Preparation method of mu-conotoxin disulfide bond substitute

Abstract

The invention provides a preparation method of a [mu]-conotoxin disulfide bond substitute, which comprises the following steps: on the basis of Fmoc solid-phase synthesis and DADA module embedding technology, coupling amino acid residues under the assistance of automatic microwaves to prepare a linear precursor peptide of [mu]-conotoxin, and carrying out in-situ oxidation and thiolysis activation on the linear precursor peptide to obtain the [mu]-conotoxin disulfide bond substitute. And carrying out folding oxidation on the obtained cyclized peptide by using natural chemical connection to assist cyclization so as to form the mu-conotoxin disulfide bond substitute. According to the method, the three-dimensional conformation and biological activity of the natural peptide are kept, meanwhile, efficient and rapid synthesis of the large-span disulfide bond substituted bridging peptide is achieved, the structural stability of the peptide product is remarkably improved, the reaction condition is mild and easy to control, the process is simple, the separation yield of the target product is stable, and the method can be used for rapidly preparing various variants and has wide application prospects. Different production and research requirements are met, and good market application prospects are achieved.

Description

Technical Field

[0001] This invention relates to the field of chemical synthesis technology of polypeptides, and specifically to a method for preparing a disulfide bond substitute for μ-conotoxin. Background Technology

[0002] In the synthesis and application of peptides, disulfide-containing peptides often regulate protein function through high-affinity and high-specificity binding to targets, attracting widespread attention as therapeutic agents (such as the FDA-approved analgesic ziconopeptide), diagnostic reagents (such as chlortoxin for cancer imaging), and research tools (such as reagents for capturing different conformations of membrane proteins). However, naturally occurring disulfide-containing peptides are often structurally unstable due to disulfide bond reduction and rearrangement, easily exhibiting poor pharmacodynamic properties. To address this issue, various disulfide bond substitutes containing metabolically stable bridging structures have been developed. These substitutes maintain the structure and function of the natural peptide while preserving stability and show potential for drug development. For example, the long-acting drug carbetocin—a synthetic disulfide bond substitute used to control postpartum and obstetric hemorrhage—is an analogue of natural oxytocin. Furthermore, recent studies have shown that disulfide bond substitution can bring other benefits to peptides, such as regulating receptor antagonism or modulating receptor subtype selectivity.

[0003] One strategy for constructing disulfide-bonded peptide substitutes involves cyclizing the side chains of amino acids at specific sites on the peptide through specific reactions such as thiol alkylation, azido-alkyne cycloaddition, or olefin metathesis. However, this method can only generate a limited variety of alternative bridging structures. Another strategy involves pre-synthesizing diamino diacid (DADA) building blocks containing disulfide-bonded alternative bridging structures, followed by insertion into specific sites on the peptide via solid-phase peptide synthesis. The DADA strategy offers greater flexibility, allowing for the synthesis of more diverse alternative bridging structures. Furthermore, the amide condensation reaction it relies on is relatively convenient, user-friendly, and easily automated. More importantly, because DADA modules can construct an unlimited number of structures, this strategy allows for the generation of more diverse disulfide-bonded alternative peptides by controlling the structure and properties.

[0004] Although this diamino acid strategy has been repeatedly validated in the development of peptide drugs and chemical tools, current technologies are unable to synthesize disulfide-substituted peptide analogs with a span of more than 10 amino acids using this strategy. For example, when Wright et al. attempted to synthesize a Cys2-Cys16 disulfide-substituted version of ProTx-II, a tarantula toxin with a voltage-gated sodium channel Nav1.7 inhibitor function, the pre-designed lanathione module failed to form a lactam structure. 【1】 Similarly, the diaminodic acid cyclization step also failed in the synthesis of the Cys10-Cys23 disulfide substitute for the long-acting spider toxicant PcTx1. 【2】Furthermore, side-chain cyclization methods, such as the azido-yne cycloaddition method, also face similar challenges. Therefore, current synthetic techniques are unable to achieve the preparation of disulfide bond substitution-bridged peptides with large spans.

[0005] μ-conotoxin KIIIA is derived from Conocarpus stolonifera, a snail native to the Indo-Pacific region. Conus kinoshitai The polypeptide toxin isolated from [the source] consists of 16 amino acids and is the shortest known μ-conotoxin, with the sequence: CCNCSSKWCRDHSRCC-NH2. Six Cys amino acid residues can form three disulfide bonds, possessing a specific -CC-CC-CC- cysteine ​​framework, exhibiting a precise blocking effect on the pores of sodium channels. Different isomers forming different disulfide bonds have been found in the synthetic and oxidative folding products of μ-KIIIA. Structural analysis of the μ-KIIIA complex with human sodium channel hNav1.2, containing C1-C15, C2-C9, and C4-C16 disulfide bonds, shows that this polypeptide is the most promising hNav1.2 channel blocker, providing a basis for the development of subtype-specific pore-blocking agents. 【3】 Currently, research on μ-KIIIA mainly relies on solid-phase synthesis. The formation of three disulfide bonds requires three oxidation steps, a cumbersome and difficult-to-success-prone process. In particular, adjacent Cys residues are prone to misalignment and ring formation. These misaligned structures are difficult to separate and purify due to their similar properties. This method of synthesizing μ-KIIIA is complex, costly, and its activity is unstable, making it unsuitable for industrial application. Furthermore, experiments have shown that natural KIIIA is prone to disulfide bond rearrangements, posing challenges to biochemical and pharmacological experiments. 【4】 Therefore, it is necessary to develop a method for preparing disulfide bond-substituted bridging peptides with a wide span for μ-conotoxin KIIIA, in order to obtain stable substitutes and chemical synthesis processes suitable for industrial production. Summary of the Invention

