A method for preparing a snake venom-like tripeptide

Abstract

The application relates to the technical field of peptide synthesis, and discloses a preparation method of a snake venom-like tripeptide, which comprises the following steps: condensing Boc-beta-Ala-OH and H-Pro-OMe, hydrolyzing the methyl ester groups to obtain Boc-beta-Ala-Pro-OH; condensing Boc-Gln-OH and benzylamine, and removing Boc to obtain H-Gln-NHBzl; then condensing Boc-beta-Ala-Pro-OH and H-Gln-NHBzl to prepare a polypeptide; and after the Gln in the polypeptide is subjected to Hofmann rearrangement, the Boc protection group in the polypeptide is removed to obtain the snake venom-like tripeptide. The preparation method adopts liquid-phase synthesis of the snake venom-like tripeptide, the synthesis route is short, the reaction condition is mild, special equipment and reagents are not needed, the operation steps are simplified, the process cost is reduced; the reaction reagents used are cheap and easy-to-obtain industrial reagents, the raw material cost is reduced; and the product purity can reach more than 99%.

Description

Technical Field

[0001] This invention relates to the field of peptide synthesis technology, and specifically to a method for preparing a snake venom-like tripeptide. Background Technology

[0002] Snake venom-like tripeptides are small molecule polypeptides that mimic the activity of the snake venom toxin Waglerin I. Like botulinum toxin and argirelin, they reduce wrinkles by inhibiting muscle nicotinic acetylcholine receptors (nmAChR), exhibiting excellent skin-smoothing and rapid wrinkle-reducing properties, and are widely used in high-end cosmetics. However, they are safer and more effective than botulinum toxin. The peptide sequence of the snake venom-like tripeptide is H-β-Ala-Pro-Dab-NHBzl, and its molecular structure is shown in formula (VIII). (VIII).

[0003] Currently, most conventional methods for synthesizing snake venom-like tripeptides employ a combination of solid-phase and liquid-phase methods, resulting in complex and costly processes. To simplify the process to some extent, researchers have developed all-liquid-phase synthesis methods for snake venom-like tripeptides. For example, patent application CN117486968A discloses the following synthetic route: starting from commercially available L-glutamine, it proceeds through Fmoc-protected amine groups, key steps of amide condensation with EDCI and Hofmann rearrangement with PIFA, Boc-protected amine groups, de-Fmoc removal, and branching from commercially available Boc-β-alanine, followed by DCC-catalyzed L-proline amide condensation, then N-hydroxysuccinimide activation of the carboxyl group, amide condensation of the two fragments, and finally de-Boc removal to obtain the final product, the snake venom-like peptide. Although this patent achieves kilogram-scale production, the synthetic route is long, involves many steps, is cumbersome, and has a low product yield.

[0004] Existing technologies also include the synthesis of compounds with the peptide sequence H-β-Ala-Pro-Gln-NHBn, followed by Hofmann rearrangement to remove the carbonyl group from the Gln group to obtain the Dab group, thus solving the problem of high Dab price. For example, the journal "Tetrahedron Letters 55 (2014) 5745-5747" reported a method using proline, 3-chloropropionyl chloride, and 5-glutamic acid methyl ester as raw materials to obtain H-β-Ala-Pro-Gln-NHBn in 5 steps, followed by Hofmann degradation to obtain a snake venom-like tripeptide. Although this method solves the problem of high Dab price, the yield of each step is not high, especially the yield of the key step (i.e., the Hofmann rearrangement yield) is only 54%, resulting in a low overall yield. Moreover, this method requires preparative liquid-phase purification, which is very costly. Summary of the Invention

[0005] In view of this, the purpose of the present invention is to provide a method for preparing a snake venom-like tripeptide, which aims to replace Dab raw material with inexpensive raw material, reduce raw material cost, shorten its liquid phase synthesis route, simplify operation steps, reduce process cost, and further improve the overall yield.

