Label-assisted reverse liquid phase polypeptide synthesis method

Abstract

The invention discloses a label-assisted reverse liquid phase polypeptide synthesis method (ILPPS). According to the method, a hydrophobic benzyl alcohol (HBA) label is used for anchoring an N terminal (amino terminal) of transient protection amino acid through a carbamate bond, the reactivity of a C terminal (carboxylic acid terminal) is retained, extension of a peptide chain from the N terminal to the C terminal is realized in liquid phase polypeptide synthesis (LPPS), and repeated amino acid N terminal protection and de-protection steps are avoided. Based on HBA tag hydrophobicity, the purpose of separating and purifying polypeptide is achieved by changing a solvent. According to the method, the repeated Fmoc / Boc protection and deprotection processes in the traditional polypeptide synthesis process are effectively avoided, and the synthesis efficiency and atom economy of the polypeptide compound are improved.

Description

Technical Field

[0001] The present invention belongs to the technical field of polypeptide synthesis, and particularly relates to a hydrophobic tag-assisted reverse liquid-phase polypeptide synthesis method, which is particularly suitable for efficient and atom-economic polypeptide synthesis. Background Art

[0002] Polypeptides are a class of compounds with important biological activities formed by amino acids linked by peptide bonds. In recent years, polypeptide drugs have become a hot topic in drug research globally due to their high safety, wide range of indications, significant efficacy, and obvious advantages in industrial development. Traditional polypeptide synthesis methods (such as solid-phase synthesis SPPS and classical liquid-phase synthesis CSPS) have problems such as cumbersome steps, low atom economy, and large solvent consumption. For example, solid-phase synthesis requires excessive Fmoc / Boc-protected amino acids and repeated de-Fmoc / Boc protection steps, while traditional liquid-phase synthesis requires intermediate purification and has low synthesis efficiency. Existing tag-assisted liquid-phase polypeptide synthesis (LPPS) combines the advantages of solid-phase synthesis and liquid-phase synthesis, but still needs to fix the C-terminus of the peptide chain on the tag through a carboxylic ester bond. Similar to traditional solid-phase / liquid-phase polypeptide synthesis, it requires repeated protection and deprotection operations, which limits its wide application. Therefore, it is of great significance to develop a polypeptide synthesis method that does not require repeated Fmoc / Boc protection and deprotection and has low solvent consumption. (Journal of Combinatorial Chemistry, 2007, 9(6): 924-925; ACS Combinatorial Science, 2013, 15(5): 217-228; Journal of the American Chemical Society, 1963, 85(19): 3045-3046; The Journal of Organic Chemistry, 2019, 84(8): 4615-4628; Journal of Peptide Science, 2004, 11(2): 69-73) Summary of the Invention

[0003] The object of the present invention is to provide a reverse liquid-phase polypeptide synthesis method with high efficiency, high atom economy and simple operation, which comprises the following steps: 1) preparing a hydrophobic benzyl alcohol (HBA) tag; 2) preparing an HBA tag active ester: the HBA tag is activated twice with CDI and HOSu respectively to obtain an HBA tag active carbonate with higher reactivity towards amino groups; 3) preparing an HBA tag-amino acid: the amino acid is transiently protected with a silylating reagent, and the transiently protected amino acid is then coupled with the active carbonate of the HBA tag. The product is washed with a citric acid aqueous solution to remove the transient protection, and at the same time, excess amino acids and impurities are removed; 4) preparing an HBA tag-polypeptide: repeating the operation of step 3), coupling the HBA tag-amino acid with the HBA tag active carbonate to achieve the reverse extension of the peptide chain from the N-terminus to the C-terminus; 5) removing the HBA tag to obtain the target polypeptide.

[0004] In order to achieve the above object, the technical solution adopted by the present invention is:

[0005] The present invention provides a tag-assisted reverse liquid-phase polypeptide synthesis method, which comprises the following steps:

[0006] (1) Preparing a hydrophobic benzyl alcohol (HBA) tag, and the synthesis route is as follows:

[0007]

[0008] The HBA tags PC1 and PC2 are prepared, and their structures are as follows:

[0009]

[0010] Methyl 3,5-dihydroxybenzoate is alkylated in solvent 1 to obtain compound 1, and the methyl ester bond of compound 1 is reduced to a hydroxyl group by a reducing agent in solvent 2 to obtain compound 2 of formula 2.

[0011] In this step, due to the solubility difference between the substrate methyl 3,5-dihydroxybenzoate and the bromoalkane, solvent 1 is selected as a mixture of two different polar solvents.

[0012] In the present invention, a hydrophobic benzyl alcohol derivative is used to anchor the N-terminus of the amino acid / peptide chain. Based on the hydrophobic property of the HBA tag, the whole liquid-phase synthesis of the polypeptide can be carried out in a non-polar solvent, and the reaction solvent is changed, and precipitation separation (using PC1) or extraction and liquid separation (using PC2) is carried out.

[0013] (2) Preparing an HBA tag active ester, and the synthesis route is as follows;

[0014]

[0015] Compound 2 reacts with N,N'-carbonyldiimidazole (CDI) in solvent 3 to obtain intermediate 3 after a reaction time. Intermediate 3 reacts with N-hydroxysuccinimide (HOSu) in solvent 3 to obtain intermediate 4 after a reaction time.

[0016] Considering that the carbamate bond can stably exist in weak acid and weak base environments and can be removed by hydrogenolysis, in the present invention, an HBA-label active carbonate with high reactivity towards amino groups is prepared by a two-step activation method, and the HBA label is coupled with an amino acid through a carbamate bond. During the two-step activation process, due to the existence of a reverse reaction between intermediate 3 and intermediate 4, an acidifying agent is added to precipitate the imidazole shed from intermediate 3 into a salt, inhibiting the reverse reaction and increasing the yield of intermediate 4.

[0017] (3) Preparation of HBA-labeled amino acid, the synthesis route is as follows:

[0018]

[0019] Intermediate 4 reacts with compound 5 in solvent 3, and after acidification, compound 6 is obtained.

[0020] Among them, R2 includes an alkyl group, a heterocyclic alkyl group, an aryl group, a heteroaryl group or a polypeptide fragment.

[0021] (4) Preparation of HBA-labeled polypeptide, the synthesis route is as follows:

[0022]

[0023] Compound 6 is activated in solvent 3, and then coupled with compound 5 in one pot. After acidification, compound 7 is obtained. The operation of "in-situ activation of carboxyl - coupling of transiently protected amino acid - removal of transient protection" is repeated to obtain compound 8 of the formula.

[0024] Among them, R2 is as described in step (3).

[0025] (5) Removal of the HBA label, the specific route is as follows:

[0026]

[0027] Compound 8 undergoes hydrogenolysis in solvent 4 to remove the HBA label, obtaining compound 9.

[0028] Among them, R2 is as described in step (3).

[0029] In some embodiments of the present invention, the phase transfer catalyst is one or more of benzyltriethylammonium chloride (TEBA), tetrabutylammonium chloride (TBAC), and tetrabutylammonium bromide (TBAB).

[0030] In some embodiments of the present invention, the solvent 1 is one or more of 2-MeTHF, THF, DMF, DMA, in any proportion mixture.