[0006] To address the aforementioned shortcomings of existing technologies and to expand the application prospects of disulfide-bonded proteins and improve their flexibility in modification, the inventors have developed a diamino diacid (DADA) strategy based on native chemical linkage (NCL) assistance for the stable synthesis of disulfide-substituted analogs of μ-conotoxin KIIIA. This strategy transfers the cyclization system from a solid phase to a more flexible liquid phase, successfully synthesizing previously difficult-to-obtain disulfide-substituted analogs required for biochemical and pharmacological studies. This enhanced disulfide bond modification capability has driven in-depth research into disulfide bond function. Furthermore, the inventors have discovered that stable chemical bond substitution of disulfide bonds does not always lead to increased peptide stability; priority should also be given to replacing solvent-exposed disulfide bonds, which typically span more amino acid residues. This highlights the importance of the modification method described in this invention. The technical solution adopted in this invention is as follows: This invention provides a method for preparing a disulfide bond substitute for μ-conotoxin, comprising the following steps: Step S1: Under microwave assistance, a linear precursor peptide of μ-conotoxin was prepared by solid-phase synthesis using Fmoc and the embedding of a diamino acid module, followed by sequential coupling of amino acid residues. The sequence of this precursor peptide is as follows: Cys1-Cys2-Asn3-Cys4-Ser5-Ser6-Lys7-Trp8-Cys9-Arg10-Asp11-His12-Ser13-Arg14-Cys15-Cys16-NH2, wherein the diamino diacid module is embedded between one or more pairs of amino acid residues selected from Cys1, Cys2, Cys4, Cys9, Cys15, and Cys16; Step S2: The linear precursor peptide obtained in step S1 is subjected to in-situ oxidation and thiolysis activation, and cyclization reaction is carried out with the assistance of natural chemical linkage to obtain cyclized peptide. Step S3: The cyclized peptide obtained in step S2 is placed in a redox buffer for folding and oxidation to form a μ-conotoxin disulfide bond substitute.

[0007] Preferably, in the preparation method of the μ-conotoxin disulfide bond substitute of the present invention, the diamino diacid module is embedded in one or more pairs selected from Cys1, Cys2, Cys4, Cys15, and Cys16; more preferably, it is selected from Cys1-Cys15 or Cys4-Cys16.

[0008] Furthermore, in the preparation method of the μ-conotoxin disulfide bond substitute of the present invention, the diaminodioic acid module insertion in step S1 includes: after the diaminodioic acid module is inserted, the allyloxycarbonyl group and allyl group on it are removed, and then amino acid coupling is continued.

[0009] Furthermore, in the preparation method of the μ-conotoxin disulfide bond substitute of the present invention, the removal of the allyloxycarbonyl and allyl groups includes: orthogonally protecting the N-terminal amino group of the peptide chain with tert-butoxycarbonyl, and then removing the groups using a phenylsilane / tetra(triphenylphosphine)palladium system.

[0010] Furthermore, in the preparation method of the μ-conotoxin disulfide bond substitute of the present invention, step S1 further includes the step of adding a trifluoroacetic acid mixture to remove the polypeptide from the solid phase after the linear precursor peptide is assembled.

[0011] Furthermore, in the preparation method of the μ-conotoxin disulfide bond substitute of the present invention, the cyclization reaction in step S2 is carried out in a liquid phase system, including: dissolving the linear precursor peptide obtained in step S1 in a buffer solution, adding sodium nitrite for in-situ oxidation, adding 4-mercaptophenylacetic acid for thiolysis, and then reducing the reaction system with a neutral tris(2-carboxyethyl)phosphine solution.

[0012] Preferably, in the preparation method of the μ-conotoxin disulfide bond substitute of the present invention, the linear precursor peptide solution concentration is 0.05-0.5 mM, the buffer solution is 0.1-0.5 M phosphate, 5-10 M urea, pH 1-4, the 4-mercaptophenylacetic acid is 80-120 equivalents, and the neutral tris(2-carboxyethyl)phosphine solution is 0.05-0.5 M, 80-120 equivalents.

[0013] Preferably, the linear precursor peptide solution has a concentration of 0.1 mM, the buffer solution is 0.2 M phosphate, 8 M urea, pH 2.3, the 4-mercaptophenylacetic acid is about 100 equivalents, and the neutral tris(2-carboxyethyl)phosphine solution is 0.1 M, about 100 equivalents.

[0014] Furthermore, in the preparation method of the μ-conotoxin disulfide bond substitute of the present invention, steps S2 and S3 respectively include the step of purifying the peptide products obtained in each step by reversed-phase high-performance liquid chromatography or characterizing them by electrospray ionization mass spectrometry.

[0015] Furthermore, in the preparation method of the μ-conotoxin disulfide bond substitute of the present invention, the disulfide bond substitution is a bridging of more than 10, preferably more than 12, and more preferably 10 to 15 amino acid residues. More preferably, it spans 10, 11, 12, 13, 14, or 15 amino acid residues.

[0016] On the other hand, the present invention provides a disulfide substitute for μ-conotoxin prepared by any of the above methods.

[0017] Furthermore, in the disulfide bond substitutes of μ-conotoxin prepared by the method of the present invention, the Cys1-Cys15, Cys2-Cys9 and / or Cys4-Cys16 disulfide bonds are replaced with chemically stable linkages such as thioether linkages.

