Mu-conopeptide analogue and preparation method thereof

Abstract

The invention provides a mu-conopeptide analogue and a preparation method thereof, and belongs to the technical field of polypeptide drugs. The C terminal of the [mu]-conopeptide analogue is amidated, and the [mu]-conopeptide analogue has the following sequence: H-Pyr-Gly-Cys-Cys-Asn-Gly-Pro-Lys-Gly-Ala-Ser-Lys-Trp-Cys-Arg-Asp-His-Ala-Arg-C-Ala-NH2, has two pairs of disulfide bonds, and is positioned at the positions of Cys3-Cys15 and Cys4-Cys21. The invention provides the preparation method of the [mu]-conopeptide analogue, a Cys cross protecting group strategy is adopted, and the [mu]-conopeptide analogue can be used for preparing the [mu]-conopeptide analogue. A first pair of disulfide bonds is formed through solid-phase oxidation, a second pair of disulfide bonds is formed through liquid-phase oxidation, and two pairs of correct disulfide bonds are constructed. The method is simple to operate, low in cost and suitable for industrial mass production.

Description

Technical Field

[0001] The present invention relates to the technical field of polypeptide drugs, and in particular to a μ-conopeptide analog and a preparation method thereof. Background Art

[0002] Cone snails are a family of venomous tropical marine gastropod molluscs that vary widely in size and are named for their conical appearance. Cone snails can secrete venom through their venom tubes or venom glands on the inner wall of their venom sacs to capture prey or defend themselves.

[0003] In the 1980s, Olivera and others isolated conotoxin from cone snails for the first time. Due to its unique functions, few side effects, and clear targeting, it has become a focus of attention in the fields of pharmacology and biomedicine. In 2012, Swiss scientists developed cono peptides, opening the "magic door to wrinkle removal" in the field of skin care and medical beauty. So far, scientists have identified 5 types of active conotoxins, namely α, δ, κ, μ, and ω. α-Conotoxin can inhibit nicotinic acetylcholine receptors; δ-Conotoxin inhibits voltage-dependent Na + Channel; κ-conotoxin inhibits K + Channels; μ-conotoxin inhibits voltage-dependent Na in muscle + Channels; ω-conotoxin inhibits N-type voltage-dependent Ca 2+ aisle.

[0004] Cono peptide actually simulates the biological effect of the neurotoxin μ conotoxin. It can restrain the excessive expression of facial muscles under the condition of paralyzing muscle activity, and can provide effective muscle relaxation. Its effect is similar to that of botulinum toxin, which helps to relieve wrinkles and ultimately achieve the effect of reducing wrinkles. Cono peptide blocks the current conduction of neuromuscular and instantly reduces wrinkles. Cono peptide blocks voltage-dependent sodium ion channels, especially the channel for muscles-NaV1.4, which selectively blocks sodium ion channels. The blocking rate is above 90%, which can not only play a blocking role, but also maintain a certain flow of electrical signals. The expression is greatly improved, but not tight and stiff, not completely stiff and paralyzed, so it provides a very natural wrinkle removal and firming effect. It can not only be used in functional cosmetics, but also directly as a drug, which is of great significance to the research and development of new drugs.

[0005] At present, conotoxins are mainly obtained through three methods: genetic engineering, chemical solid phase synthesis, and extraction from natural cone snails. Natural extraction is the most primitive and earliest used method, but the amount of conotoxins obtained by this method is very small, and with the destruction of marine ecology in recent years, the number of wild cone snails has dropped sharply. Relying solely on natural extraction methods will cause further environmental deterioration. Therefore, it is not realistic to extract a large number of toxin peptides from natural cone snails. This method can only be used to extract a small amount of toxin peptides for scientific research. However, one advantage of this method is that scientists can obtain the amino acid sequence of a single toxin peptide through a series of instrumental analysis methods, and use the amino acid sequence as a template for artificial synthesis. The second method is to synthesize conotoxins by genetic engineering methods, that is, to transform the gene of conotoxins into microorganisms to express them naturally, and to separate and purify the products after the expression is completed. However, since the prokaryotic expression system cannot perform deep processing on the expression product, and the molecular weight of conotoxins is small, there are many basic amino acids, and it is difficult to form an active conformation, making it difficult to separate the expression products obtained. The process is not mature, it is difficult to realize the industrialization of conotoxins, and it cannot meet the needs of the market. The third method is to obtain conotoxin by chemical solid phase synthesis, that is, to obtain its amino acid sequence by isolating natural conotoxin, and then obtain a large number of toxin peptides by chemical solid phase synthesis. At present, with the continuous emergence of research results on peptide drugs, more and more complex peptide drug molecules have been successfully prepared for commercial mass production through chemical solid phase synthesis technology. The chemical solid phase synthesis technology of peptides is already very mature, and it is entirely possible to synthesize them in large quantities through solid phase peptide synthesis, reducing costs and meeting scientific research purposes and the growing market demand.

