Preparation method of itecatide

Abstract

The invention relates to a preparation method of Itecartide, and belongs to the technical field of biology. According to the method, by adjusting the cracking temperature, impurities generated by acetyl removal through hydrolysis of one-site [Ac-D-Cys] 1 are effectively reduced (the retention time of the impurities is very close to that of a main peak in HPLC (High Performance Liquid Chromatography), and the purification efficiency is very influenced); in addition, one-site [Ac-D-Cys] 1 sulfydryl modification is firstly carried out, then Pbf protecting group removal is carried out, and a reaction endpoint is monitored through an Ellman reagent, so that the generation of sulfonated impurities can be reduced. In addition, different solvents are used for washing for multiple times, so that the generation of homodimers can be effectively controlled, and the product yield can be improved. In conclusion, according to the preparation method, acetylation impurities, sulfonation impurities and homodimers are effectively reduced, the difficulty of purifying the impurities is reduced, and the preparation method has important application value in industrial production.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, in particular to a method for preparing eterkatide. Background Art

[0002] Etelcalcetide is primarily used to treat secondary hyperparathyroidism (SHPT). For adult chronic kidney disease (CKD) patients undergoing hemodialysis, etelcalcetide can inhibit PTH synthesis by regulating the calcium-sensing receptor in the parathyroid gland, increasing extracellular calcium sensitivity. It can also upregulate the transcription and translation of parathyroid calcium-sensing receptor mRNA and vitamin D receptor mRNA, thereby inhibiting PTH synthesis.

[0003] The peptide sequence of eterkatide is: Ac-D-Cys 1 -D-Ala 2 -D-Arg 3 -D-Arg 4 -D-Arg 5 -D-Ala 6 -D-Arg 7 -NH2. The main chain D-Cys1 side chain thiol group is connected to the L-Cys side chain thiol group in the form of a disulfide bond.

[0004] The preparation method disclosed in the patent publication number CN115490756A is that the peptide resin and H-Cys(S-Py)-OH are added together to a high concentration of TFA lysis solution to react, and the side chain protecting group pbf of D-Arg is cleaved to form a positive ion, which is easily 1 The thiol group captures the D-Cys 1 Sulfonated impurities. In addition, the exposed amino groups after the main chain peptide resin is cleaved also have the potential to react with H-Cys(S-Py)-OH.

[0005] The preparation method disclosed in the patent publication number CN108218957A is to remove a D-Cys 1 When the side chain protecting group and the resin are cut, due to the long reaction time required, some of the side chain protecting groups pbf of D-Arg will be cleaved, and D-Cys 1 Sulfonated impurities, similarly, the exposed amino groups after cleavage of the peptide resin also have the potential to react with H-Cys(S-Py)-OH.

[0006] In the preparation method disclosed in the patent publication number CN106795201A, [D-Cys(S-Py)] 1The side chain is not stable enough in strong acid and may break off, preventing the reaction from proceeding and affecting the yield. It also reacts with the cation formed by the cleaved D-Arg side chain protecting group pbf to produce a series of impurities.

[0007] In summary, several currently disclosed solid-liquid combined methods for preparing eterkatide will form acetylated impurities and sulfonated impurities that are difficult to separate and have low yields. Therefore, a method for preparing eterkatide with high yield and easy separation of impurities is urgently needed. Summary of the Invention

[0008] Therefore, the technical problem to be solved by the present invention is to overcome the problems of low yield and difficulty in separating impurities in the prior art preparation method of eterkatide.

[0009] To solve the above technical problems, the present invention provides a method for preparing eterkadide. The present invention uses amino resin as a solid phase carrier and gradually couples to prepare the main chain peptide resin: Ac-D-Cys(Mmt)-D-Ala-D-Arg(Pbf)-D-Arg(Pbf)-D-Arg(Pbf)-D-Ala-D-Arg(Pbf)-resin. Then, one D-Cys is removed in the solid phase system. 1 The side chain is protected by Mmt, and Boc-Cys(S-Py)-OH is added to react to obtain etelkatide peptide resin. Etelkatide is obtained through cleavage, purification, salt conversion, lyophilization and other steps. Specifically, the side chain protecting group pbf of D-Arg will be cleaved to form a positive ion, which is easily absorbed by D-Cys 1 To solve the problem of sulfhydryl capture and formation of sulfonated impurities, the present invention firstly 1 The side chain was deprotected by Mmt treatment and ligated with Boc-Cys(S-Py)-OH to avoid D-Cys 1 The sulfhydryl group of D-Arg is exposed, and then the side chain protecting group pbf of D-Arg is removed, thereby avoiding the 1 The exposed side chain thiol groups react with PBF, producing difficult-to-separate sulfonated impurities. Furthermore, the present invention controls the pyrolysis temperature to reduce the generation of difficult-to-separate deacetylated impurities. Furthermore, increasing the number of washes with different solvents reduces the generation of homodimer impurities. As a result, the eterkatide of the present invention achieves a purity of 90.66% without producing difficult-to-separate deacetylated, sulfonated, or homodimer impurities.