[0018] Specifically, the preparation method of the μ-conotoxin disulfide bond substitute of the present invention preferably includes the following steps: First, solid-phase peptide synthesis (SPPS) based on 9-fluorenylmethoxycarbonyl (Fmoc) is performed by sequentially coupling amino acid residues in an automated microwave peptide synthesizer. Single coupling is carried out at 85–90°C, preferably 89°C, for 1 minute. Coupling of histidine (His) and cysteine ​​(Cys) is carried out at 45–55°C, preferably 50°C, for 5 minutes. Deprotection of the Fmoc group is carried out at 85–90°C, preferably 89°C, for 45 seconds. A 20% piperidine / dimethylformamide (DMF) solution containing 0.05–0.5 M, preferably 0.1 M, of ethyl 2-oxime cyanoacetate (Oxyma) is added to the reaction system, and the reaction is carried out for 1 minute.

[0019] The hydrazide resin was then swollen in DMF for 5–10 minutes. Each coupling cycle consisted of two core reactions: first, removing the Fmoc protecting group using a 20% piperidine / DMF solution containing 0.05–0.5 M, preferably 0.1 M Oxyma, at 89 °C for 45 seconds; second, performing an amino acid coupling reaction in DMF with the protected amino acid (4 equivalents), Oxyma (4 equivalents), and N,N-diisopropylcarbodiimide (DIC, 8 equivalents), wherein the coupling reaction of His and Cys was carried out at 50 °C for 5 minutes, and the coupling reaction of other amino acid residues was carried out at 89 °C for 1 minute. After each reaction, the resin was washed with DMF.

[0020] During solid-phase synthesis, diaminodioic acid (DADA) is inserted into a specific site corresponding to the disulfide bond to be replaced. After the synthesis of the intercalated peptide, the allyl (Alloc) and allyl groups carried on DADA are removed. This includes: orthogonal protection of the N-terminal amino group of the peptide chain with tert-butoxycarbonyl, followed by suspension in a resin (0.1–0.5 mM, preferably 0.25 mmol) with dichloromethane (DCM), and then addition of 400–800 μL, preferably 600 μL, of phenylsilane (PhSiH3). 40–80 mg, preferably 60 mg, of tetrakis(triphenylphosphine)palladium (Pd(PPh3)4) is dissolved in 1–5 mL, preferably 2 mL, of DCM and added to the above reaction system. The mixture is shaken at room temperature for 6 hours to remove the Alloc and Allyl groups. After further orthogonal protection of the terminal, solid-phase peptide extension continues until completion.

[0021] After peptide chain assembly, the resin is transferred to a solid-phase peptide synthesis vessel, and a cleavage mixture containing trifluoroacetic acid is added, preferably at a volume ratio of anisole:water:1,2-ethylenedithiol:trifluoroacetic acid = 5:5:3:87. The mixture is treated for 3 hours to cleave the peptides from the resin. The crude peptides are precipitated with cold diethyl ether and collected by centrifugation at 3000–5000 rpm, preferably 4000 rpm, to obtain the crude peptide product.

[0022] The crude peptide product was purified by reversed-phase high-performance liquid chromatography (RP-HPLC) using a C18 semi-preparative column. A gradient elution system of 10%-50% or 20%-60% buffer A (acetonitrile containing 0.1% trifluoroacetic acid) in buffer B (deionized distilled water containing 0.1% trifluoroacetic acid) was used for 30 minutes. The purified peptide was characterized by electrospray ionization mass spectrometry (ESI-MS) and subsequently freeze-dried to obtain the target precursor peptide product.

[0023] Secondly, cyclization was achieved using an NCL reaction. The purified peptidylhydrazine was dissolved in an aqueous buffer solution, preferably 0.2 M phosphate, 8 M urea, pH 2.3. The peptidylhydrazine was oxidatively activated at -15°C for 30 min, followed by the addition of 4-mercaptophenylacetic acid (MPAA) to the reaction system to generate the corresponding thioester, which was used for subsequent intramolecular natural chemical linking reactions. The reaction was carried out at pH 7, and the reaction progress was monitored by analytical high-performance liquid chromatography (HPLC). The target product was identified by ESI-MS. Finally, the reaction system was reduced using a neutral tris(2-carboxyethyl)phosphine (TCEP) solution, and the product was purified by semi-preparative RP-HPLC.

[0024] Finally, the cyclized peptide was dissolved in a redox buffer, preferably 0.1 M tris(hydroxymethyl)aminomethane (Tris), 10 equivalents of reduced glutathione (GSH): 10 equivalents of oxidized glutathione (GSSG), and oxidative folding was performed at pH 8. The final peptide concentration was preferably 0.01 mM. The folding reaction was carried out under gentle stirring at room temperature. The main peak product was purified by RP-HPLC and identified by ESI-MS. This demonstrates the folding of disulfide-bonded peptides and their DADA-substituted analogs.

[0025] The μ-conotoxin natural peptides described in this invention include, but are not limited to, μ-conotoxin peptides prepared by natural extraction, microbial fermentation, chemical synthesis, and their isomers with different disulfide bond linkages.

[0026] The disulfide bond substitutes described in this invention include, but are not limited to, peptide derivatives formed by structurally replacing the disulfide bonds (-SS-) between specific cysteine ​​(Cys) residues in natural peptides through chemical synthesis methods. These are also known as disulfide bond-substituted bridging peptides, disulfide bond-substituted peptides, etc.

