Synthesis method of snake venom-like tetrapeptide

Abstract

The invention provides a synthetic method of snake venom-like tetrapeptide. Specifically, the invention provides a method for preparing the snake venom-like tetrapeptide H-beta-Ala-Pro-Dab-D-Tyr-NH-Bzl in a reaction mode of combining a liquid phase and a solid phase. According to the preparation method, racemization of Pro and Dab can be avoided, the process route is stable and simpler, and the high-purity snake venom-like tetrapeptide can be conveniently prepared at lower cost.

Description

Technical Field

[0001] The present invention relates to the field of polypeptide preparation, and particularly to a preparation method of a snake venom-like tetrapeptide H-β-Ala-Pro-Dab-D-Tyr-NH-Bzl. Background Art

[0002] H-β-Ala-Pro-Dab-NH-Bzl is a small molecule polypeptide (snake venom-like tripeptide) that mimics the activity of snake venom toxin Waglerin I. Clinical trials have shown that this polypeptide can inhibit the binding of acetylcholine to acetylcholine receptors, thereby inhibiting muscle contraction and smoothing skin wrinkles, and has excellent properties of smoothing the skin and quickly removing wrinkles. D-tyrosine can act on tyrosinase and regulate the production of melanin, thereby achieving the effect of whitening. Modifying D-tyrosine onto the above snake venom-like tripeptide can achieve both whitening and anti-wrinkle effects, and the structure of the obtained modified molecule is shown in Formula A below:

[0003]

[0004] Common chemical synthesis methods for polypeptide synthesis include liquid-phase synthesis and solid-phase synthesis. For small molecule polypeptides, generally polypeptides with less than 10 amino acids, the common method is to use liquid-phase synthesis. However, the liquid-phase synthesis method requires continuous coupling and hydrolysis deprotection, with cumbersome steps, lower yield and purity in the later process, and Pro and Dab are prone to racemization. Using solid-phase synthesis, the obtained crude peptide has a higher purity, but it is more difficult and costly. Completely using solid-phase synthesis or completely using liquid-phase synthesis will inevitably have the limitations of each method.

[0005] Therefore, there is an urgent need in the art to develop a synthesis method of snake venom-like tetrapeptides that can avoid racemization, has a stable process route and is simpler. Summary of the Invention

[0006] The purpose of the present invention is to provide a preparation method of a snake venom-like tetrapeptide H-β-Ala-Pro-Dab-D-Tyr-NH-Bzl that is superior to the prior art.

[0007] In the first aspect of the present invention, a preparation method of a snake venom-like tetrapeptide H-β-Ala-Pro-Dab-D-Tyr-NH-Bzl is provided, and the preparation method includes the steps of:

[0008] (S1) Provide Fmoc-D-Tyr(tBu)-2-CTC resin;

[0009] (S2) Use a deprotection reagent to deprotect the amino terminus of the Fmoc-D-Tyr(tBu)-2-CTC resin;

[0010] (S3) Using Fmoc-Dab(Boc)-OH, Fmoc-Pro-OH, and Boc-β-Ala-OH as raw materials, the activated amino acids are successively coupled with the deprotected resin to obtain Boc-β-Ala-Pro-Dab(Boc)-D-Tyr(tBu)-2-CTC resin;

[0011] (S4) Using a cleavage reagent, perform a cleavage reaction on the Boc-β-Ala-Pro-Dab(Boc)-D-Tyr(tBu)-2-CTC resin, and precipitate to obtain the crude peptide Boc-β-Ala-Pro-Dab(Boc)-D-Tyr(tBu)-OH;

[0012] (S5) Using an activation system, couple the crude peptide Boc-β-Ala-Pro-Dab(Boc)-D-Tyr(tBu)-OH with an activating reagent to obtain a crude peptide activated ester;

[0013] (S6) Using benzylamine as a raw material, couple the crude peptide activated ester with benzylamine to obtain Boc-β-Ala-Pro-Dab(Boc)-D-Tyr(tBu)-NH-Bzl; and

[0014] (S7) Using a deprotection reagent, deprotect the amino terminus of the Boc-β-Ala-Pro-Dab(Boc)-D-Tyr(tBu)-NH-Bzl to obtain the snake venom-like tetrapeptide H-β-Ala-Pro-Dab-D-Tyr-NH-Bzl.

[0015] In another preferred example, the step (S1) includes sub-steps:

[0016] (S1a) Provide 2-CTC resin; and

[0017] (S1b) Connect Fmoc-D-Tyr(tBu)-OH with 2-CTC resin to prepare Fmoc-D-Tyr(tBu)-2-CTC resin.

[0018] In another preferred example, the step (S1b) includes sub-steps:

[0019] (S1b1) Swell the 2-CTC resin with a solvent;

[0020] (S1b2) Dissolve Fmoc-D-Tyr(tBu)-OH in an organic solvent and add it to the swollen 2-CTC resin, and add an organic base to adjust the pH value, and react for a period of time; and

[0021] (S1b3) After the reaction is completed, it is blocked with MeOH to obtain Fmoc-D-Tyr(tBu)-2-CTC resin.

[0022] In another preferred example, the solvent is selected from the group consisting of: DMF, DCM, NMP, or a combination thereof; preferably, the solvent is selected from: DCM.

[0023] In another preferred example, the organic base is selected from the group consisting of: NMM, TEA, DIEA, or a combination thereof; preferably, the organic base is selected from: DIEA.

[0024] In another preferred example, in step (S1b2), the molar ratio of the 2-CTC to the Fmoc-D-Tyr(tBu)-OH is 2:1 - 1:5, preferably 1:1 - 1:3.

[0025] In another preferred example, the conditions of the reaction include stirring the reaction with nitrogen bubbling.

[0026] In another preferred example, the temperature of the reaction is 10 - 40 °C, preferably 20 - 30 °C.

[0027] In another preferred example, the period of time is 1 - 5 hours, preferably 1.5 - 3 hours, more preferably 2 hours.

[0028] In another preferred example, the blocking time is 10 - 20 min, preferably 15 min.

[0029] In another preferred example, the deprotection reagent is selected from the group consisting of: Piperazine / DMF, Piperidine / DMF, diethylamine / DMF, or a combination thereof; preferably, the deprotection reagent is selected from: Piperazine / DMF.

[0030] In another preferred example, the Piperazine / DMF is 5% Piperazine / DMF (mass ratio).

[0031] In another preferred example, step (S3) includes sub-steps:

[0032] (S3a) Using Fmoc-Dab(Boc)-OH as a raw material, the activated amino acid is coupled with the deprotected Fmoc-D-Tyr(tBu)-2-CTC resin to obtain Fmoc-Dab(Boc)-D-Tyr(tBu)-2-CTC resin;

[0033] (S3b) Using Fmoc-Pro-OH as a raw material, the activated amino acid is coupled with the deprotected Fmoc-Dab(Boc)-D-Tyr(tBu)-2-CTC resin to obtain Fmoc-Pro-Dab(Boc)-D-Tyr(tBu)-2-CTC resin; and

[0034] (S3c) Using Boc-β-Ala-OH as a raw material, the activated amino acid is coupled with the deprotected Fmoc-Pro-Dab(Boc)-D-Tyr(tBu)-2-CTC resin to obtain Boc-β-Ala-Pro-Dab(Boc)-D-Tyr(tBu)-2-CTC resin.