[0031] In some embodiments of the present invention, the solvent 2 is one of DCM, 2-MeTHF, THF.

[0032] In some embodiments of the present invention, the acidifying agent in step (2) is one or more of TFA, TfOH, MsOH.

[0033] In some embodiments of the present invention, the reaction temperature in step (2) is 0°C - 45°C; preferably 0°C - 25°C.

[0034] In some embodiments of the present invention, the molar ratio of CDI to the compound of formula 2 is (1 - 6):1; preferably (1 - 3):1; the molar ratio of the compound HOSu to the compound of formula 3 is (1 - 6):1; preferably (2 - 6):1

[0035] 10. According to a label-assisted reverse liquid-phase polypeptide synthesis method as claimed in claim 1, wherein the solvent 3 in steps (2), (3) and (4) is one or more of MeCN, DCM, 2-MeTHF, THF.

[0036] In the present invention, since the unprotected amino acid exists in the form of an intramolecular zwitterion under neutral conditions, it results in poor solubility and the amino group does not have nucleophilicity, making it difficult to carry out condensation reactions. Using a silylating reagent as a transient protecting reagent can temporarily protect the carboxyl group in the unprotected amino acid to form a silyl ester compound (compound 5), while freeing up its amino group, and then being able to undergo a nucleophilic substitution reaction with the active ester intermediate of compound 6 to form a peptide bond. The preparation method of the transiently protected amino acid is as follows:

[0037]

[0038] In some embodiments of the present invention, the silylating reagent includes one or more of TMSA, BSA, HMDS.

[0039] In some embodiments of the present invention, the activating agent is one or more of HOPO, HOBT, HOAT, HOSu, HBTU, HATU, PyAOP, PyOxim, COMU.

[0040] In some embodiments of the present invention, the solvent 4 is one or more of 2-MeTHF, THF, DCM, EA.

[0041] The beneficial effects of the present invention are:

[0042] The present invention provides a label-assisted reverse liquid-phase polypeptide synthesis method. In this method, a hydrophobic benzyl alcohol label is used to anchor the N-terminus of an amino acid / peptide chain, endowing the peptide chain with hydrophobic properties, and enabling the total liquid-phase synthesis of polypeptides in a non-polar solvent. By changing the solvent used in the post-treatment, the polypeptide is purified by precipitation separation or extraction separation. The reaction solvent consumption is less, reducing environmental pollution. The strategy of transiently protecting amino acids is adopted, and the N-terminus of the amino acid / peptide chain is anchored with a hydrophobic benzyl alcohol label to reverse-extend the peptide chain, avoiding the use of excessive reagents and repeated Fomc / Boc protection and de-Fomc / Boc protection steps. Compared with traditional solid-phase polypeptide synthesis, classical liquid-phase polypeptide synthesis, and label-assisted liquid-phase polypeptide synthesis, this strategy improves the synthesis efficiency and atom economy. Detailed implementation manners

[0043] The following examples are used to further illustrate the present invention in detail, so that those skilled in the art can implement it according to the description in the specification.

[0044] It should be understood that terms such as "having", "comprising", and "including" used herein do not exclude the presence or addition of one or more other elements or their combinations.

[0045] Unless otherwise specified, the test methods used in the following examples are all conventional methods. The materials and reagents used in the following examples, unless otherwise specified, can all be obtained from commercial sources. For those not specified in the following examples, they are carried out under conventional conditions or the conditions recommended by the manufacturer. For reagents or instruments without indicating the manufacturer, they are all conventional products that can be obtained through commercial purchase.

[0046] Example 1

[0047] The following method is used to synthesize the HBA label PC1, and its specific synthesis route is as follows:

[0048]

[0049] Methyl 3,5-dihydroxybenzoate (5.0 g, 30 mmol) was added to DMA (180 mL). While stirring, K2CO3 (20.7 g, 150 mmol) was added, and TBAB (1.9 g, 0.6 mmol) was added. After stirring for 15 minutes, 1-bromodocosane (29.2 g, 75 mmol) was added. The mixture was reacted at 90 °C for 10 hours. After the temperature in the flask dropped to room temperature, water (300 mL) was added, and the mixture was stirred for 20 minutes. Then, filtration was carried out, and the precipitate was collected. The filter cake was washed twice with water and twice with methanol, and finally intermediate 1-1a (22.1 g, separation yield 94%) was obtained. After drying, it was used for the next reaction.

[0050] Intermediate 1-1a (15.7 g, 20.0 mmol) was dissolved in 200 mL of DCM. Diisobutylaluminum hydride (DIBAL-H) (1.0 mol / L THF solution, 60 mL, 60 mmol) was added dropwise under a nitrogen atmosphere at 0 °C. After stirring at room temperature for 6 hours, 0.2 M hydrochloric acid (50 mL) was added dropwise at 0 °C to quench the reaction. 200 mL of DCM was added to the reaction solution for dilution and extraction. The extract was washed three times with 0.2 M hydrochloric acid (200 mL), once with 5% aqueous sodium bicarbonate solution (200 mL), once with 20% brine (200 mL), and then dried over sodium sulfate. Concentration under reduced pressure gave PC1 (14.9 g, yield 97%). Characterization data of PC1:

[0051] 1 H NMR (400 MHz, Chloroform-d) δ 6.50 (d, J = 2.2 Hz, 2H), 6.39 - 6.35 (t, J = 2.3 Hz, 1H), 4.62 (d, J = 5.3 Hz, 2H), 3.97 - 3.89 (t, J = 6.6 Hz, 4H), 1.81 - 1.71 (m, 4H), 1.48 - 1.39 (m, 4H), 1.36 - 1.20 (s, 72H), 0.92 - 0.84 (t, J = 6.8 Hz, 6H).

[0052] Examples 2 - 4

[0053] HBA label PC2 was synthesized by the following method, and its specific synthetic route was:

[0054]

[0055] Methyl 3,5-dihydroxybenzoate (5.0 g, 30 mmol) was added to DMF (120 mL). K2CO3 (20.7 g, 150 mmol) was added with stirring, and TEBA (1.4 g, 0.6 mmol) was added. After stirring for 15 minutes, a solution of Phy-Br (26.0 g, 75 mmol) in THF (60 mL) was added. The reaction was refluxed and stirred at 90 °C for 10 hours. After the temperature in the flask dropped to room temperature, THF was removed by distillation under reduced pressure at low temperature. Deionized water (300 mL) was added, and the mixture was stirred for 10 minutes. After the solid was completely dissolved, extraction was performed with ethyl acetate (3 x 100 mL), and washing was carried out with saturated NaCl (3 x 100 mL). Drying was done over anhydrous NaSO4. Intermediate 1-2a (19.9 g, yield 90%) was obtained.