[0010] The first object of the present invention is to provide a method for preparing eterkatide, comprising the following steps:

[0011] S1. Fmoc-D-Ala-OH, Fmoc-D-Arg(R)-OH, and Fmoc-D-Cys(X)-OH are mixed with a deprotecting agent to obtain D-Ala-OH, D-Arg(R)-OH, and D-Cys(X)-OH from which Fmoc protection has been removed. In the presence of a condensing agent, D-Ala-OH, D-Arg(R)-OH, and D-Cys(X)-OH are linked to an amino resin according to the peptide sequence from the C-terminus to the N-terminus of the eterkatide backbone to obtain a first eterkatide resin, wherein X is selected from a Trt, Mmt, or tBu protecting group, and R includes a Pbf protecting group;

[0012] S2, adding a mixed solution of trifluoroacetic acid and dichloromethane to the first eterkatide resin for deprotection treatment and washing to obtain a second eterkatide resin;

[0013] S3, adding Boc-Cys(S-Py)-OH to the second etelkatide resin prepared in S2 in the presence of a catalyst to react to obtain a thiol-modified third etelkatide resin;

[0014] S4, performing a cleavage reaction on the third eterkatide resin, and obtaining eterkatide after purification, from which the R and the amino resin are removed;

[0015] Wherein, the temperature of the cracking reaction in step S4 is 15-25°C.

[0016] Furthermore, the deprotecting agent is a mixed solution of piperidine and DMF.

[0017] Furthermore, Fmoc-D-Cys(X)-OH is preferably Fmoc-D-Cys(Mmt)-OH (ie, X is an Mmt protecting group) whose side chain is less acid-resistant.

[0018] Furthermore, the amino resin includes Rink Amide-AM Resin, wherein the resin substitution degree is 0.5 mmol / g.

[0019] Further, in step S1, in the presence of a condensing agent and a racemization protecting agent, D-Cys(X)-OH, D-Ala-OH, D-Arg(R)-OH, D-Arg(R)-OH, D-Arg(R)-OH, and D-Ala-OH are sequentially coupled to an amino resin to obtain the eterkatide resin.

[0020] Further, the condensing agent is selected from one or more of dicyclohexylcarbodiimide (DCC), N,N'-diisopropylcarbodiimide (DIC) and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide, and the racemization protecting agent is selected from one or more of 1-hydroxybenzotriazole (HOBt), 4-dimethylaminopyridine and benzotriazole-1-yloxytris (dimethylamino) phosphoric triamide. DCC (dicyclohexylcarbodiimide) easily forms O-isourea intermediate in the reaction, and this intermediate is easily converted into racemic oxazolone. In order to prevent this situation, HOBt (1-hydroxybenzotriazole) can be added to react with it to form another intermediate, and the aminolysis speed of this intermediate is faster, thereby effectively suppressing the formation of oxazolone active intermediate.

[0021] DMAP (4-dimethylaminopyridine) is a commonly used catalyst that accelerates amide bond formation while reducing racemization. It is often used with DCC to form the DCC / DMAP system, which is widely used in peptide synthesis and other organic syntheses.

[0022] TBTU (benzotriazol-1-yloxytris(dimethylamino)phosphoric triamide) is a highly effective condensation reagent that promotes amide bond formation under mild conditions with minimal racemization. It is often used with DIPEA (diisopropylethylamine) to form a TBTU / DIPEA system, which is suitable for condensation reactions of various amino acids.

[0023] Furthermore, the first etelkatide resin is acetylated by adding acetic anhydride and pyridine to the first etelkatide resin. The main reason for acetylation of the etelkatide resin is to block the N-terminus of the polypeptide to prevent degradation and enhance its stability.