[0035] In another preferred example, the raw materials Fmoc-Dab(Boc)-OH, Fmoc-Pro-OH, and Boc-β-Ala-OH are dissolved in a solvent selected from the group consisting of DMF, DCM, NMP, or a combination thereof; preferably, the solvent is selected from: DMF.

[0036] In another preferred example, the coupling system is selected from the group consisting of DIC / HOBt, HBTU / DIEA, HATU / DIEA, DCC / HOBt, or a combination thereof; preferably, the coupling system is selected from: DIC / HOBt.

[0037] In another preferred example, when the coupling system is DIC / HOBt, the activation in step (S3a), (S3b), or (S3c) includes the steps of: respectively adding DIC to the dissolved Fmoc-Dab(Boc)-OH, Fmoc-Pro-OH, or Boc-β-Ala-OH to activate the amino acid.

[0038] In another preferred example, when the coupling system is DIC / HOBt, the coupling in step (S3a), (S3b), or (S3c) further includes the step of: adding HOBt.

[0039] In another preferred example, the reaction time of the coupling is 1 - 5 hours, preferably 1.5 - 2 hours.

[0040] In another preferred example, the molar ratio of the 2-CTC resin to Fmoc-Dab(Boc)-OH is 1:2 - 1:3.

[0041] In another preferred example, when the coupling system is DIC / HOBt, the molar ratio of the 2-CTC resin to HOBt to DIC is 1:(2 - 3):(2 - 3).

[0042] In another preferred embodiment, the cleavage reagent is selected from the group consisting of: TFE / DCM, TFA / DCM, or a combination thereof; preferably, the cleavage reagent is selected from: TFE / DCM.

[0043] In another preferred embodiment, the concentration of the cleavage reagent is: 1 g / 8 ml - 1 g / 15 ml; preferably 1 g / 10 ml.

[0044] In another preferred embodiment, the cleavage reagent is selected from: 15% TFE / DCM cleavage solution with a concentration of 1 g / 10 ml.

[0045] In another preferred embodiment, the precipitation is carried out using MTBE.

[0046] In another preferred embodiment, the cleavage time is 1 - 8 hours, preferably 3 - 6 hours; more preferably 4 hours.

[0047] In another preferred embodiment, the cleavage reaction is carried out at room temperature.

[0048] In another preferred embodiment, the activation system in the step (S5) is selected from: DCC / HOSu, DCC / HONB or TSTU / DIEA.

[0049] In another preferred embodiment, the activation system in the step (S5) is selected from: DCC / HOSu.

[0050] In another preferred embodiment, the crude peptide activated ester is selected from: Boc-β-Ala-Pro-Dab(Boc)-D-Tyr(tBu)-OSu, Boc-β-Ala-Pro-Dab(Boc)-D-Tyr(tBu)-ONB, or a combination thereof.

[0051] In another preferred embodiment, the crude peptide activated ester is Boc-β-Ala-Pro-Dab(Boc)-D-Tyr(tBu)-OSu.

[0052] In another preferred embodiment, the step (S5) includes sub-steps:

[0053] (S5a) Dissolve the crude peptide Boc-β-Ala-Pro-Dab(Boc)-D-Tyr(tBu)-OH using a solvent; and

[0054] (S5b) Add HOSu and DCC respectively, and after reacting for a period of time, couple the crude peptide Boc-β-Ala-Pro-Dab(Boc)-D-Tyr(tBu)-OH with the activation reagent HOSu to obtain Boc-β-Ala-Pro-Dab(Boc)-D-Tyr(tBu)-OSu.

[0055] In another preferred example, the solvent is selected from the group consisting of: THF, Diox, DCM, or a combination thereof; preferably, the solvent is selected from: THF.

[0056] In another preferred example, the solubility of the solvent is 40 - 50 mg / ml.

[0057] In another preferred example, the molar ratios of the crude peptide to DCC and HOSu are both 1:(1.2 - 1.5):(1.5 - 2), preferably 1:1.5:2.

[0058] In another preferred example, the molar ratio of the crude peptide to DCC is 1:(1.2 - 1.5), preferably 1:1.5.

[0059] In another preferred example, the molar ratio of the crude peptide to HOSu is 1:(1.5 - 2), preferably 1:2.

[0060] In another preferred example, the reaction includes two steps:

[0061] (R1) Reacting in an ice bath for 0.5 - 3 hours (preferably 1 hour); and

[0062] (R2) Reacting at room temperature for 3 - 6 hours (preferably 4 hours).

[0063] In another preferred example, the step (S5) further includes the steps of: filtration, precipitation, and drying.

[0064] In another preferred example, the step (S6) includes sub - steps:

[0065] (S6a) Dissolving benzylamine in a solvent and adjusting the pH to alkaline; and

[0066] (S6b) Adding the crude peptide activated ester to react with the benzylamine for a period of time, and maintaining the pH value with an organic base during the reaction.

[0067] In another preferred example, the solvent in the step (S6a) is selected from the group consisting of: DMSO, DMF, THF, or a combination thereof; preferably, the solvent is selected from the group consisting of: DMF.

[0068] In another preferred example, the pH is adjusted with an organic base in the step (S6a).

[0069] In another preferred example, the organic base is selected from the group consisting of: NMM, TEA, DIEA, or a combination thereof; preferably, the organic base is selected from: DIEA.

[0070] In another preferred example, the organic base in the step (S6b) is selected from the group consisting of: NMM, TEA, DIEA, or a combination thereof; preferably, the organic base is selected from: DIEA.

[0071] In another preferred example, the equivalent of the organic base is 1.2 - 1.5.

[0072] In another preferred example, the pH value in the step (S6b) is 7 - 8.

[0073] In another preferred example, the deprotection reagent in the step (S7) is selected from the group consisting of: HCl / EA, TFA / DCM, or a combination thereof; preferably, the deprotection reagent in the step (S7) is selected from: TFA / DCM.

[0074] In another preferred example, the deprotection reagent in the step (S7) is selected from: 90 - 95% TFA / DCM (volume ratio).

[0075] In another preferred example, the reaction temperature for deprotection in the step (S7) is 20 - 30 °C.

[0076] It should be understood that within the scope of the present invention, the above - mentioned technical features of the present invention and the technical features specifically described below (such as in the examples) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be elaborated one by one here. Description of the Drawings

[0077] Figure 1 Shows the chemical formula for the preparation of the tetrapeptide resin in Example 4 of the present invention.

[0078] Figure 2 Shows the chemical formula for the removal of the resin during the preparation of the tetrapeptide fragment in Example 4 of the present invention.

[0079] Figure 3 Shows the chemical formula for the preparation of the tetrapeptide modification in Example 4 of the present invention.

[0080] Figure 4 Shows the flow chart of the synthesis method of the snake - venom - like tetrapeptide of the present invention.