[0056] Two other phase transfer catalysts were used, and the specific operations were similar to the above and will not be elaborated. The separation yields of intermediate 1-2a are as follows:

[0057] Table 1

[0058]

[0059] 1-2a (14.6 g, 20.0 mmol) was dissolved in 200 mL of DCM. Diisobutylaluminum hydride (DIBAL-H) (1.0 mol / L THF solution, 60 mL, 60 mmol) was added dropwise under a nitrogen atmosphere at 0 °C. After stirring at room temperature for 6 hours, 0.2 M hydrochloric acid (50 mL) was slowly added dropwise at 0 °C to quench the reaction. The residue was diluted and extracted with 200 mL of DCM added. The extract was washed three times with 0.2 M hydrochloric acid (200 mL), once with 5% aqueous sodium bicarbonate solution (200 mL), once with 20% brine (200 mL), and then dried over anhydrous Na2SO4. Evaporation of the solvent gave PC2 (13.6 g, yield 97%). Characterization data of PC2:

[0060] 1 H NMR (400 MHz, Chloroform-d) δ 7.16 (d, J = 2.1 Hz, 2H), 6.63 (t, J = 2.1 Hz, 1H), 4.61 (d, J = 2.3 Hz, 2H), 3.92 (q, J = 6.6 Hz, 4H), 1.81 (t, J = 12.6 Hz, 2H), 1.70 - 1.64 (m, 2H), 1.56 - 1.46 (m, 4H), 1.37 (s, 3H), 1.26 (t, J = 11.0 Hz, 22H), 1.18 - 1.11 (m, 7H), 1.11 - 1.03 (m, 8H), 0.94 (d, J = 6.5 Hz, 6H), 0.85 (d, J = 14.7 Hz, 24H).

[0061] Example 5

[0062] The active ester of PC1 was prepared by the following method, and the specific route was as follows:

[0063]

[0064] PC1 (7.57 g, 10 mmol) was dissolved in 80 mL of anhydrous THF. CDI (2.43 g, 15 mmol) was added with stirring. The reaction was stirred at room temperature for 10 minutes. The solvent was evaporated at low temperature, 400 mL of DCM was added, and then it was washed three times with saturated brine. The organic layer was dried over anhydrous Na2SO4, and the solvent was rotary evaporated under vacuum to obtain 8.16 g of white solid 2-1a, and the isolated yield was 94%.

[0065] Characterization data of 2-1a:

[0066] . 11H NMR (400 MHz, Chloroform-d) δ 6.50 (d, J = 2.3 Hz, 2H), 6.46 - 6.36 (m, 1H), 5.25 - 5.21 (s, 1H), 4.64 - 4.57 (s, 1H), 3.97 - 3.89 (t, J = 6.6 Hz, 4H), 2.88 - 2.77 (s, 2H), 1.80 - 1.72 (m, 4H), 1.48 - 1.40 (m, 5H), 1.35 - 1.22 (m, 72H), 0.91 - 0.85 (t, J = 6.7 Hz, 6H).

[0067] Intermediate 2-1a (0.87 g, 1 mmol) was dissolved in 8 mL of anhydrous DCM, and TfOH (0.225 g, 1.5 mmol) was added dropwise at room temperature. The reaction was stirred at room temperature for 10 min. HOSu (0.35 g, 3 mmol) was dissolved in 1.5 ml of MeCN and added dropwise to the reaction vessel. The reaction was transferred to a 25 °C oil bath and stirred for 8 h. A large amount of white precipitate was formed in the reaction solution. The solution was concentrated under reduced pressure, 20 mL of DCM was added to dissolve the mixture, and the mixture was washed three times with saturated brine. The organic layer was dried over anhydrous Na2SO4, the solvent was removed at low temperature, and the solid was washed three times with MeCN (10 mL x 3). Column chromatography was used to obtain 0.70 g of white solid 2-1b, and the separation yield was 81%.

[0068] Characterization data of 2-1b:

[0069] 1 1H NMR (400 MHz, Chloroform-d) δ 6.50 (d, J = 2.2 Hz, 2H), 6.46 - 6.42 (t, J = 2.3 Hz, 1H), 5.24 - 5.20 (s, 2H), 3.96 - 3.89 (t, J = 6.6 Hz, 4H), 2.88 - 2.78 (s, 4H), 1.80 - 1.72 (m, 4H), 1.46 - 1.42 (m, 4H), 1.32 - 1.23 (m, 72H), 0.90 - 0.86 (m, 6H).

[0070] Examples 6 - 8

[0071] The active ester of PC2 was prepared by the following method, and the specific route was as follows:

[0072]

[0073] PC2 (7.01 g, 10 mmol) was dissolved in 50 mL of anhydrous DCM, and CDI was added under stirring

[0074] (2.43 g, 15 mmol). The reaction was stirred at room temperature for 10 minutes. The mixture was diluted to 200 mL with DCM and then washed three times with saturated brine. The organic layer was dried over anhydrous Na2SO4 and the solvent was rotary evaporated under vacuum to give 7.63 g of a pale yellow liquid 2-2a with an isolated yield of 96%.

[0075] The characterization data of 2-2a are as follows:

[0076] 1 1H NMR (400 MHz, Chloroform-d) δ 8.18 - 8.13 (s, 1H), 7.45 - 7.43 (s, 1H), 7.07 - 7.05 (s, 1H), 6.54 (d, J = 2.2 Hz, 2H), 6.48 - 6.44 (t, J = 2.3 Hz, 1H), 5.35 - 5.29 (s, 2H), 4.04 - 3.91 (m, 4H), 1.89 - 1.76 (m, 2H), 1.72 - 1.65 (m, 2H), 1.60 - 1.46 (m, 4H), 1.38 - 1.04 (m, 40H), 0.94 (d, J = 6.5 Hz, 6H), 0.90 - 0.81 (m, 24H).

[0077] Intermediate 2-2a (0.80 g, 1 mmol) was dissolved in 4 mL of anhydrous THF, and methanesulfonic acid (0.07 mL, 1.5 mmol) was added dropwise at room temperature. The reaction was stirred at room temperature for 10 min, HOSu (0.35 g, 3.0 mmol) dissolved in 1.5 ml of MeCN was added dropwise to the reaction, and the reaction was transferred to a 35 °C oil bath and stirred for 8 h. A large amount of white imidazole methanesulfonate precipitate was formed in the reaction solution. The solvent was rotary evaporated at low temperature under vacuum, 20 mL of DCM was added to dissolve the mixture, and it was washed three times with saturated brine. The organic layer was dried over anhydrous Na2SO4 and the solvent was removed at low temperature. Column chromatography separation gave 0.74 g of a pale yellow liquid 2-2b with an isolated yield of 88%.

[0078] Using different acidifying agents, the specific operations were similar to the above and will not be elaborated. The yields of intermediate 2-2b are as follows:

[0079] Table 2

[0080]

[0081] The characterization data of 2-2b are as follows:

[0082] 11H NMR (400 MHz, Chloroform-d) δ 6.50 (d, J = 2.2 Hz, 2H), 6.46 - 6.42 (m, 1H), 5.29 - 5.10 (s, 2H), 4.02 - 3.89 (m, 4H), 2.97 - 2.93 (s, 1H), 2.91 - 2.87 (s, 1H), 2.85 - 2.80 (s, 2H), 1.85 - 1.77 (m, 2H), 1.70 - 1.63 (m, 2H), 1.58 - 1.47 (m, 4H), 1.38 - 1.06 (m, 40H), 0.96 - 0.91 (m, 6H), 0.89 - 0.82 (m, 24H).