[0024] Furthermore, in step S2, the deprotection treatment is to add a mixed solution of trifluoroacetic acid as a solute and dichloromethane as a solvent to the first eterkatide resin for reaction, wherein the concentration of the mixed solution is 5%-20%, the number of deprotection treatments is 4-5 times, and the time of each deprotection treatment is 4-5 minutes.

[0025] Furthermore, in step S2, the washing is to add DCM solution to the first eterkatide resin after deprotection treatment, wash it three times, then add a mixed solution of 10%-20% N,N-diisopropylethylamine and dichloromethane to wash for 1-2 minutes, repeat 2-5 times, and finally add N,N-dimethylformamide to wash the resin 3-6 times.

[0026] Furthermore, in step S2, the Ellman reagent is used to monitor the reaction progress during the deprotection treatment. When the Ellman reagent changes color, the deprotection treatment is completed.1 The reaction endpoint was monitored by detecting the exposed thiol groups using Ellman's reagent before and after side chain Mmt protection and before and after docking Boc-Cys(S-Py)-OH. The use of Ellman's reagent is more efficient than the commonly used HPLC method. 1 By changing whether the side chain thiol group is exposed or not and introducing a special amino acid into the side chain, the reaction endpoint can be displayed by the Ellman reagent colorimetric reaction during solid-phase reaction control without relying on HPLC, thus simplifying the detection of the reaction endpoint.

[0027] Furthermore, the catalyst in step S3 includes DIPEA.

[0028] Furthermore, the cleavage reaction in step S4 is performed by adding a cleavage solution to eterkatide, wherein the cleavage solution is a mixture of trifluoroacetic acid, triisopropylsilane, and water, and the volume ratio of trifluoroacetic acid, triisopropylsilane, and water is (90-96): (2-5): (2-5). The trifluoroacetic acid in the cleavage solution can remove the Pbf protecting group from the peptide chain.

[0029] Beneficial effects of the present invention:

[0030] The present invention effectively reduces one [Ac-D-Cys] by adjusting the pyrolysis temperature. 1 The impurities generated by the removal of acetyl groups due to hydrolysis (the retention time of these impurities in HPLC is very close to that of the main peak, which greatly affects the purification efficiency); In addition, the present invention first performs a [Ac-D-Cys] 1 The present invention is a sulfhydryl modification, followed by removal of the Pbf protecting group, to reduce the generation of sulfonated impurities. In addition, the present invention utilizes different solvents to carry out multiple washings, which can effectively control the generation of homodimers to improve product yield. Therefore, the present invention effectively reduces the generation of acetylated impurities, sulfonated impurities and homodimers, reduces the difficulty of purifying impurities, and has important application value in industrial production. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] In order to make the content of the present invention more clearly understood, the present invention is further described in detail below based on specific embodiments of the present invention in conjunction with the accompanying drawings, wherein

[0032] Figure 1 It is the main flow chart of the present invention;

[0033] Figure 2 is the HPLC chromatogram of the crude eterkatide peptide prepared in Example 2;

[0034] Figure 3 is the HPLC chromatogram of the crude eterkatide peptide prepared in Example 3;

[0035] Figure 4 This is the HPLC chromatogram of the crude eterkatide peptide prepared in Comparative Example 1;

[0036] Figure 5 It is the HPLC chromatogram of the crude eterkatide peptide prepared in Comparative Example 2. DETAILED DESCRIPTION

[0037] The present invention will be further described below with reference to the accompanying drawings and specific embodiments so that those skilled in the art can better understand the present invention and implement it. However, the embodiments are not intended to limit the present invention.

[0038] Example 1: Synthesis of eterkatide resin

[0039] (1) Preparation of Deprotected Amino Resin: 384.6 g of Rink Amide AM resin (200 mmol, degree of substitution 0.52 mmol / g) was weighed and added to DMF, stirring and swelling for 30 min. The Fmoc protection was then removed with a 20% piperidine / DMF solution. After deprotection was complete, the liquid was removed and the resin was washed five times with DMF. A Kaiser test indicated that the resin was blue, indicating complete deprotection.

[0040] (2) Preparation of deprotected amino acid: Weigh Fmco-D-Arg(pbf)-OH (3eq, 389.3g) and HOBt (3.6eq, 97.3g), dissolve in 3L DMF, then add DIC (3.6eq, 111.3mL), stir and activate, then add to the deprotected amino resin, and stir to react. During the reaction, take a small amount of resin for ninhydrin detection. The resin is transparent and colorless, and the condensation reaction is complete. Wash the resin 3 times with DMF. Remove Fmoc protection with 20% piperidine / DMF solution (the deprotection treatment of Fmoc-D-Ala-OH and Fmoc-D-Cys(Mmt)-OH is similar to that of Fmco-D-Arg(pbf)-OH). After the deprotection is completed, remove the liquid and wash with DMF 5 times. Ninhydrin detection shows that the resin is blue, indicating complete deprotection.