[0081] Figure 5 Shows the HPLC chromatogram of the final product polypeptide obtained by the synthesis method in Comparative Example 3.

[0082] Figure 6 Shows the HPLC chromatogram of the final product polypeptide obtained by the synthesis method of the present invention. Detailed Description of the Invention

[0083] After extensive and in-depth research and a large number of screenings, the present inventor unexpectedly discovered for the first time that choosing the reaction mode combining liquid phase and solid phase has certain advantages for the preparation of snake venom-like tetrapeptide H-β-Ala-Pro-Dab-D-Tyr-NH-Bzl. First, the C-terminus of the short peptide is connected to the chlororesin, and then the peptide is gradually coupled. After the peptide coupling is completed, the fully protected polypeptide is cleaved from the resin with a weak acid. First, the crude peptide activated ester is obtained with an activation system, and then benzylamine is coupled by a liquid phase method. Finally, the protection groups are removed to obtain the crude peptide. Compared with the prior art, the above preparation method can avoid racemization, and the process route is stable and simpler, and high-purity and high-yield snake venom-like tetrapeptide can be prepared at a lower cost. On this basis, the present invention is completed.

[0084] Term

[0085] To more easily understand the present disclosure, certain terms are first defined. As used in this application, unless otherwise expressly specified herein, each of the following terms shall have the meaning given below.

[0086] The term "about" may refer to a value or a component within an acceptable error range of a specific value or component determined by a person of ordinary skill in the art, which will depend in part on how the value or component is measured or determined.

[0087] Chinese-English name comparison table of abbreviations

[0088] Fmoc 9-Fluorenylmethyloxycarbonyl Boc N-tert-Butyloxycarbonyl tBu tert-Butyl Bzl Benzylamino 2-CTC Resin 2-Chlorotrityl Chloride Resin DCM Dichloromethane DMF N,N-Dimethylformamide MeOH Methanol THF Tetrahydrofuran Diox 1,4-Dioxane DMSO Dimethyl Sulfoxide EA Ethyl Acetate MTBE Methyl tert-Butyl Ether NMP N-Methylpyrrolidone NMM N-Methylmorpholine DIEA N,N-Diisopropylethylamine TEA Triethylamine TFE Trifluoroethanol TFA Trifluoroacetic Acid DIC N,N'-Diisopropylcarbodiimide DCC N,N'-Dicyclohexylcarbodiimide TBTU 2-(1H-Benzotriazol-1-yl)-1,1,3,3-tetramethyluronium Tetrafluoroborate TSTU 2-Succinimidyloxy-1,1,3,3-tetramethyluronium Tetrafluoroborate HOBt 1-Hydroxybenzotriazole HOSu N-Hydroxysuccinimide HONB N-Hydroxy-5-norbornene-2,3-dicarboximide Piperazine Piperazine Piperidine Piperidine

[0089] Snake venom-like tripeptide

[0090] Snake venom-like tripeptide (Snake venom tripeptide, Snake tripeptide, SYN-AKE), also known as Dipeptide Diaminobutyroyl Benzylamide Diacetate, molecular weight: 370.50, molecular formula C 19 H 29 N 5 O 3 , polypeptide sequence H-β-Ala-Pro-Dab-NH-Bzl.

[0091] The structure of snake venom-like tripeptide is shown in formula B below:

[0092]

[0093] D-Tyrosine

[0094] D-Tyrosine, also known as D-Thr-OH, molecular weight: 181.19, molecular formula C 9 H11 NO 3 。

[0095] The structure of D-tyrosine is shown in Formula C below:

[0096]

[0097] The chemical synthesis method of snake venom-like tripeptides in the prior art

[0098] Currently, the chemical synthesis method of snake venom-like tripeptides in the prior art is as follows:

[0099] In the method mentioned in the invention patent with the publication number CN107936108A and the patent name "A liquid-phase synthesis method of snake venom-like tripeptides", two hydrolysis reactions are used, which are prone to the racemization of Dab and pose a great risk to the product quality.

[0100] In the method mentioned in the invention patent with the publication number CN107857797A and the patent name "A liquid-phase fragment synthesis method of snake venom-like tripeptides", in the reaction process of preparing Fmoc-Pro-OSu from Fmoc-Pro-OH and reacting it with H-Dab(Boc)-OH to form Fmoc-Pro-Dab(Boc)-OH, due to the large steric hindrance of Pro amino acid, the reaction is difficult and the yield is relatively low; in addition, there is also a risk of racemization of Dab in this route.

[0101] In view of the deficiencies of the prior art, the present invention provides a solid-liquid phase synthesis method of snake venom-like tetrapeptides that can avoid the racemization of Dab during the above reaction process, has a stable process route, and can improve the yield and purity.

[0102] The preparation method of the present invention

[0103] The present invention provides a preparation method of snake venom-like tetrapeptide H-β-Ala-Pro-Dab-D-Tyr-NH-Bzl, and the preparation method includes the steps of:

[0104] (S1) Provide Fmoc-D-Tyr(tBu)-2-CTC resin;

[0105] (S2) Use a deprotection reagent to deprotect the amino terminus of the Fmoc-D-Tyr(tBu)-2-CTC resin;

[0106] (S3) Using Fmoc-Dab(Boc)-OH, Fmoc-Pro-OH, and Boc-β-Ala-OH as raw materials, sequentially couple the activated amino acids with the deprotected resin to obtain Boc-β-Ala-Pro-Dab(Boc)-D-Tyr(tBu)-2-CTC resin;

[0107] (S4) Use a cleavage reagent to perform a cleavage reaction on the Boc-β-Ala-Pro-Dab(Boc)-D-Tyr(tBu)-2-CTC resin, and precipitate to obtain the crude peptide Boc-β-Ala-Pro-Dab(Boc)-D-Tyr(tBu)-OH;

[0108] (S5) Use an activation system to couple the crude peptide Boc-β-Ala-Pro-Dab(Boc)-D-Tyr(tBu)-OH with an activation reagent to obtain a crude peptide activated ester;

[0109] (S6) Using benzylamine as a raw material, couple the crude peptide activated ester with benzylamine to obtain Boc-β-Ala-Pro-Dab(Boc)-D-Tyr(tBu)-NH-Bzl; and

[0110] (S7) Use a deprotection reagent to deprotect the amino terminus of the Boc-β-Ala-Pro-Dab(Boc)-D-Tyr(tBu)-NH-Bzl to obtain the snake venom-like tetrapeptide H-β-Ala-Pro-Dab-D-Tyr-NH-Bzl.

[0111] Preferably, for step (S1), it may include sub-steps:

[0112] (S1a) Provide 2-CTC resin;

[0113] (S1b) Connect Fmoc-D-Tyr(tBu)-OH to the 2-CTC resin to obtain Fmoc-D-Tyr(tBu)-2-CTC resin.