[0006] To achieve the above objectives, the present invention provides a method for preparing a snake venom-like tripeptide, the preparation method being carried out according to the appendix. Figure 1 The liquid-phase synthesis route shown is followed, in detail, see attached. Figure 1 The liquid-phase synthesis route shown includes the following reaction steps: (1) Under the action of a condensing agent, Boc-β-Ala-OH and H-Pro-OMe undergo a condensation reaction to obtain Boc-β-Ala-Pro-OMe; (2) Under the action of alkaline reagent, Boc-β-Ala-Pro-OMe undergoes a hydrolysis reaction to obtain Boc-β-Ala-Pro-OH; (3) Under the action of the condensing agent, Boc-Gln-OH and NHBzl undergo a condensation reaction to obtain Boc-Gln-NHBzl; (4) Under the action of an acidic reagent, the Boc protecting group in Boc-Gln-NHBzl is removed to obtain H-Gln-NHBzl; (5) Under the action of the condensing agent, Boc-β-Ala-Pro-OH and H-Gln-NHBzl undergo a condensation reaction to obtain Boc-β-Ala-Pro-Gln-NHBzl; (6) Under the action of an oxidizing agent, Boc-β-Ala-Pro-Gln-NHBzl is degraded by Hoffmann to obtain Boc-β-Ala-Pro-Dab-NHBzl; (7) Under the action of an acidic reagent, the BOC protecting group in Boc-β-Ala-Pro-Dab-NHBzl is removed to obtain the snake venom-like tripeptide.

[0007] In the liquid-phase synthesis route provided by this invention, firstly, in the presence of a condensing reagent, the carboxyl group of Boc-β-Ala-OH is activated, and the lone pair electrons on the nitrogen atom of H-Pro-OMe act as a nucleophile to attack the activated carboxyl carbon, resulting in an amide condensation reaction to obtain Boc-β-Ala-Pro-OMe. Then, by alkaline hydrolysis of the methyl ester group, the Boc-β-Ala-Pro-OH fragment is obtained. Parallel, in the presence of a condensing reagent, the carboxyl group of Boc-Gln-OH is activated and undergoes an amide condensation reaction with benzylamine to obtain Boc-Gln-NHBzl. Then, by acid desorption, the Boc group is removed to obtain the H-Gln-NHBzl fragment. Subsequently, the Boc-β-Ala-Pro-OH fragment, acting as the carboxyl component, and the H-Gln-NHBzl fragment, acting as the amino component, undergo an amide condensation reaction under the action of a condensing reagent to obtain Boc-β-Ala-Pro-Gln-NHBzl. This is then followed by Hoffmann rearrangement and Boc removal to yield a snake venom-like tripeptide. Because the amide bonds (-CO) of β-alanine (β-Ala) and glutamine (Gln) are easily degraded during the Hoffmann rearrangement, leading to changes in the peptide chain structure and reducing the rearrangement yield, this invention employs an amino protection strategy for Boc-β-Ala to reduce side reactions and improve the rearrangement yield.

[0008] Furthermore, the condensing agent is selected from at least one of DCC, DIC, EDCl, HBTU, HATU, TBTU, HOBt, HOAt, and Oxyma.

[0009] Furthermore, the alkaline reagent is selected from at least one of sodium hydroxide, potassium hydroxide, lithium hydroxide, sodium carbonate, sodium bicarbonate, potassium carbonate, and potassium bicarbonate.

[0010] Furthermore, the acidic reagent is selected from at least one of hydrochloric acid, acetic acid, trifluoroacetic acid, methanesulfonic acid, p-toluenesulfonic acid, sulfuric acid, a mixture of hydrochloric acid and 1,4-dioxane (HCl / 1,4-dioxane), a mixture of hydrochloric acid and ethyl acetate (HCl / ethyl acetate), and a mixture of hydrochloric acid and tetrahydrofuran (HCl / THF).

[0011] Furthermore, the oxidizing agent is selected from at least one of PIDA, PIFA, PhI(OH)Ots, sodium hypochlorite, and sodium hypobromite.

[0012] Furthermore, in some embodiments of the present invention, the Hofmann degradation reaction of Boc-β-Ala-Pro-Gln-NHBzl in reaction step (6) is carried out at pH 5.0 to 6.0. By controlling the reaction system to be weakly acidic and combining it with the amino protection strategy of Boc-β-Ala, the occurrence of side reactions can be further reduced, and the rearrangement yield can be increased to over 90%.