[0041] (3) Peptide chain condensation: Fmoc-D-Ala-OH, Fmoc-D-Arg(Pbf)-OH, Fmoc-D-Arg(Pbf)-OH, Fmoc-D-Arg(Pbf)-OH, Fmoc-D-Ala-OH, and Fmoc-D-Cys(Mmt)-OH were sequentially condensed from the C-terminus to the N-terminus according to the peptide sequence, and the Fmoc protection was removed.

[0042] (4) Acetylation of the first eterka peptide resin: 3 L of DMF was used as the solvent. Acetic anhydride (5 eq, 94.0 ml) and pyridine (5 eq, 80.7 ml) were added and stirred. The mixture was then added to the peptide resin and reacted at room temperature for 30 minutes. The resin was washed five times with DMF. A sample was taken for ninhydrin analysis, and the resin was colorless and transparent. The resin was washed three times with 3 L of anhydrous methanol for 10 minutes each time. The resin was vacuum dried at room temperature for 15 hours to obtain 871.9 g of the main chain peptide resin.

[0043] Example 2: Synthesis of etercalcetide

[0044] Weigh 21.8 g (5 mmol) of the main-chain peptide resin obtained in Example 1. Add DMF and stir to swell for 30 min. Prepare 100 mL of 10% trifluoroacetic acid / DCM solution, add it to the peptide resin, stir and react for 5 min, filter the reaction solution, and repeat this process 5 times. Wash with DCM 3 times. Prepare 10% DIPEA / DCM solution and wash the resin twice, each for 2 min. Wash with DMF 5 times. Take a small amount of resin and add Ellman's reagent for detection; the resin turns purple-red. Add Boc-Cys(S-Py)-OH (3 eq, 5.0 g) to 100 mL of DMF and dissolve it. Then add 5.2 mL of DIPEA (6 eq). Stir and add to the peptide resin to react. During the reaction, take a small amount of resin and add Ellman's reagent for detection; the resin is transparent and colorless, indicating the end point of the reaction. Wash with DMF 5 times, wash with anhydrous methanol 3 times, and vacuum dry to obtain 21.6 g of etka peptide resin.

[0045] Prepare 172.8mL of lysis solution (TFA:TIS:water=95:2.5:2.5). The volume of lysis solution is 8 times the weight of the resin (8mL / g). After cooling to 2-8℃, add it to the peptide resin and stir the reaction for 3 hours. The temperature is controlled at 25℃. Filter, add the filtrate to 1036.8mL of methyl tert-butyl ether (6mL / mL lysis solution, about 0℃), centrifuge, wash 3 times with methyl tert-butyl ether, dry the wet product under vacuum at room temperature for 12 hours, and weigh 5.2g of crude peptide. The purity detected by HPLC is 90.66%, and the homodimer is 1.15%. The HPLC chromatogram is as follows Figure 2 shown.

[0046] Example 3

[0047] Weigh 21.8 g (5 mmol) of the main-chain peptide resin obtained in Example 1. Add DMF and stir to swell for 30 min. Prepare 100 mL of 10% trifluoroacetic acid / DCM solution, add it to the peptide resin, stir and react for 5 min, filter the reaction solution, and repeat this process 5 times. Wash with DCM 3 times. Prepare 10% DIPEA / DCM solution and wash the resin twice, each for 2 min. Wash with DMF 5 times. Take a small amount of resin and add Ellman's reagent for detection; the resin turns purple-red. Add Boc-Cys(S-Py)-OH (3 eq, 5.0 g) to 100 mL of DMF and dissolve it. Then add 5.2 mL of DIPEA (6 eq). Stir and add to the peptide resin to react. During the reaction, take a small amount of resin and add Ellman's reagent for detection; the resin is transparent and colorless, indicating the end point of the reaction. Wash with DMF 5 times, wash with anhydrous methanol 3 times, and vacuum dry to obtain 21.6 g of etka peptide resin.