[0114] For step (S1b), it may include sub-steps:

[0115] (S1b1) Swell the 2-CTC resin with a solvent;

[0116] Preferably, the solvent is selected from the group consisting of: DMF, DCM, NMP, or a combination thereof; more preferably, the solvent is selected from: DCM;

[0117] (S1b2) Dissolve Fmoc-D-Tyr(tBu)-OH in an organic solvent and add it to the swollen 2-CTC resin, and add an organic base to adjust the pH value, and react for a period of time;

[0118] Preferably, the organic base is selected from the group consisting of: NMM, TEA, DIEA, or a combination thereof; more preferably, the organic base is selected from: DIEA;

[0119] Preferably, the molar ratio of the 2-CTC to the Fmoc-D-Tyr(tBu)-OH is 2:1 - 1:5, more preferably 1:1 - 1:3;

[0120] Preferably, the reaction conditions include stirring the reaction with nitrogen bubbling; the reaction temperature is 10 - 40°C, more preferably 20 - 30°C; the period of time is 1 - 5 hours, more preferably 1.5 - 3 hours, even more preferably 2 hours;

[0121] (S1b3) After the reaction is completed, it is blocked with MeOH to obtain Fmoc-D-Tyr(tBu)-2-CTC resin;

[0122] Preferably, the blocking time is 10 - 20 min, more preferably 15 min.

[0123] For step (S2), preferably, the deprotection reagent is selected from the group consisting of: Piperazine / DMF, Piperidine / DMF, diethylamine / DMF, or a combination thereof; more preferably, the deprotection reagent is selected from: Piperazine / DMF.

[0124] Preferably, the Piperazine / DMF is 5% Piperazine / DMF (mass ratio).

[0125] For step (S3), it may include sub-steps:

[0126] (S3a) Using Fmoc-Dab(Boc)-OH as a raw material, the activated amino acid is coupled with the deprotected Fmoc-D-Tyr(tBu)-2-CTC resin to obtain Fmoc-Dab(Boc)-D-Tyr(tBu)-2-CTC resin; (S3b) Using Fmoc-Pro-OH as a raw material, the activated amino acid is coupled with the deprotected Fmoc-Dab(Boc)-D-Tyr(tBu)-2-CTC resin to obtain Fmoc-Pro-Dab(Boc)-D-Tyr(tBu)-2-CTC resin;

[0127] (S3c) Using Boc-β-Ala-OH as a raw material, the activated amino acid is coupled with the deprotected Fmoc-Pro-Dab(Boc)-D-Tyr(tBu)-2-CTC resin to obtain Boc-β-Ala-Pro-Dab(Boc)-D-Tyr(tBu)-2-CTC resin;

[0128] Preferably, the raw materials Fmoc-Dab(Boc)-OH, Fmoc-Pro-OH, and Boc-β-Ala-OH are dissolved in a solvent selected from the group consisting of DMF, DCM, NMP, or a combination thereof; preferably, the solvent is selected from DMF;

[0129] Preferably, the coupling system is selected from the group consisting of DIC / HOBt, HBTU / DIEA, HATU / DIEA, DCC / HOBt, or a combination thereof; preferably, the coupling system is selected from DIC / HOBt;

[0130] For example, when the coupling system is DIC / HOBt, the activation in step (S3a), (S3b), or (S3c) includes the steps of: respectively adding DIC to the dissolved Fmoc-Dab(Boc)-OH, Fmoc-Pro-OH, or Boc-β-Ala-OH to activate the amino acid; the coupling in step (S3a), (S3b), or (S3c) further includes the step of adding HOBt.

[0131] Preferably, the reaction time for the coupling is 1 - 5 hours, preferably 1.5 - 2 hours;

[0132] Preferably, the molar ratio of the 2-CTC resin to Fmoc-Dab(Boc)-OH is 1:2 - 1:3;

[0133] Preferably, when the coupling system is DIC / HOBt, the molar ratio of the 2-CTC resin to HOBt to DIC is 1:(2 - 3):(2 - 3).

[0134] For step (S4), preferably, the cleavage reagent is selected from the group consisting of TFE / DCM, TFA / DCM, or a combination thereof; preferably, the cleavage reagent is selected from TFE / DCM;

[0135] Preferably, the concentration of the cleavage reagent is: 1 g / 8 ml - 1 g / 15 ml; preferably 1 g / 10 ml;

[0136] Preferably, the cleavage reagent is selected from: 15% TFE / DCM cleavage solution with a concentration of 1 g / 10 ml;

[0137] Preferably, the precipitation is carried out using MTBE;

[0138] Preferably, the cleavage time is 1 - 8 hours, preferably 3 - 6 hours; more preferably 4 hours; the cleavage reaction is carried out at room temperature.

[0139] For step (S5), preferably, the activation system in step (S5) is selected from: DCC / HOSu, DCC / HONB or TSTU / DIEA; more preferably, the activation system is selected from: DCC / HOSu;

[0140] Preferably, the crude peptide activation ester is selected from: Boc-β-Ala-Pro-Dab(Boc)-D-Tyr(tBu)-OSu, Boc-β-Ala-Pro-Dab(Boc)-D-Tyr(tBu)-ONB, or a combination thereof; more preferably, the crude peptide activation ester is Boc-β-Ala-Pro-Dab(Boc)-D-Tyr(tBu)-OSu;

[0141] Preferably, step (S5) may include sub-steps:

[0142] (S5a) Dissolve the crude peptide Boc-β-Ala-Pro-Dab(Boc)-D-Tyr(tBu)-OH in a solvent;

[0143] Preferably, the solvent is selected from the following group: THF, Diox, DCM, or a combination thereof; more preferably, the solvent is selected from: THF;

[0144] Preferably, the solubility of the solvent is 40 - 50 mg / ml; and

[0145] (S5b) Add HOSu and DCC respectively, and after reacting for a period of time, couple the crude peptide Boc-β-Ala-Pro-Dab(Boc)-D-Tyr(tBu)-OH with the activation reagent HOSu to obtain Boc-β-Ala-Pro-Dab(Boc)-D-Tyr(tBu)-OSu;

[0146] Preferably, the molar ratio of the crude peptide to DCC and HOSu is 1:(1.2 - 1.5):(1.5 - 2), preferably 1:1.5:2; the molar ratio of the crude peptide to DCC is 1:(1.2 - 1.5), more preferably 1:1.5; the molar ratio of the crude peptide to HOSu is 1:(1.5 - 2), more preferably 1:2;

[0147] In this step (S5b), preferably, the reaction includes two-step reactions:

[0148] (R1) React in an ice bath for 0.5 - 3 hours (preferably 1 hour); and

[0149] (R2) React at room temperature for 3 - 6 hours (preferably 4 hours);

[0150] Furthermore, step (S5) may further include steps: filtration, precipitation, and drying.

[0151] For step (S6), it may include sub-steps:

[0152] (S6a) Dissolve benzylamine in a solvent and adjust the pH to alkaline;

[0153] Preferably, the solvent is selected from the group consisting of DMSO, DMF, THF, or a combination thereof; more preferably, the solvent is selected from the group consisting of DMF;

[0154] Preferably, an organic base is used to adjust the pH in step (S6a); the organic base is selected from the group consisting of NMM, TEA, DIEA, or a combination thereof; more preferably, the organic base is DIEA; and

[0155] (S6b) Add the crude peptide activated ester to react with the benzylamine for a period of time, and maintain the pH value with an organic base during the reaction process;

[0156] Preferably, the pH value in step (S6b) is 7-8;

[0157] Preferably, the organic base in step (S6b) is selected from the group consisting of NMM, TEA, DIEA, or a combination thereof; more preferably, the organic base is DIEA;

[0158] For the above-mentioned organic base, its equivalent is preferably 1.2-1.5.