[0013] Furthermore, in some embodiments of the present invention, the preparation method includes the following steps: Step 1: Preparation of Boc-β-Ala-Pro-OMe: Add Boc-β-Ala-OH, H-Pro-OMe·HCl and condensation reagent to an organic solvent and stir until dissolved. After the reaction is complete, extract the solute from the reaction solution and purify it to obtain an oily substance. Step 2, Preparation of Boc-β-Ala-Pro-OH: Add oily substance one to an organic solvent and stir until dissolved. Then add an alkaline reagent dropwise. After the reaction is complete, extract the solute from the reaction solution and purify it to obtain solid substance two. Step 3, Preparation of Boc-Gln-NHBzl: Add Boc-Gln-OH, benzylamine, and condensation reagent to an organic solvent and stir until dissolved. After the reaction is complete, crystallize out the solute in the reaction solution to obtain solid three. Step 4: Preparation of H-Gln-NHBzl: Add solid three to an organic solvent and stir until dissolved. Then add an acidic reagent dropwise. After the reaction is complete, evaporate and concentrate the reaction solution to obtain solid four. Step 5: Preparation of Boc-β-Ala-Pro-Gln-NHBzl: Add solids II and IV and the condensation reagent to an organic solvent and stir until dissolved. After the reaction is complete, extract the solute from the reaction solution and purify it to obtain oily substance V. Step 6: Preparation of Boc-β-Ala-Pro-Dab-NHBzl: Add oily substance 5 to acetonitrile solution and stir until dissolved. Then add oxidizing agent dropwise. After the reaction is complete, extract the solute from the reaction solution and purify it to obtain solid substance 6. Step 7: Preparation of snake venom-like tripeptide: Add solid 6 to an organic solvent and stir until dissolved. Then add an acidic reagent dropwise. After the reaction is complete, evaporate and concentrate the reaction solution to obtain solid 7. Solid 7 is purified by crystallization to obtain the snake venom-like tripeptide. The organic solvent is selected from at least one of N,N-dimethylformamide, dichloromethane, tetrahydrofuran, and 1,4-dioxane.

[0014] Preferably, the reaction temperature of steps (1), (3), and (5) is -5 to 20°C, the reaction temperature of step (2) is 0 to 20°C, the reaction temperature of step (6) is -10 to 5°C, and the reaction temperature of steps (4) and (7) is 20 to 40°C.

[0015] Preferably, in step (1), the molar ratio of Boc-β-Ala-OH to H-Pro-OMe·HCl is 1:(1.1~1.5), the molar ratio of Boc-β-Ala-OH to the condensing reagent is 1:(1.1~1.5), and the molar ratio of Boc-β-Ala-OH to the organic solvent is 1g:(5~10)mL; In step (2), the ratio of the amount of oily substance 1 to the amount of organic solvent is 1 g: (5-10) mL, and the molar ratio of the amount of oily substance 1 to the alkaline reagent is 1: (1.5-2.5). In step (3), the molar ratio of Boc-Gln-OH to benzylamine is 1:(1.1-1.5), the molar ratio of Boc-Gln-OH to organic solvent is 1g:(5-10)mL, and the molar ratio of Boc-Gln-OH to condensing reagent is 1:(1.1-1.5). In step (4), the ratio of solid substance 3 to organic solvent is 1g:(5-10)mL, and the ratio of solid substance 3 to acidic reagent is 1g:(3-7)mL. In step (5), the molar ratio of solid 2 to solid 4 is 1:(1.1-1.5), the amount ratio of solid 2 to organic solvent is 1g:(5-10)mL, and the molar ratio of solid 2 to condensing reagent is 1:(1.1-1.5). In step (6), the ratio of oily substance 5 to acetonitrile solution is 1 g: (5-10) mL, and the molar ratio of oily substance 5 to oxidizing agent is 1: (1.1-2.5). In step (7), the ratio of solid VI to organic solvent is 1g:(2-5)mL, and the ratio of solid VI to acidic reagent is 1g:(1-3)mL.