[0027] The advantages and beneficial effects of this invention are as follows: (1) The method of this invention achieves efficient synthesis of large-span disulfide bond-substituted bridged peptides, which can cover peptide chains with more than 15 amino acid residues. It solves the technical problem of complex and difficult-to-purify products in the synthesis of macrocyclic peptides by the traditional resin-based DADA intercalation / cyclization strategy. Furthermore, by using NCL-assisted DADA, the specificity and quantitative conversion of the cyclization reaction are ensured, with almost no intermolecular linker byproducts generated. Thus, it overcomes the limitations of the resin-based DADA intercalation / cyclization strategy in the synthesis of large-span bridged disulfide bond-substituted peptides. (2) The disulfide bond substitutes prepared in this invention maintain the three-dimensional conformation and biological activity of the natural peptides, while significantly improving structural stability. They are not prone to disulfide bond rearrangement in the GSH environment, providing reliable tool molecules for biochemical and pharmacological research and possessing the potential to become therapeutic candidates.

[0028] (3) The preparation method described in this invention is based on the mature Fmoc solid-phase peptide synthesis technology, combined with microwave-assisted synthesis, selective deprotection and standardized separation and purification process. It does not require complex special equipment, the reaction conditions are mild and easy to control, and the process is simple and highly operable.

[0029] (4) The linear peptide precursor synthesis, cyclization and folding processes in the preparation method of the present invention can be completed quickly through efficient reactions. The cyclization reaction is carried out quantitatively within 1-6 hours, the folding reaction forms the main product within 1 hour, and the target product separation yield is stable at about 70%, which greatly improves the production efficiency. In addition, it can also quickly prepare a variety of mutants to meet different production and research needs and has good market application prospects. Attached Figure Description

[0030] Figure 1 This is a schematic diagram of the NCL-assisted synthesis strategy for disulfide bond substitutes of DADA described in this invention.

[0031] Figure 2 The diagram shows the chemical structures of compounds 2 to 4 prepared in Examples 1 to 3 of this invention.

[0032] Figure 3 This is a graph showing the folding process and molecular weight identification of compounds 1 to 4 described in this invention.

[0033] Figure 4This is a diagram of the protein database (PDB) showing the NMR structure of compound 2 prepared in Example 1 of the present invention.

[0034] Figure 5 The diagram shows the results of the reduction condition stability test of compounds 1 to 4 described in this invention. Detailed Implementation

[0035] The present invention will be further described below with reference to the accompanying drawings and preferred embodiments. It should be understood that the preferred embodiments described herein are for illustration and explanation only and are not intended to limit the invention. The raw materials, reagents, equipment, etc., used in the following embodiments are all commercially available or conventionally obtainable. Experimental methods without specific conditions are generally performed under conventional conditions or according to the manufacturer's recommendations. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those skilled in the art.

[0036] The μ-conotoxin KIIIA polypeptide described in this embodiment of the invention consists of 16 amino acids, and its natural sequence is as follows: Cys1-Cys2-Asn3-Gly4-Ser5-Ser6-Lys7-Trp8-Cys9-Arg10-Asp11-His12-Ser13-Arg14-Cys15-Cys16-NH2, wherein 6 Cys amino acid residues can form three pairs of disulfide bonds. Different isomers of μ-KIIIA exist in the synthesis and oxidative folding products, forming different disulfide bonds. This invention selects μ-KIIIA, which has a precise blocking effect on the pores of the human sodium channel hNav1.2. 【3】 As compound 1, its chemical structural formula is shown in Formula I below:

[0037] Formula I Compound 1 of Formula I contains three pairs of disulfide bonds linked in the following manner: Cys1-Cys15, Cys2-Cys9, and Cys4-Cys16, obtained through conventional solid-phase synthesis and oxidative folding. Using the preparation method described in this invention, the inventors synthesized disulfide-substituted analogs of μ-conotoxin KIIIA at the Cys1-Cys15 (compound 2), Cys2-Cys9 (compound 3), and Cys4-Cys16 (compound 4) sites, respectively. The modified disulfide bonds are stable chemical bonds such as thioethers, which can resist disturbances under reducing conditions.

[0038] The RP-HPLC testing process described in this embodiment of the invention uses a Shimadzu Prominence LC-20AT high-performance liquid chromatograph. The analytical column is a Welch Xb-C18 (250 mm × 4.6 mm, 5 μm), and the semi-preparative column is a Welch Xb-C18 (250 mm × 10 mm, 5 μm). Mobile phase A is acetonitrile containing 0.1% trifluoroacetic acid (TFA), and mobile phase B is deionized water containing 0.1% TFA. Before use, the mobile phases are degassed by sonication for 30 minutes. The elution gradient is 10%-50% of mobile phase A in mobile phase B, with linear gradient elution for 30 minutes. The flow rate is 1.0 mL / min for the analytical column and 5.0 mL / min for the semi-preparative column. The detection wavelength is 214 nm.

[0039] The mass spectrometry operation described in this embodiment of the invention is ESI-MS, which uses a Shimadzu LC / MS-2020 electrospray ionization mass spectrometer, with positive ion mode, a scan range of m / z 400-3000, an ion source temperature of 350℃, a capillary voltage of 4.5 kV, and a nebulizer gas pressure of 0.3 MPa.