[0006] By simulating the natural μ-conotoxin, μ-cono peptide was artificially synthesized. The peptide is composed of 22 amino acids and contains three disulfide bonds. The sequence is Pyr-Gly-Cys-Cys-Asn-Gly-Pro-Lys-Gly-Cys-Ser-Ser-Lys-Trp-Cys-Arg-Asp-His-Ala-Arg-Cys-Cys-NH2. The disulfide bonds are located at Cys3-Cys15, Cys4-Cys21 and Cys10-Cys22. The process route for the artificial synthesis of cono peptide includes linear peptide synthesis, cleavage, oxidative folding and crude peptide purification. The oxidative folding of linear peptides is an important step in the synthesis of cono peptides. Only by folding to form the correct three pairs of disulfide bonds can μ-cono peptide have biological activity. The construction of three pairs of disulfide bonds has the problems of high difficulty in synthetic preparation technology and high production cost. Developing a method for preparing conopeptide analogs that is simple in process, stable and controllable, can achieve correct disulfide bond construction, retains the biological activity of conopeptide, and is suitable for large-scale industrial production to meet market demand is an issue that needs to be urgently addressed in this field. Summary of the invention

[0007] The purpose of the present invention is to provide a μ-conopeptide analog and a preparation method thereof, which adopts a Cys cross-protecting group strategy, solid phase oxidation to form a first pair of disulfide bonds, liquid phase oxidation to form a second pair of disulfide bonds, and constructs two correct pairs of disulfide bonds. The operation is simple, the cost is low, and it is suitable for industrial mass production.

[0008] The technical solution of the present invention is achieved in this way:

[0009] The invention provides a μ-conopeptide analog. The μ-conopeptide analog is amidated at the C-terminus and has the following sequence: H-Pyr-Gly-Cys-Cys-Asn-Gly-Pro-Lys-Gly-Ala-Ser-Ser-Lys-Trp-Cys-Arg-Asp-His-Ala-Arg-Cys-Ala-NH2, and has two pairs of disulfide bonds located at the positions of Cys3-Cys15 and Cys4-Cys21.

[0010] The present invention further protects a method for preparing the above μ-conopeptide analog, comprising the following steps:

[0011] S1. Preparation of linear peptide resin: According to the peptide sequence, select Fmoc amino resin as the carrier for solid phase synthesis; put in the resin to swell, react with Fmoc deprotection agent to remove Fmoc protecting group, wash the resin with solvent, put in activated amino acid with protecting group, and under the action of coupling system, carry out amino acid coupling and washing in sequence from carboxyl end to amino end according to peptide sequence to obtain peptide resin;

[0012] S2. Solid phase synthesis of the first pair of disulfide bonds: oxidizing the linear peptide resin obtained in step S1 with an oxidant to form the first pair of disulfide bonds, thereby obtaining a cono peptide analog precursor resin containing a single disulfide bond;

[0013] S3. Preparation of crude single disulfide bond peptide: using a cleavage solution to cut the resin in the single disulfide bond-containing cono peptide analog precursor resin obtained in step S2;

[0014] S4. Liquid phase synthesis of a second pair of disulfide bonds: oxidizing the single disulfide bond crude peptide obtained in step S3 with an oxidant to form a second pair of disulfide bonds to obtain a double disulfide bond crude peptide;

[0015] S5. Purification: Use a preparative reverse-phase high performance liquid phase device to perform gradient preparation and purification, collect components with a purity of ≥99%, concentrate, and lyophilize to obtain μ-conopeptide analogs.