[0016] The preparation method provided by this invention requires only seven reaction steps to prepare snake venom-like tripeptides. The synthetic route is short, and the reaction conditions throughout the route are mild. It does not require special equipment or reagents, and the operation is simple. At the same time, the reactants used are all existing, inexpensive raw materials, which are readily available and inexpensive, effectively reducing raw material costs and process costs.

[0017] Compared with the prior art, the present invention has the following beneficial effects: (1) Innovative Hoffmann rearrangement strategy: This invention shortens the synthetic route of snake venom-like tripeptides and simplifies the process steps by designing the Hoffmann rearrangement after peptide synthesis; by converting Gln to Dab through Hoffmann rearrangement, the use of expensive Dab raw materials is avoided, and the occurrence of β-Ala side reactions is reduced and the rearrangement yield is improved by using the Boc-β-Ala amino protection strategy. Furthermore, by controlling the rearrangement reaction system to weakly acidic reaction conditions, the occurrence of Gln side reactions is further reduced. Combined with the Boc-β-Ala amino protection strategy, the rearrangement yield can be increased to 90%, and the overall yield can reach more than 50%, which greatly reduces the preparation cost of snake venom-like tripeptides.

[0018] (2) Excellent product quality: After recrystallization and purification, the final product of this invention has a purity of over 99% as determined by HPLC, with a single impurity of less than 1%, which meets the quality requirements of cosmetic raw materials. Moreover, the product purification method is simple and easy to operate, without the need for expensive preparative liquid phase purification.

[0019] (3) Suitable for large-scale industrial production: The reaction temperature of each step of the present invention is between 0 and 30°C, and no special equipment with high temperature and high pressure is required. The reaction conditions are mild and the operation is simple. The intermediates are mostly obtained in solid form, which is convenient for transportation and storage. The reaction reagents and principles are easy to obtain and inexpensive, which meets the requirements of industrial production, is suitable for large-scale industrial production, and has great promotional value.

[0020] In summary, this invention has the advantages of low cost, high quality, high safety, simple operation, and suitability for industrial production. Attached Figure Description

[0021] Figure 1 A liquid-phase synthesis route diagram for a snake venom-like tripeptide provided by the present invention; Figure 2 The HPLC chromatogram of the snake venom-like tripeptide prepared in Example 1 of this invention (VWD1 A, wavelength = 215 nm). Specific implementation methods To better understand the present invention, the present invention will be further described below with reference to the embodiments. However, the content of the present invention is not limited to the following embodiments. All changes or equivalent substitutions that do not depart from the concept of the present invention are included within the protection scope of the present invention.

[0022] The Chinese names corresponding to the abbreviations used in the specification and claims are shown in Table 1.

[0023] Table 1. Correspondence of Name Abbreviations

[0024] See Figure 1As shown, this invention provides a liquid-phase synthetic route for a snake venom-like tripeptide, specifically including the following reaction steps: (1) Under the action of a condensing agent, Boc-β-Ala-OH and H-Pro-OMe undergo a condensation reaction to obtain Boc-β-Ala-Pro-OMe; (2) Under the action of alkaline reagent, Boc-β-Ala-Pro-OMe undergoes a hydrolysis reaction to obtain Boc-β-Ala-Pro-OH; (3) Under the action of the condensing agent, Boc-Gln-OH and NHBzl undergo a condensation reaction to obtain Boc-Gln-NHBzl; (4) Under the action of an acidic reagent, the BOC protecting group in Boc-Gln-NHBzl is removed to obtain H-Gln-NHBzl; (5) Under the action of the condensing agent, Boc-β-Ala-Pro-OH and H-Gln-NHBzl undergo a condensation reaction to obtain Boc-β-Ala-Pro-Gln-NHBzl; (6) Under the action of an oxidizing agent, Boc-β-Ala-Pro-Gln-NHBzl is degraded by Hoffmann to obtain Boc-β-Ala-Pro-Dab-NHBzl; (7) Under the action of an acidic reagent, the BOC protecting group in Boc-β-Ala-Pro-Dab-NHBzl is removed to obtain the snake venom-like tripeptide.