[0040] Example 1 A method for preparing a μ-conotoxin disulfide bond substitute (compound 2) includes the following steps: Step S1: Microwave-assisted solid-phase synthesis of linear precursor peptides Fmoc Deprotection: 5-10 mL of DMF was added to 2-chlorotriphenylmethylhydrazine resin (2-Cl-Trt-NHNH2 resin, 0.25 mmol) for swelling. Then, 8-10 mL of 20% piperidine-DMF solution containing 0.1 M Oxyma was added. The reaction was carried out at 89 °C for 45 seconds in a Liberty Blue 2.0 microwave peptide synthesizer to remove the Fmoc protecting group from the resin and expose free amino groups. After deprotection, the resin was washed three times with DMF to remove residual piperidine and byproducts.

[0041] DADA module coupling: A DADA module was embedded at the Cys15 site. The resin, DADA amino acid, DIC, and Oxyma were mixed in a molar ratio of 1:4:8:4. The Fmoc-DADA, DIC, and Oxyma were dissolved in DMF and added to the reaction vessel. The reaction was carried out at 50°C for 5 minutes under microwave assistance to complete the covalent connection between the DADA module and the resin. After the reaction, the resin was washed three times with DMF to remove unreacted reagents.

[0042] Removal of Alloc and Allyl groups: During the solid-phase synthesis described above, DADA was intercalated into Cys15 corresponding to the disulfide bond to be replaced. After completing the synthesis of the intercalated peptide, the Alloc and Allyl groups carried on DADA were removed: First, the N-terminal amino group of the peptide chain was orthogonally protected with a tert-butoxycarbonyl group. Then, DCM was added to 0.25 mmol of resin to suspend the peptide, followed by the addition of 600 μL of PhSiH3. 60 mg of Pd (PPh3)4 was dissolved in 2 mL of DCM and poured into the above reaction system. The reaction was carried out at room temperature with shaking for 6 hours to remove the Alloc and Allyl groups. Subsequently, the terminal was orthogonally protected again, and solid-phase peptide extension was continued until completion.

[0043] Amino acid sequence assembly: Following the scheme of replacing the Cys1-Cys15 disulfide bonds in the amino acid sequence of the target peptide μ-conotoxin KIIIA, the above-described "Fmoc deprotection-amino acid coupling-washing" cycle was repeated sequentially. The coupling reaction of His and Cys was carried out at 50°C for 5 minutes, and the coupling reaction of other amino acids was carried out at 89°C for 1 minute to ensure efficient linkage of each amino acid residue, forming a linear peptide precursor.

[0044] Linear peptide precursor cleavage and separation: After the prepared linear peptide chains were assembled, a cleavage mixture was added to the reaction vessel at a volume ratio of anisole:water:1,2-ethylenedithiol:trifluoroacetic acid = 5:5:3:87. The reaction was carried out at room temperature for 3 hours to cleave the linear precursor from the resin. Subsequently, the crude peptide was washed with 50 mL of cold diethyl ether for precipitation, and the precipitate was collected by centrifugation at 4000 rpm / min to obtain the crude linear peptide precursor.

[0045] Step S2: NCL-assisted intramolecular cyclization The linear peptide obtained in step S1 was dissolved at a concentration of 0.1 mM in a buffer solution of 0.2 M phosphate, 8 M urea, and pH 2.3. Ten equivalents of NaNO2 were added for in-situ oxidation at -15°C, activating the acylhydrazine at the Trp8 position to an acyl azide intermediate. Subsequently, 100 equivalents of MPAA were added for thiolysis to activate the peptidylhydrazine, converting it into a reactive peptide thioester. The activated thioester immediately undergoes intramolecular natural chemical linkage with the only exposed N-terminal Cys residue, forming a new amide bond and completing the intramolecular cyclization reaction. The reaction process was monitored by RP-HPLC. After the reaction was completed, the cyclized product was purified, and its molecular weight was verified by ESI-MS.

[0046] Step S3: Oxidative folding to form disulfide bond substitutes The cyclic peptide (0.01 mM) purified in step S2 was placed in redox buffer (50 mM Tris, pH 8) for folding. The folding conditions were: peptide:GSH:GSSG molar ratio of 1:10:10, pH 8, and temperature 30°C. The folding process was monitored by RP-HPLC. After folding, the peptide was purified again by HPLC.

[0047] Structural identification: Mass spectrometry analysis was performed on target compound 2 obtained in step S3. The mass spectra before and after folding showed a molecular weight decrease of 3.9 Da, which corresponds precisely to the loss of 4 hydrogen atoms when 4 thiol groups (-SH) form two pairs of disulfide bonds (-SS-), indicating the formation of correct disulfide bonds. The chemical structural formula of compound 2 is attached. Figure 2 As shown.

[0048] Example 2 A method for preparing a μ-conotoxin disulfide bond substitute (compound 3) includes the following steps: Step S1: Microwave-assisted solid-phase synthesis of precursor peptides Following the preparation process described in Example 1, step S1, involving Fmoc deprotection, DADA module coupling, and amino acid sequence assembly, the peptide chain was assembled through a cyclic process of "Fmoc deprotection-amino acid coupling-washing." The difference is that Example 2 employed a conventional solid-phase cyclization method. Following the scheme of replacing Cys2-Cys9 disulfide bonds in the amino acid sequence, the DADA module was embedded into Cys2. DADA coupling was completed by microwave reaction at 50°C for 5 minutes, with a resin:DADA:DIC:Oxyma molar ratio of 1:4:8:4. Other amino acids were reacted under conventional Fmoc-SPPS conditions.