[0083] Example 9

[0084] The following method was used to anchor amino acids. Taking the anchoring of L-alanine with the active ester 2-1b of PC1 as an example, the specific route is as follows:

[0085]

[0086] (1) Place L-Ala-OH (0.18 g, 2 mmol) in a 20 mL pressure-resistant tube, add it to anhydrous MeCN (8 mL), and dropwise add N-trimethylsilylacetamide (TMSA) (1.2 mL, 4.8 mmol) at room temperature. Heat the pressure-resistant tube in an oil bath at 40 °C for 2 h, and L-alanine completely dissolves in MeCN. The reaction is not treated, and the reaction solution is directly added to the subsequent reaction.

[0087] (2) Dissolve 2-1b (0.70 g, 0.8 mmol) in 6 mL of DCM, add the solution of transiently protected L-Ala-OH prepared in step (1) (4.6 mL, 1.0 mmol), and stir at room temperature for 30 min. Remove the solvent, add 10% citric acid (10 mL) and stir for 10 min. Filter out the filter cake and wash it three times repeatedly. Wash it once with MeCN (10 mL), filter and separate the filter cake, and dry to obtain 0.58 g of white solid, and the separation yield is 94%.

[0088] The characterization data of PC1-Ala-OH are as follows:

[0089] 11H NMR (400 MHz, Chloroform-d) δ 6.45 (d, J = 2.2 Hz, 2H), 6.40 - 6.37 (t, J = 2.2 Hz, 1H), 5.06 - 4.94 (m, 2H), 4.38 - 4.19 (s, 1H), 3.94 - 3.87 (t, J = 6.6 Hz, 4H), 1.79 - 1.70 (m, 4H), 1.46 - 1.39 (m, 4H), 1.31 - 1.20 (s, 75H), 0.89 - 0.86 (t, J = 6.5 Hz, 6H).

[0090] 13 13C NMR (101 MHz, CDCl3) δ 172.6, 160.4 (2C), 156.1, 138.1, 106.2 (2C), 101.0, 68.1 (2C), 41.1, 39.4, 31.9 (2C), 29.7 (32C), 29.6 (2C), 29.4 (2C), 26.1, 24.9, 22.7 (2C), 21.8, 14.2 (2C).

[0091] Example 10

[0092] The following method was used to anchor amino acids. Taking the anchoring of L-phenylalanine with the active ester 2-2b of PC2 as an example, the specific route is as follows:

[0093]

[0094] Place L-Phe-OH (0.33 g, 2 mmol) in a 20 mL pressure-resistant tube, add it to anhydrous DCM (8 mL), and dropwise add N,O-bis(trimethylsilyl)acetamide (BSA) (1.2 mL, 4.8 mmol) at room temperature. Heat the pressure-resistant tube in an oil bath at 40 °C for 2 h until L-phenylalanine is completely dissolved in DCM. Without further treatment, directly add the reaction solution to the subsequent reaction.

[0095] Dissolve 2-2b (7.19 g, 8.5 mmol) in 20 mL of DCM, add the transiently protected L-Ala-OH solution prepared in step (1) (48.9 mL, 10.5 mmol), and stir at room temperature for 30 min. Dilute with DCM to 200 mL, add 10% citric acid (100 mL × 3), separate and retain the organic layer. Dry the organic layer over anhydrous Na2SO4 and remove the solvent to obtain 7.36 g of a light yellow liquid with a separation yield of 95%.

[0096] The characterization data of PC2-Phe-OH are as follows:

[0097] 11H NMR (400 MHz, Chloroform-d) δ 7.32 - 7.24 (m, 3H), 7.16 (d, J = 7.1 Hz, 2H), 6.49 - 6.44 (s, 2H), 6.43 - 6.39 (m, 1H), 5.25 (d, J = 7.7 Hz, 1H), 5.12 - 4.92 (m, 2H), 4.74 - 4.63 (q, J = 6.4 Hz, 1H), 4.04 - 3.87 (m, 4H), 3.27 - 3.04 (m, 2H), 1.90 - 1.75 (m, 2H), 1.74 - 1.63 (m, 2H), 1.61 - 1.48 (m, 4H), 1.39 - 1.03 (m, 40H), 0.95 (d, J = 6.5 Hz, 6H), 0.91 - 0.77 (m, 24H).

[0098] 13 13C NMR (101 MHz, CDCl3) δ 175.9, 160.4 (2C), 155.8, 138.1, 135.6, 129.4 (2C), 128.7 (2C), 127.2, 106.3 (2C), 101.1, 67.2, 66.4 (2C), 54.7, 39.4 (2C), 37.5 (6C), 37.3 (6C), 36.3, 32.8 (4C), 29.9 (2C), 28.0 (2C), 24.8 (2C), 24.5 (2C), 24.4 (2C), 22.7 (4C), 19.8 (6C).

[0099] Example 11

[0100] The following method was used to anchor amino acids. Taking the anchoring of L-alanine with the active ester 2-2b of PC2 as an example, the specific route is as follows:

[0101]

[0102] L-Ala-OH (0.18 g, 2 mmol) was placed in a 20 mL pressure-resistant tube and added to anhydrous 2-MeTHF (8 mL). At room temperature, N,O-bis(trimethylsilyl)acetamide (BSA) (1.2 mL, 4.8 mmol) was added dropwise. The pressure-resistant tube was heated in an oil bath at 40 °C for 2 h, and L-alanine was completely dissolved in 2-MeTHF. The reaction was not treated, and the reaction solution was directly added to the subsequent reaction.

[0103] Dissolve 2-2b (0.67 g, 0.8 mmol) in 6 mL of 2-MeTHF, add the transiently protected L-alanine solution (4.6 mL, 1.0 mmol) prepared in step (1), and stir at room temperature for 30 min. Dilute with 2-MeTHF to 20 mL, wash with 10% citric acid (10 mL × 3), separate the layers and retain the organic layer. Dry the organic layer over anhydrous Na2SO4, remove the solvent to obtain 0.62 g of a pale yellow liquid, and the isolation yield is 95%.

[0104] The characterization data of PC2-Ala-OH are as follows:

[0105] 1 H NMR (400 MHz, Chloroform-d) δ 6.49 - 6.45 (s, 2H), 6.42 - 6.38 (t, J = 2.3 Hz, 1H), 5.38 - 5.32 (m, 1H), 5.11 - 4.95 (m, 2H), 4.48 - 4.35 (m, 1H), 4.02 - 3.89 (m, 4H), 1.86 - 1.75 (m, 2H), 1.71 - 1.62 (m, 2H), 1.61 - 1.49 (m, 4H), 1.46 (d, J = 7.2 Hz, 3H), 1.38 - 1.02 (m, 40H), 0.93 (d, J = 6.5 Hz, 6H), 0.88 - 0.83 (m, 24H).