[0016] As a further improvement of the present invention, the resin substitution degree in step S1 is controlled at 0.5-0.8.

[0017] As a further improvement of the present invention, the activated amino acid with a protecting group in step S1 is Fmoc-Ala-OH, Fmoc-Cys(Acm)-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Ala-OH, Fmoc-His(Trt)-OH, Fmoc-Asp(OtBu)-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Cys(Trt)-OH, Fmoc-Trp(Boc)-OH, Fmoc-Lys(Trt)-OH, s(Boc)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Ala-OH, Fmoc-Gly-OH, Fmoc-Lys(Boc)-OH, Fmoc-P ro-OH, Fmoc-Gly-OH, Fmoc-Asn(Trt)-OH, Fmoc-Cys(Acm)-OH, Fmoc-Cys(Trt)-OH, Fmoc-Gly-OH, Boc-Pyr-OH.

[0018] As a further improvement of the present invention, the oxidant in step S2 is selected from at least one of H2O2 and N-chlorosuccinimide.

[0019] As a further improvement of the present invention, step S3 specifically comprises: preparing a lysate, adding a peptide resin, carrying out a cleavage reaction, concentrating the lysate after the reaction, vacuum filtering the lysate resin with a sand core filter funnel, washing the resin with trifluoroacetic acid, draining, slowly adding the filtered lysate to isopropyl ether, precipitating the polypeptide, and centrifuging to separate the solid. Then, washing the solid precipitate with isopropyl ether, centrifuging to separate the solid, blowing the solid precipitate dry with nitrogen, and vacuum drying to obtain a crude monodisulfide bond peptide.

[0020] As a further improvement of the present invention, the oxidant in step S4 is a methanol solution of iodine.

[0021] As a further improvement of the present invention, the mobile phase A used in the reverse phase high performance liquid phase equipment in step S5 is prepared pure acetonitrile, and the mobile phase B is a pure aqueous solution of 0.1% trifluoroacetic acid.

[0022] As a further improvement of the present invention, the chromatographic column in the reverse phase high performance liquid chromatography equipment in step S5 is Yuexu DAC-100.

[0023] The present invention has the following beneficial effects: the present invention provides a method for preparing a μ-conopeptide analog, adopts a Cys cross-protecting group strategy, solid phase oxidation forms a first pair of disulfide bonds, liquid phase oxidation forms a second pair of disulfide bonds, and constructs two correct pairs of disulfide bonds. The operation is simple, the cost is low, and it is suitable for industrial mass production. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative labor.

[0025] Figure 1 This is the HPLC spectrum of the target compound μ-conopeptide analog. DETAILED DESCRIPTION

[0026] The technical solutions in the embodiments of the present invention are described clearly and completely below. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0027] RinkAM resin: starting resin for peptide synthesis; HOBt: 1-hydroxybenzotriazole; DIC: diisopropylcarbodiimide; DMF: N,N-dimethylformamide; DCM: dichloromethane; Piperidine: piperidine; TFA: trifluoroacetic acid; Tis: triisopropylsilane; NCS: N-chlorosuccinimide; Cys: cysteine; Arg: arginine; Ala: alanine; His: histidine; A sp: aspartic acid; Trp: tryptophan; Ser: serine; Lys: lysine; Gly: glycine; Pro: proline; Asn: asparagine; Pyr: pyroglutamic acid; Fmoc: fluorenylmethoxycarbonyl; Boc: tert-butoxycarbonyl; Trt: trityl; Acm: acrylate; Pbf: 2,2,4,6,7-pentamethyldihydrobenzofuran-5-sulfonyl; tBu: tert-butyl; OtBu: tert-butoxy.