[0025] Preferably, the condensing agent is selected from at least one of DCC, DIC, EDCl, HBTU, HATU, TBTU, HOBt, HOAt, and Oxyma.

[0026] Preferably, the alkaline reagent is selected from at least one of sodium hydroxide, potassium hydroxide, lithium hydroxide, sodium carbonate, sodium bicarbonate, potassium carbonate, and potassium bicarbonate.

[0027] Preferably, the acidic reagent is selected from at least one of hydrochloric acid, acetic acid, trifluoroacetic acid, methanesulfonic acid, p-toluenesulfonic acid, sulfuric acid, a mixture of hydrochloric acid and 1,4-dioxane (HCl / 1,4-dioxane), a mixture of hydrochloric acid and ethyl acetate (HCl / ethyl acetate), and a mixture of hydrochloric acid and tetrahydrofuran (HCl / THF).

[0028] Preferably, the oxidizing agent is selected from at least one of PIDA, PIFA, PhI(OH)Ots, sodium hypochlorite, and sodium hypobromite.

[0029] Furthermore, in some embodiments of the present invention, the Hofmann degradation reaction of Boc-β-Ala-Pro-Gln-NHBzl in reaction step (6) above is carried out under weakly acidic conditions. In the present invention, the weakly acidic condition refers to the pH value of the Hofmann degradation reaction system being in the range of 5.0 to 6.0.

[0030] The preparation method of the snake venom tripeptide of the present invention will be specifically described below with reference to the embodiments.

[0031] Unless otherwise specified, the raw materials, reagents, equipment, etc. used in the following examples can all be obtained through conventional market channels.

[0032] In the following examples, the reaction yields of each step are calculated according to the following formula (IX). The overall yield is the product of the yields of each step (in decimal form) and then multiplied by 100%.

[0033] (IX) Example 1: The preparation method of the snake venom-like tripeptide provided in this embodiment specifically includes the following steps: Step 1: Prepare Boc-β-Ala-Pro-OMe according to the synthetic route provided by formula (Ⅰ):

[0034] (I); Add reaction reagent 1 "Boc-β-Ala-OH" (95.9 g, 507 mmol, 1.0 eq), reaction reagent 2 "H-Pro-OMe·HCl" (109.2 g, 660 mmol, 1.3 eq), catalyst "NMM" (3 eq), condensation reagent 1 "Oxyma" (1.2 eq), and organic solvent "DMF" (500 mL) to the reaction vessel. Stir until dissolved, add condensation reagent 2 "EDCl" (1.2 eq) in portions, and control the temperature at 0℃ until the reaction is complete as monitored by TLC. Add water to the reaction solution, extract with ethyl acetate, wash the organic layer with sodium carbonate aqueous solution, and concentrate to obtain 151.5 g of oil (Boc-β-Ala-Pro-OMe), with a reaction yield of 98.5%.

[0035] The purity of the above-mentioned oily substance was determined to be 98.2% by high performance liquid chromatography (HPLC).

[0036] Step 2: Prepare Boc-β-Ala-Pro-OH according to the synthetic route provided by formula (II):

[0037] (II); The oily substance (Boc-β-Ala-Pro-OMe, 151.5 g, 504 mmol, 1.0 eq) obtained in step 1 and the organic solvent "THF" (1500 mL) were added to the reaction vessel and stirred until dissolved. The reaction solution was cooled to 10 °C, and the alkaline reagent "LiOH" (1.8 eq) was added dropwise. After the addition was complete, the reaction was allowed to proceed for 3 h. The reaction was monitored by TLC until it was complete. The reaction solution was extracted with ethyl acetate, dried over anhydrous sodium sulfate, and concentrated to obtain an oily substance. Heptane was then crystallized to give 134.32 g of white solid (Boc-β-Ala-Pro-OH), with a reaction yield of 93%.

[0038] The purity of the white solid was determined to be 99.3% by high performance liquid chromatography (HPLC).