[0106] 13 C NMR (101 MHz, CDCl3) δ 177.3, 160.4 (2C), 155.8, 138.1, 106.3 (2C), 101.1, 67.2, 66.4 (2C), 49.5, 39.4 (2C), 37.4 (6C), 37.3 (6C), 36.3, 32.8 (4C), 29.9, 28.0 (2C), 24.8 (2C), 24.5 (2C), 24.4 (2C), 22.7 (4C), 19.8 (6C), 18.4.

[0107] Example 12

[0108] In this example, the hydrophobic dipeptide PC2-Phe-Phe-OH was prepared.

[0109]

[0110] The specific process is as follows:

[0111] L-Phe-OH (0.11 g, 1.2 mmol) was placed in a 20 mL pressure-resistant tube and added to anhydrous DCM (5.6 mL). At room temperature, hexamethyldisilazane (HMDS) (1.2 mL, 4.8 mmol) was added dropwise. The pressure-resistant tube was heated in an oil bath at 40 °C for 2 h, and L-alanine was completely dissolved in 2-MeTHF. The reaction was not treated, and the reaction solution was directly added to the subsequent reaction.

[0112] At room temperature, PC2-Phe-OH (0.89 g, 1 mmol) was dissolved in the solvent (5 mL) DCM. A solution of HOSu (0.17 g, 1.5 mmol) in MeCN (1 mL) was added. After stirring for 10 min, EDCI (0.23 g, 1.2 mmol) was added. After continuing to stir for 50 min, transiently protected L-Phe-OH (5.6 mL, 1.2 mmol) was added. After the mixture was stirred for 1 h, it was diluted to 20 mL with DCM and washed with 10% citric acid (10 mL × 3). The organic layer was separated and retained, and the organic layer was dried over anhydrous Na2SO4. After removing the solvent, 0.96 g of a light yellow liquid was obtained, and the separation yield was 92%.

[0113] The characterization data of PC2-Phe-Phe-OH are as follows:

[0114] 1 H NMR (400 MHz, Chloroform-d) δ 7.26 - 7.12 (m, 8H), 7.06 (d, J = 5.5 Hz, 2H), 6.76 - 6.68 (m, 1H), 6.50 - 6.44 (s, 2H), 6.43 - 6.40 (m, 1H), 5.64 (d, J = 8.3 Hz, 1H), 5.04 - 4.93 (m, 2H), 4.83 - 4.75 (q, J = 6.4 Hz, 1H), 4.56 - 4.46 (m, 1H), 4.02 - 3.88 (m, 4H), 3.19 - 3.10 (m, J = 13.9, 5.5 Hz, 1H), 3.07 - 2.91 (m, 3H), 1.89 - 1.78 (m, 2H), 1.73 - 1.63 (m, 2H), 1.63 - 1.48 (m, 4H), 1.40 - 1.05 (m, 40H), 0.95 (d, J = 6.5 Hz, 6H), 0.91 - 0.85 (m, 24H).

[0115] 1313C NMR (101 MHz, CDCl3) δ 174.0, 171.1, 160.5 (2C), 156.1, 138.0, 136.2, 135.7, 129.4 (2C), 129.3 (2C), 128.6 (2C), 128.5 (2C), 127.1, 127.0, 106.3 (2C), 101.0, 67.2, 66.4 (2C), 56.0, 53.4, 39.4 (2C), 38.4, 37.5 (6C), 37.3 (6C), 36.3, 36.2, 32.8 (4C), 29.9 (2C), 28.0 (2C), 24.8 (2C), 24.5 (2C), 24.4 (2C), 22.7 (4C), 19.8 (6C).

[0116] Example 13

[0117] The hydrophobic dipeptide PC2-Phe-Leu-OH was synthesized by the following method, and the specific process is as follows:

[0118]

[0119] L-Leu-OH (0.16 g, 1.2 mmol) was placed in a 20 mL pressure-resistant tube and added to anhydrous DCM (5.6 mL). At room temperature, N,O-bis(trimethylsilyl)acetamide (BSA) (1.2 mL, 4.8 mmol) was added dropwise. The pressure-resistant tube was heated in an oil bath at 40 °C for 2 h, and L-alanine was completely dissolved in 2-MeTHF. The reaction was not treated, and the reaction solution was directly added to the subsequent reaction.

[0120] At room temperature, PC2-Phe-OH (0.89 g, 1 mmol) was dissolved in solvent (5 mL) DCM. A solution of HOSu (0.17 g, 1.5 mmol) in MeCN (1 mL) was added. After stirring for 10 min, EDCI (0.23 g, 1.2 mmol) was added. After continuing to stir for 50 min, the transiently protected L-leu-OH (5.6 mL, 1.2 mmol) prepared in step (1) was added. After the mixture was stirred for 1 h, it was diluted to 20 mL with DCM and washed with 10% citric acid (10 mL × 3). The organic layer was separated and retained, and the organic layer was dried over anhydrous Na2SO4. After removing the solvent, 0.90 g of a light yellow liquid was obtained, and the separation yield was 90%.

[0121] The characterization data of PC2-Phe-Leu-OH are as follows:

[0122] 11H NMR (400 MHz, Chloroform-d) δ 7.28 - 7.22 (m, 2H), 7.22 - 7.15 (t, J = 7.2 Hz, 3H), 6.72 - 6.54 (s, 1H), 6.47 - 6.42 (m, 2H), 6.42 - 6.38 (m, 1H), 5.74 - 5.57 (s, 1H), 5.04 - 4.93 (m, 2H), 4.61 - 4.48 (m, 2H), 4.03 - 3.87 (m, 4H), 3.05 (d, J = 7.0 Hz, 2H), 1.87 - 1.76 (m, 2H), 1.71 - 1.61 (m, 3H), 1.60 - 1.47 (m, 6H), 1.38 - 1.03 (m, 40H), 0.94 (d, J = 6.5 Hz, 6H), 0.92 - 0.89 (m, 6H), 0.89 - 0.83 (m, 24H).

[0123] 13 13C NMR (101 MHz, CDCl3) δ 175.7, 171.2, 160.4 (2C), 156.1, 138.0, 136.2, 129.4 (2C), 128.7 (2C), 127.1, 106.2 (2C), 101.0, 67.2, 66.4 (2C), 56.0, 51.0, 41.2, 39.4 (2C), 38.5, 37.5 (6C), 37.3 (6C), 35.9, 32.8 (4C), 29.9 (2C), 28.0 (2C), 24.8 (2C), 24.5 (2C), 24.4 (2C), 22.7 (4C), 21.9 (2C), 19.8 (6C).

[0124] Example 14

[0125] The hydrophobic dipeptide PC2 - Phe - Trp - OH was synthesized by the following method.

[0126]

[0127] The specific process is as follows:

[0128] L - Trp - OH (0.25 g, 1.2 mmol) was placed in a 20 mL pressure - resistant tube and added to anhydrous DCM (5.6 mL). At room temperature, N,O - bis(trimethylsilyl)acetamide (BSA) (1.2 mL, 4.8 mmol) was added dropwise. The pressure - resistant tube was heated in an oil bath at 40 °C for 2 h, and L - alanine was completely dissolved in 2 - MeTHF. The reaction was not treated, and the reaction solution was directly added to the subsequent reaction.