[0049] Step S2: Solid-phase cyclization on the resin When the preparation process in step S1 condenses to the W8 site, solid-phase cyclization on the resin is performed. Subsequently, PhSiH3 and Pd(PPh3)4 are added to the resin (Rink amide AM Resin, 0.25 mmol) to remove Alloc and Allyl groups, similar to that in Example 1. The cyclizing reagent is dissolved in 5 mL of DMF solution at a molar ratio of resin: (7-azabenzotriazole-1-oxy)tripyrrolidinephosphine (PyAOP): N-hydroxy-7-azabenzotriazole (HOAT): N-methylmorpholine (NMM) = 1:5:5:10. The above cyclizing reagent is mixed with the resin, and the reaction is carried out at 37°C for 3 hours, and this process is repeated once. After the reaction is complete, the mixture is cleaved using a method similar to that in Example 1, and the crude mixture containing the cyclized product is collected. The crude cyclized product is purified by semi-preparative RP-HPLC and then freeze-dried to obtain the purified cyclized product.

[0050] Step S3: Oxidative folding to form disulfide bond substitutes The 2 mg precyclized peptide obtained in step S2 was dissolved in 100 mL of 50 mM Tris, pH 8 redox buffer. The peptide was gently stirred and folded at pH 8 and 30 °C for 1 hour at a molar ratio of peptide:GSH:GSSG = 1:10:10. The main peak was then separated and purified by RP-HPLC to obtain target compound 3, whose chemical structure is shown in the attached figure. Figure 2 As shown.

[0051] Example 3 A method for preparing a μ-conotoxin disulfide bond substitute (compound 4) includes the following steps: Step S1: Microwave-assisted solid-phase synthesis of linear precursor peptides As described in Example 1, using 2-chlorotriphenylmethylhydrazine resin (2-Cl-Trt-NHNH2 resin, 0.25 mmol) as the starting support, DADA was inserted into the peptide chain via DADA-DIC-Oxyma coupling, with a molar ratio of resin (equivalent):DADA (equivalent):DIC (equivalent):Oxyma (equivalent) = 1:4:8:4. The reaction was carried out at 50°C for 5 minutes under microwave assistance to obtain a solid-phase linear peptide.

[0052] Unlike Example 1, a DADA module was embedded at the Cys16 site of the linear peptide. Fmoc-DADA, DIC, and Oxyma were dissolved in DMF according to the above-mentioned molar ratio of resin:DADA amino acid:DIC:Oxyma = 1:4:8:4, and added to the reaction vessel. The reaction was carried out at 50°C for 5 minutes under microwave assistance to complete the covalent connection between the DADA module and the resin. After the reaction, the resin was washed three times with DMF to remove unreacted reagents.

[0053] After DADA was inserted into the Cys16 site, the Alloc and Allyl protecting groups were removed using the same Pd(PPh3)4 / PhSiH3 system as in Example 1. Subsequently, a mixture of 400 mg of 9-fluorenemethylsuccinimino carbonate Fmoc-OSu, 200 μL of N,N-diisopropylethylamine (DIEA), and 5 mL of DMF was added to further protect the amino group released from DADA using Fmoc orthogonal protection. Then, using a coupling system with a molar ratio of DIC:Oxyma:N,N-dimethylpyridine (DMAP) of 8:4:0.1, the free carboxyl group of DADA was coupled with 0.2 mL of allyl alcohol to obtain a solid-phase branched peptide. The peptide chain was then further extended using Fmoc solid-phase peptide synthesis technology until completion.

[0054] Finally, 15-20 mL of cleavage mixture (aniline sulfide:water:1,2-ethylenedithiol:trifluoroacetic acid = 5:5:3:87, volume ratio) was added to the resin to cleave the peptidylhydrazine precursor from the resin. The crude precursor peptide, analyzed by RP-HPLC, showed a relatively single main peak. After purification by semi-preparative RP-HPLC, a linear precursor peptide with DADA substitution at positions Cys4-Cys16 was obtained. Based on the resin loading, the separation yield was 14%.

[0055] Step S2: NCL-assisted intramolecular cyclization Intramolecular cyclization was performed following steps similar to those in Example 1. The linear peptide obtained in step S1 was dissolved at a concentration of 0.1 mM in a buffer solution of 0.2 M phosphate, 8 M urea, and pH 2.3. 10 equivalents of NaNO2 were added for in-situ oxidation at -15°C, followed by the addition of 100 equivalents of MPAA for thiolysis to activate the peptidyl hydrazide, and then intramolecular cyclization was carried out.

[0056] RP-HPLC monitoring results showed that the cyclization reaction was successfully and quantitatively completed within 6 hours, generating the cyclized product with almost no intermolecular linker byproducts. The clear main peak further confirmed the NCL-mediated cyclization reaction. The cyclized product was purified by semi-preparative RP-HPLC with a separation yield of 68%, and its molecular weight was verified by ESI-MS.

[0057] Step S3: Oxidative folding to form disulfide bond substitutes Take 2 mg of the cyclized product obtained in step S2 (concentration 0.01 mM, 0.02 mg / mL), add 100 mL of redox buffer (peptide:GSH:GSSG = 1:10:10, molar ratio; 100 mM tris(hydroxymethyl)aminomethane (Tris), pH 8), and carry out the folding reaction at 30 °C.

[0058] The reaction progress was monitored by RP-HPLC. Results showed that the folding product was successfully generated after 1 hour of folding reaction, and ESI-MS identified the main peak as the target folding product. The folding reaction solution was purified using a C18 column to obtain target compound 4, whose chemical structure is shown in the attached figure. Figure 2 As shown.