[0028] Example 1

[0029] 1. Preparation of Boc-Pyr-Gly-Cys(Trt)-Cys(Trt)-Asn(Trt)-Gly-Pro-Lys(Boc)-Gly-Ala-Ser(tBu)-Ser(tBu)-Lys(Boc)-Trp(Boc)-Cys(Trt)-Arg(Pbf)-Asp(OtBu)-His(Trt)-Ala-Arg(Pbf)-Cys(Trt)-Ala-Peptide Resin

[0030] S1. Swelling of the starting resin

[0031] Add 100g of RinkAM resin with a substitution degree of 0.65 into the solid phase synthesis column, add 500ml DMF to completely immerse the resin, then introduce nitrogen, start stirring at 100r / min, stir for 5min, and then drain the solvent for 5min. Add another 500ml DMF, completely immerse the resin, then introduce nitrogen, start stirring at 100r / min, stir for 30min, and drain the solvent. Continue to add 500ml DMF, completely immerse the resin, then introduce nitrogen, start stirring at 100r / min, and drain the solvent for 5min to complete the swelling.

[0032] S2. Removal of Fmoc

[0033] Add 500 ml of 20% piperidine / DMF, introduce nitrogen, stir for 5 minutes, and drain for 5 minutes. Add another 500 ml of 20% piperidine / DMF, introduce nitrogen, stir for 10 minutes, and drain for 3 minutes. Then add 500 ml of DMF, introduce nitrogen, stir for 1 minute, and drain for 3 minutes. Repeat the resin washing 6 times.

[0034] S3. Feeding reaction

[0035] Add the weighed amino acids and HOBt to a 2L conical flask, add 500ml DMF, shake for 2 minutes to dissolve, and put it in a ±5℃ refrigerator for refrigeration. After 10 minutes, take out the conical flask from the refrigerator and add DIC, shake for 10 minutes. Add the mixed activation solution to the reaction column, introduce nitrogen and stir, and time the reaction for 2 hours. Control the temperature at around 25℃.

[0036] S4. Washing the resin after the reaction

[0037] Add 500ml DMF, introduce nitrogen, start stirring, mix the resin and solvent evenly, stir for 1min, and drain for 3min. Wash the resin repeatedly with 500ml DMF and drain the solvent until the filtered solvent is clear and transparent.

[0038] S5. Sequential coupling of amino acids

[0039] Repeat steps S2-S4 to sequentially couple Fmoc-Ala-OH, Fmoc-Cys(Acm)-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Ala-OH, Fmoc-His(Trt)-OH, Fmoc-Asp(OtBu)-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Cys(Trt)-OH, Fmoc-Trp(Boc)-OH, Fmoc-Lys(Boc)-OH , Fmoc-Ser(tBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Ala-OH, Fmoc-Gly-OH, Fmoc-Lys(Boc)-OH, Fmoc-Pro-OH , Fmoc-Gly-OH, Fmoc-Asn(Trt)-OH, Fmoc-Cys(Acm)-OH, Fmoc-Cys(Trt)-OH, Fmoc-Gly-OH, Boc-Pyr-OH.

[0040] S6. Shrinkage and drying of resin

[0041] Add 700ml DCM, start stirring, pass nitrogen, time 3min, and drain for 3min. Add 700ml methanol, start stirring, pass nitrogen, time 5min, and drain for 3min. Add 700ml methanol again, start stirring, pass nitrogen, time 5min, and drain for 3min. Pour the resin onto a tray to dry, grind it to make it into quicksand, then weigh, seal and refrigerate to obtain Boc-Pyr-Gly-Cys(Trt)-Cys(Acm)-Asn(Trt)-Gly-Pro-Lys(Boc)-Gly-Ala-Ser(tBu)-Ser(tBu)-Lys(Boc)-Trp(Boc)-Cys(Trt)-Arg(Pbf)-Asp(OtBu)-His(Trt)-Ala-Arg(Pbf)-Cys(Acm)-Ala-linear peptide resin.

[0042] 2. Preparation of Boc-Pyr-Gly-Cys-Cys(Acm)-Asn(Trt)-Gly-Pro-Lys(Boc)-Gly-Ala-Ser(tBu)-Ser(tBu)-Lys(Boc)-Trp(Boc)-Cys-Arg(Pbf)-Asp(OtBu)-His(Trt)-Ala-Arg(Pbf)-Cys(Ac m)-Ala-peptide resin (Cys3-Cys15 disulfide bond) single disulfide bond peptide resin.