[0129] At room temperature, PC5-Phe-OH (0.89 g, 1 mmol) was dissolved in the solvent (5 mL) DCM, and a solution of HOSu (0.17 g, 1.5 mmol) in MeCN (1 mL) was added. After stirring for 10 min, EDCI (0.23 g, 1.2 mmol) was added, and after continuing to stir for 50 min, the transiently protected L-Trp-OH (5.6 mL, 1.2 mmol) prepared in step (1) was added. After the mixture was stirred for 1 h, it was diluted to 20 mL with DCM and washed with 10% citric acid (10 mL × 3). The organic layer was separated and retained, and the organic layer was dried over anhydrous Na2SO4, and the solvent was removed to obtain 0.90 g of a light yellow liquid, and the separation yield was 81%.

[0130] The characterization data of PC2-Phe-Trp-OH are as follows:

[0131] 1 1H NMR (400 MHz, Chloroform-d) δ 8.23 - 8.11 (s, 1H), 7.44 (d, J = 7.9 Hz, 1H), 7.24 (d, J = 12.8 Hz, 1H), 7.21 - 6.99 (m, 7H), 6.81 - 6.73 (s, 2H), 6.45 (d, J = 10.1 Hz, 3H), 5.55 (d, J = 8.4 Hz, 1H), 4.98 - 4.87 (s, 2H), 4.87 - 4.78 (q, J = 6.0 Hz, 1H), 4.53 - 4.41 (q, J = 7.4 Hz, 1H), 4.02 - 3.89 (m, 4H), 3.33 - 3.16 (m, 2H), 3.03 - 2.85 (m, 2H), 1.88 - 1.77 (m, 2H), 1.73 - 1.62 (m, 2H), 1.62 - 1.49 (m, 4H), 1.41 - 1.06 (m, 40H), 0.94 (d, J = 6.6 Hz, 6H), 0.91 - 0.83 (m, 24H).

[0132] 1313C NMR (101 MHz, CDCl3) δ 174.3, 171.3, 160.4 (2C), 156.1, 138.2, 136.1, 136.0, 129.3 (2C), 128.6 (2C), 127.6, 127.0, 123.5, 122.0, 119.6, 118.5, 111.4, 109.3, 106.4 (2C), 100.9, 67.1, 66.5 (2C), 56.0, 53.1, 39.4 (2C), 38.4, 37.5 (6C), 37.3 (6C), 36.2, 32.8 (4C), 29.9 (2C), 28.0 (2C), 24.8 (2C), 24.5, (2C) 24.4 (2C), 22.7 (4C), 19.8 (6C).

[0133] Example 15

[0134] The hydrophobic dipeptide PC2-Phe-Met-OH was synthesized by the following method.

[0135]

[0136] The specific process is as follows:

[0137] L-Met-OH (0.18 g, 1.2 mmol) was placed in a 20 mL pressure-resistant tube and added to anhydrous DCM (5.6 mL). At room temperature, N,O-bis(trimethylsilyl)acetamide (BSA) (1.2 mL, 4.8 mmol) was added dropwise. The pressure-resistant tube was heated in an oil bath at 40 °C for 2 h, and L-alanine was completely dissolved in 2-MeTHF. The reaction was not treated, and the reaction solution was directly added in the subsequent reaction.

[0138] At room temperature, PC5-Phe-OH (0.89 g, 1 mmol) was dissolved in solvent (5 mL) DCM. A solution of HOSu (0.17 g, 1.5 mmol) in MeCN (1 mL) was added. After stirring for 10 min, EDCI (0.23 g, 1.2 mmol) was added. After continuing to stir for 50 min, the transiently protected L-Met-OH prepared in step (1) (5.6 mL, 1.2 mmol) was added. After the mixture was stirred for 1 h, it was diluted to 20 mL with DCM and washed with 10% citric acid (10 mL × 3). The organic layer was separated and retained, and the organic layer was dried over anhydrous Na2SO4. After removing the solvent, 0.93 g of a light yellow liquid was obtained, and the separation yield was 89%.

[0139] The characterization data of PC2-Phe-Met-OH are as follows:

[0140] 11H NMR (400 MHz, Chloroform-d) δ 7.28 - 7.22 (m, 2H), 7.22 - 7.15 (t, J = 7.9 Hz, 3H), 7.07 (d, J = 7.6 Hz, 1H), 6.46 - 6.42 (s, 2H), 6.42 - 6.38 (t, J = 2.2 Hz, 1H), 5.76 (d, J = 7.9 Hz, 1H), 5.06 - 4.88 (m, 2H), 4.69 - 4.62 (q, J = 7.4 Hz, 1H), 4.62 - 4.51 (m, 1H), 4.03 - 3.86 (m, 4H), 3.06 (d, J = 7.1 Hz, 2H), 2.51 - 2.39 (m, 2H), 2.24 - 2.10 (m, 1H), 2.06 - 2.02 (s, 3H), 2.02 - 1.94 (m, 1H), 1.88 - 1.76 (m, 2H), 1.73 - 1.62 (m, 2H), 1.62 - 1.48 (m, 4H), 1.39 - 1.05 (m, 40H), 0.94 (d, J = 6.5 Hz, 6H), 0.90 - 0.83 (m, 24H).

[0141] 13 13C NMR (101 MHz, CDCl3) δ 174.3, 171.6, 160.4 (2C), 156.2, 138.0 136.1, 129.4 (2C), 128.7 (2C), 127.1, 106.3 (2C), 101.0, 67.3, 66.4 (2C), 56.1, 51.8, 39.4 (2C), 38.5, 37.5 (6C), 37.3 (6C), 36.3, 32.8 (4C), 31.2, 29.9 (2C), 28.0 (2C), 24.8 (2C), 24.5 (2C), 24.4 (2C), 22.7 (4C), 19.8 (6C), 15.3.

[0142] Example 16

[0143] The hydrophobic dipeptide PC2 - Phe - His(Trt) - OH was synthesized by the following method,

[0144]

[0145] The specific process is as follows:

[0146] Place L-His(Trt)-OH (0.11 g, 1.2 mmol) in a 20 mL pressure-resistant tube, add it to anhydrous DCM (5.6 mL), and dropwise add N,O-bis(trimethylsilyl)acetamide (BSA) (1.2 mL, 4.8 mmol) at room temperature. Heat the pressure-resistant tube in an oil bath at 40 °C for 2 h, and L-alanine is completely dissolved in 2-MeTHF. The reaction is not treated, and the reaction solution is directly added in the subsequent reaction.