[0159] For step (S7), preferably, the deprotection reagent in step (S7) is selected from the group consisting of HCl / EA, TFA / DCM, or a combination thereof; more preferably, the deprotection reagent in step (S7) is selected from TFA / DCM;

[0160] Preferably, the deprotection reagent in step (S7) is selected from 90-95% TFA / DCM (volume ratio);

[0161] Preferably, the reaction temperature for deprotection in step (S7) is 20-30 °C.

[0162] Through the careful design of the solid-liquid phase synthesis method of the snake venom-like tetrapeptide in the present invention, the limitations of completely using the solid-phase synthesis method or completely using the liquid-phase synthesis method can be avoided, thereby establishing a preparation method that can avoid the racemization of Pro and Dab, and has a stable and simpler process route, and can conveniently prepare high-purity snake venom-like tetrapeptide at a lower cost.

[0163] The main advantages of the present invention

[0164] 1. Based on the original snake venom-like tripeptide, D-Tyr is added to produce a new type of beauty peptide. This invention avoids the racemization of Dab, has a high yield and low cost. The selected D-Tyr increases the purity and yield of the product, and is easy to improve the overall yield and purity of the short peptide.

[0165] 2. In addition, compared with the prior art, by adopting a solid-liquid phase synthesis method with a pre-activated system, the purity and yield of the final product polypeptide β-Ala-Pro-Dab-D-Tyr-NH-Bzl obtained by the present invention are both very high, and there is no need for subsequent high-cost and high-threshold enrichment, purification and other steps. It not only simplifies the process, but also can obtain a large amount of high-purity final products at a low cost. Therefore, the preparation method of the present invention is more suitable for large-scale industrial production.

[0166] The present invention will be further described below in conjunction with specific embodiments. It should be understood that these embodiments are only used to illustrate the present invention and not to limit the scope of the present invention. The experimental methods without specific conditions noted in the following embodiments are usually carried out under conventional conditions, such as the conditions described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or the conditions recommended by the manufacturer. Unless otherwise specified, percentages and parts are weight percentages and weight parts.

[0167] Example 1: Preparation of Fmoc-D-Tyr(tBu)-2-CTC Resin

[0168] Weigh 48.19 g (40 mmol, Sub = 0.83 mmol / g) of 2-CTC Resin and add it to the solid-phase reaction column for swelling and washing with DCM. Then add a mixed solution of 36.76 g (80 mmol) of Fmoc-D-Tyr(tBu)-OH, 26.44 ml (160 mmol) of DIEA, and 350 ml of DCM, and stir and react for 2 h. Add 106 ml of methanol and block for 15 min. Drain the solvent and wash three times with DMF, 150 ml each time.

[0169] Example 2: Preparation of Boc-β-Ala-Pro-Dab(Boc)-D-Tyr(tBu)-2-CTC Resin

[0170] React the Fmoc-D-Tyr(tBu)-2-CTC Resin prepared in Example 1 with 150 ml of 5% Piperazine / DMF solution for 5 min. Drain the solvent, and wash once with 150 ml of MeOH, 150 ml of DCM, and 150 ml of DMF respectively. Drain the solvent, add 150 ml of 5% Piperazine / DMF solution and react for 15 min. Drain the solvent and wash 6 times with DMF, 150 ml each time.

[0171] Weigh 42.28 g (96 mmol) of Fmoc-Dab(Boc)-OH, 12.96 g (96 mmol) of HOBt, and 17.84 ml (115.2 mmol) of DIC, add them to 100 ml of DMF for dissolution, and activate in an ice bath for 5 min. Add the activated solution to the reactor, react for 1.5 h, and drain the solvent. Add DMF for washing three times, 150 ml each time.

[0172] Repeat the above steps. According to the amino acid sequence, sequentially add Fmoc-Pro-OH and Boc-β-Ala-OH for coupling reaction. After the coupling reaction is completed, drain the solvent, add DMF for washing 3 times, 150 ml each time. Drain the solvent, add 150 ml of MeOH for washing twice, 150 ml of DCM for washing twice, 150 ml of MeOH for washing twice, and dry in vacuo to obtain 75.96 g of peptide resin Boc-β-Ala-Pro-Dab(Boc)-D-Tyr(tBu)-2-CTC Resin.

[0173] Example 3: Preparation of Boc-β-Ala-Pro-Dab(Boc)-D-Tyr(tBu)-OH

[0174] Weigh 75.69 g of the dry Boc-β-Ala-Pro-Dab(Boc)-D-Tyr(tBu)-2-CTC Resin prepared in Example 2, add 15% TFE / DCM cleavage solution (1 g / 10 ml) for cleavage reaction for 4 h. After the reaction is completed, precipitate with methyl tert-butyl, and a white solid will precipitate to obtain the fully protected crude peptide. Place it in a vacuum dryer for drying to obtain 21.17 g of the product Boc-β-Ala-Pro-Dab(Boc)-D-Tyr(tBu)-OH, with a yield of 75%.

[0175] Example 4: Preparation of Boc-β-Ala-Pro-Dab(Boc)-D-Tyr(tBu)-OSu

[0176] Weigh 10.15 g (14.38 mmol) of Boc-β-Ala-Pro-Dab(Boc)-D-Tyr(tBu)-OH obtained in Example 3, dissolve it in 203 ml of THF, add 2.48 g (21.57 mmol) of HOSu, stir in an ice bath for 10 min, then add 5.93 g (28.76 mmol) of DCC, continue to stir in the ice bath for 1 hour, remove the ice bath and react at room temperature for another 4 hours. Filter the reaction solution, evaporate the filtrate to dryness, add it to 406 ml of MTBE solution, dissolve and filter, add the filtrate to 1.2 L of n-heptane, let it stand for 30 minutes, a large amount of precipitate will form, filter, wash with n-heptane 3 times, and dry in vacuum to obtain 11.46 g of Boc-β-Ala-Pro-Dab(Boc)-D-Tyr(tBu)-Osu (crude peptide activated ester), with a purity of 80.76% and a yield of 99.3%.

[0177] Example 5: Preparation of Boc-β-Ala-Pro-Dab(Boc)-D-Tyr(tBu)-NH-Bzl

[0178] Dissolve 11.46 g (12.45 mmol) of Boc-β-Ala-Pro-Dab(Boc)-D-Tyr(tBu)-OSu obtained in Example 4 in 114.6 ml of DMF solution, add 1.83 g (14.94 mmol) of benzylamine, adjust the pH of the reaction solution to 7 - 8 with DIEA, react at room temperature for 2 h, add 345 ml of MTBE and 230 ml of purified water to the reaction solution, let it stand for liquid separation, wash the organic phase with 230 ml of purified water and 230 ml of saturated sodium chloride aqueous solution, dry the organic phase with anhydrous sodium sulfate, filter, evaporate under reduced pressure, dissolve the residue in 100 ml of MTBE, then add it to 600 ml of n-heptane, let it stand for 30 minutes, a large amount of precipitate will form, filter, wash with n-heptane, and dry in vacuum to obtain 11.13 g of Boc-β-Ala-Pro-Dab(Boc)-D-Tyr(tBu)-NH-Bzl, with a purity of 95.50% and a yield of 98.10%.