[0039] Step 3: Prepare Boc-Gln-NHBzl according to the synthetic route provided in formula (III):

[0040] (III); Add reagent 3 "Boc-Gln-OH" (195.4 g, 794 mmol, 1.0 eq), reagent 4 "Benzylamine" (110.5 g, 1032 mmol, 1.3 eq), NMM (2.0 eq), Oxyma (1.2 eq), and DMF (1000 mL) to the reaction vessel, stir until dissolved, add EDCl (1.5 eq) in portions, and control the temperature at 0 °C until the reaction is complete as monitored by TLC. Add a large amount of ice water to the reaction solution, stir to induce crystallization, and obtain 239.5 g of white solid (Boc-Gln-NHBzl), with a reaction yield of 90%.

[0041] The purity of the solid was determined to be 98.6% using high performance liquid chromatography (HPLC).

[0042] Step 4: Prepare H-Gln-NHBzl according to the synthetic route provided by formula (Ⅳ):

[0043] (Ⅳ); The solid obtained in step 3 (Boc-Gln-NHBzl, 239.5 g, 714 mmol, 1.0 eq) and 1,4-dioxane (1198 mL) were added to the reactor and stirred until dissolved. 2 mol / L HCl / 1,4-dioxane (1198 mL) was added in portions and the reaction was carried out for 2 h. The mixture was then concentrated to obtain 178.6 g of solid (H-Gln-NHBzl hydrochloride), with a reaction yield of 92%.

[0044] The purity of the above solid was determined to be 92.3% by high performance liquid chromatography (HPLC).

[0045] Step 5: Prepare Boc-β-Ala-Pro-Gln-NHBzl according to the synthetic route provided in formula (V):

[0046] (V); The white solid (Boc-β-Ala-Pro-OH, 134.4 g, 469 mmol, 1.0 eq) prepared in step 2, the solid obtained in step 4 (H-Gln-NHBzl hydrochloride, 178.6 g, 657 mmol, 1.4 eq), NMM (3.0 eq), Oxyma (1.2 eq), and DMF (1100 mL) prepared in step 2 were added to the reaction vessel and stirred until dissolved. EDCl (1.5 eq) was added in portions, and the reaction was carried out at 0 °C until the reaction was complete as monitored by TLC. Water was added to the reaction solution, and the mixture was extracted with ethyl acetate. The organic layer was washed with sodium carbonate aqueous solution and concentrated to obtain an oily substance (Boc-β-Ala-Pro-Gln-NHBzl) of 212.73 g, with a reaction yield of 90%.

[0047] The purity of the oily substance was determined to be 98.3% using high performance liquid chromatography (HPLC).

[0048] Step 6: Prepare Boc-β-Ala-Pro-Dab-NHBzl according to the synthetic route provided in formula (VI):

[0049] (VI); The oily substance (Boc-β-Ala-Pro-Gln-NHBzl, 212.7 g, 422 mmol, 1.0 eq) obtained in step 5 and acetonitrile solution (1600 mL) were added to the reaction vessel and stirred until dissolved. PIDA (2.0 eq) was added in portions. After all PIDA was added, the pH of the reaction solution was measured to be 5.6. The reaction was controlled at -5℃ until the reaction was complete as monitored by TLC. Water was added to the reaction solution, and the mixture was extracted with ethyl acetate. The organic layer was washed with sodium carbonate aqueous solution, dried with anhydrous sodium sulfate, concentrated, and crystallized with n-heptane to obtain 206.7 g of solid (Boc-β-Ala-Pro-Dab-NHBzl). The reaction yield was 90.0%.

[0050] The purity of the solid was determined to be 85.0% using high performance liquid chromatography (HPLC).

[0051] Step 7: Prepare H-β-Ala-Pro-Dab-NHBzl according to the synthetic route provided in formula (VII):

[0052] (VII) The solid obtained in step 6 (Boc-β-Ala-Pro-Dab-NHBzl, 206.7 g, 435 mmol, 1.0 eq) and THF (800 mL) were added to the reactor and stirred until dissolved. 18% hydrochloric acid (400 mL) was added in portions and the reaction was carried out for 4 h. The temperature was controlled at 30 °C until the reaction was complete as monitored by TLC. The mixture was concentrated to dryness and crystallized with methanol / ethyl acetate to obtain 106.3 g of white solid (snake venom-like tripeptide). The reaction yield was 88% and the overall yield was 63.4%.