[0147] Dissolve PC5-Phe-OH (0.89 g, 1 mmol) in solvent (5 mL) DCM at room temperature, add a solution of HOSu (0.17 g, 1.5 mmol) in MeCN (1 mL), stir for 10 min, then add EDCI (0.23 g, 1.2 mmol), continue to stir for 50 min, and then add the transiently protected L-His(Trt)-OH prepared in step (1) (5.6 mL, 1.2 mmol). After the mixture is stirred for 1 h, dilute it to 20 mL with DCM, and wash it with 10% citric acid (10 mL × 3). Separate and retain the organic layer, dry the organic layer over anhydrous Na2SO4, remove the solvent to obtain 1.17 g of a light yellow liquid, and the separation yield is 84%. The characterization data of PC2-Phe-His(Trt)-OH are as follows:

[0148] 1 1H NMR (400 MHz, Chloroform-d) δ 7.86 - 7.74 (s, 1H), 7.45 - 7.27 (m, 7H), 7.22 - 7.13 (m, 2H), 7.13 - 7.01 (m, 8H), 6.98 - 6.86 (s, 1H), 6.79 - 6.62 (s, 1H), 6.49 - 6.22 (m, 3H), 5.29 (d, J = 7.6 Hz, 1H), 4.93 (d, J = 12.2 Hz, 1H), 4.76 (d, J = 12.5 Hz, 1H), 4.57 - 4.48 (q, J = 4.9 Hz, 1H), 4.24 - 4.15 (m, 1H), 4.01 - 3.86 (m, 4H), 3.34 - 3.12 (m, 2H), 3.07 - 2.87 (m, 2H), 1.89 - 1.77 (m, 2H), 1.72 - 1.63 (m, 2H), 1.62 - 1.48 (m, 4H), 1.40 - 1.05 (m, 40H), 0.94 (d, J = 6.6 Hz, 6H), 0.90 - 0.83 (m, 24H).

[0149] 1313C NMR (101 MHz, CDCl3) δ 173.6, 170.3, 160.4 (2C), 155.5, 141.2 (3C), 138.3, 136.9, 136.2, 132.9, 129.7 (6C), 129.3 (3C), 128.6 (4C), 128.4 (6C), 126.9, 121.4, 106.3 (2C), 100.9, 66.9, 66.4 (2C), 56.0, 52.9, 39.4 (2C), 38.6, 37.5 (6C), 37.3 (6C), 36.3, 32.8 (4C), 29.9 (2C), 28.0 (2C), 24.8 (2C), 24.5 (2C), 24.4 (2C), 22.8 (4C), 19.8 (6C).

[0150] Examples 17 - 25

[0151] The hydrophobic tripeptide PC2 - Phe - His(Trt) - Val - OMe was synthesized by the following method, and the specific route is as follows:

[0152]

[0153] At room temperature, PC2 - Phe - His(Trt) - OH (0.70 g, 0.5 mmol) was dissolved in the solvent (3 mL) DCM, and a solution of HOSu (0.09 g, 0.75 mmol) in MeCN (0.5 mL) was added. After stirring for 10 min, EDCI (0.12 g, 0.6 mmol) was added, and after continuing to stir for 50 min, L - Phe - OMe (0.08 g, 0.6 mmol) was added. After the mixture was stirred for 1 h, it was diluted to 20 mL with DCM and washed with 10% citric acid (10 mL × 3). The organic layer was separated and retained, dried over anhydrous Na2SO4, and the solvent was removed to obtain 0.57 g of a light yellow liquid, with a separation yield of 81%.

[0154] Using different combinations of activators, the specific operations were similar to those above and will not be elaborated. The yields of PC2 - Phe - His(Trt) - Val - OMe are as follows:

[0155] Table 3

[0156]

[0157] Note (EDCI: 1 - (3 - dimethylaminopropyl) - 3 - ethylcarbodiimide hydrochloride; DIPEA: N,N - diisopropylethylamine; DIEA: N,N - diisopropylethylamine)

[0158] The characterization data of PC2 - Phe - His(Trt) - Val - OMe are as follows:

[0159] 1 1H NMR (400 MHz, Chloroform-d) δ 8.26 (d, J = 7.0 Hz, 1H), 7.54 (d, J = 8.5 Hz, 1H), 7.25 - 7.18 (m, 9H), 7.18 - 7.04 (m, 6H), 7.03 - 6.94 (m, 6H), 6.60 - 6.55 (s, 1H), 6.30 - 6.25 (s, 3H), 5.38 (d, J = 6.8 Hz, 1H), 4.93 (d, J = 12.5 Hz, 1H), 4.71 - 4.58 (m, 2H), 4.45 - 4.37 (q, J = 6.9 Hz, 1H), 4.37 - 4.31 (m, J = 8.5, 5.2 Hz, 1H), 3.89 - 3.77 (m, 4H), 3.61 - 3.54 (s, 3H), 3.16 (d, J = 14.1 Hz, 1H), 3.06 - 2.90 (m, 2H), 2.86 - 2.74 (m, J = 15.0, 6.0 Hz, 1H), 2.09 - 1.98 (m, 1H), 1.78 - 1.65 (m, 2H), 1.64 - 1.52 (m, 2H), 1.52 - 1.37 (m, 4H), 1.30 - 0.95 (m, 40H), 0.85 (d, J = 6.5 Hz, 6H), 0.81 - 0.73 (m, 30H).

[0160] 13 13C NMR (101 MHz, CDCl3) δ 170.9, 169.8, 159.3 (2C), 155.0, 141.2 (3C), 137.2, 137.0, 135.8, 135.4, 128.7 (6C), 128.2 (2C), 127.7 (2C), 127.1 (3C), 127.0 (6C), 125.9, 118.5, 105.0 (2C), 99.9, 74.3, 65.9, 65.3 (2C), 56.6, 55.4, 52.4, 50.9, 38.3 (2C), 37.2, 36.4 (6C), 36.3 (6C), 35.3, 31.8 (4C), 29.8, (2C) 28.8 (2C), 26.9, (2C) 23.8 (2C), 23.5 (2C), 23.4 (2C), 21.6 (2C), 18.8 (6C), 18.1, 17.0.

[0161] Examples 26 - 29

[0162] The HBA tag was removed by the following method. Taking the removal of the HBA tag PC2 from the hydrophobic tripeptide PC2 - Phe - His(Trt) - Val - OMe as an example, the specific route is as follows:

[0163]

[0164] Dissolve PC2-Phe-His(Trt)-Val-OMe (0.28 g, 0.2 mmol) in THF (0.6 mL) and place it in a 5 mL reaction flask. Bubble nitrogen into the reaction flask to expel the air in the solvent THF (bubble nitrogen for 5 min). Place the reaction flask in a 100 mL hydrogenation autoclave and use nitrogen to displace the air in the autoclave (repeat 5 times). Introduce 2 MPa of hydrogen into the hydrogenation autoclave. Place the hydrogenation autoclave in a 25 °C water bath and stir the reaction for 12 h. Add THF to dilute the reaction to 5 mL, filter off the Pd / C, and evaporate the solvent to obtain a light brown liquid mixture. Pulp the mixture with MeCN (1 mL), let the mixture stand for 10 min to separate into layers, and use a syringe to take the upper clear liquid (repeat the above operation three times). Collect the upper clear liquid and remove the solvent to obtain the tripeptide compound H-Phe-His(Trt)-Val-OMe (0.12 g, yield 91%).