[0179] Example 6: Preparation of β-Ala-Pro-Dab-D-Tyr-NH-Bzl

[0180] Add 11.13 g (14.00 mmol) of Boc-β-Ala-Pro-Dab(Boc)-D-Tyr(tBu)-NH-Bzl obtained in Example 5 to 111.3 ml of 90% TFA / DCM mixed solution, react at room temperature for 1 h, add the reaction solution to 670 ml of MTBE solution for precipitation, filter, wash with MTBE, and dry in vacuum to obtain 7.29 g of snake venom-like tetrapeptide β-Ala-Pro-Dab-D-Tyr-NH-Bzl, with a purity of 97.81% and a yield of 96.83%.

[0181] Comparative Example 1: Total Liquid Phase Synthesis of Snake Venom-like Tetrapeptide H-β-Ala-Pro-Dab-D-Tyr-NH-Bzl

[0182] (1) Weigh 11.56 g (50 mmol) of Fmoc-β-Ala-OH and 6.33 g (55 mmol) of HOSu into a container, add 110 mL of THF and stir to dissolve. Cool in an ice bath, then add 12.38 g (60 mmol) of DCC, and stir and react at 0 - 10 °C for 4 hours. Filter, precipitate the filtrate in 600 ml of MTBE, a white precipitate will separate out. Let it stand for 30 minutes, filter, and dry with forced air to obtain 18.24 g of Fmoc-β-Ala-OSu, with a yield of 89.32% and a purity of 94.37%.

[0183] (2) Dissolve 18.24 g (44.66 mmol) of Fmoc-β-Ala-OSu in 180 mL of DMF, add 11.67 ml (67 mmol) of DIEA, cool in an ice bath for 5 min, add 6.17 g (53.59 mmol) of H-Pro-OH, stir and react at room temperature for 3 hours. Adjust the pH to 3 - 5 with 1M citric acid solution, add 1 L of ethyl acetate, and extract three times with 200 ml of distilled water. Let it stand for layering, dry the organic phase with anhydrous magnesium sulfate, filter, concentrate by rotary evaporation, perform recrystallization with stirring at 0 - 5 °C, filter, and dry in vacuo to obtain 12.21 g of Fmoc-β-Ala-Pro-OH, with a yield of 66.94% and a purity of 80.31%.

[0184] (3) Weigh 12.21 g (29.89 mmol) of Fmoc-β-Ala-Pro-OH and 4.13 g (35.87 mmol) of HOSu into a container, add 120 mL of THF and stir to dissolve. Cool in an ice bath, then add 12.33 g (59.78 mmol) of DCC, and stir and react at 0 - 10 °C for 5 hours. Filter, precipitate the filtrate in 600 ml of MTBE, a white precipitate will separate out. Let it stand for 30 minutes, filter, and dry with forced air to obtain 13.66 g of Fmoc-β-Ala-Pro-OSu, with a yield of 90.40% and a purity of 79.63%.

[0185] (4) Dissolve 13.66 g (27.01 mmol) of Fmoc-β-Ala-Pro-OSu in 130 mL of DMF, add 7.06 mL (40.52 mmol) of DIEA, ice-bath for 5 min, add 11.03 g (32.42 mmol) of H-Dab(Fmoc)-OH, stir and react at room temperature for 5 hours, adjust the pH to 3 - 5 with 1 M citric acid solution, add 1 L of ethyl acetate, extract three times with 200 mL of distilled water, let it stand for layer separation, dry the organic phase with anhydrous magnesium sulfate, filter, concentrate by rotary evaporation, stir for recrystallization at 0 - 5 °C, filter, and dry in vacuum to obtain 8.30 g of Fmoc-β-Ala-Pro-Dab(Fmoc)-OH, with a yield of 42.05% and a purity of 77.36%.

[0186] (5) Weigh 8.30 g (11.36 mmol) of Fmoc-β-Ala-Pro-Dab(Fmoc)-OH and 1.57 g (13.64 mmol) of HOSu into a container, add 80 mL of THF and stir to dissolve, ice-bath, then add 3.52 g (17.04 mmol) of DCC, stir and react at 0 - 10 °C for 5 hours, filter, precipitate the filtrate in 480 mL of MTBE, white precipitate appears, let it stand for 30 minutes, filter, and dry by blowing air to obtain 7.39 g of Fmoc-β-Ala-Pro-Dab(Fmoc)-OSu, with a yield of 78.62% and a purity of 75.24%.

[0187] (6) Dissolve 7.39 g (8.93 mmol) of Fmoc-β-Ala-Pro-Dab(Fmoc)-OSu in 70 mL of DMF, add 2.33 mL (13.39 mmol) of DIEA, ice-bath for 5 min, add 11.03 g (32.42 mmol) of H-D-Tyr-OH, stir and react at room temperature for 5 hours, adjust the pH to 3 - 5 with 1 M citric acid solution, add 1 L of ethyl acetate, extract three times with 200 mL of distilled water, let it stand for layer separation, dry the organic phase with anhydrous magnesium sulfate, filter, concentrate by rotary evaporation, stir for recrystallization at 0 - 5 °C, filter, and dry in vacuum to obtain 8.30 g of Fmoc-β-Ala-Pro-Dab(Fmoc)-D-Tyr-OH, with a yield of 42.05% and a purity of 77.36%.

[0188] (7) Weigh 8.30 g (9.28 mmol) of Fmoc-β-Ala-Pro-Dab(Fmoc)-D-Tyr-OH obtained in the previous step, dissolve it in 80 ml of THF, add 1.28 g (11.14 mmol) of HOSu, stir in an ice bath for 10 min, then add 3.83 g (18.56 mmol) of DCC, continue to stir in the ice bath for 1 hour, remove the ice bath and react at room temperature for another 4 hours. Filter the reaction solution, rotary evaporate the filtrate, add it to 400 ml of MTBE solution, dissolve and filter, add the filtrate to 1.2 L of n-heptane and let it stand for 30 minutes. A large amount of precipitate will form. Filter it, wash it with n-heptane three times, and dry it under vacuum to obtain 8.54 g of Fmoc-β-Ala-Pro-Dab(Fmoc)-D-Tyr-OSu, with a yield of 92.85% and a purity of 70.32%.