[0043] 102.5g of the linear peptide resin obtained above was swollen with 1L DMF solution for 1 hour, the solution was removed under reduced pressure, and washed twice with DCM (500ml / time). The resin was washed with 250ml of 2% TFA / DCM (v / v) solution for 2 minutes each time until the color of the resin changed from red to colorless, then washed twice with DCM (500ml / time), washed twice with DMF (500ml / time), and the solution was removed under reduced pressure. 1L DMF was added, and then NCS (3 times equivalent) was added. After reacting for half an hour, the solution was removed under reduced pressure, washed 3 times with DMF (500ml / time), 500ml of methanol was added to shrink for 30 minutes, methanol was removed, 500ml of DCM was added to wash for 30 minutes, DCM was removed, and vacuum dried to obtain 92.5g of resin.

[0044] 3. Preparation of crude single disulfide bond peptide H-Pyr-Gly-Cys-Cys-Asn-Gly-Pro-Lys-Gly-Ala-Ser-Ser-Lys-Trp-Cys-Arg-Asp-His-Ala-Arg-Cys-Ala-NH2 (Cys3-Cys15 disulfide bond)

[0045] Add 92.5 g of the peptide resin obtained above to a 2L three-necked flask, add 900 ml of pre-prepared TFA: H2O: Tis = 90: 5: 5 (V: V), react at room temperature for 2 hours, filter the resin under reduced pressure, and collect the filtrate. Wash the resin with a small amount of TFA, and combine the filtrate. Slowly add the filtrate to 10L of ice isopropyl ether for precipitation, centrifuge, wash 3 times with ice isopropyl ether (5L / time), and dry under reduced pressure to obtain 32.5 g of a single disulfide bond crude peptide.

[0046] 4. Preparation of H-Pyr-Gly-Cys-Cys-Asn-Gly-Pro-Lys-Gly-Ala-Ser-Ser-Lys-Trp-Cys-Arg-Asp-His-Ala-Arg-Cys-Ala-NH2 (disulfide bonds located at Cys3-Cys15 and Cys4-Cys21)

[0047] 32.5 g of the crude monodisulfide peptide obtained above was dissolved in 500 ml of a 50% acetic acid / water (v / v) solution, and 0.1 mol of I2 / MeOH (15 equivalents) solution was added dropwise. After the addition was complete, the reaction mixture was stirred for 30 minutes, and saturated ascorbic acid was added dropwise until the solution was clear. The mixture was directly loaded onto a preparative column for purification.

[0048] 5. Purification of crude μ-conopeptide analogs

[0049] (I) Pretreatment

[0050] The crude μ-conopeptide buffer was filtered through an aqueous filter membrane with a pore size of 0.80 μm, and then filtered through an aqueous filter membrane with a pore size of 0.45 μm.

[0051] (II) Purification and preparation

[0052] The preparation and purification were carried out by high performance liquid chromatography.

[0053] Mobile phase A: prepared pure acetonitrile; Mobile phase B: 0.1% trifluoroacetic acid in pure water;

[0054] Chromatographic column: Yuexu DAC-100;

[0055] Prepare the gradient (Table 1):

[0056] Table 1

[0057] Time (min) Flow rate (mL / min) Mobile phase A (%) Mobile phase B (%) 0 200 3 97 5 200 5 95 16 200 20 80 21 200 30 70 31 200 30 70 34 200 5 95 41 200 3 97

[0058] (1) Collecting components with a purity of 99%, about 90%, and about 70% in stages;

[0059] (2) recovering and repurifying the 70% purity collected in step (1), and collecting the components with a purity of 90% and about 70% in stages;

[0060] (3) recovering and repurifying the 90% purity collected in step (1) and step (2), and collecting components with a purity of 99%, about 90%, and about 70% in stages;

[0061] (4) recovering and repurifying the 70% purity collected in step (2) and step (3), and collecting the components with a purity of 90% and about 70% in stages;

[0062] (5) recovering and repurifying the 90% purity collected in step (3) and step (4), and collecting components with a purity of 99%, about 90%, and about 70% in stages;

[0063] (6) Recover and purify the 90% purity fraction in step (5) to collect a fraction with a purity of 99%.

[0064] (III) Pure concentrated freeze-dried

[0065] The pure product solution was added to a 20L rotary evaporator, set at 35°C and 60r / min, and concentrated by rotary evaporation to remove the organic solvent and water in the solution. After concentrating to a volume of ≤1L, it was transferred to a 2L round-bottom bottle. Freeze-dried to obtain a white powdery solid, that is, the pure μ-cono peptide analog was obtained, such as Figure 1 , total yield 30%, purity 99%.