[0059] Product testing and conclusion analysis The inventors have synthesized analogs of the natural polypeptide μ-conotoxin KIIIA by substituting the disulfide bonds of each of its three pairs. Compound 1 was compared with compounds 2 to 4 obtained in Examples 1 to 3, respectively. The correctness of the synthesized products was verified by HPLC, ESI-MS, and NMR structural analysis.

[0060] Specifically, the inventors selected Trp8-Cys9 in the KIIIA sequence as the site for natural chemical linkage during cyclization, and prepared the cyclization intermediate on an acylhydrazine resin using a Fmoc-SPPS synthesis strategy. Natural chemical linkage of the acylhydrazine was employed for the cyclization of the intermediate. To improve cyclization efficiency, the inventors optimized the peptide concentration, linkage system, reaction temperature, and reaction pH. Finally, after in-situ oxidation and thiolysis activation, intramolecular cyclization was carried out at a peptide concentration of 0.1 mM, 8 M urea, a reaction pH of 7, and a reaction temperature of 30°C. The cyclization reaction was successfully completed within 6 hours, with almost no intermolecular linkage byproducts. The purified separation yield reached 70%, and ESI-MS verified the accuracy of its molecular weight. For the folding and refolding conditions, folding was performed under redox conditions, i.e., peptide:GSH:GSSG = 1:10:10 (molar ratio), pH 8.0, and 25°C. HPLC monitoring showed that a single, pure target product was formed after 1 hour. The resulting compound decreased in mass by 3.9 Da after folding, confirming that the four free thiol groups formed two pairs of disulfide bonds. The results indicate that disulfide bond substitution at the above sites can significantly improve folding performance.

[0061] 1. NMR structure analysis A Varian 700 MHz nuclear magnetic resonance spectrometer was used at a testing temperature of 298 K. The natural peptide of compound 1 and the disulfide substitutes of compounds 2 to 4 were dissolved in 50 mM PBS buffer (pH 6.5) containing 10% D₂O to prepare sample solutions with a concentration of approximately 3.0 mM, which were then placed in 5 mm NMR sample tubes. Based on the nuclear overhauser effect (NOE) signal in the nuclear overhauser effect spectra (NOESY), 226 distance constraints (including 25 long-range NOE constraints) were obtained. Structural calculations were performed using XPlor-NIH software, and the 20 lowest-energy conformations were selected from the 200 calculated structures. The root mean square deviation (RMSD) of all atoms in 20 preferred conformations was analyzed. The RMSD for skeletal atoms was 0.246 Å, and the RMSD for all heavy atoms was 0.968 Å. The conformational rationality was verified by Ramachandran plots (57.5% of residues were in the most favorable region, with no forbidden regions). Comparison of chemical shifts and disulfide bond connections between compounds 1 and 2 confirmed that compound 2 maintained the correct Cys2-Cys9 and Cys4-Cys16 disulfide bond connections. Its three-dimensional conformation was highly similar to the natural peptide structure of KIIIA, indicating that the CS substitution of the Cys1-Cys15 disulfide bond had minimal impact on the structure and biological activity of KIIIA. These results are attached. Figure 4 As shown.

[0062] 2. Stability Test Prepare 100 mM PBS buffer (pH 7.5) in advance, degas it by sonication for 10 minutes, and ensure that the buffer is free of bubbles and the pH is stable. Weigh an appropriate amount of GSH, dissolve it in the above PBS buffer, and prepare a 0.25 mM GSH stock solution. Prepare and use immediately to avoid oxidation and deactivation. Weigh the natural KIIIA of compound 1 and the disulfide substitutes of compounds 2, 3, and 4, respectively, dissolve them in PBS buffer, and prepare 0.25 mM sample stock solutions. Calibrate the concentration by UV spectrophotometry (280 nm) to ensure that the initial concentration of each sample is consistent.

[0063] Mix 1 mL of sample stock solution with 1 mL of GSH stock solution to obtain a reaction system with a final concentration of 0.25 mM (1:1 molar ratio). Gently invert the centrifuge tube three times to ensure thorough mixing and avoid vigorous shaking that could alter the sample structure. Incubate the reaction system in a 25°C incubator, avoiding light exposure to prevent light from affecting the stability of disulfide bonds.

[0064] Samples were taken at 0 h (initial time), 0.5 h, 1 h, 3 h, and 6 h of incubation, with a sample volume of 20 μL each time. The sample was quickly added to 80 μL of buffer B (an aqueous solution containing 0.1% TFA) in an ice bath, vortexed for 10 seconds, and then stored at -20°C to terminate the reaction. After sampling at all time points, RP-HPLC and ESI-MS analyses were performed.

[0065] The test results are attached. Figure 5 As shown, the natural peptide of compound 1 exhibits low tolerance to reducing conditions. Significant disulfide rearrangement occurs after 0.5 h of incubation, reaching a rearrangement rate of 15%. ESI-MS verification confirmed that the new peak was consistent with the molecular weight of natural KIIIA (theoretical molecular weight 1884.2 Da), confirming it as an isomerized product. After 3 h of incubation, the rearrangement rate increased to 35%, and multiple impurity peaks appeared in the chromatogram, indicating its susceptibility to structural instability in a reducing environment. In contrast, the disulfide substitute of compound 2, under the same testing conditions, retained over 97% of the main product after 6 h.