[0053] The purity of the prepared snake venom-like tripeptide was determined to be 99.85% using high-performance liquid chromatography (HPLC) (see chromatogram). Figure 2 (As shown).

[0054] Example 2: Same as Example 1, except that 42.5 mL of triethylamine was added to the reaction vessel in step 6, and the pH of the reaction solution was 7.1 after all reagents were added. The concentrations of reaction products, reaction yields, and overall yields for each step are shown in Table 2 below.

[0055] Example 3: Same as Example 1, except that 28.0 mL of glacial acetic acid was added to the reaction vessel in step 6, and the pH of the reaction solution was 4.6 after all reagents were added. The concentrations of reaction products, reaction yields, and overall yields for each step are shown in Table 2 below.

[0056] Table 2. Concentration and Yield of Reaction Products

[0057] As can be seen from Table 2 above, the overall yield of Examples 1 to 3 is all above 30%, which greatly reduces the preparation cost of snake venom tripeptide.

[0058] The above are merely preferred embodiments of the present invention. It should be noted that the above preferred embodiments should not be considered as limitations on the present invention, and the scope of protection of the present invention should be determined by the scope defined in the claims. For those skilled in the art, any improvements and modifications made based on the core ideas of the present invention without departing from the spirit and scope of the present invention should be considered within the scope of protection of the present invention.

Claims

1. A method for preparing a snake venom-like tripeptide, characterized in that: The preparation method adopts the following liquid-phase synthesis route: (1) Under the action of a condensing agent, Boc-β-Ala-OH and H-Pro-OMe undergo a condensation reaction to obtain Boc-β-Ala-Pro-OMe; (2) Under the action of alkaline reagent, Boc-β-Ala-Pro-OMe undergoes a hydrolysis reaction to obtain Boc-β-Ala-Pro-OH; (3) Under the action of a condensing agent, Boc-Gln-OH reacts with benzylamine to give Boc-Gln-NHBzl; (4) Under the action of an acidic reagent, the Boc protecting group in Boc-Gln-NHBzl is removed to obtain H-Gln-NHBzl; (5) Under the action of the condensing agent, Boc-β-Ala-Pro-OH and H-Gln-NHBzl undergo a condensation reaction to obtain Boc-β-Ala-Pro-Gln-NHBzl; (6) Under the action of an oxidizing agent, Boc-β-Ala-Pro-Gln-NHBzl is degraded by Hoffmann to obtain Boc-β-Ala-Pro-Dab-NHBzl; (7) Under the action of an acidic reagent, the Boc protecting group in Boc-β-Ala-Pro-Dab-NHBzl is removed to obtain the snake venom-like tripeptide.

2. The preparation method according to claim 1, characterized in that: The condensation reagent is selected from at least one of DCC, DIC, EDCl, HBTU, HATU, TBTU, HOBt, HOAt, and Oxyma.

3. The preparation method according to claim 1, characterized in that: The alkaline reagent is selected from at least one of sodium hydroxide, potassium hydroxide, lithium hydroxide, sodium carbonate, sodium bicarbonate, potassium carbonate, and potassium bicarbonate.

4. The preparation method according to claim 1, characterized in that: The acidic reagent is selected from at least one of hydrochloric acid, acetic acid, trifluoroacetic acid, methanesulfonic acid, p-toluenesulfonic acid, sulfuric acid, a mixture of hydrochloric acid and 1,4-dioxane, a mixture of hydrochloric acid and ethyl acetate, and a mixture of hydrochloric acid and tetrahydrofuran.

5. The preparation method according to claim 1, characterized in that: The oxidizing agent is selected from at least one of PIDA, PIFA, PhI(OH)Ots, sodium hypochlorite, and sodium hypobromite.

6. The preparation method according to claim 1, characterized in that: The Hofmann degradation reaction of Boc-β-Ala-Pro-Gln-NHBzl in route (6) was carried out under conditions of pH 5.0 to 6.