[0048] Prepare 172.8mL of lysis solution (TFA:TIS:water=95:2.5:2.5). The volume of the lysis solution is 8 times the weight of the resin (8mL / g). After cooling to 2-8°C, add it to the peptide resin, stir the reaction for 3 hours, and control the temperature at 15°C. Filter, add the filtrate to 1036.8mL of methyl tert-butyl ether (6mL / mL lysis solution, about 0°C), centrifuge, wash 3 times with methyl tert-butyl ether, dry the wet product under vacuum at room temperature for 12 hours, and weigh 5.2g of crude peptide. The purity detected by HPLC was 65.33%, and the homodimer was 1.26%. When the lysis temperature is low, it is not easy to produce dimer impurities, but a series of peaks with different amino acid side chain protecting groups are produced. These impurities are far away from the main peak of eterka peptide and are therefore easier to separate. The HPLC chromatogram is as follows Figure 3 shown.

[0049] Comparative Example 1

[0050] Weigh 21.8 g (5 mmol) of the main chain peptide resin obtained in Example 1. Add DMF and stir to swell for 30 minutes. Prepare 100 ml of 10% trifluoroacetic acid / DCM solution, add it to the peptide resin, stir and react for 5 minutes, filter out the reaction solution, and repeat the operation 5 times. Wash with DMF 5 times. Take a small amount of resin and add Ellman's reagent for detection. The resin turns purple-red. Add Boc-Cys(S-Py)-OH (3 eq, 5.0 g) to 100 ml of DMF and dissolve it, then add 5.2 ml of DIPEA (6 eq). Stir and add to the peptide resin to react. During the reaction, take a small amount of resin and add Ellman's reagent for detection. The resin is transparent and colorless, which is the end point of the reaction. Wash with DMF 5 times, wash with anhydrous methanol 3 times, and then vacuum dry to obtain 19.8 g of etka peptide resin.

[0051] Prepare 158.4 ml of lysis solution (TFA:TIS:water=95:2.5:2.5). The volume of the lysis solution is 8 times the weight of the resin (8 ml / g). After cooling to 2-8 ° C, add it to the peptide resin, stir and react for 3 hours, and control the temperature at 25 ° C. Filter, add the filtrate to 950.4 ml of methyl tert-butyl ether (6 ml / ml lysis solution, about 0 ° C), centrifuge, wash 3 times with methyl tert-butyl ether, dry the wet product in vacuum at room temperature for 12 hours, and weigh 4.8 g of crude peptide. HPLC detection purity is 63.94%, and homodimer is 6.65%. The HPLC chromatogram is as follows Figure 4 shown.

[0052] Example 2 added multiple washes with DCM and 10% DIPEA / DCM after Mmt removal. The crude peptide purity was 81.74% and 63.94%, respectively. The purity of Example 2 was significantly higher than that of Comparative Example 1. The homodimer impurity in the two experiments was 2.39% and 6.65%, respectively. Example 2 showed a significant reduction.

[0053] Comparative Example 2

[0054] Weigh 21.8 g (5 mmol) of the main-chain peptide resin obtained in Example 1. Add DMF and stir to swell for 30 min. Prepare 100 mL of 10% trifluoroacetic acid / DCM solution, add it to the peptide resin, stir and react for 5 min, filter the reaction solution, and repeat this process 5 times. Wash with DCM 3 times. Prepare 10% DIPEA / DCM solution and wash the resin twice, each for 2 min. Wash with DMF 5 times. Take a small amount of resin and add Ellman's reagent for detection; the resin turns purple-red. Add Boc-Cys(S-Py)-OH (3 eq, 5.0 g) to 100 mL of DMF to dissolve, then add 5.2 mL of DIPEA (6 eq). Stir and add to the peptide resin to react. During the reaction, take a small amount of resin and add Ellman's reagent for detection; the resin is transparent and colorless, indicating the end point of the reaction. Wash with DMF 5 times, wash with anhydrous methanol 3 times, and then vacuum dry to obtain 20.5 g of etka peptide resin.

[0055] Prepare 164.0mL of lysis solution (TFA:TIS:water=95:2.5:2.5). The volume of lysis solution is 8 times the weight of the resin (8mL / g). After cooling to 2-8°C, add it to the peptide resin and stir the reaction for 3 hours. The temperature is controlled at 35°C. Filter, add the filtrate to 984mL of methyl tert-butyl ether (6mL / mL lysis solution, about 0°C), centrifuge, wash 3 times with methyl tert-butyl ether, dry the wet product under vacuum at room temperature for 12 hours, and weigh 5.0g of crude peptide. HPLC detection purity is 58.36%, and deacetylated impurities are 15.02%. The HPLC chromatogram is as follows Figure 5 shown.