[0066] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A μ-conopeptide analogue, characterized in that: The μ-conopeptide analog is C-terminally amidated and has the following sequence: H-Pyr-Gly-Cys-Cys-Asn-Gly-Pro-Lys-Gly-Ala-Ser-Ser-Lys-Trp-Cys-Arg-Asp-His-Ala-Arg-Cys-Ala-NH2, and has two pairs of disulfide bonds located at positions Cys3-Cys15 and Cys4-Cys21.

2. A method for preparing the μ-conopeptide analogue according to claim 1, characterized in that: The following steps are involved: S1. Preparation of linear peptide resin: According to the peptide sequence, select Fmoc amino resin as the carrier for solid phase synthesis; put in the resin to swell, react with Fmoc deprotection agent to remove Fmoc protecting group, wash the resin with solvent, put in activated amino acid with protecting group, and under the action of coupling system, carry out amino acid coupling and washing in sequence from carboxyl end to amino end according to peptide sequence to obtain peptide resin; S2. Solid phase synthesis of the first pair of disulfide bonds: oxidizing the linear peptide resin obtained in step S1 with an oxidant to form the first pair of disulfide bonds, thereby obtaining a cono peptide analog precursor resin containing a single disulfide bond; S3. Preparation of crude single disulfide bond peptide: using a cleavage solution to cut the resin in the single disulfide bond-containing cono peptide analog precursor resin obtained in step S2; S4. Liquid phase synthesis of a second pair of disulfide bonds: oxidizing the single disulfide bond crude peptide obtained in step S3 with an oxidant to form a second pair of disulfide bonds to obtain a double disulfide bond crude peptide; S5. Purification: Use a preparative reverse-phase high performance liquid phase device to perform gradient preparation and purification, collect components with a purity of ≥99%, concentrate, and lyophilize to obtain μ-conopeptide analogs.

3. The preparation method according to claim 2, characterized in that: The resin substitution degree in step S1 is controlled at 0.5-0.

8.

4. The preparation method according to claim 2, characterized in that: The activated amino acid with a protecting group in step S1 is Fmoc-Ala-OH, Fmoc-Cys(Acm)-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Ala-OH, Fmoc-His(Trt)-OH, Fmoc-Asp(OtBu)-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Cys(Trt)-OH, Fmoc-Trp(Boc)-OH, Fmoc-Lys(Boc) -OH, Fmoc-Ser(tBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Ala-OH, Fmoc-Gly-OH, Fmoc-Lys(Boc)-OH, Fmoc-Pro- OH, Fmoc-Gly-OH, Fmoc-Asn(Trt)-OH, Fmoc-Cys(Acm)-OH, Fmoc-Cys(Trt)-OH, Fmoc-Gly-OH, Boc-Pyr-OH.

5. The preparation method according to claim 2, characterized in that: The oxidant described in step S2 is selected from at least one of H2O2 and N-chlorosuccinimide.

6. The preparation method according to claim 2, characterized in that: Step S3 specifically includes: preparing a lysate, adding a peptide resin, and performing a cleavage reaction. After the reaction, the lysate is concentrated, the lysate resin is vacuum filtered using a sand core filter funnel, the resin is washed with trifluoroacetic acid, and dried, the filtered lysate is slowly added to isopropyl ether, the polypeptide is precipitated, and the solid is separated by centrifugation. The solid precipitate is then washed with isopropyl ether, the solid is separated by centrifugation, the solid precipitate is dried with nitrogen, and vacuum dried to obtain a single disulfide bond crude peptide.

7. The preparation method according to claim 2, characterized in that: The oxidant in step S4 is a methanol solution of iodine.

8. The preparation method according to claim 2, characterized in that: The mobile phase A used in the reverse phase high performance liquid phase apparatus in step S5 is prepared pure acetonitrile, and the mobile phase B is a pure aqueous solution of 0.1% trifluoroacetic acid.

9. The preparation method according to claim 2, characterized in that: The chromatographic column in the reverse phase high performance liquid chromatography equipment in step S5 is Yuexu DAC-100.

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