[0189] (8) Weigh 8.54 g (8.62 mmol) of Fmoc-β-Ala-Pro-Dab(Fmoc)-D-Tyr-OSu and dissolve it in 85 ml of DMF solution. Add 1.11 g (10.34 mmol) of benzylamine, adjust the pH of the reaction solution to 7 - 8 using DIEA, and react at room temperature for 2 h. Add 255 ml of MTBE and 170 ml of purified water to the reaction solution, let it stand for liquid separation. Wash the organic phase with 170 ml of purified water and 170 ml of saturated sodium chloride aqueous solution. Dry the organic phase with anhydrous sodium sulfate, filter it, rotary evaporate under reduced pressure. Dissolve the residue in 80 ml of MTBE, then add it to 480 ml of n-heptane and let it stand for 30 minutes. A large amount of precipitate will form. Filter it, wash it with n-heptane, and dry it under vacuum to obtain 5.42 g of Fmoc-β-Ala-Pro-Dab(Fmoc)-D-Tyr-NH-Bzl, with a yield of 63.95% and a purity of 80.32%.

[0190] (9) Weigh 5.42 g (8.62 mmol) of Fmoc-β-Ala-Pro-Dab(Fmoc)-D-Tyr-NH-Bzl, dissolve it in 50 ml of DMF solution, add 2.37 g of piperazine, react at room temperature for 30 min. Add the reaction solution to 300 ml of MTBE solution to precipitate, filter it, wash it with MTBE, and dry it under vacuum to obtain 2.78 g of snake venom-like tetrapeptide β-Ala-Pro-Dab-D-Tyr-NH-Bzl, with a yield of 95.63% and a purity of 83.63%.

[0191] From the comparison between the complete liquid-phase synthesis method and the method of the present invention, it can be seen that:

[0192] The steps of the complete liquid-phase synthesis method are cumbersome and the process is more complex than that of the method of the present invention.

[0193] Pro and Dab are prone to racemization in the complete liquid-phase synthesis method, which is also one of the reasons for the low yields and purities of steps (2) and (4) in the complete liquid-phase synthesis method;

[0194] The yields and purities of the final product β-Ala-Pro-Dab-D-Tyr-NH-Bzl in the complete liquid-phase synthesis method are also lower than those of the method of the present invention.

[0195] Comparative Example 2: Total Solid Phase Synthesis of Snake Venom-like Tetrapeptide H-β-Ala-Pro-Dab-D-Tyr-NH-Bzl

[0196] Weigh 1.0 mol of AM Resin (1.0 mmol / g), add it to a reactor, add 5% piperazine / DMF solution, and swell for 0.5 h. After the swelling is completed, perform suction filtration and wash with DMF.

[0197] Weigh 3 mol of 4-hydroxymethylphenylacetic acid and 3 mol of HOBT, add them to a beaker, add DMF to dissolve them clearly, cool to 0 - 5 °C in an ice bath, slowly add 3.6 mol of DIC, control the temperature to remain at 0 - 5 °C, after the addition is completed, activate at 0 - 5 °C for 5 min, add the activation solution to the reactor, start the reaction by bubbling nitrogen, react for 3 h, perform suction filtration, wash 3 times with DMF, and then wash 2 times with DCM.

[0198] Weigh 2 mol of Boc-D-Tyr-OH, add it to the reactor, add DCM, bubble nitrogen, add 4 mol of DIEA, dropwise add 4 mol of DCB, after the dropwise addition is completed, control the temperature of the reaction solution at 25 - 30 °C, react for 3 h, perform suction filtration, and wash 3 times with DMF.

[0199] Measure DMF, add DIEA, stir and mix evenly, then add Ac 2 O, stir and mix well, add it to the reactor, block the end for 0.5 h, perform suction filtration, wash 3 times with DMF, and then wash 2 times with DCM. Add 50% TFA / DCM, bubble nitrogen and react for 0.5 h, perform suction filtration, and wash 5 times with DMF.

[0200] Weigh 3 mol of Boc-Dab(Fmoc)-OH and 3 mol of HOBT, add them to an activation cup, add DMF to dissolve them clearly, cool to 0 - 5 °C in an ice bath, slowly add 3.6 mol of DIC, control the temperature to remain at 0 - 5 °C, after the addition is completed, activate at 0 - 5 °C for 5 min, add the activation solution to the reactor, start the reaction by bubbling nitrogen, react for 2 h, perform suction filtration, wash 3 times with DMF, and then wash 2 times with DCM. Add 50% TFA / DCM, bubble nitrogen and react for 0.5 h, perform suction filtration, and wash 5 times with DMF.

[0201] Connect Boc-Pro-OH and Boc-β-Ala-OH successively according to the connection method of Boc-Dab(Fmoc)-OH. Wash the resin twice with 50% DMF / THF. Weigh 1.5 mol of benzylamine, add it to a beaker, dissolve it in 50% DMF / THF and then add it to the reactor. React under nitrogen bubbling for 3 h, filter, and wash the resin twice with 50% DMF / THF solution. After combining the filtrates and drying them by evaporation, slurry with MTBE, wash, and dry to obtain β-Ala-Pro-Dab-D-Tyr-NH-Bzl with a yield of 15.23% and a purity of 76.81%.

[0202] From the comparison between the complete solid-phase synthesis method and the method of the present invention, it can be seen that:

[0203] The yields and purities of the products cleaved by the complete solid-phase synthesis method (for example, using MBHA / AM resin to connect amino acids as mentioned in Patent CN112409445A) are both low, and high-yield and high-purity products can only be obtained after enrichment and purification. Adding a purification process increases the cost of the complete solid-phase synthesis method. The method of the present invention has a lower cost and combines high purity and high yield.

[0204] Comparative Example 3: Other Solid-Liquid Phase Synthesis Methods of Snake Venom-like Tetrapeptide H-β-Ala-Pro-Dab-D-Tyr-NH-Bzl

[0205] In this comparative example, the solid-liquid phase synthesis method of the present invention was compared with the solid-liquid phase synthesis method in CN115521356A.

[0206] Specifically, the main difference between the synthesis method in CN115521356A and the present invention is that after obtaining Boc-β-Ala-Pro-Dab(Boc)-D-Tyr(tBu)-OH through step (10) in CN115521356A and performing cleavage in step (11), NHBzl (dissolved in DMF solvent) was directly added dropwise slowly without prior activation for reaction, thereby obtaining Boc-β-Ala-Pro-Dab(Boc)-D-Tyr(tBu)-NHBzl.

[0207] In the synthesis method of the present invention, a prior activation system (such as the DCC / HOSu activation system) was specifically adopted. After activating Boc-β-Ala-Pro-Dab(Boc)-D-Tyr(tBu)-OH in THF solvent first to obtain the crude peptide activation ester Boc-β-Ala-Pro-Dab(Boc)-D-Tyr(tBu)-OSu which precipitated out in large amounts, it was then reacted with benzylamine to obtain Boc-β-Ala-Pro-Dab(Boc)-D-Tyr(tBu)-NH-Bzl.

[0208] Through the comparison of the above synthesis methods, the inventor of the present invention found that:

[0209] The purity of the final product polypeptide β-Ala-Pro-Dab-D-Tyr-NH-Bzl obtained by using the synthesis method (steps (1)-(12)) in CN115521356A is 49%, and the yield is 50%. Both its purity and yield are much lower than those of the synthesis method of the present invention.