[0056] Comparing Example 2 with Comparative Example 2, Example 2 maintained the cleavage temperature at 25°C, while Comparative Example 2 reached 35°C. The crude peptide purities were 90.66% and 58.36%, respectively. The purity of Example 2 was significantly higher than that of Comparative Example 2. Comparative Example 2 produced a significant amount of deacetylated impurities, accounting for 15.02%. This was not the case in Example 2.

[0057] Obviously, the above embodiments are merely examples for clarity of explanation and are not intended to limit the implementation methods. Those skilled in the art will appreciate that other variations or modifications can be made based on the above description. It is not necessary and impossible to enumerate all implementation methods here. Obvious variations or modifications arising therefrom remain within the scope of protection of the present invention.

Claims

1. A method for preparing eterkatide, characterized in that: The following steps are involved: S1. Fmoc-D-Ala-OH, Fmoc-D-Arg(R)-OH, and Fmoc-D-Cys(X)-OH are respectively mixed with a deprotecting agent to obtain D-Ala-OH, D-Arg(R)-OH, and D-Cys(X)-OH from which Fmoc protection has been removed; D-Ala-OH, D-Arg(R)-OH, and D-Cys(X)-OH are connected to an amino resin in the presence of a condensing agent according to the peptide sequence from the C-terminus to the N-terminus of the etecarotene backbone, and acetylated to obtain a first etecarotene resin, wherein X is selected from a Trt, Mmt, or tBu protecting group, and R includes a Pbf protecting group; S2, deprotecting and washing the first eterkatide resin to obtain a second eterkatide resin from which X is removed; S3, adding Boc-Cys(S-Py)-OH to the second etelkatide resin prepared in S2 in the presence of a catalyst to react to obtain a thiol-modified third etelkatide resin; S4, performing a cleavage reaction on the third eterkatide resin, and obtaining eterkatide after purification, from which the R and the amino resin are removed; Wherein, the temperature of the cracking reaction in step S4 is 15-25°C.

2. The preparation method according to claim 1, characterized in that In step S1, in the presence of a condensing agent and a racemization protecting agent, D-Cys(X)-OH, D-Ala-OH, D-Arg(R)-OH, D-Arg(R)-OH, D-Arg(R)-OH, and D-Ala-OH are sequentially coupled to an amino resin to obtain the eterkatide resin.

3. The preparation method according to claim 2, characterized in that The condensing agent is selected from one or more of dicyclohexylcarbodiimide, diisopropylcarbodiimide and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide, and the racemic protecting agent is selected from one or more of 1-hydroxybenzotriazole, 4-dimethylaminopyridine and benzotriazol-1-yloxytris(dimethylamino)phosphoric triamide.

4. The preparation method according to claim 1, characterized in that The amino resin includes Rink Amide-AM resin.

5. The preparation method according to claim 1, characterized in that The acetylation treatment in step S1 is performed by adding acetic anhydride and pyridine to the first etercalpeptide resin.

6. The preparation method according to claim 1, characterized in that In step S2, the deprotection treatment is to add a mixed solution of trifluoroacetic acid as a solute and dichloromethane as a solvent to the first eterkatide resin for reaction, wherein the concentration of the mixed solution is 5%-20%, the number of deprotection treatments is 4-5 times, and the time of each deprotection treatment is 4-5 minutes.

7. The preparation method according to claim 1, characterized in that In step S2, the washing is to add dichloromethane to the first eterkatide resin after the deprotection treatment and wash it three times, then add a mixed solution of 10%-20% N,N-diisopropylethylamine and dichloromethane to wash for 1-2 minutes, repeat 2-5 times, and finally add N,N-dimethylformamide to wash 3-6 times.

8. The preparation method according to claim 1, characterized in that In step S2, the deprotection treatment is performed using Ellman's reagent to monitor the reaction progress. When the Ellman's reagent changes color, the deprotection treatment is completed.

9. The preparation method according to claim 1, characterized in that The catalyst in step S3 includes N,N-diisopropylethylamine.

10. The preparation method according to claim 1, characterized in that The cleavage reaction in step S4 is to add a cleavage solution to etercaltide, wherein the cleavage solution is a mixture of trifluoroacetic acid, triisopropylsilane and water, and the volume ratio of trifluoroacetic acid, triisopropylsilane and water is (90-96): (2-5): (2-5).

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