[0066] From the appendix Figure 5 It can also be seen that the disulfide bond substitutes of compound 2 and compound 4 prepared in Examples 1 to 3 have significantly better structural stability than the natural μ-conotoxin of compound 1. However, the stability of compound 3, which has the smallest disulfide bond span obtained by solid-phase cyclization synthesis, is actually reduced. This indicates that the substitution of stable chemical bonds of disulfide bonds can not always improve the stability of peptides. It is also necessary to give priority to replacing disulfide bonds that are exposed by the solvent and usually span more amino acid residues.

[0067] On the other hand, the preparation method described in this invention achieves the replacement of large-span disulfide bonds, which is difficult to achieve in solid-phase cyclization. Figure 3 Experimental results show that it achieves a more significant improvement in folding efficiency compared to small-span disulfide bond substitution. The liquid-phase modification system provides greater freedom for cyclization, reducing the influence of hydrogen bonds and steric hindrance in the solid phase on cyclization. Compared to the free folding of natural toxic peptides, the pre-formed large-span disulfide bonds can spatially confine linear peptides, guiding the toxic peptides to the correct conformation, thereby improving folding efficiency. This indicates that the present invention achieves unexpected technical effects.

[0068] Finally, it should be noted that the above specific embodiments are only used to illustrate the technical solutions of the present invention and not to limit it. Although the present invention has been described in detail with reference to examples, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention, and all such modifications or substitutions should be covered within the scope of the claims of the present invention.

[0069] References: 1. M. Cemazar, D. J. Craik et al., Curr. Top. Med. Chem. 2012, 12, 1534–1545. 2. G. Cohen, J. A. Frank et al., Toxins 2018, 10, 496. 3. Xiaojing Pan, Nieng Yan et al., “Molecular basis for pore blockade of human Na+ channel Nav1.2 by the μ-conotoxin KIIIA”, Science 363, 1309–1313 (2019) 22 March 2019. 4. J. V. Lin King, D. Julius et al., Cell 2019, 178, 1362–1374.

Claims

1. A method for preparing a μ-conotoxin disulfide bond substitute, characterized in that: Includes the following steps: Step S1: Under microwave assistance, a linear precursor peptide of μ-conotoxin was prepared by solid-phase synthesis using Fmoc and the embedding of a diamino acid module, followed by sequential coupling of amino acid residues. The sequence of this precursor peptide is as follows: Cys1-Cys2-Asn3-Cys4-Ser5-Ser6-Lys7-Trp8-Cys9-Arg10-Asp11-His12-Ser13-Arg14-Cys15-Cys16-NH2, wherein the diamino diacid module is embedded between one or more pairs of amino acid residues selected from Cys1, Cys2, Cys4, Cys9, Cys15, and Cys16; Step S2: The linear precursor peptide obtained in step S1 is subjected to in-situ oxidation and thiolysis activation, and cyclization reaction is carried out with the assistance of natural chemical linkage to obtain cyclized peptide. Step S3: The cyclized peptide obtained in step S2 is placed in a redox buffer for folding and oxidation to form a μ-conotoxin disulfide bond substitute.

2. The method for preparing the μ-conotoxin disulfide bond substitute according to claim 1, characterized in that, The diaminodioic acid module embedding in step S1 includes: after the diaminodioic acid module is embedded, the allyloxycarbonyl group and allyl group on it are removed, and then amino acid coupling is continued.

3. The method for preparing the μ-conotoxin disulfide bond substitute according to claim 2, characterized in that, The removal of the allyloxycarbonyl and allyl groups includes: orthogonally protecting the N-terminal amino group of the peptide chain with tert-butoxycarbonyl, and then removing the groups using a phenylsilane / tetra(triphenylphosphine)palladium system.

4. The method for preparing the μ-conotoxin disulfide bond substitute according to claim 1, characterized in that, Step S1 also includes the step of adding a trifluoroacetic acid mixture to remove the polypeptide from the solid phase after the linear precursor peptide is assembled.

5. The method for preparing the μ-conotoxin disulfide substitute according to claim 1, characterized in that, The cyclization reaction described in step S2 is carried out in a liquid phase system, including: dissolving the linear precursor peptide obtained in step S1 in a buffer solution, adding sodium nitrite for in-situ oxidation, adding 4-mercaptophenylacetic acid for thiolysis, and then reducing the reaction system with a neutral tris(2-carboxyethyl)phosphine solution.

6. The method for preparing the μ-conotoxin disulfide bond substitute according to claim 5, characterized in that, The linear precursor peptide solution has a concentration of 0.05–0.5 mM, the buffer solution is 0.1–0.5 M phosphate, 5–10 M urea, pH 1–4, the 4-mercaptophenylacetic acid is 80–120 equivalents, and the neutral tris(2-carboxyethyl)phosphine solution is 0.05–0.5 M, 80–120 equivalents.

7. The method for preparing the μ-conotoxin disulfide bond substitute according to claim 1, characterized in that, Steps S2 and S3 further include the purification of the peptide products obtained in each step by reversed-phase high-performance liquid chromatography or the characterization by electrospray ionization mass spectrometry.

8. The method for preparing the μ-conotoxin disulfide bond substitute according to claim 1, characterized in that, The disulfide bond substitution mentioned above is a disulfide bond substitution bridging that spans more than 10 amino acid residues.

9. The μ-conotoxin disulfide substitute prepared by the method according to any one of claims 1 to 8.

10. The μ-conotoxin disulfide substitute prepared according to the method of claim 9, characterized in that, In the μ-conotoxin disulfide bond substitutes, the Cys1-Cys15, Cys2-Cys9, and / or Cys4-Cys16 disulfide bonds are replaced with chemically stable linkages such as thioether linkages.

Images