0.

7. The preparation method according to any one of claims 1 to 6, characterized in that: The preparation method includes the following steps: Step 1: Preparation of Boc-β-Ala-Pro-OMe: Add Boc-β-Ala-OH, H-Pro-OMe·HCl and condensation reagent to an organic solvent and stir until dissolved. After the reaction is complete, extract the solute from the reaction solution and purify it to obtain an oily substance. Step 2, Preparation of Boc-β-Ala-Pro-OH: Add oily substance one to an organic solvent and stir until dissolved. Then add an alkaline reagent dropwise. After the reaction is complete, extract the solute from the reaction solution and purify it to obtain solid substance two. Step 3, Preparation of Boc-Gln-NHBzl: Add Boc-Gln-OH, benzylamine, and condensation reagent to an organic solvent and stir until dissolved. After the reaction is complete, crystallize out the solute in the reaction solution to obtain solid three. Step 4: Preparation of H-Gln-NHBzl: Add solid three to an organic solvent and stir until dissolved. Then add an acidic reagent dropwise. After the reaction is complete, evaporate and concentrate the reaction solution to obtain solid four. Step 5: Preparation of Boc-β-Ala-Pro-Gln-NHBzl: Add solids II and IV and the condensation reagent to an organic solvent and stir until dissolved. After the reaction is complete, extract the solute from the reaction solution and purify it to obtain oily substance V. Step 6: Preparation of Boc-β-Ala-Pro-Dab-NHBzl: Add oily substance 5 to acetonitrile solution and stir until dissolved. Then add oxidizing agent dropwise. After the reaction is complete, extract the solute from the reaction solution and purify it to obtain solid substance 6. Step 7: Preparation of snake venom-like tripeptide: Add solid 6 to an organic solvent and stir until dissolved. Then add an acidic reagent dropwise. After the reaction is complete, evaporate and concentrate the reaction solution to obtain solid 7. Solid 7 is purified by crystallization to obtain the snake venom-like tripeptide. The organic solvent is selected from at least one of N,N-dimethylformamide, dichloromethane, tetrahydrofuran, and 1,4-dioxane.

8. The preparation method according to claim 7, characterized in that: The reaction temperature of steps (1), (3), and (5) is -5 to 20°C, the reaction temperature of step (2) is 0 to 20°C, the reaction temperature of step (6) is -10 to 5°C, and the reaction temperature of steps (4) and (7) is 20 to 40°C.

9. The preparation method according to claim 7, characterized in that: In step (1), the molar ratio of Boc-β-Ala-OH to H-Pro-OMe·HCl is 1:(1.1~1.5), the molar ratio of Boc-β-Ala-OH to the condensing reagent is 1:(1.1~1.5), and the volume ratio of Boc-β-Ala-OH to the organic solvent is 1g:(5~10)mL. In step (2), the ratio of the amount of oily substance 1 to the amount of organic solvent is 1 g: (5-10) mL, and the molar ratio of the amount of oily substance 1 to the alkaline reagent is 1: (1.5-2.5). In step (3), the molar ratio of Boc-Gln-OH to benzylamine is 1:(1.1-1.5), the molar ratio of Boc-Gln-OH to organic solvent is 1g:(5-10)mL, and the molar ratio of Boc-Gln-OH to condensing reagent is 1:(1.1-1.5). In step (4), the ratio of solid substance 3 to organic solvent is 1g:(5-10)mL, and the ratio of solid substance 3 to acidic reagent is 1g:(3-7)mL. In step (5), the molar ratio of solid 2 to solid 4 is 1:(1.1-1.5), the amount ratio of solid 2 to organic solvent is 1g:(5-10)mL, and the molar ratio of solid 2 to condensing reagent is 1:(1.1-1.5). In step (6), the ratio of oily substance 5 to acetonitrile solution is 1 g: (5-10) mL, and the molar ratio of oily substance 5 to oxidizing agent is 1: (1.1-2.5). In step (7), the ratio of solid VI to organic solvent is 1g:(2-5)mL, and the ratio of solid VI to acidic reagent is 1g:(1-3)mL.

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