[0165] Prepare H-Phe-His(Trt)-Val-OMe using different solvents. The operation is similar to the above and will not be elaborated here. The yields of H-Phe-His(Trt)-Val-OMe are as follows:

[0166] Table 4

[0167]

[0168] The characterization data of H-Phe-His(Trt)-Val-OMe are as follows:

[0169] 11H NMR (500 MHz, DMSO-d6) δ 8.23 (d, J = 8.3 Hz, 1H), 8.16 (d, J = 8.3 Hz, 1H), 7.40 - 7.35 (m, 9H), 7.26 - 7.22 (m, 3H), 7.18 - 7.14 (m, 3H), 7.07 - 7.04 (m, J = 8.0, 1.8 Hz, 6H), 6.65 (d, J = 1.4 Hz, 1H), 4.69 - 4.62 (m, 1H), 4.22 - 4.16 (m, J = 8.3, 6.0 Hz, 1H), 3.51 - 3.48 (s, 3H), 3.38 - 3.35 (m, J = 8.9, 4.2 Hz, 1H), 2.94 - 2.88 (m, 1H), 2.87 - 2.81 (m, J = 14.8, 4.7 Hz, 1H), 2.80 - 2.72 (m, J = 14.7, 8.1 Hz, 1H), 2.49 - 2.42 (m, J = 13.5, 8.9 Hz, 1H), 2.08 - 1.98 (m, 1H), 0.86 - 0.83 (m, J = 6.9, 4.2 Hz, 6H).

[0170] Anchoring the N-terminus of the peptide chain with an HBA tag, gradually extending the peptide chain backward step by step in a fully liquid phase environment using different amino acid blocks, and removing the HBA tag to obtain the target peptide chain. The specific preparation process is similar to the above and will not be elaborated here. The characterization data of the synthesized peptide chains are shown as follows:

[0171] H-Phe-Leu-Arg-Leu-His-Val-OH: Isolated yield 75%, HRMS (ESI) m / z calcd for C 38 H 61 N 11 O7[M + H] + : 784.4755, found: 784.4762.

[0172] H-Asp-Arg-Val-Tyr-Ile-His-Pro-Phe-His-OH: Isolated yield 56%, HRMS (ESI) m / z calcd for C 56 H 78 N 16 O 13 [M + H] + : 1183.5934, found: 1183.5939.

[0173] Although the embodiments of the present invention have been disclosed as above, they are not limited to the applications listed in the specification and embodiments. It can be fully applied to various fields suitable for the present invention. For those skilled in the art, additional modifications can be easily made. Therefore, without departing from the general concept defined by the claims and the equivalent scope, the present invention is not limited to specific details.

Claims

1. A label-assisted reverse liquid-phase polypeptide synthesis method, characterized in that It includes the following steps: (1) Prepare hydrophobic benzyl alcohol (HBA) tags. The synthesis route is as follows: Obtain HBA tags PC1 and PC2, and their structures are as follows: Methyl 3,5-dihydroxybenzoate is alkylated in Solvent 1 to obtain Compound 1. The methyl ester bond of Compound 1 is reduced to a hydroxyl group by a reducing agent in THF to obtain Compound 2; (2) Prepare HBA tag active esters. The synthesis route is as follows; At the reaction temperature, Compound 2 and N,N'-carbonyldiimidazole (CDI) are mixed in Solvent 3 to react to obtain Intermediate 3; at the reaction temperature, Intermediate 3 and N-hydroxysuccinimide (HOSu) are mixed in Solvent 3 to react to obtain Compound 4; (3) Prepare HBA tag-amino acids. The synthesis route is as follows: Compound 4 and Compound 5 are mixed and reacted in Solvent 3, and after acidification, Compound 6 is obtained; wherein, R2 includes alkyl, heterocycloalkyl, aryl, heteroaryl; (4) Prepare HBA tag-polypeptides. The synthesis route is as follows: Compound 6 is activated by an activator in Solvent 3, and then coupled in one pot with Compound 5, and after acidification, Compound 7 is obtained; using Compound 7 as a substrate, repeat the operations of "in-situ activation of carboxyl-coupling transiently protected amino acids-removal of C-terminal silyl protection" to obtain polypeptide Compound 8; Wherein, R2 is as described in step (3); (5) Remove the HBA tag. The specific route is as follows: Compound 8 undergoes hydrogenolysis in Solvent 4 to remove the HBA tag to obtain Compound 9; Wherein, R2 is as described in step (3).

2. The label-assisted reverse liquid-phase polypeptide synthesis method according to claim 1, wherein The phase transfer catalyst in step (1) is one of benzyltriethylammonium chloride (TEBA), tetrabutylammonium chloride (TBAC), and tetrabutylammonium bromide (TBAB).

3. A label-assisted reverse liquid-phase polypeptide synthesis method according to claim 1, characterized in that, The Solvent 1 in step (1) is one or a mixture of any proportion of 2-methyltetrahydrofuran (2-MeTHF), tetrahydrofuran (THF), N,N-dimethylformamide (DMF), and N,N-dimethylacetamide (DMA).

4. A label-assisted reverse liquid-phase polypeptide synthesis method according to claim 1, characterized in that The acidifying reagent in step (2) is one or more of trifluoroacetic acid (TFA), trifluoromethanesulfonic acid (TfOH), and methanesulfonic acid (MsOH).

5. A label-assisted reverse liquid-phase polypeptide synthesis method according to claim 1, wherein The reaction temperature in step (2) is 0°C - 45°C.

6. A label-assisted reverse liquid-phase polypeptide synthesis method according to claim 1, characterized in that The molar ratio of CDI to Compound 2 in step (2) is (1 - 6):1; the molar ratio of HOSu to Compound 3 is (1 - 6):

1.

7. A label-assisted reverse liquid-phase polypeptide synthesis method according to claim 1, characterized in that The Solvent 3 in steps (2), (3), and (4) is one or more of acetonitrile (MeCN), dichloromethane (DCM), 2-methyltetrahydrofuran (2-MeTHF), and tetrahydrofuran (THF).

8. A label-assisted reverse liquid-phase polypeptide synthesis method according to claim 1, characterized in that The preparation methods of Compound 5 and Compound 7 in steps (3) and (4) are as follows: Wherein the silylating reagent is one or more of N-trimethylsilylacetamide (TMSA), bis(trimethylsilyl)acetamide (BSA), and hexamethyldisilazane (HMDS).

9. A label-assisted reverse liquid-phase polypeptide synthesis method according to claim 1, wherein The activator in the step (4) is one or more of hydroxypyridone (HOPO), 1-hydroxybenzotriazole (HOBT), 1-hydroxy-7-azabenzotriazole (HOAT), N-hydroxysuccinimide (HOSu), O-benzotriazol-1-yl-N,N,N',N'-tetramethyluronium hexafluorophosphate (HBTU), (7-azabenzotriazol-1-yloxy)tripyrrolidinophosphonium hexafluorophosphate (HATU), (7-azabenzotriazol-1-yloxy)tripyrrolidinophosphonium hexafluorophosphate (PyAOP), pyridinium chlorochromate ( PyOxim), [(1-cyano-2-ethoxy-2-oxoethylideneaminooxy)dimethylamino]morpholinocarbon hexafluorophosphate (COMU).

10. A tag-assisted reverse liquid-phase polypeptide synthesis method according to claim 1, characterized in that, The Solvent 4 in step (5) is one or more of 2-methyltetrahydrofuran (2-MeTHF), tetrahydrofuran (THF), dichloromethane (DCM), and ethyl acetate (EA).

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