[0210] In addition, due to the low purity and yield of the synthesis method in CN115521356A, after obtaining the final product polypeptide β-Ala-Pro-Dab-D-Tyr-NH-Bzl through step (12) in CN115521356A, step (13) is still required to purify and separate impurities by HPLC.

[0211] However, in the synthesis method of the present invention, since both the purity and yield of the obtained final product polypeptide β-Ala-Pro-Dab-D-Tyr-NH-Bzl are very high, a large amount of final product with high purity can be obtained without HPLC purification.

[0212] To further demonstrate that the final product polypeptide obtained by the synthesis method of the present invention has reached a very high purity without HPLC purification, the inventors of the present invention respectively performed HPLC analysis on the final product polypeptide obtained by using the synthesis method (steps (1)-(12)) in CN115521356A and the final product polypeptide obtained by using the synthesis method of the present invention.

[0213] As Figure 5 shown, the HPLC chromatogram of the final product polypeptide obtained by using the synthesis method (steps (1)-(12)) in CN115521356A is shown.

[0214] As Figure 6 shown, the HPLC chromatogram of the final product polypeptide obtained by using the synthesis method of the present invention is shown.

[0215] Obviously, the types and contents of impurities in the final product polypeptide obtained by using the synthesis method of the present invention are lower than those of the final product polypeptide obtained by using the synthesis method (steps (1)-(12)) in CN115521356A. Especially for CN115521356A, Figure 5 there is impurity 1 at about 11 minutes, its content is relatively high and very close to the peak time of the final product polypeptide. Therefore, impurity 1 will cause great interference to the purification of the final product polypeptide. While in the synthesis method of the present invention, the contents of each impurity are very low, further proving that the purity of the final product polypeptide obtained by using the synthesis method of the present invention is already very high, and a large amount of final product with high purity can be obtained without HPLC purification.

[0216] Therefore, compared with the synthesis method of CN115521356A, the synthesis method of the present invention simplifies the production process on the one hand and reduces the production cost and threshold on the other hand, making it more suitable for large-scale industrial production.

[0217] All documents mentioned in the present invention are cited in this application as references, as if each document was cited separately as a reference. In addition, it should be understood that after reading the above teachings of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the appended claims of this application.

Claims

1. Preparation method of snake venom-like tetrapeptide H-β-Ala-Pro-Dab-D-Tyr-NH-Bzl, characterized in that, the preparation method includes the steps: (S1) Provide Fmoc-D-Tyr(tBu)-2-CTC resin; (S2) Use a deprotection reagent to deprotect the amino terminus of the Fmoc-D-Tyr(tBu)-2-CTC resin; (S3) Using Fmoc-Dab(Boc)-OH, Fmoc-Pro-OH, and Boc-β-Ala-OH as raw materials, sequentially couple the activated amino acids with the deprotected resin to obtain Boc-β-Ala-Pro-Dab(Boc)-D-Tyr(tBu)-2-CTC resin; (S4) Use a cleavage reagent to perform a cleavage reaction on the Boc-β-Ala-Pro-Dab(Boc)-D-Tyr(tBu)-2-CTC resin, and precipitate to obtain the crude peptide Boc-β-Ala-Pro-Dab(Boc)-D-Tyr(tBu)-OH; (S5) Use an activation system to couple the crude peptide Boc-β-Ala-Pro-Dab(Boc)-D-Tyr(tBu)-OH with an activation reagent to obtain a crude peptide activation ester; (S6) Using benzylamine as a raw material, couple the crude peptide activation ester with benzylamine to obtain Boc-β-Ala-Pro-Dab(Boc)-D-Tyr(tBu)-NH-Bzl; and (S7) Use a deprotection reagent to deprotect the amino terminus of the Boc-β-Ala-Pro-Dab(Boc)-D-Tyr(tBu)-NH-Bzl to obtain the snake venom-like tetrapeptide H-β-Ala-Pro-Dab-D-Tyr-NH-Bzl.

2. The preparation method according to claim 1, characterized in that, the deprotection reagent is selected from the group consisting of: piperazine / DMF, piperidine / DMF, diethylamine / DMF, or a combination thereof; preferably, the deprotection reagent is selected from: piperazine / DMF.

3. The preparation method according to claim 1, characterized in that, the step (S3) includes sub-steps: (S3a) Using Fmoc-Dab(Boc)-OH as a raw material, couple the activated amino acid with the deprotected Fmoc-D-Tyr(tBu)-2-CTC resin to obtain Fmoc-Dab(Boc)-D-Tyr(tBu)-2-CTC resin; (S3b) Using Fmoc-Pro-OH as a raw material, couple the activated amino acid with the deprotected Fmoc-Dab(Boc)-D-Tyr(tBu)-2-CTC resin to obtain Fmoc-Pro-Dab(Boc)-D-Tyr(tBu)-2-CTC resin; and (S3c) Using Boc-β-Ala-OH as a raw material, the activated amino acid was coupled with the deprotected Fmoc-Pro-Dab(Boc)-D-Tyr(tBu)-2-CTC resin to obtain Boc-β-Ala-Pro-Dab(Boc)-D-Tyr(tBu)-2-CTC resin.

4. The preparation method according to claim 3, characterized in that, the coupling system is selected from the group consisting of: DIC / HOBt, HBTU / DIEA, HATU / DIEA, DCC / HOBt, or a combination thereof; preferably, the coupling system is selected from: DIC / HOBt.

5. The preparation method according to claim 1, characterized in that, the cleavage reagent is selected from the group consisting of: TFE / DCM, TFA / DCM, or a combination thereof; preferably, the cleavage reagent is selected from: TFE / DCM.

6. The preparation method according to claim 1, characterized in that, the activation system in the step (S5) is selected from: DCC / HOSu, DCC / HONB or TSTU / DIEA.

7. The preparation method according to claim 1, characterized in that, the step (S5) includes sub-steps: (S5a) Dissolving the crude peptide Boc-β-Ala-Pro-Dab(Boc)-D-Tyr(tBu)-OH with a solvent; and (S5b) Adding HOSu and DCC respectively, and after reacting for a period of time, coupling the crude peptide Boc-β-Ala-Pro-Dab(Boc)-D-Tyr(tBu)-OH with the activating reagent HOSu to obtain Boc-β-Ala-Pro-Dab(Boc)-D-Tyr(tBu)-OSu.

8. The preparation method according to claim 7, characterized in that, the molar ratio of the crude peptide to DCC and HOSu is 1:(1.2 - 1.5):(1.5 - 2), preferably 1:1.5:

2.

9. The preparation method according to claim 1, characterized in that, the step (S6) includes sub-steps: (S6a) Dissolving benzylamine with a solvent and adjusting the pH to alkaline; and (S6b) Adding the crude peptide activated ester and reacting with the benzylamine for a period of time, and maintaining the pH value with an organic base during the reaction process.

10. The preparation method according to claim 1, characterized in that, the deprotection reagent in the step (S7) is selected from the group consisting of: HCl / EA, TFA / DCM, or a combination thereof; preferably, the deprotection reagent in the step (S7) is selected from: TFA / DCM.

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