Boc)-Cys(X)-Arg(pbf)-Asp(tBu)-His(trt)-Ala-Arg(pbf)-Cys(X)-Cys(X)-Y, wherein X is selected from one of trt, Acm, tBu, and Mob, and Y is a resin selected from Rink amide AM-Resin; S12: using a cleavage reagent prepared with TFA, TIS, EDT, phenol and water in a volume ratio of 95:2.5:2.5:0.5:0.5 to perform cleavage to prepare a μ-conopeptide main chain linear peptide.

3. The method for preparing the purified μ-conopeptide according to claim 1, characterized in that: The solvent of the 1-butyl-3-methylimidazolium dihydrogen phosphate ion solution in step S2 is 0.1M phosphate buffer, and the concentration of 1-butyl-3-methylimidazolium dihydrogen phosphate ion is 0.1-0.5M.

4. The method for preparing the purified μ-conopeptide according to claim 1, characterized in that: The 0.1M phosphate buffer described in step S2 is a phosphate buffer with sodium dihydrogen phosphate: disodium hydrogen phosphate = 1:9, and the concentration of the 1-butyl-3-methylimidazole dihydrogen phosphate ion is 0.2M.

5. The method for preparing the purified μ-conopeptide according to claim 1, characterized in that: In step S2, after the μ-cono peptide linear peptide is dissolved in 1-butyl-3-methylimidazole dihydrogen phosphate ion solution, the concentration of the μ-cono peptide linear peptide is 10 mg / mL to 200 mg / mL, and after the sodium sulfate is added, the concentration of the sodium sulfate is 0.01 to 0.1 M, the reaction pH is 6.5 to 8.5, the reaction temperature is 16 to 36° C., and the reaction time is 12 to 24 h.

6. The method for preparing the purified μ-conopeptide according to claim 5, characterized in that: In step S2, after the μ-cono peptide linear peptide is dissolved in 1-butyl-3-methylimidazole dihydrogen phosphate ion solution, the concentration of the cono peptide linear peptide is 5 mg / mL, and after the sodium sulfate is added, the final concentration of the sodium sulfate is 0.05 M, the reaction pH is 7.4±0.5, and the reaction temperature is 25-36° C.

7. The method for preparing a purified μ-conopeptide according to claim 1, characterized in that: In step S3, during the ultrafiltration process, the pressure is set to 0.2-0.4 MPa, the flow rate is set to 20 mL / min, the retained liquid is collected, and the refined peptide is obtained after liquid phase purification.

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